Book of Abstracts

Transcription

Book of Abstracts

ISSY32nd
Book
of Abstracts
32nd INTERNATIONAL
SPECIALIZED SYMPOSIUM
ON YEASTS
YEAST BIODIVERSITY
AND BIOTECHNOLOGY
IN THE TWENTY-FIRST
CENTURY
SEPTEmBER 13-17, 2015
HOTEL GIò - PERUGIA CONGRESS CENTRE,
PERUgIA, ITALY
www.issy32.com
32nd International Specialized
Symposium on Yeasts
Yeasts Biodiversity and Biotechnology
in the Twenty-First Century
Perugia, Italy
September 13-17, 2015
BOOK OF ABSTRACTS
Edited by:
Pietro Buzzini
Lisa Granchi
Patrizia Romano
Benedetta Turchetti
Published by the Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, University of Perugia, Borgo XX
Giugno 74, I-06121, Perugia, Italy.
All right reserved.
ISBN 978-88-99407-00-1
http://www.unipg.it/
http://www.agr.unipg.it/
MAIN SPONSORS
CONTRIBUTING SPONSORS
SUPPORTING SPONSORS
OTHER SPONSORS
UNDER THE PATRONAGE OF
TABLE OF CONTENTS
Welcome to Perugia............................................................................................. I
From The Organizing Committee........................................................................ II
From The International Commission on Yeasts (ICY)........................................ III
From The University of Perugia.......................................................................... IV
Plenary Lecture and Key Notes Speakers............................................................ V
Programme ........................................................................................................ XIII
Gala Dinner and Tours......................................................................................... XXI
Perugia Informations............................................................................................ XXII
Maps of Perugia................................................................................................... XXIII
Congress Informations......................................................................................... XXV
Committes ........................................................................................................ XXVI
ORAL LECTURES ABSTRACTS...................................................................... 1
Opening Lecture................................................................................................... 2
Session 1A Yeasts in the environment: ecology and taxonomy........................ 3
Session 1B Yeasts in the environment: physiology and stress response........... 11
Session 2A Yeasts in food biotechnology:
biodiversity and ecology in foods and beverages........................... 19
Session 2B Yeasts in food biotechnology:
detection methods and strain improvement.................................... 33
Session 3
Yeasts in no-food biotechnology:
biofuels, new molecules and enzymes............................................ 39
Session 4 Yeasts genetic and genomic............................................................ 53
Session 5
Non-conventional yeasts................................................................. 67
Session 6
Yeasts Culture Collections.............................................................. 75
POSTERS ABSTRACTS.................................................................................... 81
Session 1A Yeasts in the environment: ecology and taxonomy........................ 83
Session 1B Yeasts in the environment: physiology and stress response........... 99
Session 2A Yeasts in food biotechnology:
biodiversity and ecology in foods and beverages........................... 115
Session 2B Yeasts in food biotechnology:
detection methods and strain improvement.................................... 171
Session 3 Yeasts in no-food biotechnology:
biofuels, new molecules and enzymes............................................ 187
Session 4
Yeasts genetic and genomic............................................................ 213
Session 5
Non-conventional yeasts................................................................. 233
Session 6
Yeast Culture Collections................................................................ 251
INDEX OF AUTHORS....................................................................................... 256
WELCOME TO PERUGIA
Dear attendees of the 32nd ISSY,
Welcome to Perugia!
Perugia the capital of the beautiful Umbria is one of the goals more evocative of the region, a city of
artists (Perugino, Pinturicchio and Raffaello, the contemporary art of Burri and Beuys). This city built
on a hill in the valley of the Tevere river is with the its history and its artistic and cultural heritage one
of the more favorite tourist destinations.
Starting from its Etruscan walls and walking the streets of Old Town, Perugia is a succession of
monuments.
Perugia is also an important cultural center, with its historic University and the oldest and most
prestigious University for Foreigners in Italy.
In addition to the artistic and cultural riches, Perugia offers tasty itineraries to try food and wine with
all the flavor of simple regional cuisine.
Perugia offers also many occasions for fun with pubs and discos or visiting it on the occasion of its
major events such as the renowned international festival Eurochocolate, which transforms Perugia
in October in the most coveted destination for lovers of the food of the gods, and even at the Umbria
Jazz Festival in July that gives ten days of the show, and jazz, ranking among the most important jazz
events in Europe.
Besides, the beauty of the surrounding towns (Assisi, Todi) and nature (Trasimeno lake, Marmore
falls) and the rich cultural heritage of the region will provide an excellent opportunity for scientific
presentations and discussions as well as for the informal encounters.
I
FROM THE ORGANIZING COMMITTEE
Dear Friends and Colleagues,
on behalf of the Organizing Committee, I cordially welcome you to the 32nd Specialized Symposium
on Yeasts (ISSY32), Perugia, Italy. This Symposium back to Perugia after 27 years: in fact ISSY7
was held in the same city in 1988. ISSY32 is organized by the joint collaboration of a few prestigious
Italian Universities: the University of Perugia, the University of Basilicata and the University of
Florence. The Symposium is also patronized by the International Commission on Yeasts (ICY), by the
Department of Agricultural, Food and Environmental Science (University of Perugia) and by some
other important Italian and local Institutions.
Yeasts are a group of eukaryotic organisms belonging to the Kingdom of Fungi, which are widely
distributed in worldwide microbiome. Their manifest ubiquity in the Earth’s biosphere is however
balanced by their great diversity and specificity for different habitats. Besides, yeasts are probably
one of the most relevant microbial groups in both traditional fermentations and biotechnological
innovative applications. Accordingly, ISSY32 has been designed to provide an overview on the latest
research developments on yeast ecology, physiology, taxonomy, food and non-food biotechnology,
genetic and genomic.
Distinguished senior and young scientists from over 40 Countries will present their recent results
during ISSY32. We hope that the interaction among worldwide Colleagues in our Symposium will
stimulate a creative exchange of new ideas that ultimately results in new scientific advances in the
fascinating yeast world.
We would like to thank all Colleagues, the talented staff and support personnel whose efforts make
the organization of ISSY32 possible and successful. We are especially grateful to all Companies for
their generous financial support, without which the organization of ISSY32 would not have been
possible. Finally, ISSY32 is dedicated to Prof. Alessandro Martini, Chair of ISSY7, eminent yeast
biologist and great teacher of science and life.
Pietro Buzzini
Chair of the Organizing Committee of ISSY32.
Dipartimento di Scienze Agrarie, Alimentari ed Ambientali
Industrial Yeasts Collection DBVPG (www.dbvpg.unipg.it)
University of Perugia, Perugia (Italy)
II
FROM THE INTERNATIONAL COMMISSION ON YEASTS (ICY)
On behalf of the International Commission on Yeasts (ICY), I am pleased to welcome you to the 32nd
ISSY symposium. The theme of this year’s specialized meeting is yeast biodiversity and biotechnology
in the 21st century. The meeting will highlight how yeasts are important contributors to the biodiversity
of Earth’s ecosystem. The meeting will cover some of the commercial applications of yeasts in foods,
beverages, biofuels, bio-based chemicals, and medicines. These topics will occupy a central part
of this symposium’s talks, posters, and discussions. Furthermore, the role of yeast taxonomy and
ecology will be featured in the keynote talk by our colleague MARC-ANDRÉ LACHANCE, and in a
session dedicated to these topics that are central to the discovery and understandingof all new genera
of yeasts.
The role of yeasts in producing foods and beverages has long been recognized, and it is fitting that
we meet here in the heart of Italy, in the country long known for its food culture from bread, cheeses,
aged meats and wines to. . . chocolates that would not be possible without yeast. Some of us who have
worked and studied the cocoa fermentation are aware of the key role yeasts play in the fermentation
of the cocoa beans. As such yeast are important contributors to the quality of the raw cocoa beans
used to produce the fine chocolates made in Perugia.
I wish you all a good week full of lively discussions as you engage one another in advancing knowledge
of yeast research and the contributions yeasts have made over the many centuries to the well being
of all mankind.
Charles A. Abbas
Chair of the International Commission on Yeasts (ICY)
III
FROM THE UNIVERSITY OF PERUGIA
Dear ISSY32 participants,
It is with great pleasure that, on behalf of the Department of Agricultural, Food and Environmental
Sciences of the University of Perugia, I welcome you in our University for the 32nd edition of the
International Specialized Symposium on Yeasts (ISSY32).
The Department of Agricultural, Food and Environmental Sciences was born at the beginning of 2014
as a legacy from the Royal High School of Agriculture founded in 1896 then transformed into the
Faculty of Agriculture in 1936. The mission of the Department is the higher education, the research
and the dissemination of innovation in the fields of biology, ecology, agronomy, biochemistry, (bio)
technology, engineering and economy related to both agricultural and industrial Companies.
Among the most promising researches ongoing in our Department, it is possible to mention those
related to yeast taxonomy, biodiversity and biotechnology, which demonstrated that there is
considerable value contained in the yeast biodiversity, which is unanimously considered of great value
to a variety of industries (including food and non-food products, i.e. pharmaceuticals, fine chemicals,
cosmetics, etc.). The Department of Agricultural, Food and Environmental Science also is the hosting
Institution of the Industrial Yeasts Collection DBVPG, which is affiliated to the European Culture
Collection Organization and to the World Federation of Culture Collections. The DBVPG Collection
is specialized in the study and ex-situ conservation of yeasts and yeast-like microorganisms, distributes
strains and offers services to the international scientific community and to other private Institutions.
Therefore, the organization of the 32nd edition of the International Specialized Symposium on Yeasts
(ISSY32) is considered one of the prominent events in 2015 supported by our Department.
I wish to all ISSY32 participants to find new scientific inputs, fruitful exchanges of scientific
information and a valuable development of the subject.
Prof. Francesco Tei
Director of the Dipartimento di Scienze Agrarie, Alimentari ed Ambientali
University of Perugia, Perugia, Italy
IV
PLENARY LECTURE SPEAKER
Some Big Questions of Yeast Ecology
September 13, 2015, 17:15
MARC-ANDRÉ LACHANCE
University of Western Ontario (Canada) Marc-André Lachance obtained his PhD in Microbiology at the University of California, Davis,
under Herman Phaff’s mentorship. After a postdoctoral fellowship at the Institut Pasteur in Paris,
where he strayed from his main path to work on the molecular systematics of cyanobacteria, he joined
the University of Western Ontario, where he began his exploration of yeast biodiversity. Lifelong
collaborations with Herman Phaff and Tom Starmer led him first to study yeasts from necrotic cacti
of the Caribbean Islands, the Sonoran Desert, Hawaii, Argentina, and Australia. His interest gradually
shifted to the yeast community of flower beetles and in particular the large-spored Metschnikowia
species, of which he discovered 22 species. These studies were enhanced by collaborations with
Carlos Rosa and other colleagues, not to neglect the important field contributions made by the late Jane
Bowles, his field partner of 34 years. Noteworthy are studies of spontaneous Tequila fermentations
in Mexico, as well as natural yeast communities of Brazil, Costa Rica, some South Pacific islands,
or Malaysia. More recently, his collecting range has vicariously extended to Africa, in partnership
with several colleagues, but in particular, members of Carlos Herrera’s research team. Lachance
has served as Editor and Publisher of the Yeast Newsletter since 1988 and Associate Editor for the
International Journal of Systematic and Evolutionary Microbiology since 2001. He is a passionate and
celebrated teacher. His classroom activities have covered a broad array of topics, from microbiology
to mycology, genetics, ecology, evolutionary genetics, and the theory and application of systematics.
V
KEYNOTE SPEAKER
Biodiversity and Ecological Interactions of Yeasts in Neotropical Environments
September 14, 2015, 8:30
LEDA MENDONÇA-HAGLER
Universidade Federal do Rio de Janeiro (Brazil)
Leda Cristina Mendonca-Hagler obtained her B. Sc. Degree in Chemistry from the Federal
University of Sergipe and the Ph. D. in Biological Sciences (Microbiology) from the Federal University
of Rio de Janeiro, (UFRJ). She did her postdoctoral training on Yeast Biology at the University of
California, Davis, USA, under the supervision of Prof. Herman Phaff, and on Molecular Microbial
Ecology, at the Institute for Soil Fertility, (NL), with Prof. Jan D. van Elsas. Professor L. MendoncaHagler served on the faculty of the UFRJ for over four decades, where she was Head of the General
Microbiology Department, Chair of the Microbiology Graduate Program, Dean for Health Sciences
Graduate Programs and co-founder of the Plant Biotechnology and Ecology Graduate Programs.
She supervised 44 graduate students and published over 120 scientific papers and book chapters on
yeast taxonomy, microbial ecology, biotechnology and biosafety. Her research group investigated the
microbial diversity associated with tropical environments and the relationships between microbial
community structures and ecosystem functions, applying multidisciplinary approaches. She was a
Guest Researcher at Georgia State University (US), working on yeast taxonomy with Dr. S. Meyer
and developed cooperation projects on environmental microbiology with Prof A. Martini (IT) and
Prof. K. Smalla (DE). She organized several scientific meetings, has been member of advisory
committees and a consultant for several companies. Prof. Mendonca-Hagler received the award
“Honor of the Scientific Merit” and the title of “Comendador” from the Brazilian Government. The
genus Hagleromyces and 3 yeast species were named in honor of A. Hagler and L. Mendonça-Hagler,
in recognition of their contribution to yeast research. She was the Chair of the Int. Committee on
Yeasts/IUMS, and currently is ICY Honorary member. She is Scientific Director of the Brazilian
Biosafety Association and Ambassador at the International Society for Microbial Ecology.
VI
KEYNOTE SPEAKER
Yeasts and the Fermented Food Renaissance
September 14, 2015, 14:00
GRAHAM FLEET
University of New South Wales (Australia)
Graham Fleet is an Emeritus Professor at the University of New South Wales ( UNSW), Sydney,
Australia. He has BSc ( 1966) and MSc degrees ( 1969) in microbiology/biochemistry from the
University of Queensland and completed his PhD at the University of California , Davis ( 1973)working on yeast cell wall biochemistry. After postdoctoral studies at Heriot Watt University,
Edinburgh, he joined UNSW in 1975 where he was responsible for teaching and research programs
in food microbiology/ biotechnology until his retirement in 2007. During this time, he has published
many research papers and several books on the microbiology/biotechnology of fermented foods
and beverages, with specialized interest in the contributions of yeasts. He was Chairperson of
the International Commission on Yeasts ( 1996-2000) and served on the Executive Board of the
International Union of Microbiological Societies ( IUMS) and as Chairperson of the Mycology
Division ( IUMS) during 2002-2008. Since 2010, he has been a member of the Executive Board of the
International Committee on Food Microbiology and Hygiene. He has served on the editorial boards
of several journals associated with yeasts, wine and foods and has been an editor of the International
Journal of Food Microbiology since 2008. His most recent book is Schwan, R and Fleet, G ( eds)
Cocoa and Coffee Fermentations CRC Press, 2015.
VII
KEYNOTE SPEAKER
Recent Advances in the Development of Yeasts for Biofuels
September 15, 2015, 8:30
CHARLES ABBAS
Yeast and Renewables Reseach Archer Daniels Misland (ADM) (USA)
Charles Abbas is the Chair of the International Commission on Yeasts (ICY). He has served in this
role since being elected in August 2012 for a 4-year term. He also has two academic appointments
as an adjunct Professor in the Dept. of Bioproducts and Biosystems Engineering (BBE) at the U. of
Minnesota in St. Paul, MN and as an adjunct Faculty in the Dept. of Food Science & Human Nutrition
(FSHN) at the U. of Illinois in Champaign-Urbana.
Charles received a B.S. in Microbiology from the U of Minnesota (Twin Cities), an M.S. in
Microbiology from the U. of Montana (Missoula), completed the Ph.D. Biochemistry coursework
requirements at the U. of Minnesota (St. Paul) and received a Ph.D. in Microbiology and Cell Science
from the U. of Florida (Gainesville). After working as a post doctoral student at the U. of Florida
(Gainesville), he began a 27 year career in industrial biotechnology first working at Difco R & D in
Ann Arbor, MI as a senior scientist, and later as a group leader, manager and most recently as Director
of Yeast and Renewables Research at Archer Daniels Midland (ADM) in Decatur, IL, U.S.A.
Dr. Abbas is the author of over 100 abstracts, scientific articles, book chapters and reviews, patents and
patent applications. He is considered a leading expert in yeast, large-scale industrial fermentations,
and biorefining.
VIII
KEYNOTE SPEAKER
A Population Genomics View of Saccharomyces Natural History and Domestication
September 16, 2015, 8:30
JOSÉ PAULO SAMPAIO
Universidade Nova de Lisboa (Portugal) Jose Paulo Sampaio has worked mainly on yeast systematics, evolutionary ecology and biogeography.
His more recent research interests concern the use of population and comparative genomics to
understand microbe domestication using the genus Saccharomyces as a model. He coordinates the
Portuguese Yeast Culture Collection (PYCC).
IX
KEYNOTE SPEAKER
Non-Conventional Yeasts as Promising Producers of Biofuels and Chemicals
September 16, 2015, 14:00
ANDREI A. SIBIRNY
National Academy of Science (Ukraine)
Andriy A. Sibirny received a B.S. in biology from the I. Franko Lviv State University (Ukraine),
and a M.S. in biochemistry from the Lviv Division of Institute of Biochemistry, National Academy
of Sciences of Ukraine. Since 1973 he was scientist, senior scientist, chief scientist of Lviv Division
of Institute of Biochemistry, National Academy of Sciences of Ukraine, and since 1988 he is Head
of the Department of Biochemical Genetics (since 2000, Department of Molecular Genetics and
Biotechnology), above mentioned Institute (transformed in 2000 to Institute of Cell Biology). From
2000 to present he is Director of Institute of Cell Biology, National Academy of Sciences of Ukraine,
Lviv and since 1990s he is Professor of the Department of Genetics and Biotechnology, Lviv State
University, Professor of Biotechnology, Częstochowa Technical University (Poland) and Professor of
Microbiology and Genetics, University of Rzeszów (Poland). From 2005 to present (simultaneously)
he is Head of Department of Biotechnology and Microbiology, University of Rzeszów (Poland).
He received the follows Awards and Memberships: Honored Worker of Science and Technology of
Ukraine (2008); Laureate of the State Award in the field of Science and Technology (2012); Decored
with award “Honored for Wasraw University of Life Sciences, SGGW: with diploma and badge;
Corresponding member of National Academy of Sciences of Ukraine (field: Cell Biology) since 2003;
Full member of National Academy of Sciences of Ukraine (field: Yeast Biology) since 2012; Laureate
of O.V. Palladin and Ilya Mechnikov awards of NAS of Ukraine for the series works in the field of
biochemistry and molecular biology (2004) and molecular microbiology (2015), respectively; Chair
of International Commission on Yeast of International Union of Microbiological Societies during
2008-2012 and Member of this Commission since 1987; Member of Finance and Policy Committee of
the Community on Yeast Genetics and Molecular Biology since 1992; President of Ukrainian Society
for Cell Biology, Member of Presidium of Ukrainian Biochemical Society, Member of Presidium
and FEMS delegate of Society of Microbiologists of Ukraine, Member of Central Committee of
Ukrainian Society of Geneticists and Breeders since 1987.
He was awarded by long-term research grant of G. Soros International Science Foundation (19941995); two NATO linkage grants (1995-1996, 2004-2005); two Fogarty International Research
Collaboration Awards (1995-1998, 2002-2005); two CRDF grants (2002-2004, 2006-2008); eight
INTAS grants (1994-1995; 1995-1998; 1996-1999; 2000-2002; 2001-2003, 2002-2004, 2004-2006,
2006-2008), two STCU grants (2007-2009, 2008-2009).
He was the Head of the Organizing Committee of 21st International Specialized Symposium on
Yeasts “Biochemistry, Genetics, Biotechnology and Ecology of Non-conventional Yeasts”, Lviv,
X
Ukraine, 2001; 12th International Congress on Yeasts (Kyiv, Ukraine, 2008) and 1st International
Symposium on Non-conventional Yeasts (Lviv, Ukraine, 2011).
• Sibirny is co-author of more than 220 full-length scientific articles including three
monograph and 100 papers in peer reviewed International scientific journals, 28 Patent
Applications, 100 abstracts of oral and poster communications of conferences, including
40 plenary lectures on domestic and International scientific meetings. He is specialist in
the field of biochemistry, genetics and biotechnology of non-conventional yeasts. The
main topics of research activities: riboflavin and flavin nucleotide synthesis; regulation
of methanol metabolism; the pathways of hydrogen peroxide and formaldehyde
detoxification; glutathione metabolism; catabolite regulation; peroxisome biogenesis and
degradation; biosensors.
He presented many seminars and invited lectures at many domestic and International laboratories and
scientific meetings.
XI
KEYNOTE SPEAKER
The Importance of the Budapest Treaty and IDAs for the Protection of Biotechnological
Inventions
September 17, 2015, 9:00
EWALD GLANTSCHNIG
World Intellectual Property Organization (Switzerland)
Ewald Glantschnig
Nationality: Austrian
Academic degree: Dr. Jur. Salzburg 1984
Actual Function: Head, Budapest Treaty Section, Patent Law Division, World Intellectual Property
Organization (WIPO).
Former positions held:
• Permanent Mission of Austria in Geneva: Adviser for intellectual property matters.
• Ministry for Economic Affairs, Vienna: Adviser; cooperation with Austrian Patent Office,
preparatory work for TRIPs-implementation.
• CA-Leasing GmbH, Vienna: Marketing Manager.
• Austrian Trade Mission in Chicago: Deputy Trade Commissioner.
• Austrian Trade Mission in Lisbon: Deputy Trade Commissioner.
• Elektronische Haustechnik Laimer GmbH, Salzburg: Consultant for export-planning.
• Civil Court, Salzburg: Assistant to the judge.
XII
PROGRAMME
Sunday, September 13, 2015
10:00 – 16:00 Registration
16:00 – 16:30 Welcome cocktail
16:30 – 17:15 Welcome introduction
- Rector of the University of Perugia
- Director of the Department of Agricultural, Food and Environmental
Science, University of Perugia
- Chair of the International Commission on Yeasts
- Chair of the Organizing Committee of ISSY32
OPENING SESSION. Chairs: C. ABBAS (USA), A. E. VAUGHAN (Italy)
17:15 – 18:00 Opening Lecture:
MARC-ANDRÉ LACHANCE (Canada): Some Big Questions of Yeast Ecology
19:00 – 20:30 Concert of the Assisi Chorus at the Sala dei Notari Hall, Perugia
20.30
Get together party at the Cloister of the S. Lorenzo Cathedral, Perugia
Monday, September 14, 2015
Session 1A: YEASTS IN THE ENVIRONMENT: ECOLOGY AND TAXONOMY. Chairs: T.
BOEKHOUT (The Netherlands), D. LIBKIND (Argentina)
08:30 – 09:00 Keynote Lecture:
LEDA MENDONÇA-HAGLER (Brazil): Biodiversity and ecological interactions of yeasts
in neotropical environments
09:00 – 10:30 Selected Lectures:
09:00 – 09:15 R. NASANIT (Thailand): Diversity of epiphytic yeasts in phyllosphere of
corn in Thailand by culture-independent approach
XIII
09:15 – 09:30 I. STEFANINI (Italy) Social wasps are mating nests for yeasts
09:30 – 09:45 E. S. NAUMOVA (Russia) Phylogenetics, ecology and biogeography of
Komagataella yeasts: molecular and genetic analysis
09:45 – 10:00 D. LIBKIND (Argentina) Yeast diversity in extreme environments of
southern South America
10:00 – 10:15 N. ČADEŽ (Slovenia) Yeast communities in Slovenian virgin olive oil and
their taxonomic placement
10:15 – 10:30 R. FOTEDAR (Qatar) Diversity of yeasts from marine waters in Arabian
Gulf surrounding Qatar
10:30 – 10:50 Coffee/Tea break
Session 1B: YEASTS IN THE ENVIRONMENT: PHYSIOLOGY AND STRESS RESPONSE.
Chairs: H. TAKAGI (Japan), M. PENTTILÄ (Finland)
10:50 – 11:15 H. TAKAGI (Japan): Quality control of plasma membrane proteins by yeast
Nedd4-like ubiquitin ligase Rsp5 under environmental stress conditions
11:15 – 11:30 E. VAUDANO (Italy): Transcriptional and metabolic responses of different
Saccharomyces cerevisiae strains to hyperosmotic stress caused by inoculation
in grape must
11:30 – 11:45 S. GUYOT (France): Heterogeneity of response to heat stress in Saccharomyces
cerevisiae
11:45 – 12:00 L. BENEY (France): The unexpected role of ergosterol in yeast adaptation to
hydric fluctuations
12:00 – 12:15 A. RAPOPORT (Latvia) Anhydrobiosis in yeast: unique state of live organisms
and its possible non-conventional applications
12:15 – 12:40 N. PEREIRA MIRA (Portugal) Genetic adaptive mechanisms mediating
response and tolerance to acetic acid stress in the human pathogen Candida
glabrata: role of the CgHaa1-dependent signalling pathway
12:40 – 14:00 Lunch at the Restaurant of Hotel Giò – Perugia Centro Congressi
Session 2A: YEASTS IN FOOD BIOTECHNOLOGY: YEASTS IN FOODS AND BEVERAGES.
Chairs: G. FLEET (Australia), P. ROMANO (Italy)
GRAHAM FLEET (Australia): Yeasts and the fermented food renaissance
XIV
14:30 – 14:45 K. S. HOWELL (Australia) Yeast species associated with Drosophila in
vineyard ecosystems
14:45 – 15:00 S. BENITO (Spain) Combine use of selected Schizosaccharomyces pombe
and Lachancea thermotolerans yeast strains as an alternative to the traditional
malolactic fermentation in red wine production
15:00 – 15:15 A. CEUGNIEZ (France) Fungal flora of a traditional French cheese,
the “Tomme d’Orchies” showed strains of Kluyveromyces with atypical
antagonistic properties
15:15 – 15:30
N. GUARAGNELLA (Italy) Comparative study of Saccharomyces cerevisiae
indigenous wine strains to identify potential marker genes involved in
desiccation stress resistance
15:30 – 15:45 N. JOLLY (South Africa) Non-Saccharomyces yeast biodiversity on
Chardonnay grapes: a comparison 15 years later
16:15 – 16:30
G. PERPETUINI (Italy) Study of ATG1, ATG17 and ATG29 genes expression
in Saccharomyces cerevisiae sparkling wine yeasts
16:30 – 16:45 H. M. DANIEL (Belgium) Yeast diversity of Cuban cocoa bean heap
fermentations and their environments
16:45 – 17:00
L. CANONICO (Italy) Contribution of Torulaspora delbrueckii yeast in mixed
fermentation with different commercial starter strains of Saccharomyces
cerevisiae to improve the quality of craft beer
17:00 – 17:15 H. ERTEN (Turkey) Yeasts microbiota of naturally fermented black olives
made from Cv. Gemlik grown in various districts of turkey
17:15 – 17:30 F. CARRAU (Uruguay) Synthesis of phenolic aroma compounds by
Hanseniaspora vineae yeast strains contribute to increase flavor diversity of
wines
17:30 – 17:45 D. A. MILLS (USA) Yeast and bacterial landscapes within food production:
A case for microbial terroir
17:45 - 18:00 Short Coffee/Tea break
Session 2B: YEASTS IN FOOD BIOTECHNOLOGY: DETECTION METHODS AND STRAIN
IMPROVEMENT. Chairs: I. S. PRETORIUS (Australia), L. GRANCHI (Italy)
18:00 – 18:20 I. S. PRETORIUS (Australia) From Bach to Bacchus: yeast genomics and
wine symphonics
XV
18:20 – 18:40 S. DEQUIN (France) Improvement of wine yeast strains using adaptive
evolution
18:40 – 19:00 B. TRINDADE DE CARVALHO (Belgium) Polygenic analysis of
phenylethyl acetate production in yeast for aroma production improvement
in alcoholic beverages
19:00 – 19:20 T. OTA (Japan) Crossbreeding of bottom-fermenting yeast strains with a
novel method for high throughput screening of mating-competent cells
19:20 – 19:40
T. THERY (Ireland) Antifungal activity of defensins from different organisms
and potential applications in cereal-based products
19:40 – 21.00
Dinner at the Restaurant of Hotel Giò – Perugia Centro Congressi
Satellite Event: Dinner and Meeting of the International Commission for
Yeasts
21:00 – 23.00
Poster Session
Tuesday, September 15, 2015
Session 3:
YEASTS IN NO-FOOD BIOTECHNOLOGY: BIOFUELS, NEW MOLECULES
AND ENZYMES. Chairs: C. ABBAS (USA), V. PASSOTH (Sweden)
C. ABBAS (USA); Recent Advances in the Development of Yeasts for Biofuels
09:00 – 09:20 D. MATTANOVICH (Austria): Organic acids from lignocellulose: Candida
lignohabitans as a novel microbial cell factory
09:20 – 09:40
L. OLSSON (Sweden): Cellular robustness in lignocellulose derived streams
09:40 – 10:00 T. DESFOUGERES (France): Phenotypic and genotypic analysis of
engineered xylose consuming Saccharomyces cerevisiae strains adapted to
industrial medium for second-generation biofuel
10:00 – 10:20 A. J. A. VAN MARIS (The Netherlands): Engineering cytosolic Acetyl
Coenzyme A supply in Saccharomyces cerevisiae
XVI
10:50 – 11:05 E. NEVOIGT (Germany): Towards industrial glycerol fermentation by
Saccharomyces cerevisiae
11:05 – 11:20 N. S. PARACHIN (Brazil): Metabolic engineering of Pichia pastoris for
l-lactic acid production using glycerine as carbon source
11:20 – 11:35 D. PIROZZI (Italy): Use of oleaginous yeasts for the exploitation of
lignocellulosic biomasses
11:35 – 11:50 P. POLBUREE (Thailand): Selection and optimization of oleaginous yeast
for lipid production from biodiesel-derived crude glycerol
11:50 – 12:05 L. FAVARO (Italy): Engineering industrial yeast strains for consolidated
bioprocessing of starchy substrates and by-products to ethanol
12:05 – 12:20 A. S. ZAKY (UK): The potential and advantages of using marine yeast and
seawater-based media in bioethanol production
12:20 – 12:35 F. BISCHOFF (Germany): Characterization of three new cutinases from the
yeast Arxula adeninivorans
15:00
Excursion to Assisi or Perugina Chocolate Factory (optional)
Wednesday, September 16, 2015
Session 4: YEASTS GENETIC AND GENOMIC. Chairs: S. DEQUIN (France), G. LITI (France)
JOSÉ PAULO SAMPAIO (Portugal): A Population Genomics View of Saccharomyces
Natural History and Domestication
09:00 – 09:15
J. L. LEGRAS (France) New insights into the adaptation of yeast to anthropic
environment using comparative genomics
09:15 – 09:30 F. Y. BAI (China) The origin and domestication of lager beer yeast
09:30 – 09:45 A. NICOLAS (France) Reversion of meiotic progression allows extensive
recombination of Saccharomyces cerevisiae hybrid diploids
09:45 – 10:00 A. GOOVAERTS (Belgium) Polygenic analysis of ethanol tolerance and
maximal ethanol accumulation capacity in Saccharomyces cerevisiae
10:00 – 10:15 P. DARAN-LAPUJADE (The Netherlands) Pathway swapping: a new
approach to simply and efficiently remodel essential native cellular functions
XVII
10:15 – 10:30
F. A. CUBILLOS (Chile) Natural variation in non-coding regions underlying
phenotypic diversity in budding yeast
11:00 – 12:30
Selected Lectures:
11:00 – 11:15 C. BRION (France) Genomic and transcriptomic landscapes within a
protoploid yeast species
11:15 – 11:30 C. CURTIN (Australia) Genomic and transcriptomic landscape of the
industrial yeast species Brettanomyces bruxellensis
11:30 – 11:45
T. JEFFRIES (USA) Comparative genomics of biotechnologically important
yeasts
11:45 – 12:00 J. MORRISSEY (Ireland) Genetics of lactose utilisation in Kluyveromyces
marxianus
12:00 – 12:15 A. GONZÁLEZ (Mexico) BAT1 and BAT2 Functional diversification
through subfunctionalization of chromatin organization and transcriptional
regulation in Saccharomyces cerevisiae
12:15 – 12:30 O. P. ISHCHUK (Sweden) The haploid nature of Candida glabrata is
advantageous under harsh conditions
Session 5:
NON-CONVENTIONAL YEASTS. Chairs: A. A. SIBIRNY (Ukraine, Poland), D.
MATTANOVICH (Austria)
ANDRIY A. SIBIRNY (Ukraine, Poland): Non-Conventional Yeasts as Promising Producers
of Biofuels and Chemicals
14:30 – 14:50
B. GASSER (Austria): The impact of degradative pathways on recombinant
protein secretion in Pichia pastoris
14:50 – 15:10 G. KUNZE (Germany): Arxula adeninivorans – a suitable biocatalyst for
new biotechnological products
15:10 – 15:30 R. LEDESMA-AMARO (France): Yarrowia lipolytica, a model for lipid
metabolism and a platform for lipid production
15:30 – 15:50
V. PASSOTH (Sweden): Lipid production from lignocellulose by oleaginous
yeasts for biodiesel and animal feed
15:50 – 16:10 H. YURIMOTO (Japan): Transcription factors involved in regulation of
methanol-inducible gene expression in the methylotrophic yeast
XVIII
16:40 – 19.30
Poster Session
Satellite Event: Discussion meeting on Taxonomy of Yeasts, including the
future of “The Yeasts”
20.00
Gala dinner at the Restaurant Posta dei Donini, San Martino in Campo,
Perugia (optional)
Thursday, September 17, 2015
Session 6:
YEASTS CULTURE COLLECTIONS. Chairs: K. BOUNDY-MILLS (USA),
P. BUZZINI (Italy)
EWALD GLANTSCHNIG (Switzerland): The Importance of the Budapest Treaty and IDAs
for the Protection of Biotechnological Inventions
09:30 – 09:50 K. BOUNDY-MILLS (USA): Yeasts of yesterday and today, preserved for
discoveries of tomorrow
09:50 – 10:10
B. TURCHETTI (Italy): Yeasts culture collections: how to transform sleeping
beauties in real opportunities
10:10 – 10:30 I. ROBERTS (UK): Yeast collecting for fun and fortune
10:30 – 10:50 A. YURKOV (Germany): Yeast biodiversity in culture collections: old
sources and new challenges
10:50 – 11:20
Coffee/Tea break
11:20 – 11:30
Awarding of the best posters
11:30 – 12:30
Farewell considerations and introduction of future ISSY & ICY meetings
- Chair of the Organizing Committee of ISSY32
- Chair of the Organizing Committee of ICY14
XIX
- Chairs of the Organizing Committee of future ISSY & ICY meetings
- Chair of the International Commission on Yeasts
11:30
XX
End of the Conference and Departure
GALA DINNER
Wednesday, September 16, 2015
20:00
Gala dinner at the restaurant ALLA POSTA DEI DONINI , San Martino in Campo, Perugia.
Just outsite of Perugia, in the little town of San Martino in Campo, the historic residence of Alla Posta
dei Donini offers exclusive comfort and services of exquisite quality. This prestigious building dates
to the XVII century.
TOURS
Tuesday, September 15, 2015
15:00
We’re glade to invite registered participants and accompanying persons to participate in one of
the following excursion on Tuesday September 15, in the afternoon. The cost of the excursion
is not included in the fee and you can book and pay it through the registration procedure, during
the registration or in the following days. Deadline for excursion booking: July 31st, 2015.
The cost will cover the bus transport, the tickets and the guide.
-
Assisi: placed close to Perugia on the western flank of Monte Subasio, it is the birthplace of St.
Francis, who founded the Franciscan religious order and St. Clare founder of the Order of Poor
Clare. Known for the noteworthy churches, in particular the beautiful Basilica of San Francesco
d’Assisi decorated by Giotto and Cimabue, Assisi is identified as the most important artistic
place of Umbria.
-
Discover Perugina Chocolate Factory: Perugina is the Italian confectionery company based
in Perugia. The company produces a wide array of chocolate and food products but the most
important is undoubtedly the Bacio (Kiss). You will have the possibility to enter where the
chocolate processes take place and spot the secret of the chocolate manufacturing. The visit to
the close Chocolate Museum will close the excursion.
XXI
PERUGIA INFORMATIONS
PASSPORT REGULATIONS
A visa is not required for US or Canadian citizens holding a valid passport unless they expect to stay
in Italy for more than 90 days or are entering the country for study or employment reasons. Anyone
who decides to stay over 90 days once they have entered the country should make an application
once only to any police station for an additional 90-day extension. Generally, permission is granted
immediately. Non-American citizens should check current visa requirements with the nearest Italian
Embassy or Consulate before departure.
PETS
A traveller entering Italy with a dog or cat must have a veterinary certificate stating that the animal
is in good health and has been vaccinated against rabies between 20 days and 11 months prior to
entry into Italy. The certificate is valid for 30 days. Forms are obtainable at all Italian Embassies and
Consulates and from the Italian Government Travel Office. Parrots, parakeets, rabbits and hares also
require health certificates and in addition are subject to an examination upon entering Italy. Dogs
must be on a leash or muzzled when in public. Customs officials may require a health examination of
any pet if they suspect that it is ill or has come directly from tropical regions.
HEALTHCARE AND MEDICAL ASSISTANCE
Tourists requiring urgent medical care should go to the nearest hospital emergency ward (airports and
many railway stations also have medical teams and first aid facilities). Those with seriousillnesses or
allergies should always carry a special note from their physicians giving detailed information on the
treatments they are following or that may be necessary. Pharmacies generally follow shop opening
times (approximately from 8.30 a.m. to 12.30 p.m. and from 3.00 to 7.00 pm, Monday to Saturday,
but some are open throughout the day (Centre of Perugia). Night time service is provided on a few
pharmacies (Farmacia Sodalizio di San Martino, Piazza Matteotti 26, Perugia).
Opening hours are displayed outside each pharmacy and are published in local newspapers. It is
advisable to dispose of a document certifying coverage by the national health care service before
departure.
HEALTH SERVICES AND INSURANCE POLICY
Italy has no medical program covering citizens from the US and Canada. US and Canadian tourists
are therefore advised to take out an insurance policy before travelling. First Aid Services are available
in airports, ports, railway stations and in all hospitals. Medicines, be they prescription or over the
counter, can be obtained only in pharmacies.
PERUGIA WEATHER INFORMATION
Umbria is a landlocked, mountainous region with a typically Mediterranean climate: hot, dry summers
and cold winters. In recent years winters have been considerably drier than in the past. The Apennine
mountains acts as a protection barrier from both the climatic influences of the Adriatic Sea to the
North West and from the cold air currents descending from the North East. Although Perugia is a
perfectly functional tourist destination all year round, it is preferable to arrange visits from the spring
(February -March) until the late fall (October). The city of Perugia is located at about 400 m a.s.l. and
can be cool in the early morning and late evening, even during summer.
XXII
XXIII
XXIV
CONGRESS INFORMATIONS
ORAL PRESENTATIONS GUIDELINES
Speakers are required to report to the technical support staff at least three hours prior to the start of
their presentations with a PowerPoint document saved in USB Flash Drive. If combining video films
with PowerPoint, please make sure to check it in the session hall where your lecture will taking place.
Important note for Macintosh users: If Speakers want to use Mac presentations, please note that
they must inform technical support staff in advance. However they need to prepare it according to
the instructions below:
- Use a common font, such as Arial, Times New Roman, Verdana etc. (special fonts might be
changed to a default font on a PowerPoint based PC).
- Insert pictures as JPG files (and not TIF, PNG or PICT - these images will not be visible on
a PowerPoint based PC ).
- Use a common movie format, such as AVI and WMV (MOV files from QuickTime will not
be visible on a PowerPoint based PC).
The Organizing Committee would like to post all PowerPoint files on the congress website after the
event. If you do not want your file to be posted on the congress website, return to the technical support
staff after your session and ask that the file be removed from the list of congress files.
POSTER PRESENTATIONS GUIDELINES
- Posters may be prepared on one sheet (preferred method) or alternatively on several smaller
sheets.
- The suggested dimensions of the poster are 70 cm wide by 100 cm tall. - Assign the top of the poster for the Session (number and title of the session), title and authors
as stated on the submitted abstract.
- The text, illustrations, etc should be bold enough to be read from a distance of two meters. - Before the starting day of the Conference you will receive by email a list with the number of
the poster board allocated to you. Please use the board with the same number.
- Double sided tape, tacks and technical equipment will be available for the mounting of posters.
Staff will also be in the poster area to assist you. The Organising Committee will not be responsible for posters that are not removed by the end of the
Conference.
XXV
COMMITTEES
ITALIAN ORGANIZING COMMITTEE
Pietro Buzzini (University of Perugia) - Chair
Patrizia Romano (University of Basilicata) - Vice-Chair
Lisa Granchi (University of Florence) - Vice-Chair
Gianluigi Cardinali (University of Perugia)
Benedetta Turchetti (University of Perugia)
Angela Capece (University of Basilicata)
Maurizio Ciani (University of Marche)
Marilena Budroni (University of Sassari)
Ilaria Mannazzu (University of Sassari)
Ciro Sannino (University of Perugia)
Simone Di Mauro (University of Perugia)
Sara Filippucci (University of Perugia)
INTERNATIONAL SCIENTIFIC COMMITTEE
Charles Abbas - USA
Feng-Yan Bai - China
Teun Boekhout - The Netherlands
Pietro Buzzini - Italy
Sylvie Dequin - France
Graham Fleet - Australia
Lisa Granchi - Italy
Thomas Jeffries - USA
Marc-André Lachance - Canada
Patricia Lappe Oliveras - Mexico
Diethard Mattanovich - Austria
Diego Libkind - Argentina
Anna Maraz - Hungary
Leda Mendonça-Hagler - Brazil
Gennadi Naumov - Russia
Bernard Prior - South Africa
Isak Pretorius - Australia
Amparo Querol - Spain
Alexander Rapoport - Latvia
Peter Raspor - Slovenia
Doris Rauhut - Germany
Patrizia Romano - Italy
Josè-Paulo Sampaio - Portugal
Andriy Sibirny - Ukraine
Hana Sychrova - Czech Republic
Hiroshi Takagi - Japan
Ann Vaughan-Martini - Italy
Graeme M. Walker - U.K.
XXVI
ORAL LECTURES ABSTRACTS
1
Opening session - Opening lecture
Some big questions of yeast ecology
Marc-André Lachance
University of Western Ontario, London, Ontario, Canada N6A 5B7
[email protected]
Yeasts, in particular a handful of model species, have played a foundational role in the development
of modern biochemistry and genetics. Yeasts as a whole have blazed the trail in the systematics of
eukaryotic microbes, as exemplified by a fully functional DNA barcode by the turn of the 21st century
and an enviable tradition known as “The Yeasts, a Taxonomic Study”. But what about ecology? In
spite of a number of inspiring studies of a small array of species, a unifying view of the place of yeasts
in nature would appear to remain elusive. For those concerned with yeast ecology, there seems to be
an epistemic divide that opposes Bass Becking’s “alles is overal: maar het milieu selecteert”(De Wit
& Bouvier 2006) to the biogeographer’s ‘Everything is endemic’(Williams 2011), and more recently
to an ultra-neutral model (Goddard & Duncan 2015) that would deny, at least for baker’s yeast, both a
biogeography and a niche. The fact that the search for a habitat for baker’s yeast remains inconclusive
may be due to the in part in the highly typological mindset that pervades yeast ecology, which seeks
to identify in nature the equivalent of selective agar plates where yeast populations boom and crash.
A more flexible, probabilistic approach may be preferable. Having discussed these theoretical and
historical considerations, I shall review some of the “big” questions facing yeast ecologists today. I
shall also provide highlights of some of my own work on yeasts associated with floricolous beetles,
their phylogenetics, their distribution at various spatial scales, and some attempts to identify the
adaptive basis for their ecological specificity.
KEYWORDS: Yeast ecology, Yeast biogeography, Yeast habitat, Yeast ecological niche, Spatial scale
REFERENCES:
De Wit R, Bouvier T (2006). ‘Everything is everywhere, but, the environment selects’; what did Baas Becking and
Beijerinck really say? Environmental Microbiology 8:755–758
Williams DM (2011). Historical biogeography, microbial endemism and the role of classification: everything is
endemic. In Fontaneto D. Biogeography of microscopic organisms. Cambridge pp. 11-31
Goddard MR , Duncan G (2015). Saccharomyces cerevisiae: a nomadic yeast with no niche? FEMS Yeast Research
15:1-6
2
Session 1A
Yeasts in the environment:
ecology and taxonomy
3
Session 1A: Yeasts in the environment: ecology and taxonomy - Key note
Biodiversity and ecological interactions of yeasts from neotropical environments
Leda Mendonca-Hagler, Allen N. Hagler
U. Fed. Rio de Janeiro, Brazil
[email protected]
Biodiversity assessments have been mostly focused on plants and animals, but recently the less visible
microbial diversity has received some attention. Tropical biomes include threatened ecosystems that
are rich in biodiversity and with high levels of endemism. During the last four decades over one
hundred habitats were studied in Brazil and neighboring countries. These included soil, plant, and
animal microhabitats in rain forests, coastal ecosystems and agro-ecosystems. An overview of yeast
biodiversity and ecological associations in neotropical ecosystems will be presented. The use of DNA
sequencing technologies has had a strong impact on accuracy and time needed for identifications. There
are few reports on the assessment of yeast diversity by cultivation-independent methods. Because of
the degree of diversity, no single cultivation or molecular genetic method reveals all the species in
a habitat. Yeast diversity studies in neotropical habitats have shown distinct communities including
many new species. Most studies detected the prevalent species of communities, but detection of
rare species is typically incomplete. In ephemeral habitats it is important to consider the succession
of species resulting in temporal changes on community structure. The prevalent species associated
with plant surfaces were mostly basiodiomycetous yeasts and Aureobasidium spp. Green fruits have
similar species to plant surfaces, but during ripening they attract insect vectors of ascomycetous
species that dominate in a succession during deterioration. Rotting cacti have associated insects and
characteristic yeast communities comparable with those of other regions. Drosophilids of tropical
forests have similar yeast groups to temperate forests with higher proportion of Hanseniaspora and
Pichia spp and less Kluyveromyces and Saccharomyces spp. Flower nectars are visited by insects that
vector ascomycetous yeasts, such as Metschinikovia spp. Yeasts from various habitats had different
spectra of mycocinogenic activity against other species. Soils yeasts were involved in nutrient cycles,
maintenance of soil structures, and symbioses. Yeast community structures in tropical habitats are
becoming known but their interactions in these habitats needs more study.
KEYWORDS: Yeast diversity, Tropical environments, Yeast ecology
REFERENCES:
Kurtzman CP, Fell JW, Boekhout T (2011). The Yeasts. A Taxonomic study. Elsevier, Amsterdam
Rosa CA, Gabor P (2006). Biodiversity and Ecophysiology of Yeasts, Springer, Berlin
4
Session 1A: Yeasts in the environment: ecology and taxonomy
Diversity of epiphytic yeasts in phyllosphere of corn in Thailand by culture-independent approach
Rujikan Nasanit1, Sopin Jaibangyang1, Manee Tantirungkij2, Savitree Limtong3,4
Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, Sanamchandra
palace campus, Nakorn Pathom 73000, Thailand; 2Central Laboratory and Greenhouse Complex, Faculty of Agriculture,
Kasetsart University, Kamphaeng Sean Campus, Nakhon Pathom 73140, Thailand; 3Department of Microbiology,
Faculty of Science, Kasetsart University, Jatujak, Bangkok 10900, Thailand; 4Center for Advanced Studies in Tropical
Natural Resources, National Research University-Kasetsart
University, Bangkok 10900, Thailand
1
[email protected]
The epiphytic yeast diversity in corn phyllosphere in Thailand was investigated by cultureindependent technique based on the sequence of the D1/D2 domain of the large subunit rRNA
gene. Thirty-seven samples of corn leaf were collected randomly from 10 provinces in Thailand.
The DNA was extracted from leaf washing samples and the D1/D2 domain was amplified using
PCR technique. The PCR products were cloned and then screened by colony PCR. The restriction
analysis was used to preliminarily cluster the PCR products from each clone library. Of total 1,041
clones, 357 clones (33.7%) revealed the D1/D2 domain sequences closely related to sequences of
yeasts in GenBank, and they were clustered into 108 operational taxonomic units (OTUs) at 99%
homology cut off. The majority of yeasts presented in corn leaf surfaces were members of phylum
Basidiomycota (98.0%). Of total yeast related clones, 98 clones (27.5%, 14 OTUs) were identified
as 12 known yeast species including Bullera derxii, B. oryzae, Cryptococcus flavescens, Hannaella
sinensis, Jaminaea angkoriensis, Pseudozyma antarctica, P. aphidis, P. hubeiensis, P. prolifica,
Sporidiobolus pararoseus, Sporobolomyces carnicolor and S. odoratus. The D1/D2 sequences
(259 clones) that could not be identified as known yeast species were closest to 1 and 26 species in
Ascomycota and Basidiomycota, respectively, some of which may be new yeast species. Interestingly,
yeasts in subphylum Ustilaginomycotina were mostly detected (72.3%). Pseudozyma was the most
prevalent yeast genus and it seems to represent the asexual counterpart of Ustilaginales that parasitize
monocotyledonous plants. Some species of this genus has been found in the gut of several pests of
corn. The most common yeast species detected were P. antarctica and P. hubeiensis with 24.3 %
frequency of occurrence in corn phyllosphere samples.
KEYWORDS: Corn, Phyllosphere, D1/D2 domain, Epiphytic yeast, Culture-independent
REFERENCES:
Kurtzman CP, Fell JW, Boekhout T, Robert V (2011). Methods for isolation, phenotypic characterization and
maintenance of yeasts. In: Kurtzman CP, Fell JW, Boekhout T (eds) The Yeasts. A Taxonomic study. Elsevier,
Amsterdam, pp 87–110
Molnar O, Wuczkowski M, Prillinger H (2008). Yeast biodiversity in the guts of several pests on maize; comparison of
three methods: classical isolation, cloning and DGGE. Mycological Progress 7:111-123
5
Social wasps are mating nests for yeasts
Irene Stefanini1, Leonardo Dapporto2, Luisa Berná3, Mario Polsinelli4, Stefano Turillazzi4,5,
Duccio Cavalieri1,6
Centre for Research and Innovation, Fondazione Edmund Mach, San Michele all’Adige, Trento, Italy; 2Department of
Biological and Medical Sciences, Oxford Brookes University, Headington, Oxford, OX3 0BP, UK; 3Molecular Biology
Unit, Institut Pasteur, Montevideo, Uruguay; 4Department of Biology, University of Florence, Italy; 5Centro di Servizi
di Spettromeria di Massa, University of Florence, Florence, Italy; 6Department of Neuroscience, Psychology, Drug
Research and Child’s health, University of Florence, Florence, Italy
1
[email protected]
Saccharomyces cerevisiae (Sce) is largely used as a model for a wealth of purposes. The recent
availability of genome sequences of a large number of S. cerevisiae and S. paradoxus (Spa) strains
representing the widest known genetic, phenotypic and geographical diversity renewed the interest
in the use of these yeasts as models for evolution and ecology studies. Nevertheless, one of the
still unanswered questions is whether genetically diverse yeasts mate and recombine in the wild.
The yeasts outcrossing was estimated to occur only once every 105 mitotic division, thus confining
their reproduction to mitosis and to occasional intra-ascus breeding (inbreeding). Although, the
recent observation on larger set of strains of unexpectedly high levels of genetic heterozygosity and
prions diffusion called the rarity of outcrossing into question. To outbreed at least two conditions
have to occur: i) different strains has to simultaneously inhabit the same area, ii) they have to face
environmental oscillations favouring sporulation (because natural yeasts are usually diploid) followed
by germination. Social wasps have been shown to bear yeast cells all year long and feeding on sources
that are potentially inhabited by different Saccharomyces spp. strains, thus representing a potential
incubator for different yeast cells to meet and mate. Here we show that the intestine of social wasps
favours the mating of different yeast strains and species by providing a
sequentiality of environmental conditions prompting the sporulation and germination of S. cerevisiae
and making heterospecific mating the only option for S. paradoxus to survive. Our results open a new
perspective introducing insects as unaware players in the evolution of Saccharomyces spp. yeasts.
Saccharomyces spp. yeasts could prefer sexual reproduction to react to the environment changes
occurring within the wasp intestine and in the continuous flux from the wasp to the environment and
vice-versa.
KEYWORDS : Saccharomyces cerevisiae, Saccharomyces paradoxus, Ecology, Mating, Evolution
6
Phylogenetics, ecology and biogeography of Komagataella yeasts: molecular and
genetic analysis
Elena S. Naumova1, Kyria Boundy-Mills2, Gennadi I. Naumov1
1
State Institute for Genetics and Selection of Industrial Microorganisms, I Dorozhnyi proezd, 1, Moscow 117545,
Russia; 2Phaff Yeast Culture Collection, Department of Food Science and Technology, University of California, One
Shields Avenue, Davis, CA 95616, USA
[email protected]
The methanol-assimilating genus Komagataella currently includes six phenotypically similar
species: K. pastoris, K. pseudopastoris, K. phaffii, K. populi, K. ulmi and K. kurtzmanii (Kurtzman
2011; Naumov 2013). The first two species have European origin, while the other four NorthAmerican. Three species (K. pastoris, K. phaffi and K. kurtzmanii) are used in genetic engineering
and biotechnology for recombinant protein production. The genus Komagataella is a good object to
study evolutionary genetics, ecology and taxonomy of ascomycetous yeasts.
We have conducted a molecular-genetic study of big collection of strains maintained at Phaff’s Yeast
Culture Collection (Davis, USA). The strains were initially identified as Komagataella (Pichia)
pastoris.Our approach combines phylogenetic analysis of the D1/D2 LSU rRNA and ITS1-5.8SITS2 sequences with classical genetic hybridization. Comparative D1/D2 and ITS analyses revealed
four species (K. pastoris, K. phaffi, K. populi and K. ulmi) among 38 strains studied. According to
the phylogenetic analysis, some strains may represent novel Komagataella species. Using induced
complementary auxotrophic mutants and selective growth of prototrophic hybrids on minimal
medium, hybridization of the type culture of K. kurtzmanii VKPM Y-727 with the type strains of K.
pastoris NRRL Y-1603, K. phaffii NRRL Y-7556, K. populi NRRL YB-455, K. pseudopastoris NRRL
Y-27603 and K. ulmi NRRL YB-407 was demonstrated. However, due to postzygotic isolation, the
resulting interspecies hybrids were sterile, having non-viable ascospores. The data obtained suggest
that the genus Komagataella, established earlier by phylogenetic analysis, corresponds well to the
concept of genetic genus in ascomycetous fungi. According to this concept (Naumov 1979), a genetic
genus is a group of hybridized species having a common mating type system. Application of the
concept of genetic genus for different yeast genera is discussed.
KEYWORDS: Komagataella, Pichia pastoris, Sibling species, Interspecies hybridization
REFERENCES:
Kurtzman CP (2011). Komagataella Y. Yamada, Matsuda, Maeda & Mikata (1995). In: Kurtzman CP, Fell JW, Boekhout
T (eds) The Yeasts. A Taxonomic study. Elsevier, Amsterdam, pp 491–495
Naumov GI, Naumova ES, Tyurin OV, Kozlov DG (2013).Komagataella kurtzmanii sp. nov., a new sibling species
of Komagataella (Pichia) pastoris in accordance with multigene sequence analysis. Antonie van Leeuwenhoek
104:339–347
Naumov GI (1979).Genetic concept of genus in fungi. Doklady Biological Sciences 241:345–347
7
Yeast diversity in extreme environments of southern South America
Virginia de García, Martín Moliné, Diego Libkind
Laboratorio de Microbiología Aplicada y Biotecnología, INIBIOMA, UNComahue – CONICET, Bariloche, Argentina
[email protected]
Yeasts that constantly live under stress conditions evolve adaptive mechanisms destinated to
minimize or resist their negative effects and thus still permit survival and reproduction. The
study of yeasts inhabiting extreme environments is still limited, particularly in pristine habitats of
South America. Here we resume numerous yeast diversity studies performed in non-conventional
environments, mainly of Argentina, which are exposed to one or more of the following factors: high
UV irradiance, desiccation, low temperatures, very high (> 10) or low pH (< 2), presence of heavy
metals (volcanic origin), ultraoligotrophicity. Over 1000 yeasts and dimorphic fungi were collected,
molecularly identified, and when possible relevant secondary metabolites were screened, as well
as ability to tolerate several types of stress in laboratory conditions. The latter include carotenoid
pigments, mycosporines (UV sunscreens), psycroactive enzymes, among others. In some cases these
activities could be correlated to habitat characteristics and for such (ex. mycosporines, carotenoid
pigments, heavy metal tolerance) their potential role in the adaptive mechanisms to specific stress
factors was evaluated. At least 100 different yeast species were identified and 25 novel taxa were
detected. Genome sequencing and analysis was performed for biotechnologically relevant isolates of
Phaffia rhodozyma, Saccharomyces eubayanus, S. uvarum, Giraudozyma gen. nov. and Guehomyces
pullulans. The present work represents an overview of our findings related to the biodiversity, ecology,
physiology and genetics of extremophilic and polyextromophilic yeasts in southern South America.
KEYWORDS: Extremophilic yeasts, UV radiation, Cold habitats, Adaptation
8
Yeast communities in slovenian virgin olive oil and their taxonomic placement
Neža Čadež1, Mateja Mervič1, Gábor Péter2
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; 2National Collection of
Agricultural and Industrial Microorganisms, Faculty of Food Sciences, Corvinus University of Budapest, Somlói út
14-16. H-1118 Budapest, Hungary
1
[email protected]
Due to the extremely low aw value of olive oil, it is uncommon as substrate for growth of microorganisms.
Nevertheless, we found that yeasts are the predominant microorganisms which can be found growing
in it. The extra virgin olive oil is obtained from the fruit of the olive tree (Olea europaea L.) solely
by milling and cold-pressing of olive flesh. Yeasts as predominant microorganisms in olive oil have
either a positive role by de-bittering of the olive oil or negative roles by causing undesired acidity
due to their lipolytic activity and spoilage of olive fermentations by their pectinolytic activity. During
a four years survey of yeasts associated with extra virgin olive oils of different geographical origins
like Slovenia, Croatia and Italy, four groups of isolates showing a distinct physiological profile were
found. By an extent of divergence in the D1/D2 region of the large-subunit rDNA we predicted that
they represented four novel yeast species, two of which belonged to Nakazawaea and Cyberlindnera
clades while three other species were found to be methylotrofic yeasts belonging to the genus
Ogataea. The three novel species O. kolombanensis, O. histrianica and O. deakii were found to
belong to three closely related species in Minnimum Spanning Tree based on the comparisons of the
concatenated gene sequences from the SSU, ITS/5.8S, LSU D1/D2 domains of the rRNA and the
translation elongation factor-1α. Interestingly, the high EF-1α gene sequence divergence among and
between the type strains of the three novel species is suggesting the presence of heterozygous diploids
or the presence of paralogs of the elongation factor gene that amplify with applied primer sequences.
Furthermore, by cloning of the PCR product of EF-1α gene, up to six variants of EF-1alpha genes
were recovered from individual strains indicating mating between polymorphic haploid strains. By
analysis of the sequence variance the population structure of Ogataea species will be discussed.
KEYWORDS: Yeast diversity, Olive oil, New species, Phylogenetic marker
REFERENCES:
Valenčič V, Bandelj Mavsar D, Bučar-Miklavčič M, Butinar B, Čadež N, Golob T, Raspor P, Smole Možina S (2010).
Food Technology and Biotechnology 48:404-410
Čadež N, Raspor P, Turchetti B, Cardinali G, Ciafardini G, Veneziani G. & Péter G (2012). International Journal of
Systematic and Evolutionary Microbiology 62:2296–2302
9
Diversity of yeasts from marine waters in arabian gulf surrounding Qatar
Rashmi Fotedar1, Aisha Zeyara1, Anna Kolecka2, Amina Al Malaki1, Jack W. Fell3, Sabine
Filker4, Hans-Werner Breiner4, Sayed J. Bukhari5, Mohamed A. Abdel-Moati6, Eric Febbo7,
Saad J.Taj-Aldeen8, Thorsten Stoeck4 , Masoud Al Marri1, Teun Boekhout2
1
Department of Genetic Engineering, Biotechnology Centre, Ministry of Environment, Doha, State of Qatar; 2CBS
Fungal Biodiversity Centre (CBS-KNAW), Utrecht, The Netherlands; 3Rosenstiel School of Marine and Atmospheric
Sciences, University of Miami, Key Biscayne, Florida, USA; 4University of Kaiserslautern, Ecology Group, Erwin
Schroedinger Str. 14, D-67663 Kaiserslautern, Germany; 5Environment Information System Department, Ministry of
Environment, Doha, State of Qatar; 6Environmental Assessment Department, Ministry of Environment, Doha, State
of Qatar; 7ExxonMobil Research Qatar (EMRQ), Doha, State of Qatar; 8Department of Lab Medicine and Pathology,
Hamad Medical Corporation, Doha, State of Qatar
[email protected]
The Arabian Gulf surrounding Qatar is distinct from other marine ecosystems due to its high salinity
(39-57 psu) and extreme water temperature fluctuations (18-39°C). Furthermore in the last decade,
Qatar has been witnessing an industrial boom as well as extensive infrastructure construction activities.
During the first year of a 3 year study, we investigated the diversity of marine yeasts in Qatar. Water
samples were collected during two seasons (winter 2013 and summer 2014) from 14 different
sites along the coastal waters of the Arabian Gulf surrounding Qatar. Yeast species were isolated
and identified by sequence analyses of the internal transcribed spacers (ITS1/ITS2) and the D1/D2
domains of the large subunit (LSU) of the ribosomal DNA (rDNA). A total of 262 yeast isolates of
belonging to 27 genera of Ascomycetes and Basidiomycetes were identified during the two sampling
campaigns. Species distribution depicted seasonal and geographical differences. Candida spp. (27%),
Rhodotorula spp. (12%), Kondoa aeria (8%), and Aureobasidium spp (7%) were among the most
frequently identified yeast species. Some species (Kondoa aeria, Knufia petricola, Sakaguchia
dacryoidea, Pseudozyma spp.) were only isolated during summer, whereas Clavispora lusitaniae,
Debaryomyces spp., Delphinella strobiligena, Erythrobasidium hasegawianum, Geotrichum spp.,
Issatchenkia orientalis, Kazachstania servazzii, Kodamaea ohmeri, Phaeococcomyces chersonesos,
Pichia spp., Saccharomycopsis crataegensis and Trichosporon c.f. asahii complex were only isolated
during winter. The highest number of yeast isolates was recovered from sites impacted by landbased activities, especially fishing harbors along the Eastern coast of Qatar. Konda aeria, Hortaea
werneckii, Delphinella strobiligena and Erythrobasidium hasegawianum were isolated from the high
salinity areas (range 44-57 psu). This report is the first study on the diversity of yeasts from the
marine environment surrounding Qatar.
Acknowledgements This Research was supported by grant (NPRP-6-647-1-127) from the Qatar
National Research Fund (a member of Qatar Foundation) to Rashmi Fotedar, Teun Boekhout, Jack.
W. Fell, and Thorsten Stoeck.
10
Session 1B
Yeasts in the environment: physiology and stress
response
11
Session 1B: Yeasts in the environment: physiology and stress response
Quality control of plasma membrane proteins by yeast Nedd4-like ubiquitin
ligase Rsp5 under environmental stress conditions
Takeki Shiga, Daisuke Watanabe, Hiroshi Takagi
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan
[email protected]
In eukaryotic cells, plasma membrane proteins with limited conformational defects can escape
endoplasmic reticulum (ER), localize at the plasma membrane, and be eliminated by lysosomal
degradation. However, it is poorly understood how the post-ER quality control is involved in
degradation of aberrant plasma membrane proteins generated by environmental stresses. In the yeast
Saccharomyces cerevisiae, when a rich nitrogen source such as ammonium is added to the culture
medium, the general amino acid permease Gap1 is ubiquitinated by the yeast Nedd4-like ubiquitin
ligase Rsp5, followed by its endocytosis to the vacuole. The arrestin-like Bul1/2 adaptors for Rsp5
specifically mediate this process. Here, to investigate the downregulation of Gap1 in response
to environmental changes, we analyzed the intracellular trafficking of Gap1 under various stress
conditions.
An increase in the extracellular ethanol concentration induced ubiquitination and trafficking of Gap1
from the plasma membrane to the vacuole in wild-type cells, whereas Gap1 remained stable on the
plasma membrane under the same conditions in rsp5A401E and ∆end3 cells. A 14C-labelled citrulline
uptake assay using a non-ubiquitinated form of Gap1 (Gap1K9R/K16R) revealed that ethanol stress caused
a dramatic decrease of Gap1 activity. These results suggest that Gap1 is inactivated and ubiquitinated
by Rsp5 for endocytosis when S. cerevisiae cells are exposed to a high concentration of ethanol. This
endocytosis occurs in a Bul1/2-independent manner, whereas ammonium-triggered downregulation
of Gap1 was almost completely inhibited in ∆bul1/2 cells. We also found that other environmental
stresses, such as high temperature and H2O2, also promoted endocytosis of Gap1. Similar intracellular
trafficking caused by ethanol occurred in other plasma membrane proteins (Agp1, Tat2, and Gnp1).
Our findings suggest that stress-induced quality control is a common process requiring Rsp5 for
plasma membrane proteins.
KEYWORDS: Saccharomyces cerevisiae, The ubiquitin ligase Rsp5, The general amino acid
permease Gap1, Stress response, Protein quality control
REFERENCES:
Shiga T, Yoshida N, Shimizu Y, Suzuki E, Sasaki T, Watanabe D, Takagi H (2014). Quality control of plasma
membrane proteins by yeast Nedd4-like ubiquitin ligase Rsp5p under environmental stress conditions. Eukaryotic
Cell 13:1191-1199
12
Transcriptional and metabolic responses of different Saccharomyces cerevisiae
strains to hyperosmotic stress caused by inoculation in grape must
Enrico Vaudano, Olta Noti, Antonella Costantini, Francesca Doria,
Emilia Garcia-Moruno
Consiglio per la Ricerca in Agricoltura e l’analisi dell’economia agraria - Centro di Ricerca per l’Enologia,
Via Pietro Micca 35, 14100 Asti, Italy
[email protected]
During the winemaking process, glycerol synthesis represents the first adaption response of
Saccharomyces cerevisiae to osmotic stress after inoculation in grape must. With the aim to monitor
this response, we studied the early metabolites production and we have implemented an RT-qPCR
(Real Time-quantitative PCR) methodology to study the expression of metabolites related genes.
The transcription intensity in three strains, previously selected on the basis of different metabolite
production at the end of fermentation, was monitored in the first 120 min from inoculation into
natural grape must with a preventive evaluation of candidate reference genes. We studied six target
genes related to glycerol synthesis (GPD1, GPD2, GPP2 and GPP1) and flux (STL1 and FPS1),
and three ALD genes coding for aldehyde dehydrogenase involved in redox equilibrium via acetate
production. At the same time the production of intra/extra glycerol, acetate and ethanol was followed.
Expression analysis showed a transient response of genes GPD1, GPD2, GPP2, GPP1 and STL1 with
differences among strains in term of mRNA abundance, while FPS1 was expressed constitutively.
The metabolite analysis showed a quick response in term of glycerol accumulation where an
“optimum” of concentration was reached in few minutes; afterward glycerol in excess freely flow
through aquaglyceroporin Fps channels. The transient response and different expression intensity
among strains, in relation to the intracellular glycerol accumulation pattern, prove the negative
feedback control via the HOG (High Osmolarity Glycerol) signalling pathway in S. cerevisiae wine
strains under winemaking conditions. Among the ALD genes, only ALD6 was moderately induced
in the hyperosmotic in two of three strains tested, while ALD3 and ALD4 were drastically glucose
repressed. The intensity of transcription of ALD6 and ALD3 seems to be related to different acetate
production found among the strains.
KEYWORDS: Saccharomyces cerevisiae, Glycerol, Hyperosmosis, RT-qPCR
REFERENCES
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, ShipleyGL,
Vandesompele J, Wittwer CT (2009). The MIQE guidelines: minimum information for publication of quantitative
real-time PCR experiments. Clinical Chemistry 55:611–622
Hohmann S (2002). Osmotic stress signaling and osmoadaptation in yeasts. Microbiology and Molecular Biology
Review 66:300–372
Jiménez-Martí E, Gomar-Alba M, Palacios A, Ortiz-Julien A, Del Olmo ML (2011). Towards an understanding of the
adaptation of wine yeasts to must: Relevance of the osmotic stress response. Applied Microbiology and Biotechnology
89:1551–1561
Petelenz-Kurdziel E, Kuehn C, Nordlander B, Klein D, Hong KK, Jacobson T, Dahl P, Schaber J, Nielsen J, Hohmann
S, Klipp E (2013). Quantitative analysis of glycerol accumulation, glycolysis and growth under hyper osmotic stress.
PLOS Computational Biology 9(6): e1003084
13
Heterogeneity of response to heat stress in Saccharomyces cerevisiae
Jennifer Dumont1, Stéphane Guyot1, Patrick Gervais1, Michael Young2, Pascale Winckler3,
Hazel M. Davey2
UMR PAM Procédés Alimentaires et Microbiologiques, Equipe Procédés Microbiologiques et Biotechnologiques,
Université de Bourgogne / Agrosup Dijon, 1 Esplanade Erasme 21000 Dijon, France; 2Institute of Biological,
Environmental and Rural Sciences, Aberystwyth University, Penglais, Aberystwyth, Wales, U.K. SY23 3DA; 3Spectral
Imaging Resource Center, DIMACELL, AgroSup Dijon, 1 Esplanade Erasme 21000 Dijon, France
1
[email protected]
Microbial populations have to cope with a continuously changing environment both in Nature and
in the food industry. Indeed, they are exposed to multiple stresses impacting on their physiology.
As previously shown, their ability to survive depends on the stress kinetic. When Saccharomyces
cerevisiae is submitted to a heat slope from 25 °C to 50 °C (gradual heating 0.5 °C.min-1), it acquires
a certain degree of thermal resistance that is not achieved during a sudden heat shock (applied within
30 s). The mechanisms involved in thermal resistance are still not well understood.
Using single-cell methods (e.g. flow cytometry) compared to analysis at the whole population level
(for instance using two-photon fluorescence microscopy), the heterogeneity of the heat stress response
within populations of yeast cells has been studied and subpopulations have been characterized. The
impact of heat stress kinetics on the physico-chemical properties of the plasma membrane has been
investigated both at the cell and the population levels. The plasma membrane fluidity of yeast cells
exposed to heat shock and heat slope has also been measured at the cell and population scales after
recovery at 25 °C.
Results showed that plasma membrane fluidity was not a key factor involved in this type of resistance
whereas the major role of membrane permeability/potential in cell survival or death has been
highlighted.
These results help to characterize survival populations which are directly related to the capacity of
growth and regrowth of populations facing environmental heat stress. This work gives new insight
in our understanding of heat stress impacts at the level of the single cell compared to the whole
population. The increasing importance of microbes in biotechnological processes such as biofuel
production and bioremediation, makes a thorough understanding of the impact of stress responses of
populations and individuals necessary and valuable.
KEYWORDS: Heat stress, Kinetic, Heterogeneity, Plasma membrane, Thermoresistance
REFERENCES:
Guyot S, Gervais P, Young M, Winckler P, Dumont J, Davey HM (2015). Surviving the heat: Heterogeneity of response
in Saccharomyces cerevisiae provides insight into thermal damage to the membrane. Environmental Microbiology (in
press)
14
The unexpected role of ergosterol in yeast adaptation to hydric fluctuations
Sébastien Dupont, Céline Lafarge, Philippe Cayot, Patrick Gervais, Laurent Beney
UMR Procédés Alimentaires et Microbiologiques, Université de Bourgogne/AgroSup Dijon, Dijon, France
[email protected]
Sterols are represented by three predominant forms: cholesterol in vertebrates, phytosterols in
plants, and ergosterol in fungi. The specificity of sterols, in each life kingdom, could be related to
biological evolution but the origin of the ramification into distinct ways after common early steps
is an intriguing fact that remains unclear. Especially, the reason why ergosterol was preferred by
fungi is not elucidated since its synthesis requires more energy than cholesterol and the structurefunction studies have failed to show advantages of ergosterol. We hypothesized that the origin of the
ergosterol biosynthetic pathway (EBP) could be related to some fungi’s life specificities that could
have constituted the main force that drove the biochemical evolution of this pathway. Fungi are well
adapted to interfacial habitats where they experience relative humidity fluctuations and anhydrobiosis
(Dupont et al 2014). As ergosterol is known to increase the mechanical resistance of cell’s membrane
to osmotic dehydration (Dupont et al 2011), the EBP may have been selected under the constraints
caused by transitions between wet and dry conditions. Survivals of Saccharomyces cerevisiae mutants
of the EBP were compared after transitions from aquatic to aerial medium. The resistance was found
to depend on the progression in the EBP. Early mutants in the pathway (erg6Δ, erg2Δ, erg3Δ) were
found to be highly sensitive and the resistance of the next two mutants (erg5Δ and erg4Δ) was higher.
The wild type strain, accumulating ergosterol, exhibited the best survival rate. This survival is linked
to the role of ergosterol in plasma membrane protection against mechanical and oxidative constraints
(Dupont et al 2012). Therefore, evolution of the EBP may have been a key element in the conquest
of solid-aerial interfacial habitats by fungi. This study suggests answers to the unanswered question
“Why ergosterol in Fungi?” and proposes an unexpected role of ergosterol in yeast adaptation to
hydric fluctuations.
KEYWORDS: Ergosterol, Dehydration, Cell resistance, Plasma membrane, Oxidation
REFERENCES:
Dupont S, Rapoport A, Gervais P, Beney L (2014). Survival kit of Saccharomyces cerevisiae for anhydrobiosis. Applied
Microbiology and Biotechnology 98(21): 8821-34
Dupont S, Beney L, Ferreira T, Gervais P (2011). Nature of sterols affects plasma membrane behavior and yeast
survival during dehydration. Biochimica et Biophysica Acta 1808(6): 1520-8
Dupont S, Lemetais G, Ferreira T, Cayot P, Gervais P, Beney L (2012). Ergosterol biosynthesis: a fungal pathway for
life on land? Evolution 66(9):2961-8
15
Anhydrobiosis in yeast: unique state of live organisms and its possible nonconventional applications
Alexander Rapoport1, Diana Borovikova1, Linda Rozenfelde1, Silvia Lisi2,
Pietro Buzzini2
Laboratory of Cell Biology, Institute of Microbiology and Biotechnology, University of Latvia, Riga, Latvia;
Department of Agricultural, Food and Environmental Science & Industrial Yeasts Collection DBVPG, University of
Perugia, Perugia, Italy
1
2
[email protected]
Anhydrobiosis is a unique state of live organisms when their metabolism is temporary reversibly
delayed as the result of very essential dehydration of cells. It is revealed that yeast cells transfer
into this state is followed by structural-and-functional changes in all organelles. These changes are
found at the level of cell wall, plasma membrane, nucleus, vacuoles and peroxisomes. The minimum
changes take place in mitochondria. A number of intracellular protective reactions occur at the early
stages of dehydration process. Main factors which determine the preservation of yeast cells viability
at their transfer into anhydrobiosis are revealed at the moment. One of these factors is linked with
the maintenance of molecular organization of plasma membrane when it loses significant amounts
of “bound” water. The conclusion is made on the changes and importance of both components of
intracellular membranes (proteins and lipids). A new model system based on the comparison of
characteristics of close wild strains grown in similar or different conditions for further studies of
mechanisms of anhydrobiosis has been developed during last years. The possibility to reach the state
of anhydrobiosis for yeast cells grown in anaerobic conditions has been found recently. It is shown
that besides traditional applications of active dry yeasts (which are in the state of anhydrobiosis) in
bread baking and winemaking there are also some other interesting possibilities to use knowledge on
this unique state of live nature in biotechnology. One of them is linked with the development of new
test-system for the evaluation of natural and chemical compounds for cosmetic and pharmaceutical
industry. It is shown the efficiency of viable dry yeast use for the production of biofilters for the
purification of waste waters and protection of the environment. Knowledge on anhydrobiosis gives
the approach for the obtaining of new stable, cheap and efficient immobilized yeast preparations.
KEYWORDS: Anhydrobiosis, Dehydration, Cell resistance, Plasma membrane
16
Genetic adaptive mechanisms mediating response and tolerance to acetic acid
stress in the human pathogen Candida glabrata: role of the CgHaa1-dependent
signaling pathway
Ruben T. Bernardo1, Diana V. Cunha1, Can Wang2, Hiroji Chibana3, Sónia Silva4, Isabel SáCorreia1,5, Joana Azeredo4, Geraldine Butler2, Nuno P. Mira1,5
iBB, Institute for Bioengineering and Biosciences, Av. Rovisco Pais, 1049-001 Lisboa; 2School of Biomolecular and
Biomedical Sciences, Conway Institute, University College of Dublin, Belfield; 3Medical Mycology Research Center,
Chiba University, Chiba, Japan; 4CEB, Centre of Biological Engineering, LIBRO – Laboratório de investigação em
Biofilmes Rosário Oliveira, University of Minho, Campus de Gualtar 4710-057, Braga, Portugal; 5Instituto Superior
Técnico, Department of Bioengineering, Universidade de Lisboa, Avenida Rovisco Pais, 1049-001 Lisbon, Portugal
1
[email protected]
C. glabrata is a commensal found in the human genitourinary tract but under certain conditions this
harmless colonization evolves to a mucosal infection and, in more serious cases, to disseminated
mycosis. To thrive in the acidic vaginal tract C. glabrata has to cope with the presence of a competing
commensal microbiota known to restrain the overgrowth of pathogens through the production of acetic
and lactic acids, among other interference effects. The persistent emergence of C. glabrata strains
resistant to currently used antifungals demands the implementation of novel therapeutic strategies
based on non-conventional targets. Genes contributing to increase C. glabrata competitiveness in the
vaginal tract by mediating tolerance to the organic acids found therein are a cohort of interesting and
yet unexplored therapeutic targets.
Tolerance mechanisms of C. glabrata to acetic acid at low pH are poorly studied but much knowledge
was gathered in Saccharomyces cerevisiae (Mira et al 2010a; 2010b; 2011; 2010c). In particular,
the central role of the ScHaa1 transcription factor in mediating S. cerevisiae tolerance to acetic acid
stress was demonstrated (Mira et al 2010b; 2011; 2010c). In this work it is shown that CgHaa1, an
orthologue of ScHaa1, controls an acetic acid-responsive system in C. glabrata. The mechanisms by
which the CgHaa1 pathway mediate tolerance to acetic acid in C. glabrata were further dissected,
exploring a transcriptomics approach, being of notice the involvement of this regulatory system in
the control of internal pH and in reducing the internal accumulation of the acid. In the presence of
acetic acid CgHaa1 enhanced adhesion and colonization of reconstituted vaginal human epithelium
by C. glabrata, this correlating with a positive effect of CgHaa1 over the expression of adhesinencoding genes. The results obtained show similarities, but also remarkable differences, in the way
by which the ScHaa1 and CgHaa1 pathways mediate tolerance to acetic acid in S. cerevisiae and in
C. glabrata, indicating a “functional expansion” of the network in the later species. The role of the
CgHaa1-pathway in the extreme acetic acid-tolerance exhibited by vaginal C. glabrata isolates will
also be discussed, along with other uncovered mechanistic insights.
KEYWORDS: Acetic acid stress, Candida glabrata, Vaginal candidiasis, Stress response, Targets for
antifungal therapy
REFERENCES:
Mira NP et al (2010a). OMICS 14: 587-60
Mira NP et al (2010b). OMICS 14 :525-40
Mira NP et al (2011). Nucleic Acids Research 16 :6896-907
Mira NP et al (2010c). Microbial Cell Factories 9-79
17
18
Session 2A
Yeasts in food biotechnology:
biodiversity and ecology in foods and beverages
19
Session 2A: Yeasts in food biotechnology: biodiversity and ecology in foods and beverages - Key note
Yeasts and the fermented food renaissance
Graham H. Fleet
Food Science Group. School of Chemical Engineering, University of New South Wales, Sydney, Australia
[email protected]
There is a renaissance in the ancient art and process of fermenting food, recently popularized by titles
such as “ a festive ferment” (Mc Gee 2013) or “the new fermented food culture” (Despain 2014). A
diversity of factors are driving this movement. Scientific understanding of their microbiology and
chemistry has enabled the development of simplified processes that give safe, consistent quality
products and can be managed to enrich the intensity, complexity and enjoyment of the sensory
experience. Greatly expanded awareness of the globality of fermented foods and beverages has
revealed new diversity in the raw materials that can be exploited and transformed into novel and
valuable products. New insights into the linkages between diet, nutrition , the gut microbiome and
human health reveal fermented foods as natural functional foods with in situ probiotic organisms and
bioactive components that have the potential to positively impact on human well-being including a
vast range of physiological, immunological and mental / psychological conditions. What is the role of
yeasts? Yeasts are far more prevalent in many fermented foods and beverages than previously thought.
They contribute directly to the chemical, physical, sensory, nutritional and bioactive properties of the
product but, indirectly, they also modulate the contributions of bacteria and filamentous fungi through
microbial teamwork. This presentation describes the diversity of yeasts in fermented foods and
beverages and how they impact on product quality, safety, bioactive potential, and dietary/ nutritional
functionality. Emphasis will be given to some of the less well known products.
KEYWORDS: Fermented foods, Yeast diversity, Impact
REFERENCES:
Despain D (2014). The new fermented food culture. Food Technology ( September issue) 40-45 McGee H (2013). A
festive ferment. Nature 504: 372-374
20
Session 2A: Yeasts in food biotechnology: biodiversity and ecology in foods and beverages
Yeast species associated with Drosophila in vineyard ecosystems
Samuel S.T.H Lam, Kate S. Howell
Faculty of Veterinary and Agricultural Sciences, University of Melbourne Parkville Victoria 3010, Australia
[email protected]
The activity of yeasts in wine fermentations directly contributes to wine quality, but the source and
movement of these yeasts in vineyard and winery environments has not been resolved. This study
investigates the yeast species associated with a insect vector to help understand yeast dispersal
and persistence. Drosophila are commonly found in vineyards and Drosophila and yeasts have a
known mutualistic relationship in other ecosystems. Drosophilids were collected from vineyards,
marc piles and wineries during the grape harvest. Captured flies were identified morphologically
to and their associated yeasts were identified. Of the 296 Drosophila flies captured in this study
the species identified were Drosophila melanogaster, Drosophila simulans, Drosophila hydei, and
Scaptodrosophila lativittata. These flies were associated with the yeasts Metschnikowia pulcherrima,
Hanseniaspora uvarum, Torulaspora delbrueckii and Hanseniaspora valbyensis. The diversity of
yeasts and Drosophila species differed between collection locations (vineyard and marc; R=0.588
for Drosophila and R= 0.644 for yeasts). Surprisingly, the primary wine fermentation yeast,
Saccharomyces cerevisiae was not isolated in this study. Drosophila flies are preferentially associated
with different species of yeasts in the vineyard and winery environment and this association may help
movement and dispersal of yeast species in the vineyard and winery ecosystem.
KEYWORDS: Drosophila, Vineyard, Yeast diversity, Biogeography, Wine
21
Combine use of selected Schizosaccharomyces pombe and Lachancea
thermotolerans yeast strains as an alternative to the traditional malolactic
fermentation in red wine production
Santiago Benito
Departamento Química y Tecnología de Alimentos, Universidad Politécnica de Madrid, Ciudad Universitaria S/N,
28040, Spain
[email protected]
Most red wines that are commercialized in market develop the malolactic fermentation process in
order to be stabilized from a microbiological point of view. In this second fermentation, malic acid
is converted into L-lactic. However such process is not free from possible collateral effects that on
some occasions produce off flavors, wine quality loss and human health problems such as biogenic
amines production.
This manuscript develops a new red winemaking methodology that consists on combining the use
of Lachancea thermotolerans and Schizosaccharomyces pombe as an alternative to the traditional
malolactic fermentation. In this method, malic acid is totally consumed by Schizosaccharomyces
pombe reaching the microbiological stabilization objective while Lachancea thermotolerans produces
lactic acid in order not to reduce and even increase the acidity of wines produced from low acidic
musts. Several fermentations involving selected Lachancea thermotolerans, Schizosaccharomyces
pombe, Saccharomyces cerevisiae and Oenococus oene strains were performed. Final results were
compared from a chemical and sensorial point of view.
The result from the proposed technique were more fruity wines without any acidity loss that contained
less acetic acid and biogenic amines than the traditional controls that perform classical malolactic
fermentation.
REFERENCES:
Kapsopoulou K, Mourtzini A, Anthoulas M, Nerantzis E (2007). Biological acidification during grape must
fermentation using mixed cultures of Kluyveromyces thermotolerans and Saccharomyces cerevisiae. World Journal of
Microbiology and Biotechnology 23:735–739
Gobbi M, Comitini F, Domizio P, Romani C, Lencioni L, Mannazzu I, Ciani M (2013). Lachancea thermotolerans and
Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve
the overall quality of wine. Food Microbiology 33:271–281
Benito S, Palomero P, Calderón F, Palmero D, Suárez-Lepe JA (2014a). Schizosaccharomyces. In: Batt CA, Tortorello
ML (eds) Encyclopedia of Food Microbiology. Elsevier, Amsterdam, vol 3, pp 365–370
Benito S, Palomero P, Calderón F, Palmero D, Suárez-Lépe JA (2014b). Selection of Appropriate Schizosaccharomyces
strains for winemaking. Food Microbiology 42:218-224
22
Fungal flora of a traditional french cheese, the “Tomme d’Orchies” showed
strains of Kluyveromyces with atypical antagonistic properties
Alexandre Ceugniez, Françoise Coucheney, Philippe Jacques, Djamel Drider
University of Lille 1. Charles Viollette Institute: Regional Laboratory of Research on Food and Biotechonology. 59650
Villeneuve d’Ascq, France
[email protected]
The aim of this study was to investigate and evaluate the competitive and antagonistic properties of
yeasts isolated from Tomme d’Orchies, a traditional raw milk French cheese.
To this end, culture-dependent of yeasts microbiota of Tomme d’orchies was performed on the crust
and core. Thus colonies with yeasts characteristics were grouped molecularly using REP-PCR, and
then identified by sequencing the 26S rDNA and ITS1-5.8S-ITS2 region.
Overall, we isolated 185 yeast colonies, and at least six species were identified: Debaryomyces hansenii,
Yarrowia lipolytica, Saturnispora mendoncae, Clavispora lusitanie, Kluyveromyces marxianus and
K. lactis. Among these strains, only K. marxianus and K. lactis displayed inhibitory activities against
Kocuria rhizophila, Candida albicans and some Bacillus sp. producer of biosurfactant.
This study underscores the presence of frequent species such as D. hansenii, Y. lipolytica, K. lactis and
K. marxianus, and unusual species such as S. mendoncae and C. lusitaniae. The antagonistic strains
are characterized by their cell-cell contact antagonism; inhibition of prokaryotic and eukaryotic cells
and antagonism directed against strains which produce lipopeptides.
KEYWORDS: Cheese, Ecology, Atypical antagonism, rDNA sequencing
23
Comparative study of Saccharomyces cerevisiae indigenous wine strains
to identify potential marker genes involved in desiccation stress resistance
Marianna Zambuto1, Sonia Votta1, Nicoletta Guaragnella2,1, Patrizia Romano1,
Angela Capece1
Scuola di Scienze Agrarie, Forestali, Alimentari ed Ambientali, Università degli Studi della Basilicata, Potenza, Italy;
2
Istituto di Biomembrane e Bioenergetica, CNR, Bari, Italy
1
[email protected]
The industrial production of Active Dry Yeast (ADY), commonly used in food industry, is characterized
by environmental changes resulting in metabolic modifications and changes in gene expression to
counteract desiccation stress. The exploitation of indigenous yeast strains for resistance to desiccation
process represents a valuable biological heritage for discovering unique genetic and molecular
properties conferring survival advantage, potentially useful for biotechnological applications.
In this work a comparative study of Saccharomyces cerevisiae indigenous wine strains, previously
selected for technological traits, has been carried out to evaluate desiccation stress tolerance and to
identify potential marker genes related to cell stress resistance. For desiccation treatment, cells were
incubated at 37°C. Viability of the indigenous strains and the commercial strain EC1118, used as
control, was analyzed after 20 and 40 min of heat treatment by plate viable count. Results obtained
revealed significant differences in cell survival and death rates for the analyzed strains and two strains
have been selected for their higher resistance or sensitivity to desiccation with respect to the control.
The impact of desiccation on cells can potentially be considered as a complex effect of several stresses,
mainly thermal, hyperosmotic and oxidative. Thus, for each selected strain, group of genes belonging
to these functional categories of stresses were analyzed by real-time RT-PCR for their expression
before and up to 40 min of desiccation treatment. Our results demonstrate that drying process elicits a
time-dependent up-regulation of specific genes mostly related to the general stress response pathway.
Interestingly, some of these genes were potentially correlated to the relative cell survival measured
in the indigenous wine strains.
Acknowledgements This work was supported by the project PIF-LIELUC (Misura 124 PIF Vini di
Lucania, PSR Basilicata 2007-2013-N. 94752044753)
KEYWORDS: Saccharomyces cerevisiae wine strains, Desiccation, Cell survival, Gene expression,
Real-time RT-PCR
24
Non-Saccharomyces yeast biodiversity on Chardonnay grapes: a comparison 15
years later
Neil Jolly1, Justin Hoff1, Grant Harold1, Louisa Beukes1, Bulelwa Fass1, Dudley Rowswell2
ARC Infruitec-Nietvoorbij, Private bag X5026, Stellenbosch 7699, South Africa; 2AgroClimatology, Institute for Soil
Climate and Water, Private Bag X79 Pretoria, South Africa
1
[email protected]
There is increasing evidence in the positive manner in which some non-Saccharomyces yeasts affect
wine quality. Commercial non-Saccharomyces yeasts have also become available for use in coinoculated or sequential fermentations with the standard wine yeast Saccharomyces cerevisiae. The
main source of non-Saccharomyces yeasts are the grapes in the vineyard from where they are carried
over to the grape must during crushing. The most robust and fermentative non-Saccharomyces yeasts
contribute to the fermentation dynamics and wine quality.
An investigation into the presence of non-Saccharomyces yeast on Chardonnay grapes from four
distinct geographical areas in South Africa was conducted over three vintages from 1997 to 2000
(Jolly et al 2003). Tank samples of the clarified grape must were also collected from the corresponding
commercial cellars. Yeast identification was done by electrophoretic karyotyping and biochemical
profiling. Over the three vintages certain species were dominant (> 50%) in the collected samples. These
included Hanseniaspora uvarum/Kloeckera apiculata, Lachancea thermotolerans/Kluyveromyces
thermotolerans, Zygosaccharomyces bailii, Rhodotorula sp., Metschnikowia pulcherrima/Candida
pulcherrima, Torulaspora delbrueckii/Candida colliculosa, and Candida zemplinina/ Starmerella
bacillaris.
In 2015 (fifteen years later) the investigation was repeated on grapes from the same vineyard, or a
Chardonnay vineyard in close proximity in cases where the original vineyard had been removed.
In the current study PCR-ITS (internally transcribed spacer regions) was used to assist in yeast
identification. The investigation is ongoing but preliminary indications are that there has been a
change in yeast biodiversity. The rainfall of the 2015 pre-harvest period was very low compared to
the earlier vintages. This, together with viticultural practices, may have contributed to changes in
yeast biodiversity.
KEYWORDS: Non-Saccharomyces yeasts, Geographic location
REFERENCES:
Jolly NP, Augustyn OPH, Pretorius IS (2003). The occurrence of non-Saccharomyces cerevisiae yeast strains over three
vintages in four vineyards and grape musts from four production regions of the Western Cape, South Africa. South
African Journal of Enology and Viticulture 24 :35-42
25
Study of ATG1, ATG17 and ATG29 genes expression in Saccharomyces cerevisiae
sparkling wine yeasts
Giorgia Perpetuini, Paola Di Gianvito, Rosanna Tofalo, Giuseppe Arfelli,
Maria Schirone, Aldo Corsetti, Giovanna Suzzi
Faculty of BioScience and Technology for Food, Agriculture and Environment, University of Teramo, Mosciano
Sant’Angelo, TE, Italy
[email protected]
Sparkling wine production, according to traditional method, is characterized by a secondary base
wine fermentation which requires the addition of sucrose and yeast strains. Yeasts involved in this
process should show specific criteria and among them flocculation and autolysis are the main. Under
enological conditions autolysis is slow. Recently, it has been postulated that autophagy may contribute
to autolysis outcome (Cebollero & Gonzalez 2007). Flocculent wine Saccharomyces cerevisiae strains
were previously investigated to improve sparkling wine production (Tofalo et al 2014). In this study
the same strains were characterized for their autolytic and autophagic activities in synthetic medium
and in base wine. The autolysis process was monitored through the determination of amino acid
nitrogen (AAN) and total protein content. A new qRT-PCR method was developed to evaluate the
expression of ATG1, ATG17 and ATG29 genes involved in autophagy process. Twelve strains were
selected for their flocculation degree and ATG genes expression levels: strains showing the highest,
the lowest and no expression were considered. These strains were inoculated in a base wine and after
30, 60 and 180 days the autolytic process and ATG gene expression were studied. Total proteins and
AAN contents differed, suggesting that the time at which autolysis takes place is strain specific. A
relationship between autophagy and yeast autolysis was established. ATG1 was the most expressed
gene followed by ATG17 gene in both synthetic medium and base wine, wheras ATG29 gene was the
less expressed in both conditions. After 6 months ATG29 gene was not expressed, but a contemporary
increase of AAN was determined suggesting a relationship between autophagy outcome and autolysis.
ATG29 gene could be considered as a biomarker for autolysis in flocculent wine strains. The study
of ATG genes has to be further investigated to select and modulate the autolytic activity of sparkling
wine yeasts.
KEYWORDS: Autophagy, ATG genes, Autolysis, Sparkling wine, Yeasts
REFERENCES :
Tofalo R, Perpetuini G, Gianvito P, Schirone M, Corsetti A, Suzzi G (2014). Genetic diversity of FLO1 and FLO5 genes
in wine flocculent Saccharomyces cerevisiae strains. International Journal of Food Microbiology 191:45–52
Cebollero E, Gonzalez R (2007). Autophagy: From basic research to its application in food biotechnology.
Biotechnology Advances 25:396–409
26
Yeast diversity of cuban cocoa bean heap fermentations and their environments
Yurelkys Fernandez Maura1, Tom Balzarini2, Luc De Vuyst2, Heide-Marie Daniel3
Facultad de Agronomía y Forestal, Departamento de Ciencias Básicas, Universidad de Guantánamo, Cuba; 2Research
Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of Sciences and Bio-Engineering Sciences,
Vrije Universiteit Brussel, Brussels, Belgium; 3Université catholique de Louvain, Earth and Life Institute, Applied
Microbiology, Laboratory of Mycology BCCM/MUCL, Louvain-la-Neuve, Belgium
1
[email protected]
The yeast diversity of spontaneous cocoa bean heap fermentations carried out in the east of Cuba was
investigated. Yeasts were isolated from eight fermentation processes and their environments such
as surfaces, tools, insects, and plants. The basic fermentation process parameters temperature and
pH were recorded. About 350 yeast isolates were grouped by M13-PCR-fingerprinting of genomic
DNA. Representative isolates were identified using sequences of the ITS and the D1/D2 region of
the large subunit rRNA gene, as well as partial actin gene sequences if required. Hanseniaspora
opuntiae, Pichia manshurica and Pichia kudriavzevii were the major components of the fermentation
yeast community of more than 30 species. Saccharomyces cerevisiae was detected occasionally.
Hanseniaspora opuntiae and Meyerozyma guilliermondii/Candida carpophila were the most frequent
species in the fermentation environments among more than 50 detected species.
KEYWORDS: Cocoa bean fermentation, Yeast, Cuba
27
Contribution of Torulaspora delbrueckii yeast in mixed fermentation with
different commercial starter strains Saccharomyces cerevisiae to improve the
quality of craft beer
Laura Canonico, Francesca Comitini, Lucia Oro, Maurizio Ciani
Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce
Bianche, 6013, Ancona, Italy
[email protected]
In craft beer production, the searching for distinctive flavor is an aspect widely researched. Many
aromatic compounds which characterize the various styles of beer, coming from raw materials.
However, yeast plays a central role, in the brewing process, metabolizing sugars into ethanol, carbon
dioxide and several other aromatic compounds (Lodolo et al 2008; Pires et al 2014). Currently, the
request of fermented alcoholic beverages with peculiar features has led the researchers looking for new
selection criteria for yeast strains. In winemaking, several studies have documented the positive role
of non-Saccharomyces yeasts that influenced the final profile of wine (Comitini et al 2011; Sadoudi et
al 2012). The possible use of non-Saccharomyces yeasts is less investigated in the brewing industry
where most of the beers are obtained by the use of a single yeast strain. For these reason, the aim of the
study was to assess the influence of T. delbrueckii selected strain in mixed culture with three different
S. cerevisiae starter strains. The biomass evolution, the fermentation behavior of these mixed cultures
as well as the analytical and sensorial profile of the resulting beers were evaluated. Preliminary results
showed that T. delbrueckii had a limited effect on the fermentation kinetics of S. cerevisiae starter
strain. Conversely, T. delbrueckii strain showed a significant influence on the biomass evolution of
the S. cerevisiae strains. All beers showed differences as regards the main aromatic compounds and
sensorial profiles underlining how each fermentation is able to give different products, characterized
by distinctive aromatic notes.
KEYWORDS: Craft beer, Torulaspora delbrueckii, Mixed fermentations, Saccharomyces cerevisiae
REFERENCES:
Comitini F, Gobbi M, Domizio P, Romani C, Lencioni L, Mannazzu I, Ciani M (2011). Selected non-Saccharomyces
wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiology 28: 873-882
Lodolo E, Kock JLF, Axcell BC, and Brooks M (2008). The yeast Saccharomyces cerevisiae – the main character in
beer brewing. FEMS Yeast Research 81018-1036
Pires EJ, Teixeira JA, Branyik T, Vicente AA (2014). Yeast: the soul of beer´s aroma - a review of flavour-active esters
and higher alcohols produced by the brewing yeast. Applied Microbiology and Biotechnology 98:1937-1949
Sadoudi M, Tourdot-Maréchal R, Rousseaux S, Steyer D, Gallardo-Chacón JJ, Ballester J, Vichi S, Guérin-Schneider R,
Caixach J, Alexandre H (2012). Yeast-yeast interactions revealed by aromatic profile analysis of sauvignon blanc wine
fermented by single or co-culture of non-Saccharomyces and Saccharomyces yeasts. Food Microbiology 32:243-53
28
Yeasts microbiota of naturally fermented black olives made from cv. Gemlik
grown in various districts of Turkey
Huseyin Erten, Sezgi Leventdurur, Bilal Agirman, C. Pelin Boyaci Gunduz,
Akram B. Ghorbal
Cukurova University, Faculty of Agriculture, Department of Food Engineering, 01330 Adana, Turkey
[email protected]
Mediterranean countries of Italy, Spain, Greece and Turkey are responsible for 75% of the total
worldwide olive production. Turkey lies in the second place for world table olives production after
Spain. Black table olives are account for 70-85% of total table olive production in Turkey and the
best quality table olives are produced from Gemlik variety (Erten et al 2014). Table olives with the
exception of California style are obtained by lactic acid fermentation. In addition to lactic acid
bacteria, yeasts can play a double role in production of table olives, acting as desirable and detrimental
organisms (Arroya-Lopéz et al 2012; Tofalo et al 2013). The aim of the present study was to identify
yeast microbiota during the spontaneous fermentation of olives produced from Gemlik variety grown
in various districts of Turkey.
After harvesting black olives at suitable ripening stage, olive fruits were fermented in 10% brine
solution. During the fermentation, microbiological and physicochemical characteristics were studied.
Yeasts were identified by a combination of PCR-RFLP of the 5.8S ITS rRNA gene and sequencing of
the D1/D2 domain of the 26S rRNA gene.
During the fermentation, high numbers of lactic acid bacteria and yeasts were counted. The main
yeast species identified were Wickerhamomyces anomolus Candida boidinii, and Saccharomyces
cerevisae. In addition, Candida aaseri, Candida butyrii, Zygoascus hellenicus, Pichia mexicana were
also found in table olives fermentations at subdominant levels.
Rich yeast community were identified in present study, showing good agreement with the reports of
Arroya-Lopéz et al 2012;Tofalo et al 2013.
Acknowledgments This work is part of the project supported by grant from TUBITAK – TOVAG
(Project no: 112O164).
KEYWORDS: Table olives, Cv. Gemlik, Fermentation, Microbiota, Yeast
REFERENCES:
Erten H, Bircan S, Sert S, Ağırman B, Boyacı-Gündüz CP, (2014). Microbiological and physicochemical changes
during the different ripening stages of cv. Gemlik for the production of naturally fermented black olives. Food
Microbiology 192
Arroya-Lopéz FN, Romero-Gil V, Bautista-Gallego J, Rodriguez-Gomez F, Jimenez-Diaz R, Garcia-Garcia P, Querol
A, Garrido-Fernandez A (2012). Yeasts in table olive processing: Desirable or spoilage microorganisms? International
Journal of Food Microbiology 160:42-49
Tofalo R,Perpetuini G, Schiorone M, Suzzi G, Corsetti A (2013). Yeast biota associated to naturally fermented table
olives from different Italian cultivars. International Journal of Food Microbiology 161:203-208
29
Syntheses of phenolic aroma compounds by Hanseniaspora vineae yeast strains
contribute to increase flavor diversity of wines
Valentina Martin1, Eduardo Boido1, Facundo Giorello1, Albert Mas2, Eduardo Dellacassa3,
Francisco Carrau1
Sección Enología, Catedra de Ciencia y Tecnología de Alimentos and 3 Laboratorio de Biotecnología de Aromas,
Departamento de Quimica Orgánica, Facultad de Quimica, Universidad de la Republica, 11800 Montevideo, Uruguay;
2
Departamento de Bioquímica y Biotecnología. Faculty of Oneology. University Rovira i Virgili. 43007 Tarragona,
Spain
1
[email protected]
Although non-Saccharomyces yeast strains, which account for more than 99% of the grape native
flora, have a well-recognized genetic diversity the understanding of their impact on wine flavor
richness is still incipient (Carrau et al 2015; Bomeman et al 2013).
In several grape varieties the dominating aryl alkyl alcohols found are the volatile group of benzenoid/
phenylpropanoid related compounds that contribute significantly to wine aroma after being hydrolyzed
during fermentation and/or barrel aging. Within this group, β-phenylethyl alcohol and benzyl alcohol
have been identified in grape must and wine contributing with floral and fruity flavors to wines.
Previously, we have found increased levels of benzyl alcohol in Tannat and Chardonnay wines when
compared to grape juices, suggesting de novo formation of this metabolite during vinification (Medina
et al 2013).
In this work, a chemically defined simil-grape fermentation medium, resembling the nutrient
composition of grape juice but devoid of grape derived secondary metabolites was used. GC-MS
analysis was performed to determine volatile compounds in the produced wines. Our results showed
that benzyl alcohol, 4-hydroxybenzyl alcohol and β-phenylethyl acetate can be synthetized de novo by
the wine yeast H. vineae, in the absence of grape derived precursors. Levels of this compound found
in fermentations with 11 H. vineae different strains were 1-2 orders of magnitude higher than those
measured in fermentations with known Saccharomyces cerevisiae wine strains (Medina et al 2013).
These results show that H. vineae strains contribute to flavor diversity, increasing a varietal aroma
concentration in a standard wine. Genomic analysis (Giorello et al 2014) indicates that alternative
pathways to the phenylalanine ammonia lyase, are used by yeast, compared to plants and some fungi,
to generate benzyl alcohol and the 4-hydroxybenzyl alcohol. Consequently, alternative pathways
derived from chorismate through phenylpiruvate and 4-hydroxyphenylpiruvate, intermediates of the
synthesis of phenylalanine and tyrosine are discussed.
KEYWORDS: Benzenoid aroma compounds, Wine yeast fermentation, Hanseniaspora vineae
genome
REFERENCES:
Carrau F et al (2015).Yeast diversity and native vigor for flavor phenotypes. Trends in Biotechnology 33:148-154
Borneman AR et al (2013). Comparative genomics: a revolutionary tool for wine yeast strain development. Current
Opinion in Biotechnology 24:192-199
Medina K et al (2013). Increased flavour diversity of Chardonnay wines by spontaneous fermentation and cofermentation with Hanseniaspora vineae. Food Chemistry 141:2513-2521
Giorello FM et al (2014). Genome sequence of the native apiculate wine yeast Hanseniaspora vineae T02/19AF.
Genome Announcements 2(3):e00530-14
30
Yeast and bacterial landscapes within food production:
a case for microbial terroir
David A. Mills
Department of Food Science and Technology, Department of Viticulture and Enology,
University of California, USA
[email protected]
Wineries and breweries are a useful models for studying food ecosystem dynamics, as these
fermented products illustrate opposing roles of adventitious microbes in beverage production—as
spoilage agents and as beneficial members of the microbial consortium—both of which influence
final product quality. Recently, application of ribosomal RNA marker gene surveys to define the
modes of microbial transmission across space and time in wine and beer production has provided
unique insight into these two important commercial fermentations. Wine fermentations are well
known to be initiated by grape-associated yeast and bacteria, however recent studies reveal that this
grape-associated microbiota is strongly influenced by the regional, environmental and viticultural
factors thus potentially contributing to the “regional character” often attributed to specific wines.
During harvest, grape- and fermentation-associated yeast and bacteria populate most winery
surfaces contacting the fermentation, acting as potential reservoirs for microbial transfer between
fermentations. This early stage microbial consortium, composed from vineyard and winery sources,
also exhibits numerous links with the chemical composition of the finished wines suggesting that
grape-associated yeast and bacteria are possible mechanism for regional attributes in wines. A similar
analysis of brewery operations show that distinct microbial populations associate both with beer
production style and specific substrates and surfaces in breweries. Mapping of these populations
over time illustrates patterns of dispersal and identifies potential contaminant reservoirs within the
brewery environment. Importantly, exposure to beer is linked to increased abundance of hop-resistance
spoilage genes on brewery equipment, correlating with lactic acid bacterial populations and predicting
greater contamination risk. Elucidating the microbial ecosystems and spatial characteristics present
in beverage production environments identifies the fundamental drivers of microbial biogeography
and provides important practical implications for winemakers, brewers and food-production systems
in general.
KEYWORDS: Yeast, Next-Generation Sequencing, Microbiome, Wine, Beer
31
32
Session 2B
detection methods and strain improvement
33
Session 2B: Yeasts in food biotechnology: detection methods and strain improvement
From Bach to Bacchus: yeast genomics and wine symphonics
Isak S. Pretorius
Chancellery, Macquarie University, Balaclava Road, Sydney, NSW 2109, Australia
[email protected]
A perfectly balanced wine can be said to create a symphony in the mouth. To achieve the sublime, both
in wine and music, requires skilled orchestration. For wine, it starts in the vineyard. Grapegrowers
(composer) produce grapes to specification. Different varieties allow the creation of wine of different
genres. Winemakers (conductor) decide what genre to create and consider resources required to
exploit the grape’s potential. A primary consideration is the yeast: inoculate the grape juice or leave
it ‘wild’; which specific or combined Saccharomyces strain(s) should be used; or proceed with a nonSaccharomyces species? Whilst the various Saccharomyces and non-Saccharomyces yeasts perform
their role during fermentation, the performance is not over until the ‘fat lady’ (S. cerevisiae) has
sung (i.e. the grape sugar has been fermented to specified dryness and alcoholic fermentation is
complete). Is the wine harmonious or discordant? Has the consumer demanded an encore and made
a repeat purchase? Understanding consumer needs lets winemakers orchestrate different symphonies
(i.e. wine styles) using single- or multi-species ferments. Some consumers will choose the sounds of
a philharmonic orchestra comprising a great range of diverse instrumentalists (as is the case with wine
created from spontaneous fermentation); some will prefer to listen to a smaller ensemble (analogous
to wine produced by a selected group of non-Saccharomyces and Saccharomyces yeast); and others
will support the well-known and reliable superstar soprano (i.e. S. cerevisiae). But what if a music
synthesizer (a synthetic yeast) becomes available that can produce any music genre with the purest
of sounds by the touch of a few buttons? Will synthesizers spoil the character of the music and lead
to the loss of the much-lauded romantic mystique? Or will music synthesizers support composers
and conductors to create novel compositions and even higher quality performances that will thrill
audiences?
34
Improvement of wine yeast strains using adaptive evolution
Axelle Cadiere, Valentin Tilloy, Carole Camarasa, Frédéric Bigey, Stéphane Guézenec,
Virginie Galeote, Sylvie Dequin
INRA, UMR1083 SPO, F-34060 Montpellier, France
[email protected]
The wine industry continuously faces news challenges that require the development of yeast strains
with novel, specialized traits. Non-targeted approaches such as adaptive evolution have recently been
used to develop Saccharomyces cerevisiae wine yeast strains with new properties. We will present
the development of a wine yeast strain producing increased levels of esters, which are important
determinants of the fruity character of wines, obtained after 70 generations on gluconate as sole carbon
source (Cadiere et al 2011; 2012). Another example is the selection, by a combination of experimental
evolution and conventional breeding, of an evolved strain that produce up to 1.3% v/v less ethanol
in wine obtained in pilot-scale trials (Tilloy et al 2014). This strain produces substantially more
glycerol and 2,3-butanediol, less acetate and more succinic acid. To identify the genetic determinants
of the evolved phenotypes, we used a combination of genome-wide approaches including whole
genome sequencing. We identified a loss-of-function mutation of BCY1, a gene controlling the
cAMP-dependent protein kinase (PKA) nutrient signaling pathway, as a causative mutation of the
aroma phenotype of the gluconate-evolved strain. These examples highlight the potential of adaptive
evolution to reprogram yeast metabolic network and to generate industrial strains with new abilities
for the food industry.
KEYWORDS: Wine yeast, Adaptive evolution, Esters, Ethanol, Glycerol
REFERENCES:
Cadiere A, Camarasa C, Julien A, Dequin S (2011). Evolutionary engineered Saccharomyces cerevisiae wine yeast
strains with increased in vivo flux through the pentose phosphate pathway. Metabolic Engineering 13: 263-271
Cadiere A, Aguera E, Caille S, Ortiz-Julien A, Dequin S (2012). Pilot-scale evaluation the enological traits of a novel,
aromatic wine yeast strain obtained by adaptive evolution. Food Microbiology 32:332-7
Tilloy V, Ortiz-Julien A, Dequin S (2014). Reduction of ethanol yield and improvement of glycerol formation
by adaptive evolution of the wine yeast Saccharomyces cerevisiae under hyperosmotic conditions. Applied
Environmental Microbiology 80:2623-32
35
Polygenic analysis of phenylethyl acetate production in yeast for aroma
production improvement in alcoholic beverages
Bruna T. Carvalho1,2, Maria Foulquié-Moreno1,2, Ben Souffriau1,2, Johan M. Thevelein1,2
Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven, Belgium;
2
Laboratory of Molecular Cell Biology, Department of Molecular Microbiology, VIB, Leuven, Belgium
1
[email protected]
Selected yeast strains are commonly used to achieve a desirable flavour profile in fermented
beverages. However, little is known about the genetic basis of the variance in aroma production by
different strains. As a result, all new yeast strains must be obtained empirically either by selection or
by classical breeding methods. In this study, we applied pooled-segregant whole-genome sequence
analysis to elucidate the genetic basis of phenylethyl acetate production (2-PEAc), a rose-like aroma.
A hybrid strain was constructed by crossing two random parent strains. Meiotic segregants were
obtained, screened by fermentation and 2-PEAc was analyzed by Gas Chromatography (GC-FID).
From a total of 576 segregants, 24 superior segregants producing a high level of phenylethyl acetate
in small-scale fermentations were pooled and sequenced. SNP variant frequency plotted against the
chromosomal position revealed four major loci involved in the superior phenotype. Fine-mapping
and allele exchange analysis are being applied to identify the causative genes. To date, novel superior
genes are being identified through polygenic analysis by using a superior parent (Trait +) and inferior
parent (Trait-). Our results show that identification of QTLs involved in complex traits can be
successfully accomplished by crossing haploid segregants of two random parent strains, eliminating
time-consuming screening for selection of appropriate parent strains and extending the range of
strains useful for identifying causative alleles of superior traits.
KEYWORDS: Polygenic analysis, Fermented beverages, Flavour, Esters, Phenylethyl acetate
REFERENCES:
Cordente AG, Curtin CD, Varela C, Pretorius IS (2012). Flavour-active wine yeasts. Applied Microbiology and
Biotechnology 96(3): 601-618
Etschmann MM W, Sell D, Schrader J (2005). Production of 2-Phenylethanol and 2-Pheynlethylacetate from
L-Phenylalanine by Coupling Whole-Cell Biocatalysis with Organophilic Pervaporation”. Wiley InterScience
92(5):624-634
Swinnen S, Thevelein JM, Nevoigt E (2012). Genetic mapping of quantitative phenotypic traits in Saccharomyces
cerevisiae. FEMS Yeast Research 12(2):215-27
36
Crossbreeding of bottom-fermenting yeast strains with a novel method for high
throughput screening of mating-competent cells
Taku Ota1, Keiko Kanai1, Hisami Nishimura1, Satoshi Yoshida2, Hiroyuki Yoshimoto1,
Osamu Kobayashi1
Research Laboratories for Alcoholic Beverage Technologies; 2Central Laboratories for Key Technologies; Kirin
Company, Limited, Japan
1
[email protected]
Crossbreeding is an effective approach to construct novel yeast strains with improved traits. However,
there are few reports on crossbreeding of brewer’s yeast, especially Saccharomyces pastorianus
known as bottom-fermenting yeast. Although yeast crossbreeding depends on mating between spores,
it is quite inefficient to obtain mating-competent spores from bottom-fermenting yeast without DNA
recombination technique. This background inspired us to develop a novel screening method to isolate
mating-competent cells (MCCs).
First, we noticed characteristics of mutants supersensitive to mating factor, Dbar1 and Dsst2 strains,
that showed a severe growth defect when they were inoculated around mating-competent yeast strains
on plates. We then spread yeast strains to be tested on plate, followed by selection of MCCs that
gave the growth defect to Dbar1 or Dsst2. Particularly, this method was made use of to obtain nongenetically modified MCCs from industrial yeast strains (brewer’s, wine and sake yeast). Furthermore,
the mating-competent bottom-fermenting yeast strains (meiotic segregants) got hybridized with each
other. To examine the brewing characteristics of the hybrids, fermentation test in pilot fermenter scale
was performed, resulting that they had enough brewing ability for practical use.
The present method developed in this study is expected to be useful for easy crossbreeding between
brewer’s yeasts to construct novel strains with unique brewing traits.
KEYWORDS: Crossbreeding, Mating factor, Bottom-fermenting yeast
37
Antifungal activity of defensins from different organisms and potential
applications in cereal-based products
Thibaut Thery1, James C. Tharappel 2, Joanna Kraszewska2, Michael Beckett2, Ursula Bond2,
Elke Arendt1
School of Food and Nutritional Sciences, University College Cork, Ireland;
Moyne Institute for Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, College
Green, Dublin, Ireland
1
2
[email protected]
Fungal spoilage is an important cause of economic losses in the baking industry and might be a source
of mycotoxins which can be a potential health danger for consumers. The use of preservatives is
subject to controversy and rejected more and more by consumers. Furthermore, due to specific action
of many antibiotics, a development of microbial resistance is noticed.
Defensins are antimicrobial peptides of the innate immune system with direct action against microbes,
they are also involved in adaptive immunity. Present in many living organisms, these molecules and
their activity arouse more and more interest in pharmaceutical applications. The antifungal activity
of eleven recombinant lyophilised defensins and defensin-like peptides was studied against common
bakery products contaminants, including Fusarium culmorum, Aspergillus niger, Penicillium
expansum and Penicillium roqueforti. Eight defensins clearly show a partial or total inhibition of
fungal growth at different µg.ml-1 ranges in vitro. Among these host defense peptides, Human
β-Defensin-3 (HBD3) has attracted much attention since its discovery. In the study, HBD3 appears
to be the peptide showing the strongest potency of inhibition, with heat and salt stability. All these
characteristics make HBD3 a good candidate for bread making process.
To test potential baking applications, the novel lager yeast Saccharomyces pastorianus strain CMINT-51, able to express the gene encoding HBD3, was created from the parental strain CMBS-33.
The use of the strain CM-INT-51 allows to extend the shelf life of the bread by three days compared
to the strain CMBS-33. Furthermore, appriopriate concentration of synthetic HBD3 delays the arrival
of fungal colony until bread gets stale. These results have direct applications to the baking and food
industry. In synthetic form or produced in situ by genetically modified yeasts, the use of defensins
may be the new means to fight microbial spoilage.
KEYWORDS : Defensins, HBD3, Antifungal activity, Saccharomyces pastorianus, Baking
REFERENCES :
James T C, Gallagher L, Titze J, Bourke P, Kavanagh J, Arendt E, Bond U (2013). In situ production of human B
defesin-3 in lager yeasts provides bactericidal activity against beer-spooiling bacteria under fermentation conditions.
Journal of Applied Microbiology 116 : 368-379
Rezaei MN, Dornew E, Jacobs P, Parsi A, Verstrepen KJ, Courtin CM (2013). Harvesting yeast (Saccharomyces
cerevisiae) at different physiological phases significantly affects its functionality in bread dough. Food Microbiology
39 :108-115
38
Session 3
biofuels, new molecules and enzymes
39
Session 3: Yeasts in no-food biotechnology: biofuels, new molecules and enzymes - Key note
Yeasts for biofuels production
Charles A. Abbas
Director of Yeast and Renewables Research. Archer Daniels Midland Research, JRRRC, 1001 Brush College Road,
Decatur, IL 62526, USA
[email protected]
The development of new yeast strains of Saccharomyces cerevisiae by genetic engineering to produce
biofuels has been the focus of research efforts. This coincides with parallel efforts to develop other
genera of yeasts that can utilize a broader range of feedstocks. The increased worldwide interest
in biofuel production has also accelerated the commercial development of new improved strains of
Saccharomyces cerevisiae. Among these are new strains that can be used for 1st generation ethanol
production that have been selected and/or genetically engineered for more stress tolerance or that
harbor enzymes that provide greater access to carbohydrate such as starch derived maltose. Other new
Saccharomyces cerevisiae strains are also commercially available for 2nd Gen ethanol that can produce
ethanol from xylose. Some of these strains have been engineered for greater tolerance to inhibitors
that are present in lignocellulosic hydrolysates. The pursuit of other nonconventional xylose utilizing
yeasts, has continued to move in parallel with the characterization of new genera of yeast.
In addition to 1st and 2nd generation ethanol, there have been other attempts to engineer yeast that can
produce advanced biofuels such as the higher alcohols (1-butanol and isobutanol), the sesquiterpenes
(farnesene and bisabolene), and fatty acid ethyl esters (biodiesel) for use as drop in fuels. In spite of
the expanded interest in nonconventional yeasts, the yeast Saccharomyces cerevisiae still offers many
advantages as a platform cell factory. This yeast is easy to genetically engineer with its physiology,
metabolism and genetics well understood. The robustness of this yeast and its tolerance to harsh
industrial conditions is documented by its current worldwide use for the industrial scale production
of bioethanol. The introduction of novel pathways and optimization of its native cellular processes by
metabolic engineering are rapidly expanding its range of cell-factory applications.
KEYWORDS: Biofuels, Yeasts
REFERENCES:
Demeke MM, Dietz H, Li Y, Foulquié-Moreno MR, Mutturi S, Deprez S, Den Abt T, Bonini BM, Liden G, Dumortier
F, Verplaetse A , Boles E, Thevelein JM (2013). Development of a D-xylose fermenting and inhibitor tolerant
industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and
evolutionary engineering. Biotechnology for Biofuels 6:89
Nielsen J, Larsson C, van Maris A, Pronk J (2013). Metabolic engineering of yeast for production of fuels and
chemicals. Current Opinion ion in Biotechnology 24(3):398-404
Nyguyen NH, Suh SO, Marshall CJ, Blackwell M (2006). Morphological and ecological similarities: wood-boring
beetles associated with novel xylose-fermenting yeasts, Spathaspora passalidarum gen. sp. nov. and Candida jeffriesii
sp. nov. Mycological Research 110:1232-41
40
Session 3: Yeasts in no-food biotechnology: biofuels, new molecules and enzymes
Organic acids from lignocellulose: Candida lignohabitans as a novel microbial
cell factory
Martina Bellasio1, Diethard Mattanovich1,2, Michael Sauer1,2, Hans Marx1
Department of Biotechnology, BOKU – University of Natural Resources and Life Sciences, Vienna, Austria;
2
ACIB – Austrian Centre of Industrial Biotechnology, Vienna, Austria
1
[email protected]
Lignocellulose based biorefineries require processes that convert hexoses and pentoses derived from
cellulose and hemicelluloses into value-added chemicals or fuels. For this purpose microorganisms
must be employed that can efficiently utilize at least glucose, xylose and arabinose, are tolerant to
inhibitors derived from biomass pretreatment and to acidic conditions, and are accessible to genetic
modifications. We tested various yeast species for growth on lignocellulosic sugar monomers like
glucose, galactose, mannose, arabinose and xylose. Of these yeasts, Candida lignohabitans grew
and fermented best on all these substrates in pure and mixed form. Furthermore, C. lignohabitans
performed very well on hydrolysates of miscanthus, sawdust, hard- and softwood chips, thereby
accumulating biomass and varying amounts of ethanol as a natural fermentation product. Genetic
engineering of C. lignohabitans was established successfully. Expression of lactate dehydrogenase
(L-LDH) and cis-aconitate decarboxylase (CAD) resulted in stable production of lactic acid and
itaconic acid, respectively. The desired organic acids were produced both on pure sugars as well as on
lignocellulosic hydrolysates. In addition, C. lignohabitans proved to be very tolerant to low pH, which
is an important feature when organic acids are the desired product. Based on the genome sequence
we will discuss specific features concerning metabolic traits and cell physiology of C. lignohabitans.
KEYWORDS: Lignocellulose, Itaconic acid, Lactic acid, Biorefinery
41
Cellular robustness in lignocellulose derived streams
Lisbeth Olsson, Lina Lindahl, Peter Adeboye, Christian Marx, Maurizio Bettiga
Department of Biology and Biological Engineering, Division of Industrial Biotechnology, Chalmers University of
Technology, SE-412 96 Gothenburg, Sweden
[email protected]
In designing efficient biorefinery concepts for a biobased economy, a major challenge will be to
design cell factories that efficiently can convert the substrate stream to the targeted product. Not only
should the cell factory be metabolically engineered for product formation, it should also be well suited
to work in biorefinery streams. Working with lignocellulose derived streams, robustness towards
inhibitory compounds; furans, weak acids and phenolics, found in such streams, is a prerequisite for
efficient conversion. With the interest in creating more efficient processes, better water economy and
cheaper and more energy efficient down stream processing calls for high substrate loadings in the
biorefinery. High substrate loading puts further challenges on the microbial performances (Koppram
et al 2014).
Cellular robustness, which relates to the cells ability to perform well also under industrially challenging
conditions is an attractive property that we attempt to modulate through metabolic engineering,
evolutionary engineering and by design of fermentation conditions. In our research group we focus
on using Saccharomyces cerevisiae as a cell factory for usage in different lignocellulose biorefinery
concepts. In the presentation examples to approach and understand cellular robustness will be given,
including:
• Metabolic conversion of phenolics
• Acetic acid tolerance and diffusion over the cell membrane
• Modulation of the production of the cellular protectant glutathione
KEYWORDS: Phenolic, Acetic acid, Glutathione, Saccharomyces cerevisiae
REFERENCES:
Koppram R, Tomas-Pejo E, Xiros C, Olsson L (2014). Lignocellulosic ethanol production at high-gravity: Challenges
and perspectives. Trends in Biotechnology 32:46-53
42
Phenotypic and genotypic analysis of engineered xylose consuming
Saccharomyces cerevisiae strains adapted to industrial medium for secondgeneration biofuel
Thomas Desfougeres, Georges Pignede, Jean M. Bavouzet, Emilie Fritsch,
Didier Colavizza
Lesaffre International
[email protected]
The lignocellulosic hydrolysates are a major source of carbon, which has, among others, the interest
of coming from plant sources that are not intended for human food or animal feed. However,
there are two major limitations to its use. First, a certain number of microorganisms are unable to
metabolize 5 carbons sugars, which consists in large part of hemicellulose. The second aspect is that
the hemicellulose can be rich in acetyl groups, which become powerful microbial inhibitors after
hydrolysis. Many yeast strains have been proposed as being able to metabolize 5 carbons sugars into ethanol.
However, many of those strains have phenotypic characteristics that are not suitable for industrial
fermentation media. Lesaffre has been working for nearly 10 years to understand the prerequisites to
effectively implement the xylose consumption pathway in strains compatible with these environments.
Thus, we have been able to show that the results obtained in laboratory strains are not directly
and systematically transposable to industrial strains. This work was the opportunity to pay a little
more attention to the special metabolism of these last strains.
The other aspect of our work was to improve the strength of the strains obtained, regarding acetic
acid, which is a powerful inhibitor of sugars fermentation. In this study, we were able to identify ten
QTLs conferring improved glucose fermentation kinetics. We were also able to demonstrate that the
impact of acetic acid on the fermentation is different, depending on the nature of the sugar used.
KEYWORDS: Industrial yeast, Xylose, Biofuel, Inhibitor, QTL
43
Engineering cytosolic Acetyl Coenzyme A supply in Saccharomyces cerevisiae
Antonius J.A. van Maris
Industrial Microbiology, Department of Biotechnology, Delft University of Technology, The Netherlands
[email protected]
Cytosolic acetyl coenzyme A (Ac-CoA) is a key precursor for biosynthesis in eukaryotes and for many
industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae,
such as isoprenoids or lipids. In this yeast, synthesis of cytosolic Ac-CoA via acetyl-CoA synthetase
(ACS) involves hydrolysis of ATP to AMP and pyrophosphate, which constrains maximum yields
of Ac-CoA -derived products. Therefore, this study explores replacement of ACS by three ATPindependent pathways for cytosolic Ac-CoA synthesis: acetylating acetaldehyde dehydrogenase
(A-ALD), pyruvate-formate lyase (PFL) and pyruvate dehydrogenase (PDH).
After evaluating expression of different bacterial genes encoding A-ALD and PFL, acs1Δ acs2Δ S.
cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALDdependent strains, aerobic growth rates of up to 0.27 h-1 were observed, while anaerobic growth rates
of PFL-dependent S. cerevisiae (0.20 h-1) were stoichiometrically coupled to formate production. In
glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences
between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALDand PFL-dependent strains were lower than those of the reference strain.
Next, it was investigated whether expression in the yeast cytosol of an ATP-independent PDH from
Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic Ac-CoA synthesis.
In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding
its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of
lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at nearwild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic
conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain
showed small differences in biomass yields and metabolic fluxes.
44
Towards industrial glycerol fermentation by Saccharomyces cerevisiae
Martina Carrillo, Mathias Klein, Steve Swinnen, Zia Ul Islam, Ping-Wei Ho,
Elke Nevoigt
Jacob University, Bremen, Germany
[email protected]
Most wild-type strains of S. cerevisiae grow poorly or not at all in defined minimal medium with
glycerol as the sole source of carbon. Improving the efficiency of glycerol utilization in baker’s yeast
would enhance the versatility of this platform microbial production host. Using rational and inverse
metabolic engineering strategies (including QTL mapping), we were able to improve the lab strain
CEN.PK113-7D from zero glycerol growth to a maximum specific growth rate of 0.08 h-1. Moreover,
the growth rate of a previously selected natural S. cerevisiae isolate (Swinnen et al 2013) able to
naturally grow on glycerol at a growth rate of 0.12 h-1 was improved to 0.18 h-1 by the expression of
a single gene encoding a heterologous glycerol transporter.
Beside the fact that valorization of glycerol would increase the economic viability of oil-plant
biorefineries (including biodiesel production) the use of glycerol as a feedstock has additional
advantages. Firstly, this carbon source does not induce the Crabtree-effect during respiratory growth
and secondly, it has a higher degree of reduction compared to sugars, i.e. its catabolism provides more
reducing equivalents when related to carbon equivalents. In order to make use of this reducing power
in the form of NADH, we sought to replace the native FAD+-dependent glycerol catabolic pathway
in S. cerevisiae by an NAD+-dependent one. The resulting rationally engineered strain based on our
above-mentioned natural isolate bearing the heterologous transporter showed a maximum specific
growth rate of 0.11 h-1.
KEYWORDS: Biodiesel, Crude glycerol, Glycerol utilization, NADH, Metabolic engineering
REFERENCES:
Swinnen S, Klein M, Carrillo M, McInnes J, Nguyen HTT, Nevoigt E (2013). Re-evaluation of glycerol utilization
in Saccharomyces cerevisiae: characterization of an isolate that grows on glycerol without supporting supplements.
Biotechnology Biofuels 6:157-168
45
Metabolic engineering of Pichia pastoris for l-lactic acid production using
glycerin as carbon source
Nádia S. Parachin
Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brazil
[email protected]
The worldwide growth of the bioplastic market is 5-8% per year with the increase in this share
expected to go up to 30% in 2020 what would represent a market of 20 billion dollars. Moreover
the utilization of bioplastics allows the reduction of plastics derived from petrochemicals and
consequently the amount of waste deposited in the environment. Thus the economically feasible
production of L-lactic acid, a precursor for the bioplastic poly (lactic acid) will result in a lower
demand for plastics derived from petrochemicals. One of possibility to reduce production cost is to
use industrial residues as substrate. Among these, an excellent candidate is the crude glycerol, the
main residue produced during the biodiesel synthesis. The yeast Pichia pastoris is able to utilize
glycerol as a carbon source reaching high cell densities, but is not able to produce lactic acid. Thus
genetic engineering has been used to over produce the gene that encodes an enzyme able to catalyze
the conversion of pyruvate to lactate. It has been previously shown in other microorganisms that
the choice of enzyme lactate dehydrogenase (LDH) from different species will determine the yield
and purity of produced lactic acid. Furthermore, other genetic engineering strategies can be used to
increase lactic acid production such as deletion of gene that encodes Pyruvate decarboxylase and
introduction of a lactate transporter. Concomitant, different fermentation parameters were tested
aiming at process optimization. Our results show that genetically modified Pichia pastoris is able to
produce L-lactic acid with a yield of 0.4 g.g. Moreover the introduction of lactate transporter lead to
an increase of 20% secreted lactic acid in the supernadant. Finally process optimization have shown
that this yeast increases L-lactic production under oxygen limited conditions and high cell densities.
Overall Pichia pastoris had shown that is is a promising candidate for the successful production o
L-lactic acid using crude glycerol as carbon source.
KEYWORDS: Metabolic engineering, Pichia pastoris, Lactic acid, Crude glycerol
REFERENCES:
Iles A, Martin A N (2013). Expanding bioplastics production: sustainable business innovation in the chemical industry.
Journal of Cleaner Production 45:38-49
Luengo JM, Garcìa B, Sandoval A, Naharro G, Olivera ER (2003). Bioplastics from microorganisms. Current Opinion
in Microbiology 6:251–260
Fernando S, Adhikari S, Chandrapal C, Murali N (2006). Biorefineries: Current satatus, challenges, and future direction.
Energy & Fuels 20:1727-1737
Upadhyaya BP, DeVeaux, LC, Christopher LP (2014). Metabolic engineering as a tool for enhanced lactic acid
production. Trends in Biotechnology 32:637-644
46
Use of oleaginous yeasts for the exploitation of lignocellulosic biomasses
Domenico Pirozzi1, Maria Abagnale1, Gerardo Caputo1, Massimo Fagnano2, Nunzio
Fiorentino2, Ciro Florio1, Biancamaria Pietrangeli3, Filomena Sannino2, Giuseppe Toscano1,
Gaetano Zuccaro1
Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale (DICMAPI)
Università Federico II di Napoli, P.le Tecchio, 80, 80125 Napoli, Italia;
2
Dipartimento di Agraria Università Federico II di Napoli, via Università, 100 – 80055 Portici (NA), Italia;
3
Dipartimento Innovazioni Tecnologiche e Sicurezza degli Impianti, Prodotti ed Insediamenti Antropici. INAIL Ricerca,
Via Alessandria, 220/E, 00198 Roma, Italia
1
[email protected]
The production of bioplastics and of second-generation biodiesel, based on the exploitation of
agricultural lignocellulosic biomasses through the use of oleaginous microorganisms, can ensure
significant environmental benefits and increase our energy security.
In this view, oleaginous yeasts offer an useful alternative to microalgae, due to their ability to use
several agroforestry wastes as feedstock and their simple cultural requirements (Li et al 2013; Liu
et al 2007; Papanikolaou et al 2009). In addition, the microbial oils obtained from yeasts have a
composition quite similar to that of vegetable oils (Papanicolaou et al 2011). Yet, their full industrial
exploitation is still prevented by the high cost of the II-generation biodiesel, still exceeding that of
the mineral diesel.
The exploitation of lignocellulosic biomasses from contaminated soil could improve of the economical
balance of the process. In this view, the fate and the effect of contaminants have been followed along
the different steps of the process.
In addition, specific tests have been carried out to integrate different stages of the process (enzymatic
hydrolysis and growth of oleaginous yeasts) in a single reactor, and to minimize the effect of the
inhibitors of the microbial growth using synergically biological and physical methods.
KEYWORDS: Oleginous yeasts, Arundo donax, Soil contaminants, II generation biodiesel
REFERENCES:
Li et al (2013). Journal of Agricultural and Food Chemistry. 61:646–654
Liu et al (2007). Journal of Agricultural and Food Chemistry 82:775–780
Papanikolaou et al (2009). Lipid Technology 21:83–87
Papanikolaou et al (2011). European Journal of Lipid Science and Technology 113:1031–1051
47
Selection and optimization of oleaginous yeast for lipid production from
biodiesel-derived crude glycerol
Pirapan Polburee1, Wichien Yongmanitchai1, Noppon Lertwattanasakul1, Takao Ohashi2,
Kazuhito Fujiyama2, Savitree Limtong1
Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, Thailand;
2
International Center for Biotechnology, Osaka University, Osaka, Japan
1
[email protected]
Microbial lipid produced by oleaginous microorganisms has been suggested as a potential feedstock
for biodiesel production due to their similar composition of fatty acids to that of vegetable oils. In
biodiesel production, glycerol is generated as a byproduct. It is consider being a low cost raw material
and can be used as a carbon substrate by many types of yeast. Therefore, this study aims to use
oleaginous yeast for microbial lipid production from crude glycerol. A total of yeast 323 strains were
isolated from 142 samples. Based on two-step screening, 34 yeast strains accumulated lipid in the
range of 15.3-71.0% of biomass when pure glycerol was used as a carbon source. Determination of
lipid accumulation by these 34 strains cultivated in nitrogen-limited medium containing 70 g/L crude
glycerol (70 g/L crude glycerol, 0.55 g/L (NH4)2SO4, 0.75 g/L yeast extract, 2 g/L MgSO4•7H2O, 0.4
g/L KH2PO4 and pH 5.5) revealed that strain DMKU-RK253 accumulated the highest lipid of 65.2%
of dry biomass. Therefore, this strain was selected and optimized for lipid production by the respond
surface method (RSM). The result showed that strain DMKU-RK253 produced the highest lipid of
10.7 g/L and 16.2 g/L of biomass which calculated to be lipid content of 66.4% of dry biomass, at
216 h of cultivation in the nitrogen-limited medium containing 70 g/L crude glycerol but using 1
g/L monosodium glutamate instead of yeast extract (Fig 1). The major fatty acids of lipid produced
by strain DMKU-RK253 were stearic acid (C18:0), palmitic acid (C16:0) and oleic acid (C18:1),
respectively. On the basis of molecular taxonomy by analysis of the D1/D2 region of the large subunit
(LSU) rRNA gene sequence, strain DMKU-RK253 was identified to be Rhodosporidium fluviale
DMKU-RK253. The lipid produce by this yeast strain has potential to be used as a feedstock for
biodiesel production.
Fig 1 Lipid production by strain DMKU-RK253 under optimal nutrient
KEYWORDS: Oleaginous yeast, Lipid production, Crude glycerol, Respond surface method (RSM)
48
Engineering industrial yeast strains for consolidated bioprocessing of starchy
substrates and by-products to ethanol
Lorenzo Favaro1, Marko J. Viktor2, Shaunita H. Rose2, Marina Basaglia1, Lorenzo Cagnin1,
Rosemary Cripwell2, Marinda Viljoen-Bloom2, Sergio Casella1, Willem H. Van Zyl2
Department of Agronomy Food Natural resources Animals and Environment, Dafnae, Università di Padova, Agripolis,
Viale dell’Università 16, 35020 Legnaro (PD), Italy; 2Department of Microbiology, Stellenbosch University, Private
Bag X1, 7602 Matieland, Stellenbosch, South Africa
1
[email protected]
Commercial bioethanol is currently produced from various starchy substrates, with a relatively mature
technology for corn established in the USA. However, starch-to-ethanol processes are still expensive
and the development of Consolidated Bioprocessing (CBP) technologies through amylolytic yeast
could considerably reduce commercial costs (van Zyl et al 2012). This report reviews our recent
research on the construction of efficient amylolytic CBP Saccharomyces cerevisiae strains for
industrial ethanol production from starchy feedstock.
Ten fungal glucoamylase and alpha-amylase genes, native and codon-optimized, were screened in
different combinations for activity in the laboratory strain S. cerevisiae Y294. The most proficient
sequences were δ-integrated into industrial yeast strains (van Zyl et al 2011; Favaro et al 2012; Favaro
et al 2015).
So far, the most effective raw starch-hydrolyzing combination was found to be the codon-optimized
glucoamylase of Thermomyces lanuginosus (TLG1) and α-amylase of Saccharomycopsis fibuligera
(SFA1). These genes were δ-integrated into the industrial S. cerevisiae strains M2n and MEL2, with
the recombinants displaying high amylolytic activities on raw starch and producing about 70 g/L
from 200 g/L raw corn starch in a bioreactor. The starch conversion efficiencies were even higher
on sorghum and triticale grains. Moreover, both recombinant strains were effective for the CBP of
starchy by-products, such as wheat bran and rice husk, where the starch content is about 10-30% of
the biomass. Supplementing the CBP with recombinant cellulases ensured the additional hydrolysis
of the cellulose of the agricultural residues, thus increasing the overall ethanol yield.
The research demonstrated the first CBP from natural starchy substrates and by-products using
industrial yeast strains co-secreting a fungal glucoamylase and α-amylase. The high ethanol yields
achieved at bioreactor scale with the recombinant strains pave the way for their commercial CBP
applications.
KEYWORDS: Bioethanol, Consolidated bioprocessing, Starchy substrates, Industrial yeast
REFERENCES:
Favaro L, Jooste T, Basaglia M, Rose SH, Saayman M, Görgens JF, Casella S, van Zyl WH (2012). Codon-optimized
glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast. Applied Microbiological
Biotechnology 95:957-968
Favaro L, Viktor MJ, Rose SH, Viljoen-Bloom M, van Zyl WH, Basaglia M, Cagnin L, Casella S (2015). Consolidated
bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal
amylases. Biotechnol Bioeng (in press)
van Zyl WH, Jooste T, Görgens JF, Saayman M, Favaro L, Basaglia M, Casella S (2011). Biofuel production. PCT/
WO/2011/128712
van Zyl WH, Bloom M, Viktor MJ (2012). Engineering yeasts for raw starch conversion. Applied Microbiology and
49
The potential and advantages of using marine yeast and seawater-based media
in bioethanol production
Abdelrahman S. Zaky1,2,3, Gregory Tucker1, Chenyu Du1,2
Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham,
U.K. LE12 5RD; 2School of Applied Sciences, University of Huddersfield, Huddersfield, UK. HD1 3DH;
3
Department of Microbiology, Faculty of Agriculture, Cairo University, Giza, Egypt. 12613
1
[email protected]
The majority of fermentations -including bioethanol production- have been carried out using distilled
or tap water. During fermentations, producing one litter of bioethanol consumes 5-10L of freshwater.
With biofuels projected to increase as a transportation fuel, there are concerns over use of freshwater
resources. Seawater, which is free and accounts for about 97% of the world’s water, can be a promising
alternative especially in arid zones where freshwater is increasingly precious (Zaky et al 2014). In
addition, seawater composition -which is not favourable for terrestrial microorganisms- may play a
role as a selective agent against microbial contamination in bio-refineries. Hence, the development of
seawater based media along with marine yeast in bioethanol production can make a valuable impact
on overcoming both the freshwater crisis and energy crisis. Therefore, a new isolation method has
been developed in order to isolate pure marine yeast strains in 9-10 day through 3 simple steps. 122
pure isolates of marine yeasts were obtained from 14 samples that were collected from different
marine environments in Egypt, UK and USA. The results of the metabolic measurements obtained
using biolog indicated that many marine yeast isolates could utilise glucose and/or galactose quicker
than the reference strain (terrestrial yeast S. cereviae NCYC2592) in media made up using either
seawater or freshwater. In addition, many marine yeast isolates show higher tolerance to different
inhibitors comparing with the reference strain. Using a 15L bioreactor, one of our new marine yeast
strains could produce around 70g/L of ethanol from seawater-based media with 20% (w/v) glucose
in 20h. These results indicate the potential of marine yeasts and seawater-based media in bioethanol
production at the industrial level.
KEYWORDS: Marine yeast, Seawater media, Osmo-halo-tolerant yeast
REFERENCES:
Zaky AS, Tucker GA, Daw ZY and Du C (2014). Marine Yeast Isolation and Industrial Application. FEMS Yeast
Research 14:813-825
50
Characterization of three new cutinases from the yeast Arxula adeninivorans
Felix Bischoff1, Katarzyna Litwińska1, Sebastian Worch1, Arno Cordes2, Klaus Krüger3, Sonja
Bischoff3, Gotthard Kunze1
1
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany;
2
ASA Spezialenzyme GmbH, Am Exer 19c, D-38302 Wolfenbüttel, Germany; 3Gesellschaft zur Förderung von
Medizin-, Bio- und Umwelttechnologien e. V. Erich-Neuß-Weg 5, D-06120 Halle (Saale)
[email protected]
In the last decades cutinases have become a promising group of enzymes for research and industrial
applications. Their biochemical properties make them useable for hydrolysis reactions in dairy,
food and oleochemical industry. Furthermore several synthesis reactions, transesterification and
stereo selective esterification were reported (Carvalho 1998). The genes, ACUT1, ACUT2 and
ACUT3, encoding putative cutinases or cutinase-like-enzymes were identified in genome of Arxula
adeninivorans LS3. The alignment of amino acid sequences with other cutinases or cutinase-likeenzymes from different fungi showed that they all contained the catalytic triad S-D-H with a conserved
G-Y-S-Q-G domain. Recombinant His-tagged Acut1-6hp, Acut2-6hp and Acut3-6hp were purified by
immobilized metal ion affinity chromatography and subsequently biochemically characterized. The
well known cutinase from Fusarium solani f. sp. pisi (FsCut-6hp) was expressed and purified as
well to serve as positive control when comparing activity with natural cutin substrates. The substrate
spectra for Acut1-6hp, Acut2-6hp and Acut3-6hp were quite similar with highest activity for short
chain length fatty acid esters of p-nitrophenol and glycerol. Additionally, they were found to have
polycaprolactone degradation activity and cutinolytic activity against cutin from apple peel which
indicates that they are true cutinases. Based on current data, these cutinases show high potential for
the use in industrial applications due to their high expression levels in A. adeninivorans.
KEYWORDS: Arxula adeninivorans, Cutinase
REFERENCES:
Carvalho CML, Aires-Barros MR, Cabral JMS (1998). Cutinase structure, function and biocatalytic applications.
Electronic Journal of Biotechnology 1:160–173
51
52
Session 4
Yeasts genetic and genomic
53
Session 4: Yeasts genetic and genomic - Key note
A population genomics view of Saccharomyces natural history and domestication
José P. Sampaio
UCIBIO, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal
[email protected]
Saccharomyces yeasts are used since the dawn of civilization to ferment a myriad of foods and
beverages. Although it is usually assumed that such microbes have underwent a domestication
process equivalent to those already known for plant crops and livestock, the genomic underpinnings
of Saccharomyces domestication remain unknown. Moreover, the natural history and distribution of
wild populations from which the domesticated lineages likely derive is also poorly known. NGS-based
comparative and population genomics allow an unprecedented fine scale resolution of population
dynamics and organismic evolution thus providing a powerful tool to study the evolutionary ecology,
natural phylogeography and domestication history of Saccharomyces. An update of a large scale survey
of whole-genome sequence variation using an improved dataset of Saccharomyces representatives
that includes a less human-biased collection of strains will be presented. The relationship between
putatively domesticated and wild lineages will be discussed and recently revealed new patterns of
domestication will be highlighted.
This work was supported by research grants PTDC/BIA-EVF/118618/2010 and PTDC/AGRALI/118590/2010.
54
Session 4: Yeasts genetic and genomic
New insights into the adaptation of yeast to anthropic environment using
comparative genomics
Jean-Luc Legras 1, Anna L. Coi2, Frédéric Bigey1, Virginie Galeote1, Souhir Marsit1, Arnaud
Couloux3, Julie Guy3, Ricardo Franco-Duarte4, Dorit Schuller4, José P. Sampaio5, Marilena
Budroni 2, Sylvie Dequin 1
INRA, UMR 1083 Sciences pour l’Oenologie, 2 place Viala – F-34060 Montpellier – France ; 2Dipartimento di
Agraria, Università di Sassari, Sassari, Italy; 3Genoscope – Centre National de Séquençage, UMR CNRS 8030,
2 Gaston Crémieux, CP 5706, 91507 Evry, France ; 4Universidade do Minho , Braga, Portugal; 5 CREM, DCV Universidade Nova de Lisboa, Portugal
1
[email protected]
The yeast Saccharomyces cerevisiae is one of the most important microorganisms for food and drink
production and it is as well a model for biology. Surprisingly, the bases of yeast population structure
have been unraveled only recently (Fay & Benavides 2005; Lagras et al 2007) and the first genomic
population approaches have failed to point adaptation to ecological niches. However, as many groups
present highly contrasted lifestyle (e.g. anaerobic growth on grape must for wine yeast, aerobic
growth as a biofilm on ethanol and glycerol for flor strains, growth on low amount of sugars for oak
strains) different adaptations are expected.
In this project we have obtained high quality genome sequences of 82 yeast strains: 35 from wine
environment (27 wine strains, 8 flor strains) as well as 47 other strains from rum, fermented dairy
product, bakery including 8 from oaks trees.
Our genomic data enable us to delineate specific genetic groups corresponding to the different
ecological niches, and indicate different life cycles. We first detected the amplification of several
genes with critical function in their environment (e.g. CUP1 for wine yeast, IMA2 and SUC2 for
bakery yeast…). We then established a catalog of genes potentially impacted per population. Several
tests revealed a non-neutral evolution at several loci, and especially the presence of selective sweep
for wine yeast, suggesting positive selection. The comparison of flor and wine yeast revealed allelic
variation associated to growth under velum, as well as the fructophily of flor strains, or a specific
metal homeostasis. Last among the three large chromosomal regions originated from horizontal gene
transfer (HGT) from distant yeasts first discovered in EC1118 (Novo et al 2009) were encountered
mainly in wine or flor yeasts but the complete region C was only found among flor strains.
These genomic data represent a unique resource for understanding the adaptation of yeast to wine
making niches and thus for elucidating the bases of technological properties.
KEYWORDS: Saccharomyces cerevisiae, Diversity, Adaptation
REFERENCES:
Fay JC, Benavides JA (2005). PLoS Genetics 1(1):66-71
Legras JL, Merdinoglu D, Cornuet JM, Karst F(2007). Molecular Ecology 16(10): 2091–102
Novo M, Bigey F, Beyne E, Galeote V, Gavory F, Mallet S, Cambon B, Legras JL, Wincker P, Casaregola S, Dequin S
(2009). PNAS 106(38): 16333–8
55
The origin and domestication of lager beer yeast
Jian Bing, Pei-Jie Han, Wan-Qiu Liu, Qi-Ming Wang, Feng-Yan Bai
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
[email protected]
Lager-brewing at low temperature arose in 15th century Bavaria and has become the most popular
technique for alcoholic beverage production in the world. The lager yeast Saccharomyces pastorianus
is a domesticated microbe through the hybridization between an ale yeast S. cerevisiae and a
cryotolerant wild yeast S. eubayanus. The latter firstly discovered from Patagonia, Argentina exhibits
99.5% genome sequence identity with the non-ale subgenome of S. pastorianus. Consequently, a
Patagonian hypothesis for the origin of lager yeast has been proposed. Here we show that S. eubayanus
commonly occurs in the Tibetan Plateau and adjacent high altitude regions in west China and exhibits
surprisingly high genetic and phenotypic diversity. Three distinct lineages with over 6% inter-lineage
sequence divergence were identified from the S. eubayanus strains from China based on multiple
gene sequence analyses. A Tibetan population of S. eubayanus exhibits the closest known match
(99.8%) to the non-ale subgenome of S. pastorianus. Our results suggest that S. eubayanus is native
to Far East Asia and that the Tibetan S. eubayanus population is the progenitor of lager yeast. Tibetan
S. eubayanus strains also exhibit diversified phenotypic characters and are usually more cryophilic.
Sequence, structure and function comparison showed that the functional maltose transporter genes in
lager yeast were mainly contributed from S. cerevisiae MTY1 and S. eubayanus AGT1, with some
degree of variations and recombination. We are performing comparative genomic and transcriptomic
analyses to reveal the contribution of S. eubayanus to the psychrophilic brewing nature of lager yeast.
KEYWORDS: S. pastorianus, S. eubayanus, Domestication, Biogeography, Population genetics
56
Session 4: Yeast genetic and genomic
Reversion of meiotic progression allows extensive recombination
of S. cerevisiae hybrid diploids
Raphaëlle Laureau1, Sophie Loeillet1, Francisco Salinas2, Anders Bergström2, Gianni Liti2,
Alain Nicolas1
Recombination and genetic instability, Institut Curie Centre de Recherche, CNRS UMR3244, Université Pierre et
Marie Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France ; 2Institute of Research on Cancer and Ageing of Nice
(IRCAN), CNRS UMR 7284-INSERM U1081, Faculté de Médecine, Université de Nice Sophia Antipolis, 28 Avenue
de Valombrose, 06107 NICE Cedex 2, France
1
[email protected]
Inter-homolog recombination in mitotically growing yeast diploids is rare but would be of major
interest if one wishes to rapidly conduct phenotype/genotype analyses of polymorphic complex
traits in diploid cells. For this purpose, we used a phenomenon unique to the yeast Saccharomyces
cerevisiae, i.e. meiosis reversibility and Return-to-Growth (RTG), to generate genetically diversified
diploids. Genome-wide sequencing of 36 RTG cells demonstrates that these cells remained diploid
and were diversely recombined, comprising a large variety of loss of heterozygosity (LOH) regions.
Phenotype/genotype analyses of these RTG cells provides a powerful way to map QTLs of complex
traits in diploid cells, without going through sexual reproduction.
Recombination between the homologous pairs of chromosomes is rare in somatic cells, thus preserving
the genetic integrity. In contrast, it is rather frequent during meiosis, allowing the production of
recombinant gametes that contributes to the genetic diversity of sexually reproducing organisms. I
will summarize our recent methodological advances to (i) target and modify meiotic recombination
in S. cerevisiae and (ii) generate recombinant yeast diploid cells that can be used to identify the QTL
of complex traits.
In contrast to meiosis, inter-homologs recombination is strongly repressed in vegetative growth.
The benefit is to preserve the integrity of the genome, as inter-homolog mitotic recombination can
induce Loss Of Heterozygosity (LOH); This can be deleterious if the parental chromosomes carry
heterozygous mutations. However, in order to dissect the genetic information of polymorphic diploid
cells, a method allowing to recombine diploid genomes will be useful. For this purpose, we used
the unique feature of S. cerevisiae diploid cells to reversibly enter meiosis (Return-To-Growth).
Upon genome-wide sequencing of a series of 36 RTG cells issued from the S288C/SK1 hybrid, we
observed that the regimen of RTG generates a large variety of genetically diversified diploid cells that
will be presented. RTG cells can be conveniently used to map yeast Quantitative Trait Loci (QTL) of
complex traits.
57
Polygenic analysis of ethanol tolerance and maximal ethanol accumulation
capacity in Saccharomyces cerevisiae
Annelies Goovaerts1,2, Steve Swinnen1,2, Thiago Pais1,2, Maria Foulquié-Moreno1,2,
Johan M. Thevelein1,2
Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium; 2Department of
Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001, Leuven, Belgium
1
[email protected]
Most traits of industrial importance in yeast are polygenic, therefore complex and difficult to study.
In our laboratory, we have developed a technology called ‘pooled-segregant whole-genome sequence
analysis’, which has been successfully applied to complex traits such as high ethanol tolerance
(Swinnen et al 2012)and maximal ethanol accumulation capacity (Pais et al 2013).The aim of this
study was to identify new genes underlying high ethanol tolerance and maximal ethanol accumulation
capacity by combining both pooled-segregant whole-genome sequence analysis and pooled-segregant
RNA expression analysis.
A haploid strain displaying the superior trait of interest was crossed with a haploid inferior lab BY
strain. Segregants of this cross showing the phenotype of the superior parent were selected and pooled
together. The genomic DNA of the pool and the parents was extracted and sequenced by Illumina
HiSeq2000. The SNP variant frequency of the pooled DNA was plotted against the SNP chromosomal
position, to map the quantitative trait loci (QTLs). Reciprocal hemizygosity analysis (RHA) was
applied to identify the causative genes in the QTLs. Genome-wide gene expression analysis at a
fermentation time-point where the ethanol concentration was 13.8% (v/v) was performed for the
superior pool and parent strains via RNA-Seq.
Several QTLs were identified for the traits ethanol tolerance and maximal ethanol accumulation.
Fine-mapping and RHA identified several specific causative genes for these traits of interest. RNA
sequence analysis revealed 37 genes that were overexpressed in the pool of segregants and superior
parent in comparison with the inferior parent. Most of these genes have a biological function related
to stress tolerance. Our results reveal that a combination of pooled-segregant whole-genome sequence
analysis and gene expression analysis is a promising approach to understand the genetic basis of
complex traits.
KEYWORDS: Ethanol tolerance, Ethanol accumulation capacity, Saccharomyces cerevisiae,
Polygenic analysis, RNA-Seq
REFERENCES:
Swinnen S, Schaerlaekens K, Pais T, Claesen J, Hubmann G, Yang Y, Demeke M, Foulquié-Moreno MR, Goovaerts A,
Souvereyns K, Clement L, Dumortier F, Thevelein JM (2012). Identification of novel causative genes determining the
complex trait of high ethanol tolerance in yeast using pooled-segregant whole-genome sequence analysis. Genome
Research 22(5):975-84
Pais TM, Foulquié-Moreno MR, Hubmann G, Duitama J, Swinnen S, Goovaerts A, Yang Y, Dumortier F, Thevelein
JM (2013). Comparative polygenic analysis of maximal ethanol accumulation capacity and tolerance to high ethanol
levels of cell proliferation in yeast. PLoS Genetics 9(6): e1003548
58
Pathway swapping: a new approach to simply and efficiently remodel essential
native cellular functions
Niels G.A. Kuijpers, Daniel Solis-Escalante, Marijke A.H. Luttik , Jack T. Pronk, Jean-Marc
Daran, Pascale Daran-Lapujade
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628BC, Delft, The Netherlands
[email protected]
Spectacular developments in the field of synthetic biology have enabled the introduction of complete
new pathways to living cells. However, the mosaic design of microbial genomes hinders the largescale remodeling of their core machinery required to obtain a profound understanding of its governing
regulatory principles and thereby to enhance the performance of industrial microbes. To overcome this
limitation, we introduce the concept of ‘pathway swapping’. Using as paradigm glycolysis, nearlyubiquitous and essential metabolic highway for sugar utilization, we constructed a Saccharomyces
cerevisiae platform enabling the quick and easy replacement of the 27-isoenzymes native glycolysis by
simplified, heterologous synthetic versions. Yeasts carrying synthetic glycolyses from Saccharomyces
kudriavzevii and mosaic glycolysis mixing yeast and human genes were successfully constructed and
were able to grow in chemically defined minimal media. This work paves the way for a modular
approach to engineering of central metabolism.
59
Natural variation in non-coding regions underlying phenotypic diversity in
budding yeast
Francisco Salinas1, Carl G. de Boer2, Valentina Abarca3,4, Verónica García3,4, Mara Cuevas3,4,
Felipe Herbage3,4, Luis F. Larrondo1, Claudio Martínez3,4, Francisco A. Cubillos3,4
Millennium Nucleus for Fungal Integrative and Synthetic Biology, Departamento de Genética Molecular y
Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile; 2Broad
Institute of MIT and Harvard, Cambridge, MA, United States; 3Departamento de Ciencia y Tecnología de los Alimentos,
Universidad de Santiago de Chile (USACH), Santiago, Chile; 4Centro de Estudios en Ciencia y Tecnología de
Alimentos (CECTA), Universidad de Santiago de Chile (USACH), Santiago, Chile
1
[email protected]
Determining the different sources of heritable variation underlying quantitative traits in nature is
currently at the forefront of genetic studies. To this end, molecular profiling studies in S. cerevisiae
have shown that individual gene expression levels are subject to genetic control and this variation can
mediate genetic differences on phenotype. Thus, determining how natural variation influences allele
specific expression (ASE) and ultimately complex traits represents a useful tool to determine the
mechanisms leading to yeast niche adaptation. Here, in order to test the hypothesis that allele-specific
expression differences between isolates contributes to the phenotypic diversity in natural populations,
we evaluated ASE levels in a grid of six F1 hybrids from the cross of four representative founder
strains from major lineages. Genome-wide and across hybrids we quantified ASE for 3,320 genes.
We found evidence for abundant genome-wide expression differences between alleles, with levels
ranging between 27% up to 61% of the evaluated genes, depending on the cross. We observed that
ASE can be explained by allele-specific differences in transcription factor binding to cis-regulatory
regions and differences in strain-specific trans-activation can be detected by taking advantage of
the shared trans environment of F1 hybrids. Furthermore, modules of genes under cis-regulatory
variation with related function are enriched within the different genetic backgrounds, supporting the
premise of intraspecies directional regulatory selection in yeast. Finally, we were able to identify two
genes, GDB1 and ASN1 exhibiting high expression levels in the Wine/European strain and underlying
phenotypic differences for oenological phenotypes due to polymorphisms within non-coding regions,
providing direct evidence of the importance of regulatory variation in natural trait diversity.
60
Genomic and transcriptomic landscapes within a protoploid yeast species
Christian Brion, Anne Friedrich, David Pflieger, Joseph Schacherer
Department of Genetics, Genomics and Microbiology, University of Strasbourg, CNRS, UMR7156, Strasbourg,, France
[email protected]
Exploration of genetic diversity and gene expression variations between isolates of the same species
are both essential to obtain an overview of the evolution of genomes and regulatory networks, which
underlie phenotypic diversity. Numerous studies have characterized intraspecific variations and
focused on a large number of individuals using Saccharomyces cerevisiae as a model organism. While
extremely important, this wealth of data stands in contrast to our limited knowledge in other yeast
species. In this perspective, we sought to have a view of the genomic and transcriptomic landscapes in
the protoploid species (i.e. which diverged from the S. cerevisiae lineage prior to its ancestral whole
genome duplication): Lachancea kluyveri (formerly Saccharomyces kluyveri). We first performed a
population genomic analysis on a large number of isolates. Our results clearly showed that distinct
recombination and substitution regimes coexist and lead to different evolutionary patterns. More
precisely, we revealed that a 1-Mb region on the chromosome C is a relic of an introgression event
and is characterized by a higher GC content, a higher sequence diversity as well as a large set of
unique unannotated genes. In this context, we then explored the transcriptomic landscape within
this collection of isolates by RNA-seq. Interestingly, our comparative transcriptomic analysis clearly
showed a link between gene evolutionary history and expression behavior. Indeed, genes recently
acquired (such as genes present in the introgressed region) or under function relaxation tend to be
less transcribed, show a higher intraspecific variation and are less involved in network. Moreover,
utilizing this approach in L. kluyveri also highlighted specific regulatory network signatures in aerobic
respiration, amino-acid biosynthesis and glycosylation, presumably due to its different lifestyle. Our
datasets shed an important light on the genome evolution in yeast and its impact on transcription.
KEYWORDS: Population genomics, Comparative transcriptomics, Intraspecific variation, Gene
evolution, Protoploid yeast
61
Genomic and transcriptomic landscape of the industrial yeast species
Brettanomyces bruxellensis
Chris Curtin1, Lucy Joseph2, Ryan Zeppel1, Warren Albertin3, Isabelle Masneuf- Pomarede3,
Linda Bisson2, Anthony Borneman1
Australian Wine Research Institute, Urrbrae, SA 5064, South Australia, Australia; 2Department of Viticulture and
Enology, University of California, Davis, 595 Hilgard Lane, Davis, CA 95616; 3Univ. de Bordeaux, ISVV, EA 4577,
Unité de recherche Œnologie, F-33140 Villenave 13 d’Ornon, France
1
[email protected]
Brettanomyces bruxellensis, like its wine yeast counterpart Saccharomyces cerevisiae, is intrinsically
linked with industrial fermentations. In wine, B. bruxellensis has what are generally considered
negative influences on wine quality, whereas for some styles of beer it is an essential contributor. B.
bruxellensis also plays a role in bioethanol fermentation – sometimes beneficial, but in other systems
detracting from production efficiency by outcompeting S. cerevisiae. We previously investigated the
level of inter-strain variation that is present within this economically important species, by comparing
the genomes of four diverse B. bruxellensis isolates. Two of these genomes were predicted to be
triploid, comprising a core diploid set of chromosomes and a third divergent haploid set, reminiscent
of allotriploids within the Saccharomyces sensu- stricto. Re-sequencing of a further 38 isolates from
around the world revealed that triploidy is relatively common for B. bruxellensis. Our data suggest
at least five independent ‘hybridisation’ events have occurred to generate these triploid lineages that
now populate brewing, winemaking and soft-drink related niches. It is unclear what fitness benefits
polyploidy has conferred, although the most common triploid lineage recovered from Australian
wineries exhibits greater tolerance to the common preservative sulfite. In order to better understand the
basis of this phenotypic advantage, comparative transcriptomics was performed for two strains during
growth in model-wine conditions and when exposed to sulfite. Similar to S. cerevisiae, relatively
few transcripts were differentially expressed in response to sulfite. A clear case of allele-specific
expression (ASE) for the sulfite efflux pump BbSSU1 was detected, and the global instance of ASE
across the triploid genome examined to infer genes for which the divergent allele may be beneficial.
KEYWORDS: Non-conventional yeast, Wine, Spoilage, Genome evolution, Transcriptomics
62
Comparative genomics of biotechnologically important yeasts
Thomas Jeffries1, Robert Riley2, Sajeet Haridas2, Asaf Salamov2, Kyria Boundy-Mills3,
Markus Göker4, Chris Hittinger5, Hans-Peter Klenk6, Mariana Lopes5, Jan P. Meier-Kolthoff4,
Antonis Rokas7, Carlos A. Rosa8, Carmen Scheuner4, Marco Soares5, Benjamin Stielow9,
Jennifer H. Wisecaver7, Ken Wolfe10, Meredith Blackwell11, Clete Kurtzman12, Igor Grigoriev2
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI; 2US Department of Energy Joint Genome
Institute, Walnut Creek, CA; 3Department of Food Science and Technology, University of California Davis, One Shields
Ave, Davis, CA; 4Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany; 5Laboratory of Genetics, Genetics/Biotechnology Center, Madison, WI; 6School of Biology, Newcastle
University, Newcastle upon Tyne, UK; 7Department of Biological Sciences, Vanderbilt University; 8Instituto de Ciências
Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 9CBS-KNAW Fungal Biodiversity Centre,
Utrecht, Netherlands; 10Conway Institute, University College Dublin, Dublin, Ireland; 11Department of Biological
Sciences, Louisiana State University, Baton Rouge, LA; 12USDA, ARS, MWA, NCAUR, BFPM, Peoria, IL
1
[email protected]
Saccharomyces cerevisiae, is used in the
vast majority of the world’s bioprocesses.
Its
economic
significance
is
unchallenged. It, however, represents
only a small slice of yeast physiological
diversity. Many other yeasts, are used in
lesser known, but commercially
important processes that take advantage
of their unique physiological and
biochemical properties. Through a
project conducted with the DOE Joint
Genome
Laboratory
(JGI),
we
sequenced, annotated and compared 18
new yeast genomes to 20 previously
sequenced yeasts and other fungi
belonging to the Pizizomycotina,
Taphrinomycotina, Basidiomycota, and
other more distant taxa. Whole genome alignment of these 38 fungi revealed four distinct clades of
ascomycetous yeasts and confirmed the monophyletic nature of the Taphrinomycotina (Fig. 1). Native
capacities for fermenting xylose and cellobiose were confined almost entirely to the CTG yeast clade.
Highly lipogenic yeasts were found in the Dipodascascacae, and the methylotrophic yeasts clearly
exhibited higher capacities for respiration. The Saccharomycetaceae, some of which were clearly
adapted for fermentative metabolism, were in a distinct but heterogeneous clade. Highly divergent
yeast species showed marked losses of many enzymatic activities, which apparently occurred during
their evolutionary speciation. Lipomyces starkeyi has the largest genome, 21x106 bp, of all the yeasts
studied. Pneumocystis jirovecii has the smallest genome, 8.15x106 bp. Introns were more prevalent in
the basal species. TY5 elements are more prevalent than the Ty1 and Ty2 elements found in S.
cerevisiae, which also had 99% of all the TY-LTR’s identified. Methylotrophic and galactose
fermenting yeasts could be predicted based on genomic features. Strongly expressed genes for selected
traits were often found in functional clusters.
63
Genetics of lactose utilisation in Kluyveromyces marxianus
Javier Varela1, Ralph van der Ploeg1, Damhan Scully1, Raul Ortiz2, Kenneth Wolfe2,
Eckhard Boles3, John Morrissey1
School of Microbiology, University College Cork, Ireland; 2Conway Institute, University College Dublin, Ireland;
3
Institut für Molekulare Biowissenschaften, Goethe-Universität Frankfurt, Germany
1
[email protected]
Kluyveromyces lactis and Kluyveromyces marxianus are the best-studied species of the Kluyveromyces
genus. K. lactis has been adopted as a model for non–Saccharomyces yeasts whereas K. marxianus is
more widely used for industrial applications. Recently genome sequences for a number of different K.
marxianus strains have been generated and comparison to K. lactis reveals some interesting metabolic
differences between the two species and highlights the significant structural differences between the
genomes. Some key genomic differences arise in sub-telomeric regions, where different genes are
duplicated at each end of the chromosome, or at the ends of separate chromosomes, in each yeast.
For example, the LAC12 gene responsible for lactose assimilation is duplicated in the subtelomeric
regions of K. marxianus but not in K. lactis. The duplication of the LAC12 permease reflects a theme
of expansion of gene families encoding sugar transporters in K. marxianus – which may explain the
rapid growth kinetics of this species. We are focusing in particular on the assimilation of lactose
as although it is important for growth on industrial substrates, the capacity to rapidly assimilate
lactose is quite variable between K. marxianus strains. Four conserved alleles of LAC12 are found
in all K. marxianus genomes compared to one in K. lactis. Analysis of the gene regulatory regions
suggests that the different alleles of LAC12 are differentially expressed both within a strain and
between isolates. Expression analysis via RT-PCR confirms this and detailed analysis of expression
of each LAC12 allele under a range of different carbon sources is underway. Furthermore, LAC12
alleles are being expressed in a S. cerevisiae strain lacking endogenous monosaccharide transporters
to establish the substrate range of each Lac12p protein. These data will yield new knowledge on the
evolution and function of this important gene family in K. marxianus and will inform commercial
strain development.
KEYWORDS: Kluyveromyces marxianus, Lactose, Genome, Gene expression, Permease
64
BAT1 and BAT2 functional diversification through subfunctionalization
of chromatin organization and transcriptional regulation
in Saccharomyces cerevisiae
James González1, Joseph Strauss2,3, Geovani López1, Alicia González1
Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma
de México, Ciudad de México, México; 2Fangal Genetics and Genomics Unit, Department of Applied Genetics and Cell
Biology, BOKU-University of Natural Resources and Life Sciences, Campus Tulln, Austria; 3Health and Environment
Department, AIT – Austrian Institute of Technology GMBH, Campus Tulln, Tulln, Austria
1
[email protected]
Paralogous gene expression divergence, results in differential expression profiles that determine
functional diversification of the encoded proteins. Here, we analyze the role of chromatin organization
and trans/cis-acting elements on the diversification of BAT1 and BAT2 paralogous genes encoding
branched chain amino acid aminotransferases, generated from the Whole-Genome Duplication
(WGD) of S. cerevisiae.
Standard molecular biology techniques, mutant construction and NuSA.
Our results show that BAT1 and BAT2 chromatin organization of the paralogous promoter regions have
diverged from the ancestral-type KlBAT1 and LkBAT1. Furthermore, BAT1 transcriptional activation
is determined through the action of Gcn4, Leu3-α-IPM, and indirectly by Gln3, while repression is
triggered by Put3. In addition, BAT2 transcription activation is achieved through the action of the
Swi/Snf complex, Leu3, Gln3 and Put3. Interestingly, BAT2 expression is repressed in glutamine,
through the action of Leu3, a novel Gln3-independent regulatory mechanism. Surprisingly, Leu3
can act as negative or positive modulator under the same physiological condition, suggesting that
this capacity is independent of the intracellular concentration of the co-regulator α-isopropylmalate
(Brisco & Kohlhaw 1990).
Expression divergence plays a major role in paralogous functional diversification, dependent on
chromatin promoter organization, such as nucleosome sliding or displacement. On the other hand,
subfunctionalization could be triggered by positive selection of transcriptional factors which could
act as either repressors, or activators, under the same physiological conditions, resulting on opposed
regulation of the target paralogous genes.
KEYWORDS: Paralogous diversification, Chromatin organization, Gene expression
REFERENCES:
Brisco PR, Kohlhaw GB (1990). Regulation of yeast LEU2. Total deletion of regulatory gene LEU3 unmasks GCN4dependent basal level expression of LEU2. Journal of Biological Chemistry 265:11667-75
65
The haploid nature of Candida glabrata is advantageous under harsh conditions
Olena P. Ishchuk1, Silvia Polakova1,5, Khadija M. Ahmad1, Praveen Chakravarthy1, Sofia
M. Wisén1, Sofia Dashko1, Maryam Bakhshandeh1, Leif Søndergaard2, Victoria Rydengård3,
Artur Schmidtchen3, John Synnott4, Can Wang4, Sarah Maguire4, Geraldine Butler4,
Wolfgang Knecht1,6, Jure Piškur1
Department of Biology, Lund University, Sölvegatan 35, Lund SE-223 62, Sweden; 2Center for Functional and
Comparative Insect Genomics, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100,
Copenhagen, Denmark; 3Section of Dermatology and Venereology, Department of Clinical Sciences, Lund University,
Biomedical Center, Lund, Sweden; 4UCD School of Biomolecular and Biomedical Science, Conway Institute,
University College Dublin, Belfield, Dublin 4, Ireland; 5Max F. Perutz Laboratories, University of Vienna, Dr. BohrGasse 9, Vienna, Austria, A-1030; 6Lund Protein Production Platform, Sölvegatan 35, Lund SE-223 62, Sweden
1
[email protected]
Candida glabrata is the second most prevalent yeast pathogen in humans. Systemic infections
caused by this pathogenic yeast have high mortality rates and are difficult to treat because it readily
develops resistance in response to drug exposure during treatment. In contrast to other human yeast
pathogens and the closely related Saccharomyces yeasts, C. glabrata has only been found haploid
and asexual yeast. We asked if its haploid nature and the observed genome rearrangements could be
an advantage for C. glabrata to survive in vivo. To address this question, the competition between
haploid and artificially created diploid strains of C. glabrata was studied in vivo (in a fly and a mouse
model) and in vitro under normal and stress conditions (fluconazole, high temperature). Experimental
populations (competition groups) of 2 haploid parental strains and one diploid (a fusion product of
the corresponding parental haploids) were used in competition experiments, and the outcome was
analyzed. We showed that after few days in most cases haploid strains outcompeted the diploid one
in infected flies and mice. The haploid fraction increased but the diploid cells decreased in number
in vivo. When this experiment was done competed in vitro, the diploid strains always prevailed
under non-stressed conditions. However, with increasing fluconazole concentrations and at elevated
temperatures the haploid strains outcompeted the diploid one more often. Thus, the haploid nature
seems to provide an advantage in the competition under harsh conditions. Some of the prevailing
strains were analyzed for their gene expression, showing that several genes drastically changed their
expression.
KEYWORDS: Candida glabrata, Haploid nature, Stress conditions, Competition
66
Session 5
Non-conventional yeasts
67
Session 5: Non-conventional yeasts - Key note
Non-conventional yeasts as promising producers of biofuels and chemicals
Andriy A. Sibirny1,2, Olena Kurylenko1, Justyna Ruchala2, Mariana Yurkiv1, Oleksiy Lyzak1,
Kostyantyn Dmytruk1
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005 Ukraine; 2Rzeszow University,
Zelwerowicza 4, Rzeszow 35-601, Poland
1
[email protected]
Non-conventional yeasts attract increasing attention of researchers due to many unusual peculiarities
of growth physiology and metabolism. Methylotrophic yeast Hansenula polymorpha is apparently the
most thermotolerant yeast organism known with maximal growth temperature of 50 oC. It ferments
major sugars of lignocellulosic hydrolyzates including xylose (Ryabova et al 2003). Using combination
of the methods of metabolic engineering and classical selection, the strain of H. polymorpha was
constructed which accumulats elevated amounts of ethanol from xylose under 45 oC relative to the
wild-type strain (Kurylenko et al 2014). It was found that overexpression of genes coding the initial
enzymes of xylose catabolism along with peroxisomal transketolase and transaldolase and knock
out of transcriptional activator CAT8 led to 25 fold increase in ethanol production from xylose.
Overproducers of glutathione were isolated in H. polymorpha due to overexpression of transcriptional
activator MET4 and glutathione biosynthesis gene GSH2. Flavinogenic yeasts overproduce riboflavin
under iron deprivation and the mutants isolated from one of such species, Candida famata belong to
the most flavinogenic organisms known (Abbas & Sibirny 2011). However yeast industrial producers
of riboflavin appeared to be very unstable. We have identified putative transcriptional activator
SEF1 which is involved in regulation of riboflavin synthesis. Multiple integration of SEF1 and
overexpression of structural genes of riboflavin biosynthesis RIB1 and RIB7 led to non-reverting
riboflavin overproducers which accumulated more than 16 g of riboflavin/L during fed batch
cultivation in the laboratory bioreactor. Perspectives of the further increase of biofuel and chemical
production by non-conventional yeasts will be discussed.
KEYWORDS: Non-conventional yeasts; Xylose, Ethanol, Gutathione, Riboflavin
REFERENCES:
Abbas CA, Sibirny AA (2011). Microbiology and Molecular Biology Review 75:321-360
Kurylenko OO, Ruchala J, Hryniv OB, Abbas CA, Dmytruk KV, Sibirny AA (2014). Metabolic engineering and
classical selection of the methylotrophic thermotolerant yeast Hansenula polymorpha improvement of hight
temperature xylose alcoholic fermentation. Microbial Cell Factories 13:122
Ryabova OB et al.(2003). FEMS Yeast Research 3:157-164
68
Session 5: Non-conventional yeasts
The impact of degradative pathways on recombinant protein secretion in Pichia
pastoris
Richard Zahrl1,2, Martin Pfeffer1, Diethard Mattanovich1,2, Brigitte Gasser1,2
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria;
2
Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria
1
[email protected]
Recombinant protein production is an expanding branch of biotechnology with increasing economic
importance. Many proteins are efficiently secreted by yeast systems, reaching product titers in the g
L-1 range. The expression of more complex proteins, however, overwhelms the folding and secretion
capacity of the host cells. This triggers cellular stress responses, mainly the unfolded protein response
(UPR), which in turn activates the endoplasmic-reticulum-associated protein degradation (ERAD)
in order to decrease the load of unfolded or misfolded proteins in the endoplasmic reticulum (ER).
After retro-translocation to the cytosol, ERAD-client proteins are finally degraded by the proteasome.
However, the impact of ERAD on recombinant protein secretion in yeast has not been investigated
in detail yet.
By using sulfur isotope 34S labeling, we have recently modelled and measured intracellular fluxes of
secreted antibody Fab fragments in the yeast Pichia pastoris. The results have shown that about 60%
of the newly synthesized recombinant proteins are intracellularly degraded, instead of being secreted
(Pfeffer et al 2011). Additionally the interactome of the Fab revealed several proteasomal proteins
(Pfeffer et al 2012). These findings have led to the speculation of a possibly overshooting ER quality
control. Based on the results obtained, we targeted ERAD for inhibition by using chemical inhibitors
or by knocking-out genes associated with the ERAD complex. The fate of the recombinant secreted
protein was followed intracellularly, the impact on the host’s UPR response was measured and the
amounts of secreted product were determined. Unexpectedly, we detected a possible involvement of
the recently described pre-insertional degradation complex (Ast et al 2014) on protein secretion.
Taken together, these studies aimed at investigating the impact of recombinant protein production on
the ER homeostasis. Strategies on manipulating the host’s response in order to efficiently adapt its
secretory capacity will be discussed.
KEYWORDS: Heterologous protein production, Secretion, ER quality control, Degradation, Pichia
pastoris
REFERENCES:
Pfeffer M, Maurer M, Köllensperger G, Hann S, Graf AB, Mattanovich D (2011). Modeling and measuring intracellular
fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling procedure. Microbial Cell
Factories 10:47
Pfeffer M, Maurer M, Stadlmann J, Grass J, Delic M, Altmann F, Mattanovich D (2012). Intracellular interactome
of secreted antibody Fab fragment in Pichia pastoris reveals its routes of secretion and degradation. Applied
Microbiology and Biotechnology 93(6):2503-12
Ast T, Aviram N, Chuartzman SG, Schuldiner M (2014). A cytosolic degradation pathway, prERAD, monitors preinserted secretory pathway proteins. Journal of Cell Science 127(14):3017-23
69
Arxula adeninivorans – a suitable biocatalyst for new biotechnological products
Martin Giersberg1, Dagmara Jankowska1, Urs Hähnel1, Jan Riechen1, Alexandre Chamas1,
Jakub Kasprzak1, Marion Rauter2, Felix Bischoff1, Sebastian Worch1, Mateuzs Biernacki1,
Anke Trautwein1, Keith Baronian3, Gotthard Kunze1
1
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr.3, D-06466 Gatersleben, Germany;
Orgentis Chemicals GmbH, Bahnhofstr. 3, D-06466 Gatersleben, Germany; 3School of Biological Sciences, University
of Canterbury, Private Bag 4800, Christchurch, New Zealand.
2
[email protected]
The industrially important yeast Arxula adeninivorans is an asexual hemiascomycete phylogenetically
very distant from Saccharomyces cerevisiae. Its unusual metabolic flexibility allows it to use a wide
range of carbon and nitrogen sources, while being thermotolerant, xerotolerant and osmotolerant.
Based on the completely sequenced and annotated Arxula genome combined with gene expression
data, numerous so far non-described pathways in yeasts were explored and exploited such as the
metabolism of n-butanol, 2,3-butanediol and tannic acid. The obtained data provide new knowledge
to the exceptional broad substrate spectrum and robustness of this yeast. In addition A. adeninivorans
is used as suitable host for synthesis of special products such as recombinant functional human
receptors or glycosylated secretory tannases and it serves as a suitable biocatalyst for the synthesis
of biotechnologically interesting products such as n-butanol and 2,3-butanediol, because all essential
prerequisites and components for heterologous gene expression are available. A lot of special protocols
have been established (transformation/expression platform Xplor®2, gene disruption, protoplast
fusion, mitotic segregation) and industrial strains were constructed. These strains are suitable
producers of enzymes and enzyme mixes to degrade plastic material and lignocellulose (Kunze et
al 2014), to synthesize enantiometrically pure alcohols (Jankowska et al 2014) and to produce food
with low purine content (Rauter et al 2014). Other biotechnological application fields for Arxula
cells are bioremediation by accumulation of metal ions and production of biobutanol. Furthermore
A. adeninivorans is used as microbial sensor compound to detect hormone activities (estrogenic,
androgenic, glucocorticoidic, gestagenic activities), dioxins as well as pharmaceuticals in tap water,
mineral water, waste water, urine and blood serum.
KEYWORDS: Arxula adeninivorans, Biocatalyst, n-butanol, 2,3-butanediol
REFERENCES:
Kunze G, Gaillardin C, Czernicka M et al (2014). The complete genome of Blastobotrys (Arxula) adeninivorans LS3 - a
yeast of biotechnological interest. Biotechnology for Biofuels 7:66
Jankowska DA, Trautwein-Schult A, Cordes A, Boden R, Baronian K, Kunze G (2014). A novel enzymatic approach
in the production of food with low purine content using Arxula adeninivorans endogenous and recombinant purine
degradative enzymes. Bioengineered 6:1,1-6
Rauter M, Kasprzak J, Weniger M, Becker K, Baronian K, Bode R, Piontek M, Kunze G, Vorbrodt HM (2014).
Reusuability of ADH and GDH producing Arxula adeninivorans cells and cell extract for the production of 1-(S)phenylethanol. Journal of Molecular Catalysis B: Enzymatic 108:72-76
70
Yarrowia lipolytica, a model for lipid metabolism
and a platform for lipid production
Rodrigo Ledesma-Amaro 1,2, Thanos Beopoulos1,2, Anne M. Crutz-Le Coq1,2,
Rémi Dulermo1,2, Thierry Dulermo1,2, Zbigniew Lazar1,2,3, Cecile Neuveglise1,2,
Heber Gamboa-Meléndez1,2, Tristan Rossignol1,2, Jean M. Nicaud1,2
INRA, UMR1319, MICALIS, Domaine de Vilvert, F-78352 Jouy-en-Josas, France; 2AgroParisTech, UMR Micalis,
Jouy-en-Josas, France; 3Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental
and Life Sciences, Chełmońskiego 37/41, 51-630 Wroclaw, Poland
1
[email protected]
Yarrowia lipolytica is a non-conventional oleaginous yeast model for fundamental and applied studies
on lipid metabolism. Currently, the most popular oleaginous yeast species for lipid production are
Lipomyces starkeyi, Rhodosporidium toruloides and Yarrowia lipolytica. Among these species, Y.
lipolytica is the only yeast for whom a large outfit of tools is available: a well-curated genome,
efficient genetic tools, a recent lipid metabolism model of fatty acid transport and activation, and
several genome scale models.
Key genes involved in lipid synthesis and remobilization as well as in sugar utilization and central
metabolic pathways have been characterized. A review on these key genes will be presented, including
genes involved in glycerol-3-P synthesis (GUT2, GPD1), genes governing citric acid and fatty
acid synthesis (ACL1/ACL2, PHD1, ACC1), as well as the ones responsible for TAG synthesis and
remobilization e.g. the diacyl-glycerol:acyl-transferases (DGA1, DGA2, LRO1) and the triglyceride
lipases (TGL3, TGL4), and genes involved in fatty acid transport and activation (FAA1, FAT1, PXA1,
PXA2, ANT1, …).
Production of biofuels using microorganisms is the most promising alternative to petroleum-based
chemistry. Several groups have focused on both, the genetic improvement of the Y. lipolytica ability
to accumulate high amounts of biolipids in the form of triacylglycerols (TAG) and on the modification
of its fatty acid profile. Recent studies revealed the important roles of the glucokinase (GLK1,
YALI0E15488g) and hexokinase (HXK1, YALI0B22308g), key enzymes in central metabolism for
improving lipid production on glucose and fructose media (Lazar et al 2014).
Thereby Y. lipolytica emerge as an efficient host for the production of usual and unusual lipids.
Thus, understanding its fatty acid transport and activation mechanisms is essential. We found that Y.
lipolytica has homologous genes involved in fatty acid transport and activation similar to those of S.
cerevisiae (FAA1, FAT1, PXA1, PXA2, ANT1 …). However, our current model for fatty acid transport
and activation differs significantly from the one of S cerevisiae (Dulermo et al 2015).
KEYWORDS: Yeast, Metabolic engineering, Lipid production,
Biolipid, Lipid metabolism
REFERENCES:
Lazar Z, Dulermo T, Neuvéglise C, Crutz Le Coq AM, Nicaud JM, (2014). Hexokinase—A
limiting factor in lipid production from fructose in Yarrowia lipolytica, Metabolic
Engineering 26:89-99
Dulermo R, Gamboa-Melendez H, Ledesma R, Thevenieau F, Nicaud JM (2015). Unraveling fatty acid transport and
activation mechanisms in Yarrowia lipolytica. BBA Molecular and Cell Biology of Lipids (in press)
71
Lipid production from lignocellulose by oleaginous yeasts for biodiesel and
animal feed
Volkmar Passoth 1, Jule Brandenburg2, Johanna Blomqvist2, Hanna Karlsson3, Jana Pickova4
Department of Microbiology, 2Department of Biotechnology and Biochemistry, 3Department of Energy and
Technology, 4Department of Food Science, Swedish University of Agricultural Sciences (SLU), Sweden
1
[email protected]
Biodiesel is currently generated from first generation resources- oil plants, including soya, oil palms
or rape, which can also be used as food. Moreover, their energy yield per covered area is low and
cutting of rain forest area for oil plant production has been reported. In contrast, oleaginous yeasts can
accumulate lipids to more than 50% of their biomass, and their fermentation does not compete with
the utilisation of arable land.
A variety of oleaginous yeast strains were screened for lipid production from lignocellulose substrate,
extraction and analyses methods were established and fermentation protocols for lipid production
from lignocellulose hydrolysates optimised. A general energy balance of lipid production was also
calculated and compared to other scenarios.
A number of ascomycetous and basidiomycetous yeasts were identified as promising to convert
lignocellulose hydrolysate to lipids. In fed-batch fermentations 7 g lipids per l and more were
reached. Ascomycetous yeasts e.g. Lipomyces starkeyi showed a better capacity to produce lipids
from xylose than basidiomycetes e.g. Rhodotorula glutinis. On the other hand, R. glutinis generated
more unsaturated fatty acids (10% and more of linolenic acid- C18:3). Analysing the general energy
balance indicated that lipid production can have a comparable or in some scenarios even better energy
output than ethanol production depending on conversion efficiencies and co-product substitution.
KEYWORDS: Lignocellulose, Biofuels, Biodiesel, Lipids, Oleaginous yeasts
72
Transcription factors involved in regulation of methanol-inducible gene
expression in the methylotrophic yeast
Hiroya Yurimoto, Saori Oda, Zhai Zhenyu, Yu Sasano, Yasuyoshi Sakai
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan
[email protected]
Methylotrophic yeasts, that can utilize methanol as the sole source of carbon and energy, have been
studied intensively in terms of both physiological activities and potential applications. During growth
on methanol, the enzymes involved in methanol metabolism are massively produced in these yeasts,
indicating that the gene promoters of these enzymes are strong methanol-inducible promoters. Using
these promoters, high-level heterologous gene expression systems have been developed in several
methylotrophic yeast strains, such as Pichia pastoris, Hansenula polymorpha, and Candida boidinii.
To achieve efficient industrial use of methanol and efficient protein production by methylotrophic
yeasts, it is important to elucidate the molecular basis of methanol-inducible gene expression in these
yeasts.
Methanol-inducible gene expression is assumed to be controlled under three distinct modes of
regulation: glucose repression, glucose derepression and methanol-specific induction (Yurimoto
2009; 2011). In preceding studies, we identified several transcription factors responsible for methanolinducible gene expression in C. boidinii, and revealed the specific function of each transcription
factor, i.e., CbMig1p in glucose repression, CbTrm2p in glucose derepression, and CbTrm1p in
methanol-specific induction, respectively (Sasano et al 2008; 2010; Zhai et al 2012). Recently, we
identified three genes, CbHAP2, CbHAP3 and CbHAP5, encoding homologs of three components of
the Hap complex in C. boidinii (Oda et al 2015). Unexpectedly, CbHap2p, CbHap3p, and CbHap5p
were found to be responsible for methanol-specific induction rather than for glucose derepression.
A proposed molecular mechanism for transcriptional regulation of methanol-inducible gene will be
discussed.
KEYWORDS: Methanol, Methylotrophic yeast, Gene expression, Transcription factor
REFERENCES:
Yurimoto H (2009). Molecular basis of methanol-inducible gene expression and its application in the methylotrophic
yeast Candida boidinii. Bioscence,Biotechnology and Biochemistry 73:793-800
Yurimoto H, Oku M, Sakai Y (2011). Yeast methylotrophy: metabolism, gene regulation and peroxisome homeostasis.
International Journal of Microbiology 2011:101298
Sasano Y, Yurimoto H, Yanaka M, Sakai Y (2008). Trm1p, a Zn(II)2Cys6-type transcription factor, is a master regulator
of methanol-specific gene activation in the methylotrophic yeast Candida boidinii. Eukaryotic Cell 7:527-536
Sasano Y, Yurimoto H, Kuriyama M, Sakai Y (2010). Trm2p-dependent derepression is essential for methanol-specific
gene activation in the methylotrophic yeast Candida boidinii. FEMS Yeast Research 10:535-544
Zhai Z, Yurimoto H, Sakai Y (2012). Molecular characterization of the Candida boidinii MIG1 and its role in regulation
of methanol-inducible gene expression. Yeast 29:293-301
Oda S, Yurimoto H, Nitta N, Sasano Y, Sakai Y (2015). Molecular characterization of Hap complex components
responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. Eukaryotic Cell
14:278-285
73
74
Session 6
Yeasts Culture Collections
75
Session 6: Yeast Culture Collections - Key note
The importance of the Budapest Treaty and IDAs for the protection of
biotechnological inventions
Ewald Glantschnig
World Intellectual Property Organization, 34, chemin des Colombettes, CH-1211 Geneva 20, Switzerland
[email protected]
The main feature of the Treaty (http://www.wipo.int/budapest) is that a Contracting State which allows
or requires the deposit of microorganisms for the purposes of patent procedure must recognize, for such
purposes, the deposit of a microorganism with any “international depositary authority”, irrespective of
whether such authority is on or outside the territory of the said State. Disclosure of the invention is a
requirement for the grant of patents. Normally, an invention is disclosed by means of a written description.
Where an invention involves a microorganism or the use of a microorganism, disclosure is not possible
in writing but can only be effected by the deposit, with a specialized institution, of a sample of the
microorganism. In practice, the term “microorganism” is interpreted in a broad sense, covering biological
material the deposit of which is necessary for the purposes of disclosure, in particular regarding inventions
relating to the food and pharmaceutical fields. It is in order to eliminate the need to deposit in each
country in which protection is sought, that the Treaty provides that the deposit of a microorganism with
any “international depositary authority” suffices for the purposes of patent procedure before the national
patent offices of all of the Contracting States and before any regional patent office (if such a regional
office declares that it recognizes the effects of the Treaty).
What the Treaty calls an “international depositary authority” is a scientific institution - typically a “culture
collection” - which is capable of storing microorganisms. Such an institution acquires the status of
“international depositary authority” through the furnishing by the Contracting State in the territory of
which it is located of assurances to the Director General of WIPO to the effect that the said institution
complies and will continue to comply with certain requirements of the Treaty. On June 1, 2015, there were
44 such authorities: seven in the United Kingdom, four in the Republic of Korea, three in Italy, Russia
and the United States of America, two each in Australia, China, India, Japan, Poland and Spain, and one
each in Belgium, Bulgaria, Canada, Chile, the Czech Republic, Finland, France, Germany, Hungary,
Latvia, the Netherlands and Slovakia. The Treaty makes the patent system of the contracting State more
attractive because it is primarily advantageous to the depositor if he is an applicant for patents in several
contracting States; the deposit of a microorganism under the procedures provided for in the Treaty will
reduce his costs and increase his security. It will reduce his costs because, instead of depositing the
microorganism in each and every Contracting State in which he files a patent application referring to
that microorganism, he will deposit it only once, with one depositary authority. The Treaty increases the
security of the depositor because it establishes a uniform system of deposit, recognition and furnishing of
samples of microorganisms. The Budapest Treaty was concluded in 1977 and entered into force on August
19, 1980.
KEYWORDS: Budapest Treaty, Budapest Union, International depositary authority
REFERENCES:
Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent
Procedure World Intellectual Property Organization, Geneva. http://www.wipo.int/budapest
76
Session 6: Yeast Culture Collections
Yeasts of yesterday and today, preserved for discoveries of tomorrow
Kyria Boundy-Mills
Phaff Yeast Culture Collection, Food Science and Technology, University of California, Davis CA 95616, USA
[email protected]
Thousands of yeast strains were isolated by Herman Phaff from the 1940s through 1990s, and are
preserved in the Phaff Yeast Culture Collection at the University of California Davis. They continue
to be used at UC Davis and by researchers around the world, thanks in part to a recent award from the
US National Science Foundation to validate species ID, and for remote cryopreservation. Valuable
characterization data will be made available to users of the collection via a new database and website,
using BioloMICS. Deposit of ribosomal sequences in GenBank will make the strains more useful and
visible to researchers globally.
Phaff isolated yeasts from around the world for his foundational studies of yeast ecology and taxonomy.
The yeasts continue to be utilized by researchers around the world in innovative ways. Recent uses
include major multi-institutional projects such as the Thousand Fungal Genomes project and biofuels
research at government agency research labs, as well as smaller projects on ecology, comparative
genomics, taxonomy, yeast-insect associations, food fermentations, and food spoilage.
The yeasts are also used for research at UC Davis. Access to the fourth largest yeast collection in
the world allows research approaches that are possible in very few labs. The advantage of screening
numerous strains to identify those with valuable combinations of characteristics has been demonstrated
repeatedly. Recent publications from our lab describe screening of large numbers of Phaff collection
strains, from 39 to 180 yeast strains, and have resulted in discovery of yeasts able to utilize specific
carbon sources, tolerate inhibitors (Sitepu et al 2014a), or tolerate ionic liquids (Sitepu et al 2014b).
Of the 70 known oleaginous (high lipid) yeast species, 17 were discovered in the last 3 years at
UC Davis, using Phaff collection yeasts (Sitepu et al 2014c). A particularly exciting development
is discovery of yeasts that can synthesize and secrete sophorolipids, natural biosurfactants that have
industrial value.
These exciting discoveries demonstrate the importance of preserving yeasts in pubic repositories, and
the importance of supporting those repositories to ensure that innovative discoveries continue.
KEYWORDS: Culture Collections, Sophorolipids, Oleaginous yeasts
REFERENCES:
Sitepu I, Selby T, Lin T, Zhu S, Boundy-Mills K (2014a). Carbon source utilization and inhibitor tolerance of 45
oleaginous yeast species. Journal of Industrial Microbiology and Biotechnology. 41:1061-1070
Sitepu, I, Shi S, Simmons BS, Singer SW, Boundy-Mills K, Simmons CW (2014b). Yeast tolerance to the ionic liquid
1-ethyl-3-methylimidazolium acetate. FEMS Yeast Research 14(8):1286-1294
Sitepu, I, Geray LA, Sestric R, Levin D, Block DE, German JB, Boundy-Mills KL (2014c). Oleaginous yeasts for
biodiesel: Current and future trends in biology and production. Journal of Biotechnology Advances 32(7):1336-1360
77
Session 6: Yeasts Culture Collections
Yeasts Culture Collections: an interface to transform sleeping beauties in real
opportunities
Benedetta Turchetti, Ciro Sannino, Simone Di Mauro, Sara Filippucci, Pietro Buzzini
Department of Agricultural, Food and Environmental Science & Industrial Yeasts Collection DBVPG (www.dbvpg.
unipg.it), University of Perugia, Italy
[email protected]
The strategic importance to conserve microbial diversity in Culture Collections has been internationally
recognized in innumerable researches. The spin-off of these studies, particularly those investigating
metabolic expression, is the discovery and exploitation of useful properties of the microorganisms
(including yeasts) conserved ex situ. This can be realized by performing large-scale screening surveys
for the selection of strains expressing metabolic features of commercial interest. Yeasts are too
frequently exemplified by the model species Saccharomyces cerevisiae, even though this domesticated
microorganism represents only a fragment of the vast biodiversity and biotechnological potential
occurring into the yeast world. The exploration and ex-situ conservation of yeast biodiversity is an
important contribution towards the selection of strains exhibiting specific phenotypes. Accordingly,
some thousands of yeast cultures have been screened in the last decades based on their ability to give
satisfactory biotechnological performances (Wolf et al 2003; Buzzini & Vaughan-Martini 2006).
Nevertheless, a significant number of yeast strains cited in worldwide research articles are not so far
deposited in Culture Collections. This represents a severe limitation for their possible utilization for
further studies by other researchers or for their potential exploitation by third parties. This limitation
reduces enormously the possibility to scale-up scientific results from the laboratory to the industrial
scale (Boundy-Mills 2012; Stackebrandt et al 2014). Therefore, the risk that a consistent part of yeast
diversity remains in their laboratories in the role of “hidden treasury” for years, often for decades,
sometimes forever, is undoubtedly very high. In this context, Culture Collections could implement
their role of “interface” between research laboratories worldwide and industries transforming
“sleeping beauties” in real opportunities.
KEYWORDS: Yeast Culture Collections, Yeast biodiversity, ex-situ conservation
REFERENCES:
Boundy-Mills K (2012). Yeast culture collections of the world: meeting the needs of industrial researchers.
Biotecnology Journal of Industrial Microbiology 39: 673-680
Buzzini P, Vaughan-Martini A (2006). Yeast Biodiversity and Biotechnology. In: Rosa CA, Peter G (eds) Biodiversity
and Ecophysiology of Yeasts, Springer, Berlin, pp. 533-559
Stackebrandt E, Smith D, Casaregola S, Varese GC, Verkleij G, Lima N, Bridge P (2014). Deposit of microbial strains
in public service collections as part of the publication process to underpin good practice in science. SpringerPlus
3:208
Wolf K, Breunig K, Barth G (2003). Non Conventional Yeasts in Genetics, Biochemistry and Biotechnology, Springer:
Berlin
78
Yeast collecting for fun and fortune
Ian Roberts1, Adam Elliston2, Gwenaelle LeGall3, Darren Heavens4, Steve James1, Jo Dicks1 ,
Keith Waldron2
National Collection of Yeast Cultures; 2Biorefinery Centre; 3Analytical Sciences Unit Institute of Food Research,
Norwich Research Park, Norwich NR4 7UA, UK; 4The Genome Analysis Centre, Norwich Research Park, Norwich
NR4 7UH, UK
1
[email protected]
Growing concerns about climate change, energy and food security, and the rising human population
have accelerated the call for economic growth without environmental damage. In response to the
need to reduce fossil fuel usage there is an increasing demand for production of alternative fuels and
chemicals from renewable sources.
Yeast has shown considerable potential as a production vehicle for such compounds. Collecting
yeast from different habitats is fun. Mining their genomes computationally generates fascinating new
knowledge of species diversity and its origins. Linked to phenotypic screening it facilitates discovery
of surprising biological novelty which can lead to commercial innovations of high market value.
In this presentation we will (i) report progress on a project to genome sequence all 4,000 strains
assembled by the NCYC yeast collection since its origins in 1948, (ii) describe a high-throughput NMR
screen which detects approximately 50 yeast metabolites relevant to agri-food waste exploitation in
an experimental biorefinery setting, and (iii) discuss ongoing computational tool development for
exploratory mining of these data.
We will also speculate on the changing role of yeast culture collections in an era of synthetic biology
and growing opportunities in the area of industrial biotechnology and bioenergy.
79
Yeast biodiversity in Culture Collections: old sources and new challenges
Andrey Yurkov1, José P. Sampaio2
Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany;
PYCC -Portuguese Yeast Culture Collection, UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e
Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal
1
2
[email protected]
Environmental studies established the foundation of our knowledge of the microbial biodiversity.
Furthermore, cultures originating from these assays remain often the only source of microbial resources
available to the society through open culture collections. Collections are expected to (a) provide safe
preservation of cultures, (b) ensure identity of microbial resources, (c) enable easy distribution of
strains, (d) offer isolates of different origin and (e) perform research. These tasks represent also the
major challenges for a collection of microorganisms, including yeast culture collections.
(a) Since yeasts are phylogenetically heterogeneous, a combination of different preservation
techniques should be used. (b) Methods for yeast taxonomy and identification have rapidly evolved
in the last decades, and consequently yeast classification and nomenclature had to be updated. Among
methods facilitating routine identification and quality control in collections, rDNA sequencing and
MALDI-TOF are the most promising tools. (c) Unlike plants and animals microorganisms require
labor-intensive handling. Therefore, yeasts are distributed mainly through public repositories in
accordance with the Convention on Biological Diversity and the recently ratified by EU Nagoya
protocol. (d) Recent changes to data availability polices driven through major microbiological journals
force, among other requirements, free access to cultures through a national culture collection. Despite
limited funding, culture collections may still suit well for biodiversity assessments of different scales,
however they were not conceived to cover large population studies involving tens or hundreds of
strains of the same species. (e) Apart from performing pure taxonomic studies, culture collections can
be successfully linked to scientific projects to provide necessary taxonomic expertise to the scientific
community. With a few ongoing projects we exemplify how biodiversity assessments enlarge our
knowledge on yeast diversity.
This work was partly supported by Fundação para a Ciência e a Tecnologia (Portugal), projects PTDC/
BIA- MIC/113051/2009, PTDC/BIA-BIC/4585/2012.
KEYWORDS: Culture Collections, Biodiversity, Identification, Preservation, Conservation
80
POSTERS ABSTRACTS
81
82
Session 1A
ecology and taxonomy
83
Diversity of cultivable yeast in sugarcane phyllosphere
in Thailand, a tropical country
Savitree Limtong1,2, Janjira Surussawadee1, Nantana Srisuk1,2
1
Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand and Center for
Advanced Studies in Tropical Natural Resources; 2 National Research University-Kasetsart University, Bangkok 10900,
Thailand
[email protected]
The phyllosphere, which is usually referred to the external surface of plant leaf, has been recognized
as an important habitat for epiphytic microorganisms that are capable of surviving, growing and
reproducing in it (Fonseca and Inacio 2006). This study aimed to investigate the diversity of
cultivable yeast in the phyllosphere of sugarcane in Thailand. Yeasts were isolated by plating of
leaf washing from 84 leaf samples and 262 strains that revealed yeast type colonies were collected.
On the basis of sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene or the
D1/D2 and internal transcribed space (ITS) regions, 168 strains were found to be yeasts while 94
strains were fungi. Among yeasts strains, 83 strains were identified to be 24 known species in 11
genera of Basidiomycota viz. Bullera sinensis, Cryptococcus flavescens, C. flavus, C. heveanensis,
C. rajasthanensis, Dioszegia zsoltii, Hannaella pagnoccae, Occutifur externus, Pseudozyma
alboarmeniaca, P. aphidis, P. hubeiensis, P. jejuensis, P. rugulosa, P. siamensis, P. vetiver, Rhodotorula
benthica, R. mucilaginosa, R. taiwanensis, Rhodosporidium paludigenum, Sporobolomyces blumeae,
S. carnicolor, S. vermiculatus, Jaminaea angkoriensis and Tremella globispora, and four species
in three genera of Ascomycota viz. and Candida parapsilosis, C. nymphaea, Kodamaea ohmeri and
Meyerozyma caribbica. As many as 85 strains represented new or may be new yeast species. Seventeen
strains were confirmed to be 11 new species in seven genera viz. Cryptococcus (2 species), Hannaella
(2 species), Occultifur (1 species), Papiliotrema (1 species), Pseudozyma (2 species), Rhodotorula
(1 species), Tremealla (1 species) and Wickerhamilla (1 species). Sixty-eight strains were possible
to be new yeast species in the phylum Basidiomycota, however, further analysis is needed. Number
of basidiomycetous yeast strains and species were found to be higher than that of ascomycetous
strains and species. The most prevalent species among known species was M. caribbica with a 22.9%
frequency of occurrence.
KEYWORDS: Yeast, Phyllosphere, Sugarcane, Thailand, Meyerozyma caribbica
REFERENCES:
Fonseca A, Inacio J (2006.) Phylloplane yeasts. In: Rosa C, Peter G (eds) Biodiversity and ecophysiology of yeasts.
Springer-Verlag, Berlin Heidelberg 263–301
Table 1 Strains confirmed to be new yeast species in phylum Basidiomycota
Strain
DMKU-SP67
DMKU-SP105
DMKU-SP423
DMKU-SP85
DMKU-SP56
DMKU-SP385
DMKU-SP71
DMKU-SP76
84
Closest Species
(GenBank Accession number)
Cryptococcus laurentii (AF075469)
Cryptococcus nemorosus (AF472625)
Hannaella coprosmaensis (AF363660)
Occultifur externus (AF131062)
Pseudozyma churashimaensis (AB548955)
Pseudozyma rugulosa (AJ235300)
% nt
substitution
D1/D2
ITS
2.1
1.7
1.3
4.6
2.2
3.5
1.2
2.5
5.5
0.8
1.7
3.4
1.5
0.5
1.2
0.6
Strain
Closest Species with
(GenBank Accession number)
DMKU-SP427
DMKU-SP200
DMKU-SP213
DMKU-SP273
DMKU-SP322
DMKU-SP332
DMKU-SP23
DMKU-SP40
Rhodotorula calyptogenae (AB025996)
Rhodotorula marina (AF189944)
Tremella globispora (AF189869)
DMKU-SP403
% nt substitution
D1/D2
0.4
1.1
0.9
1.3
1.1
0.9
1.8
1.7
ITS
5.6
4.3
3.5
3.2
3.5
3.5
3.3
3.5
1.3
3.4
Yeast culturable diversity in the soils of the Urmia Lake National Park, Iran
Lachin Mokhtarnejad1, Mahdi Arzanlou1, Asadollah Babai-Ahari1, Pietro Buzzini2, Simone Di
Mauro2, Benedetta Turchetti2
Department of plant protection, Faculty of agriculture, University of Tabriz, Tabriz, Iran; 2Department of Agricultural,
Environmental and Food Science & Industrial Yeasts Collection DBVPG, University of Perugia, Perugia, Italy
1
[email protected]
The National Park of Urmia Lake (Iran) is a protected area, which represents a unique ecosystem
owing its special ecological conditions. The present study reports the identification of halotolerant
yeast diversity in the hypersaline soils of the Urmia Lake National Park. Soil samples were collected
from eight sites in Urmia lake basin and six islands insides the lake and isolations were subsequently
made by using Dichloran Rose Bengal agar medium. A total number of 141 yeast strains were
isolated from sampled areas. Yeast strains were identified by sequencing the D1/D2 domains of the
26S rDNA gene and the ITS region and comparing the sequences obtained in GenBank database
(BLASTN freeware from www.ncbi.nlm.nih.gov/BLAST).
The strains isolated belonged to species of the genera Candida, Cryptococcus, Debaryomyces,
Holtermanniella, Metschnikowia, Meyerozyma, Rhodosporidium, Rhodotorula, Trichosporon and
Torulaspora. In addition, seven strains which presumably represent a new species were also isolated.
The genus Cryptococcus represented the dominant genus with an isolation frequency of 84%; in
particular Cryptococcus aerius were isolated from almost all of the sampling sites. The ability of
growing at high concentration of NaCl (10% and 15%) was checked for all of the isolates; the majority
of the strains grew at 10% while 18 isolates could grow in medium with 15% NaCl. All yeast strains
showed a psychrotolerant aptitude and grew at 4°C and 30°C (40°C for a few strains).
KEYWORDS: Yeasts diversity, D1/D2, ITS, Hyper saline soils, Extreme environments
85
Diversity of culture-independent endophytic yeasts from sugarcane leaves in
Thailand
Manee Tantirungkij1, Rujikan Nasanit2, Savitree Limtong3
Central Laboratory and Greenhouse Complex, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University,
Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand; 2Department of Biotechnology, Faculty of Engineering
and Industrial Technology, Silpakorn University, Sanamchandra Palace Campus, Nakhon Pathom 73000, Thailand;
3
Department of Microbiology, Faculty of Science, Kasetsart University, Jatujak, Bangkok 10900, Thailand
1
[email protected]
Endophytic microorganisms inhabit internal plant tissues without causing any symptoms or negative
effects in the host plant. Diversity of endophytes has been evaluated due to their ability to produce
various beneficial bioactive, however, only a few reports have focused on yeast community. Therefore,
in this study culture-independent method was used to investigate the endophytic yeasts associated
with sugarcane leaves in Thailand via semi-nested PCR and amplified rDNA restriction analysis
(ARDRA) techniques. Based on sequence analysis of the D1/D2 domain of the LSU rDNA, the results
indicated that the colonization frequency (CF) and the relative species frequency (RF) of endophytic
yeast phylotypes were 0.52 and 0.21, respectively. The closest related species to the clone sequences
determined by BLAST search showed eight (77.2% RF) and nine (22.8% RF) phylotypes belonged
to the Ascomycota and Basidiomycota, respectively. The phylotypes were designated as three known
species (Candida palmioleophila, Debaryomyces hansenii and Kodamaea ohmeri), together with ten
phylotypes closest to D. hansenii, Cryptococcus flavus, Malassezia restricta, Pseudozyma aphidis,
Rhodotorula cassiicola, R. marina and Sporobolomyces vermiculatus. The most prevalent phylotypes
were K. ohmeri (44.2% RF) followed by D. hansanii (20.5% RF) and C. palmioleophila (11.6% RF).
Moreover, the most of novel phylotypes were the phylotypes in the Basidiomycota. Consequently, our
findings suggest that sugarcane leaves are a potential choice for the discovery of novel yeast species.
KEYWORDS: Endophyte, Yeast, Sugarcane, Culture-independent diversity, PCR
86
A highly resolved phylogenetic tree of Trichosporonales species based on
multiple gene sequence analysis
Masako Takashima1, Ri-ichiroh Manabe2, Wataru Iwasaki3, Akira Ohyama4, Moriya
Ohkuma1, Takashi Sugita5
Japan Collection of Microorganisms, RIKEN BioResource Center, Tsukuba, Ibaraki; 2Division of Genomic
Technologies, RIKEN Center for Life Science Technologies, Yokoham; 3Department of Biological Sciences, Graduate
School of Science, the University of Tokyo, Tokyo; 4Planning, in silico biology, inc., Yokohama; 5Department of
Microbiology, Meiji Pharmaceutical University, Tokyo, Japan
1
[email protected]
The order Trichosporonales (Tremellomycotina, Basidiomycota) includes various species that have
clinical, agricultural and biotechnological value. According to “The Yeasts, A Taxonomic Study” 5th
ed., it includes the genera Trichosporon (37 species) and Cryptotrichosporon (1 species), and some
species of highly polyphyletic genera, Bullera (3 of 41 species listed) and Cryptococcus (10 of 70
species listed). Since the type species of the genera Bullera and Cryptococcus were assigned to the
Tremellales, species within these two genera in the Trichosporonales are misleading taxonomically.
Thus, understanding why and how evolutionary diversification occurred within this order is extremely
important. This study was performed to clarify the phylogenetic relationships among Tricosporonales
species, especially those occurring at the basal position of the tree. First, we determined the draft
genomes of selected Trichosporonales species, and selected 30 orthologous genes for construction of
a highly resolved phylogenetic tree from genomic data. The coding regions of genes from T. asahii
and T. faecale were determined using a BLAST search against the respective mRNA data. Multiple
alignment of respective genes was first performed using the CDSs of T. asahii and T. faecale employing
those of C. neoformans as an outgroup, and those of other species were added and aligned based on
codons. The phylogenetic trees were constructed based on each gene and a concatenated alignment.
Resolution of the maximum-likelihood trees estimated from the concatenated dataset based on both
nucleotide and amino acid sequences were greater than in previous reports. Now, our study proposes a
set of genes suitable for constructing a phylogenetic tree with high resolution to examine evolutionary
diversification in Trichosporonales. These can also be used for epidemiological and biogeographical
studies. It also might provide a platform for a comprehensive reclassification of pleomorphic fungi.
87
Diversity and properties of yeasts colonizing fruit trees
Renáta Vadkertiová, Jana Molnárová
Culture Collection of Yeasts, Institute of Chemistry, SAS, 845 38 Bratislava, Slovakia
[email protected]
Yeasts form significant and diverse part of the phyllosphere microbiota. The diversity and density of
yeasts in this environment depend on various factors such as geographical locality, climatic conditions,
season, plant species, and plant organs.
This study focused on the diversity of yeasts and yeast-like organisms associated with matured fruits
and fully opened blossoms of apple (Malus domestica Borkh.), plum (Prunus domestica) and pear
(Pyrus communis) trees. Samples of fruits and blossoms were harvested in three localities of southwest
Slovakia during two consecutive years. The ability to produce extracellular enzymes (proteases, betaglucosidase, lipases and polygalacturoneses) as well as the physiological profile of yeasts associated
with the latter plant organs were also examined.
The occurrence of yeasts and yeast-like organisms associated with fruits was 2.5 times higher than that
in blossom samples. The species Aureobasidium pullulans and Metschnikowia pulcherrima occurred
regularly in blossoms samples, whereas Galactomyces candidus, Hanseniaspora guilliermondii,
Hanseniaspora uvarum, M. pulcherrima, Pichia kluyveri, Pichia kudriavzevii and Saccharomyces
cerevisiae formed a major part of yeast microbiota related to fruit samples. The species Aureobasidium
pullulans showed the largest spectrum of activities of all the species tested. It exhibited proteolytic,
beta-glucosidase, lipolytic and polygalacturonase extracellular enzymatic activities. The majority
of strains tested exhibited beta-glucosidase activity. However, only a small number of yeast strains
produced polygalacturonases and lipases. Yeasts isolated from blossoms assimilated saccharose and
D-xylose more frequently than the fruit yeasts, whereas the fruit yeasts were more osmophilic than
the blossom yeasts.
Some intraspecies and interspecies variations were found among the yeasts exhibiting proteolytic,
beta-glucosidase and lipolytic activities.
This work was supported by a grant VEGA No. 2/0023/14 from the Slovak Grant Agency and Ministry
of Education.
KEYWORDS: Blossoms, Fruits, Enzymatic activities, Physiological properties
88
Ogataea uvarum sp. nov., a new yeast species of the Ogataea clade from italian
grape bunches
Mariana Tristezza1*, Luca Roscini2*, Laura Corte2, Claudia Colabella2, Carla Perrotta3,
Patrizia Rampino3, Gianluigi Cardinali2, Francesco Grieco1
Istituto di Scienze delle Produzioni Alimentari, Consiglio Nazionale delle Ricerche, Unità Operativa di Supporto di
Lecce, Lecce, Italy; 2Dip. di Scienze Farmaceutiche-Microbiologia, Università di Perugia, Perugia, Italy; 3Dip. di
Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Lecce, Italy
*
Both authors equally contributed to this work
1
[email protected]
Indigenous yeasts are present on the surfaces of grapes and their success in surviving and driving
fermentation depends on the sum of various physical, chemical and biotic factors. The analysis of
berry-resident strain diversity and the relationship between genotype and phenotype can be used in
the development and identification of specific non-Saccharomyces strains provided with interesting
technological properties. In this contribution, during a large-scale study on vineyard-associated yeast
strains from Apulia (Southern Italy), we isolated a new yeast species from “Negroamaro” grape
berries.
The morphological and physiological characteristics of the strain were determined by using
conventional methods. The D1/D2 and the ITS domains of rDNA belonging to the isolated strain
were amplified using respectively the primer pairs NL1/NL4 and ITS1/ITS4 and then sequenced.
The generated DNA sequence was submitted to the GenBank and aligned using the BLAST database
search program
On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and
on the basis of the sequence analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene
and the internal transcribed spacer region, the isolated strain was assigned to be a novel species of the
Ogatea genus. The two marker sequence indicated that the strain could not be attributed to any known
species and is described as the type strain of Ogatea uvarum sp.nov.
The evidence produced in this study of a novel species from grape berries may be a consequence
of the fact that these yeasts being carried to healthy and rotten berries by visiting insects. Therefore,
grape berries can represent a promising source for further investigations of yeasts belonging to
different clades including the Ogatea one.
Acknowledgments This research was supported by the Italian MIUR - Project S.I.Mi.S.A. PON02_00186_3417512/1
KEYWORDS: Autochthonous yeast, rDNA, Ogatea clade, Negroamaro
89
Heterogeneity of the genus Barnettozyma: towards reinstatement of
Zygowilliopsis Kudriavzev (1960)
Gennadi I. Naumov1, Elena S. Naumova1, Ching-Fu Lee2
1
Russia; 2Department of Applied Science, National Hsinchu University of Education, 521 Nanda Rd., Hsinchu 30014,
Taiwan
[email protected]
Current classification of ascomycetous yeasts is substantially based on multigene phylogenetic
analysis, including the D1/D2 domain of the 28S rRNA gene sequences (Kurtzman et al 2011).
Despite obvious achievements in the yeast taxonomy, many yeast genera accepted in the last Yeast
Monograph (Kurtzman et al 2011) are still heterogeneous, one of them is the genus Barnettozyma
Kurtzman, Robnett & Basehoar-Powers (2008) with weak bootstrap support of 63%. The genus was
created while revising the yeast genera Pichia, Issatchenkia and Williopsis based on the concatenated
gene sequences of SSU rRNA, LSU rRNA and EF-1a (Kurtzman et al 2008). The genus Barnettozyma
included B. californica (Zygowilliopsis californica), B. hawaiiensis (Pichia hawaiiensis), B.
pratensis (Williopsis pratensis), B. populi (P. populi), B. salicaria (P. salicaria) and B. wickerhamii
(P. wickerhamii). Later, two new species B. vustenii and B. sucrosica have been described (Yurkov et
al 2010, Imanishi et al 2010).
Using D1/D2 26S rDNA sequencing, we have conducted a molecular screening of a big collection of
Barnettozyma strains isolated in Taiwan and other world regions and found three novel taxa: two in
Taiwan and one in North America. Phylogenetic analysis of 18S rDNA, 26S rDBA and EF-1a sequences
of all known Barnettozyma species and three novel taxa clearly revealed that the taxonomic genus
Barnettozyma Kurtzman, Robnett, Basehoar-Powers (2008) is highly heterogeneous and does not
reflect the evolutionary relatedness of its member-species. On the other hand, within the heterogeneous
clade there is a group of closely related species (100% bootstrap support): B. californica, B. populi, B.
vustenii, B. sucrosica, Barnettozyma sp. 1, Barnettozyma sp. 2, Barnettozyma sp. 3 and B. hawaiiensis,
which we refer to as the Zygowilliopsis clade. We propose to reinstate the genus Zygowilliopsis
Kudriavzev (1960) with the type species Z. californica. The member species of this genus have the
same mating type system and can be crossed (Naumov et al 2010), and phylogenetically separate
from B. pratensis, B. salicaria and B. wickerhamii.
KEYWORDS: Zygowilliopsis, Barnettozyma, Phylogeny, Genetic hybridization
REFERENCES:
Kurtzman CP, Robnett CJ, Basehoar-Powers E (2008). Phylogenetic relationships among species of Pichia, Issatchenkia
and Williopsis determined from multigene sequence analysis, and the proposal of Barnettozyma gen. nov., Lindnera
gen. nov. and Wickerhamomyces gen. nov. FEMS Yeast Research 8:939–954
Yurkov A, Schäfer AM, Begerow D (2010). Barnettozyma vustinii A. Yurkov, A.M. Schäfer & Begerow, sp. nov.
Fungal Planet 38:1–2
Imanishi Y, Yamazaki A, Nakase T (2010). A new Barnettozyma species forming hat-shaped ascospores isolated from
soil in Japan. The Journal of General and Applied Microbiology 56: 447–453
Naumov GI, Kondratieva VI, Naumova ES (2009). Taxonomic genetics of Zygowilliopsis yeasts. Russian Journal of
Genetics 45:1422–1427
90
Identification, characterization and enzymes activities of cold-adapted yeasts
from antarctic soil
Katarzyna M. Szulczewska, Joanna Krysiak, Aneta Białkowska, Marianna Turkiewicz
The Institute of Technical Biochemistry, Technical University of Lodz, Poland
[email protected]
Psychrophilic yeasts are diverse group of eukaryotic microorganisms characterized by variety of
nutritional preferences and ability to survive in extreme environments that differ significantly in
geochemical and physical parameters. Hitherto the highest number of cold-adapted yeasts were
isolated from soil, water, ice and snow samples collected in Antarctica. Among over 70 yeast species
were such genus as Candida, Dioszegia, Rhodotorula, Mrakia, Mrakiella, Sporobolomyces and
Cryptococcus.
In this study cold-adapted yeasts were isolated from soil collected in the vicinity of Arctowski
Polish Antarctic Station at King George Island. The isolates were subjected to the physiological and
biochemical characteristics. Taxonomy identification was carried out by sequences D1/D2 domains
of 26S rRNA gene and ITS1-5,8S-ITS2 regions analysis. Furthermore, DNA content in yeast cells
and ploidy were examined by flow cytometry. Ability of psychrotolerant yeasts to produce interesting
for biotechnology psychrozymes as amylases, pectinases, cellulases, xylanases, β‑galactosidases,
phytases, lipases and proteinases was determinated by plate tests.
Molecular analyses based on rDNA sequences revealed that 18 tested strains belong to 9 species from
Debaryomyces, Candida, Cryptococcus and Rhodotorula genera, which grew in temperature range
4-20°C. The most popular was production lipases and proteases. However activity of xylanases and
cellulases was not observed in plate tests. Cr. albidus strain D62 displayed 6 of 8 enzyme activities.
Analysis of DNA content in yeast cell indicated that the majority of isolated have the haploid genome
size in range 11,5-13,5 Mb.
Over 70% of isolated strains were classified to phylum of Basidiomycota, mainly belonging to
Cryptococcus species. It confirms numerous reports on Antarctic soil biodiversity.
KEYWORDS: Cold-adapted yeasts, Antarctic soils, Enzyme activities, Genome size
REFERENCES:
Buzzini P, Branda E, Goretti M, Turchetti B (2012). Psychrophilic yeasts from worlwide glacial habitats: diversity,
adaptation strategies and biotechnological potential. FEMS Microbiology Ecology 82:217-241
Carrasco M, Rozas JM, Barahona S, Alcaino J, Cifuentes V, Baeza M (2012). Diversity and extracellular enzymatic
activities of yeasts isolated from King George Island, the sub-Antarctic region. BMC Microbiology 12:251
91
Species and genetic diversity of yeasts inhabited in mangrove ecosystems in
Taiwan
Sing-Yi Huang1,Yii-Cheng Chou2, Hsiu-Chuan Chou1, Zhenming Chi3, Ching-Fu Lee1
Department of Applied Science, National Hsinchu University of Education, 521 Nanda Rd.,Hsinchu 30014, Taiwan;
2
Department of Medical Laboratory Science and Biotechnology, Chu Hwa University of Medical Technology, 91
Wenhua 1st Street, Zender, Tainan, 717 Taiwan; 3Unesco Chinese Center of Marine Biotechnology, Ocean University of
China, 5 Yushan Road, Qingdao, China
1
[email protected]
Currently, most scientist and ecologist pay highly attention to mangrove ecosystem because of
its rich microbial resources (Chi et al 2012). In this study, the species diversity of soil and plantinhabited yeasts in mangrove system were investigated on wetland in Taiwan. Totally, hundreds of
the yeasts were isolated from 120 samples of soil and leaves of mangroves (Lumnitzera racemosa,
Avicennia marina , Kandelia candel, Rhizophora stylosa ) collected on wetland area in Taiwan. All
the representative colonies with their characteristic morphology were picked on DRBC agar plates
( Dichloran rose Bengal chloramphemicol agar, pH 6.5) or AYM agar ( acidified yeast extract-malt
extract agar, pH 3.5 ) (Kurtzman et al 2011), then conspecific strains possibly derived from a single
clone were eliminated with identical DNA fingerprinting profiles based on RAPD with primers
(GTG)5. The yeast strains isolated were identified based on morphology, physiology, and molecular
characteristics including the sequence analysis of D1/D2 LSU and ITS fragment of ribosomal DNA
(Chen et al 2013). Two hundred and thirty-eight of several yeasts have been identified and were
classified into 28 genus and 67 species based on the traditional and molecular approaches. While,
about approximately 15 species of 67 cannot be classified into currently recognized species, indicating
that these species may be proposed novel taxa, respectively. In this study, Aureobasidium pullulans
complex (Li et al 2013), Meyerozyma guilliermondii, and Cryptococcus sp. were most commonly
isolated species from mangrove leaves. While, Debaryomyces and Kluyveromyces species were
frequently isolated from soil. The results demonstrated the yeast flora showed significant diversity in
mangrove ecosystem, and different yeast species dominate can be found on different substrates.
KEYWORDS: Yeast diversity, Mangrove, Genetic diversity, Phylogeny
REFERENCES:
Chi ZM, Liu TT, Chi Z, Liu GL, Wang ZP (2012). Occurrence and diversity of yeasts in the mangrove ecosystems in
Fujian, Guangdong and Hainan Provinces of China. Indian Journal of Microbiology 52(3):346–353
Chen SF, Lo SF, Chang CF, Lee CF (2013). Tetrapisispora taiwanensis sp. nov., and Tetrapisispora pingtungensis
sp. nov. two ascosporogenous yeast species isolated from soil in Taiwan. International Journal of Systematic and
Evolutionary Microbiology 63:2351–2355
Li Y, Chi Z, Wang GY, Wang ZP, Liu GL, Lee CF, Ma ZC, Chi ZM (2013). Taxonomy of Aureobasidium spp. and
biosynthesis and regulation of their extracellular polymers. Critical Reviews in Microbiology 41(2):228-37
92
The yeast and bacteria in various types of sourdough
Miloslava Kavkova, Marketa Lizalova, Jaroslava Markova
Dairy Research Institute Ltd. Sobeslavka 841, Tabor CZ-39001, Czech Republic
[email protected]
The sourdoughs comprise heterogenic and nutritive environment for biota such as yeast and lactic
acid bacteria. The utilisation of various cereal flours in sourdough is moderated by demand for
well-balanced bread inquired by various nutrition groups of consumers. In present study, five paste
sourdoughs were analysed to obtain information about yeast and bacteria composition. Totally, 16
isolates of seven yeast species and five bacteria strains were isolated, cultured and identified by using
phenotypic and molecular genetic methods.
Microbiota was isolated from commercially produced sourdoughs by using specific cultured media
enriched by selective antibiotics. Identification of yeast strains was based on their morphology and
physiological properties. The cultured yeast were analysed by using molecular method. Phylogenetic
tree was constructed based on sequences of three genes ITS 5.8 S rDNA, 18S rDNA (NS1/NS4) and
(D1/D2 region) 26S rDNA. Lactic acid bacteria were identified based on 16S gene sequencing data.
The type of cereal flour with acidity employed as co-factor influenced species composition of
co-existing lactic acid bacteria and enterococci significantly (R2=0,62, F(6,28) = 10,05, p ≤ 0,001).
The microbial diversity was significantly highest in spelt and wheat sourdoughs corresponding to
Saccharomyces cerevisiae, Kazachstania unispora, Pichia fermentans, Rhotodorula mucilaginosa as
dominant species co-existed with Lactobacillus paralimentarius. Wheat sourdough contained several
bacterial species (Lactobacillus plantarum, Leuconostoc citreum, L. holzapfelii and Enterococcus
faecium). Saccharomyces cerevisiae and Candida humillis was found in rye sourdough without
presence of any bacterial species.
Yeast showed to be dominant organisms of sourdoughs with respect to acidity and flour composition
Saccharomyces cerevisiae and Naumovozyma castellii occurred in the most of sourdoughs except
to spelt sourdough where Kazachstania unispora and Pichia fermentans appeared. Identified yeast
species corresponded to species occurring in sourdough (Di Cagno et al 2014; Vrancken et al 2010).
Rhotodorula mucilaginosa is terrestrial and aquatic species with wide ecological amplitude.
KEY WORDS: Sourdough, Sequencing, Biodiversity, Yeast
REFERENCES:
Di Cagno R, Pontonio E, Buchin S, De Angelis M, Lattanzi A, Valerio F, Gobbetti M, Calasso M (2014). Diversity of
the lactic acid bacteria and yeast microbiota in the Awitch from firm to liquid sourdough fermentation. Applied and
Environmental Ecology 80(10):3161-3172
Vrancken G, Rimaux T, Veckx S, Leroy F, De Vuyst L (2011). Influence of temperature and backslopping time on the
microbiota of a type I propagated laboratory wheat sourdough fermentation. Applied and Environmental Microbiology
77(8):2716-2726
93
A new obligate osmophilic yeast species from bee associated substrates
Neža Čadež1, László Fülöp2, Dénes Dlauchy3, Gábor Péter3
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; 2Department of Chemistry
and Biochemistry, Szent István University, Páter Károly u. 1. H-2103 Gödöllő, Hungary; 3National Collection of
Agricultural and Industrial Microorganisms, Faculty of Food Science, Corvinus University of Budapest, Somlói út 1416. H-1118 Budapest, Hungary
1
[email protected]
Some currently recognized Zygosaccharomyces species are characterized by extreme osmotolerance,
but they are also able to grow with high water activity culture media. Only one obligate osmophilic
yeast species, Candida glucosophila, was treated in the latest edition of The Yeasts, a Taxonomic
Study (Kurtzman et al 2011).
Using an isolation medium containing 50% glucose five obligate osmophilic yeast strains were
recovered from bee bread and honey originating from Hungary. For the phenotypic characterization
of the strains several standard methods were modified to fulfil the requirements of the strains unable to
grow on/in high water activity culture media. The sequence based species delineation and phylogenetic
placement of the strains were achieved by analysis of the sequences for D1/D2 domains of the LSU
rRNA and translation elongation factor-1α (EF-1 α) genes and the ITS regions. Due to intragenomic
sequence variability in ITS regions, the attempt to sequence directly the amplicons obtained with Taq
DNA polymerase enzyme by the primer pair ITS1 and ITS4 was unsuccessful. Therefore, the ITS
sequences were determined following cloning of the amplified PCR products into pGEM-T vector.
The analysis of the sequences of the LSU rRNA gene D1/D2 domain placed the strains in the
Zygosaccharomyces clade and their phenotypic characters matched the diagnosis of genus
Zygosaccharomyces. Sequence comparisons of the D1/D2, ITS and EF-1 α sequences revealed that
the five strains represented a new Zygosaccharomyces species, closely related to Z. gambellarensis.
Zygosaccharomyces favi sp. nov. (type strain: NCAIM Y.01994T) was proposed for this new yeast
species (Čadež et al 2015). Three to eight different ITS copies were detected in each strain. However,
the majority of the intragenomic ITS variability was restricted to the long homopolymer regions of
the sequences. The secondary structure of the ITS region was predicted as well.
KEYWORDS: New yeast species, Obligate osmophilic yeast, Zygosaccharomyces favi, Intragenomic
DNA, Sequence variability, Honey bee
REFERENCES:
Kurtzman CP, Fell JW, Boekhout T (2011). The Yeasts, a Taxonomic Study. Elsevier, Amsterdam
Čadež N, Fülöp L, Dlauchy D, Péter G (2015). Zygosaccharomyces favi sp. nov., an obligate osmophilic yeast species
from bee bread and honey. Antonie van Leeuwenhoek 107:645–65
94
The migratory birds: novel ecological niche of fungal diversity?
Nicola Francesca1,2, Cláudia Carvalho2, Marco Alexandre Guerreiro2,3, Antonio Alfonzo1,
Raimondo Gaglio1, Luca Settanni1, José Paulo Sampaio2, Giancarlo Moschetti1
Dipartimento Scienze Agrarie e Forestali, Università degli Studi di Palermo, Viale delle Scienze 4, 90128 Palermo,
Italy; 2UCiBio Applied Molecular Biosciences Unit, Departamento de Ciências da Vida, Faculdade de Ciências
e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal; 3Geobotany, Faculty for Biology and
Biotechnology, Ruhr-Universität Bochum, Bochum, Germany
1
[email protected]
Francesca et al (2010) studied the ecology of wine yeasts associated to birds caught in vineyards. The
same authors were able to prove that migratory birds might carry living pro-technological yeasts for
about 12 hours from the ingestion of inoculated feed (Francesca et al 2012). In subsequent studies,
they tried to demonstrate that microorganisms are not only transported for a short period by birds,
but microorganisms might be adapted to the specific conditions (body temperature of about 42 °C
and low pH) of the intestinal tract of birds. Hence, it was demonstrated that the majority of isolates
carried by birds are thermotolerant. The most interesting results were the isolation of two new species
of thermotolerant yeasts, isolated from birds (Francesca et al 2013, 2014). Presently, the main scope
of this work is to investigate an additional number of seven new species of thermotolerant yeasts
isolated from migratory birds.
Bird’s cloacae were analyzed for the presence of yeasts (Francesca et al 2012). All isolates were
subjected to phylogenetic and phenotypic analyses as reported by Francesca et al (2014).
Twenty four cultures belonging to the genera Candida and Aureobasidium were isolated from birds.
The phylogenetic analysis of D1/D2 domain of 26S and ITS region of 5.8S rRNA genes placed the
cultures of Candida and Aureobasidium in new lineages that differed conspicuously from their closest
relatives, C. verbasci and A. pullulans, respectively. For our Candida isolates the phenotypic analyses
showed several discrepancies in assimilation tests between our cultures and C. verbasci, as well as
notable growth up to 42 °C. Thus, additional evidence supporting the hypothesis that migratory birds
represent a novel ecological niche of new species of thermotolerant yeasts gathered.
KEYWORDS: Novel species, Thermotolerant yeasts, Phylogenetic analysis, Migratory birds
REFERENCES:
Francesca N, Chiurazzi M, Romano R, Settanni L, Moschetti G (2010). Indigenous yeast communities in the
environment of “Rovello bianco” grape variety and their use in commercial white wine fermentation. World Journal of
Microbiology and Biotechnology 26:337–351
Francesca N, Canale DE, Settanni L, Moschetti G (2012). Dissemination of wine related yeasts by migratory birds.
Environmental Microbiology Rep 4:105–112
Francesca N, Carvalho C, Miguel Almeida P, Sannino C, Settanni L, Sampaio JP, Moschetti G (2013).
Wickerhamomyces sylviae f.a., sp. nov., an ascomycetous yeast species isolated from migratory birds in Sicily, Italy.
International Journal of Systematic and Evolutionary Microbiology 63:4824–4830
Francesca N, Carvalho C, Sannino C, Guerreiro MA, Almeida PM, Settanni L, Massa B, Sampaio JP, Moschetti
G (2014). Yeasts vectored by migratory birds collected in the Mediterranean island of Ustica and description of
Phaffomyces usticensis f.a. sp. nov., a new species related to the cactus ecoclade. FEMS Yeast Research 6:910–921
95
To be an isolate or a strain: this is the question
Claudia Colabella1, Luca Roscini1, Matteo Tiecco1, Laura Corte1, Duong Vu3, Wieland Meyer4,
Vincent Robert3, Gianluigi Cardinali1,2
Department of Pharmaceutical Sciences; 2CEMIN– University of Perugia- Perugia Italy Via Borgo XX Giugno, 74
– Perugia – ITALY;3 Centraalbureau voor Schimmelcultures CBS-KNAW, The Netherlands,4 Westmead Millennium
Institute for Medical Research-University of Sidney –NSW, Australia
1
[email protected]
One of the basic questions in microbiology is how to tell that an isolate is an independent strain or just
a replica of other known strains. The introduction of modern molecular and spectroscopic techniques
promises a huge increase in the “taxonomic resolution”. At the same time a theoretical question raises:
how different must two strains be to be considered different? “Dereplication” is the complex of
analytical and interpretative steps deployed to assess the difference between isolates and to determine
which group of identical isolates represents a strain. On the other hand, an effective dereplication
discriminates between strains considered identical. The efficacy of dereplication depends on the
variability, on the independence of the employed markers and on their processing with bioinformatics
tools. The assessment of the statistical probability of identity between two strains description and
the development of a high-throughput analytical pipelines are necessary for effective dereplication.
The aim of this work was to improve the ability of the ITS barcode to discriminate between Candida
isolates and strains, isolated in several wards of two different Italian Hospitals. This approach was
taken in order to investigate on the variability within cluster of isolates with identical ITS sequences.
The internal heterogeneity of these clusters was investigated by FT-IR spectroscopy and with a new
potential barcode.
The results showed that the isolates sharing the same ITS sequence could be separated with different
degrees of statistical significance, giving the opportunity to medical microbiologists to track really
identical isolates within the same ward and hospital, in order to assess their origin and spread-out.
These findings point out the importance of the simultaneous use of molecular biology, spectroscopy
and statistical analysis to define if a new isolate is just a replica of a known strain or a new strain.
96
Session 1A: Yeasts in the environment: Ecology and Taxonomy
Yeast diversity of sugar cane plants and organically managed soil and
persistance of soil killer yeasts in soil and roots of growing corn plants.
Jose R. de Assis Ribeiro, Anderson Cabral, Leda Cristina Mendonça-Hagler,
Allen N. Hagler
U. Fed. Rio de Janeiro, Brazil
[email protected]
A protocol was developed for isolating yeasts from samples with many mold propagules in order to
survey populations on rhizoplane, phyloplane, and in bulk soil of an organically cultivated sugar cane
field. The best results for yeast isolations from agricultural soil were from spread plates using YM agar
or PDA with chloramphenicol and incubation at low temperature to slow growth. Over 700 cultures
were screened with differential media reducing the redundant isolates. Different yeast community
structures were found for bulk soil, root and leaf surface. Prevalent in soil were Torulaspora globosa,
Pichia caribbica and Cryptococcus podzolicus. Aureobasideum pullulans and basideomicetes yeasts
of Cryptococcus spp., Rhodotorula marina and Pseudozyma spp., with P. aff. pruni and P. jejuensis,
were prevalent on leaves. On roots ascomicetic yeasts shared prevalence, especially P. caribbica and
Candida aff. azyma, with basidiomicetes species Cryptococcus laurentii and C. podzolicus. During
the rainy season the ascomicetous yeast guild increased by several fermentative species, indicating
a rhizosphere effect. Over 700 cultures including 24 ascomycetes and 45 basidiomycetes species
were isolated from sugar cane and associated soil. More than 40 % of species isolated had D1/D2
rDNA sequences with less than 99 % of similarity to the type cultures of known species. Two broad
spectrum killer yeasts from soil, Williopsis saturnus (IMUFRJ/51938) and a Candida yuanshanicus
(IMUFRJ/51934), were inoculated separately on maize seeds and planted in pots with soil from
organically managed farm. After 100 days of cultivation in a greenhouse, the inoculated yeasts were
prevalent, in rhizosphere and roots (endophytic), compared to an uninoculated control. Cryptococcus
flavescens from roots, and Candida maltosa, Cryptococcus laurentii e Torulaspora globose from
rhizosphere were also isolated from the soil-maize microcosms using low nutrient level medium.
97
98
Session 1B
physiology and stress response
99
A new antioxidant role for sterols: the case of ergosterol
Sébastien Dupont, Céline Lafarge, Cécile Jouffrey, Philippe Cayot, Patrick Gervais,
Laurent Beney
UMR Procédés Alimentaires et Microbiologiques, Université de Bourgogne/AgroSup Dijon, Dijon, France
[email protected]
Sterols are one of the most abundant plasma membrane constituents of eukaryotic cells. In
mammalian cells, the major sterol present in the plasma membrane is cholesterol, whereas ergosterol
and phytosterol predominate in fungi and plant cells, respectively. Through their interactions with
phospholipids and sphingolipids, sterols confer important properties on the plasma membrane, and
they play an essential role in the stability of membranes by affecting rigidity, fluidity, and permeability.
In a previous work, we observed that ergosterol is important for yeast resistance to dehydration/
rehydration cycles (Dupont et al 2012, 2014). Results suggested that ergosterol could be involved in
the protection of lipids from oxidation. The aim of the present study was to test this hypothesis and to
characterize the effect of ergosterol on lipid oxidation. For that, we examined the effect of different
sterols (zymosterol, cholesterol, and ergosterol) on the kinetic of lipid oxidation induced by freeradical reactions or by production of singlet oxygen. Antioxidant capacity of sterols was assessed
in three systems with different complexity and organization: non-organized matrix (DPPH method),
in liposomes (lipids organized in bilayers), and in whole cells (S. cerevisiae wild-type and mutants
accumulating different kinds of sterols). We observed that sterols display antioxidant property and
allow the protection of phospholipids from oxidative stress. Moreover, we showed that ergosterol was
the best antioxidant in comparison with the other tested sterols. For the first time, this study revealed
that sterols exhibit an antioxidant role in addition to their role of membrane stabilizer and organizer.
KEYWORDS: Ergosterol, Cell resistance, Plasma membrane, Oxidation
REFERENCES:
Dupont S, Lemetais G, Ferreira T, Cayot P, Gervais P, Beney L (2012). Ergosterol biosynthesis: a fungal pathway for
life on land? Evolution 66(9):2961-8
Dupont S, Rapoport A, Gervais P, Beney L (2014). Survival kit of Saccharomyces cerevisiae for anhydrobiosis. Applied
Microbiology and Biotechnology 98(21): 8821-34
100
Regulation of fatty acids biosynthesis and the general cellular fitness in
Kluyveromyces lactis: role of the transcription factor KlMga2
Rosa Santomartino1, Lorenzo De Angelis1, Paola Ballario1, Teresa Rinaldi1, Massimo
Reverberi2, Cristiano Bello2, Alberto Amaretti3, Luca Brambilla4, Michele M. Bianchi1
Dept. Biology and Biotechnology C. Darwin, Sapienza University, Rome, Italy; 2Dept. Environmental Biology
Sapienza University, Rome, Italy; 3Dept. Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 4Dept.
Biotechnology and Biosciences, University of Milano Bicocca, Milano, Italy
1
[email protected]
In K. lactis glucose repression is not the predominant regulation of carbon metabolism. Instead,
regulation of glycolytic and fermentative genes is significantly dependent on oxygen availability in
addition to glucose induction; also lipid biosynthesis is regulated by hypoxia. We have studied in
detail the role of the hypoxic regulatory gene KlMGA2.
Strains were constructed in Rome. Cells were grown in flask or bioreactor under hypoxia and on
plates in anaerobic jar for hypoxia. Transcription analysis was performed by Northern blotting. Lipids
were determined by gas chromatography or photometrically. Cells were visualized by fluorescence an
light microscopy. Respiration was measured with Clark electrode. Catalase was measured by activity.
We have found that the deletion of KlMGA2 causes the loss of the hypoxic induction of some genes,
especially genes involved in lipid biosynthesis, and the reduction of transcription of other metabolic
genes. Also transcriptional response to low temperature was affected in the mutant strain. The mutant
strain also showed reduced growth rate and rag- phenotype, typical of glycolytic/fermentative defects.
This phenotype was suppressed by unsaturated fatty acids (UFAs). The mutant strain also showed
defects in mitochondrial morphology, respiration and catalase expression.
Hypoxic shift in K. lactis generates induction of transcription of many genes. We showed that
the hypoxic regulator KlMga2 has a role in this response. However, KlMga2 is also involved in
mitochondrial/respiratory functions suggesting a general role of this protein in regulating cellular
fitness. The suppression by UFAs of the defects of the mutant strain indicate the importance of
membrane functions in the mechanisms controlled by KlMga2.
This work was partially funded by Ministero degli Affari Esteri e della Cooperazione Internazionale,
Direzione Generale per la Promozione del Sistema Paese.
KEYWORDS: Lipids, Hypoxia , Mitochondria
REFERENCES:
Micolonghi C, Wésolowski-Louvel M, Bianchi MM (2011). The Rag4 glucose sensor is involved in the hypoxic
induction of KlPDC1 gene expression in the yeast Kluyveromyces lactis. Eukaryot Cell 10:146-148
Micolonghi C, Ottaviano D, Di Silvio E, Damato G, Heipieper HJ, Bianchi MM (2012). A dual signaling pathway
for the hypoxic expression of lipid genes, dependent on the glucose sensor Rag4, is revealed by the analysis of the
KlMGA2 gene in Kluyveromyces lactis. Microbiology 158:1734-1744
Ottaviano D, Montanari A, De Angelis L, Santomartino R, Visca A, Brambilla L, Rinaldi T, Bello C, Reverberi M,
Bianchi MM (2015). Unsaturated fatty acids-dependent linkage between respiration and fermentation revealed by
deletion of hypoxic regulatory KlMGA2 gene in the facultative anaerobe-respiratory yeast Kluyveromyces lactis.
FEMS Yeast Research (accepted)
101
The ability of individual Saccharomyces cerevisiae cells to resume proliferation
upon acetic acid stress is determined by their cytosolic pH in the non-stress
condition
Miguel Fernández-Niño1, Maribel Marquina2, Steve Swinnen1, Boris Rodríguez-Porrata2,
Elke Nevoigt1, Joaquín Ariño2
Deparment of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Bremen, Germany; 2Institut de
Biotecnologia i Biomedicina & Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona,
Spain
1
[email protected]
It has been recently shown that individual cells of an isogenic S. cerevisiae population show variability
in acetic acid tolerance, and this variability affects the quantitative manifestation of the trait at the
population level. In the current study, we investigated whether the cell-to-cell variability in acetic
acid tolerance could be explained by the observed differences in the cytosolic pH of individual cells
immediately before exposure to the acid. The cytosolic pH changes of individual S. cerevisiae cells
were recorded using a pH-sensitive GFP protein (i.e. pHluorin). The initial cytosolic pH and the
cytosolic pH drop were recorded by microfluidics-based time-lapse fluorescence microscopy after
shifting the cells from medium without to acetic acid containing medium. For each cell, these
values were then correlated with the individual cell’s ability to proliferate in the presence of the
acid. Data obtained with cells of the strain CEN.PK113-7D in synthetic medium containing 96 mM
acetic acid (pH 4.5) showed a direct correlation between the initial cytosolic pH and the cytosolic
pH drop after exposure to the acid. Moreover, only those cells with a low initial cytosolic pH, which
experienced a less severe drop in cytosolic pH, were able to proliferate after exposure to the acid.
Our results emphasize the relevance of studying weak acid tolerance at the single-cell level instead of
the population level, and could serve as a starting point for developing industrially useful weak acid
tolerant strains.
102
The role of nitrogen for acclimation and yeast fitness for sparkling wine
production
Maria Martí-Raga1,2, Philippe Marullo2,3, Gemma Beltran1, Albert Mas1
Universitat Rovira i Virgili, Fac. Enologia, Tarragona, Spain; 2Université de Bordeaux, ISVV, Villenave d’Ornon,
France; 3Biolaffort, Bordeaux, France
1
[email protected]
Microbial acclimation is used in different biotechnological industries as a means to obtain an inoculum
already adapted to a given stress. For the production of sparkling wine by the traditional method, a
second fermentation inside the bottle is required. Fermenting yeast will face stresses including of high
ethanol concentration, low temperature and high pressure. To succeed in the second fermentation,
yeast cells must previously undergo an acclimation process (pied-de-cuve). In this study, we
investigated the role of the nitrogen composition during this acclimation phase by measuring growth
and fermentative parameters during the whole second fermentation process.
We used eight Saccharomyces cerevisiae strains with different origins to analyze the genetic
background on the efficiency of the acclimation process. The acclimation process was carried out
with different nitrogen sources.
The nitrogen source used in the acclimation media had the strongest impact on yeast growth during
this phase as well as the yeast fermentation kinetics during the second fermentation. A nitrogen source
based on amino acids precursors of fusel alcohols resulted in slow yeast growth during the acclimation
phase but increased yeast viability through the second fermentation. The yeast strain origin has also
a strong effect in particular for the second fermentation kinetics. Overall, we demonstrated how the
nitrogen composition of the acclimation phase affects yeast fitness and viability. The modification of
the nitrogen composition of the acclimation phase is proposed as a tool to optimize yeast performance
during the second fermentation.
KEYWORDS: Saccharomyces cerevisiae, Second fermentation, Viability, Cava
103
Session 1B: Yeast in the environment physiology and stress response
Melatonin effect on oxidative stress in Saccharomyces cerevisiae
Jennifer Vázquez, Beatriz González, Albert Mas, Maria Jesús Torija, Gemma Beltran
Dept. Bioquímica i Biotecnologia, Universitat Rovira i Virgili. C/Marcel·lí Domingo 1, 43007 Tarragona, Spain
[email protected]
Melatonin (N-acetyl-5-methoxytryptamine) can be found in small quantities in wine. This ubiquitous
indolamine is synthesized from tryptophan metabolism via serotonin and exhibits various biological
activities in humans. One of them is its antioxidant effect by which it protects various biomolecules
against damage provoked by free radicals and reactive oxygen species (ROS). Recent studies have
shown that melatonin is formed during alcoholic fermentation, with an unknown role for yeast in
winemaking process. The aim of this study was to evaluate melatonin effect on oxidative stress in
Saccharomyces cerevisiae exposed to different stressing agents, such as plumbagine or hydrogen
peroxide (H2O2). Therefore, different strains of S. cerevisiae were grown in rich medium with
or without melatonin. Cultures in exponential phase were exposed to H2O2, and free intracellular
radicals were detected using dihydrorhodamine 123, and quantified by flow cytometry. In another
experiment, the protection against plumbagine was tested on agar plates, measuring the diameter
of the inhibition. The results showed that low doses of melatonin supplementation (5 µM) causes a
significant reduction in the intracellular content of ROS when cells are exposed to H2O2, with similar
results to those obtained with ascorbic acid. The presence of melatonin also increased the strains
resistance to superoxide anions generated by plumbagine. The reduction of exogenous ROS source
when melatonin was added could indicate its antioxidant properties on yeast, probably acting as a
direct free radical scavenger and stimulating antioxidant enzymes.
KEYWORDS: Yeast, Antioxidant, ROS, Plumbagine, H2O2
104
Changes in the sterol composition of yeast plasma membrane affect membrane potential, salt
tolerance and the activity of multidrug resistance pumps
Marie Kodedov, Hana Sychrová
Department of Membrane Transport, Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic
[email protected]
The impact of the deletions of genes from the final steps in the biosynthesis of ergosterol on the
physiological function of yeast plasma membrane was studied using a combination of biological
tests and the diS-C3(3) fluorescence assay. Most of the erg mutants were more sensitive than the wild
type to salt stress or cationic drugs, their susceptibilities were proportional to the hyperpolarization
of their plasma membranes. The different sterol composition of the plasma membrane played an
important role in the short-term and long-term processes that accompanied the exposure of erg strains
to a hyperosmotic stress (effect on cell size, pH homeostasis and survival of yeasts), as well as in the
resistance of cells to antifungal drugs. The pleiotropic drug-sensitive phenotypes of erg strains were,
to a large extent, a result of the reduced efficiency of the Pdr5 efflux pump, which was shown to be
more sensitive to the sterol content of the plasma membrane than Snq2. In summary, the erg4Δ and
erg6Δ mutants exhibited the most compromised phenotypes. As Erg6 is not involved in the cholesterol
biosynthetic pathway, it may become a target for a new generation of antifungal drugs.
This work was supported by GA CR 15-03708S, TA CR TA04010638 and within the scientific
programme of the project „BIOCEV – Biotechnology and Biomedicine Centre of the Academy
of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), financed by the European Regional
Development Fund.
KEYWORDS: Ergosterol synthesis, Drug tolerance, MDR pumps, Osmotic stgress, Plasmamembrane hyperpolarization
105
Cobalt chloride resistance mechanism in Rhodotorula mucilaginosa
1
Ceren Goral1,2, Sara Landolfo1, Mario Deroma1, Annalisa Coi1, Petek Cakar2,
Ilaria Mannazzu1
Dipartimento di Agraria, Università degli Studi di Sassari, Viale Italia 39, Sassari, Italy; 2Department of Molecular
Biology and Genetics, Istanbul Technical University, Istanbul, Turkey
[email protected]
Cobalt chloride is a transition metal that, at millimolar doses, causes hypoxic stress and generates
a whole spectrum of ROS that can be co-responsible of cobalt toxicity. Cobalt toxicity has been
investigated in several living organisms but at present the understanding of cobalt uptake and
detoxification mechanisms in living organism is still rather incomplete. Here, we describe the
utilization of an evolutionary engineering approach to obtain CoCl2 resistant mutants of the red
yeast Rhodotorula mucilaginosa. The four mutants, selected after thirty-eight cycles of growth in the
presence of increasing concentration of the transition metal, show cross resistance to other metals and
differences in the sensitivity to NaCl, hydrogen peroxide and heat shock, as compared to the parental
strain. Since transition metals have stimulatory effect on carotenogenesis and act as cofactors of
several enzymes involved in biosynthetic pathways, total carotenoid production in term of β-carotene
equivalents were evaluated in the four mutants during growth in the absence and presence of cobalt
chloride. In parallel cobalt chloride absorption was evaluated in the mutants and the parental strains.
Project financially supported by Regione Autonoma della Sardegna (Legge Regionale 7- 2007
Annualità 2010, PI IM).
KEYWORDS: Rhodotorula mucilaginosa, Cobalt chloride, Stress resistance, Carotenoids
106
Session 1B: Yeasts in environment: physiology and stress response
Exoglucanase genes (WaEXG1 and WaEXG2) in Wickerhamomyces anomalus
respond to the nutritional environment and differentially to postharvest
pathogens
1
Lucia Parafati1, Cristina Restuccia1, Gabriella Cirvilleri1, Michael Wisniewski2
Di3A- Dipartimento di Agricoltura, Alimentazione e Ambiente, University of Catania, Italy; 2United States Department
of Agriculture, Agricultural Research Service (USDA-ARS), Kearneysville, WV 25430 USA
[email protected]
Wickerhamomyces anomalus, a yeast that can be used as a postharvest biocontrol agent is known
to produce killer toxins that have been demonstrated to be exoglucanases, coded by the genes
WaEXG1 and WaEXG2 (Mucilli et al 2013). Among a variety of mechanisms, glucanase production
by antagonistic yeast have been reported to play a role in inhibiting other fungi. The aim of the present
study was to examine the expression of these genes using RT-qPCR.
WaEXG1 and WaEXG2 gene expression in W. anomalus was determined when the yeast was grown
in wounds made in oranges either non-inoculated or inoculated with spores of Penicillium digitatum,
or in minimal salt (MS) media supplemented with cell walls of P. digitatum or Botrytis cinerea, and
presented as fold-change relative to the expression of the genes at T0 in NYDB (nutrient broth, yeast
extract, glucose). Expression was quantified by RT-qPCR utilizing gene-specific primers over 48h of
incubation.
Expression of WaEXG1 increased over a 48 h period when the yeast was grown in NYDB, noninoculated and inoculated wounds, while it was stable over 48 h when the yeast was grown on MS
media supplemented with cell walls of P. digitatum or B. cinerea. In contrast, the expression of
WaEXG2 was stable over a 48 h period of incubation when the yeast was grown in NYDB, noninoculated or inoculated wounds, but it increased dramatically over the same period when the yeast
was grown in MS media with P. digitatum cell walls, with the highest level of induction observed at 24
and 48 h. A much smaller induction of WaEXG2 expression occurred in MS media supplemented with
B. cinerea cell walls, although the yeast population decreased dramatically in MS media containing
cell walls of either pathogen.
These results suggest that while WaEXG2 responds to the nutritional environment, it may also be
responsive to the presence of a specific pathogen (P. digitatum).
KEYWORDS: Lytic enzymes, Killer toxins, Tritrophic interactions, Biological control
REFERENCES:
Muccilli S, Wemhoff S, Restuccia C, Meinhardt F (2013). Exoglucanase-encoding genes from three Wickerhamomyces
anomalus killer strains isolated from olive brine. Yeast 30:33-43
107
A FLO1 paralog which yields a NewFlo phenotype
Johan O. Westman1, Jonas Nyman2, Rich Manara2, Valeria Mapelli1, Carl J. Franzén1
Biology and Biological Engineering – Division of Industrial Biotechnology, Chalmers University of Technology,
Gothenburg, Sweden; 2Chemistry, University of Southampton, Southampton, UK
1
[email protected]
Flocculation is often utilised as means of separation of yeast cells from the product in alcoholic
beverage production. Brewery type strains generally start to flocculate towards the end of the
fermentation process, when sugars in the wort are depleted. In Saccharomyces cerevisiae, flocculation
is governed by the FLO gene family, with FLO1 generally being the main contributor to strong, Flo1
phenotype, flocculation.
S. cerevisiae CCUG 53310, isolated from a spent sulphite liquor plant, has high tolerance to
fermentation inhibitors typically present in lignocellulose hydrolysates (Westman et al 2012).
Furthermore, CCUG 53310 flocculates constitutively with a Flo1 phenotype that is only marginally
affected by the presence of high concentrations of mannose (see figure: circles).
Using primers designed for FLO1, we isolated a flocculin gene from the genome of CCUG 53310.
However, constitutive expression of the gene in the otherwise non-flocculating S. cerevisiae CEN.PK
113-7D, resulted in a strain with NewFlo phenotype flocculation, being inhibited by various sugars
(see figure: squares, triangles, diamonds and stars). Nonetheless, the protein was phylogenetically
closely related to Flo1p and by inverse PCR we could also show that the gene is a paralog of
FLO1. Homology modelling of the N-terminal part of the protein structure revealed high structural
similarities to the reported structure of the Flo5p N-terminal domain. Closer examination revealed
differences in certain positions that have been reported to be important for carbohydrate binding
by flocculins. Not previously reported, but of special interest due to its position in a loop flanking
the carbohydrate binding site, was a glutamate residue that in the corresponding position in Flo1, 5
and 9p is a glycine. We hypothesise that this glutamate residue contributes to the observed NewFlo
phenotype flocculation.
KEYWORDS: Flocculation, Cell wall protein, Homology modelling, Bioethanol
REFERENCES:
Westman JO, Taherzadeh M, Franzén CJ (2012). Inhibitor tolerance and flocculation of a yeast strain suitable for 2nd
generation bioethanol production. Electron Journal of Biotechnology 15:3
108
FTIR stress response assay could lead the development of industrial yeast strains
with high tolerance to lignocellulose-to-ethanol inhibitors
Luca Roscini1,2, Lorenzo Favaro3, Laura Corte1,2, Lorenzo Cagnin3, Matteo Tiecco1,2, Claudia
Colabella1,2, Marina Basaglia3, Gianluigi Cardinali1,2, Sergio Casella3
Department of Pharmaceutical Sciences-Microbiology, University of Perugia, Borgo XX Giugno 74,I-06121 Perugia,
Italy; 2CEMIN, Centre of Excellence on Nanostructured Innovative Materials, Department of Chemistry, Biology and
Biotechnology, University of Perugia, via Elce di Sotto 8, I-06123 Perugia, Italy; 3Department of Agronomy Food
Natural resources Animals and Environment, DAFNAE, Università di Padova, Agripolis, Viale dell’Università 16,
35020 Legnaro (PD), Italy
1
[email protected]
Robust yeast strains with high inhibitors tolerance remain a critical requirement for the production
of lignocellulosic bioethanol. These stress factors are known to severely hinder yeast growth and
fermentation performance (Almeida et al 2007). Fourier Transform InfraRed Spectroscopy (FTIR),
recently applied in bioassays to obtain the metabolomic fingerprint of cells challenged with different
chemicals (Corte et al 2010), has never been used for the phenotypical characterization of industrial
yeast strains under stressing conditions.
A FTIR-based bioassay was employed to explore the yeast metabolomic and viability responses to
four inhibitors commonly found in lignocellulosic hydrolyzates: acetic acid, formic acid, furfural
and 5-hydroxymethyl-2-furaldehyde. Among the 160 previously screened for inhibitors tolerance,
three different Saccharomyces cerevisiae strains were selected as representative for the uppermost,
medium and low robustness, respectively (Favaro et al 2013b, 2014). The strains were assessed for
their ability to withstand increasing concentrations of single inhibitors as well as binary, ternary and
quaternary mixtures.
The yeasts reacted with a strain-specific metabolomic and viability response to increasing levels of
single inhibitors. For the first time, this study highlighted antagonistic interactions between inhibitors,
confirmed by both metabolomic and vitality responses. The antagonism of these mixtures on yeast
metabolism revealed to be strain-specific and was measured by qualitative and quantitative parameters.
FTIR analysis characterized the selected strains in agreement with the results obtained in previous
investigations, demonstrating that FTIR-based assay is a powerful tool for screening bioethanol
industrial fitness in yeasts. The antagonistic effects of the described mixtures are worth of further
studies to understand the related mechanism and to assist strain selection towards the development
of highly tolerant yeast strains.
KEYWORDS: Lignocellulosic ethanol, Inhibitors, Inhibitor-tolerance, FTIR, Industrial yeast strains;
Strain selection
REFERENCES:
Almeida JRM, et al (2007). Journal of Chemical Technology and Biotechnology 82:340-349
Corte L et al (2010). Analytica Chimica Acta 1-2:258-265
Favaro L et al(2013b). Biotechnology for Biofuels 6:168
Favaro et al (2014). Annals of Microbiology 64:1807-1818
109
Inactivation of FeS enzymes as a marker of oxidative stress in the yeast
Izabela Sadowska-Bartosz, Agnieszka Kozdraś, Grzegorz Bartosz
Department of Biochemistry and Cell Biology, University of Rzeszów, Zelwerowicza 4, 35-601 Rzeszów, Poland
[email protected]
FeS enzymes, seemig to be biochemical fossils from the anaerobic stages of evolution, are expected
to be especially sensitive to oxidative stress and be useful biomarkers of exposure to oxidative
stress. Yeast growth on non-fermentable media leads to intensification of respiration, which imposes
oxidative stress on cells. The aim of the study was to compare the activity of two FeS enzymes:
succinate dehydrogenase (SDH) and aconitase in the baker’s yeast S. cerevisiae grown on different
media.
Yeast strains deficient in superoxide dismutase were a generous gift of Dr. E.B. Gralla (Gralla and
Valentine 1991). The yeast was grown on standard YPD medium with 2% glucose, YPE medium with
2% ethanol, YPG medium with 3% glycerol and YPA medium with 2% sodium acetate. SDH activity
was estimated by a whole-cell assay with NitroBlue Tetrazolium (Kregiel et al 2008). Aconitase
activity was assayed with an aconitase assay kit (Cayman).
The non-fermentable media impose oxidative stress on yeast cells, which was more pronounced in
strains deficient in superoxide dismutases. SDH activity was most sensitive to oxidant stress in cells
deficient in both superoxide dismutases (SODs).
WT
Δsod 1
Δsod 2
Δsod1
Δsod 2
SDH
YPD
100
100
100
100
YPE
72.2 +
11.7
48.0 +
3.1
47.8 +
3.2
48.0 +
7.5
YPG
86.0 +
29.5
73.9 +
29.1
57.9 +
9.5
47.5 +
15.7
YPA
99.7 +
11.8
60.1 + 5.3
55.1 + 2.6
44.6 + 4.3
Aconitase
YPD
100
100
100
100
YPE
77.9 +
10.3
62.5 +
8.3
82.3 +
13.7
82.0 +
15.4
YPG
77.4 +
10.3
62.5 +
8.3
82.3 +
13.7
82.0 +
15.4
YPA
100.7 +
10.3
93.5 +
13.9
78.3 +
14.8
76.6 +
10.9
KEYWORDS: S. cerevisiae, Oxidative stress, Aconitase, Succinate dehydrogenase
REFERENCES:
Gralla EB, Valentine JS (1991). Null mutants of Saccharomyces cerevisiae Cu,Zn superoxide dismutase:
characterization and spontaneous mutation rates. Journal of Bacteriology 173:5918-5920
Kregiel D, Berlowska J, Ambroziak W (2008). Succinate Dehydrogenase Activity in S. cerevisiae, Food Technology
and Biotechnology 46:376–380
110
Effect of oxidative stress on the survival of Phaffia rhodozyma and in the
production of carotenoid pigments and mycosporine-glutaminol-glucoside
1
Ana L. Pajarola1, Virginia de Garcia1, Diego Libkind 1, Victor Cifuentes2,
Martin Moliné1
Laboratorio de Microbiología Aplicada y Biotecnología (MABB), INIBIOMA-UNComahue, San Carlos de Bariloche,
Argentina; 2Departamento de Ciencias Ecológicas, Centro de Biotecnologia, Facultad de Ciencias, Universidad de
Chile, Santiago, Chile
[email protected]
Phaffia rhodozyma is a basidiomycetous yeast capable of synthesizing two compounds of
biotechnological value: carotenoid pigments (astaxanthin) and mycosporine-glutaminol-glucoside
(MGG, UV sunscreen). Both metabolites exert protection against UV radiation and probably against
reactive oxygen species (ROS). In this work, we studied for the first time the effect of ROS on
the accumulation of carotenoids and MGG in Phaffia rhodozyma and evaluated the role of both
compounds in the survival of this yeast against oxidative stress.
Six strains including an hyperproducer mutant of MGG and carotenoids, two wild type strains, and 3
mutants deficient for the production of one of the two compounds were exposed to hydrogen peroxide,
superoxide anion (generated with duroquinone) and singlet oxygen (generated with rose bengal). The
survival of the strains was estimated by CFU. The effect of ROS on the production of MGG and
carotenoids was assessed by adding sub-inhibitory concentrations of each ROS at the beginning of
the cultures, and the accumulation of each metabolite was determined after 5 days exposure.
The results obtained in this work demonstrated that both, carotenoids and MGG, exerts a protecting
role in P. rhodozyma against hydrogen peroxide. The hyperproducer strain was more resistant to stress
induced by H2O2 (81% survival after 90 minutes) than the parental strains and the strains deficient
for the production of these metabolites (less than 2% survival). On the other hand, no differences in
survival could be detected for strains exposed to superoxide anion, suggesting that these compounds
do not protect against this radical. On the contrary when exposed to singlet oxygen the strains
producing high concentrations of MGG presented the lowest survival suggesting that the presence of
this compound was detrimental. Only exposure to singlet oxygen induced the accumulation of MGG
while the accumulation of carotenoid pigments was stimulated by the three ROS studied.
KEYWORDS: Oxidative stress, Carotenoids, Mycosporine, Xanthophyllomyces
111
Improving the representation of respiration in yeast metabolic models
Duygu Dikicioglu, Ayca Cankorur-Cetinkaya, Stephen G. Oliver
Cambridge Systems Biology Centre & Department of Biochemistry, University of Cambridge, Cambridge, UK
[email protected]
Iron is a key transition metal co-factor involved in the aerobic respiration of yeast cells, yet even
the well-established genome-scale model of the metabolic network of Saccharomyces cerevisiae has
an inadequate representation of iron metabolism. Genome-scale models are useful tools with which
to investigate metabolic networks since they allow us to scan a broad landscape of physiologically
feasible metabolic states and so provide a focus for strain design or further investigations. The precise
representation of respiration-related phenomena in such models is critical to the generation of accurate
predictions concerning aerobic growth and production. For these reasons, we have investigated the
representation of iron metabolism in the most recent version of the S.cerevisiae metabolic model. We
identified its shortcomings and the problems these shortcomings caused for the model’s predictions.
We will present an extended metabolic network, in which we have incorporated the pathways of iron
transport and storage, as well as the formation and utilization of iron-containing complexes including
the iron-sulphur clusters. Further, we propose an approach to account for the regulatory mechanism
controlling iron uptake via the iron regulon. The incorporation of a complete representation of iron
metabolism into the yeast metabolic network expanded its gene coverage by more than 5%, allowing
an in-depth exploration of the flux distribution landscape in a variety of metabolic states concerning
iron. We believe this to be a useful proof-of-principle study that should be extended to include the
metabolic models of non-conventional yeasts, such as Komagataella (Pichia) pastoris, which rely
solely on respiratory metabolism.
112
Wine yeast biofilms and quorum sensing
Ee Lin Tek1, Jennie Gardner1, Joanna Sundstrom1, Stephen G. Oliver2, Vladimir Jiranek1
Dept. Wine & Food Science, University of Adelaide, Australia; 2Dept. Biochemistry & Cambridge Systems Biology
Centre, University of Cambridge, UK
1
[email protected]
Wine yeast exhibit an extraordinary ability to survive in the harsh environment of wine. The ability to
form biofilms may have a role as it enables yeast to colonise and persist in various stressful ecological
niches. This study aims to investigate biofilm formation and regulation of wine yeast and whether the
yeast quorum-sensing molecules (tryptophol and 2-phenylethanol) influence this process.
The ability of three commercial wine yeasts and the laboratory strain Σ1278b to form biofilms (mats)
on rich or nitrogen-limiting low-agar media were compared. Cell morphologies were examined and
compared between different parts of the mats, as well as with and without the addition of tryptophol,
2-phenylethanol, or ethanol.
Each wine yeast strain formed mats with unique structures. Within the biofilm, cells from the mat
rim showed a uniform actively growing population whereas those from the mat body showed a
variety of morphologies. Under nitrogen-limitation, a subset of cells switched from surface growth to
filamentous and invasive growth. Ethanol enhanced this filamentous invasive growth for a wine yeast,
yet the effect was suppressed by the aromatic alcohols.
The morphological complexity of wine yeast mats, make them suitable models to study cellular
differentiation and organisation. Nutrient availability and the presence of signaling molecules could
influence strategies for colonisation and survival.
KEYWORDS: Biofilm, Mat, Wine yeast, Quorum sensing, Filamentation
113
114
Session 2A
biodiversity and ecology in foods and beverages
115
Schizosaccharomyces isolation and selection for winemaking
Santiago Benito
Departamento Química y Tecnología de Alimentos, Universidad Politécnica de Madrid, Ciudad Universitaria S/N,
28040, Spain
[email protected]
Genus Schizosaccharomyces have occasionally been described as spoilage. However, it also has been
used for industrial purposes due to their deacidifying properties (Benito et al 2014a). On the other
hand, during the last years new uses of this genus has been developed (Benito et al 2014a). Some
of them are applications in ageing over lees and polysaccharide release, gluconic acid reduction and
color improvement. However the number of commercial strains is very limited, probably due to
the low incidence of this genus compared to other microorganisms (Benito et al. 2013, Benito et al
2014b). Efforts should therefore focus on the isolation and selection of strains from this species for
industrial applications.
100 hundred strains were isolated following the methodology described in figure 1. It was based in
a selected-differential media containing actidione, benzoic acid, high glucose level and malic acid.
Those isolated strains were fermented in sterilized must and final fermentation results were compared.
Just a few strains were selected for no presenting collateral effects in winemaking. Nevertheless they
showed a high ability to reduce malic acid content in acidic musts.
Figure 1. Schizosaccharomyces isolation method summary
REFERENCES:
Benito S, Palomero P, Calderón F, Palmero D, Suárez-Lepe JA (2014a). Schizosaccharomyces. In: Batt CA, Tortorello
ML (eds) Encyclopedia of Food Microbiology. Elsevier, Amsterdam 3:365–370
Benito S, Palomero P, Calderón F, Palmero D, Suárez-Lépe JA (2014b). Selection of Appropriate Schizosaccharomyces
strains for winemaking. Food Microbiology 42:218-224
Benito S, Gálvez L, Palomero F, Calderón F, Morata A, Suárez-Lepe JA (2013). Schizosaccharomyces selective
differential media. African Journal of Microbiology Research 7(24):3026-3036
116
Session 2A: Yeasts in food biotechnology: biodiversity and ecology in foods and beverage
Wild yeasts of Slovenia wine region and their potential for food industry
Sofia Dashko 1,2, Uros Petrovic 2, Jure Piskur 1,4, Justin Fay 3
University of Nova Gorica, Wine Research Centre, Glavni trg 8, Vipava, Slovenia, SI 5271; 2Jožef Štefan Institute,
Department of Molecular and Biomedical Sciences, Jamova 39, Ljubljana, SI 1000, Slovenia; 3Washington University,
Center for Genome Sciences and System Biology, 4444 Forest Park Pkwy, St. Louis, MO 63108; 4Lund University,
Solvegatan 35, Lund, Sweden
1
[email protected]
Species’ natural habitat shapes the phenotypes they acquire. Therefore, wild yeast isolates with
good fermentation capacity are expected to be found in the vineyard and associated areas. While the
origin of wine yeasts is not yet known, current research aims to relate yeasts ecology to geographical
locations and habitats.
In our study, we assessed the phenotypic diversity of the yeast isolates of the wine region of Slovenia
and identified the genotypes of the species with promising industrial properties. A collection of
1,5 thousand yeast isolates were characterized for their basic microbiological properties, source of
isolation and their genotypes were partially identified. Quantitative measurements of fitness were
assessed using the robotic manipulator and automated analysis pipeline for 30 different physiological
conditions. We focused our phenotype analysis on the traits, which are mainly important for the wine
industry. As expected, S. cerevisiae wine strains performed well under different physical and chemical
stresses that occur during the winemaking process. But, surprisingly, S. cerevisiae commercial control
strains were comparable with a number of wild isolates of different genera obtained from within and
outside the vineyard. Another interesting observation is the sympatric relationship of S. cerevisiae
and S. paradoxus, which are abundant in both vineyard and on the oak trees. This result suggests that
S. paradoxus, which is considered as an exclusive wild species, to have possible industrial potential
for fermentation industry. We conclude that the present collection consists of isolates with promising
potential for winemaking. At the same time, the collection is being the source of knowledge for
discovering phenotype – genotype relations and population structure of yeasts in Slovenia.
117
Screening and identification of yeast flora from natural sandstone pit and their
involvement during Italian Fossa cheese ripening
Francesca Comitini, Claudia Biagiotti, Maurizio Ciani
Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131,
Ancona, Italy
[email protected]
Fossa cheese is a traditional Italian cheese which name, literally “cheese of the pit”, derived
from the process of ripening in special natural underground pits. The maturation in pit (over four
months) is the phase that organoleptically characterizes this product with a considerable decrease
in weight, irregular shape, hardness (Gobbetti et al 1999). The secondary flora that originate during
pit maturation, is composed of bacteria, yeasts and molds that coexist in a complex equilibrium
and play a fundamental role to confer the typical characteristic to the final product. A lot of studies
investigated on the contribution of bacteria and filamentous fungi but little is known on the role of
yeasts. In this work, we carried out a double yeast isolation campaign in a natural pit before and
after fossa cheese maturation. Before the ageing molecular tools allowed to identify eight different
yeasts biotypes belonging to: Candida zeylanoides, Candida sorbosa, Candida norvegica, Pichia
guillermondii, Pichia jadinii, Cryptococcus albidus, Cryptococcus skinneri and Sporobolomyces
roseus. Only C. zeylanoides was also found after ripening stage, together with Wickerhamomyces
anomalus, Saccharomyces cerevisiae, Debaryomyces hansenii and Candida humilentoma. With the
aim to evaluate the effective contribution of these autochthonous yeasts during ripening, the isolated
yeasts were used to inoculate fresh cheese in different modalities: in the milk with LAB starter, in the
curd and on the surface of ripening cheese. Cheeses were matured in artificial pit and then evaluated
by sensorial panel test. Results showed that C. zeylanoydes and W. anomalus drastically reduced
the mold colonization of cheese surface, exhibiting excellent results in the panel test evaluation.
Indeed, cheeses inoculated with C. zeylanoides and W. anomalus did not exhibited defects, showing a
homogeneous structure and adequate softness. GC-SPME analysis of the best product, compared with
the control (uninoculated cheese) showed higher concentrations of metilchetoni, hexanoic, butanoic
and octanoic acids that typically enhance the taste of highly matured pit cheese.
KEYWORDS: Pit cheese, Yeast microbiota, Cheese ripening, Candida zeylanoides, Wickerhamomyces
anomalus
REFERENCES:
Gobbetti M, Folkertsma B, Fox PF, Corsetti A, Smacchi E, De Angelis M, Rossi J, Kilcawley K, Cortini M (1999).
Microbiology and Biochemistry of Fossa (pit) cheese. International Dairy Journal 9:763–773
118
Use of immobilized non-Saccharomyces yeasts for the reduction of alcohol
content in wine
Laura Canonico, Francesca Comitini, Lucia Oro, Maurizio Ciani
Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 6013, Ancona,
Italy
[email protected]
Over the last few decades, there has been a progressive increase in the ethanol content in wines due
to global climate change and to the new wine styles that are associated with increased grape maturity.
For these reasons, several studies are aimed at the reduction of ethanol content in wines. In the context
of a microbiological approach several strategies that use genetically modified (GM) Saccharomyces
cerevisiae yeasts or evolution-based strategies have been proposed for the production low-alcohol
wines. Another approach to reduce the ethanol content in wine could be the use of non-Saccharomyces
wine yeasts. The use of non-Saccharomyces yeasts in combination with Saccharomyces cerevisiae
has been proposed to improve the quality and enhance the complexity of wine. Recently, sequential
inoculations has been suggested for the potential reduction of ethanol content in wine (Contreras et
al 2014; Morales et al 2015).
In the present study we investigated on the effect of sequential fermentation of immobilized nonSaccharomyces yeast with S. cerevisiae starter strain. Fermentation trials were carried out using
yeast species belonging to Hanseniaspora, Starmerella, Metschnikowia, Zygosaccharomyces genera
and selected for grape juice fermentation. Synthetic grape juice or natural grape juice were inoculated
with immobilized non-Saccharomyces yeast strains. After 48 h or 72 h of fermentation, the beads
were removed and S. cerevisiae starter strain was inoculated. The fermentation behavior of these
mixed cultures as well as the analytical profiles (main enological compounds, volatile compounds,
alcohol content) of the resulting wines were evaluated. Results showed that mixed fermentations
exhibited significant reduction of ethanol content from 0.6 to 1.4 % vol. if compared with pure S.
cerevisiae fermentation trials. Differences in the analytical profile of wines were also detected.
KEYWORDS: Non-Saccharomyces yeast, Mixed fermentations, Ethanol reduction, Immobilized
yeast
REFERENCES:
Contreras A, Hidalgo C, Henschke PA, Chambers PJ, Curtin C, Varela C (2014). Evaluation of non-Saccharomyces yeasts
for the reduction of alcohol content in wine. Applied and Environmental Microbiology 80:1670-1678
Morales P, Rojas V, Quirós M, Gonzalez R (2015). The impact of oxygen on the final alcohol content of wine fermented
by a mixed starter culture. Applied Microbiology and Biotechnology 99: 3993-400
119
Survey, molecular characterization and comparison of Brettanomyces
bruxellensis strains coming from grape surfaces and winery
Lucia Oro, Francesca Comitini, Laura Canonico, Maurizio Ciani
Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131,
Ancona, Italy
[email protected]
Brettanomyces bruxellensis is considered a major spoilage yeast in the last stages of fermentation
process and during wine ageing, while its presence on the surface of the grape berries was detected
with extreme difficulty (Renouf et al 2007). To find the possible origin of spoilage yeast is crucial
to control the Brettanomyces yeast contamination. To detect and identify B. bruxellensis, different
selective media and several molecular methods such as random amplified polymorphism DNA
(RAPD), mtDNA restriction analysis, amplified fragment length polymorphism (AFLP), restriction
enzyme analysis and pulse field gel electrophoresis (REA-PFGE) have been proposed (Miot-Sertier
& Lonvaud-Funel 2007). In the first instance, it was evaluated the presence of B. bruxellensis on the
surface of the grape berries and in the winery environment through enrichment and selective media.
Afterwards, isolated strains were submitted to fingerprinting procedures through RAPD (M13, M14)
and minisatellites (PIR1, PIR3) analysis. Then a comparison, between B. bruxellensis strains coming
from grape surface and those isolated from winery, was carried out. Results of typing procedures
showed that the 15 strains of B. bruxellensis, coming from the vineyard, were grouped into six
clusters, while the 28 strains, isolated from the cellar, were grouped in ten biotypes. The comparison
of the biotypes indicated that three biotypes exhibited a full correspondence between grape surface
and winery strains. Interestingly, one of these, includes 5 strains coming from grapes and 14 from
winery strains indicating that this genotype is dominant in this specific ecological niche (44% of total
strains).
KEYWORDS: Brettanomyces bruxellensis, Grape berry, Winery, Molecular characterization
REFERENCES:
Renouf V, Lonvaud-Funel A (2007). Development of an enrichment medium to detect Dekkera/Brettanomyces
bruxellensis, a spoilage wine yeast, on the surface of grape berries. Microbiology Research 162:154-167
Miot-Sertier C, Lonvaud-Funel A (2007). Development of a molecular method for the typing of Brettanomyces
bruxellensis (Dekkera bruxellensis) at the strain level. Journal of Applied Microbiology 102:555-562
120
Exploring genetic diversity and metabolic profile of Kluyveromyces marxianus
dairy yeast
Giuseppe Fasoli1, Rosanna Tofalo1, Francesca Patrignani2, Rosalba Lanciotti2, Giorgia
Perpetuini1, Maria Schirone1, Luigi Grazia2, Aldo Corsetti1, Giovanna Suzzi1
Faculty of BioScience and Technology for Food, Agriculture and Environment, University of Teramo, Mosciano
Sant’Angelo, TE, Italy; 2Department of Agricultural and Food Sciences, University of Bologna, P.zza
Goidanich 60, I-47521 Cesena (FC), Italy
1
[email protected]
Kluyveromyces marxianus plays an important role in the ripening of a wide variety of cheeses and
in the production of volatile organic compounds (VOC) that improve the characteristics of the final
product. In a previous study K. marxianus strains were characterized in terms of genetic and metabolic
diversity (Tofalo et al., 2014). In this study to determine genetic polymorphysm of this species,
karyotype profile of 39 K. marxianus strains from Pecorino di Farindola cheese was compared to
that of strains of other origin (Parmigiano Reggiano cheese, fermented milk and cow whey) and two
type strains K. marxianus CBS 834T and K. lactis CBS 683T. In order to demonstrate the role of K.
marxianus in flavour modulation and to select starter cultures with particular aromatic potential, 12
strains, selected on the basis of some their dairy properties and chromosomal patterns, were tested in
raw cheese whey and ricotta cheese whey. PFGE analysis showed 11 patterns that differed in size and
number of the chromosomal bands and 70% of strains displayed a basic set of 6 chromosomal bands.
The 12 selected strains were tested under limited oxygen condition in raw cheese whey and ricotta
cheese whey. Growth kinetics distinguished four biotypes that showed different growth patterns
in both media. In general, all biotypes had the best performance in raw cheese whey. The main
VOC compounds found were alcohols, acids, esters, ketones and aldehydes. Ethanol was the main
compound produced while esters were qualitatively and quantitatively more present in raw cheese
than in ricotta whey, with ethyl acetate as the highest one. Butanoic, decanoic and octanoic acids
were present only in raw cheese whey whereas acetic acid in both media. The results suggested a
high biodiversity at genetic and metabolic levels indicating a heterogeneous contribution to VOC
production and the feasibility of strain selection to modulate cheese ﬂavour and aroma.
KEYWORDS: Kluyveromyces marxianus, Dairy products, Flavour, PFGE
REFERENCES:
Tofalo R, Fasoli G, Schirone M, Perpetuini G, Pepe A, Corsetti A, Suzzi G (2014) The predominance, biodiversity
and biotechnological properties of Kluyveromyces marxianus in the production of Pecorino di Farindola cheese.
International Journal of Food Microbiology 187:41–49
121
From the grape berries to the cellar: intraspecific biodiversity of two nonSaccharomyces yeasts belonging to the genera Candida and Hanseniaspora
Cédric Grangeteau1, Sandrine Rousseaux1, Daniel Gerhards2, Christian von Wallbrunn2,
Hervé Alexandre1, Michèle Guilloux-Benatier1
UMR Procédés Alimentaires et Microbiologiques, Equipe VAlMiS, AgroSup Dijon Université de Bourgogne, IUVV,
rue Claude Ladrey, BP 27877, 21000 Dijon, France; 2Institut für Mikrobiologie und Biochemie Zentrum Analytische
Chemie und Mikrobiologie Hochschule Geisenheim University, Geisenheim, Germany
1
[email protected]
The origin of the non-Saccharomyces (NS) strains implicated in grape must alcoholic fermentation
(AF) is not determined. Furthermore, the persistence of these NS strains in the cellar for several years
is unknown. This work had 2 objectives: to determine the origin (berries or cellar) of NS strains
isolated in must and to demonstrate or not their persistence in cellar. We focused on 2 common
genera Candida and Hanseniaspora for which discrimination at strain level was possible by FT-IR
spectroscopy. So, yeasts isolated from musts and during AF were compared to those isolated on
berries and in cellar environment during 2 vintages (4049 isolates).
In 2012, 214 yeasts of C. zemplinina species were isolated (berries, must, AF and cellar) and identified
as 33 different strains. Among these strains, 3 were isolated from berries. 19 strains were isolated in
must, among which 1 isolated on berries. During AF, 13 strains were identified: 1 also presents on
berries and 1 another in must. In 2013, no Candida was isolated.
For the genus Hanseniaspora, 1084 isolates belonging to 174 different strains were identified during
the 2 vintages. In 2012, 61 strains were isolated from berries among which, 14 were also found in must.
During AF, 100 strains were identified, 12 strains were also present in must and 8 on berries. These
results reflect that most of the non-Saccharomyces under study are from cellar origin and demonstrate
the higher fitness of cellar Candida and Hanseniaspora compared to grape berries. Among all the
Hanseniaspora strains isolated in 2012, 4 of them were also present in 2013.
These results show a great intra-specific biodiversity among NS genera of oenological interest. Like
for Saccharomyces strains, the NS strains present on berries are minority in must or during AF (10%
of Candida and 14% of Hanseniaspora). For the first time, we demonstrate also the ability of NS
yeasts to persist in cellar environment and to implant in musts the following year.
KEYWORDS: Non-Saccharomyces, Intraspecific biodiversity, Cellar, Grape berries, Wine
122
Central carbon metabolic flux diversity for forties S.cerevisiae strains
Thibault Nidelet, Pascale Brial, Isabelle Sanchez, Carole Camarasa, Sylvie Dequin
INRA, UMR1083 Sciences Pour l’Oenologie, F-34060, Montpellier, France
[email protected]
System biology has emerged as a key approach to provide a quantitative description of cellular
processes and ultimately, to predict how cells operate. Knowing how metabolic fluxes are modulated
by genetic and/or environmental perturbations is a central question to understand yeast physiology.
In order to identify the metabolic and evolutionary constraints that shape metabolic fluxes and to
highlight the most robust and variable nodes, we used a dedicated constraint-based model to quantify
intracellular fluxes in 43 strains of S.cerevisae of various origins: “bread”, “flor”, “oak”, “wine”
and “rum”. We used metabolite concentration and biomass production at the end of the exponential
growth phase of an oenological fermentation to constraint our model and predict for each strain the
central carbon metabolism fluxes’ distribution. By analyzing them among strains we first highlighted
correlations between fluxes like the trade-off between the flux through the pentose phosphate pathway
(PPP) and the synthesis of acetate that we can link to the synthesis of NADPH. The PPP is also
positively correlated to the biomass flux linked to biomass precursors’ synthesis. We also pointed
out a highly contrasted situation in fluxes’ variability with quasi-constancy of the glycolysis and
ethanol synthesis yield and on the contrary a high flexibility of the PPP. These fluxes with broad
distributions show bimodal behaviors that can be explained by strains’ origins. Indeed strains display
contrasted distribution based on their origins, showing a convergence between genetic origins and
flux phenotypes. Overall this study allowed us to highlight the constraints shaping the operative
central carbon network during yeast fermentation and will provide clues for the design of strategies
for strain improvements.
KEYWORDS: Metabolic fluxes, Modeling, S.cerevisae, Flux balance analysis
123
Gas bubble discovery in fermenting yeasts
Khumisho Dithebe1, Carolina Pohl1, Hendrik Swart2, Elizabeth Coetsee2, Pieter van Wyk3,
Elizabeth Lodolo4, Chantel Swart1,3
UNESCO-MIRCEN: Department of Microbial, Biochemical and Food Biotechnology; 2Department of Physics;
Centre for Microscopy, University of the Free State, P.O. Box 339, Bloemfontein, 9300; 4SABLtd Brewing Centre of
Excellence, PO Box 123902, Alrode 1451, South Africa
1
3
[email protected]
Fermentation exploits the ability of yeasts to produce increased ethanol and carbon dioxide (CO2).
Since yeasts vigorously release CO2 into the surrounding medium during fermentation, it is expected
that yeast cells would be filled with gas bubbles. Regardless of how well established yeast fermentation
is, no reports of CO2 bubbles inside yeast cells have been made resulting in a missing link. Therefore
the aim of the study was to find the link between intracellular CO2 production and eventual release
from the cells.
Saccharomyces pastorianus and S. cerevisiae were grown in fermentable and non-fermentable
media respectively and analysed with various microscopic techniques to investigate the presence of
intracellular gas bubbles.
Light microscopy revealed an increased number of light scattering granules inside cells grown in
fermentable media as opposed to non-fermentable media. Transmission electron microscopy and
Nano scanning Auger microscopy results confirmed the presence of a large number of intracellular
gas bubbles filling a significant part of the cells when grown in fermentable media as opposed to nonfermentable media.
The missing link has therefore been uncovered and further studies should now be performed to
characterize these inclusions.
KEYWORDS: Fermentation, NanoSAM
REFERENCES:
Swart CW, Dithebe K, Pohl CH, Swart HC, Coetsee E, Van Wyk PWJ, Swarts JC, Lodolo EJ, Kock JLF (2012). Gas
bubble formation in the cytoplasm of a fermenting yeast. FEMS Yeast Research 12:867–869
124
Sensory profiling of Touriga Nacional wines obtained from
non-Saccharomyces yeasts
Andreia Teixeira1, Ilda Caldeira1,2, Filomena L. Duarte1
Instituto Nacional de Investigação Agrária e Veterinária, INIAV-Dois Portos, Quinta da Almoínha, 2565-191 Dois
Portos, Portugal; 2ICAAM – Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Pólo
da Mitra, Ap. 94, 7002-554 Évora, Portugal
1
[email protected]
The increasing demand for quality and innovative wines challenges the producers and researchers to
be creative. Yeasts are the main microorganisms involved in wine production and non conventional
yeast species have showed to improve the complexity of wines and their use is promising regarding
the diversification of wine characteristics. Descriptive sensory analysis is a major tool to obtain
sensory profiling of food and beverages which will be better characterized from a consumption point
of view. The aim of the present work was to characterize the sensory profile of Touriga Nacional
wines obtained from non-Saccharomyces yeasts.
Yeasts were isolated from spontaneous Touriga Nacional (TN) grape must from different vineyards.
A total of twenty three yeast isolates belonging to ten species, were inoculated in TN grape must and
solids and the wines obtained were evaluated by descriptive sensory analysis. The tasting panel was
composed of 13 tasters, nine female and four male, aged between 24 and 59 years. A score sheet was
generated and the sensory descriptors were trained. In all tasting sessions a reference wine obtained
from a commercial strain of Saccharomyces cerevisiae was also presented in order to evaluate
individual taster’s performance. ANOVA was performed to the sensory results obtained.
A significant species effect was obtained for the majority of the descriptors evaluated. The similarity
of the profiles obtained within each species and genera will be graphically highlighted. Though
negative aromas were observed, some yeasts originated wines with quite interesting aromas like
passion fruit, dried fruits, wild fruits, honey, jam, chocolate and clove. The isolates of the species
Starmerella bacillaris and Candida diversa produced the higher quality wines, with higher balance
and more intense and diverse aroma, the former enhancing the aromas characteristic of TN grape
variety, bergamot, violet and rock-rose, being the most promising for improving TN wines.
KEYWORDS: non-Saccharomyces, Wine, Sensory profiling, Touriga Nacional
125
Influence of culture conditions on the carotenoids production by Rhodotorula
glutinis
Ayerim Hernández-Almanza1, Julio Montañez-Sáenz2, Alejandro Aguilar-Jiménez3,
Juan C. Contreras-Esquivel1, Cristóbal N. Aguilar1
Department of Food Research. School of Chemistry. Universidad Autónoma de Coahuila. Saltillo, 25280, Coahuila,
México; 2Department, of Chemical Engineering School of Chemistry. Universidad Autónoma de Coahuila. Saltillo,
25280, Coahuila, México; 3Biotechnology Center FEMSA, Instituto Tecnológico de Monterrey, 64849, Nuevo León,
México
1
[email protected]
Carotenoids are colored terpenoids with important biological properties. These bioactive compounds
are found in plants, animals, fungi and photosynthetic and non-photosynthetic microorganisms
(Garrido-Fernández et al 2010). Lycopene is a liposoluble carotenoid with beneficial properties for
human health such as cancer and cardiovascular diseases prevention (Colle et al 2013). Due to these
properties, biotechnological alternatives for obtaining this kind of pigments are required. The aim
of this study was to evaluate the influencing factors on the carotenoids production by Rhodotorula
glutinis. Cells of the strain P4M422 of R. glutinis were inoculated in YM medium. A Plackett-Burman
design was used to evaluated the influence of light, nitrogen and carbon sources, temperature, time,
and inoculum. Carotenoid production by R. glutinis was totally intracellular, therefore cell disruption
to free the carotenoids produced was necessary. Ultrasonication and microwave methods were used
for pigment extraction (Cheung et al 2015). The results showed that light is an important factor in
carotenoid production (Table 1). Zhang et al (2014) reported that maximum carotenoids concentration
(2.6 mg/L) was in three LED lamps batch by R. glutinis. Carotenoids are important in protecting of
photo-oxidative damage.
Table 1. Factors to evaluate in Plackett-Burman design.
Factor
pH
Temperature (°C)
Carbon (g/L)
Nitrogen (g/L)
Time (h)
Inoculum (cel/mL)
Light
Level (-)
5
25
20
3
48
106
0
Level (+)
6
30
30
5
96
108
1
KEYWORDS: Lycopene, Rhodotorula glutinis, Ultrasonication, Cell disruption, Carotenoids
REFERENCES:
Cheung YC, Liu XX, Wang WQ, Wu JY (2015). Ultrasonic disruption of fungal mycelia for efficient recovery of
polysaccharide-protein complexes from viscous fermentation broth of a medicinal fungus. Ultrason Sonochem
22:243-248
Colle IJP, Lemmens L, Buggenhout SV, Loey AMV, Hendrickx ME (2013). Modeling lycopene degradation and
isomerization in the presence of lipids. Food Bioprocess Technology 6:909-918
Garrido-Fernández J, Maldonado-Barragán A, Caballero-Guerrero B, Hornero-Méndez D, Ruiz-Barba JL (2010).
Carotenoid production in Lactobacillus plantarum. International Journal of Food Microbiology 140:34-39
Zhang Z, Zhang X, Tan T (2014). Lipid and carotenoid production by Rhodotorula glutinis under irridation/hightemperature and dark/low-temperature cultivation. Bioresource Technology 157:149-153
126
Hanseniaspora guilliermondii–Saccharomyces cerevisiae mixed-culture
interactions during wine fermentation
Catarina Barbosa1, Patrícia Lage1, Isabel Vasconcelos2, Arlete Mendes-Faia1,4, Nuno P. Mira3,
Ana Mendes-Ferreira1,4
Universidade de Trás-os-Montes e Alto Douro, Escola de Ciências da Vida e Ambiente; Vila Real, Portugal; 2CBQF/
Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto,
Portugal; 3IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa,
Lisboa, Portugal; 4BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, Lisboa, Portugal
1
[email protected]
The introduction of yeast starter cultures consisting in a blend of Saccharomyces cerevisiae and nonSaccharomyces yeast strains is emerging as an interesting option for production of wines with improved
complexity of flavor. In this study, single- and mixed-culture of Hanseniaspora guilliermondii and
Saccharomyces cerevisiae were used to ferment natural grape-juice, under two nitrogen regimes. In
mixed-culture, that strain negatively interfered with the growth and fermentative performance of S.
cerevisiae, resulting in lower fermentation rate and longer fermentation length, irrespective of the
initial nitrogen concentration. The impact of co-inoculation on the volatile compounds profile was
more evident in the wines obtained from DAP-supplemented musts, characterized by increased levels
of ethyl and acetate esters, associated with fruity and floral character of wines. Moreover, the levels
of fatty acids and sulphur compounds which are responsible for unpleasant odors that depreciate wine
sensory quality were significantly lower. In addition, gene expression profiles of S. cerevisiae were
monitored along time in parallel fermentations either in single or in mixed-culture. The integration of
transcriptome and physiological data allowed an improved understanding of S. cerevisiae in mixedculture fermentation. Our results demonstrate that nutrient availability, mainly nitrogen and vitamins,
is a determinant factor of population dynamics, fermentative activity and by-product formation during
mixed-culture fermentations.
The research presented was financially supported by FEDER through COMPETE (FCOMP-01-0124FEDER-014043(PTDC/AGR-ALI/111224/2009) and Project ENOEXEL - FROM VINEYARD TO
WINE: TARGETING GRAPE AND WINE EXCELLENCY - NORTE-07-0124-FEDER-000032,
financed by the North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under
the National Strategic Reference Framework (QREN), through the European Regional Development
Fund (FEDER), as well as by National Funds (PIDDAC) through the Portuguese Foundation for
Science and Technology (FCT/MEC).
KEYWORDS: Mixed-culture, Yeast-yeast interaction, Wine fermentation, Aroma compounds,
Transcriptome
127
Session 2A: Yeasts in food biotechnology: biodiversity and ecology in food and beverages
Yeasts in Smen, a fermented farm butter
Clarisse Iradukunda, Fatiha Ben Mellouk, Ahmed Tadlaoui Ouafi, Abdellatif Boussaid
Bioprocess Engineering team, Department of Biology, Faculty of Sciences and Technology Marrakech, Cadi Ayyad
University, Marrakech, Marocco
[email protected]
Smen is a traditional fermented Moroccan product prepared from farm butter fermented up to a
few years in closed earthy jars. Lipolysis has been described as the main mechanism that leads to
the formation of smen. This work is oriented towards the understanding of the role of yeasts in the
fermentation of smen and the production of aromas along with the analysis of the quality of this
commercial product.
Hence, 17 samples of commercial final products of smen collected in the area of Marrakech were
analyzed for total flora; yeasts and moulds; lactic acid bacteria; lipolytic flora; proteolytic flora;
coliforms, fecal streptococci and pathogenic staphylococci.
Physicochemical and microbiological compositions of smen varied widely. Average fat content,
water, dry matter, salt, lactose and proteins are respectively 81%, 10-20%, 2.73 to 7.36%, 0.9 to
3.4%, 1.2% and 3.25%. Present in 59% of samples, Lactobacillus were the main lactic acid bacteria
at 3.105 CFU/g of smen. Fecal coliforms and presumed pathogenic staphylococci were both found in
3 samples with concentrations above the safety standards for fermented food.
Average concentrations of yeasts and fermenting flora of smen (in CFU/g of smen)
Fermenting flora
Total flora
(All samples)
Yeasts
2,18.105
Yeasts and moulds
( In 82% of samples)
3,89.10
Lipolytic flora
(In 82% of samples)
3,48.104
Proteolytic flora
(In 41% of samples)
2,54.104
4
Total yeasts
(In 70% of samples)
Lipolytic yeasts
(In 57% of samples containing
lipolytic flora)
Proteolytic yeasts
(In 28% of samples containing
proteolytic flora)
3,49.104
5,97.104
1,09.103
Comparing the different floras, yeasts were the predominant microorganisms and their contribution
in the fermentation and maturation of smen should be important as for they constitute the major part
of lipolytic and proteolytic floras. This may be linked to their metabolic activity and ability to resist
to physicochemical stresses as it is the case of smen environment. Many strains were observed such
as Geotrichum sp., Yerrowia sp., Debaryomyces sp., Saccharomyces sp., Klyveromyces sp., Candida
sp., and Rhodotolura sp.
The presence and role of yeast in smen have been overlooked. However, we think that their lipolytic
and proteolytic properties, formation of aromas, probiotic effect and their killer factors towards
undesired bacteria contribute to smen maturation and safety.
KEYWORDS: Yeasts, Smen, Butter, Fermentation, Safety
128
Kluyveromyces marxianus fragilis B0399®, a new generation probiotic strain,
improves the quality of fermented milk beverage, named Jogofir
Ana Backović1, Anka Kasalica2, Tijana Lopičić-Vasić3, Goran Grubješić3, Boris Milović4,
Anka Papović-Vranješ3
International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy and Turval Biotechnologies,
Udine, Italy; 2Dairy Institute, New Belgrade, Serbia; 3Dairy Laboratory at the Dept. of Animal Science, Faculty of
Agriculture, University of Novi Sad, Serbia; 4Union University, Belgrade, Serbia
1
[email protected]
Introduction: In an effort to follow contemporary trends in the dairy industry, we made a novel
fermented milk product named Jogofir that contains new generation probiotic strain: Probiotic Lactic
Yeast®, Kluyveromyces marxianus fragilis B0399®. We investigated its particular biochemical and
physical properties. Yeast Kluyveromyces m.f. B0399 has the ability to decompose lactose with
the enzyme beta-galactosidase, giving rise to the lactic acid as the end product. The utilisation of
this probiotic in the human diet counteracts the negative effects of antibiotics, maintains the gut
homeostasis, improves immunity and regulate cytokines production.
Materials and Methods: The preparation of the strain Kluyveromyces m.f. B0399 was produced and
patented by Turval Laboratories srl. of Udine. The test production of Jogofir, microbiological and
the biochemical analysis was performed in: Faculty of Agriculture, Univ. of Novi Sad, Serbia; Dairy
Institute of Novi Beograd and ICGEB Center in Trieste. For yogurt production, Yeast Kluyveromyces
m.f. B0399 was added to milk in two ways: before the fermentation start at room temperature-23.5°C
and at the end of the fermentation, in the cooled yogurt-4°C. We analyzed physical and microbiological
characteristics; amino acid content, fatty acids, proteins, lactose and vitamins in all Jogofir variants.
These results were compared with the yogurt fermented with bacterial probiotic cultures only (control
yogurt).
Conclusion: Jogofir fermented with yeast culture had superior quality properties comparing to the
control yogurt. This novel probiotic beverage meets the Serbian legal provision on the quality of dairy
products and starter cultures, containing more than 106/ml or 106/g live cells of probiotic cultures
during processing and storage life and having pH superior than 3.8. Sensory characteristics, smell,
taste and color of Jogofir are inherent to the probiotic yogurt.
KEYWORDS: Yogurt, Kluyveromyces m.f. B0399, Probiotic cultures
129
Co-fermentation with non-Saccharomyces and Saccharomyces strains increases
acetaldehyde accumulation. Effect on anthocyanin derived pigments in tannat
red wines
Karina Medina1, Eduardo Boido1, Laura Fariña1, Eduardo Dellacassa2,
Francisco Carrau 1
Sección Enología, Cátedra de Ciencia y Tecnología de Alimentos; 2Cátedra de Farmacognosia y Productos Naturales,
Departamento de Quimica Orgánica, Facultad de Quimica, Universidad de la Republica, 11800 Montevideo, Uruguay
1
[email protected]
Although it is well known that phenolic compounds have great impact in the sensory characteristics of
quality wines, yeast and phenolic compound interactions are some of the least studied wine processes
during vinification. It has been demonstrated that during fermentation, S. cerevisiae releases secondary
metabolic products into the medium, such as pyruvic acid and acetaldehyde, some of which react
with anthocyanins to produce vitisin A, vitisin B, and ethyl-linked anthocyanin-flavanol pigments.
However, very limited reports are found about non-Saccharomyces effects in grape fermentation.
In this work, six non-Saccharomyces yeast strains, belonging to the genera Metschnikowia and
Hanseniaspora were screened for their effect on red wine color and fermentation capability for
winemaking in pure and mixed culture conditions with Saccharomyces. An artificial red grape must
was prepared containing a polyphenol extract of Tannat grapes that allow monitoring changes of key
phenol parameters during fermentation without skin solids in the medium. When fermented in pure
cultures S. cerevisiae result in highest concentration of acetaldehyde and vitisin B compared to M.
pulcherrima M00/09G, Hanseniaspora guillermondii T06/09G, Hanseniaspora opuntiae T06/01G,
Hanseniaspora vineae T02/05F, and Hanseniaspora clermontiae (A10/82F and C10/54F). However,
co-fermentation of H.vineae and H. clermontiae species with S. cerevisiae result in significant higher
concentration of acetaldehyde in mixed culture treatments compared to the pure S. cerevisiae control.
Analysis with HPLC-DAD-MS, confirmed increase formation of Vitisin B (acetaldehyde reaction
dependent) in co-fermentation treatments compared to the pure Saccharomyces fermentation,
suggesting the key role of acetaldehyde.
Further studies with mixed cultures in this chemical defined medium, could explain this increase
acetaldehyde formation.
KEYWORDS: Non-Saccharomyces yeasts, Anthocyanins, Anthocyanin-derived pigments, Wine
color, Tannat
130
Yeast-aided purification of levansucrase-produced
prebiotic fructooligosaccharides
Eerik Jõgi1, Katrin Viigand1, Kati Metsla1 , Triinu Visnapuu1, Heiki Vija2, Tiina Alamäe1
1 University of Tartu, Institute of Molecular and Cell Biology, Riia 23, Tartu, Estonia;
2 National Institute of Chemical Physics and Biophysics, Akadeemia 23, Tallinn, Estonia
[email protected]
We have described a catalytically powerful levansucrase Lsc3 from Pseudomonas syringae pv.
tomato which produces from sucrose not only levan, but also potentially prebiotic levan-type
fructooligosaccharides (FOS) effects of which are yet poorly studied. At levan precipitation, residual
sucrose, some fructose and lots of glucose stay in the supernatant. For prebiotic potency assay of
the FOS, glucose, fructose and sucrose should be removed from the product. Selective fermentation
of sugar mixtures by yeasts can be used to remove monosugars. Invertase-negative mutant Y02321
of Saccharomyces cerevisiae from Euroscarf collection was used by us for 24 h treatment of the
FOS mixture under static or aerobic batch conditions. Sugars, organic acids and alcohols in yeasttreated product were quantified by HPLC. Viability and metabolic status of the yeast was estimated
by staining of cells with methylene blue and fluoresceine diacetate.
Fructose and glucose, but not sucrose, were effectively removed under all studied conditions and
some glycerol, succinate, ethanol and acetate were produced as by-products. The static treatment
yielded less organic acids and a more glycerol than aerobic one. Metabolic proficieny of the yeasts
temporarily dropped in the beginning of the treatment till the cells adapted to osmotic stress. The yeast
cells stayed viable during the entire treatment, althought their metabolic activity largely decreased
after glucose exhaustion.
Fermentative removal of monosaccharides from oligosaccharidic mixtures by microbes is often
performed with no attention to physiological status of the microbe or by-products produced. We have
addressed this issue and show that our yeast-based procedure is feasible to yield a FOS preparation
for prebiotic efficiency studies on bacterial pure cultures and fecal consortia.
KEYWORDS: Levansucrase, FOS, Invertase-negative yeast
FUNDING: ERC 9072 and ERF 3.2.0701.12-0041 to TA
131
Session 2A- Yeasts in food biotechnology: biodiversity and ecology in foods and beverages
Dynamic behavior of inoculated yeast starters during cocoa fermentations and
their effect on sensory characteristics of chocolate
Nadia N. Batista, Cíntia L. Ramos, Disney D. Ribeiro, Ana C. M. Pinheiro, Rosane F.
Schwan
Department of Biology, Federal University of Lavras, 37.200-000, Lavras, MG, Brazil
[email protected]
The dynamic of Saccharomyces cerevisiae, Pichia kluyveri and Hanseniaspora uvarum during
spontaneous and inoculated cocoa fermentations and their effect on sensory characteristics of
chocolate were investigated. Yeast populations were assessed by qPCR. S. cerevisiae was predominant
during spontaneous (average 5.4 log cell/g) and inoculated (average 7.2 log cell/g) fermentations.
The H. uvarum seemed to be suppressed by the other two yeasts, as it showed similar population
(approximately 4.0 log cell/g) even in the inoculated assay. Carbohydrates were consumed quickly
at inoculated fermentation (68% and 42% were consumed in the inoculated and control assays
respectively, at 24 h). Ethanol content was higher in the inoculated (8.3 g/kg at 48 h) than in the
control (4.6 g/kg at 96 h) fermentation. Consumers did not report a significant preference for either
chocolate (p<0.5). However, differences in the flavor attributes were noted, as consumers reported
stronger coffee and sour attributes in the inoculated assay. This is the first time qPCR has been used
to assess the dynamic of yeasts during the complex fermentation of cocoa beans. The inoculation
accelerated the process. S. cerevisiae and P. kluyveri likely contributed coffee, sour and bitter flavors
to the inoculated chocolate
KEYWORDS: Cocoa fermentation, Starter culture, Chocolate, qPCR, Sensory analysis
132
Coffee mucilage degraded by yeasts that present hydrolytic activity
Silvana Arreola1, Mirely Guevara1, Rubén Moreno-Terrazas1, Lorena Pedraza1,
Patrícia Lappe-Oliveras2, Rebeca Romo1
Universidad Iberoamericana, Dpto. de Ing. y C. Químicas; 2Instituto de Biología, Universidad Autónoma de México,
Mexico
1
[email protected]
The degradation of the coffee mucilage occurs during the fermentation of the coffee cherries, there are
around 5-9x10^6 of microorganisms/mL that are present in wet fermentation and help to degrade the
mucilage, mainly they are yeast. The main of this work was identify the yeasts involve in the coffee
mucilage degradation and their hydrolytic activity. The samples were obtained from a organic artisanal
farm in Xomotla-Ver. Mexico, at 0 h, 12 h and 24 h, from the coffee cherries wet fermentation. The yeasts
obtained, were quantified, isolated and purified in order to identify through the PCR amplification by the
sequencing of the D1/D2 rDNA. The yeasts that presented pectinolytic activity and with possibilities
to present another hydrolytic activity, were grown in different agar media with xylan, starch, lignin
and cellulose, as a sole carbon source. The behavior of the microorganisms in wet fermentation had
growth from 10^4 to 10^5 UFC/mL at the end of the process. It was obtained 12 different species
(Candida glabrata, Debaryomyces hansenii, Galactomyces geotrichum, Hanseniaspora uvarum,
Hyphopichia burtonii, Kodamaea ohmeri, Meyerozyma guilliermondii, Pichia kluyveri, Pichia
kudriavzevii, Rhodotorula mucilaginosa, Torulaspora delbrueckii, Wickerhamomyces anomalus) of
which 6 present another hydrolytic activity, T. delbrueckii presented activity in all the carbohydrates
polymers evaluated and W. anomalus only in cellulose did not have. Presence of these indigenous
yeasts in wet fermentation, with hydrolytic activity may degrade faster and efficiently the mucilage
of coffee cherries and with possibility to improve the process using these microorganisms as starters
in organic coffee and also to degrade byproducts.
KEYWORDS: Yeasts, Pectinolytic, Xylanolytic, Amilolytic, Cellulolytic
REFERENCES:
de Melo Pereira GV, Soccol VT, Pandey A, Medeiros AB, Andrade Lara JM, Gollo AL, Soccol CR (2014). Isolation,
selection and evaluation of yeasts for use in fermentation of coffee beans by the wet process. International Journal of
Food Microbiology 1(188):60-6
Avallone Sylvie, Brillouet Jean M, Guyot Bernard, Olguin Eugenia, Guiraud Joseph P (2002). Involvement of pectolytic
micro – organisms in coffee fermentation. International Journal of Food Science and technology
37: 191-198
Pereira Rodarte Marian, Ribeiro Dias Disney, Marques Vilela Danielle, Freitas Schwan Rosane (2011). Proteolytic
activities of bacteria, yeasts and filamentous fungi isolated from coffee fruit. Acta Scientiarum Agronomy
33 (3): 457-464
133
Microevolution in FLO and MAL genes of industrial brewers’ yeasts
Anna Misiewicz, Anna Goncerzewicz
Prof. Wacław Dabrowski Institute of Agricultural and Food Biotechnology, Microbiology Department, Culture
Collection of Industrial Microorganisms, Warsaw, Poland
[email protected]
The exact knowledge of the genetic bases of the variation in industrial brewer’s yeast strain of
Saccharomyces cerevisiae is the primary criterion for selecting and matching the strain for the
biotechnological process.
The objective of the research was to carry out a comparative analysis of populations of brewer’s yeast
strains, determine the degree of polymorphism of multigene nucleotide sequences of the subtelomeric
gene families FLO and MAL.
20 brewers’ strains from Culture Collection of Industrial Microorganisms, prof Wacław Dabrowski
Institute of Agricultural and Food Biotechnology, PCR analysis: Perkin Elmer 2400 (USA) and
Thermo Px2 (USA). Sequencing: CEQ 8000 Genetic Analysis System (Beckman Coulter, USA),
Bioinformatics: BLAST (www.ncbi.nlm.nih.gov/BLAST), SGD (www.yeastgenome.org).
The intrastrain variation was significantly higher among amplicons of the FLO and MAL genes in
the strains KKP 171, KKP 199, and KKP 211, detected by MSSCP method. The differences in the
structure of the FLO and MAL genes result in the production of various protein products responsible
for the processes of flocculation and degradation of sugar and thus for the interstrain diversity of
biotechnological properties. The existence of 120 polymorphic, syntenic active sites with a different
nucleotide variation in the FLO1 gene at the same nucleotide positions in relation to the gene reference
strain S288c.
The polymorphism of industrial yeast strain s may reflect their adaptative evolution extending the
possibilities of using those strains in technological processes.
134
Influence of a selected Hanseniaspora uvarum strain in multistarter grape must
fermentations with Saccharomyces cerevisiae
Mariana Tristezza1, Corallo Daniela2, Giovanni Cantele2, Maria Tufariello1, Giovanni Mita1,
Giuseppe Spano3, Francesco Grieco1
Istituto di Scienze delle Produzioni Alimentari, Consiglio Nazionale delle Ricerche, Unità Operativa di Supporto
di Lecce, Lecce, Italy; 2Azienda Vinicola Cantele, Guagnano, Lecce, Italy; 3 Dipartimento di Scienze Agrarie, degli
Alimenti e dell’Ambiente, Università di Foggia, Foggia, Italy
1
[email protected]
In conventional winemaking, grape must fermentations are carried out by a succession of different
yeast species. The Saccharomyces cerevisiae completes the process, whereas its initial stage is
dominated by non-Saccharomyces strains, whose by-products contribute to the composition of the
wine bouquet. In this study, we evaluated the performance of two selected strains of Hanseniaspora
uvarum and S. cerevisiae as multistarters for inoculation of Negroamaro must vinification.
Two previously described H. uvarum ITEM8795 (De Benedictis et al 2011) and S. cerevisiae
ITEM6920 (Tristezza et al 2012) strains were used. Their were used in composite culture to inoculate
microvinifications of Negroamaro must at lab (500mL), pilot (100L) and industrial (70-100q) scale.
Fermentations were constantly monitored and produced wines were chemically and microbiologically
analyzed.
Although the reliable production of secondary products by single culture of H uvarum ITEM8795,
desirable amounts of them were detected in mixed culture. The kinetics of fermentation and yeast
strains growth and the analytical profiles of the wines produced showed that the ITEM8795 strain
can be used with S. cerevisiae starter cultures to enhance the volatile composition and to reduce the
volatile acidity of the final product.
The results of this investigation corroborate the concept that non-Saccharomyces yeasts play a essential
role in wine-bouquet formation and their action could be enhanced by the selection of suitable strains
able to appropriately cooperate with S. cerevisiae.
Acknowledgments This research was supported by the Italian MIUR - Project S.I.Mi.S.A. PON02_00186_3417512/1
KEYWORDS: Wine, Autochthonous yeast, Multistarter, Hanseniaspora uvarum, Negroamaro
REFERENCES:
De Benedictis M, Bleve G, Grieco F, Tristezza M, Tufariello M, Grieco F (2011). An optimized procedure for the
enological selection of non-Saccharomyces starter cultures. Antonie Van Leeuwenhoek 99:189-200
Tristezza M, Vetrano C, Bleve G, Grieco F, Tufariello M, Quarta A, Mita G, Spano G, Grieco F (2012). Autochthonous
fermentation starters for the industrial production of Negroamaro wines. Journal of Industrial Microbiology and
135
Filter media evaluation for the removal of Brettanomyces bruxellensis from wine
Filomena L. Duarte, Luis Coimbra, Margarida Baleiras-Couto
Instituto Nacional de Investigação Agrária e Veterinária, INIAV-Dois Portos, Quinta da Almoínha, 2565-191 Dois
Portos, Multifiltra – Filtração e Equipamentos Industriais, Lda., 2735-003
Cacém, Portugal
[email protected]
The presence of Brettanomyces bruxellensis is of great concern for wine producers due to off-flavor
production, particularly 4-ethylphenol and 4-ethyl guaiacol, as well as tetrahydropyridines, acetic
acid, ethyl acetate and isovaleric acid. One of the available tools for eliminating contamination with
these yeasts is filtration. (Suarez et al 2007) stated that effective removal of Brettanomyces cells
is achieved using membranes with a pore size smaller than 0.45 μm. In the present work different
filtration media, which differ in terms of composition and micron rating, were compared for the
efficacy of B. bruxellensis removal from wine.
The FDA Guidelines on aseptic processing, which are intended to provide a margin of safety well
beyond what would be expected in wine production, were followed. Filter removal capacity was
evaluated by plate counting. Red wine inoculated with B. bruxellensis was used to perform duplicate
laboratory tests with filters from different production lots, whenever possible. The tested filters
(Amazon Filters) were of different media and micron rating: borosilicate glass microfiber (X, V),
polypropylene (PP 0.6 and PP 1.0 mm) and polyethersulphone (PES) (0.45, 0.65, 1.0 mm).
The results obtained showed low retention (lower than 104) of B. bruxellensis in V grade borosilicate
glass microfiber filters, while X grade presented high retention efficacy, as no cells were detected in
the filtrated wine analyzed. Polypropylene filters showed poor efficacy as the wine maintained a high
microbial charge with PP 1.0 filtering discs while with PP 0.6 a reduction of around 104 was observed.
Regarding polyethersulphone filters PES 1.0, PES 0.65 and PES 0.45 no B. bruxellensis cells were
detected in the filtrated wine analyzed considering the different lots and respective duplicates tested.
Thus, PES filters showed high B. bruxellensis removal efficacy in all micron rating tested. Similar
efficacy was achieved for X grade borosilicate glass microfiber filter. On the opposite, low retention
was obtained with polypropylene and V grade borosilicate glass microfiber filters. Different filter
media with similar micron rating showed different retention of B. bruxellensis, highlighting the
relevance of filter media on removal mechanisms.
KEYWORDS: Brettanomyces bruxellensis, Filtration, Wine
REFERENCES:
Suárez R, Suárez-Lepe JA, Morata A, Calderón F (2007). The production of ethylphenols in wine by yeasts of the
genera Brettanomyces and Dekkera: A review. Food Chemistry 102:10–21
136
Session 2A :Yeasts in food biotechnology: biodiversity and ecology in foods and beverages
The impact of the adaptation to different nutritional environments reveals
diverse strategies in nitrogen consumption between Saccharomyces cerevisiae
strains
Claire Brice1,3, Francisco A. Cubillos1,2, Sylvie Dequin3,4,5, Carole Camarasa3,4,5,
Claudio Martinez1,2
Departamento de Ciencia y Tecnologıa de los Alimentos, Universidad de Santiago de Chile (USACH), Santiago, Chile;
2
Centro de Estudios en Ciencia y Tecnologıa de Alimentos (CECTA), Universidad de Santiago de Chile (USACH),
Santiago, Chile; 3INRA, UMR1083, F-34060 Montpellier, France; 4SupAgro, UMR1083, Montpellier, France;
5
Universite´ Montpellier 1, UMR1083, Montpellier, France
1
[email protected]
The strains populations of Saccharomyces cerevisiae are derived from different geographical origins,
corresponding to extreme living environments. These adaptive pressures to various ecological niches
generated behavioral differences between these strains, particularly in the fluxes of nitrogen sources
consumption and consequently impact in their fermentation capacities. This phenotypic variability
has been exploited by man for the production of diverse beverages (wine, sake, rum) from various
matrices with different nitrogen composition. Nowadays the molecular mechanisms underlying these
differences remain poorly elucidated.
In this context, this project aims to determine the genomic adaptations of yeast strains responsible
for the phenotypic diversity regarding their ability to use the nitrogen sources in wine fermentation
environments. To this end, we implemented a combined approach that includes a physiological
characterization of a panel of strains from different food processes, and transcriptomic analysis,
focusing our attention on the capacity of strains to use nitrogen sources”.
First results on physiological characterization of strains deriving from different geographical origins,
show differences in the total amount of consumed nitrogen, reflecting differences in their ability
to use nitrogen. The composition of the residual nitrogen appeared to be in line with the nitrogen
content of the ecological niches of which each strain originated. Moreover, changes in the dynamic of
assimilation of individual amino acids were evidenced, suggesting differences between strains in the
regulatory mechanisms of nitrogen transporters.
137
Pilot scale assay of co-fermentation with Metschnikowia pulcherrima using
different grape varieties
Filomena L. Duarte1, Fernando Pedrosa2, Nuno Alves 2, Patrícia Marques1, Margarida
Baleiras-Couto1
Instituto Nacional de Investigação Agrária e Veterinária, INIAV - Dois Portos, Quinta da Almoínha, 2565-191 Dois
Portos, Portugal; 2Proenol-Indústria Biotecnológica, Lda., Vila Nova de Gaia, Portugal
1
[email protected]
The benefits of adding non-Saccharomyces yeasts in wine production are the focus of a great deal
of studies among wine yeast researchers (Teixeira et al 2015). The sensory diversification achieved
with the use of these yeasts motivated the industry to bring to the market a large number of products
with non-Saccharomyces yeasts. The present work reports a pilot-scale assay of co-fermentation
with commercial Metschnikowia pulcherrima, FLÁVIA® (Lallemand), tested in different grapevine
varieties. The addition of this commercial yeast has been recommended primarily for fermentation of
white grape varieties. The study presented here was the first comparative assay using this commercial
yeast for the production of elementary wines from white and red varieties.
Vinification of five white and nine red grape varieties took place in stainless steel vats of 50-60 L.
For each grape variety two modalities were assayed, one with the addition of M. pulcherrima at time
zero and addition of Saccharomyces cerevisiae after 24 h (Flavia), and another one using only S.
cerevisiae at time zero (Control). Fermentation was monitored by daily measurement of density and
temperature. Triangle and ranking tests were performed to the wines obtained, using an experienced
sensory panel. Sensory characteristics were also described.
For both modalities alcoholic fermentation proceeded smoothly until complete consumption of the
sugars. In the red grape varieties, malolactic fermentation took place without defects. Physicochemical
results were similar between modalities. The results of the triangle tests performed to the wines from
each modality of the varieties Alvarinho, Seara Nova, Verdelho, Cabernet Sauvignon, Caladoc and
Tinta Barroca revealed sensory differences at a significance level of 10 %. With the ranking tests
performed to Touriga Nacional, Aragonês and Syrah no differences between Flavia and Control wines
were detected. A summary of the sensory characteristics performed in four open sessions will be
presented, highlighting the description notes of the wines obtained for each modality. This work
emphasizes the grape variety as an important factor, not always considered in studies with nonSaccharomyces yeasts.
KEYWORDS: Metschnikowia pulcherrima, Fermentation, Wine, Sensory analysis
REFERENCES:
Teixeira A, Caldeira I, Duarte FL (2015). Molecular and enological characterization of Touriga Nacional nonSaccharomyces yeasts. Journal of Applied Microbiology 118:658-671
138
Antioxidant enzyme activity response to selenium in wine yeasts
Margarida Baleiras-Couto1, Mónica Assunção1,2, Luísa L. Martins2, Miguel P. Mourato2
Instituto Nacional de Investigação Agrária e Veterinária, I.P., Dois Portos, Quinta da Almoínha, 2565-191 Dois Portos,
Portugal; 2LEAF, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisbon,
Portugal
1
[email protected]
Selenium is an essential micronutrient with an antioxidant and anti-carcinogenic role in human
and animal health. The use of supplements in the diet is a common practice to address nutritional
deficiencies. Some of these supplements contain selenium salts, such as sodium selenate or sodium
selenite, but the most commonly used are prepared based on selenium-enriched yeasts (Ponce de León
et al 2002). The production of such cells may hamper selenium toxicity problems. The possibility of
producing wine with selenium-enriched yeasts, led us to investigate the selenium tolerance of wine
related yeasts. Antioxidant response mechanisms with different concentrations of sodium selenite
were evaluated.
Yeast strains of Starmerella bacillaris, Hanseniaspora guilliermondiiT, Hanseniaspora uvarum,
Lachancea thermotoleransT, Saccharomyces cerevisiae and Torulaspora delbrueckii species were
tested. Cell growth was monitored following optical density (640 nm) in YEPD broth with two
concentrations of sodium selenite: 5 and 100 µg mL-1. Preparation of cell extract for determination
of antioxidant enzyme activity was performed in cells grown in YEPD with three concentrations of
Na2SeO3 (0, 5, 100 or 250 µg mL-1). Statistical analysis of variance with one factor (ANOVA) was
performed to assess the effect of the three concentrations of selenium in each of the strains tested.
Viability assays demonstrated that the yeast strain Torulaspora delbrueckii showed the highest
tolerance for the tested levels of 100 µg mL-1 of sodium selenite. The evaluation of antioxidant
enzyme activities showed the best performance for concentrations of 250 µg mL-1 and 100 µg mL1
, respectively for the yeast species Saccharomyces cerevisiae and Hanseniaspora guilliermondii.
These results encourage future studies on the possibility of using pre-enriched yeast cells as selenium
supplement in wine production. The possible use of pre-enriched yeast strains to produce wine
supplemented with selenium will be investigated by testing the behavior of these yeast strains in pilot
scale vinification processes.
KEYWORDS: Wine-yeast, Selenium, Antioxidant enzymes
REFERENCES:
Ponce de León CA, Bayón MM, Paquin C, Caruso JA (2002). Selenium incorporation into Saccharomyces cerevisiae
cells: a study of different incorporation methods. Journal of Applied Microbiology 92:602-610
139
Comparison of sensory characteristics of non-alcoholic beverage kvass
fermented with Saccharomyces cerevisiae and Kluyveromyces marxianus
Ivo Lidums, Daina Karklina
Latvia University of Agriculture, Faculty of Food Technology, Latvia
[email protected]
Kvass is a non-alcoholic beverage produced by fermenting kvass mash with yeast (traditionally
Saccharomyces cerevisiae); alcohol content in kvass must be less than 1.2% alcohol by volume.
Since dairy yeast Kluyveromyces marxianus is positive for glucose and sucrose fermentation, it
could possibly be applied to beverage production. The aim of this research was to compare sensory
characteristics of kvass fermented with S. cerevisiae and K. marxianus. Strain K. marxianus DSM
5422 was obtained from Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell
Cultures and maintained on agar plates containing 2% glucose and recultivated in semi synthetic
medium containing lactose 50 g/L, yeast extract 5 g/L, MgSO4·7H2O 1.4 g/L, KH2PO4 1.0 g/L, K2HPO4
0.1 g/L, (NH4)2SO4 5.0 g/L at 30 ̊C with agitation 180 rpm. Naturally fermented bread kvass was
made from dried rye bread rusks which were soaked in hot water to obtain kvass mash; fermentation
of kvass mash took 9 h at 29 ± 1 °C. Kvass was matured for 12 h at 6 ± 1 °C, total production time
was 25 h. Dry matter content (ISO 2173:2003) and active acidity (ISO 10523:2012) were determined
kvass samples. Hedonic evaluation and line scale (ISO 4121:2003) were used to determine sensory
characteristics of kvass samples (26 trained panellists): kvass fermented with S. cerevisiae (sample A)
and K. marxianus (sample B). Sample A had higher dry matter content (8.6%) and lower pH (3.88)
compared to sample B (7.0% and 4.60, respectively). Hedonic evaluation showed that there were
not significant differences (p>0.05) between the preference of kvass samples. The intensity of colour
and acidity was significantly more pronounced in kvass fermented with S. cerevisiae, the intensity of
aroma and flavour were similar in both kvass samples. The results suggest that K. marxianus DSM
5422 is suitable for kvass fermentation and production as the most important sensory parameters –
flavour and aroma – were acceptable.
KEYWORDS: kvass, Saccharomyces cerevisiae, Kluyveromyces marxianus, sensory evaluation
REFERENCES:
Hugenholtz J (2013). Traditional biotechnology for new foods and beverages. Current Opinion in Biotechnology
24:155–159
López-Alvarez A, Díaz-Pérez AL, Sosa-Aguirre C, Macías-Rodríguez L, Campos-García J (2012). Ethanol yield
and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing
with Saccharomyces cerevisiae baker’s yeast used in tequila production. Journal of Bioscience and Bioengineering
113(5):614–618
140
Gas bubble formation in fermenting and non-fermenting yeasts
1
Evodia Kgotle1, Khumisho Dithebe1, Carolina Pohl1, Hendrik Swart2, Elizabeth Coetsee2,
Pieter van Wyk3, Chantel Swart1,3
UNESCO-MIRCEN: Department of Microbial, Biochemical and Food Biotechnology; 2Department of Physics; 3Centre
for Microscopy, University of the Free State, P.O. Box 339, Bloemfontein, 9300, South Africa
[email protected]
During fermentation ethanol and carbon dioxide (CO2) is produced and excreted into the environment.
It is therefore expected that CO2 would be present in the cytoplasm of cells before it is released.
Although previous studies (Hemmingsen & Hemmingsen 1979) indicated that gas bubbles cannot be
formed in the cytoplasm of cells, Swart and co-workers (Swart et al 2012) discovered gas bubbles
in the Crabtree positive yeast, Saccharomyces. This study investigates the conserved status of gas
bubble formation in Crabtree negative and strictly respiring yeasts.
Crabtree positive, Crabtree negative and strictly respiring yeasts were grown on fermentable and nonfermentable media and analysed with various microscopic techniques to determine the gas bubble
status.
Light microscopy indicated that Crabtree positive yeasts contained a large number of gas bubbles
compared to Crabtree negative and strictly respiring yeasts. Results were verified with transmission
electron microscopy and nano scanning Auger microscopy.
The gas bubble phenomenon seems to be conserved in yeasts, however the amount of gas bubbles
formed is affected by the mode of CO2 production, i.e. fermentation, respiration or both.
KEYWORDS: Fermentation, Gas bubbles
REFERENCES:
Hemmingsen EA, Hemmingsen BB (1979). Lack of intracellular bubble formation in microorganisms at very high gas
supersaturations. Journal of Applied Physiology Respiratory Environmental Exercise Physiology 47:1270–1277
Swart CW, Dithebe K, Pohl CH, Swart HC, Coetsee E, Van Wyk PWJ, Swarts JC, Lodolo EJ, Kock JLF (2012) Gas
bubble formation in the cytoplasm of a fermenting yeast. FEMS Yeast Research 12:867–869
141
Exposing gas bubbles in the fungus Rhizopus oryzae
Susanna Saaiman1, Carolina Pohl1, Pieter van Wyk2, Chantel Swart1,2
UNESCO-MIRCEN: Department of Microbial, Biochemical and Food Biotechnology;
Centre for Microscopy, University of the Free State, P.O. Box 339, Bloemfontein, 9300,
South Africa
1
2
[email protected]
Fermentation produces carbon dioxide and ethanol that is excreted into the environment. Therefore it is
expected that fermenting microorganisms will contain intracellular gas bubbles that have not yet been
released into the environment. However, no gas bubbles have been observed in cells (Hemmingsen
et al 1990) until 2012, when gas bubbles were discovered in Saccharomyces (Swart et al 2012). This
study investigates the conserved status of gas bubble formation in the fermenting fungus Rhizopus
oryzae which is commonly used in industry for the production of tempeh, fumaric acid, lactic acid,
ethanol and enzymes via fermentation (Ghosh et al 2011).
R. oryzae was cultivated in fermentable and non-fermentable media and analysed using Light
Microscopy (LM), Transmission Electron Microscopy (TEM) and Nano Scanning Auger Microscopy
(NanoSAM).
The results indicated that gas bubbles are present in R. oryzae in large numbers during fermentation
as observed by LM and verified by TEM and NanoSAM.
Since gas bubbles are present in R. oryzae, we concluded that gas bubble formation may be conserved
in fungi.
KEYWORDS: Fermentation, NanoSAM
REFERENCES:
Hemmingsen BB, Ducoeur LC, Grapp SJ, Skaug V, Hemmingsen EA (1990). Gas supersaturation tolerances in
amoeboid cells before and after ingestion of bubble-promoting particles. Cell Biophysics 7:37-51
Swart CW, Dithebe K, Pohl CH, Swart HC, Coetsee E, van Wyk PWJ, Swarts JC, Lodolo EJ, Kock JLF (2012). Gas
bubble formation in the cytoplasm of a fermenting yeast. FEMS Yeast Research 12(7):867-869
Ghosh B, Ray RR (2011). Current commercial perspective of Rhizopus oryzae: A review. Journal of Applied Science
11(14):470-486
142
Extracellular fructanase production by Kluyveromyces marxianus var.
drosophilarum from Agave tequilana fructan
Zazil Escalante1, Rosa Corona1 Amador Campos1, Rosa Camacho2, Enrique Arriola1,
Guadalupe Guatemala2, Carlos Pelayo1
Departamento de Ingeniería Química, Universidad de Guadalajara, C.P. 44430, Guadalajara, Jalisco, México; 2Unidad
de Tecnología Agroalimentaria, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C.
(CIATEJ), A.C., C.P. 44270, Guadalajara, Jalisco, México
1
[email protected]
Nowadays fructose and fructooligosaccharides (FOS) are in great demand by the nutraceutical and
food industries, the development of prebiotics and high fructose syrups (HFS). Fructose and FOS
can be obtained from Agave tequilana Weber var. Blue fructans (ATF) by enzymatic hydrolysis
by fructanase. The ATF are highly branched structures with fructose waste residues between links
predominant type β(2-1) and β(2-6) (Arrizon et al 2011). The aim of this study was to evaluate
the production of fructanases from K. marxianus strain isolated from tequila (worldwide known
traditional Mexican drink) using ATF as inductor.
K. marxianus var. drosophilarum a new strain isolated from tequila wort and K. marxianus ATCC
26548 were used. The effect of pH (4-8) and temperature (24-36°C) was studied with a culture medium
components (g/L): ATF (5-35), (NH4)2HPO4 (0.5-10.25), yeast extract (10-47.5) and NH4NO3 (1-10).
A unifactorial design with central composite design (CCD) with five central points was used, and
Statgraphics Centurion XV analyzed results.
The highest production of fructanases for K. marxianus var. drosophilarum and K. marxianus ATCC
26548 was at 30°C, while the pH between 4 and 5 was best for K. marxianus var. drosophilarum with
fructanase activity of 15.2 U/ml. For K. marxianus ATCC 26548 the best pH was between 6 and 7
with fructanase activity of 11.14 U/ml. These results show that even in the case of the same gender
and species, their behavior is different and is likely to also produce enzymes that are. The CCD
indicated that the most influential components in the production of fructanases of K. marxianus var.
drosophilarum were ATF and yeast extract, and highest fructanases production was obtained with
22.5 g/L of yeast extract and 32.5 g/L of ATF getting 23 U/ml. Components of the culture medium
for production of inulinases have been evaluated before, but in this study the type of substrate is ATF
which can induce the production of levan-type enzymes.
KEYWORDS: Fructanase, K. marxianus var. drosophilarum, Agave tequilana
REFERENCES:
Arrizon J, Morel S, Gschaedler A, Monsan P (2011). Purification and substrate specificities of a fructanase from
Kluyveromyces marxianus isolated from the fermentation process of Mezcal. Bioresource Technology 102:3298–3303
143
Influence of the yeast specie on the volatile compounds in cider production
Laura Iñiguez, Anne Gschaedler-Mathis, Manuel Kirchmayr, Melchor Arellano-Plaza
Centro de investigación y asistencia en tecnología y diseño del estado de Jalisco A.C. Biotecnología industrial. Av.
Normalistas #800, Guadalajara, Jalisco, México. C.P. 44270
[email protected]
Cider is an alcoholic beverage produced using apple juice which is fermented by yeast and a malolactic
bacteria biotransforming the malic acid (Valles et al 2007). The aroma compounds produced in cider
fermentations vary considerably depending on: the apple specie, the yeast strain, the temperature
of fermentation, among others factors. The main yeast used in cider production is Saccharomyces
cerevisiae (Madrera et al 2008); however, other yeast species could be employed. In this work, nonSaccharomyces yeast (Pichia membranaefaciens, Zygosaccharomyces rouxii and Kluyveromyces
marxianus) and a commercial Saccharomyces cerevisiae were used for the alcoholic fermentation of
fresh apple juice (16°Bx). The fermentative capacity and major volatile compounds were evaluated
by gas chromatography coupled with head-space. The non-Saccharomyces yeasts were not able to
yield high ethanol levels and achieved only 50% of the ethanol produced by commercial S. cerevisiae.
Higher alcohols, principally 1-propanol and isobutanol, were produced at higher levels (40% more
than S. cerevisiae), isoamyl acetate and ethyl acetate were produced at highest levels by K. marxianus
(380 and 41 mg/L, respectively in the fermented must). Malic acid was not consumed during the
alcoholic fermentation then it seems necessary to achieve malolactic fermentation in order to decrease
its concentration in the final product. Lactic and acetic acids were produced by the non-Saccharomyces
but were not detected in S. cerevisiae. The results obtained show that non-Saccharomyces are necessary
in the cider production to increase the volatile quality but, the ethanol production is not sufficient;
it is necessary to modify the fermentation to raise the ethanol concentration maybe by mixing a
S. cerevisiae with a non-Saccharomyces or adding some nutrients to the apple juice necessary to
warranty the yeast growth and the success of the fermentation.
KEYWORDS: Cider, non-Saccharomyces, Volatile compounds
REFERENCES:
Valles BS, Bedriñana RP, Tascón NF, Simón AQ, Madrera RR (2007). Yeast species associated with the spontaneous
fermentation of cider. Food microbiology 24(1):25-31
Madrera RR, Hevia AG, García NP, Valles BS (2008). Evolution of aroma compounds in sparkling ciders. LWT-Food
Science and Technology 41(10):2064-2069
144
Study of the fungal dynamics of a raw ewe’s milk Pecorino cheese traditionally
produced in a foothills area of the Marche region (central Italy)
Federica Cardinali, Manuela Taccari, Vesna Milanović, Cristiana Garofalo, Andrea Osimani,
Lucia Aquilanti, Silvia Zitti, Roberta Foligni, Massimo Mozzon, Francesca Clementi
Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, Università Politecnica delle Marche, via Brecce Bianche,
60131 Ancona, Italy
1
[email protected]
The study was aimed at investigating the fungal dynamics and diversity of a Pecorino cheese (called
Caciofiore della Sibilla) traditionally manufactured in the Marche region with raw ewe’s milk and an
aqueous extract of Carlina acanthifolia All. as a coagulating agent.
During cheese-making and ripening (20 days) of the Pecorino cheese the diversity and dynamics
of the fungal community were evaluated through a combined PCR-DGGE approach, based on the
analysis of the DNA extracted directly from the samples (raw milk, curd, cheese, basal portion of
the stem, petioles of the leaves and aqueous extract of C. acanthifolia All.) and the bulk cells of
eumycetes, prepared by harvesting colonies from Rose Bengal Agar (RBA) medium.
The PCR-DGGE analysis of the cultivable fraction revealed the dominance of Debaryomyces hansenii
and Pichia kudriavzevii during the whole ripening process (from the 1st to the 20th day of ripening).
Other species were also detected, namely: Candida zeylanoides, Rhodotorula mucilaginosa and
Candida membranafaciens at the sole 1st day of ripening; Galactomyces candidus, Yarrowia lipolytica
and Meyerozyma guilliermondii at the 3th and 20th day; Pichia kluyveri and Candida incospicua at day
1, 3 and 20.
The PCR-DGGE analysis of the DNA extracted directly from the samples revealed a lower biodiversity,
confirming the sole presence of D. hansenii from the 1st to the 20th day of ripening, C. zeylanoides (at
the sole 20th day of ripening) and G. candidus (at the 3th and 20th day).
The overall results collected onto this specialty cheese revealed a succession of the fungal species
composition along the ripening process, with P. kudriavzevii and D. hansenii being dominant. The
latter species was stably detected together with C. zeylanoides also in the raw milk, in the basal
portion of the stem, petioles of the leaves and in the aqueous extract of C. acanthifolia All.
KEYWORDS: Caciofiore della Sibilla, Carlina acanthifolia All., Yeasts, Moulds, PCR-DGGE
145
Wine grape S. cerevisiae diversity and population structure reveal possible
genetic admixture of vineyard and commercial wine strains
Marine Börlin1, Franck Salin2, Jean-Luc Legras3, Isabelle Masneuf-Pomarede1
Univ. Bordeaux, ISVV, Unité de recherche Œnologie EA 4577, USC 1366 INRA, Bordeaux INP, 33140 Villenave
d’Ornon, France ; 2UMR Biodiversité Gènes et Ecosystèmes, PlateForme Génomique INRA, Pierroton, France;
3
UMR1083 Science pour l’Œnologie, Montpellier, France
1
[email protected]
Saccharomyces cerevisiae is the main yeast species responsible for the alcoholic fermentation of
grape must. Recent studies showed that S. cerevisiae strains have been globally dispersed by humans,
supporting the importance of geography in shaping S. cerevisiae’s population structure (Goddard
et al 2010; Legras et al 2007; Schacherer et al 2009). However, the existence of close geographic
location delineations of S. cerevisiae population and the interaction between vineyard and cellar
populations are still questioned, notably concerning the microbial aspect to the terroir concept. The
objective of this work is to study the intraspecific diversity of Saccharomyces cerevisiae on Merlot
grape berries at harvest ripe from different regions of Bordeaux and Bergerac vineyards. During 2012
and 2013, a total of 194 samples of grape berries were harvested within 12 vineyards in conventional
or organic farming systems in Medoc, Pessac-Léognan, Entre-deux-mers, Bergerac and St Emilion.
From enrichment by fermentation, a total of 3369 colonies were isolated from the fermented grape
berries. After microsatellite analyses using 17 loci (Legras et al 2007), 1374 Saccharomyces cerevisiae
genotype profiles were highlighted with 389 unique profiles and 100 profiles sharing a minimum of
75% of alleles with the most representative commercial strains used in Bordeaux region. Analyses
of the population structure of the Bordeaux and Bergerac vineyards, showed reduced effects of the
farming system and geographic localization on the genetic diversity and the population structure. The
use of commercial yeast may have an impact on the diversity of yeast population by mixing up with
wild population present in the vineyards and thus generating new diversity. However, some genotypes
are still recovered in specific area creating small but not significant differences.
KEY WORDS: Saccharomyces cerevisiae, Microsatellite, Genetic diversity, Commercial strains
REFERENCES:
Goddard MR, Anfang N, Tang R, Gardner RC, Jun C (2010). A distinct population of Saccharomyces cerevisiae in
New Zealand: evidence for local dispersal by insects and human-aided global dispersal in oak barrels. Environmental
Microbiology 12(1):63-7
Legras JL, Merdinoglu D, Cornuet JM, Karst F (2007). Bread, beer and wine: Saccharomyces cerevisiae diversity
reflects human history. Molecular Ecology 16:2091-2102
Schacherer J, Shapiro JA, Ruderfer DM, Kruglyak L (2009). Comprehensive polymorphism survey elucidates
population structure of Saccharomyces cerevisiae. Nature 19:458(7236):342-5
146
Phenotypic characterization of yeasts isolated from yaghnobi fermented milk
Linnea Qvirist1, Francesco Strati2, Carlotta de Filippo2, Monica Modesto3, Thomas Andlid1,
Paola Mattarelli3, Giovanna E. Felis4, Duccio Cavalieri2
Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden;
Department of Computational Biology, Edmund Mach Fundation, San Michele all’Adige, Trento, Italy; 3Department
of Agricultural Sciences, University of Bologna, Bologna, Italy; 4Department of Biotechnology, University of Verona,
Verona, Italy
1
2
[email protected]
In the Yaghnob Valley in Tajikistan lives an isolated human population, who still utilize ancient
fermentation methods to produce fermented milk as one of their main foods. The area is very isolated,
so the inhabitants and their diet have remained protected without influence or contamination from
the surrounding world. Thus, it is interesting to study the microbial biodiversity of their fermented
food. The aim of the present work is the phenotypic characterization of yeasts isolated from Yaghnobi
fermented milk.
Thirty yeast isolates were obtained from the original Yaghnobi fermented milk and from the same one
reproduced at home for three years. The isolation was performed on M17, MRS, WL, YPD and YPD
plus chloramphenicol. Phenotypic characterization were assessed by growth i) on different carbon
sources, ii) in presence of ox bile, iii) at high temperatures, iv) in acidic condition and v) in presence
of hydrogen peroxide. Also the invasiveness in YPD and hyphae formation was studied.
The 30 yeast strains, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces
cerevisiae plus one Kazachstania unispora and one Kluyveromyces lactis. revealed different phenotypes
within the same species; e.g. invasiveness from 0 to 3 among the isolates of both S. cerevisiae and P.
fermentans, the latter species also showed large variations in thermo-tolerance (inhibition from 40°C
to 46°C), and varying tolerance to acidic conditions. Only the strain of K. lactis and about 50% of
K .marxianus strains grew at 46°C. All strains of K. marxianus grew at ox bile up to 2%. Of the S.
cerevisiae strains, 50% grew in ox bile up to 2%, while two strains were inhibited already at 0.5%.
The data obtained suggest the presence of yeasts with interesting probiotic properties in Yaghnobi
fermented milk, such as the ability to survive in harsh gastrointestinal environment. The complete
probiotic characterization of these yeasts will be investigated further.
KEYWORDS: Fermented milk, Yeast, Tajikistan, Phenotypic characterization
147
Phytase activity of yeast strains isolated from italian sourdoughs
Viola Galli, Manuel Venturi, Simona Guerrini, Massimo Vincenzini
Department of Management of Agricultural, Food and Forestry Systems (GESAAF)
University of Florence, Italy
[email protected]
Nowadays, the wholemeal bread is a fermented staple food in many countries, due to its nutritional
benefits. Nevertheless, it contains high concentrations of phytic acid [myo-inositol hexakis
(dihydrogenphosphate)] which is generally recognized as an anti-nutritional factor because of its
ability to chelate minerals, such as Fe3+, Zn2+, Ca2+ and Mg2+, forming insoluble complexes
into phytate and thus reducing their bioavailability in the human intestine. However, phytate can
be hydrolyzed by phytase, an enzyme producing available phosphate and a non-metal chelator
compound. Many studies have shown that the addition of sourdough during the bread making can
reduce the phytate content. Indeed, bread making by sourdough fermentation may result in a more
suitable pH condition for the degradation of phytic acid by endogenous phytases, and, in addition,
sourdough may be a source of microbial phytases (De Angelis et al 2003). Particularly, yeasts have
been reported as useful microorganisms for phytase production (Greppi et al 2015). Therefore, the
aim of this work was to assay yeasts isolates from 20 Italian artisan sourdoughs and 9 industrial
starters in order to select the strains displaying the highest phytase activity. In total, sixty-one
yeasts isolates, belonging to Saccharomyces cerevisiae and Candida milleri, and differentiated at
strain level by RAPD-PCR analysis, were taken into consideration. Phytase activity was measured
spectrophotometrically determining the mmol of inorganic orthophosphate released from the phytic
acid by a cell suspension (ca. 108 CFU/mL). The results showed that the phytase activity is strain
dependent, released inorganic orthophosphate ranging from 0.027 mmol to 1.701 mmol. Hence, the
selection of proper yeast strain is necessary to obtain a high phytase activity in sourdoughs to be used
in the wholemeal bread fermentation.
Research project “PANACEA” funded by the Tuscany Region within the “Bando pubblico per progetti
di ricerca nel settore Nutraceutica 2014”
KEYWORDS: Yeasts, Sourdough, Phytase activity, Bread
REFERENCES:
Greppi A, Krych L, Costantini A, Rantsiou K, Hounhouigan J, Arneborg N, Cocolin L, Jespersen L (2015). Phytaseproducing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of
Pichia kudriavzevii strains. International Journal of Food Microbiology 205: 81–89
De Angelis M, Gallo G, Corbo MR, Mc Sweeney PLH, Faccia M, Giovine M, Gobbetti M (2003). Phytase activity in
sourdough lactic acid bacteria: purification and characterization of a phytase from Lactobacillus sanfranciscensis
CB1. International Journal of Food Microbiology 87:259– 270
148
Volatile Profiles and Mannoprotein content in Saccharomyces cerevisiae strains
of enological interest
Lorenzo Siroli, Giacomo Braschi, Francesca Patrignani, Giuseppina P. Parpinello, Rosalba
Lanciotti
University of Bologna, Department of Agricultural and Food Sciences, viale Fanin 44 40127 Bologna, Italy
[email protected]
The wine flavour is the sum of varietal, pre-fermentative, fermentative and post-fermentative flavours.
However, volatiles from fermentation dominate wine flavour, since yeasts affect the quality of the grape
prior to harvest and, during fermentation, they metabolise grape sugars and other components into
alcohols, esters, organic acids and aldehydes (Fleet 2003). So the production of volatile compounds
is become one of the major technological character for yeast selection. Among the new technological
features, also the production of mannoprotein has gained interest. In this perspective, main aim of this
work was to characterize 10 strains of S. cerevisiae for their volatile molecule profiles and the release
of mannoproteins in trebbiano wines.
The strains were inoculated in Trebbiano must and incubated at 15°C at the end of fermentation the
wines were evaluated by GC/MS/SPME for their volatile profiles and mannoprotein content by FTIR.
The strains, inoculated at level of 4.9 and 6.3 log cfu/ml but only the strains L318 and 12233X6167
were able to reach values of 7.5 log cfu/ml. The volatile molecule profiles were characterized by a great
amount of alcohols and in any case, the profiles obtained can be considered as a strain fingerprinting.
According to the principal component analysis, the strains L288, L234 and L318 were characterized
by the presence of propanoic acid, butanol, octanoic acid and 3 methyl pentanol while the strain
12233_35G2 was characterized by the presence of decanoic acid ethyl ester, eptanoic acid ethyl ester,
acetic acid 2 phenetyl ester. Regarding mannoproteins, the strain12233_6167 produced 104 mg/l in
trebbiano wine.
The data permitted to select the strains endowed with the best volatile molecule profiles for Trebbiano
wine and able to release the major content of mannoproteins. Moreover, the good potential of the
infra-red spectroscopy was demonstrated for mannoprotein evaluation.
KEYWORDS: Saccharomyces cerevisiae, Trebbiano wine, Volatile molecule profile, Mannoproteins
REFERENCES:
Fleet GH (2003). Yeast interactions and wine flavour. Inernational Journal of Food Microbiology 86:11–22
149
The use of co-immobilized Saccharomyces cerevisiae and Oenococcus oeni cells
for wine fermentation
Gianluca Bleve, Maria Tufariello, Cosimo Vetrano, Mariana Tristezza, Giovanni Mita,
Francesco Grieco
Consiglio Nazionale delle Ricerche - Istituto di Scienze delle Produzioni Alimentari,
Unità Operativa di Lecce, Lecce, Italy
[email protected]
Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF), but
winemakers has shown great interest about co-inoculation of yeast and malolactic bacteria at the
beginning of AF. In recent years, the use of immobilized cell systems has been investigated in some
fermented foods. In this study we have produced a mixed starter co-immobilization of Saccharomyces
cerevisiae and Oenococcus oeni in alginate beads and used it in
microvinifications tests.
O. oeni and S. cerevisiae strains were immobilized in alginate beads and used to ferment Negroamaro
must. Molecular approaches were used to check the dominance of starters and cell leaking from
beads. The process was monitored by chemical and sensorial analyses.
Co-immolization of S. cerevisiae and O. oeni allowed to perform a efficient fermentation process,
producing low volatile acidity levels and ethanol and glycerol concentrations comparable with tjhose
obtained by cell sequential inoculum and co-inoculum in free form. Co-immobilization strategy
allowed to obtain a wine with organoleptic features improved in comparison with that produced
with the co-inoculation and the sequential inoculation strategies in free form. Co-immobilization of
yeast and bacteria produced a significant decrease of the time requested to complete AF and MLF.
The immobilized cells could be efficiently reused for the wine fermentation three times without any
apparent loss of cell metabolic activites.
Co-immobilization strategy allows to produce an integrated biocatalytic system able to perform
simultaneously alcoholic and malolactic fermentation. The use of immobilized-cell systems offers
many advantages over conventional free cell fermentations, including: (i) prolonged activity and
stability of the biocatalyst; (ii) elimination of non-productive cell growth phases; (iii) feasibility of
continuous processing; (iv) regeneration and re-use of the biocatalyst.
KEYWORDS: Wine fermentation, Saccharomyces cerevisiae, Oenococcus oeni, Co-immobilization,
biocatalyst
150
New process for production of fermented black table olives using selected
autochthonous yeasts and lactic acid bacteria
Gianluca Bleve1, Maria Tufariello1, Miriana Durante1, Francesca A. Ramires1, Francesco
Grieco1, Luca Tommasi2, Antonio F. Logrieco3, Giovanni Mita1
Consiglio Nazionale delle Ricerche - Istituto di Scienze delle Produzioni Alimentari, Unità Operativa di Lecce, Lecce,
Italy; 2Associazione “Olivicoltori di Puglia”, Lecce, Italy; 3Consiglio Nazionale delle Ricerche- Istituto di Scienze delle
Produzioni Alimentari, Bari, Italy
1
[email protected]
Table olives are one of the most important traditional fermented vegetables in Europe and their world
consumption is increasing. In the Greek system, table olives are produced by natural fermentation
process, that is not predictable and strongly influenced by the physical-chemical conditions and by the
presence of microorganisms contaminating the olives, In this study , we have developed and validated
a new procedure for table olive production based on the use of a mixed yeast/bacteria starter.
Starter-driven pilot-scale fermentation (200 kg) and the corresponding natural control fermentation
were performed on Leccino, Cellina di Nardò, Kalamàta and Conservolea table olives. Selected yeast
and Lactic Acid Bacteria (LAB) autochthonous starters were used. The process was microbiologically
and chemically monitored by molecular, chemical and sensorial analyses.
A new strategy for sequential inoculum of autochthonous starters (yeasts and LAB) was developed for
pilot-scale production of black table olives. The olive fermentation was monitored using specifically
identified chemical descriptors able to identify the different fermentation stages. The use of yeast
and LAB autochthonous starters produced a significant decrease of fermentation time (from 8-12
months to a maximum of 3 months) and an important improvement in organoleptic and sensory
characteristics of the final product.
For the first time, a couple of yeast and LAB strains, specific for the each analyzed table olive cultivar,
were used as autochthonous starter cultures elaborating a sequential inoculum strategy. This approach
allowed to obtain a fermented final product with improved organoleptic characteristics in comparison
to the same olives obtained by natural fermentation and to significantly reduce the time needed to
complete the fermentation process.
KEYWORDS: Table Olives, Yeast, Lactic acid bacteria, Mixed starter, Fermentation
151
Session 2A: Yeasts in food biotechnology : biodiversity and ecology in foods and beverages
A novel killer protein from Pichia kluyveri isolated from an algerian soil
Fatima Z. K. Labbani 1,2, Benedetta Turchetti3, Leila Bennamoun2, Scheherazad Dakhmouche2,
Rita Roberti4, Lanfranco Corazzi4, Zahia Meraihi2, Pietro Buzzini3
Department of Molecular and Cellular Biology, Natural and Life Sciences Faculty, Abbes Laghrour University of
Khenchela, Route Batna, 40004 Khenchela, Algeria; 2Department of Biochemistry, Natural and Life Sciences Faculty,
University of Constantine 1, Route Ain El bey, Constantine 25017, Algeria; 3Department of Agricultural, Environmental
and Food Science & Industrial Yeasts Collection DBVPG, University of Perugia, 06121 Perugia, Italy; 4Department of
Experimental Medicine, University of Perugia, Perugia 06132, Italy4
1
[email protected]
The aim of the present study was to purify and to characterize a novel killer protein (labelled Pkkp)
produced by a strain of Pichia kluyveri isolated from an Algerian soil in order to study its in vitro
activity against food and beverage spoilage yeasts and to check its efficacy in the control of inoculated
spoilage strains in a low-alcoholic drink and fruit juice.
The killer toxin activity was tested by agar diffusion well bioassay method. One-hundred and twentyone yeast strains (all conserved in the Industrial Yeasts Collection DBVPG, www.dbvpg.unipg.it)
belonging to 24 food and beverage spoilage species were used as susceptible strains. The crude Pkkp
was purified by the gel filtration chromatography. The purity and molecular mass of the purified Pkkp
in the active fractions were analyzed in SDS–PAGE. The MICs of Pkkp, potassium metabisulphite,
potassium sorbate and ethanol were determined in 96-well microtiter plates by the microdilution
method according to the CLSI (2002) guidelines. A 2-dimensional checkerboard micro-dilution
method was used to evaluate the interaction between Pkkp and ethanol, potassium metabisulphite or
potassium sorbate. Combinations were made in RPMI 1640 (pH 4.5) using 96-well microtiter plates.
Commercial low-alcoholic drink (pH 2.9, 5 % grapefruit juice, 6.5 % ethanol) and pear juice (pH 3.5,
total carbohydrates 143 g/L), were used for the evaluation of Pkkp activity and stability in beverages.
Pkkp was active against food and beverage spoilage yeasts of the genera Dekkera, Kluyveromyces,
Pichia, Saccharomyces, Torulaspora, Wickerhamomyces and Zygosaccharomyces. After purification
by gel filtration Pkkp revealed an apparent molecular mass of 54 kDa with SDS-PAGE. MICs of
purified Pkkp exhibited a high in vitro activity against Dekkera bruxellensis (MICs from 64,000- to
256,000-fold lower than that exhibited by potassium metabisulphite) and Saccharomyces cerevisiae
(MICs from 32,000- to 64,000- fold lower than potassium sorbate). No in vitro synergistic interactions
(calculated by FIC index) were observed when Pkkp was used in combination with potassium
metabisulphite, potassium sorbate, or ethanol. Pkkp exhibited a dose–response effect against D.
bruxellensis and S. cerevisiae in a low-alcoholic drink and fruit juice, respectively. The results of the
present study suggest that the killer protein could be proposed as a novel food-grade compound useful
for the control of food and beverage spoilage yeasts.
Keywords : Yeast killer protein, Pichia kluyveri, Susceptibility testing, Spoilage yeasts.
152
Biodiversity of Saccharomyces cerevisiae populations in spontaneous wine
fermentations carried out with different grape varieties in several wineries
Lisa Granchi, Donatella Ganucci, Giacomo Buscioni, Massimo Vincenzini
[email protected]
During the last two decades, with the development of molecular techniques able to perform intraspecific yeast characterization, many studies about the distribution of S. cerevisiae strains during
spontaneous alcoholic fermentations were carried out in numerous wine-producing regions all over
the world. Independently of the region, it was pointed out that, despite of the occurrence of a high
diversity of S. cerevisiae strains at the beginning of the fermentation, only few strains (from one
to three), dominated the process in the latter stages and, therefore, had a relevant role on the final
characteristics of the wine. In addition, it was evidenced that some predominant S. cerevisiae strains
persisted in different fermentations in the same winery from one year to another and consequently
they could be representative of a particular oenological region or terroir1. In this work with the aim
to assess the genotypic diversity within natural Saccharomyces cerevisiae populations, some surveys
were carried out, by using molecular techniques, in several Italian wineries in the course of alcoholic
fermentations of musts from different grape varieties. Results pointed out that, independently of grape
variety, a few dominant and recurrent S. cerevisiae strains became the resident microbiota of a given
winery. Indeed, when all the obtained different molecular profiles, corresponding to the different S.
cerevisiae strains, were subjected to cluster analysis with the Dice coefficient and UPGMA method,
they grouped in clusters according to the winery where they come from. Moreover, some yeast
commercial strains generally used as starter cultures, which were included in the study, grouped into
a distinct cluster indicating that they were significantly different from the indigenous S. cerevisiae
strains. The occurrence of specific dominant S. cerevisiae strains in each winery supports the potential
role of these microorganisms in determining terroir-associated wine characteristics.
Keywords: Wine yeasts, Saccharomyces cerevisiae, Terroir, Spontaneous wine fermentation
References:
Bokulich NA, Thorngate JH, Richardson PM, Mills DA (2013). Microbial biogeography of wine grapes is conditioned
by cultivar, vintage, and climate. Proceedings of the National Academy of Sciences 25:139-148
153
Enzymatic capabilities of oil-born yeasts and their impact on olive oil quality
during its storage
Lisa Granchi, Eleonora Mari, Simona Guerrini, Massimo Vincenzini
[email protected]
The olive oil microbiota is mainly composed of yeasts. Some olive oil yeasts are considered useful,
as they are able to hydrolyze the bitter tasting secoiridoid compound of the oil, whereas others are
considered harmful, as they can damage the quality of the oil (Zullo et al 2013). To assess the incidence
of these abilities in oil-born yeasts, 117 yeast isolates coming from pastes, centrifuged oil and pomaces,
collected during 35 olive oil extraction processes carried out in the same oil mill during three different
harvest years, were taken into consideration. The yeasts were at first identified by using PCR-RFLP of
rITS and sequencing rRNA genes (11 species in total) and then assayed for β-glucosidase, cellulase,
polygalacturonase, peroxidase and lipase activities. All of the isolates were peroxidase positive and
cellulase negative, while β-glucosidase, lipase and polygalacturonase activities were found in 66, 22
and 2% of the assayed yeasts, respectively. Three strains, Candida molendinolei PG194 with high
peroxidase and glucosidase activities; Candida wickeramii DM15 and Yamadazima terventina DFX3
displaying high β-glucosidase, peroxidase and lipase activities, were separately inoculated in filtered
olive oil to investigate their influence on the oil quality. After two months, the oils were analyzed
(acidity level, peroxide value, total polyphenols, yeast concentrations) and statistically compared
with the control (oil incubated without yeast inoculation). The acidity level of the oil was about 20%
higher when C. wickeramii DM15 and Y. terventina DFX3 were present. Peroxide values increased
(20%) only in the presence of Y. terventina DFX3, while total polyphenols decreased (about 10%)
independently of the inoculated yeast strain. These findings show that enzymatic activities of oil-born
yeasts may negatively affect the chemical composition of olive oil during the storage.
Keywords: Olive oil quality, Yeasts, Enzymatic activities
References:
Zullo BA, Cioccia G, Ciafardini G(2013). Effects of some oil-born yeasts on the sensory characteristics of Italian virgin
olive oil during its storage. Food Microbiology 36: 70-78
154
Reprodutive cycle and sporulation profiles of yeast strains isolated from
different cachaça distilleries in Brazil
Bruna Inez Carvalho de Figueiredo, Margarete Alice Fontes Saraiva, Paloma Patrick de
Souza Pimenta, Thalita Macedo Araújo, Anna Clara Silva Campos, Ieso de Miranda Castro,
Rogelio Lopes Brandão
Núcleo de Pesquisa em Ciências Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil
[email protected]
Extreme conditions of cachaça production contribute naturally to select robust yeasts strains with
distinct physiological characteristics, such as high ethanol tolerance, aroma production and flocculation
behavior whose are interesting for diverse biotechnological applications. However, only one yeast
does not contain all these unusual characteristics together, being important the development of
strategies to combine such characteristics in new strains, for instances using classical breeding. Yeast
can present homotalism, poor sporulation capacity and low viability of spores, becoming difficult
breeding between haploids. The main objective of this work was to study and understand the efficiency
of sporulation, spores viability and the reproductive cycle of a genetically diverse collection of yeast
strains from different cachaça distilleries in order to develop a breeding method to get new strains with
industrially relevant traits. Yeasts (120 isolates) were induced at sporulation in acetate medium until
asci were observed microscopically. Sporulation frequency and efficiency were calculated and tetrads
from each isolate were dissected using micromanipulator. The spores which presented visible colonies
were designed viable and spore viability was determined as the number of viable spores divided by
the number of spores dissected. Reproductive genetic characteristic (homotalism or heterotalism)
was determined by mating type PCR. Among all tested isolates, 94% displayed values above 50% of
sporulation frequency. However, LBCM80, LBCM96 and LBCM120 isolates presented more than
50% of sporulation efficiency. The major viability of spores was observed in fourteen strains (63%).
Heterotalic characteristic was observed only in three strains (LBCM73, LBCM78 and LBCM115),
which represent 5% of all strains analyzed. In the present work, we selected yeast strains suitable
for use in breeding methods with good sporulation rates. Stable haploid spores were obtained from
strains LBCM73, LBCM78 and LBCM115 that can be useful for future breeding.
KEYWORDS: Beverage, Cachaça, Homotalism, Sporulation, Yeast
REFERENCES:
Conceição LEFR, Saraiva MAF, Diniz RHS, Oliveira J, Barbosa GD, Alvarez F, Correa LFM, Mezadri H, Coutrim MX,
Afonso RJCF, Lucas C, Castro IM, Brandão RL (2015). Biotechnological potential of yeast isolates from cachaça: The
Brazilianspirit. Journal of Industrial Microbiology and Biotechnology 42:237-246
Alvarez F, Correa LFM, Araújo TM, Mota BEF, Conceição LEFR, Castro IM, Brandão RL (2014). Variable flocculation
profiles of yeast strains isolated from cachaça distilleries. International Journal of Food Microbiology 190:97-104
155
Determination of volatile compounds of Saccharomyces cerevisiae isolated from
cachaça fermentation vats
Fernanda Barbosa Piló1, Thalita Macedo Araújo1, Anna Clara Silva Campos1, Maurício
Xavier Coutrim2, Robson José de Cássia Franco Afonso2, Ieso de Miranda Castro1,
Rogelio Lopes Brandão1
Cell and Molecular Biology Laboratory, NUPEB, Federal University of Ouro Preto, Ouro Preto, MG, Brazil;
2
Chemistry Department, Federal University of Ouro Preto, Ouro Preto, MG, Brazil
1
[email protected]
The yeast strain is the major of multiple variables that affect the flavor of fermented beverages.
Among the main secondary compounds produced by yeasts that positively influence the flavor of
beverages are the higher alcohols and esters. Given that the sensory quality of alcoholic beverages is
strictly related to the balanced concentrations of its secondary compounds, the objective of this work
was to realize a screening of strains of Saccharomyces cerevisiae, in order to find strains with high
production capacity of higher alcohols and esters and that could be used in beer production. For the
determination of aromatic compounds were performed fermentations with two brewing commercial
strains and 62 strains of S. cerevisiae isolated from cachaça fermentation vats and belonging to the
culture collection of Federal University of Ouro Preto. The yeasts were grown in YP medium (0.27%
yeast extract and 0.54% peptone, pH 4.5) containing 10% glucose for 4 days (without agitation and
at 30°C) (Conceição et al 2015). At the end of fermentation, the headspace supernatant fraction
of samples were analyzed by gas chromatography with flame ionization detection (GC-FID). The
quantified compounds included higher alcohols, esters, diacetyl and acetaldehyde. The statistical
analyses showed that among the 62 strains analyzed, five strains showed interest, because they present
high values of desirable volatile compounds production. Isolates identified as LBCM18 and LBCM76
stood out for a higher production of isoamyl acetate and the high ratio of isoamyl acetate and isoamyl
alcohol. On the other hand, LBCM69, LBCM81 and LBCM95 strains presented a higher production
of ethyl hexanoate, ethyl octanoate, and ethyl decanoate. In beers, isoamyl alcohol and medium
chain esters are known to have banana or fruity flavors, respectively, and therefore are compounds
highly desirable. Together, our data suggest that cachaça yeast strains could be very useful for beer
production.
KEYWORDS: Cachaça, Volatile compounds, Beer production
REFERENCES:
Conceição LEFR, Saraiva MAF, Diniz RHS, Oliveira J, Barbosa GD, Alvarez F, Correa LFM, Mezadri H, Coutrim,
MX, Afonso RJCF, Lucas C, Castro IM, Brandão RL (2015). Biotecnological potential of yeast isolates from cachaça:
the Brazilian spirit. Journal of Industrial Microbiology and Biotechnology 42:237-246
156
Isolation and identification of indigenous yeasts and bacteria from the initial
steps fermentations of two grape varieties, from Querétaro, México
1
Juan M. Sánchez M.1, Mayela de la Rosa M.1, Patricia Lappe-Oliveras2,
Eduardo Hernández M.2, Lorena Pedraza1, Rubén Moreno-Terrazas1
Depto. Ing. y C. Químicas, Universidad Iberoamericana, México; 2Depto. Botánica, Instituto de Biología,
Universidad Nacional Autónoma de México
[email protected]
Yeasts and bacteria are part of the ecosystem of the vineyard sharing biotic and abiotic conditions. They
influence the wine quality, producing different effects in their chemical and sensorial characteristics.
The aim of this study was to isolate and identify indigenous microorganisms present during the
different fermentation stages of two grape varieties, from a vineyard in Tequisquiapan, Querétaro;
since there are few micro-diversity studies of this region.
Macabeo (M) and Pinot Noir (P) grapes were collected under aseptic conditions. The must of each
grape variety was elaborated, and microvinification were carried out during 168 hours. Changes in
°Brix, pH, ethanol, organic acids and total acidity were quantified. Total CFU/mL were counted every
24 hours. The microorganisms were identified by morpho-physiological and molecular tests.
The pH remained constant (P 4.2; M 3.7). Total acidity increased, P (0.7-0.73%) and M (0.6-0.9%);
°Bx decreased, P (21-14°) and M (12-3°); ethanol increased, P (2.88) and M (4.5%v/v); tartaric
remain constant, malic decreased, and lactic and acetic increased. These two acids probably were
produced by lactic and acetic acid bacteria and by W. anomalus. Yeasts population increased P (2.24
x 105-1.56 x 106) M (1.2 x 104-1.83 x 106); all bacteria groups also increased up to 106 CFU/mL, in
both fermentations. The microorganisms identified are shown in the Table 1, most of these species
have been reported previously, but are first described for the region of study. The yeasts are promising
to be used as improvements of local wine, while the bacteria are not favorable for the process.
KEYWORDS: Wine, Pinot Noir and Macabeo grapes, Fermentation, Indigenous microorganisms
References:
Teixeira A, Caldeira I, Duarte FL (2015). Molecular and oenological characterization of Touriga Nacional nonSaccharomyces yeasts. Journal of Applied Micrbiology 118(3):658-671
Ciani M., Comitini F., Mannazzu I., Domizio P. (2009). Controlled mixed culture fermentation: a new perspective on
the use of non-Saccharomyces yeasts in winemaking. FEMS Yeast Research 10:123–133
González S.S., Barrio E., Querol A. (2006). Molecular identification and characterization of wine Yeasts isolated from
Tenerife (Canary Islands, Spain). Journal of Applied Micobiology 102:1018-1025
157
Yeast diversity in commercial pulque production
Patricia Lappe-Oliveras, Valeria Jiménez S., Tania Vázquez B., Concepción León C.,
Teófilo Herrera S., Rubén Moreno-Terrazas
Depto. Botánica, Inst. De Biología, Universidad Nacional Autónoma de México, Depto. Ing. y C. Químicas,
Universidad Iberoamericana, México
[email protected]
Pulque is probably the oldest and most traditional Mexican alcoholic beverage prepared and consumed
since pre-Hispanic times. It is a milky white, viscous, slightly alcoholic and acidic beverage produced
by the fermentation of aguamiel or mead extracted from several Agave species.
Commercial fermentation of agave sap is induce by addition of a starter from a previous fermentation,
and last several days; it finishes when pulque reaches certain alcoholic and viscosity degree, and
develops particular sensorial characteristics. Although pulque has been studied for more than a
hundred years, until today the microbial succession in the commercial process is poorly known. The
objectives of the study were the determination of the yeast diversity present during the elaboration
of pulque in a tinacal (place where commercial pulque is produced) as well as of ethanol by GC and
volatile compounds GC-MS. Samples (19) were collected in the Hacienda of Xochuca, Tlaxcala,
Mexico.
From the 19 samples 229 isolates were obtained, these were identified by pheno and genotypic
characteristics as: Saccharomyces cerevisiae (46.3%), Kluyveromyces marxianus (16.6 %),
Candida boidinii (10.9%), Candida lusitaniae (8.7%), Candida parapsilosis (7.9%), Meyerozyma
guilleromondii (2.2 %), Torulospora delbrueckii (2.2%), Rhodotorula glutinis (2.2%), Candida sake
(1.7%), Debaryomyces hansenii (0.9%), Candida magnoliae (0.4%), Yamadozyma mexicana (0.4%),
Pichia membranifaciens (0.4%), and Schwanniomyces occidentalis (0.4%). Of these, S. cerevisiae
was found in 17/19 samples, followed by K. marxianus 11/19, C. lusitaniae 10/19 and C. parapsilosis
8/19. S cerevisiae is considered the dominat species in pulque fermentation.
Chemical analysis showed that phenylethyl alcohol, and fatty acids or their esters, were present in
most of the samples, while other alcohols and amines were detected only in a few samples. Final
ethanol concentration was in the limits specified by the official regulation, but it decreased when
mead was added.
KEYWORDS: Pulque, Fermentation, Yeast diversity, Chemical analysis
REFERENCES:
Escalante A et al (2012). Pulque fermentation. En: Handbook of Plant-Based Fermented Food and Beverage
Technology. Hui, Y.H. y E. Özgül (eds.). CRC Press, EUA
Lappe-Oliveras P, Moreno-Terrazas R, Arrizón-Gaviño J, Herrera-Suárez T, García-Mendoza A, Gschaedler-Mathis A
(2008). Yeasts associated with the production of Mexican alcoholic non-distilled and distilled Agave beverages. FEMS
Yeast Research 8(7):1037-1052
158
Wickerhamomyces anomalus inoculated moist maize and its applicability for
storage in Cameroon
Albina Bakeeva1, Aziwo T. Niba2, Ambe C. Sirri2, Matilda Olstorpe1, Su-lin L. Leong1
Department of Microbiology, BioCentrum, SLU, Box 7025, 75007 Uppsala, Sweden; 2College of Technology, P.O. Box
39 Bambili, University of Bamenda, Cameroon
1
[email protected]
Airtight storage of moist harvested grain, a preservation method, relies on a number of factors, in which
the population of lactic acid bacteria (LAB) and yeast naturally present on the grain are important.
Moist maize stored in this system in Cameroon, combined with biocontrol yeast Wickerhamomyces
anomalus (syn. Hansenula anomala, formerly Pichia anomala) has been shown to be a hygienic
product with reduced levels of potentially pathogenic bacteria and spoilage moulds (Niba et al 2014).
It is a cheap and energy-efficient technique that can be applied in developing countries. Two common
maize cultivars, ‘Kasai’ (white maize) and ATP (acid-tolerant population, yellow), were harvested
and stored in airtight plastic barrels, with and without inoculation of W. anomalus. We compare
field trials conducted during two seasons. LAB, yeasts and moulds were enumerated and identified
at harvest and after storage for 2, 5 and 8 months in airtight plastic barrels. High levels of LAB (108
cfu/g) were maintained throughout storage and likely contributed to the decline in Enterobacteriaceae
in both control and inoculated maize to < 10 cfu/g after 2 months. In this system of airtight stored
moist maize, mould counts were also reduced to < 100 cfu/g: only in inoculated maize one season,
but in both control and inoculated maize the second season. The biocontrol species W. anomalus
was occasionally naturally present in uninoculated maize (at low levels) of both cultivars. Longterm survival of W. anomalus was poor in trials where Dekkera bruxellensis became dominant
after 5 months (both cultivars). W. anomalus survived somewhat better (5-8 months) when Pichia
kudriavzevii (white maize) or Pichia membranifaciens (yellow maize) were next in succession (both
control/inoculated maize). Airtight storage of moist harvested maize (approx. 34–30% moisture
content) combined with biocontrol is a promising technique for minimizing mould growth and risks
for mycotoxin production during storage.
KEYWORDS: Wickerhamomyces anomalus, Biocontrol, Lactic acid bacteria, Storage, Feed hygiene
REFERENCES:
Niba AT, Leong SL, Olstorpe M (2014). Biocontrol efficacy of Wickerhamomyces anomalus in moist maize storage.
African Journal of Biotechnology 13:4208-4214
159
Pilot scale evaluation of indigenous yeast starters for the production of ‘wildferment’ Moschofilero wines
1
Dimitra Dourou1, Apostolos Spyropoulos2, Georgios Banilas3, Aspasia Nisiotou1
Hellenic Agricultural Organization-DEMETER, Institute of Technology of Agricultural Products, Sofokli Venizelou 1,
Lycovrissi 14123, Greece; 2Arkas SA, Artemisio, Ancient Mantinia, Tripoli Arcadia, Greece; 3Department of Enology
& Beverage Technology, Faculty of Food Technology & Nutrition Technological Educational Institute of Athens, Ag.
Spyridona Str., 12210 Athens, Greece
[email protected]
The use of well selected, autochthonous yeast strains in winemaking may enhance the complexity
and typicity of regional wines. In the present work, the performance of indigenous S. cerevisiae and
L. thermotolerans strains isolated from Mantineia PDO region in Greece was evaluated in pilot scale
fermentations of Moschofilero grape must. The indigenous S. cerevisiae was either singly inoculated
(ISc) or along with L. thermotolerans, in both simultaneous (CoI) and sequential (SeqI) additions.
Concomitant fermentations with commercial S. cerevisiae starter (CSc) were also conducted.
Fermentation kinetics, population dynamics, strain implantation capacity and the chemical and
sensory characteristics of the resulting wines were assessed. Fermentation was more efficient with ISc
and CoI (both 8.5 days) compared to SeqI (10.5 days). Importantly, the autochthonous S. cerevisiae
exhibited good implantation ability, consisting 21, 8 and 92 % of the dominant population at the
end of SeqI, CoI and ISc fermentations, respectively. Surprisingly, the commercial starter could not
be recovered at the end of fermentation. Non-Saccharomyces yeasts persisted for longer period in
fermentations with indigenous than with commercial starters and were maintained at higher final
populations in mixed fermentations (5.4 log cfu/ml) compared to ISc (2.5 log cfu/ml) or CSc (<1.7
log cfu/ml). L. thermotolerans dominated over the autochthonous non-Saccharomyces yeasts in both
CoI and SeqI fermentations (ca. 63 and 65 %). Chemical and sensory analysis demonstrated the
strain and inoculation scenario impact on wine flavor, with wines produced with L. thermotolerans/S.
cerevisiae mixture (CoI and SeqI) being the most appreciated by expert tasters. Results confirm the
high implantation ability and excellent oenological properties of the indigenous yeast starters and
confirm their potential to be used for the production of typical Moschofilero wines.
Acknowledgements This work has been co-financed by the European Regional Development Fund
(ERDF) of the EU and by National Resources under the Operational Program Competitiveness and
Entrepreneurship (EPAN II), Action “COOPERATION 2011”.
KEYWORDS: Wine fermentation, Yeast starter cultures, Regional wines
160
Yeasts in sourdough, microbial characterization and volatile profile
Valery Ripari, Teresa Cecchi, Enrico Berardi
Università Politecnica delle Marche, Ancona, Italy
[email protected]
Sourdough is a traditional method to produce bread. Sourdough is an ecosystem composed by a
consortium of differents species of LAB, AAB and yeasts (Minervini, et al, 2014). Microbiota of 40
central Italian sourdough has been studied.
Yeast population is been evaluate with DGGE, RAPD, RFLP and 28S sequencing. Molecular
identification has been performed. We analysed the leavening and pH change during fermentation
time. Using HS-SPME-GC-MS, a comparison between volatile profile of sourdough sample and
model dough obtained using Saccharomyces cerevisiae (a wild strain and baker’s yeast) is been made.
In all sourdough samples we found Saccharomyces cerevisiae. In same cases we found Torulaspora
delbrueckii, Wicheranomyces anomalus, Saccharomyces unisporus and Saccharomyces barnettii.
Fermentation via wild S. cerevisiae strain only gives ethanol, ethyl acetate and 3-methyl-1-butanol,
while the use of baker’s yeast results in a few more alcohols and esters such as 1-hexanol, phenylethyl
alcohol, 1-butanol-3-methyl-acetate, hexyl acetate. In yeast model doughs no acids or aldehydes and
ketones were detected.
In sourdough, yeasts have an important role in leavening and volatile compounds liberation. In our
sample the dominant yeast specie is S. cerevisiae.
KEYWORDS: Yeasts, Sourdough, HS-SPME-GC-MS, Leavening
REFERENCES:
Minervini F, De Angelis M, Di Cagno R, Gobbetti M (2014). Ecological parameters influencing microbial diversity and
stability of traditional sourdough. International Journal of Food Microbiology 171: 136–146
161
A case study – Yeasts identification in order to implement terroir and vineyard
management at Oprișor winery using the GIS technology and a proteomic
approach
Iuliana D. Bărbulescu1, Doru Mihai2, Mihaela Begea3, Radu Mudura2, Răzvan Teodorescu2,
Constanţa Mihai2, Liviu Grigorică4, Gabriel Roceanu5, Simona I. Marinescu1,
Radu Tamaian6,7
1
Pharmacorp Innovation SRL, Bucharest, Romania; 2University of Agronomic Sciences and Veterinary Medicine
Bucharest, Romania; 3University Politehnica of Bucharest, Romania; 4Bevitech SRL, Bucharest, Romania; 5Carl Reh
Winery SRL, Bucharest, Romania; 6National Institute for Research and Development for Cryogenic and Isotopic
Technologies, Râmnicu Vâlcea, Romania; 7University of Bucharest, Faculty of Physics, 3Nano-SAE Research Centre,
Bucharest-Măgurele, Romania
[email protected]
Oprisor area (Mehedinti County) is one of the most famous geographical areas in Romania for
producing quality red wines. In order to implement a system to monitor aspects of terroir and viticulture
management in the vineyard Oprisor, samples of grapes, leaves and soil have been collected from the
vineyard Oprisor (Mehedinti) during the harvest of 2014 and a study using GIS technology has been
conducted.
Samples of grapes, leaves and soil have been collected during the harvest of 2014 and have been
used for isolation and identification of useful microorganisms for wine producing, having specific
characteristic of Oprisor vineyard. Peptide mass fingerprinting using a Bruker microflex™ LT/SH
MALDI-TOF mass spectrometer with nitrogen laser was performed in order to identify and characterise
the isolated wine yeast strains. Yeasts isolates were processed using multiple measurements in order
to ensure that their true biological variability was acquired. The fingerprints were processed with the
MALDI Biotyper 3.0 software for principal component analysis and dendrograms were made in order
to illustrate the scattering plots, respectively the hierarchical clustering of isolates. Topographic base
was updated through aerial photographs taken by a drone and points determined by GPS technology.
The presented results refer to the isolated yeast strains from microflora present on the leaves, soil and
Cabernet Sauvignon, Shiraz and Merlot grape berries that were subjected to specific tests in order to
their identification.The most eloquent results have been noticed for samples of grapes. The MALDI
Biotyper software classified the majority of isolates as species included in order Saccharomycetales
with some particular strains that could not be matched with strains from database and was added as
a new entries (taxa). The sampling points for samples of grapes, leaves and soil were also taken with
GPS technology and integrated in the GIS database.
KEYWORDS: Wine yeasts, Terroir, MALDI-TOF mass spectrometry, GIS
REFERENCES:
Agustini BC, Silva LP, Bloch C Jr, Bonfim TM, da Silva GA (2014). Evaluation of MALDI-TOF mass spectrometry
for identification of environmental yeasts and development of supplementary database. Applied Microbiology and
Biotechnology 98(12):5645-54
162
Test of four generations of Saccharomyces cerevisiae concerning their effect on
antioxidant phenolic compounds in wine
Andrea Caridi, Rossana Sidari, Angelo M. Giuffrè, Teresa Pellicanò, Vincenzo Sicari, Clotilde
Zappia, Marco Poiana
Department AGRARIA, Mediterranea University of Reggio Calabria, Via Feo di Vito s/n, 89122 Reggio Calabria, Italy
1
[email protected]
Red wine quality is primarily dependent on its phenolic content that confers colour, flavour, healthy
properties, and natural antioxidant activity. The aim of this research was to study the behaviour of
70 different Saccharomyces cerevisiae strains concerning their effect on wine content in antioxidant
phenolic compounds. This research was carried out using the control strain Zymaflore F15 (Laffort
Oenologie, France), eight Italian wild types, 12 derived spore clones, 15 hybrids obtained crossing
the derived spore clones, and 34 spore clones derived from the hybrids. Black grapes of the Cabernet
variety were destemmed, crushed, cold soaked at 4 °C for 3 days and punched down twice per day.
The must obtained after pressing (27°Brix) was inoculated in triplicate at 5% with precultures of the
70 yeast strains and incubated at 25 °C. At the end of the winemaking the wines, analysed by HPLC
for their antioxidant phenolic content, showed highly significant differences, due exclusively to the
wine starter used. In details, catechin content ranged from 0 to 79.53 mg/l (mean 31.67), epicatechin
content ranged from 0 to 70.51 mg/l (mean 19.24), vanillic acid content ranged from 3.10 to 12.71
mg/l (mean 8.72), gallic acid content ranged from 2.54 to 6.77 mg/l (mean 4.82), rutin content ranged
from 0 to 11.77 mg/l (mean 3.46), quercetin content ranged from 0 to 2.09 mg/l (mean 1.62), caffeic
acid content ranged from 0 to 10.63 mg/l (mean 1.28), trans-resveratrol content ranged from 0 to 0.85
mg/l (mean 0.27). Data validate the main role that wine yeast selection plays to enhance red wine
content in antioxidant phenolic compounds.
This research was supported by POR CALABRIA FESR 2007/2013 - ASSE I - Obiettivo Specifico
1.1 - Obiettivo Operativo 1.1.1 - Linea di Intervento 1.1.1.2 Innovazione Di Processo E Nuovi Prodotti
Per La Valorizzazione Dei Vini E Passiti Da Cv Autoctone - Enotria Tellus
KEYWORDS: Antioxidant phenolic compounds, Saccharomyces cerevisiae, Spore clone selection,
Wine, Yeast hybridization
163
Isolation and fast pre-selection of yeasts from spontaneous fermentation of
Calabrian table olives
Andrea Caridi
Department AGRARIA, Mediterranea University of Reggio Calabria, Via Feo di Vito s/n, 89122 Reggio Calabria, Italy
[email protected]
Calabria is a table olive-producing area in the South Italy and natural table olive processing is an old
tradition. The aim of this research was to study the yeast microbiota in order to pre-select strains able
to improve Calabrian table olives. In details, the main traits of interest are: (1) the quality improvement
by interaction with natural phenolic compounds possessing positive sensorial characteristics; (2)
the shelf-life extension by production/preservation of natural antioxidant compounds. Eighteen
samples of Calabrian table olives produced by spontaneous fermentation were used. The olive
cultivars were: Carolea, Geracese, Nocellara, Nocellara Messinese, and Ottobratica. Using Yeast
Extract-Peptone-Dextrose (YPD) Agar a total of 54 yeast strains were isolated from the 18 table
olive samples and their brines. The new strains were collected in Microbank at -80°C and their main
phenotypic characteristics were detected. To perform a fast pre-selection from a great number of yeast
strains, a simple phenotypic-based methodology was employed to allow many different strains to be
simultaneously tested. This way, only the best strains remaining at the end of this pre-selection will
be finally selected using Oxitester CDR with a great saving of money.
This research was supported by POR CALABRIA FESR 2007/2013 - ASSE I - Obiettivo Specifico
1.1 - Obiettivo Operativo 1.1.1 - Linea di Intervento 1.1.1.2 Nuove Tecnologie Per La Valorizzazione
Della Filiera Delle Conserve – Conservo
KEYWORDS: Antioxidant compounds, Fast pre-selection, Phenolic compounds, Table olives, Yeasts
164
Saccharomyces cerevisiae biodiversity in biodynamics oriented wine farming
Tilde Labagnara1, Raffaele Guzzon2, Giancarlo Scalabrelli1, Annita Toffanin1
University of Pisa, DISAAA-a - Department of Agricultural, Food and Agro-Environmental Sciences,
via del Borghetto, 80, 56124 Pisa, Italy; 2Technology Transfer Centre, Edmund Mach Foundation,
Via E. Mach 1, 38010 San Michele all’Adige, TN, Italy
1
[email protected]
In biodynamic oriented wine farming microbial biodiversity is favoured and it is reasonable to
expect a major presence of microbes with interesting traits due to the absence o very scarce use of
invasive techniques in the vineyard and in the cellar. The aim of the work was the identification and
characterization of Saccharomyces cerevisiae wine strains from biodynamic oriented wine farms in
different vintages.
Yeasts from Syrah fermentations were isolated from different steps of winemaking in 2009, 2010 and
2013 harvests. S. cerevisiae biotypes were molecularly characterized using ITS-PCR and multiplex
PCR amplification of microsatellite loci (SC8132X, YOR267C and SCPTSY7). Micro-fermentations
of genetically diverse S. cerevisiae were set up to study fermentative traits. Sulphur dioxide, ethanol
resistance and production of hydrogen sulphide were chosen as main parameters for technological
characterization of isolates.
During the harvest of 2009 and 2010, several S. cerevisiae biotypes were individuated during
spontaneous fermentation in a biodynamic oriented wine farm located in Tuscany. Some biotypes
highlighted good oenological aptitudes allowing their use in guided fermentation. The harvest of
2013 confirmed the presence of several biotypes previously identified in 2009 and 2010.
There is a debate about the persistence of “autochthonous” wine yeast strains in the cellar and during
winemaking process in the years. The results indicate the presence of the same S. cerevisiae strain in
the examed 3 of 5 vinification processes, attesting its presence in the same terroir in different years
and overcoming a possible vintage effect.
KEYWORDS: Saccharomyces cerevisiae, Biodynamic, wine yeasts, terroir
REFERENCES:
Ciani M, Mannazzu I, Marinangeli P, Clementi F, Martini A (2004). Contribution of winery-resident Saccharomyces
cerevisiae strains to spontaneous grape must fermentation. Antonie Van Leeuwenhoek. 85(2):159-64
Muñoz-Bernal E, Rodríguez ME, Benítez P, Fernández-Acero FJ, Rebordinos JMC (2013). Molecular analysis of red
wine yeast diversity in the Ribera del Duero D.O. (Spain) area. Archives of Microbiology 195(5):297-302
Vigentini I, De Lorenzis G, Fabrizio V, Valdetara F, Faccincani M, Panont CA, Picozzi C, Imazio S, Failla O, Foschino
R (2015). The vintage effect overcomes the terroir effect: a three year survey on the wine yeast biodiversity in
Franciacorta and Oltrepò Pavese, two northern Italian vine-growing areas. Microbiology 161:362-73
165
Evaluation of microbiota in cider fermentation in northern parts of Slovenia
Neža Mandl, Tatjana Košmerl, Neža Čadež
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
[email protected]
Koroška, a region in northern Slovenia, is the country’s main producer of natural cider. Natural cider
is produced by the spontaneous fermentation of apple juice. This step involves both alcoholic and
malolactic fermentation carried out by the sequential action of different yeasts and bacteria originating
from the fruit and the cider-making equipment. Yeasts are primarily responsible for alcoholic
fermentation and hence for the taste and flavour characteristic of products. Therefore, spontaneous
fermentations are of particular interest in order to ascertain the yeast species associated with the
fermentation processes.
The goal of this study was the characterisation of indigenous microbiota of isolated spontaneous
fermentations in ten cellars in the region. On various media for specific groups of microorganisms
we isolated 576 of yeasts, 480 isolates of lactic-acid bacteria (LAB) and 192 isolates of acetic-acid
bacteria. The species diversity was determined by molecular methods. First, the genetically similar
strains were grouped based on RFLP-ITS for yeasts, and (GTG)5x fingerprinting for lactic-and acetic
acid bacteria. Further, the barcoding regions of the representatives of the genetically similar groups
were determined by sequencing. The chemical composition of the cider was determined by analytical
methods. Finally, the diversity of microbiota will be correlated to quality of cider determined by
chemical analyses.
KEYWORDS: Cider fermentation, Fermetative yeasts, Lactic acid bacteria, Acetic acid bacteria
166
Intraspecific diversity of Brettanomyces/Dekkera bruxellensis established with
microsatellite markers
Marta Avramova1, Emilien Peltier1, Monika Coton2, Emmanuel Coton2, Franck Salin3 Warren
Albertin1,4, Chris Curtin6, Isabelle Masneuf-Pomarede1,5
Univ. Bordeaux, ISVV, Unité de recherche Œnologie EA 4577, USC 1366 INRA, Bordeaux INP, 33140 Villenave
d’Ornon, France ; 2Université de Brest, EA 3882, Laboratoire Universitaire de Biodiversité et Ecologie Microbienne,
ESIAB, Technopôle Brest-Iroise, 29280 Plouzané, France; 3INRA, UMR Biodiversité Gènes et Ecosystèmes,
PlateForme Génomique, 33610 Cestas, France ; 4ENSCBP, Bordeaux INP, 33600 Pessac, France; 5Bordeaux Sciences
Agro, 33170 Gradignan, France; 6The Australian Wine Research Institute, Glen Osmond, Adelaide, SA, Australia
1
[email protected]
Brettanomyces bruxellensis is a yeast associated with industrial fermented products such as wine,
beer, cider, kombucha (fermented tea), bioethanol and others. B. bruxellensis contamination alters
organoleptic qualities of the products and often results in consumers’ rejection. This yeast is the first
cause of contamination in red wine and its development results in a phenol character known as “barnyard
smell” and “wet-horse”. Nevertheless, there is no efficient method to fight against its contamination
or to prevent it. In order to better understand the biology of B. bruxellensis, more information about
the population structure and its intraspecific diversity is needed. Characteristic markers to distinguish
strains would be a very valuable tool for this purpose. Several typing methods have been previously
published, ranging from classical molecular methods (RAPD, AFLP, REA-PFGE, mtDNA restriction
analysis) to more engineered technologies (infrared spectroscopy). However, there is still a lack of a
rapid, reliable and universal genotyping approach, mostly due to the very complex genomic structure
of B. bruxellensis for which triploidy seems to be a relatively common state.
The interest of the Single Sequence Repeats tool for genotyping B. bruxellensis at the strain level
was assessed using twelve microsatellite markers on isolates from Europe, Australia, South Africa,
North and South America, as well as from different substrates (wine, beer, cider, kombucha, etc.). Our
results suggest that B. bruxellensis is a highly disseminated species, with some strains isolated from
different continents being closely related at the genetic level. The B. bruxellensis population structure
seems to be divided in groups according to ploidy level and substrate. The importance of ploidy level
for B. bruxellensis adaptation to industrial fermentations is discussed.
KEYWORDS: Brettanomyces/Dekkera bruxellensis, Fermentation, Genotype, Single Sequence
Repeat
REFERENCES:
Albertin W, Panfili A, Miot-Sertier C, Goulielmakis A, Delcamp A, Salin F, Lonvaud-Funel A, Curtin C, MasneufPomarede I (2014). Development of microsatellite markers for the rapid and reliable genotyping of Brettanomyces
bruxellensis at strain level. Food Microbiology 42:188–195
Curtin CD, Pretorius IS (2014). Genomic insights into the evolution of industrial yeast species Brettanomyces
bruxellensis. FEMS Yeast Research 14:997–1005
167
Towards the understanding of the mechanisms of action of chitosan an
alternative to SO2 as a preservative in wine
Patrícia Lage1, Ana Lemos1, Catarina Barbosa1, Arlete Mendes-Faia1,2,
Ana Mendes-Ferreira1,2
Universidade de Trás-os-Montes e Alto Douro, Escola de Ciências da Vida e Ambiente; Vila Real, Portugal; 2BioISI Biosystems & Integrative Sciences Institute, Campo Grande, Lisboa, Portugal
1
[email protected]
Microbial spoilage is a major concern throughout the food/wine industry. To assure microbiological
stability, food and beverage industries greatly rely on the antimicrobial proprieties of SO2. However
the emergence of highly tolerant spoilage yeasts is limiting the usefulness of this chemical, leading to
the need of increasing the concentrations utilized. This increase is particularly problematic given the
growing awareness that sulfites may induce a range of adverse clinical effects in sensitive individuals.
On the other hand, the non-toxic polysaccharide chitosan has been considered as an interesting
alternative to SO2 due to its antimicrobial properties. The mechanisms by which spoilage yeasts
respond and tolerate SO2 and chitosan are not fully understood.
Here, the model yeast Saccharomyces cerevisiae, itself a spoilage yeast, was explored to assess the
mechanisms by which these two preservatives exert toxicity in yeast cells. A great strain variability
on the resistance against SO2 and chitosan was found among the yeast strains tested. Additionally,
we report the results obtained using a chemogenomics approach to systematically identify the genes
required for tolerance to inhibitory concentrations of SO2 and chitosan in S. cerevisiae. Our findings
might give a significant contribution to the design of practical applications aiming to reduce SO2
levels in the food and beverage industries by exploring chitosan as an affective antimicrobial agent.
This work was supported by FEDER through COMPETE (FCOMP-01-0124-FEDER-014043) and
by national funds by FCT through the project EXPL/AGR-TEC/1823/2013.
Keywords: Yeast, SO2, Chitosan, Genome-wide screening
168
Performance at cellar level of selected autochthonous and commercial
Saccharomyces cerevisiae strains during fermentation of “Primitivo di Matera”
grape variety
Rossana Romaniello, Margherita Sarli, Angela Capece, Patrizia Romano
Scuola di Scienze Agrarie, Forestali, Alimentari ed Ambientali, Università degli Studi della Basilicata, Potenza, Italy
[email protected]
The selection of indigenous yeast strains to be used as starters is considered the best approach to
ensure the final quality of the product, by maintaining the typical sensory properties of wine from
each area. This is particularly worrying when all winemakers in a particular region use a limited
number of commercial yeasts, which results in the production of highly homogeneous wines, with a
reduction in aromatic complexity. The aim of this study was the evaluation of fermentative fitness of
S. cerevisiae indigenous strains in comparison to the commercial starter AWRI 796. The fermentations
were performed in three different cellars (M, P and B) producing Primitivo di Matera wine. In each
cellar, two strains were inoculated in 400 l of Primitivo must: one indigenous strain, specific of
each cellar and previously selected for technological parameters, and the strain AWRI 796, common
to all the cellars. In each fermentation, the implantation level of the inoculated starters and their
influence on content of aromatic compounds were evaluated. In function of analyzed cellar, different
results were obtained. In cellar M, commercial and indigenous strains showed a high implantation
level. In cellars B and P both the indigenous strains showed an implantation level higher than AWRI
796 strain. The content of aromatic compounds detected in the six wines was variable among wines
produced in the same cellar with different strains and wines obtained in different cellars with the same
strain (AWRI 796), confirming that aromatic quality of wine is affected both by starter and grape must
composition. The use of using indigenous starter in each winery could produce wines with exclusive
aromatic properties and this would be an excellent strategy for introducing variability in a highly
competitive market.
Acknowledgements This work was supported by the project PIF-LIELUC (Misura 124 PIF Vini di
Lucania, PSR Basilicata 2007-2013 “Lieviti Indigeni per Vini Lucani” N. 94752044753)
KEYWORDS: Indigenous Saccharomyces cerevisiae strains, Implantation level, Wine aroma,
Primitivo di Matera wine
169
170
Session 2B
detection methods and strain improvement
171
The influence of non-Saccharomyces yeast strains in Tempranillo wine’s aroma
Ignacio Baselga1, Javier Calzada1, Estela Perez-Lago1, Raquel Francisco-Álvarez1,
Gemma Rodríguez-Tarduchy2, Michael Qian3, Cruz Santos1
Francisco de Vitoria University; 2Instituto de Investigaciones Biomédicas Alberto Sols; 3Oregon State University, USA
1
[email protected]
A wide variety of yeasts can be involved in the wine alcoholic fermentation process. However, these
microorganisms can be classified as Saccharomyces or non-Saccharomyces species and commercial
or autochthonous yeast strains [1]. Many studies have demonstrated that the compounds of the aroma
may vary depending on the presence or absence of certain yeast species and yeast strains. Hence,
controlling which yeasts will be part of the alcoholic fermentation process could provide specific
wine aroma profiles [2]. Spanish wineries have been able to produce signature’s wine by using nonSaccharomyces autochthonous yeast strains, allowing wineries from the same regions to distinguish
their wines by creating different aroma profiles.
To further develop this claim, the microbial biotechnology laboratory of UFV in Madrid, has analyzed
1,200 yeast samples from Díaz Bayo, a winery in Ribera del Duero, an important wine-producing area
in northern Spain. The samples were collected during four years, vintages 2010 to 2013 and were
isolated from grapes, musts, and fermentation tanks. 372 samples have been characterized through
molecular techniques based on analysis of ribosomal RNA and AFLP. Theses analyses allowed the
identification of 16 different yeast strains belonging to: Saccharomyces cerevisiae, Saccharomyces
uvarum , Metschnikowia pulcherrima , Kluyveromyces thermotolerans , Hanseniaspora uvarum and
Picchia anomala (Suárez-Lepe & Morata). The fermentation potential of these strains has been tested
individually and combining different yeasts. As a result, 10 fermentations have been carried out with
combinations of 6 selected yeast strains. The aromatic fraction of the produced wines was analyzed
at the Food Science and Technology Department of OSU by HS-SPME-GC-MS. The results obtained
indicate that the combined action of these strains influence the level of very important wine aromatic
compounds as ethyl octanoate, ethyl hexanoate, isoamil acetate and phenetyl acetate.
KEYWORDS: AFLP; Wine; Yeasts; Aroma
REFERENCES:
Gayevskiy V, Goddard MR (2012). Geographic delineations of yeast communities and populations associated with
vines and wines in New Zealand. In: ISME Journal 6: 1281-1290
Suárez-Lepe JA, Morata A (2012). New trends in yeast selection for winemaking. Trends in Food Science &Technology,
23: 39-50
172
Session 2B: Yeast and food biotechnology: detection methods and strains improvement
Saccharomyces cerevisiae as model for pathogenesis study of virulences factors
of Campylobacter jejuni
Veronica García1,2, Eugenio Scovacricchi 1, Francisco A. Cubillos1,2, Claudio Martínez1,2
1
Departamento de Ciencia y Tecnología de los Alimentos, Facultad Tecnológica, Universidad de Santiago de Chile,
(USACH), Chile; 2Centro de estudio en Ciencia y Tecnología de los Alimentos (CECTA), Universidad de Santiago de
Chile, Chile
[email protected]
Campylobacter jejuni is one of the most common pathogens associated to gastroenteritis. This
microorganism displays a large array of virulence factors, such as, effector proteins and export systems,
through which these proteins are directly injected into the host cytoplasm. The effector proteins
produce a series of changes, which promote the bacterial invasion and contribute to its prevalence by
allowing it to evade the host immune system. In this work, we make use of Saccharomyces cerevisiae
as a model eukaryote system to identify effectors proteins of C. jejuni and analyze their mechanisms
of action for the design of treatments for pathologies caused by C. jejuni. Here, we initially sought
to identify effector proteins orthologs by Reciprocal Best Hit (RBH) utilizing the genome sequence
of the C. jejuni strain M1 (highly virulent) and RM1221. Using bioinformatics tools, six possible
effector proteins were identified from the virulent strain M1 (CJM1_ 148, 203, 206, 480, 1321 and
1637). These proteins together with other four proteins obtained from the literature (CiaB, HtrA,
TssD and TssI) were cloned and expressed in S. cerevisiae, where their effect were evaluated by
measuring yeast fitness under optimal and stress growth condition such as sorbitol, salts and caffeine.
Our results show that the expression of HtrA under optimal and stress condition reduces the growth
of S. cerevisiae. Moreover the expression of other three of effectors proteins reduce the growth of S.
cerevisiae under stress conditions, indicating that effector proteins interfere with the normal functions
of eukaryote cells. Finally, the impact of the effector proteins at the trancriptional level in S. cerevisiae
was also evaluated.
KEYWORDS: Saccharomyces cerevisiae, Campylobacter jejuni, Effectors proteins, Virulences
173
Improvement of acetic acid tolerance in Saccharomyces Cerevisiae by allele
replacement using CRISPR-Cas9
Marija Stojiljkovic1,2, Ben Souffriau1,2, María R. Foulquié-Moreno1,2 , Johan M. Thevelein1,2
1
Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven; 2Department of Molecular
Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium
[email protected]
A constant debate over the use of major food crops to produce fuels moved the industry towards new
substrates, such as waste streams and energy crops. However, the disadvantage of such substrates for
second-generation bioethanol production is that they are difficult to ferment, mainly because of the
inability of yeast to ferment pentoses. Additionally, pretreatment of these materials releases very high
levels of inhibitors in the hydrolysates with acetic acid being considered one of the most important.
Industry, therefore, needs a yeast which is able to ferment a substrate with a high level of acetic acid.
In our previous work, we used pooled-segregant whole-genome sequence analysis to map QTLs
of complex traits, such as tolerance to acetic acid (Swinnen et al 2012). We identified a number of
novel causative genes and one unique novel mutation located in the previously identified HAA1 gene.
In this work, we combined the knowledge gained from pooled-segregant whole-genome sequence
analysis with the advent of CRISPR-Cas9 technology to engineer the yeast strain used for secondgeneration bioethanol production (Di Carlo et al 2013). Since pentoses are more difficult to ferment
in the presence of inhibitors, the evaluation was not only performed in YP medium with glucose but
also with xylose in the presence of a range of acetic acid conditions (1.0-1.8%) and the medium pH
corrected to 4.7.
The novel identified mutation located in the HAA1 gene was introduced into an industrial yeast strain
for second-generation bioethanol production. This mutation resulted in dramatic improvement of
acetic acid tolerance both for growth on solid nutrient plates and in liquid medium in the presence of
acetic acid, as well as in semi-anaerobic small-scale static fermentations.
The improvement observed was consistently present in all conditions. Hence, we can conclude that
the novel HAA1 mutation results in improvement of acetic acid tolerance in an industrial yeast strain.
KEYWORDS: Acetic acid tolerance, Second generation biofuels, QTL mapping
REFERENCE:
Swinnen S, Schaerlaekens K, Pais T et al. (2012). Identification of novel causative genes determining the complex
trait of high ethanol tolerance in yeast using pooled-segregant whole-genome sequence analysis. Genome Research
22(5):975-984
Di Carlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013). Genome engineering in Saccharomyces cerevisiae
using CRISPR-Cas systems. Nucleic Acids Research 41(7):4336-4343
174
Heterologous expression of a killer toxin of enological interest: the case of KpKt
1
Sara Landolfo1, Rossella Chessa1, Marilena Budroni1, Severino Zara1, Maurizio Ciani2,
Ilaria Mannazzu1
Dipartimento di Agraria, Università degli Studi di Sassari, Viale Italia 39, Sassari, Italy; 2Dipartimento di Scienze della
Vita e dell’Ambiente, Via Brecce Bianche, Università Politecnica delle Marche, Ancona, Italy
[email protected]
The yeast Tetrapisispora phaffii (formerly known as Kluyveromyces phaffii) produces a glycoprotein
of about 33 kDa (Kpkt) that shows β-1,3 glucanase and killer activities and kills wine spoilage yeasts
ascribed to the genera Kloeckera/Hanseniaspora and Zygosaccharomyces through the induction of
ultrastructural modifications on the cell wall. Furthermore, it maintains its killer activity for at least
14 days under winemaking conditions thus showing interesting potential as a bioactive compound
to be used during the prefermentative stages of alcoholic fermentation. Here, the gene coding for
Kpkt (TpBGL2) was cloned into pPIC9 plasmid vector, under the control of AOX1 promoter and
downstream the α leader sequence for secretion, for the heterologous production of the toxin in Pichia
pastoris. The vector was transformed in P. pastoris GS115 and correctly integrated in the genome
of the host generating Mut+ and MutS recombinant clones. The amplification of TpBGL2 in the
cDNA of recombinant Mut+ and MutS clones indicates that the heterologous gene is transcribed.
However, contrary to that expected, none of the clones showed killer activity against the sensitive
strain DBVPG6500 of S. cerevisiae, possibly due either to the production of low concentrations of
the heterologous proteins in the supernatant or to the activity of proteolytic enzymes produced by P.
pastoris.
Project granted by Fondazione Banco di Sardegna (Prot. U925.2014/AI.808.MGB. PI I. Mannazzu)
KEYWORDS: Pichia pastoris, Killer toxin , Heterologous production
175
A new method for yeast monitoring during wine fermentation
Maria O. C. Masiero1, Cecilia Laluce1, Angela Capece2, Patrizia Romano2
Chemistry Institute – UNESP; 2School of Agricultural, Forestry, Food and Environmental Sciences - UNIBAS, Italy
1
[email protected]
The wine fermentation is an environmental complex process involving yeasts and bacteria. It is
characterized by different stages of fermentation where there is an evolution and/or populational
dynamics. Wild yeasts and Saccharomyces cerevisiae are the predominate microorganisms of the
fermentation. Due to lower resistance to high ethanol concentration, wild strains are dominated by S.
cerevisiae that conducts the wine fermentation until the end of the process. Select the starter strain to
be used in fermentation process is critical to ensure its dominance, process reproducibility and wine
quality. Current techniques based on molecular analyses are usually employed to detect the presence
of the inoculated strain. These techniques show high reproducibility and discriminatory power.
However, it is aggregated with high costs and labor-skilled labor. Simple techniques to monitor the
yeasts evolution during fermentation were not elucidated. For this reason, solid media containing dyes
were developed to detect the proportion of three different strains of yeast (two Brazilian strains aimed
to produce ethanol from sugarcane and one isolated strain of Italian grape fermentation) during mixed
fermentation of natural grape must. The results showed that the simple monitoring technique allowed
quantifying the inoculated strains by colony color differentiation. Interdelta analysis was used in
order to correlate the polymorphic profile of each colony color and validate this new technique. This
new monitoring method resulted in a patent of invention, in which has also been successfully used for
the quantification of starter yeast during the fermentation for bioethanol production.
KEYWORDS: Saccharomyces cerevisiae, Chromogenic solid medium, Interdelta analysis, Mixed
fermentation, Fermentation monitoring
176
Using FTIR spectroscopy to characterise yeast derivatives for beverage and
food production
Marcus Laier1, Claus D. Patz2, Doris Rauhut1
Center for Analytical Chemistry and Microbiology
Department for Microbiology and Biochemistry; 2Department for Wine Chemistry and Beverage Research
Hochschule Geisenheim University, Von-Lade-Straße 1, 65366 Geisenheim, Germany
1
[email protected]
Beside yeast extracts which are broadly applied in food production, yeast derivatives are lately also
distributed for the application in beverage technology. To improve the nutrient supply for yeast
(alcoholic fermentation) or bacteria (malolactic fermentation), nutrient preparations based on inactive
yeasts have been developed several years ago. Other yeast derived products are allowed to be used as
fining agents. Especially in oenology the use of such products became a code of practice. By now a vast
number of preparations based on inactive yeasts are commercially available. Micro-nutrients should
be added in an easily available way to the must or wine. Besides their main goal of nutrient supply,
several products with additional features or different applications are also offered. For oenological
products there is not much known about the detailed composition of the manifold products based on
yeasts. In a triennial joint research project with in total nine partners from research, education and
industry the yeast derivatives should be analytically characterised. The development of a simple and
fast spectroscopic method to group the products and to control their production and application is one
main goal.
The powdery original preparations were monitored with FTMIR-ATR. Besides, aqueous eluates
(1 %) were prepared and analysed with FTIR spectroscopy in transmission. The obtained spectra
were evaluated and classified using PCA and PLS models. With reference methods a calibration of
the FTIR spectra was performed. Different methods of data preprocessing and filter selection were
applied for this. It was evaluated if a correlation between reference values and predicted values could
be found. Measuring the eluates in transmission led to more reproducible spectra compared to the
direct measurement of the powdery original preparations due to inhomogeneities of several powders
caused by the additives.
The yeast derivatives can be characterised and categorised with the applied spectroscopic procedure.
The measuring techniques could be used on the one hand for batch controlling during the production of
the preparations and on the other hand to recognise variations from the norm. In further investigations it
should be verified if the recommended application correlates with the composition of the preparations.
More analytical parameters are planned to be calibrated and modelled. Validations of the models will
also be performed.
Acknowledgements We would like to thank the Federal Ministry of Education and Research (BMBF)
for supporting this research project.
KEYWORD: Yeast derivatives, FTIR, PCA, PLS models
177
New Saccharomyces cerevisiae F1 Hybrids for Winemaking under unbalanced
nutritional Conditions
Tommaso Bonciani, Lisa Solieri, Melissa Bizzarri, Luciana De Vero, Paolo Giudici
Department of Life Sciences, University of Modena and Reggio Emilia, Italy
[email protected]
Hybridization has proved to be an efficient genetic engineering-free technique for the construction of
new fermentation starters suitable for specific contexts. This approach entails yeast modifications at
whole-genome level, leading to global changes of the metabolic networks and of the related phenotypes
(Santos & Stephanopoulos 2008). In this work we applied direct mating technique to obtain new strains
with increased tolerance for winemaking in stress-conditions. Parental Saccharomyces cerevisiae
strains of already-assessed industrial performance were genetically randomized via sporulation and
subsequent combination of haploid gametes into new F1 hybrids. Preliminary analysis of spore
viability for parental strains and mating-type for their spore progeny allowed the selection of the
parents with the highest spore viability and the highest number of either MATa or MATa haploid
gametes. Direct mating yielded six hybrids from 5 different pairs of parental strains. These were
validated by PCR-amplification of 4 genes containing highly-variable minisatellites (according to
Solieri et al 2015). During this phase three monosporic cultures with shorter minisatellite regions
than those of their parents were detected. Both the hybrids and the anomalous segregants were
tested for fermentative fitness through micro-fermentative trials in unbalanced Carbon-to-Nitrogen
conditions, intended to mimic nutritional deficiency in must. Wines were then analyzed for their
chemical composition (residual sugars, ethanol and organic acids were determined by HPLC, while
glutathione and SO2 amounts by using enzyme kits) and the obtained data were integrated into a
Principal Component Analysis. The metabolic footprinting of obtained wine yeasts allowed us to
select two superior hybrids, characterized by substantially enhanced industrial traits with respect to
their parents. All newly obtained strains were deposited in the Unimore Microbial Culture Collection
(UMCC).
KEYWORDS: Wine Yeast, Saccharomyces cerevisiae, Genetic improvement, Hybridization,
Alcoholic fermentation
REFERENCES:
Santos CNS, and Stephanopoulos G (2008). Combinatorial engineering of microbes for optimizing cellular phenotype.
Current Opinion in Chemical Biology 12:168–76
Solieri L, Verspohl A, Bonciani T, Caggia C and Giudici P (2015). Fast method for identifying inter- and intra-species
Saccharomyces hybrids in extensive genetic improvement programs based on yeast breeding. Journal of Applied
Microbiology (In Press)
178
Molecular two-step strategy to select inter-species Saccharomyces hybrids
Alexandra Verspohl, Lisa Solieri, Melissa Bizzarri, Paolo Giudici
Department of life science, University of Modena and Reggio Emilia, Italy
[email protected]
Hybridization is a common tool to improve yeast for wine industry. Using non-GE hybridization
techniques a lot of attempts lead to failed mating which is mainly caused by low spore viability
and haplo-selfing. This work proposes a two-step molecular strategy to validate inter-species
Saccharomyces hybrids rapidly. The hybrids were obtained by spore-to-spore mating between
Saccharomyces cerevisiae and Saccharomyces uvarum parental strains, attaching their gametes by
the use of a micromanipulator. Six Saccharomyces cerevisiae and four Saccharomyces uvarum strains
were included in this study. Instead of conventional 880 bp-long ITS-5.8S regions (including both
the internal transcribed spacers 1 and 2 and the 5.8S rNA gene), we implemented colony screening
PCR (csPCR) on the short ITS 1 and ITS 2 regions to increase csPCR efficiency. The set up csPCR
was effective at verifying hybrids directly from the dissection plate and discard homozygous diploid
colonies arisen from one auto-diploidized progenitor. Then, pre-selected candidates were submitted
to recursive streaking and conventional PCR in order to discriminate between the hybrids with
stable genomic background and the false-positive admixtures of progenitor cells both undergone
haplo-selfing. Following, the results were verified by karyotyping. csPCRs of internal transcribed
spacer (ITS) 1 or 2, and the subsequent digestion with diagnostic endonucleases HaeIII and RsaI,
respectively, were efficient at selecting 37 new Saccharomyces cerevisiae x Saccharomyces uvarum
hybrids from 348 crosses. Both protocols reduce significantly the number of massive DNA extractions,
prevent misinterpretations caused by one or both progenitors undergone haplo-selfing, and can be
easily implemented in yeast labs without any specific instrumentation. The study provides a method
for the marker-assisted selection of several inter-species yeast hybrids in a cost effective, rapid and
reproducible manner.
KEYWORDS: Saccharomyces cerevisiae, Saccharomyces uvarum, Inter-species hybrids
REFERENCES:
Solieri L, Verspohl A, Bonciani T, Caggia C, Giudici P (2015). Fast method for identifying inter- and intra-species
Saccharomyces hybrids in extensive genetic improvement programs based on yeast breeding. Journal of applied
microbiology 119:149-151
179
A new method for detection of spoilage yeasts and molds in dairy products
Antonia Gallo1, Massimo Ferrara2, Antonia Susca2, Vito Cinquepalmi2, Fabio Cimaglia1,
Giancarlo Perrone2, Gianluca Bleve1
Consiglio Nazionale delle Ricerche - Istituto di Scienze delle Produzioni Alimentari, Unità Operativa di Lecce, Lecce,
Italy; 2Consiglio Nazionale delle Ricerche- Istituto di Scienze delle Produzioni Alimentari, Bari, Italy
1
[email protected]
Fungi (yeasts and moulds) are recognized as one of the main contaminants of dairy products including
yogurt and sour milk. These microorganisms can also cause spoilage in a wide range of processed,
preserved and refrigerated food products. During the past years, several molecular methods based
on immunological and genotypic techniques have been developed for revealing the presence of
undesirable microorganisms, including fungi, in different food matrices. However, no commercial kit
are already available to detect viable yeasts and moulds in dairy products.
Five antibodies against yeasts and molds were selected from commercially available antibodies and
used to produce functionalized magnetic beads to be used to capture and separate microrganisms
associated to dairy products. Four yeast type species (Debaryomyces hansenii, Kluyveromyces
marxianus, Geotrichum candidum and Pichia anomala) and four mold species (Alternaria alternata,
Aspergillus niger, Penicillium italicum and Rhizopus stolonifer) were used. Milk, yogurt and soft
cheese were tested as matrices. A RT-PCR protocol was developed for detection of yeast and molds
mRNA extracted from contaminated foods.
A new method for yeast and molds enrichment from different food dairy products (milk, yogurt and
soft cheese) based on the use of antibody coated magnetic beads was developed. A new RT-PCR assay
based on a nested amplification was optimized for the detection of yeast and molds in artificially
contaminated dairy products.
The correlation between the amplification signal and the microbial count will allow to use this method
for viable contaminants quantification in dairy products. This method can avoid the labor expensive
food matrices treatments, often cause of loss of sensitivity. This approach will be transferred also in
other food matrices. This procedure can be implemented by the use of automated enrichment systems
already available for pathogen microorganisms.
KEYWORDS: Spoilage yeasts and molds, Detection, Immune-beads, RT-PCR, Dairy products
180
Cell viability and metabolic activity determinations of Xanthophyllomyces
dendrorhous through dielectric spectroscopy and fluorescence microscopy
Allan Sánchez, Enrique Herrera, Melchor Arellano-Plaza, Jesús Cervantes,
Anne Gschaedler-Mathis
Biotecnología Industrial, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C.,
Guadalajara, Jalisco C.P. 44270, Mexico
[email protected]
Bioprocess monitoring and control has become vital in the last years due to the strict quality controls
in the industrial level metabolites production (Alford 2006). Generally, in a bioprocess, variables
like pH, temperature, O2 concentration, etc. are measured, these variables permit conclude about
the culture physicochemical state. However, it is important to monitor the microorganisms’ viability
and metabolic activity. In this work the astaxanthin overproducing mutant strain Xanthophyllomyces
dendrorhous 25-2, was used. A 3L Applikon® biorreactor was utilized filled with 1.7L of a chemically
defined media culture. A FOGALE® nanotech dielectric spectroscopic probe was implemented
(viability). The experimental procedure and the measuring principle are described (Tibayrenc et al
2011) . The fluorescence marker FUN-1® (metabolic activity measurement) was obtained from Life
Technologies, the procedure for cell stain and measuring principle are portrayed (Millard et al 1997).
Marked cells were visualized in a Leica® DMRA2 microscope, with fluorescence.
In Figure 1, it is possible to observe that the permittivity started with values close to 0 pF/cm. As
the fermentation progresses the permittivity raised until 3.5 pF/cm. At the 30 hours the permittivity
reaches a steady state and later starts to decrease until 1.5 pF/cm, at the end of the fermentation (168
hours).
In Figure 2, the types of cells (metabolically active and non-active cells) through the bioprocess are
shown; at the beginning just 20% of the cells are metabolically active. As the time of the fermentation
advance the active cell percentage raised to 95 %; nevertheless, when the culture come to its steady
phase (36 hours) it decreased until values around 10% at the end of the bioprocess.
These two technics reflect different populations in the same culture; these different physiological
states could play an important role in the production of some interesting metabolites like astaxanthin.
KEYWORDS: X. dendrorhous, Cell viability, Dielectric spectroscopy, Fluorescence microscopy
REFERENCES:
Alford JS (2006). Bioprocess control: advances and challenges. Computers & Chemical Engineering 30:1464-1475
Tibayrenc P, Preziosi-Belloy L, Ghommidh C (2011). On-line monitoring of dielectrical propierties of yeast cells during
a stress-model alcoholic fermentation. Process Biochemistry 46:193-201
Millard PJ, Roth BL, Thi HP, Yue S.T, Haugland RP (1997). Development of the FUN-1 family of fluorescent probes
for vacuole labeling and viability testing of yeast. Applied and Environmental Microbiology 63(7):2897-2905
181
Peptide mass fingerprinting of red wine varieties using a MALDI-TOF mass
spectrometry approach
Mihaela Begea1, Iuliana D. Bărbulescu2, Simona I. Marinescu 2, Oana R. Dincă1,3,
Roxana E. Ionete3, Radu Tamaian3,4
University Politehnica of Bucharest, Romania; 2Pharmacorp Innovation SRL, Bucharest, Romania; 3National Institute
for Research and Development for Cryogenic and Isotopic Technologies, Râmnicu Vâlcea, Romania; 4University of
Bucharest, Faculty of Physics, 3Nano-SAE Research Centre, Bucharest-Măgurele, Romania
1
[email protected]
The authentication of natural wine is an important mater of quality control and safety in vinification.
In order to implement a shotgun type proteomic approach, which requires no prior knowledge about
the proteins to be identified (Han, Aslnian and Yates, 2008; Motoyama and Yates, 2008), to establish a
varietal identification technique for red wines were selected exclusively varietal vines with protected
designation of origin (PDO) and protected geographical indication (PGI). The sampling included
varietal wines made both from well-known and world-wide cultivated red wine grape varieties (e.g.
Merlot, Cabernet Sauvignon) and from local or not so spread varieties (e.g. Băbească and Mamaia from
Romania; Montepulciano from Italy, Tempranillo from Spain). A fast method to determine the varietal
origin of red wines was developed based on peptide mass fingerprinting using a matrix assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer from Bruker Daltonics, Inc.
and MALDI Biotyper 3.0 software, respectively. The protein extracts of each sample were processed
using multiple measurements in order to ensure that the true biological variability of each varietal
wine was acquired. Therefore, a database has been developed using the molecular fingerprints of the
red wine varietals. The acquired fingerprints (stored as multiple spectra measurements) were used for
PCA (principal component analysis) and dendrogram were performed to illustrate the scattering plots,
respectively the hierarchical clustering of varietals included in database.
The results showed that this shotgun type proteomic approach can be a rapid, simple and practical
method for determining the biological origin of red wine varietals, but suffer by two major shortcomings:
a) this technique is severely jeopardized by proteolysis (both natural proteolysis and heat exposure
proteolysis) and b) it cannot discriminate between Merlot and Cabernet Sauvignon varietals.
KEYWORDS: Wine proteomics, MALDI-TOF mass spectrometry, Varietal, PDO, PGI.
REFERENCES:
Han X, Aslanian A, Yates JR (2008). Mass spectrometry for proteomics. Curr Opinion Chem Biol 12:483–490.
Motoyama A, Yates JR (2008). Multidimensional LC separations in Shotgun Proteomics. Anal Chem 80:7187-7193.
182
Can hybrids reproduce?
Agnieszka Maslowska1, Edward J. Louis1, Samina Naseeb2, Daniela Delneri2, Chris Powell3
University of Leicester, Centre for Genetic Architecture of Complex Traits, Leicester, UK;
2
University of Manchester, Faculty of Life Sciences, Manchester, UK;
3
University of Nottingham, Bioenergy & Brewing Science, Nottingham, UK
1
[email protected]
Breeding has been utilised by men for millennia to improve various characteristics in animals and
plants. It allows for selection of enhanced individuals, which might be better adapted to harsh
conditions or produce bigger crops. Hybridisation of closely related species could generate a wide
spectrum of possible trait combinations with better or even new characteristics arising. However most
hybrids are sterile and as for example in mules (hybrid between horse and donkey) the generation of
offspring is precluded by reproductive isolation. Therefore improvement of this hybrid can only be
achieved by separate breeding in horse and donkey, mating them, and hoping for new characteristics
to manifest in newly created hybrid.
Yeast hybrids are found in different locations around the world in industrial environments as well as
in the wild. Well known is the hybrid between S. cerevisiae and S. eubayanus called S. carlsbergensis/
pastorianus, used in brewing. Furthermore, hybrids of S. cerevisiae × S. kudriavzevii, as well as a
complex hybrid of three species known as S. bayanus, are utilised in wine production.
Fertility of interspecific hybrids can be rescued by genome duplication, allowing chromosomes to
create pairs of homologues, enabling completion of meiosis. We show that existing hybrids can be
improved through breeding. Fertility of a triploid wine strain has been successfully rescued by rare
mating events with haploid strain. Therefore individuals were able to successfully complete meiosis,
giving viable progeny. Previously precluded, by reproductive isolation, genetic analysis can now
be performed on yeast hybrids revealing the genetic basis of beneficial features, allowing for the
isolation of strains with huge potential for the fermentation industry.
KEYWORDS: Saccharomyces, Hybrid, Genetic variation, Fermentation
183
Optimization of the Bioprocess for Producing Dry Yeast Biomass Enriched with
Minerals
Iuliana D. Bărbulescu1, Simona I. Marinescu1, Mihaela Begea1,2, Madalina G. Albu 3, Denisa
Duta4, Ionescu Valentin4, Radu Tamaian5,6, Mihaela V. Ghica7
Pharmacorp Innovation SRL, Bucharest, Romania; 2University Politehnica of Bucharest, Faculty of Biotechnical
Systems Engineering, Bucharest, Romania; 3Leather and Footwear Research Institute - INCDTP, Collagen Department,
Bucharest, Romania; 4National Institute of Reseach&Development for Food Bioresources - IBA Bucharest (IBA),
Romania; 5National Institute for Research and Development for Cryogenic and Isotopic Technologies, Râmnicu Vâlcea,
Romania; 6University of Bucharest, Faculty of Physics, 3Nano-SAE Research Centre, Bucharest-Măgurele, Romania;
7
“Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Bucharest, Romania
1
[email protected]
Iron and calcium enriched yeasts can provide a supplementation of these micronutrients to diet
because these minerals have a better bioavailability when bonded to yeast cells. (Gaensly et al 2011)
cultivated yeast in a 5 L-glass bioreactor, in a media supplemented with iron, and the intracellular
iron content was 8.062 ± 0.251 mg Fe.g-1 dry matter at the end of the cultivation period. (He Xiuping
2004) patented a calcium enriched yeast producing process, thereby providing a high biomass of
calcium enriched beer yeast.
This paper presents an optimization method for the enrichment with calcium and iron of a wine yeast
biomass.
Fermentation has been carried out in a Biostat B plus bioreactor (working volume 4L), initial pH 5.8,
temperature 30oC, inoculation ratio 10% using a liquid inoculum. Fe and Ca have been determined
using F-AAS spectroscopy.
The obtaining of variation ranges for some operating conditions as independent variables (time – X1,
solution of iron citrate / calcium propionate – X2, stirring – X3 and air flow – X4) which leaded to the
optimum response: maximum values for the dependent variable dry weight (Y1) was followed. The
statistical analysis has been performed using the software package Statistica StatSoft Release 8.
The influence of the operating conditions of system response was quantified by some mathematical
models. The relation between the dependent variable and the independent variables has been elucidated
using the response surface shape analysis. By contour plots superposing technique the optimum zone
for the targeted response has been obtained.
Utilization of a gradually addition of 40mL iron citrate (solution 10%) and 50mL calcium propionate
(solution 10%) in fermentation medium leaded to a concentration in dry biomass of 945.027mg/100g
iron and 238.375 mg/100g calcium.
The addition of iron and calcium has been increased to 120mL and 150mL respectively, and
consequently the determined concentration of both micronutrients in final biomass was 3642 mg/100g
Fe and 3392 mg/100g Ca respectively.
REFERENCES:
Gaensly F, de Castro Wille GMF, Brand D, Bordin Bonfim TM (2011). Iron enriched Saccharomyces cerevisiae
maintains its fermenting power and bakery properties. Ciência e Tecnologia de .Alimentos 31(4):980-983
He Xiuping (2004). Calcium enriched beer yeast strain and use. China Patent 200410032718, Application Date April 16
184
Yeast multi-stress resistance and lag-phase duration in wine
David Ferreira1,2, Virginie Galeote2, Anne Ortiz-Julien1, Sylvie Dequin2
Lallemand SAS, F-31700 Blagnac, France; 2INRA, UMR1083 Sciences pour l’œnologie, F-34060 Montpellier, France
1
[email protected]
Saccharomyces cerevisiae has been used for several millennia to perform wine fermentation due
to its endurance and unmatched qualities. Nevertheless, at the moment of inoculation, wine yeasts
must cope with specific stress factors that still challenge wine-makers nowadays by slowing down
or even compromising the fermentation process. The objective of the present work is to elucidate
the metabolic and molecular bases of multi-stress resistance during wine fermentation lag-phase.
By means of a specific experimental design, we first characterized a set of commercialized wine
yeast strains for lag-phase duration in realistic wine fermentation conditions combining several stress
factors found in red wines (osmotic stress, SO2 presence and low thiamine) or in white wines (low
temperatures, low lipid availability and SO2 presence). This set of yeasts includes S. cerevisiae strains
representing a wide diversity among the wine genetic cluster, as shown by microsatellite analysis,
and other yeast species and hybrids. We found marked differences in stress sensitivities between
yeast strains. Some strains were sensitive to all stress conditions while for other strains the lag-phase
duration was stress specific. We showed that the presence of the XVIVIII translocation in S. cerevisiae
strains positively contributes to shorter lag-phase and higher endurance by playing an important role
in SO2 resistance. Furthermore, by means of a central composite plan, differences in stress-factor
weight were identified, such as a much higher impact of osmotic stress in comparison with SO2
presence in red wine fermentation conditions.
Finally, this characterization allowed the selection of the most suitable strains for further studies.
An evolutionary engineering approach and a QTL analysis are currently underway with the aim to
develop novel yeast strains with reduced lag-phase and to provide new insights into the genetic bases
of multi-stress resistance during lag phase in wine fermentation.
Keywords: Wine fermentation, Lag-phase, Multi-stress resistance
185
186
Session 3
biofuels, new molecules and enzymes
187
Salt tolerance improvement of industrial yeast strain GSE16 by
Zygosacchoromyces rouxii cDNA library expression
Yingying Li1,2, Mekonnen Demeke1,2, Maria Foulquie Moreno1,2, Johan M. Thevelein1,2
Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium; 2Department of
Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001, Leuven, Belgium
1
[email protected]
S. cerevesiae is commonly used in the 2nd generation bio-ethanol production. The substrates used
for 2nd generation bio-ethanol production are mainly biomass, in which large amount of inhibitors
are present. Salts can be present also as inhibitors in lignocellulose hydrolysates, which decrease the
sugar consumption rates and increases byproduct formation.
Z. rouxii is a halotolerant and osmotolerant yeast species closely related to S. cerevisiae, which can
stand 18% salt. 3 ORFs which could increase the salt tolerance of salt sensitive strain GSE161 were
selected from the cDNA library of Z. rouxii by certification through spot test assay, liquid assay
and fermentation. The maximum glucose consumption rate of strain GSE16-pFL39-26 in bagasse
hydrolysate was 0.413 g•L-1•h-1, while the value for the control strain GSE16 was 0.271 g•L-1•h-1.
The S. cerevisiae gene deletion strains of these 3 ORFs were tested in liquid SCD medium supplemented
with increasing concentrations of NaCl. However, the results did not show a relationship between the
3 genes and salt tolerance. Further research about improving salt tolerance in S. cerevisiae is ongoing.
KEYWORDS: Halotolerance, Bio-ethanol, GSE16, Z. rouxii, cDNA library
REFERENCES:
Demeke M, Dumortier F, Li Y, Broeckx T, Moreno MRF, Thevelein JM (2013). Combining inhibitor tolerance and
D-xylose fermentation in industrial Saccharomyces cerevisiae for efficient lignocellulose-based bioethanol production.
Biotechnology for Biofuels 6:120
188
Engineering subcellular compartmentalization in Saccharomyces cerevisiae
Mislav Oreb, Cora Mignat, Thomas Thomik, Pia Deinhard, Joanna Tripp, Mara Reifenrath,
Eckhard Boles
Goethe University, Institute of Molecular Biosciences, Frankfurt, Germany
[email protected]
In metabolic engineering and synthetic biology approaches, a host organism is endowed with
biochemical reactions naturally not occurring in it. The yield of desired products is often limited
by diversion of substrates or intermediates through competing pathways or formation of toxic
byproducts. Naturally, such limitations can be circumvented by subcellular compartmentalization or
by the formation of enzyme complexes, in which the transfer of intermediates between the active sites
is accelerated. To improve the efficiency of engineered pathways, it would therefore be advantageous
to construct synthetic enzyme complexes or membrane-surrounded compartments. I will present two
strategies to achieve this goal in Saccharomyces cerevisiae.
189
Exploring a molecular systems biology approach to setup Saccharomyces
cerevisiae as a cell factory for the production of itaconic acid
Ana Vila Santa1, Nicole M Rodrigues1, Maria Raquel Moita1, Claudio Frazão1, Silvia F.
Henriques1, Isabel Sá-Correia1, Laura R. Jarboe2, Susana Vinga3, Nuno P. Mira1
iBB-Institute for Bioengineering and Biosciences, Departamento de Bioengenharia, Instituto Superior Técnico,
Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; 2Chemical and Biological Engineering
Department, Iowa Sate University, 3051 Sweeney Hall, 4134 Biorenewables Research Laboratory, Ames-Iowa, USA;
3
Center of Intelligent Systems – Instituto de Engenharia Mecânica, Instituto Superior Técnico, Av. Rovisco Pais, 1049001 Lisboa, Portugal
1
[email protected]
Itaconic acid (IA) is a C5-dicarboxylic acid considered a highly interesting building-block molecule as
it can be used as a precursor in chemical synthetic routes explored by a variety of industries, ranging
from plastics to pharmaceuticals (Sauer et al 2008; Steiger et al 2013). The implementation of microbial
processes to produce IA is receiving a lot of attention in this “biorefinery-oriented” era as this acid can
be produced from biomass and can replace currently used catalysts obtained from petrochemistry (Sauer
et al 2008; Steiger et al. 2013). Microbe-based processes for production of IA have been implemented,
essentially exploring Aspergillus terreus and A. niger as host systems; however, the yields obtained are
well below those theoretically predicted (Steiger et al 2013). This reduced yield is, in part, attributed
to metabolic diversion of the carbon source from IA biosynthesis and to the toxic effect exerted by the
acid on the producing cells. In this work we are using a molecular systems biology approach to explore
Saccharomyces cerevisae as a cell factory for the production of IA, taking advantage of the unique
potential of this species as an experimental system and as a host for the production of carboxylic acids
(Abbot et al 2009; dos Santos & Sá-Correia 2015). To setup production of IA in yeast the A. terreus
AtCad1 enzyme was heterologously expressed rendering cells able to produce IA in titers ranging
from 0.7 mg/L, depending on the carbon source used. To improve production of IA in S. cerevisae a
molecular systems biology approach is being implemented gathering results from in silico simulation,
metabolomics and chemogenomics (Fig.1). Using the knowledge gathered it was possible to engineer
an yeast strain that produces around 20-fold more IA than wild-type cells. The phenotypic and metabolic
characterization of this “IA over-producer strain” will be discussed in this work, along with other
indicatives obtained that are expected to advance the implementation of microbial IA production.
KEYWORDS: Itaconic acid; S. cerevisiae as a cell factory, Production of carboxylic acids; Metabolic
engineering; Biorefineries
REFERENCES:
Sauer M et al (2008). Trends in Biotechnology 26:100-108
Steiger M et al (2013). Frontiers in Microbiology 4:14-23.
Abbot et al (2009). FEMS Yeast Research 9:1123-36
dos Santos SC, Sá-Correia I (2015). Current Opinion in Biotechnology 33:183-191.
190
Targeting metal-binding oligopeptides to the inner face of plasma membrane in
Saccharoyces cerevisiae cells
Ileana C. Farcasanu1, Lavinia L. Ruta1, Ioana Nicolau1, Aurora D. Neagoe2
1
Faculty of Chemistry; 2Faculty of Biology, University of Bucharest, Bucharest, Romania
[email protected]
Heavy metal pollution represents a threat to water supplies, agriculture soils, human and animal
health, whereas the deficiency is considered equally deleterious for any form of life, or for important
human activities, such as agriculture. Heavy metals are challenging pollutants as they are natural
components of the earth’s crust, they are persistent in the environment and they are non-degradable.
Under such circumstances, removal of contaminating metals by means of (micro)organisms is often the
method of choice, and obtaining resistant species which accumulate heavy metals from contaminated
sites represents a pre-requisite for bioremediation techniques. In our laboratory we aimed to obtain
heavy metal hyperaccumulating yeast cells designed primarily for metal-related bioremediation and
bioextraction actions. In this study we present the possibility to engineer yeast cells for heavy metal
hyperaccumulation by targeting heavy metal-binding oligopeptides to the inner face of the plasma
membrane.
We designed a collection of DNA plasmids harboring sequences which encode artificial metalbinding oligopeptides (MeBPep) fused to myrGFP. The myrGFP casette introduces a myristoylation
site, allowing both directional targeting to the inner face of the plasma membrane and monitoring of
the intracellular localization. To estimate and to control the potential toxicity of the constructs, the
expression level was turned monitorizable by placing the chimeric DNA under the inducible GAL1
promoter. Metal accumulation by the transgenic yeasts obtained was assessed by inductively coupled
plasma-mass spectrometry (ICP-MS).
We generated a collection of yeast strains which (over)express artificial metal-binding oligopeptides
fused to myrGFP. This collection was investigated against an array of heavy metals in terms of
metabolic changes, growth defects and heavy metal (hyper)accumulation.
191
Heterologous expression of cellulase genes in wild Saccharomyces cerevisiae
Steffi A. Davison, Riaan Den Haan, Willem H. Van Zyl
Department of Microbiology, Stellenbosch University, Stellenbosch, Western Province, South Africa
[email protected]
Simultaneous saccharification and fermentation (SSF) involves enzymatic hydrolysis of pretreated
lignocellulosic biomass and fermentation of its glucose constituents in a single bioreactor. Although
Saccharomyces cerevisiae is the microorganism of choice for cellulosic bioethanol production, it has
a significantly inferior secretory capacity for heterologous proteins compared to other filamentous
fungi. Moreover, degradation products resulting from biomass pretreatment impairs growth and
fermentation of sugars. One approach to resolve both concerns is to utilize a strain background with
innate tolerance to inhibitory degradation products and a high secretory phenotype.
In this study, we screened a collection of 32 wild S. cerevisiae strains isolated from vineyards in South
Africa, with high inhibitor tolerance and superior secretion phenotypes serving as basis for selection.
After engineering the strains with high- and low-gene copy expression cassettes harbouring the
Talaromyces emersonii Cel7A (a cellobiohydrolase), Trichoderma reesei Cel5A (an endoglucanase)
and Saccharomycopis fibuligera Cel3A (a β-glucosidase), we compared the expression ability of these
engineered strains to the benchmark S288c strain. We also compared fermentation vigor, temperature
and osmotolerance.
During high copy plasmid expression, engineered strain YI13 showed higher enzyme activity levels
and produced a similar amount of ethanol (9,0 g/L) to the benchmark S288c strain. Furthermore,
relative to the rest of the evaluated repertoire of strains, this strain exhibited superior tolerance to
industrial stresses such as high temperature, hyperosmotic stress and secretion stress. YI13 illustrated
high growth vigour, in addition to comparatively higher native invertase secretion.
These results validate our pursuit of wild Saccharomyces cerevisiae strains with high secretion
phenotypes and high tolerance properties for lignocellulosic biofuel production. Some wild yeast
strains phenotyped in this work indicate that these hosts have the genetic background that could have
potential for fermentation of pretreated biomass.
192
γ-Decalactone production by yeasts in glycerol and castor oil medium
Géssyca P. A. Soares1, Karla S. T. Souza1, Rosane F. Schwan1, Disney R. Dias2
Department of Biology; 2Department of Food Science, Federal University of Lavras, MG, Brazil
1
[email protected]
Flavor compounds are commonly obtained from chemical synthesis or extraction from plants. These
have disadvantages, such as racemic mixtures generation, more stages, low yield and high cost,
making the microbial fermentation an alternative and potential way to obtain flavor compounds.
γ-decalactone have peach aroma, and can be obtained by biotransformation of ricinoleic acid via
yeast peroxisomal β-oxidation. The aim of this work was to use alternative substrates to produce
bioaroma from two different yeasts. Yarrowia lipolytica UFLA CM-Y9.4 and Lindnera saturnus
CCMA 0243 were grown at different concentrations (10, 20 and 30%) of substrates (castor oil and
crude glycerol) for γ-decalactone production. L. saturnus CCMA 0243 produced higher concentration
of y-decalactone ( 5g/l) in crude glycerol, while Y. lipolytica CM –Y9.4 showed maximum production
in castor oil (3g/l). Crude glycerol was an alternative substrate for the production of γ-decalactone
showing better results when compared to castor oil. L. saturnus CCMA 0243 has high potential
for higher production of γ-decalactone from crude glycerol, decreasing the cost of the process and
solving an environmental problem.
KEYWORDS: Aroma, Biotransformation, Crude glycerol, Lactone, Lindnera saturnus
193
Overexpression of native Saccharomyces cerevisiae SNARE genes increased
heterologous cellulase secretion
John H. D. Van Zyl1, Riaan Den Haan2, Willem H. Van Zyl1
Department of Microbiology, Stellenbosch University, Stellenbosch 7602, South Africa; 2Department of Biotechnology,
University of the Western Cape, Bellville 7530, South Africa
1
[email protected]
SNAREs (soluble N-ethylmaleimide-sensitive factor attachment receptor proteins) are essential
components of the yeast protein trafficking machinery and are required at the majority of membrane
and vesicle fusion events in the cell (Weber et al 1998). A major obstacle to the successful utilization
of Saccharomyces cerevisiae for the single-step hydrolysis and fermentation of cellulosic material
to second generation bio-ethanol (consolidated bio-processing) remains its inferior yields for
heterologous cellulases. We have demonstrated an increase in secretory titers for the Talaromyces
emersonii Cel7A (a cellobiohydrolase) and the Saccharomycopsis fibuligera Cel3A (a β-glucosidase)
expressed in Saccharomyces cerevisiae through single and co-overexpression of some of the ERto-Golgi SNAREs (BOS1, BET1, SEC22 and SED5). Overexpression of SED5 yielded the biggest
improvements for both of the cellulolytic reporter proteins tested, with maximum increases of 22%
for the Sf-Cel3A and 68% for the Te-Cel7A. Co-overexpression of the ER-to-Golgi SNAREs yielded
proportionately smaller increases for the Te-Cel7A (46%), with the Sf-Cel3A yielding no improvement.
Co-overexpression of the most promising exocytic SNARE components identified in literature (Van
Zyl et al 2014) for secretory enhancement of the cellulolytic proteins tested (SSO1 for Sf-Cel3A and
SNC1 for Te-Cel7A) with the most effective ER-to-Golgi SNARE components identified in this study
(SED5 for both Sf-Cel3A and Te-Cel7A) yielded variable results, with Sf-Cel3A improved by 130%
and Te-Cel7A yielding no improvement. Improvements were largely independent of gene dosage, with
episomal variance between the most improved strains shown to be insignificant. This study has added
further credence to the notion that SNARE proteins fulfil an essential role within a larger cascade
of secretory machinery components that could contribute significantly to future improvements to
Saccharomyces cerevisiae as protein production host.
KEYWORDS: SNAREs, Secretion, Cellulase, Yeast
REFERENCES:
Weber T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F, Söllner TH, Rothman JE (1998).
SNAREpins: Minimal machinery for membrane fusion Cell 92:759-772
Van Zyl JHD, Den Haan R, Van Zyl WH (2014). Over-expression of native Saccharomyces cerevisiae exocytic SNARE
genes increased heterologous cellulase secretion. Applied Microbiology and Biotechnology 98(12):5567-5578
194
Metabolic engineering of yeast for commercial production of succinic acid
Alrik Los, Ben den Dulk, Rolf Poldermans, Zheng Zhao, Rene Verwaal, Mickel Jansen,
Theo Geurts
DSM Biotechnology Center, A. Fleminglaan 1, 2613 AX Delft, the Netherlands
[email protected]
DSM and ROQUETTE have started a joint venture by the name of “Reverdia” (www.reverdia.com)
for the fermentative production and commercialization of succinic acid from renewable resources, to
be marketed under the name BiosucciniumTM. Succinic acid has been identified as a potential key
building block for deriving both commodity and specialty chemicals from biomass. While existing
markets for chemically produced succinic acid include pharmaceuticals, food, coatings and pigments,
bio-based succinic acid is envisioned to drive the emergence of new applications such as polyester
polyols for polyurethanes, polybutylene succinate (PBS), plasticizers, 1,4-butanediol and resins. The
fermentative production of succinic acid at low pH results in a substantially lower environmental
footprint compared to both the current petrochemical process, the near-neutral pH used in bacterial
fermentation processes, as well as the process for petrochemical adipic acid, which is the conventional
chemical used for production of for example polyester polyols.
We present the metabolic engineering strategy of the yeast used in this process. Heterologous genes,
optimized for expression in the host, were introduced for achieving high level production of succinic
acid from sugar. Expression of these genes was verified at the protein level with LC-MS. Systemslevel analysis (transcripts, proteins and metabolic fluxes) provided insight into the physiology and
metabolism of the strain during fermentation, and generated various new leads for strain and process
optimization.
The process for production of succinic acid has been scaled up in our demonstration plant. In this
process the strain produces succinic acid at a high titer at a low pH. Furthermore, Reverdia is the first
in the world to have a large scale facility for the commercial production of bio-based succinic acid.
The new facility, based in Cassano Italy, commenced operation in December 2012.
195
Improvement of thermotolerance of Saccharomyces cerevisiae for bioethanol
production by genome shuffling
Minetaka Sugiyama, Junyuan Wu, Yu Sasano, Satoshi Harashima
Department of Biotechnology, Graduate School of engineering, Osaka University, Japan
[email protected]
Production of ethanol from lignocellulosic biomass by Saccharomyces cerevisiae contributes
to developing sustainable society. However, in the process of simultaneous saccharification and
fermentation, the optimal temperature of saccharification of the biomass by cellulase is from 45°C
to 55°C which is much higher than that of fermentation by S. cerevisiae that is about 30°C. This
temperature gap causes increase of the production cost. Therefore, in this study, we tried to improve
thermotolerance of S.cerevisiae by modified genome shuffling. In the first round of genome shuffling,
S. cerevisiae TJ14 and C3867 strains showing thermotolerance up to 41ºC with high ethanol
productivity was used as recipient strains. Non-conventional yeast Hansenula polymorpha showing
robust thermotolerance up to 49ºC was chosen as donor strains. After transformation of recipient
strains with genomic DNA from donor strain, 3 transformats, TGS3 and TGS6 from TJ14 and CGS6
from C3867, were isolated at 42ºC. They showed pretty better growth and much better ethanol
production than their recipient strains at 42ºC. Particularly 7 times higher ethanol production with
0.513 g ethanol/ g glucose was achieved in the TGS6 strain at 42ºC. Random amplified polymorphic
DNA analysis highly suggested that genome shuffling indeed occurred in these strains. In the second
round of genome shuffling, transformation of TGS6 and CGS6 with H. polymorpha DNA gave 2
transformants TGS6-2 and TGS6-3 at 42.5ºC. They showed significantly better growth and 2 times
higher ethanol production than their recipient strains at 42.5ºC. These strains contribute to providing
better platforms for further development of thermotolerant yeast for bioethanol production.
KEYWORDS: Bioethanol, Genome shuffling, Saccharomyces cerevisiae
196
The expression of genes encoding heterologous glycerol facilitator
homologues from various yeast species improves the glycerol growth of
Mathias Klein, Martina Carrillo, Zia UI Islam, Steve Swinnen, Elke Nevoigt
Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany
[email protected]
The utilization of glycerol for yeast-based bioprocesses has several advantages over glucose. There
have been several indications for glycerol transport being a rate-controlling step for glycerol utilization
in S. cerevisiae. During utilization of glycerol by wild-type S. cerevisiae, the uptake of the substrate
is conducted by an active transport system encoded by STL1, while the glycerol facilitator encoded
by FPS1 is involved in glycerol export but not in glycerol uptake. However, it has been previously
demonstrated that the expression of the FPS2 gene from the yeast species Pachysolen tannophilus,
showing 32% homology on amino acid level to S. cerevisiae FPS1, in a stl1 deletion mutant of S.
cerevisiae is able to function as the sole glycerol uptake system during utilization of glycerol as the
sole source of carbon (Liu et al 2013). Here we show that predicted heterologous glycerol facilitators
from other yeast species with moderate or superior growth on glycerol such as Cyberlindera jadinii,
Yarrowia lipolytica and Pichia pastoris can fulfill this function with similar performance as shown for
P. tannophilus Fps2. Moreover, the sole expression of one of the above-mentioned glycerol facilitator
genes in our previously selected wild-type S. cerevisiae isolate, that is able to naturally grow on
glycerol with a maximum specific growth rate of 0.13 h-1 (Swinnen et al 2013), improved the glycerol
growth rates to values ranging from 0.17 to 0.18 h-1. These results represent a promising starting
point for the future development of S. cerevisiae-based bioprocesses using glycerol as a feedstock,
particularly since the uptake by a glycerol facilitator is ATP-independent which is in contrast to the
natural active transport system encoded by STL1.
KEYWORDS: Saccharomyces cerevisiae, Glycerol facilitator, FPS1
REFERENCES:
Liu X, Mortensen UH, Workman M (2013). Expression and functional studies of genes involved in transport and
metabolism of glycerol in Pachysolen tannophilus. Microbial Cell Factories 12: 27-36
Swinnen S, Klein M, Carrillo M, McInnes J, Nguyen HT, Nevoigt E (2013). Re-evaluation of glycerol utilization in
Saccharomyces cerevisiae: characterization of an isolate that grows on glycerol without supporting supplements.
Biotechnology for Biofuels 6: 157-168
197
Genetic mapping and inverse engineering of crucial determinants for glycerol
utilization in the yeast Saccharomyces cerevisiae
Steve Swinnen, Ping-Wei Ho, Mathias Klein, Elke Nevoigt
Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany
[email protected]
So far glycerol has been neglected as a feedstock for commercial bioprocesses employing the platform
yeast Saccharomyces cerevisiae. This can be attributed to the fact that the commonly used laboratory
and industrial strains do not or just very poorly grow on glycerol as the sole carbon source, particularly
if no growth-supporting supplements such as amino acids and nucleic bases are added to the medium.
In a previous study we have identified a S. cerevisiae strain (CBS 6412) that showed comparatively
good growth under these conditions (Swinnen et al 2013). A meiotic segregant of this strain (referred
to as CBS 6412-13A) exhibiting a maximum specific growth rate of 0.13 h-1 on glycerol was crossed
with the non-growing strain CEN.PK 113-1A, and 24 segregants exhibiting similar, good glycerol
growth (compared to CBS 6412-13A) were selected. These segregants were then applied to a genetic
mapping experiment using pooled-segregant whole-genome sequencing (Swinnen et al 2012). One
major locus contributing to the good glycerol growth phenotype was identified. Further downscaling
by fine-mapping and reciprocal hemizygosity analysis finally allowed the parallel identification of
three major genetic determinants of the glycerol growth phenotype. Parallel replacements of all three
CEN.PK 113-1A alleles by the corresponding CBS 6412-13A alleles confirmed the concerted positive
effect of the three identified genes, and resulted in a maximum specific growth rate of 0.08 h-1 in the
reverse engineered strain CEN.PK 113-1A.
KEYWORDS: Saccharomyces cerevisiae, Glycerol growth, Genetic mapping, Inverse engineering
REFERENCES:
Swinnen S, Klein M, Carrillo M, McInnes J, Nguyen HT, Nevoigt E (2013). Re-evaluation of glycerol utilization in
Saccharomyces cerevisiae: characterization of an isolate that grows on glycerol without supporting supplements.
Biotechnology for Biofuels 6: 157-168
Swinnen S, Thevelein J, Nevoigt E (2012). Genetic mapping of quantitative phenotypic traits in Saccharomyces
cerevisiae. FEMS Yeast Research 12: 215-227
198
Rapid evaluation of itaconic acid metabolic engineering strategies in
Ulrike Müller, Zheng Zhao, Ben Meijrink, Bianca Gielesen, Liang Wu, Hans Roubos
DSM Biotechnology Center, A. Fleminglaan 1, 2613 AX Delft, the Netherlands
[email protected]
The recent explosion of strain optimization algorithms based on genome-scale metabolic models
(GSMM) allows for rapid generation of a large number of metabolic engineering strategies for
maximizing metabolite production. However, implementation and validation of these strategies
remains a bottleneck in the design-build-test-learn cycle. Here we demonstrate a method for targeted
high-throughput strain transformation using exchangeable building blocks. Using this method
and a high-throughput analysis platform, we evaluated multiple itaconic acid producing strategies
simultaneously in Saccharomyces cerevisiae wild type strains. The initial design included differences
in compartmental strategy, a set of gene variants, differences in promoter strength and two strain
backgrounds. The results revealed further potential for improving the productivity of itaconic acid.
Overall, we demonstrate that rapid prototyping by targeted engineering has become a valuable tool
to quickly explore metabolic engineering scenario’s including host strains for the production of a
heterologous product.
199
An in vitro control study to test the yeast Saccharomyces cerevisiae strain BCA61
for contaminated soils bioremediation
Antonio Cristaldi1, Gea Oliveri Conti1, Alfina Grasso1, Giovanni Arena1, Chiara Copat1,
Cristina Restuccia2, Margherita Ferrante1
Environmental and Food Hygiene Laboratories (LIAA), Department of Medical, Surgery Sciences and Advanced
Technologies, G. F. Ingrassia, University of Catania, Italy; 2Laboratory of Agricultural and Food Microbiology,
Department of Agriculture, Food and Environment (Di3A), University of Catania, Italy
1
[email protected]
The bioaccumulation and bioaugmentation of heavy metals has become a very serious environmental
problem as it negatively affects microbial processes in agricultural soils and human health through
the food chain. The estimated costs for the conventional soil remediation are very high, so within the
more economic and ecofriendly technologies, bacteria, fungi and plants have been used since long
time with extremely variable results. Aim of our study was to evaluate the ability of Saccharomyces
cerevisiae BCA61 to uptake heavy metals using an in vitro study and to assess its applicability for
environmental reclamation.
The uptake capacity of S. cerevisiae BCA61 was evaluated in Malt Extract Broth with 4 increasing
concentrations, respectively of Ni (120; 150; 225 and 262 mg/300 mL of broth), Cd (3.6; 4.5; 6.75;
7.88 mg/300 mL), Cu (144; 180; 270 and 315 mg/300 mL) and As (12; 15; 22.5; 26 mg/300 mL) by
performing 3 replicates for each concentration and one control constituted by the not fortified culture
broth. The analyses were performed with Elan DRC-e Perkin Elmer ICP-MS.
The S. cerevisiae strain has absorbed moderately Ni, Cu and As and to a greater extent the Cd (Fig.
1). In particular was calculated an uptake capacity respectively of 6-5.2-4.4-4.1% for Ni; 29-21.716-33.5% for Cd; 9.6-9.5-5.3-4.9% for Cu; 9.7-7.9-5.9-7% for As. The % uptake was calculated as
average of the values obtained from the three independent replicates.
S. cerevisiae is able to grow in the medium singularly fortified with Ni, Cd, Cu and As, showing a
good ability to uptake the heavy metals. However, the yeast exposed to Cu shows a nonlinear uptake
respect to Ni, Cd and As. So, it is probable that a toxic effect for the highest doses of Cu can interfere
with cell growth, then lowering the uptake capacity (Peng et al 2010).
KEYWORDS: Absorption, Nickel, Cadmium, Copper, Arsenic
REFERENCES:
Peng Q, Liu Y, Zeng G, Xu W, Yang C, Zhang J (2010). Biosorption of copper(II) by immobilizing Saccharomyces
cerevisiae on the surface of chitosan-coated magnetic nanoparticles from aqueous solution. Journal of Hazardous
Materials 177: 676-682
200
Microbial lipid production from mixed sugars hydrolyzates by the oleaginous
yeast Cryptococcus curvatus
Nicola Di Fidio2, Silvio Mastrolitti1, Federico Liuzzi1, Maria A. Capozzi2, Isabella De Bari1
1
ENEA Italian National Agency for New Technologies, Energy and Sustanaible Economic Development, C.R. Trisaia
S.S. 106 Jonica 75026 Policoro (MT) – Italy; 2Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università
degli Studi di Bari Aldo Moro, Via E. Orabona, n° 4 - 70125 Bari, Italy
[email protected]
Biomass sugars are a versatile platform for the production of a number of biobased products. Microbial
lipids obtained by means of oleaginous microorganisms represent one of the conversion options.
Most of the lipids accumulated in oleaginous microorganisms are triglycerides which can be used for
the production of biodiesel but also in a number of oils-based processes including, for instance, the
production of bioplastics. The ability of some species to use mixed sugars is an important requirement
for the utilization of abundant residual straws or low input lignocellulosic crops such as arundo
donax. In fact, they contain both C5 and C6 sugars that should be effectively converted to make the
overall process feasible.
In the present study Cryptococcus curvatus has been investigated for the production of microbial
lipids from sugars. In particular, the yeast has been preliminarily tested in synthetic media containing
different carbohydrates and different sugars concentrations. The process has been optimized as
function of the nutrients composition by using the surface methodology analysis. Finally, a fed-batch
process has been optimized for the conversion of lignocellulosic hydrolysates obtained from steam
pretreated arundo donax.
201
Aspartyl protease from psychrotolerant yeast Sporobolomyces roseus
Joanna Krysiak, Katarzyna M. Szulczewska, Tomasz Florczak, Aneta Białkowska,
Marianna Turkiewicz
The Institute of Technical Biochemistry, Technical University of Lodz, Poland
[email protected]
One of the most common and most commercialized psychrozymes are proteases. These enzymes are
widely used in various industries such as detergent, food and dairy industry and leather processing.
Nowadays more and more popular is the use of proteases in unconventional environments for peptide
synthesis, potentially useful for the pharmaceutical industry. Particularly promising tools for the
synthesis of peptides in organic solvents, are aspartyl proteases.
Psychrotolerant yeast strain was isolated from groundwater from silver and lead mine Luiza in
Zabrze (Poland). Strain was genetically classified based on D1/D2 domains of the 26S rRNA gene
and regions ITS1-5,8S-ITS2 sequence analysis. Extracellular protease produced by tested strain was
characterized and purified using ion exchange chromatography and size exclusion chromatography.
Yeast strain isolated from environment sample was genetically classified as Sporobolomyces roseus.
This strain produce an extracellular aspartic protease. The enzyme has a high, as for psychrophilic
enzyme, activity of 0.8 U/ml. Optimal pH of enzyme activity is 4, and the optimum temperature
is 50 °C. Despite the high optimum temperature this enzyme has a relatively high activity in the
temperature range of 0-20 °C (10 to 25%) and also has a high thermolability. The use of ion-exchange
chromatography and size exclusion chromatography allowed us to obtain a highly purified enzyme
preparation which is characterized by properties identical with unpurified preparation.
So far in the literature, there are only two extracellular proteases produced by yeast strains. One of
them is aspartyl protease produced by Candida humicola – strain isolated from Antarctic soil.
KEYWORDS: Cold-adapted yeast, Aspartyl protease
REFERENCES:
Joshi S and Satyanarayana T (2013). Biotechnology of cold-active proteases. Biology 2:755-783
Ray MK, Devi KU, Kumar GS, Shivaji S (1992). Extracellular protease from the Antarctic yeast Candida humicola.
Applied and Environmental Microbiology 58(6):1918-1923
202
Effect of oxygen on the fermentation performance of Candida intermedia: a
study case for lignocellulosic bioethanol production
Antonio D. Moreno, Cecilia Geijer, Lisbeth Olsson
Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology,
Gothenburg, Sweden
[email protected]
Microbial robustness is considered one of the remaining challenges for a cost-effective lignocellulosic
bioethanol production. This concept stands for the efficient conversion of all sugars present in
lignocellulose while dealing with the inhibitory compounds generated during biomass processing
(furan derivatives, short chain organic acids and phenolic compounds) and fermentation (ethanol). In
this context, efficient xylose conversion is crucial as it represents the second most abundant sugar in
lignocellulosic biomass.
We have isolated a clone of the non-conventional xylose-fermenting yeast species Candida intermedia
that shows great potential for being a non-genetically modified alternative for lignocellulosic bioethanol
production. To understand its potential for industrial use, we performed a thorough physiological
investigation of the strain. In the present work, the fermentation performance of the isolated clone was
evaluated in glucose/xylose medium under different oxygen-limiting conditions to promote ethanol
production. When oxygen was supplied with a flow rate of 1 vvm and a concentration ranging from
1% to 21% in the gas flow, a xylose consumption rate of 0.10-0.78 g/L h was observed. After glucose
depletion, ethanol concentration remained constant and only xylitol (0.34-0.61 g/g) and cell biomass
were further produced. When no oxygen was supplied, a maximum xylose consumption rate of 0.53 ±
0.04 g/L h was observed. Furthermore, almost 90% of the initial xylose concentration was consumed
within the first 48 h and ethanol was produced continuously, even after glucose depletion. Regarding
xylitol accumulation, a yield of 0.14 ± 0.04 g/g was found at 48 h of fermentation. In brief, we can
conclude that C. intermedia requires low oxygen concentrations for triggering ethanol production,
and that the presence of even low amounts of oxygen further on in the process has a detrimental effect
in the conversion of xylose to ethanol.
203
Mannitol-metabolising yeasts for conversion of algal biomass
Niccolò Tosetto1,2, Joakim Olsson1, Eva Albers1, Jenny Veide Vilg1
Biology and Biological Engineering, Industrial Biotechnology, Chalmers University of Technology, Sweden;
2
Biotecnologie e Bioscienze, University of Milano Bicocca, Milan, Italy
1
[email protected]
Seaweeds are gaining an increasing research interest as a biorefinery substrate, since the seaweeds,
especially the brown kelps, contain high concentrations of carbohydrates that are more accessible
than those from e.g. lignocellulose (Wei et al 2013). Only a fraction of these, however, contains
sugars that can be fermented by the conventional yeast S. cerevisiae (Enquist-Newman et al 2014).
This means that there is a need for novel microorganisms, natural or engineered, to enable an efficient
conversion of the seaweed carbohydrates, e.g. mannitol.
We have screened several yeasts, both newly isolated and commercial strains, for mannitol utilization.
Selected strains are being further characterized for enzymatic activities of the mannitol metabolic
pathways and for conversion of mannitol into ethanol. At present, we have >10 strains efficiently
utilizing mannitol for growth. Of these, 2 strains are producing ethanol, albeit at low yield. To increase
the ethanol yield, methods for optimization of the fermentation processes are carefully developed
within the project.
Sustainable production of biofuels is an important societal issue and an important building block
of a future bio-based economy. Yeast strains that can convert carbohydrates from seaweeds into
ethanol can become an important tool to explore this potentially sustainable biomass as a substrate for
biorefining. The use of non-modified, natural yeasts gives increased possibilities to use the residual
yeast biomass as a protein source, which in turn could increase the overall economical value of the
seaweed biomass.
KEYWORDS: Mannitol, Mannitol dehydrogenase, Seaweed, Fermentation
REFERENCES:
Wei N, Quarterman J, Jin YS (2013). Marine macroalgae: an untapped resource for producing fuels and chemicals.
Trends in Biotechnology 31(2):70-77
Enquist-Newman M et al (2014). Efficient ethanol production from brown macroalgae sugars by a synthetic yeast
platform. Nature Letter 505:239-243
204
Expression of Phanerochaete chrysosporium β-glucosidase in industrial
Saccharomyces cerevisiae yeast for bioethanol production from lignocellulosic
biomass
Lorenzo Cagnin1, Lorenzo Favaro1, Shaunita H. Rose2, Marina Basaglia1,
Willem H.Van Zyl2, Sergio Casella1
Department of Agronomy Food Natural resources Animals and Environment, DAFNAE, Università di Padova,
Agripolis, Viale dell’Università 16, 35020 Legnaro (PD), Italy; 2Department of Microbiology, Stellenbosch University,
Private Bag X1, 7602 Matieland, Stellenbosch, South Africa
1
[email protected]
Ethanol is currently being produced mostly by fermentation of sugars obtained from commodities that
can also be used as food and feed. This competition makes first generation bioethanol unsustainable.
Lignocellulose is one of the most promising alternative raw materials, due to its low cost and wide
availability as waste product. So far, little has been done in order to select robust strains that can both
tolerate stressful industrial applications and efficiently ferment sugars. Further, to reduce production
costs, a fermenting yeast, capable of producing one or more of the enzymes required for cellulose
hydrolysis, is needed (van Rooyen et al 2005). β-glucosidases, splitting cellobiose into glucose, play a
major role in the enzymatic hydrolysis of cellulose by eliminating the inhibitory activity of cellobiose
on other enzymes involved in the saccharification of cellulose (van Rooyen et al 2005).
The fungal β-glucosidase BGL3 from Phanerochaete chrysosporium expresses high hydrolytic
activities on cellobiose, when secreted by a Saccharomyces cerevisiae laboratory strain (Njokweni
et al 2012). BGL3 was then δ-integrated into the chromosome of two industrial S. cerevisiae strains,
namely M2n and MEL2 (Viktor et al 2013; Favaro et al 2015), selected for their robustness and high
ethanol performances.
Several mitotically stable recombinants were able to grow using cellobiose as the sole carbon source
and their enzymatic activity was evaluated in vitro on p-nitrophenyl-β-D-glucopyranoside. The highest
extracellular β-glucosidase activity was about 20 nkat per ml and cell-bound activity was also detected
at high levels.
This study reports the successful construction of recombinant industrial S. cerevisiae strains capable
of growing on cellobiose as sole carbon source, by expressing the fungal β-glucosidase BGL3 from P.
crysosporium. Their fermenting abilities will be evaluated on cellobiose and native cellulosic substrates,
with the addition of customized cellulases cocktails required to complete the hydrolysis of cellulose.
KEYWORDS: Bioethanol, Lignocellulose, Cellobiose, β-glucosidase, Industrial yeast
REFERENCES:
Favaro L, Viktor MJ, Rose SH, Viljoen-Bloom M, van Zyl WH, Basaglia M, Cagnin L, Casella S (2015). Consolidated
bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal
amylases. Biotechnology and Bioengineering (Accepted)
Njokweni AP, Rose SH, van Zyl WH (2012). Fungal β-glucosidase expression in Saccharomyces cerevisiae. Journal of
Industrial Microbiology and Biotechnology 39:1445-1452
van Rooyen R, Hahn-Hägerdal B, La Grange DC, van Zyl WH (2005). Construction of cellobiose-growing and
fermenting Saccharomyces cerevisiae strains. Journal of Biotechnology 120:285-295
Viktor MJ, Rose SH, van Zyl WH, Viljoen-Bloom M (2013). Raw starch conversion by Saccharomyces cerevisiae
expressing Aspergillus tubingensis amylases. Biotechnology for Biofuels 6:167-177
205
Production of ingenol-precursors in yeast
Roberta Callari1, Christophe Folly1, Michael D. Mikkelsen2, Björn Hamberger3,
Harald Heider1
Evolva SA, Duggingerstrasse 4153 Reinach, Switzerland; 2 Evolva Biotech A/S Lersø Parkallé 42-44 DK-2100
Copenhagen, Denmark; 3 Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of
Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark
1
[email protected]
Terpenoids represent the largest class of plant secondary metabolites, with enormous structural
diversity and wide range of biological functions. They have extensive applications in the fields of
pharmaceuticals, fragrances and flavourings. However, their isolation from the natural source is often
low yielding and their chemical complexity makes them not easily tractable to chemical synthesis.
Genetic engineering of biotechnological microbial systems represents an invaluable alternative
production route. Within the PlantPower (http://plantpower.ku.dk/) project we are therefore aiming
at the development of efficient cell factories for the biosynthesis of high value diterpenoids, such as
ingenol-angelate, the active agent identified in the sap of Euphorbia peplus, FDA–approved for the
treatment of actinic keratosis, a skin precancerous condition (Fidler & Goldberg 2014).
The production of ingenol-angelate precursors in Saccharomyces cerevisiae follows two strategies:
the modulation of the native mevalonate (MVA) pathway to increase the precursor pool for diterpene
biosynthesis and the introduction of enzymes for pathway reconstitution. The expression of a
truncated form of the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) enzyme together with
a heterologous geranylgeranyl diphosphate (GGPP) synthase allows to amplify the flux through the
MVA pathway to the diterpene precursor GGPP. Multi-cyclic diterpene backbones are then produced
by cyclization of GGPP, performed by diterpene synthases. Casbene synthases from different sources
were expressed in yeast to select the ones with highest activity for the production of casbene, the
putative first intermediate to ingenol-angelate biosynthesis. Further functionalization of casbene is
achieved by mono-oxygenation reactions catalysed by cytochrome P450 enzymes, identified by data
mining conducted at the University of Copenhagen.
KEYWORDS: Cell factories, Diterpenoids, Casbene, Ingenol-angelate, Cytochrome P450 expression
REFERENCES:
“
PlantPower: Light-driven synthesis of complex terpenoids using cytochrome P450s”. http://plantpower.ku.dk
Fidler B, Goldberg T (2014) Ingenol mebutate gel (picato): a novel agent for the treatment of actinic keratosis.
Pharmacy and Therapeutics 39(1):40-6
206
Utilization of by-products of tomato manufacturing for producing
polygalacturonase by a strain of Aureobasidium pullulans isolated from
saharan soil
1
Leila Bennamoun1, Scheherazad Dakhmouche1, Fatima Z. K. Labbani1,
Amel Ait-Kaki1, Tahar Nouadri1, Zahia Meraihi1, Benedetta Turchetti2, Pietro Buzzini2,
Philippe Thonart3
Laboratoire de Génie Microbiologiques et Applications, Faculté des Sciences de la Nature et de la Vie, Département
de Biochimie et Biologie Cellulaire & Moléculaire, Université Constantine 1, Route Ain El Bey 25017, Algeria;
2
Department of Agricultural, Food and Environmental Science, Industrial Yeasts Collection DBVPG, University of
Perugia, Borgo XX Giugno 74, I-06121, Perugia, Italy; 3Walloon Center of Industrial Biology, University of Liège,
B40- Sart- Tilman , 4000 Liège, Belgium
[email protected]
The aim of the present study was to describe the production of polygalacturonase (PG) by an
Aureobasidium pullulans strain (isolated from a Saharan soil sample) on by-products of tomato
manufacturing (tomato pomace). In the last ten years, yeast pectinases have attracted a great deal of
attention from various research groups worldwide as an alternative to fungal pectinases.
Statistical optimization of PG production by A. pullulans on tomato pomace, was investigated.
Plackett-Burman (PB) Design for screening of the medium constituents and a Central Composite
Design (CCD) for optimizing significant factors.
The experimental results showed that the optimal medium constituents, which determined the
maximum PG production (22.05 U/ml) were settled as follows: concentration of tomato pomace,
lactose and CaCl2 = 40, 1.84, and 0.09 g/l, respectively and initial pH 5.16. Overall, this optimization
approach resulted in a 700 % enhancement of PG activity than that observed under the screening
conditions. This result is of significant interest due to the low cost and abundant availability of tomato
pomace waste in Mediterranean area.
KEYWORDS: Polygalacturonase production, Tomato pomace, Aureobasidium pullulans, Plackett–
Burman design, Response surface methodology
207
Factors affecting the viability of Saccharomyces cerevisiae in simultaneous
saccharification and co-fermentation of pretreated wheat straw to ethanol
Johan O. Westman1, Ruifei Wang1, Vera Novy1,2, Lisbeth Olsson1, Carl J. Franzén1
1
Biology and Biological Engineering – Division of Industrial Biotechnology, Chalmers University of Technology,
Gothenburg, Sweden; 2Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz,
Austria
[email protected]
The recalcitrance of lignocellulosic materials makes economic production of second generation
ethanol difficult and necessitates pretreatment prior to hydrolysis and fermentation. Dilution in
these steps limits the final ethanol titre reached in the fermentation, even at high yields. A higher
concentration of the raw material already in the hydrolysis step is thus required to obtain good process
economy. However, this also increases the amount of toxic compounds in the fermentation.
Through simultaneous saccharification and co-fermentation, SSCF, with feeding of pretreated solids,
higher substrate concentrations can be reached (Wang et al 2014). Yeast cells can be adapted to the
material if they are propagated in fed-batch cultivation on a medium containing the liquid fraction
from the pretreatment. Yet, even with such preadaptation, the activity of the cells added to our SSCF
process dropped over time. To overcome this issue, we added fresh cells to the SSCF at different time
points. We observed that the viability and fermentation capacity of the cells still decreased during the
process. Nutrient supplementation could not help in improving the dropping viability. However, by
adding ethanol to shake flask SSCF experiments we could see that the ethanol produced in the process
was likely a contributing factor to the low viability. Drop tests on agar plates containing ethanol and/
or pretreatment liquor, incubated at both 30°C and 35°C, further indicated that the decreased viability
was an effect of the combination of the temperature in the reactor, the inhibitors in the material, and
the ethanol produced in the process.
Decreasing the temperature in the reactor to 30°C when the ethanol concentration reached 40-50 g
L-1 resulted in rapid initial hydrolysis and maintained fermentation capacity. The residual amount of
unfermented glucose and xylose at the end of the process was reduced. With the optimized process,
ethanol concentrations of more than 60 g L-1 were reached.
KEYWORDS: Bioethanol, SSCF, Ethanol tolerance, Thermotolerance, Inhibitors
REFERENCE:
Wang R, Koppram R, Olsson L, Franzén CJ (2014). Kinetic modeling of multi-feed simultaneous saccharification and
co-fermentation of pretreated birch to ethanol. Bioresource Technology 172:303–311
208
Session 3:Yeasts in no-food biotechnology: biofuels, new molecules and enzymes
High-cell-density cultures of Saccharomyces cerevisiae: a novel process-based
model highlights self-inhibition phenomena affecting growth rate
Elisabetta de Alteriis1, Carmine Landi2, Palma Parascandola2,Stefano Mazzoleni3, Francesco
Giannino3, Fabrizio Cartenì3
Dept. di Biologia, Università degli Studi di Napoli Federico II, Via Cinthia, 80100 Napoli, Italy; 2Dept. di Ingegneria
Industriale, Università Di Salerno, Via Giovanni Paolo II 132, 84084, Fisciano (Sa), Italy;; 3Dept. di Agraria, Università
degli Studi di Napoli Federico II, via Università 100, 80055 Portici (Na), Italy
1
[email protected]
High-cell-density cultivations, mostly carried out in aerated fed-batch reactors, are a pre-requisite to
maximize bioprocess yield and productivity. S. cerevisiae cultured in fed-batch progressively lose
its proliferative capacity notwithstanding a suitable nutrient supply and optimal aeration (Landi et al
2011; Landi et al 2014). In the attempt to explain this behavior, a new process-based model, developed
according to the System Dynamics principles, is proposed for the growth of S. Cerevisiae on glucose.
The model (developed in SIMILE and MATLAB R2012b) considers: i) the occurrence of inhibition
due to the accumulation of both ethanol and other self-produced toxic compounds, ii) the metabolic
shift between respiration and fermentation, as function of the levels of glycolysis process. Two strains
of the CEN.PK family of S. cerevisiae were used to carry out aerated fed batch runs (Landi et al 2014).
The agreement between simulation curves and experimental data highlight the potential of the
proposed model to describe the growth dynamics of S. cerevisiae in fed-batch culture. According to the
model, yeast growth decline is ascribed to the accumulation of self-produced inhibitory compounds
other than ethanol. The results clarify the dynamics of yeast growth and highlight the relevance
of inhibition phenomena on the maximum cell density achieved in a bioreactor. Investigations are
ongoing to determine the chemical nature of the inhibitors involved in such phenomena and the
related mechanisms of action.
KEYWORDS: HCDC, Modelling, Metabolic shift
REFERENCES:
Landi C, Paciello L, de Alteriis E, Brambilla L, Parascandola P (2011). Effect of auxotrophies on yeast performance in
aerated fed-batch reactor. Biochemical and Biophysical Research Communications 414:604–11
Landi C, Paciello L, de Alteriis E, Brambilla L, Parascandola P (2014). High cell density culture with S. cerevisiae
CEN.PK113-5D for IL-1β production: optimization, modeling, and physiological aspects. Bioprocess and Biosystems
Engineering 38 (2): 251-261
209
Ethanol production from molasses at 40-42°C by a co-culture of Issatchenkia
orientalis and Saccharomyces cerevisiae
J.C.M. Gallardo, Cecilia Laluce
Department of Biochemistry and Technological Chemistry, Instituto de Química de Araraquara, São Paulo State
University - UNESP, Araraquara, S.P., Brazil
[email protected]
Changes in environmental stresses such as nutritional starvation, variations in the pH, temperatures
and nutrient concentrations frequently occur in nature. On the other hand, gradual increases in
temperature gives rise to favorable conditions allowing enriching the culture with organisms tolerant
to temperatures. Issatchenkia orientalis is a non-Saccharomyces yeast unable to assimilate sucrose,
but able of growing and converting glucose into ethanol at 40-42° C (Gallardo & Laluce 2011). In
this work, molasses fermentation was optimized in a co-culture of I. orientalis and Saccharomyces
cerevisiae. Increase in ethanol yields were observed at 42 °C in the co-culture of both yeasts because
of the invertase produced by S. cerevisiae cells to hydrolyze molasses sucrose into simple sugars for
fermentation. At sugar concentrations higher than 15-16% (total reducing sugars, w/v), the ethanol
production was reduced to 60-65 g/L (w/v) without significant drops in viability after 12h at 42°C.
Thus, the I. orientalis cells may predominate in a yeast population in which the sucrose is hydrolyzed
by cells of Saccharomyces cerevisiae that survive the thermal stress.
KEYWORDS: Ethanol production, High temperatures, Issatchenkia orientalis, Saccharomyces
cerevisiae
REFERENCES:
JCM Gallardo, C Laluce (2011). Enrichment of a continuous culture of Saccharomyces cerevisiae with the yeast
Issatchenkia orientalis in the production of ethanol at increasing temperatures’. Journal of Industrial Microbiology and
210
Functional screening of Non-Conventional Yeasts (NCYs) for their ability to accumulate
intracellular lipids
Simone Di Mauro, Sara Filippucci, Benedetta Turchetti, Ciro Sannino, Pietro Buzzini
Department of Agricultural, Food and Environmental Science, Industrial Yeasts Collection DBVPG, University of
Perugia, 06121 Perugia, Italy
[email protected]
Oleaginous microorganisms (including yeasts) have recently attracted significant research attentions,
due to the new trend toward green processes like biodiesel or biopolymers production. To most people,
yeasts are exemplified by the species Saccharomyces cerevisiae. This is in spite of the fact that this
domesticated microorganism represents only a fragment of the vast biodiversity and biotechnological
potential of the yeast world. In recent decades, in fact, studies of the metabolic diversity of so-called
non-conventional yeasts (NCYs) have revealed innumerable promising biotechnological properties.
In this study the lipid-accumulating ability of 770 NCYs (374 ascomycetes and 396 basidiomycetes),
all isolated from natural habitats and conserved in the Industrial Yeasts Collection of the University
of Perugia, Italy (www.dbvpg.unipg.it), was evaluated. The screening was conducted in three steps:
1) Semi-quantitative screening of lipids accumulation by fluorescence microscopy coupled with
images evaluation by the freeware ImageJ (http://imagej.nih.gov/) .
2) Quantification of intracellular lipids of iper-producing strains selected at the previous point by
batch cultivation in 250 mL flasks.
3) Check of the ability of the best strains (selected at the previous point) to synthesize lipids in 250
mL flasks at different temperatures (20°C and 25°C).
The ability to accumulate intracellular lipids in significant amount was a character mainly related to
basidiomycetes genera. A positive correlation between the lipids synthesis and the psychrotolerance of
NCYs was also noted. After 4 days growth at 20°C, the strain Leucosporidiella creatinivora DBVPG
4794 exhibited the ability to accumulate the highest intracellular amounts of lipids (71,1% DW) ,
corresponding to a volumetric production of 7,1 g/L.
Acknowledgments this work was supported by the grant BIT3G - Third generation biorafinery
integrated in the territory. Project financed by the Italian Ministry for Education and Research (MIUR
- project: CTN01_00063_49295).
211
212
Session 4
Yeasts genetic and genomic
213
BAT1 is a predicted branched chain amino acid transaminase gene in
Lachancea kluyveri
Javier Montalvo-Arredondo1, Ángel Jiménez-Benítez1, Maritrini Colón-González2, James
González-Flores2, Mirelle Flores-Villegas2, Alicia González2, Lina Riego-Ruiz1
IPICYT, División de Biología Molecular, Camino a la Presa San José no. 2055, Col. Lomas 4 Sección, San Luis Potosí,
San Luis Potosí 78216, Mexico; 2Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular,
Universidad Nacional Autónoma de México, PO 70-242, México D. F. 04510, México
1
[email protected]
Pyridoxal 5’-phosphate dependent transaminases can catalyze both the last step of biosynthesis and the
first step in catabolism of branched chain amino acids (BCAAs), L-valine, L-isoleucine and L-leucine
(VIL). In Saccharomyces cerevisiae, two paralogous genes, ScBAT1 and ScBAT2, encode branched
chain amino acid transaminases (BCAATs), and function in the metabolism of VIL. However, the
genes and mechanisms of BCAAs metabolism in the related yeast Lachancea kluyveri have yet to be
functionally identified. We analyzed the genome sequence of this yeast to identify a conserved locus,
LkBAT1, which encodes a predicted BCAAT. Unexpectedly, we also identified a second unlinked
locus, which we named LkBAT1bis, exhibiting sequence similarity to LkBAT1. To determine the
function of these putative BCAATs, L. kluyveri mutant strains lacking LkBAT1, LkBAT1bis or both
genes were generated and tested for VIL auxotrophy. Transaminase activity was detected only in
strains expressing LkBAT1. Additionally, heterologous gene complementation between S. cerevisiae
and L. kluyveri was tested to confirm that LkBAT1 functions as BCAAT. LkBat1 expression, which
can be detected in the mitochondria by fluorescence microscopy, complemented the ScBAT1 deletion
in S. cerevisiae. However, LkBAT1 was unable to complement the ScBAT2 deletion in S. cerevisiae. In
reciprocal heterologous complementation assays, expression of ScBAT1 or ScBAT2 rescued LkBAT1deficiency phenotype in L. kluyveri. LkBAT1bis failed to show function for BCAAs metabolism in
these experiments. However, when ethanol was used as carbon source, the deletion of LkBAT1bis
in the Lkbat1 null strain resulted in a large ‘lag’ growth phase, pointing to a potential function of
LkBAT1bis in aerobic metabolism in L. kluyveri. These results confirm the BCAAT function of
LkBAT1 in L. kluyveri, and provide insight into the functional divergence of paralogous genes after
the WGD event that catalyzed emergence of new biological functions.
KEYWORDS: Branched chain amino acid metabolism, Aminotransferase, Lachancea kluyveri,
Saccharomyces cerevisiae, Yeast genetics
214
Genomic MAL locus of a methylotrophic yeast Hansenula polymorpha: disclosing
the role of MAL-activator genes
Katrin Viigand, Triinu Visnapuu, Tiina Alamäe
Institute of Molecular and Cell Biology, University of Tartu, Riia St. 23, Tartu, Estonia
[email protected]
Genomic clustering of functionally related genes is not very common in yeasts. Mostly these genes
are scattered over the genome. Still, there are several gene clusters in Saccharomyces cerevisiae for
example the MAL cluster with genes of maltose metabolism. In S. cerevisiae five MAL clusters, MAL1MAL4 and MAL6, are found in subtelomeric regions of different chromosomes, each containing at
least three genes encoding a maltose permease, a maltase and an activator of these genes. The maltose
permease and maltase genes are coordinately transcribed from a bidirectional promoter region as
in H. polymorpha (Hp) (Alamäe et al 2003; Viigand et al 2005; Viigand & Alamäe 2007). We have
studied the expression and regulation of HpMAL1 (maltase) and HpMAL2 (α-glucoside transporter)
genes in Hp MAL locus. Further sequencing of the locus revealed next to HpMAL2 two potential
MAL-activator genes - HpMALAKT1 and HpMALAKT2. We suggest that Hp MAL locus consists of
four genes. To investigate the functionality of the potential MAL-activator genes we have constructed
disruption mutants of HpMALAKT1 and HpMALAKT2 genes in Hp and will report on effects of the
disruptions on growth of yeasts on disaccharides. We will also readdress some properties of the Hp
maltase protein HpMAL1. S. cerevisiae maltase protein is primarily active on maltose-like substrates.
In addition, S. cerevisiae has IMA1-5 genes for the hydrolysis of isomaltose-like sugars (Naumov
& Naumov 2012). Notably, H. polymorpha is able to grow on an isomaltose-type sugar palatinose,
thus its maltase should be capable of hydrolysis of isomaltose-type substrates as well. The HpMAL1
protein will be heterolously overexpressed, purified and its substrate specificity will be investigated
in detail to compare properties of Hp maltase with S. cerevisiae maltase and isomaltases.
Acknowledgements The project is financed by the grants GLOMR7528 and GLOMR9072.
KEYWORDS: Hansenula polymorpha, MAL-genes, Gene cluster, Mal-activator
REFERENCES:
Alamäe T, Pärn P, Viigand K, Karp H (2003). Regulation of the Hansenula polymorpha maltase gene promoter in H.
polymorpha and Saccharomyces cerevisiae. FEMS Yeast Research 4:165-173
Viigand K, Tammus K, Alamäe T (2005). Clustering of MAL genes in Hansenula polymorpha: Cloning of the maltose
permease gene and expression from the divergent intergenic region between the maltose permease and maltase genes.
FEMS Yeast Research 5:1019-1028
Viigand K, Alamäe T (2007). Further study of the Hansenula polymorpha MAL locus: characterization of the
α-glucoside permease encoded by the HpMAL2 gene. FEMS Yeast Research 7:1134-1144
Naumov GI, Naumov DG (2012). Genetic differentiation of yeasts alpha-glucosidases: maltase and isomaltase.
Mikrobiologiia:81:301-5. Review
215
Complete genome sequence of a Torulaspora delbrueckii NRRL Y-50541 strain
isolated from mezcal fermentation process
Jorge Gómez-Angulo1, Leticia Vega-Alvarado2, Zazil Escalante-García3, Ricardo Grande2,
Anne Gschaedler-Mathis1, Lorena Amaya-Delgado1, Alejandro Sanchez-Flores2,
Javier Arrizon1
Unidad de Biotecnología Industrial, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco,
AC; 2Unidad de secuenciación Masiva y Bioinformática, Instituto de Biotecnología de la UNAM, Morelos, Mexico;
3
Departamento de Ingeniería Química, Centro Universitario de Ciencias Exactas e Ingenierías de la UdeG, Jalisco,
México
1
[email protected]
Torulaspora delbrueckii, is an ascomycetes yeast with high osmotic and freezing temperature
tolerance. Due to these properties, this yeast has potential biotechnological applications. Recently,
it has been reported that T. delbrueckii yeasts isolated from mezcal fermenting process produced
β-fructofuranosidase enzymes with fructosyltransferase activity and this could be used in prebiotic
molecules synthesis (Arrizon et al 2012). Lately, the genome of T. delbrueckii CBS 1146 was obtained
(using 454 sequencing) with the main purpose of studying the sex chromosome evolution among the
Saccharomycetaceae family (Gordon et al. 2011). Therefore, in order to find the differences between
the published reference and our mezcal isolate, we sequenced, assembled and characterized it, in
order to find genes and variations associated to the fermentation process. Genomic DNA from T.
delbrueckii NRRL Y-50540 was isolated and prepared as Illumina sequencing libraries to generate
a total of 20,514,013 paired-end reads (estimated coverage ~328x) with a length of 72 bases, using
the Illumina GAIIx platform. The assembly was performed with Velvet v1.2.10 using a kmer size
of 35. An assembly of 11,236,894 bp in 374 contigs with lengths greater or equal to 1,000 bp, was
obtained with N50/N90 values of 82,617/23,849 bp, respectively. The average contig length was 30,012
bp, giving a considerable space to search for genes. Gene prediction was performed using Augustus
v2.7, we predicted 4,714 protein-coding genes by intersecting all predictions. Using CEGMA v. 2.5
we obtained a 97% of genome completeness. Finally, the contigs in the assembly were ordered and
oriented using ABACAS and the published reference, leading to a total of 8 scaffolds corresponding
to chromosomes. In contrast to the genome published by Gordon JL et al, this is an improved reference
that can be used for RNA-seq differential expression analysis.
KEYWORDS: Torulaspora delbrueckii, Genome, Sequence, Fructosyltransferase, Mezcal
fermentation
REFERENCES:
Arrizon JMS, Gschaelder A, Monsan P (2012) . Fructanase and fructosyltransferase activity of non-Saccharomyces
yeasts isolated from fermenting musts of mescal.Bioresource Technology 110:560-565
Gordon JLAD, Proux-Wéra E, ÓHÉigeartaigh SS, Byrne KP, Wolfe KH 2011 Evolutionary erosion of yeast sex
chromosomes by mating-type swutching accidents. PNAS 108:20024-20029
216
De Novo whole-genome annotation of Candida apicola NRRL Y-50540
Jorge Goméz-Angulo1, Leticia Vega-Alvarado2, Zazil Escalante-García3, Ricardo Grande2,
Anne Gschaedler-Mathis1, Lorena Amaya-Delgado1, Alejandro Sanchez-Flores2,
Javier Arrizon1
Unidad de Biotecnología Industrial, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco,
AC; 2Unidad de secuenciación Masiva y Bioinformática, Instituto de Biotecnología de la UNAM, Morelos, Mexico;
3
Departamento de Ingeniería Química, Centro Universitario de Ciencias Exactas e Ingenierías de la UdeG, Jalisco,
Mexico
1
[email protected]
Candida apicola, is a high osmotolerant ascomycetes yeast, that produces sophorolipids, membrane
fatty acids and enzymes. Usually, this yeast can be found in wine, cachaҫa and mezcal fermentation
processes. In the latter, it can express enzymes such as β-fructofuranosidases with fructosyltransferase
activity. This enzymatic activity is useful to synthesize prebiotic compounds (Souza et al 2005;
Tofalo et al 2009; Arrizon et al 2012). Therefore, the fermentation process could be characterized by
sequencing the whole genome of C. apicola to discover interesting features involved in fermentation
and potentially others with biotechnological potential.
Genomic DNA from C. apicola NRRL Y-50540 (YPD culture) was isolated and prepared as Illumina
sequencing libraries to generate a total of 13,207,584 paired-end reads (estimated coverage ~211x)
with a length of 72 bases, using the Illumina GAIIx platform. The assembly was performed with
Velvet v1.2.10 using a kmer size of 51. An assembly of 9,769,876 bp in 40 contigs with lengths
greater or equal to 1,000 bp, was obtained with N50/N90 values of 773,945/186,965 bp, respectively.
Gene prediction was performed using Augustus v2.7 using three different Candida species profiles
(albicans, guilliermondii and tropicalis) we predicted 3,818 protein-coding genes by intersecting all
three predictions. Using CEGMA v. 2.5 we obtained a 92% of genome completeness. Finally, our
whole-genome shotgun project has been deposited and can be found at the NCBI GenBank database.
This genome is to our knowledge, the first high-quality draft genome for this species and can be used
as a reference to perform further analyses such as differential gene expression of enzymes related to
the synthesis and degradation of biotechnological molecules of interest.
KEYWORDS: Candida apícola, Whole-Genome, Assembly, Fructosyltransferase, Mezcal,
Fermentation
REFERENCES:
Arrizon JMS, Gschaedler A, Monsan P (2012). Fructanse and fructosyltransferase activiy of non-Saccharomyces yeasts
isolated from fermenting musts of mezcal. Bioresource Technology 110:560-565
Souza OERC, Morgano MA, Serra GE (2005). The production of volatile compounds by yeasts isolated from small
Brazilian chachaҫa distilleries. World Journal of Microbiology & Biotechnology 21:1569-1576
Tofalo RCLC, Di Fabio F, Schirone M, Felis GE, Torriani S, Paparella A, Suzzi G (2009). Molecular identification and
osmotolerant profile of wine yeast that ferment a high sugar grape must. International Journal of Food Microbiology
130:179-187
217
RNAi as a tool to study virulence in the pathogenic yeast Candida glabrata
Olena P. Ishchuk1, Khadija M. Ahmad1, Katarina Koruza1, Klara Bojanovič1, Lydia Kasper2,
Sascha Brunke2, Bernhard Hube2, Torbjörn Säll1, Christian Brion3, Kelle Freel3, Joseph
Schacherer3, Birgitte Regenberg4, Wolfgang Knecht1,5, Jure Piškur1
Department of Biology, Lund University, Sölvegatan 35, Lund SE-223 62, Sweden; 2Department of Microbial
Pathogenicity Mechanisms, Hans Knoell Institute Jena (HKI), Beutenbergstraße 11a, D-07745 Jena, Germany;
3
Department of Molecular Genetics, Genomics and Microbiology, Strasbourg University, 28 rue Goethe, Strasbourg,
France; 4Department of Biology, Faculty of Science, University of Copenhagen, DK 2200 Copenhagen; 5Lund Protein
Production Platform, Lund University, Sölvegatan 35, Lund SE-223 62, Sweden
1
[email protected]
Candida glabrata is one of the main pathogens causing mucosal and systemic infections in human.
Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to its
intrinsic and frequently further adapted antifungal resistance. To understand and treat C. glabrata
infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance.
However, C. glabrata virulence is not well studied and gene deletion protocols are time consuming
and often inefficient and, furthermore, inappropriate for the disruption of essential genes. We have
established an RNA interference (RNAi) protocol in C. glabrata by expressing Dicer and Argonaute
genes from Saccharomyces castellii. Our results using reporter genes and putative virulence genes
show that introduced RNAi results in 75-95% gene knockdown depending on the construct type
(antisense or hairpin). The RNAi strain was further used as a basis for antisense gene library based on
a multi-copy replicative plasmid. Transformants were subjected to phenotypic profiling using highresolution quantification of growth in search of genes involved in cell integrity, antifungal drug and
ROS resistance. For example, one of the amphotericin B sensitive transformant obtained was carrying
an antisense plasmid for C. glabrata uncharacterized gene, CAGL0I00116g. The genes identified by
this approach may prove to be new potential targets for the development of anti-C. glabrata therapies.
218
Phylogenetic analysis of pectinase genes PGU in the yeast genus Saccharomyces
Maxim Yu. Shalamitskiy1, Elena S. Naumova1, Nikolay N. Martynenko2, Gennadi I. Naumov1
Russia; 2Moscow State University of Food Production, Moscow, Russia
1
[email protected]
Pectinase (endo-polygalacturonase) is one of the essential enzymes involved in hydrolysis of plant
pectins. Structural gene PGU1 (=PLG1) encoding endo-polygalacturonase has been sequenced from
13 Saccharomyces strains of different origins (Gognies et al 1999; Jia & Whwals 2000; Louw et al
2010; Eschstruth & Divol 2011). The PGU1 genes studied showed nearly identical sequences: up to
seven amino acid substitutions. The aim of our study to determine intra- and interspecific divergence
of PGU genes in the yeast Saccharomyces.
Using yeast genome databases and literature data, we have conducted a phylogenetic analysis
of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S.
arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S.
paradoxus and hybrid taxon S. pastorianus (syn. S. carlsbergensis). The superfamily of divergent
species-specific PGU genes has been found. Within the Saccharomyces species, identity of PGU gene
nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum),
94–98.3% for S. kudriavzevii and 96.8–100% for S. paradoxus/S. cariocanus. Nevertheless, natural
interspecific transfer of PGU gene from S. cerevisiae to S. bayanus and from S. paradoxus to S.
cerevisiae can occur. For the first time, a family of polymeric PGU1b (accession numbers FR847038
and AACA01000043), PGU2b (AACA01000682), PGU3b (AACA01000194) and PGU4b (Gognies
et al 1999) genes is documented for the yeast S. bayanus var. uvarum important for winemaking.
KEYWORDS: Endo-polygalacturonase, Pectinase, Pectin, Wine yeasts, Saccharomyces, Phylogenetic
analysis, PGU gene, Introgression
REFERENCES:
Gognies S, Gainvors A, Aigle M, Belarbi A (1999). Cloning, sequence analysis and overexpression of a Saccharomyces
cerevisiae endopolygalacturonase-encoding gene (PGL1). Yeast 15: 11−22
Jia J, Wheals A (2000). Endopolygalacturonase genes and enzymes from Saccharomyces cerevisiae and Kluyveromyces
marxianus. Curr Genet 38: 264−270
Louw C, Young PR, Rensburg P, Divol B (2010). Regulation of endo-polygalacturonase activity in Saccharomyces
cerevisiae. FEMS Yeast Research 10: 44−57
Eschstruth A, Divol B (2011). Comparative characterization of endo-polygalacturonase (Pgu1) from Saccharomyces
cerevisiae and Saccharomyces paradoxus under winemaking conditions. Applied Microbiology and Biotechnology
91: 623−634
219
Poligenic analysis of low temperature adaptation in wine yeast
Estéfani García-Ríos1, Leopold Parts2, Gianni Liti3, José M. Guillamón1
Institute of Agrochemistry and Food Technology-CSIC (Spain); 2 Welcome Trust Sanger Institute, UK; 3 Institute for
Research on Cancer and Aging, France
1
[email protected]
Many factors such as must composition, juice clarification, the temperature of fermentation or the
yeast strain inoculated strongly affect alcoholic fermentation and aromatic profile of wine. With the
effective control of fermentation temperature by the wine industry, low temperature fermentation (10
– 15 ºC) are becoming more frequent due to the aim of producing white and “rosé” wines with more
pronounce aromatic profile (Beltran et al 2006). Low temperatures increase not only the retention but
also the production of some volatiles compounds. In these conditions greater concentration of aroma
compounds are produced, such as esters that impart sweet and fruity aromas. Another interesting
aspect is that low temperatures notably reduce the growth of acetic and lactic acid bacteria, facilitating
the control of alcoholic fermentation. However fermentation at low temperature presents some
disadvantages: reduced growth rate, long lag phase, sluggish or stuck fermentations. These problems
can be avoided by selecting better-adapted yeasts to ferment at low temperature. Low temperature
adaptation, as most enological traits of industrial importance in yeast, is a polygenic trait, regulated by
many interacting loci. In order to address the genetic determinants of low temperature fermentation,
we implemented a QTL mapping in the F12 offspring (Parts et al 2011) of two Saccharomyces
cerevisiae industrial strains with a divergent phenotype in their performance at low temperature.
We identified four genomic regions implicated in the fermentation process at low temperature in
wine yeast. We found that subtelomeric regions play a key role in defining individual quantitative
variation, emphasizing the importance of the adaptive nature of these regions in natural populations.
Reciprocal hemizygosity analysis and deletion of the complete subtelomeric regions is underway to
confirm the causative genes in the QTLs and understanding the underlying mechanism.
KEYWORDS: Wine yeast, Cold adaptation, QTLs analysis, Phenotype-genotype association
REFERENCES:
Beltran G (2006). Integration of transcriptomic and metabolic analyses for understanding the global responses of lowtemperature winemaking fermentations. FEMS Yeast Research 6: 1167–1183
Parts L (2011). Revealing the genetic structure of a trait by sequencing a population under selection. Genome Research,
21:1131–1138
220
Genetic analysis of existing and new yeast hybrids
Alexander J. Hinks Roberts, Agnieszka Maslowska, Edward J. Louis
Centre for Genetic Architecture of complex traits, University of Leicester, UK
[email protected]
Breeding between species allows for mixing of the gene pool and under selective pressure, gives rise
to beneficial gene combinations.
Saccharomyces interspecific hybrids, both artificial and natural, have huge potential in industry. The
hybrids can inherit beneficial characteristics from each parental species. However like Mules, hybrids
are sexually sterile, inhibiting the potential for strain improvement, resulting in evolutionary deadends. In nature, particularly in plants such as wheat, hybrid sterility has been overcome through
genome duplication. Here we have utilised this principle to create genetically diverse fertile hybrids
via tetraploid intermediates.
To generate fertile hybrids we first crossed haploids of each species within the Sensu stricto clade,
generating sterile diploids. By deleting one copy of the MAT locus, the diploids become maters and
form fertile tetraploids with a diploid of opposite mating type.
High throughput phenotypic analysis is performed using high-density arrays of diverse individuals on
a plate reader to assess growth rate under various conditions.
Sequential mating of diploid progeny of tetraploid hybrids for 12 generations creates huge populations
of genetically diverse progeny, which can be used to perform our quantitative trait locus (QTL)
analysis. This can identify genomic loci that give modest contributions to a given phenotype with
high resolution (down to the single nucleotide in some cases).
We have generated hybrids, using diverse strains of each parental species to give maximum phenotypic
diversity. The F1 progeny have been phenotyped for traits of interest including tolerance to varying
heat and osmolarity stresses.
Until now, strain improvement of industrial hybrids has been limited, by the lack of sexual reproduction,
to slow and small improvements by selecting for spontaneous mutants. Our method allows for
generation of vast populations of hybrids with extensive diversity. Knowledge and understanding
of these variants will allow us to produce genetically improved strains beneficial to fermentation
industries.
KEYWORDS: Mating, QTL analysis, Strain improvement
221
Protein subcellular relocalization drives the divergence of Bat1 and Bat2 in
1
Mirelle C. Flores-Villegas1, Augusto Ortega-Granillo1, Edson Robles1, Jure Piskur2,
Alicia González-Manjarrez1
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico; 2Biology Department, Lund
University, Sweden
[email protected]
Gene duplication has been considered the most important evolutionary process for the origin of
new genes. It has been proposed that protein subcellular relocalization may contribute to functional
diversification of gene duplication products (Byun-McKay & Geeta 2007). Bat1 and Bat2 branched
chain aminotransferases are involved in the Leucine, Valine and Isoleucine metabolism. They have
differential subcellular compartmentalization as Bat1 is located in the mitochondria while Bat2 is in
the cytosol (Colón et al 2011).
To relocate Bat1 to the cytoplasm we removed the mitochondrial signal sequence by Delitto Perfetto
(Storici 2006). While Bat2 was relocated to the mitochondria inserting the Bat1 mitochondrial signal
sequence by the same methodology. We analyze the phenotype of the strains in fermenters in anaerobic
conditions.
Bat1 has a preferentially biosynthetic role, which is not affected by its subcellular localization. Bat2
has a minor role in biosynthesis which is only evident in a bat1Δ bat2Δ double mutant. Bat2 improves
its biosynthetic capacity when it is located in the mitochondria. Bat1 plays a minor role in LIV
catabolism, which it is not influenced by its localization. Bat2 plays the major role in catabolism.
When Bat2 was relocalized to the mitochondria, we observed a defect in growth rate on LIV as sole
nitrogen source and in enzymatic activity that was not explained by the presence of Bat1.
Bat1 is able to biosynthesize LIV in both compartments, Bat2 catabolic function is improved when
it is localized in its original compartment. In this conditions, relocated Bat1 and Bat2 are able to
improve their catabolic and biosynthetic roles, respectively. So there is a fundamental role of Bat1
and Bat2 subcellular compartmentalization on LIV metabolism. Our experimental results show, for
the first time, that PSR is one of the mechanisms of divergence in paralogues proteins.
REFERENCES:
Byun-McKay SA, Geeta R (2007). Protein subcellular relocalization: a new perspective on the origin of novel genes.
Trends in Ecology and Evolution 22(7):338-44
Colón, M., Hernandez F, Lopez K, Quezada H, Gonzalez J, Lopez G, Aranda C, Gonzalez A (2011). Saccharomyces
cerevisiae Bat1 and Bat2 Aminotransferases Have Functionally Diverged from the Ancestral-Like Kluyveromyces
lactis Orthologous Enzyme. PLoS One 6(1):1-13
Storici, F (2006). The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements
with synthetic oligonucleotides in yeast. Methods in Enzymology 409:329-45
222
Differential gene retention as an evolutionary mechanism to generate
biodiversity and adaptation in yeasts
Serge Casaregola1, Guillaume Morel1, Lieven Sterck2, Dominique Swennen1,
Marina Marcet-Houben3, Noémie Jacques1, Yves Van de Peer2, Patrick Wincker4,
Jean-Luc Souciet5, Toni Gabaldon3, Colin R. Tinsley1
INRA/AgroParisTech, Micalis Institute, 78850 Thiverval-Grignon, France; 2Department of Plant Systems Biology VIB,
Gent, Belgium; 3Bioinformatics and Genomics Programme, Centre for Genomic Regulation, Barcelona, Spain; 4CEA,
Institut de Génomique, Genoscope, Evry, France; 5Université de Strasbourg/CNRS, Strasbourg, France
1
[email protected]
The genetic bases and evolutionary history of many of the characters underlying the adaptation of
microorganisms to food and biotechnological uses are poorly understood. We undertook comparative
genomics to investigate genetic specificities and evolutionary relationships of the dairy yeast
Geotrichum candidum within Saccharomycotina. Surprisingly, a remarkable proportion of genes
showed discordant phylogenies, clustering with filamentous fungi subphylum (Pezizomycotina),
rather than the yeast subphylum (Saccharomycotina), of the Ascomycota. These genes appear not
to be the result of Horizontal Gene Transfer (HGT), but to have been specifically retained by G.
candidum after the filamentous fungi/yeasts split concomitant with the yeasts genome contraction.
We refer to these genes as SRAGs (Specifically Retained Ancestral Genes), having been lost by all or
nearly all other yeasts, and thus contributing to the phenotypic specificity of lineages. Indeed, SRAG
functions include several types of endoglucanases associated with degradation of plant material and
lipases consistent with a role in cheese making. Similar gene retention was observed in three other
yeasts representative of this ecologically diverse subphylum. The phenomenon thus appears to be
widespread in the Saccharomycotina and argues that, alongside neo-functionalization following gene
duplication and HGT, specific gene retention must be recognized as an important mechanism for
generation of biodiversity and adaptation in yeasts.
KEYWORDS: Geotrichum candidum, Saccharomycotina, Discordant phylogeny, Biodiversity,
Adaptation
223
The genetic basis of adaptation of flor strains:
a comparative genome and genetic analysis
Anna L. Coi1, Jean-Luc Legras2, Giacomo Zara1, Frédéric Bigey2, Virginie Galeote2, Sylvie
Dequin2 , Marilena Budroni1
1
Università degli studi di Sassari, Sassari, Italy; 2INRA, UMR1083 SPO, Montpellier, France
[email protected]
Wine fermentation and flor ageing are dissimilar processes carried out by Saccharomyces cerevisiae
strains able to stand different lifestyles. Flor strains are able to ferment grape must and to overcome
stressing conditions at the end of fermentation by forming a biofilm on the air-liquid interface. In
this context, the aim of this work was to analyze genomic and genetic peculiarities involved in flor
lifestyle.
Thus, the genomes of 8 wine strains and 10 flor strains were sequenced. Subsequent population
analysis revealed that the two groups of strains belong to pure lineages separate one from the other.
Several divergent chromosomal regions were detected and four groups of genes were found more
frequently in these regions. These genes are involved in cellular processes such as metal transport
and metal homeostasis, general metabolism, regulation of filamentous growth and cell wall assembly.
In particular, several mutations leading to the deregulation of FLO11 expression have been found,
suggesting that these mutations are the hallmark of flor strains domestication. A transcriptome analysis
comparing one flor and one wine yeast on wine synthetic medium revealed expression differences
associated to genes of cell wall, hexose and metal transporters.
A set of haploid flor and wine strains has been created for the molecular evaluation of some of these
target genes.
Acknowledgements Anna Lisa Coi gratefully acknowledges Sardinia Regional Government for
the financial support of her PhD and postdoc scholarships (P.O.R. Sardegna F.S.E. Operational
Programme of the Autonomous Region of Sardinia, European Social Fund 2007-2013 - Axis IV
Human Resources, Objective l.3, Line of Activity l.3.1.).
KEYWORDS: Flor strains, Biological ageing, Biofilm, Saccharomyces cerevisiae, Domestication
224
Ectopic recombination in sex-determination system as a source of genetic
variation in the diploid yeast Zygosaccharomyces sapae
Melissa Bizzarri, Alexandra Verspohl, Paolo Giudici, Stefano Cassanelli, Lisa Solieri
[email protected]
Sexual reproduction increases genetic variation, which is strongly advantageous under harsh
environmental conditions, as it allows natural selection to proceed more effectively. The yeasts of the
Zygosaccharomyces rouxii complex are relevant in food elaboration and spoilage due to their ability
to cope with low water activity environments and are characterized by gene copy number variation,
genome instability, and aneuploidy/allodiploidy (Solieri et al 2013). The mating-type locus (MAT)
is a hotspot for chromosome rearrangement in yeasts. Here, we investigated the genetic architecture
of sex determinants, including MAT loci and HO endonuclease in Zygosaccharomyces sapae diploid
strain ABT301T, belonging to the Z. rouxii complex. We cloned these genes through a DNA walking
strategy and a characterization of the flanking regions, while the chromosome assignment was
performed combining Southern blotting and PFGE-karyotyping. We identified three divergent mating
type-like (MTL) α-idiomorph sequences, designated as ZsMTLα copies 1, 2, and 3, which encoded
homologues of Z. rouxii CBS 732T MATα2 (aa sequence identity from 67.0 to 99.5%) and MATα1
(identity 81.5-99.5%). Cloning of MATa-idiomorph yielded one ZsMTLa locus encoding two Z.
rouxii-like proteins, MATa1 and MATa2. ABT301T possesses two divergent HO genes encoding
distinct endonucleases. Based on the cloned ZsMTLα and ZsMTLa idiomorphs flanking regions
we discovered that Z. sapae ABT301T displays an aααα genotype lacking the HMR silent cassette.
Additionally, four putative HML cassettes were identified, two harbouring the ZsMTLα copy 1 and
the remaining containing ZsMTLα copies 2 and 3. In conclusion, our results show that the matingtype switching is responsible for hyper-mutation in Z. rouxii complex. The ectopic recombination
underlying this process is an error-prone mechanism, which represents a possible source of genetic
variation providing yeast progeny with phenotypic variability and adaptation to hostile environments.
KEYWORDS: Yeast, Sex cycle, Zygosaccharomyces, Mating type
REFERENCES:
Solieri L, Dakal TC, Croce M. A, Giudici P (2013a). Unraveling genomic diversity of Zygosaccharomyces rouxii
complex with a link to its life cycle. FEMS Yeast Research 13: 245–258
225
Transcriptomic study of genetic factors involved in amino acid uptake in wine
strains of Saccharomyces cerevisiae
1
Claudio Martínez1,2, David González1, Verónica García1,2
Departamento de Ciencia y Tecnología de los Alimentos, Universidad de Santiago de Chile (USACH), Chile; 2 Centro
de Estudio en Ciencia y Tecnología de los Alimentos (CECTA), Universidad de Santiago de Chile, Chile
[email protected]
Saccharomyces cerevisiae is the main microorganism responsible of the alcoholic fermentation. In
this process, the Yeast Assimilable Nitrogen (YAN) in grape must is an important factor for an
adequate fermentation and consequently, low YAN values are the main cause of stuck or sluggish
fermentation. Ammonium ions and free amino acids are two sources of YAN inside must, representing
free amino acids about the 60% of total nitrogen. The amino acids assimilation occurs during the
early phase of fermentation and the aromatic profile of the wine could be affected by the metabolism
of amino acid due to these compounds are precursors of volatile molecules. Furthermore, from a
genetic point of view, the amino acid uptake by yeasts is a complex and polygenic trait. The aim
of this work was understand the genetic factors that underlying the amino acid consumption during
wine fermentation. With this purpose, we selected two meiotic segregants that showed the highest
differences in amino acids consumption from a cross between two strains of different lineages and
compared the transcriptomic profile of these segregants by mRNA-seq (Illumina HiSeq 2000) at 6
hours of fermentation. The results show a differential expression of 42 genes, having genes associated
with YAN consumption (LST8, GAP1 and BTN2) and transcriptional regulators (COM2) a remarkable
difference between the segregants. These results were confirmed by qPCR and reciprocal hemicygotic
analysis. Finally, the nitrogen consumption of the reciprocal hemizygotes was validated in synthetic
must.
KEYWORDS: Saccharomyces cerevisiae, Wine, YAN, Transcriptomic analysis
226
Genomic signatures of wine yeast strains
Georgios Banilas1, Aspasia Nisiotou2
Department of Enology & Beverage Technology, Faculty of Food Technology & Nutrition Technological Educational
Institute of Athens, Ag. Spyridona Str., 12210 Athens, Greece; 2Hellenic Agricultural Organization-DEMETER, Institute
of Technology of Agricultural Products, Sofokli Venizelou 1, Lycovrissi 14123, Greece
1
[email protected]
Commercial Saccharomyces cerevisiae yeasts used as starters in winemaking present significant
phenotypic discrepancies from the industrial or laboratory strains. Those differences have a genetic
basis, including gene copy number variation (CNV) in genes related to the “wine character” or certain
chromosomal rearrangements (Pérez-Ortín et al 2002; Dunn et al 2005). In this study we screened
several wine-related vineyard S. cerevisiae isolates from 2 major viticultural areas in Greece (Nemea
and Santorini) for the presence of a characteristic reciprocal translocation between the promoter
regions of SSU1and ECM34 genes in chromosomes VIII and XVI, respectively. Although this
translocation is responsible for elevated sulfite resistance of yeasts and is only present in wine strains,
we found that only 35-70% of the isolates had this translocation. Thus, the sulfite resistance of wine
yeasts may not be attributed solely to this mechanism. We also analyzed the CNV of ABZ1, PLB2
and PDR5 genes by qPCR in selected isolates of the two regions. Those genes were selected because
they are implicated in aroma compound formation in wine (Steyer et al 2012). Although the copy
number of ABZ1and PDR5 was quite constant, the PLB2 gene (lysophospholipase) showed relatively
high copy number (up to 4 copies) in several strains. Further analysis is currently conducted to show
whether the higher gene copy number is related to respective higher gene expression.
Acknowledgements This work has been co-financed by the European Regional Development Fund
(ERDF) of the EU and by National Resources under the Operational Program Competitiveness and
Entrepreneurship (EPAN II), Action “COOPERATION 2011”.
KEYWORDS: Wine fermentation, Yeast starter, Genetic signature, Gene copy number, Chromosomal
rearrangement
REFERENCES:
Dunn B, Levine RP, Sherlock G (2005). Microarray karyotyping of commercial wine yeast strains reveals shared, as
well as unique, genomic signatures. BMC Genomics 6:53
Pérez-Ortín JE, Querol A, Puig S, Barrio E (2002). Molecular characterization of a chromosomal rearrangement
involved in the adaptive evolution of yeast strains. Genome Research 12:1533-1539
Steyer D, Ambroset C, Brion C, Claudel P, Delobel P, Sanchez I, Erny C, Blondin B, Karst F, Legras JL (2012). QTL
mapping of the production of wine aroma compounds by yeast. BMC Genomics 30(13):573
227
Evolutionary dynamics of DNA transposon families in Saccharomycetaceae
Véronique Sarilar1,2, Claudine Bleykasten-Grosshans3, Cécile Neuvéglise1,2
INRA UMR1319 Micalis, Jouy-en-Josas, France; 2AgroParisTech UMR Micalis, Jouy-en-Josas, France; 3CNRS
UMR7156, Laboratoire de Génétique moléculaire, Génomique et Microbiologie, Université de Strasbourg, Strasbourg,
France
1
[email protected]
Transposable elements (TEs) are widespread in eukaryotes but uncommon in yeasts of the
Saccharomycotina subphylum, in terms of both host species and genome fraction. The class II
elements are especially scarce, but the hAT element Rover is a noteworthy exception that deserves
further investigation.
We conducted a genome-wide analysis of hAT elements in 40 ascomycota. Phylogenetic analyses
were performed on hAT elements and host species with both MrBayes and PhyML. Selection pressure
acting on the elements was estimated by dN/dS calculation.
A novel family, Roamer, was found in three species, whereas Rover was detected in 15 preduplicated
species from Kluyveromyces, Eremothecium, and Lachancea genera, with up to 41 copies per
genome. Rover acquisition seems to have occurred by horizontal transfer in a common ancestor of
these genera. The detection of remote Rover copies in N. dairenensis, and in the sole S. cerevisiae
strain AWRI1631, without synteny, suggests that additional independent horizontal transfers took
place toward these genomes. Such patchy distribution of elements prevents any anticipation of TE
presence in incoming sequenced genomes, even closely related ones. The presences of both putative
autonomous and defective Rover copies, as well as their diversification into five families, indicate
particular dynamics of Rover elements in the Lachancea genus. Especially we discovered the first
miniature inverted-repeat transposable elements (MITEs) to be described in yeasts, together with
their parental autonomous copies. Evidence of MITE insertion polymorphism among L. waltii strains
suggests their recent activity. Moreover, 40% of Rover copies appeared to be involved in chromosome
rearrangements, showing the large structural impact of TEs on yeast genome and opening the door to
further investigations to understand their functional and evolutionary consequences.
KEYWORDS: MITE, Evolution, Rover, Roamer, Horizontal transfer
228
Deciphering the genetic and metabolic bases of yeast aroma properties
Matthias Eder, Isabelle Sanchez, Peggy Rigou, Thibault Nidelet, Carole Camarasa, Jean-Luc
Legras, Sylvie Dequin
INRA, UMR1083 SPO, F-34060 Montpellier, France
[email protected]
The yeast species Saccharomyces cerevisiae plays a vital role in the conversion of non-aromatic
precursors to aromas and the formation of aroma compounds, like esters, higher alcohols and organic
acids, during wine fermentation.
The aim of this work is to identify the genomic and metabolic bases for these processes. A cross
was performed between two wine yeast strains, selected because of their difference in the need for
nitrogen during fermentation. 130 F2-segregants were genotyped by whole genome sequencing and
individually phenotyped during wine fermentation by measuring extracellular metabolites using
HPLC and GC-MS. Based on the metabolic data, the intracellular metabolic fluxes were estimated
using a constraint-based model of yeast central metabolism (Celton et al 2012). Quantitative trait
locus (QTL)-mapping was used to identify allele variations influencing the aroma profile and the
metabolic fluxes.
A normal distribution among the population was found for most observed traits, which indicates the
interaction of several genes. Most traits were transgressive, indicating the presence of alleles with
opposite effects in the parental strains. An exception was a bimodal distribution for the ratio of residual
glucose to fructose after 80% of fermentation. A QTL was detected for this trait on chromosome IV.
Within this region we found the gene HXT3, encoding a glucose transporter. An allelic variant of
HXT3, enabling a better utilization of fructose, has previously been described in a wine yeast strain
(Guillaume et al 2007).
Several QTLs were also detected for aroma compounds and are currently being dissected. This will
lead to a more profound knowledge about the links between genetic variation and industrial traits and
will enable the exploitation of yeast natural diversity to generate novel aromatic wine yeast strains
through breeding.
KEYWORDS: Wine aroma, QTL-mapping, Constraint-based model, Central carbon metabolism
REFERENCES:
Celton M, Goelzer A, Camarasa C, Fromion V, Dequin S (2012). A constraint-based model analysis of the metabolic
consequences of increased NADPH oxidation in Saccharomyces cerevisiae. Metabolic Engineering 14:366-379
Guillaume C, Delobel P, Sablayrolles JM, Blondin B (2007). Molecular basis of fructose utilization by the wine
yeast Saccharomyces cerevisiae: a mutated HXT3 allele enhances fructose fermentation. Applied Environmental
Microbiology 73:2432-2439
229
Improving functional annotation of the genomes of non-conventional yeasts: a
case study with Pichia pastoris
Duygu Dikicioglu1,Valerie Wood1, Kim M. Rutherford1,Mark D. McDowall2,
Stephen G. Oliver1
Cambridge Systems Biology Centre & Department of Biochemistry, University of Cambridge, Cambridge, UK;
European Molecular Biology Laboratory European Bioinformatics Institute (EMBL-EBI) Wellcome Trust Genome
Campus, Hinxton, UK
1
2
[email protected]
The research communities studying the model yeasts Saccharomyces cerevisiae and
Schizosaccharomyces pombe are well served by model organism databases that have extensive
functional annotation. However, this is not true of many industrial yeasts that are used widely in
biotechnology. Pichia (Komagataella) pastoris is a favourite host organism for recombinant protein
production in both industry and academia. As with many industrial species, the use of P. pastoris as
an experimental organism, and its future development as a vehicle in synthetic biology, is impeded
by shortcomings in the functional annotation of its genome. In this communication, we will consider
the resources that can be implemented in the short term both to improve Gene Ontology (GO)
annotation coverage based on annotation transfer, and to establish curation pipelines for the literature
corpus of this organism. The availability of the Canto curation tool, the inclusion of P.pastoris in
the Ensembl Genomes resource, and its adoption as a Reference Proteome in the 2014_01 release of
UniProtKB should raise the profile and utility of this organism as a model and provide a platform for
the integration of knowledge from the existing distributed resources. We trust that communities of
researchers working with other yeasts of industrial, ecological, or evolutionary importance will find
these tools of use in improving the annotation status of their species of choice.
230
De Novo genome sequencing of the yeast Candida intermedia
Cecilia Geijer, Antonio D. Moreno, Lisbeth Olsson
Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology,
Gothenburg, Sweden
[email protected]
The urgency to reduce carbon emissions and to lower our dependence on oil makes it necessary to
strive towards a more sustainable, bio-based economy where energy, chemicals, materials and food
are produced from renewable resources. Lignocellulosic biomass constitutes a great source of raw
material for such a future bio-based economy since it is widely available, relatively inexpensive and
do not compete with food and feed production. Saccharomyces cerevisiae is commonly used for
bioethanol production and displays excellent glucose fermenting skills, but metabolic engineering is
needed to allow consumption and fermentation of xylose (the second to glucose most prevalent sugar
in lignocellulose). As an alternative to S. cerevisiae, microorganisms that naturally ferment xylose
can be used. An unexpected discovery in our lab allowed us to isolate a clone of the non-conventional,
xylose fermenting yeast species Candida intermedia. The aim of this project is to sequence the
genome of C. intermedia as well as to develop a molecular toolbox to allow genetic manipulations
of this yeast. PacBio sequencing and de novo assembly of the genome revealed a haploid yeast
with a genome size of 13.2 Mb and a total of 5216 genes spread over seven chromosomes. Future
activities include identification of genes involved in uptake and fermentation of sugars derived from
lignocellulosic biomass, and subsequent deletion/overexpression of interesting candidate genes to
improve the fermentation capacity of the yeast.
KEYWORDS: Lignocellulose, Fermentation, De Novo genome assembly
231
232
Session 5
Non-conventional yeasts
233
Yeast-Bacterial competition induced new metabolic traits in L. kluyveri by
segmental duplication
Nerve Zhou1, Anne Friedrich2, Concetta Compagno3, Joseph Schacherer2, Krishna B.S
Swamy4, Michael Katz5, Samuele Bottagisi1,6, Wolfgang Knecht1, Zoran Gojkovic5,
Jure Piškur1
Department of Biology, Lund University, Lund, Sweden; 2Department of Genetics, Genomics and Microbiology,
University of Strasbourg, CNRS, UMR7156, Strasbourg, France; 3Department of Food, Environmental and Nutritional
Sciences, University of Milan, Milan, Italy; 4Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan; 5Calsberg
Laboratories, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark; 6Dipartimento di Biologia e Biotecnologie,
Università degli studi di Pavia, Pavia, Italy
1
[email protected]
Large-scale chromosomal rearrangements conferring genome plasticity have reshaped genomes
of extant yeast species. Gene regulation, genome reorganization and gene expression alterations
are among their importance in evolution. Based on this, we were eager to reconstruct molecular
mechanisms that could have led to genome and phenotypic diversity in yeasts. We adopted an
experimental evolution approach by mimicking and applying selective pressures that could have
existed in nature approximately 150 million years ago. We sequentially co-cultured L. kluyveri, in
the presence of bacteria of increasing ethanol tolerance for at least 960 generations. Here we report
the emergence and fixation of a novel “extra-banded” karyotype after 720 generations in one of the
four parallel evolution lines. Physiological studies showed that this karyotype is associated with a
significantly higher fitness in shake flasks fermentations and a higher fermentative capacity under
fully controlled aerobic conditions as compared to the ancestor. Further analysis using phenotypic
microarrays technology (PM) suggested emergence of new metabolic traits. The extra-banded strains
were more stringently glucose repressed as compared to their ancestor coupled to a growth advantage
in most carbon sources and osmolytes. We found out that the extra-banded karyotype was as a result
of a duplication and translocation event involving a 261kb segment. We propose that these changes
account for the emergence of new metabolic traits and speculate that the cross-kingdom competition
has a role in genome evolution in yeasts.
KEYWORDS: Evolution of ethanol production, Experimental evolution, Yeast-bacteria life strategy,
Segmental duplication
REFERENCES:
Dashko S, Zhou N, Compagno C and Piškur J (2014). Why, when, and how did yeast evolve alcoholic fermentation?
FEMS Yeast Research 14(6):826-832
Piskur J, Rozpedowska E, Polakova S, Merico A, Compagno C (2006). How did Saccharomyces evolve to become a
good brewer? Trends in Genetics 22:183–186
234
Hypoxic regulation of transcription of the glucose transporter gene RAG1 in
Kluyveromyces lactis
Rosa Santomartino1, Daniela Ottaviano1, Andrea Visca1, Alexandre Soulard2, Marc Lemaire2,
James González3, Alicia González3, Michele M. Bianchi1
1
Dept. Biology and Biotechnology C. Darwin, Sapienza University, Rome, Italy; 2 Génétique Moléculaire des Levures,
UMR5240 Microbiologie, Adaptation et Pathogénie, Universitè de Lyon, Lyon, France; 3 Dept. Biochemistry and
Structural Biology, Universitad Nacional Autonoma de Mexico, Mexico City, Mexico
[email protected]
The expression of the low-affinity glucose transporter gene RAG1 in Kluyveromyces lactis is regulated
by glucose signaling and glycolysis. Glucose signaling proceeds through cascades involving the
glucose sensor Rag4 and other proteins. The casein kinase Rag8 and the repressor proteins Sms1
and KlRgt1 have major roles in this regulation. Another pathway involves the chromatin remodeler
KlSnf2 and Sck1. Also signals from glycolysis are involved in RAG1 expression.
Strains were constructed and selected in Lyon and Rome. Cells were grown in flask or bioreactor
under hypoxia. Transcription analysis was performed by Northern blotting. Promoter analysis was
carried out by fusion with the reporter gene LacZ and by Nucleosome scanning. ChIP was performed
with tagged Sck1 strain.
We have found that transcription of RAG1 is induced by hypoxia and this induction requires the
presence of glucose. Molecular dissection and structural analysis of the RAG1 promoter allowed
to identify the region essential for the induction. Various mutant strains of the glucose regulation
are available. Transcription analysis of RAG1 in these mutants allowed to identify Sck1 as the
possible element involved also in oxygen signaling. Dependence of Sck1 expression on hypoxia
and binding of Sck1 to the RAG1 promoter has been also investigated. Interestingly, the level of
RAG1 transcription, but not the hypoxic induction, depended on the presence of the hypoxic regulator
KlMga2. NuSA analysis of the promoter in different conditions and mutants, indicates the role of
chromatin organization in RAG1 regulation.
Our results demonstrate that expression of the low-affinity glucose transporter gene RAG1 is
synergistically regulated by glucose and low oxygen and the glucose regulator Sck1 and the hypoxic
regulator KlMga2 probably cooperate in this mechanism.
Work partially funded by MAECI (Direzione Generale per la Promozione del Sistema Paese)
KEYWORDS: Oxygen signaling, Glycolysis, Transcription factor
REFERENCES:
Hnatova M, Wésolowski-Louvel M, Dieppois G, Deffaud J, Lemaire M (2008). Characterization of KlGRR1 and SMS1
genes, two new elements of the glucose signaling pathway of Kluyveromyces lactis. Eukaryotic Cell 7:1299-1308
Cotton P, Soulard A Wésolowski-Louvel M, Lemaire M (2012). The SWI/SNF KlSnf2 subunit controls the glucose
signaling pathway to coordinate glycolysis and glucose transport in Kluyveromyces lactis. Eukaryotic Cell 11:13821390
Cairey-Remonnay A, Deffaud J, Wésolowski-Louvel M, Lemaire M, Soulard A (2015). Glycolysis controls plasma
membrane glucose sensors to promote glucose signaling in yeasts. Molecular and Cellular Biology 35:747-757
Ottaviano D, Montanari A, De Angelis L, Santomartino R, Visca A, Brambilla L, Rinaldi T, Bello C, Reverberi M,
Bianchi MM (2015). Unsaturated fatty acids-dependent linkage between respiration and fermentation revealed by
deletion of hypoxic regulatory KlMGA2 gene in the facultative anaerobe-respiratory yeast Kluyveromyces lactis.
FEMS Yeast Research (accepted)
235
Lipid analysis: a way to improve the production of sophorolipids by
Starmerella bombicola
Marilyn De Graeve, Inge Van Bogaert, Wim Soetaert
University Ghent, Ghent, Belgium
[email protected]
The yeast Starmerella bombicola is known for the commercial production of the biosurfactant
sophorolipids. Sophorolipids are surface-active molecules consisting of a disaccharide and a fatty
acyl chain.
The yeast can utilize various lipophilic substrates such as vegetable oils, alkanes and fatty acid esters
and converts them to free fatty acids; the substrate of the sophorolipid biosynthetic pathway. In
addition, the yeast has a very active de novo fatty acid synthesis; also in the absence of a lipophilic
carbon source reasonable amounts of sophorolipids are obtained.
While the production level of sophorolipids is already high; some efforts are still necessary for the
synthesis of new-to-nature sophorolipids. The production of these molecules by modified Starmerella
bombicola strains or by using special substrates is in some cases less efficient and requires further
optimization. On the one hand, the efficiency is lower because special substrates are lost due to
catabolic pathways. On the other hand, some modified strains are less productive than the wild type
yeast. The blockage of these catabolic pathways and a better availability of lipophilic carbon sources
can be a solution.
Because uptake, transport and storage of lipophilic molecules are very important in the production
process of sophorolipids, the lipid composition of different compartments are examined and compared
with other (oleaginous) yeasts. By gaining knowledge on how these molecules are converted, new
metabolic engineering strategies can be found to target specific genes.
The fundamental knowledge about the lipid metabolism of Starmerella bombicola will hence lead
to a better production platform for the synthesis of economic valuable new-to-nature sophorolipids.
KEYWORDS: Starmerella bombicola, Sophorolipids, Lipid analysis, Metabolic engineering
236
Non-Saccharomyces in the centre of the training centre
Ana Hranilovic, Federico Tondini, Vladimir Jiranek, Paul Grbin
Department of Wine and Food Sciences, University of Adelaide, Australia
[email protected]
A globally observed increase in wine ethanol content is related to various negative technological and
financial implications, as well as adverse health and sensory aspects of the final product. As such, it
is deemed a major challenge for the wine industry; the Australian winemaking sector not being the
exception.
In the light of climate change and perturbing stylistic preferences, the newly established ARC Training
Centre for Innovative Wine Production is focusing on the development of an integrated, whole-ofproduction-chain approach for ethanol and flavour management. Projects aim to elucidate the basis
of sugar accumulation and concentration in grapes, achieve pre-fermentative sugar stripping and
optimisation of early harvest and dealcoholisation regimes. As an alternative (or rather complementary)
strategy to achieve lower alcohol wines of improved quality, a line of research within the Training
Centre focuses on exploring non-Saccharomyces diversity, with the aim of unravelling the outcomes
of un-inoculated fermentation depending on the initial sugar content, and the selection of strains
capable of diverting sugar away from ethanol production.
In the initial steps an in-house culture collection has been established and screening of nonSaccharomyces isolates for their fermentation aptitude and metabolite production has been undertaken.
Fermentation kinetics were monitored regularly and the yield of the main metabolite concentrations
was determined at the end of the fermentation. Significant differences in the parameters analysed
suggest the potential applicability of non-Saccharomyces yeasts to manage wine ethanol and sensory
compound levels. Subsequent work will focus on larger scale, real juice trials and include extensive
sensory and chemical analysis.
KEYWORDS: Non-Saccharomyces yeast, Wine, Ethanol reduction
237
Selection of suitably non-repressing carbon sources for expression of alcohol
oxidase isozyme promoters in the methylotrophic yeast Pichia methanolica
Tomoyuki Nakagawa, Keishi Wakayama, Takashi Hayakawa
Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
[email protected]
The methylotrophic yeast, Pichia methanolica, is a typical species that has nine alcohol oxidase
(AOD) isozymes, which are formed by the random oligomerization of two AOD subunits encoded by
MOD1 and MOD2. We believe that P. methanolica has an advantageous ability to develop a unique
gene-expression system using the MOD2 promoter, PMOD2, together with the MOD1 promoter,
PMOD1, compared with other major methylotrophic yeasts, such as P. pastoris, H. polymorpha and
C. boidinii. In this work, we aimed to select suitable non-repressing carbon sources for the expression
of promoters derived from the AOD isozyme genes, PMOD1 and PMOD2, during the growth of P.
methanolica. Our results revealed that xylose is the best non-repressing carbon source for heterologous
gene expression using both PMOD1 and PMOD2, and that glycerol is also a suitable carbon source
with by which the on/off of PMOD2 expression can be controlled.
KEYWORDS: Pichia methaolica, Alcohol oxidase isozymes, Gene expression, Non-repressing
carbon source, MOD promoters
REFERENCES:
Nakagawa T, Wakayama K, Hayakawa T (2015). Selection of suitably non-repressing carbon sources for expression
of alcohol oxidase isozyme promoters in the methylotrophic yeast Pichia methanolica. Journal of Bioscience and
Bioengineering 120:41-44
Nakagawa T, Inagaki A, Ito T, Fujimura S, Miyaji T, Yurimoto H, Kato N, Sakai Y, Tomizuka N (2006). Regulation of
two distinct alcohol oxidase promoters in the methylotrophic yeast Pichia methanolica. Yeast 23:15-22
Nakagawa T, Mukaiyama H, Yurimoto H, Sakai Y, Kato N (1999). Alcohol oxidase hybrid oligomers formed in vivo
and in vitro. Yeast 15:1223-1230
238
Functional screening of Non-Conventional Yeasts (NCYs)
as biocatalysts for alkenes reduction
Fabio D’Achille1, Simone Di Mauro2, Ciro Sannino2, Sara Filippucci2, Benedetta Turchetti2,
Maria R. Cramarossa1, Pietro Buzzini2, Luca Forti1
Department of Life Sciences, University of Modena and Reggio Emilia, 41125 Modena, Italy; 2Department of
Agricultural, Food and Environmental Science, Industrial Yeasts Collection DBVPG, University of Perugia,
06121 Perugia, Italy
1
[email protected]
Due to the current trend towards more “green” or sustainable chemistry, the development of new
methods for biocatalyzed alkene reductions is becoming more important in the production of fine
chemicals. Nowadays the identification of new biocatalysts is a new trend required in order to enlarge
the portfolio of microorganisms and related enzymes) to be used for these bioreduction processes.
Considering the ecological biodiversity, yeasts offer a great pool of biocatalyst already involved in
the production of fine chemicals.
27 NCYs of the genera Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Kazachstania,
Kluyveromyces, Lindnera, Meyerozyma, Nakaseomyces, Pichia, Trichosporon, Vanderwaltozyma
and Wickerhamomyces were screened for their Ene-Reductase (ER) activity. Products obtained from
bioreduction were quantified and characterized by GC-MS.
In a previous study (Goretti et al 2011) we described the bioconversion of different α,β-unsaturated
ketones and aldehydes promoted by NCYs whole-cell. In this work a structure-activity-relationship
study was successively performed.
O
O
N
O
O
O
1 [(3E)-4-phenylbut-3-en-2-one
Cl
2 methyl (2E)-3-phenylprop-2-enoate 3 (2E)-3-phenylprop-2-enenitrile 4 (3E)-4-(4-chlorophenyl)but-3-en-2-one
5 (2E)-1,3-diphenylprop-2-en-1-one
Almost all NCYs showed the ability to reduce C=C double bonds of substrates 1, 3 and 5. By
substituting the methyl group of the substrate 1 with a phenyl group (1 vs. 5), the number of active
yeasts increased considerably. The same effect, although in a minor extent, was observed with the
chlorinated ketone . Noteworthy, all the yeasts showed only ER activity, reducing exclusively the
C=C double bond. The higher conversions are generally obtained for the substrate 5.
KEYWORDS: Biocatalysis, Bioreduction, Non-Conventional Yeasts, Ene-Reductase
REFERENCES:
Goretti M; Ponzoni ; Caselli E; Marchegiani E; Cramarossa MR; Turchetti B; Forti L; Buzzini P (2011). Bioreduction
of α,β-unsaturated ketones and aldehydes by non-conventional yeast (NCY) whole-cell. Bioresource Technology
102:3993-3998
239
Influence of Lachancea thermotolerans on wine fermentation
Huseyin Erten1, Eren Kemal Balıkcı1, Hasan Tanguler1,2
Cukurova University, Faculty of Agriculture, Department of Food Engineering, 01330 Adana, Turkey; 2Present address:
Department of Food Engineering, Faculty of Engineering, Nigde University, Merkez Yerleşke 51245 Nigde, Turkey
1
[email protected]
Although it is well known that the main wine yeast is Saccharomyces cerevisiae, non-Saccharomyces
yeasts can contribute positively to the chemical and sensory characteristics of wines
(Fleet 2008; Jolly et al 2014).
Lachancea thermotolerans (Formerly Kluyveromyces thermotolerans) is one of the these yeasts
which have been reported to increase the total acidity of wines by producing L-lactic acid (Mora et al
1990; Kapsopoulou et al 2005, 2007; Gobbi et al 2013). Therefore the problems an increase in alcohol
content and a reduction in total acidity of wines due to the global climate change can be lowered using
L. thermotolerans in fermentations (Gobbi et al 2013). The present work describes the behaviour of
L. thermotolerans and S. cerevisiae in mixed and sequential fermentations in grape must.
The growth kinetics and fermentation performance of S. cerevisiae isolated from cv. Emir fermentations
and L. thermotolerans CBS 2860 yeasts during the fermentation of sterile grape must of cv. Emir
using pure, mixed and sequential cultures were studied under static conditions at 20oC. Yeast counts
and analyses of physicochemical, aroma compounds and sensory were done.
Faster fermentation rates were observed in fermentations done with pure culture of S. cerevisiae and
mixed culture of L. thermotolerans and S.cerevisiae. L. thermotolerans survived in fermentations with
high numbers. Using L. thermotolerans increased the total acidity in wines, varying in the range of
5.40-6.28 g/l as tartaric acid. Pure culture of S. cerevisiae gave the lowest level of total acidity (5g/l).
Volatile acidity levels as acetic acid ranged from 0.53 g/l - 0.73 g/l. The concentration of ethyl alcohol
in samples varied between 10.76% and 11.62% (v/v). The production of higher alcohols and esters
was higher in the sequential culture of L. thermotolerans and S. cerevisiae inoculated on first day
of fermentation. According to sensory analysis, wines obtained by the mixed culture and sequential
culture with the inoculation of S. cerevisiae after one day were preferred the most. This study shows
that using L. thermotolerans could improve the wine composition which is in good agreement with
previous studies of (Mora et al 1990; Kapsopoulou et al 2005, 2007; Gobbi et al 2013).
Acknowledgments This work was funded by a grant from Cukurova University Academic Research
Projects Unit (Project no: ZF2010YL35).
KEYWORDS: Non-Saccharomyces yeast, Lachancea thermotolerans, Saccharomyces cerevisiae,
Mixed and sequential cultures, Wine
REFERENCES:
Fleet (2008). FEMS Yeast Research 8: 979-995
Gobbi et al (2013). Food Microbiology 33:271-281
Jolly et al (2014). FEMS Yeast Research 14:215-237
Kapsopoulou et al (2005). World Journal of Microbiology and Biotechnology 21:1599-1602
Kapsopoulou et al (2007). World Journal of Microbiology and Biotechnology 23:735-739
Mora et al (1990). American Journal of Enology and Viticulture 41:156-159
240
HACking Kluyveromyces lactis UPR - functional characterization of the
transcription activator KlHAC1 and study of evolutionary conservation
Hans C. Hürlimann, Lena Munzel, Karin D. Breunig
Martin-Luther-Universität Halle, Molekulargenetik, Halle, Germany
[email protected]
The unfolded protein response (UPR) is a stress response aiming to cope with unfolded proteins
accumulating in the ER. Its sensing and activation mechanism, acting by an unconventional splicing
of the transcription activator mRNA HAC1 (homolog of XBP1), is highly conserved between species.
Interests in studying the UPR are on one hand its implication in diseases such as cancer, diabetes
and neurodegeneration and on the other hand production of heterologous proteins, needing the ER
for building up. The aim of the presented study was to analyze the UPR induction mechanism in the
non-conventional, industrial yeast K. lactis, focusing on the KlHAC1 gene, with the goal to lay the
necessary basis for improvement of recombinant protein production in this host.
Homologs of genes involved in the UPR activation (HAC1, IRE1, KAR2 & TRL1) are present in
K. lactis but for KlHAC1 (KLLA0F08976g) only the ORF encoding the unspliced mRNA variant
(KlHAC1u) was annotated as a gene in NCBI. We identified a possible intron of KlHAC1 by sequence
analysis and verified it by cloning and sequencing of cDNA from the spliced mRNA variant (KlHAC1i)
obtained after by inducing an UPR with DTT or Tunicamycin. Homologs of transcripts of five wellstudied UPR reporter genes in S. cerevisiae (KlKAR2, KlERO1, KlLHS1, KlEUG1/KlPDI1 & KlINO1)
were analyzed by semi quantitative RT-PCR and RT-qPCR. We find that KlERO1 is the most sensitive
reporter gene for UPR induction in K. lactis. The functional role of KlHAC1i in UPR induction was
confirmed via UPR-independently regulated expression of a genomically integrated KlHAC1i cDNA.
We find that induction of KlHAC1i is sufficient to induce transcription of UPR reporter genes. Finally,
evolutionary conservation of KlHAC1 was revealed, since the spliced and unspliced KlHAC1 ORF on
single copy Tet-off plasmids, are able to complement the severe growth defects under UPR inducing
conditions of the S. cerevisiae mutants hac1 and ire1.
KEYWORDS: Kluyveromyces lactis, UPR, HAC1
241
Impact of acetate on lactose bioconversion by non-conventional yeasts
Kluyveromyces marxianus
Jekaterina Lukjanenko, Juris Kibilds, Agnese Kokina, Janis Liepins, Armands Vigants
Institute of Microbiology and Biotechnology, University of Latvia, Riga, Latvia
[email protected]
Kluyveromyces marxianus is non-conventional yeast that can efficiently metabolize lactose. Due
to this advantage this yeast can be used in lactose containing substrate fermentations (Fonseca et
al 2008). Acetate is a weak acid and one of the fermentation by-products with pKa 4.76. During
fermentation medium pH value decreases and acetate undissociated forms concentration increases. In
undissociated state acetate immediately diffuse into the cell cytosole and leave up negative effect on
yeast metabolism and cell growth.
K.marxianus strains: DSM 5422, DSM 4906, DSM 5418, CBS 712 and CBS 5665. Cultivation
media: semi-synthetic medium (g/L, 5 yeast extract, 1.4 MgSO4·7H2O, 1.0 KH2PO4, 0.1 KH2HPO4,
5 (NH4)2SO4 and 20 sugar (glucose, galactose or lactose) at 30̊C. Medium pH was maintained by
acetic and citric buffers. Cell growth was monitored at 600 nm by 96-well TECAN M200Pro 96 well
reader.
Inhibition of K.marxianus biomass growth by acetate was studied. Five K.marxianus strains growth
dynamics at different pH (4.0-6.0) with 0.05M acetate concentration on glucose, galactose and lactose
were tested. Growth of all strains was inhibited in the presence of acetate on lactose medium at pH
below acetic acid pKa. In the same time, acetate effect on yeast growth differed among strains when
glucose or galactose was used as carbon source. This seemed to be related with differences in strain
sugar metabolism (Carvalho-Silva & Spencer-Martins 1990).
KEYWORDS: K.marxianus, Acetic acid, Weak acid stress
REFERENCES:
Fonseca GG, Heinzle E, Wittmann C and Gombert AK (2008). The yeast Kluyveromyces marxianus and its
biotechnological potential - Applied Microbiology and Biotechnology 79(3):339–354
Carvalho-Silva M, Spencer-Martins I (1990). Modes of lactose uptake in the yeast species Kluyveromyces marxianus.
Antonie Van Leeuwenhoek 57(2):77-81
242
Bioconversion of glycerol by the yeast Yarrowia lipolytica
Michael Egermeier1,2, Hans Marx1,2, Hannes Russmayer1,2, Diethard Mattanovich2,3,
Michael Sauer1,2,3
Christian Doppler Laboratory for Biotechnology of Glycerol, Vienna, Austria; 2University of Natural Resources and
Life Sciences, Vienna, Austria; 3Austrian Centre for Industrial Biotechnology (ACIB GmbH), Vienna, Austria
1
[email protected]
The biodiesel-byproduct glycerol represents a cheap and abounding substrate for large scale microbial
conversion processes to enhance the economic viability of biodiesel production. The oleaginous yeast
Yarrowia lipolytica readily consumes glycerol for biomass formation and is able to produce valueadded products like citric acid, lipids and polyols.
The aim of this project is to investigate the potential of Y. lipolytica for the valorization of crude
glycerol and to set up a biorefinery concept for the biodiesel production process. As a first step 20
different wild-type isolates were cultivated in shake-flasks as well as in bioreactors and screened for
their potential in the formation of lipids and polyols.
Shake flask experiments in 500mL scale with 25mL of nitrogen-limited media containing 50g/L
glycerol. Bioreactor cultivations were performed in 1.4L glass-reactors in 500mL of nitrogen-limited
media containing 100g/L glycerol. Conditions with dissolved oxygen of 50% and pH set-points of
5.5 and 3.5 were analyzed.
Up to 30g/L of polyols could be produced in shake flask cultivations. Defined conditions in bioreactor
cultivations showed that the product pattern is strongly dependent on the pH of the cultivation media.
With pH maintained at 3.5 up to 55g/L of polyols were produced, whereas pH 5.5 favors the formation
of citric acid.
Twenty wild-type isolates of Y. lipolytica were screened for their potential in the valorization of
glycerol. Non-optimized cultivation conditions resulted in polyol formation with yields of 0.5 g/g.
The availability of dissolved oxygen (50%) and the pH (3.5 and 5.5) play an important role in the
regulatory mechanisms of the bioconversion process. The potential of novel wilde-type isolates
of Y. lipolytica has been determined for value-added bioconversion of glycerol. An increase of the
conversion yield will be examined through optimized process conditions and genetic engineering.
KEYWORDS: Yarrowia lipolytica, Glycerol, Biorefinery, Polyols
243
Development of molecular tools for red, basidiomycetous oleaginous yeasts
Johanna Blomqvist1, Taras Luzhetskyi1,2, Ievgeniia Tiukova3, Andrei A. Sibirny2,4, Mats
Sandgren1, Volkmar Passoth3
Department of Chemistry and Biotechnology, Uppsala BioCenter, SLU, Box 7015, 750 07 Uppsala, Sweden;
Institute of Cell Biology, Department of Molecular Genetics and Biotechnology, Lviv, 79005 Ukraine; 3Department
of Microbiology, Uppsala BioCenter, SLU, Box 7025, 750 07 Uppsala, Sweden; 4Department of Biotechnology and
Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow, Poland
1
2
[email protected]
The basidiomycetous yeasts, Rhodototula glutinis, Rhodosporidium toruloides and their relatives are
red, oleaginous yeasts that can accumulate lipids to more than 50% of their cell mass. Moreover, they
can produce substantial amounts of the ω3-fatty acid linolenic acid, small amounts of longer chained
ω3-fatty acids have also been found. Therefore, these yeasts are of interest for biodiesel and fish feed
production. Besides lipids, those yeasts produce pigments such as β-carotenes that give them their
characteristic colour. Those pigments and their precursors can be the basis for the production of highvalue chemicals such as e.g. astaxanthin.
However, tools for molecular manipulation of these red basidiomycetous yeasts are so far underdeveloped.
In this study we are describing the development of a random integrative transformation system for
R. glutinis and the development of gene-walking methods to identify regions flanking the integrated
sequence.
KEYWORDS: Oleaginous yeasts, Lipids, Transformation, Random integration, Gene walking
244
Session5: Non-conventional yeasts
Lipid production by Lipomyces starkeyi grown on lignocellulosic hydrolysate in a
pH regulated feeding system
Jule Brandenburg1, Johanna Blomqvist1, Mats Sandgren1, Volkmar Passoth2
Department of Chemistry and Biotechnology, SLU, Box 7025, 75007 Uppsala, Sweden; 2Department of Microbiology,
SLU, Box 7025, 75007 Uppsala, Sweden
1
[email protected]
Lignocellulose is the most abundant biomass resource on earth and its hydrolysates are commonly
considered as promising substrates for biofuel production. The hemicellulose fraction is mainly built
up of xylanes and therefore its hydrolysates are more difficult to convert to bioethanol compared to
hydrolysates of the cellulose fraction.
Many oleaginous yeasts are able to convert carbohydrates present in lignocellulose material into fatty
acids under nitrogen limitation. The rate of lipid accumulation is significantly higher than in other
oleaginous microbes and the lipid content can exceed half of the total biomass. The low content of
nitrogen in hemicellulose is advantageous for lipid accumulation. On the other hand, a variety of
inhibitors are present, which can suppress growth and lipid formation. Starting with a model substrate
we have established feeding strategies to keep the level of inhibitors on a for the yeast tolerable
level. Using a pH regulated feeding, based on an observed co‑consumption of acetic acid and sugars
by Lipomyces starkeyi, we could keep intoxication on a minimum and moreover establish a selfregulating feeding system. Using this type of continuous feeding we were able to grow L. starkeyi
on the hemicellulose fraction of birch hydrolysate and reached an intracellular lipid content of more
than 40%, about 8 g/L lipid with a lipid yield of 0.12 g lipid/g xylose. Lipid analyses showed that the
degree of saturation of fatty acids increased in the late stage of cultivation.
245
Session 5:Non-conventional yeasts
Extracellular proteases produced by Yarrowia lipolytica: functional
characterization of food protein hydrolysates
Davide Gottardi1,2, Giorgia Gozzi1, Esther Lauree Moudi Koullè1, Lucia Vannini1,3
Department of Agricultural and Food Science, Alma Mater Studiorum, University of Bologna, Italy; 2ProDigest, Gent,
Belgium; 3Inter-departmental Center of Industrial Agri-Food Research (CIRI Agroalimentare), Alma Mater Studiorum,
University of Bologna, Italy
1
[email protected]
Microorganisms can produce a wide array of extracellular proteases (ES) which have been
commercially exploited to assist protein degradation in various industrial food processes. Most of the
commercial protease preparations used in Europe are produced with genetically modified Aspergillus
and Bacillus spp. strains. On the other hand, ES have been widely studied also in Yarrowia lipolytica.
(Hernández-Montañez et al 2007) sequenced the genes encoding an alkaline protease (AEP) and an
acidic protease (AXP) which are regulated by the pH of the medium (Young et al 1996). However,
investigations on strain diversity or the possible biological activity of the produced hydrolysates are
still lacking.
Therefore in this work the suitability of proteases, produced by Y. lipolytica at different pH values,
to hydrolyze various food proteins and the characterization of the derived hydrolysates have been
evaluated. In particular the proteolytic activity of 16 strains of Y. lipolytica from different habitats
(i.e. isolated from speck, salami, cheeses, “light” butter, commercial chilled foods, irradiated poultry
meat, Po river waters) was tested on gelatin, meat protein, dairy proteins (α- and β-caseins) and wheat
gluten. Moreover, the hydrolysates were characterized for their antioxidant (i.e DPPH and inhibition
of linoleic acid peroxidation assays) and antimicrobial activities.
The results showed that all strains were able to synthesizes ES active on all the tested proteins although
some differences were found in relation to the pH of the medium. The hydrolysates were endowed
with antioxidant activity. In particular ES released in cultural medium with pH 5.0 seem to have
generated peptides with the highest antioxidant activity. Moreover, among the different hydrolysates,
those obtained from caseins with Y. lipolytica strains from Po river and chilled foods displayed a
remarkable antimicrobial activity against food spoilage yeasts and fungi.
In conclusion the selected strains may have a good biotechnological potential which can be exploited
to produce functional ingredients for the food industry.
KEYWORDS: Yarrowia lipolytica, Extracellular protease, Meat proteins, Functional ingredients
REFERENCES:
Hernández-Montañez Z, Araujo-Osorio J, Noriega-Reyes Y, Chávez-Camarillo G, Villa-Tanaca L (2007). The
intracellular proteolytic system of Yarrowia lipolytica and characterization of an aminopeptidase. FEMS Microbiology
Letters 268(2):178-186
Young TW, Wadeson A, Glover DJ, Quincey RV, Butlin MJ, Kamei EA (1996). The extracellular acid protease gene of
Yarrowia lipolytica: sequence and pH-regulated transcription. Microbiology 142(10):2913-2921
246
Elucidation of the biochemical pathway for detergent production in the yeast
Rhodotorula bogoriensis
Sofie Lodens*, Sofie L. De Maeseneire*, Sophie Roelants, Inge Van Bogaert, Wim Soetaert
Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Faculty of Bioscience Engineering, Ghent University,
Coupure Links 653, 9000 Ghent, Belgium
*
These authors contributed equally to this work
[email protected]
Micro-organisms are well established producers of surfactants or detergents. Their “bio-surfactants”
are recently getting a lot of attention due to their low eco-toxicity and good biodegradability. One of
the most promising bio-surfactants are sophorolipids, which nowadays are used in several industrial
fields, e.g. household detergents, cosmetics and pharmaceuticals. Nevertheless, a lack in structural
variation of efficiently produced glycolipid bio-surfactants is hampering real breakthrough in a number
of other industries. Therefore, our laboratory started research on the yeast Rhodotorula bogoriensis,
which produces a new and interesting type of sophorolipids, containing a docosanoic acid (C22 fatty
acid), which has its hydroxyl group at the unusual C13 position, resulting in a branched hydrophobic
moiety (Van Bogaert et al 2011). As the biochemical pathway for the production of this sophorolipid
is unknown, we focus on the identification and characterization of its genes and enzymes.
We optimized a transformation protocol for R. bogoriensis via “Agrobacterium tumefaciens mediated
transformation “ (ATMT) as this had not been done till now. The new transformation protocol was
used to create an R. bogoriensis P450 knock-out strain, to confirm a candidate sophorolipid gene
cluster. Identification of the latter will allow characterization of the involved enzymes and metabolic
engineering towards production optimization. The protocol was used to create the P450 knock-out,
which was genetically confirmed. Growth and production experiments are currently ongoing.
KEYWORDS: Sophorolipids, Bio-surfactants, Transformation, P450, Rhodotorula bogoriensis
REFERENCES:
Van Bogaert INA, Zhang J, Soetaert W (2011). Microbial synthesis of sophorolipids. Process Biochemistry 46:821–833
247
Utilization of autochthonous strains of Metschnikowia fructicola and
Metschnikowia pulcherrimi to inhibit Botrytis cinerea development
Wilson J. Fernandes Lemos Junior, Selimi Emisal, Silvana Odorizzi, Francesco Favaron,
Alessio Giacomini, Viviana Corich
Department of Agronomy Food Natural Resources Animals and Environment and Interdepartmental Centre for
Research in Viticulture and Enology CIRVE),University of Padova, Italy
[email protected]
Metschnikowia fructicola and Metschnikowia pulcherrimi yeasts isolated from fruits are used
as antagonists against postharvest pathogens of fruits (Sipiczki 2006; Parafati et al 2015). These
yeasts could be used for treatments of fruit undergoing a short shelf life. In the present work 98
strains of M. fructicola and M. pulcherrimi isolated from Friularo and Friularo Passito musts, were
characterized. The molecular identification of the strains was performed by sequencing the D1/D2
region of the 26S rRNA gene. A phenotypic screening was performed evaluating several enzymatic
activities of technological interest (cellulolytic, xylan-degrading, proteolytic, β-glucosidase, lipolytic
and pectinolytic activity). Antagonistic activity of the yeast strains was determined for 5 days at 25
C on PDA dual culture against some fungal pathogens. Three M. pulcherrimi isolates showed more
than 50-60% inhibition and can be effective in controlling B. cinerea postharvest rot of grape and
apple fruits.
KEYWORDS: Non-saccharomyces, Sustainability, Natural protection
REFERENCES:
Parafati L, Vitale A, Restuccia C, Cirvilleri G (2015). Biocontrol ability and action mechanism of food-isolated yeast
strains against Botrytis cinerea causing post-harvest bunch rot of table grape. Food Microbiology 47:85–92
Sipiczki M (2006). Metschnikowia strains isolated from botrytized grapes antagonize fungal and bacterial growth by
iron depletion. Applied Environmental Microbiology 72:6716–6724
248
A consensus genome-scale metabolic network reconstruction for the industrial
yeast, Komagataella (Pichia) pastoris
Ayca Cankorur-Cetinkaya, Duygu Dikicioglu, Stephen G. Oliver
Cambridge Systems Biology Centre & Department of Biochemistry, University of Cambridge, Cambridge, UK
[email protected]
Systems biotechnology integrates omics data with in silico modelling to design cells that have
improved metabolic properties for industrial applications. For the optimization of recombinant
protein production, we need to understand how the metabolic network copes with the burden
generated by the production of the foreign protein and what changes in the distribution of fluxes
result. Flux Balance Analysis represents a useful approach to trace the re-distribution of fluxes in
silico, but this is critically dependent on a model of the metabolic network that is able to make reliable
predictions. The predictive ability of the metabolic network model for Saccharomyces cerevisiae has
been improved significantly in recent years. However, for the industrial yeast, Komagataella (Pichia)
pastoris, although there are three genome-scale metabolic network models available, they differ in
content and use different terminologies to describe the same chemical entities - making it difficult
to compare them. In this communication, we will describe our efforts to reconstruct a consensus
metabolic network for K. pastoris. This organism is by far the most commonly used yeast species
in the production of recombinant proteins, and we believe that the new metabolic model will be of
significant help in the design of industrial strains and processes.
249
250
Session 6
Yeast Culture Collections
251
Laktoflora® – Culture collection of dairy microorganisms (CCDM)
Petr Roubal, Vladimir Drab, Miloslava Kavkova, Eva Suchanova
MILCOM Company Limited, Sobeslavska 841, Tabor CZ-39001, Czech Republic
[email protected]
The Culture Collection of Diary Microorganisms (CCDM) Laktoflora® was established in 1965 as a
gen-bank of bacteria, yeast and fungi for dairy research, agriculture, medicine and food processing.
Laktoflora® collection now maintains about 1.000 strains of microorganisms. The strains are
deposited in lyophilized form, deep-frozen and/or cultured on media. The collection is registered
in WFCC No 878 belong to acronym CCDM. The operation and maintenance of the collection is
funded by The Czech National Programme on Conservation and Utilisation of Plant genetic resources
and Agrobiodiversity. (http://genbank.vurv.cz/genetic/nar_prog/default_a.htm). The collection cooperates closely with Dairy Research Institute Ltd. on various research projects funded especially by The
Ministry of Agriculture of the Czech Republic and The Technological Agency of the Czech Republic.
The important participants of project are other RTDs and SMEs as well. The Laktoflora’s research
is focused on the isolation of new strains, genetic and phenotypic identification, re-identification
and characterization of bacterial, yeast and fungal strains. Prebiotic and probiotic effect of bacterial
and fungal strains on human health is tested under laboratory conditions (for example production of
biocines, assimilation of cholesterol, production of exopolysacharides, resistance to antibiotics) as
well as in pilot operations (sourdough, milk products etc). The fundamentals of our collection are
the inquiry of microorganisms and fungi, their maintenance, recovery as well as identification and
re-identification based on biochemical, morphological and molecular/genetic methods. The service
provided by the collection is in accord to the activity and equipment of the collection. The information
about strains: http://www.vurv.cz/collections/vurv.exe/search?lang=cz.
KEYWORDS: CCDM, Collection, Yeast, Bacteria
252
Unimore microbial culture collection: a source for providing novel
yeasts for winemaking
Luciana De Vero, Tommaso Bonciani, Alexandra Verspohl, Francesco Mezzetti,
Melissa Bizzarri, Lisa Solieri, Paolo Giudici
[email protected]
The University of Modena and Reggio Emilia (UNIMORE) Microbial Culture Collection (UMCC)
supplies authenticated strains and fundamental biological data for research and biotechnological
application (www.umcc.unimore.it). More than 1600 yeast strains, isolated from must, wine, beer,
vinegar, sourdough and other fermented products are included in UMCC and are maintained by
techniques that ensure preservation and long term genetic stability. Moreover, UMCC provides critical
insights into yeast physiology and metabolism and integrates sequence data with transcriptional and
functional studies to better define complex traits and to exploit their potential industrial application.
Among the collected yeasts, there are strains of oenological interest which represent an important
biological source for the application of genetic improvement strategies, able to achieve desirable
traits in winemaking. Recently, UMCC has been implemented with novel wine yeast strains obtained
by non-GE techniques such as breeding and evolution-based strategies. The first strategy was
functional for the construction of Saccharomyces cerevisiae intra-species hybrids and S. cerevisiae
x S. uvarum inter-species hybrids, respectively suitable for fermentation in nutritional deficiencies
conditions and at lower temperatures. The second strategy was designed in order to rapidly select
evolved S. cerevisiae strains with low or nil sulfite and sulfide production and enhanced glutathione
(GSH) production (De Vero et al 2011; Mezzetti et al 2014). These phenotypes are attractive for the
winemaking field, as they reduce allergenic risks, off-flavour compounds and limit must and wine
oxidation, respectively. All the novel yeast strains have been characterized by a polyphasic approach
and the associated information has been managed in the UMCC Database (http://biolomics.umcc.
unimore.it) using the BioloMICS software (BioAware).
KEYWORDS: Wine yeast, Saccharomyces cerevisiae, S. uvarum, Breeding, Evolution-based strategy
REFERENCES:
De Vero L, Solieri L, Giudici P (2011). Evolution-based strategy to generate non-genetically modified organisms
Saccharomyces cerevisiae strains impaired in sulfate assimilation pathway. Letters in Applied Microbiology
53(5):572-575
Mezzetti F, De Vero L, Giudici P (2014). Evolved Saccharomyces cerevisiae wine strains with enhanced glutathione
production obtained by an evolution-based strategy. FEMS Yeast Research 14(6): 977-987
253
ABM Culture collection
Matti Korhola
Department of Biosciences, POB 56 (Viikinkaari 9), FIN-00014 University of Helsinki and Alkomohr Biotech Ltd.,
Lehtotie 8, FIN-00630 Helsinki, Finland
[email protected]
The ABM Culture Collection is a national node in the Finnish Microbial Resource Centre Organisation
(www.micco.fi).
The ABM culture collection of about 3600 strains consists mainly of industrial, taxonomic, and rye
sour dough, yeasts. The cultures are preserved frozen at ‒80 oC. Identification of some of the yeasts
has been confirmed by sequencing the 25S rDNA D1/D2 region and the ITS1 – 5.8S rDNA – ITS2
region.
We have determined some physiological characteristics of many yeast strains: Tmax on solid agar
surface; Fermentation of glucose / maltose / sucrose; Sugar utilization (API 32C, API 50CH);
Tolerance to lactic acid.
Karyotyping (CHEF) and hybridization to MAL, MEL, and SUC gene probes has been performed
to many Saccharomyces strains revealing polymorphic gene families. Also preservation of genetic
segregants and determination of their mating type of some industrial S. cerevisiae strains has been
made.
The focus of our current research is on finding explanations to why maltose-negative Candida milleri
and Torulaspora delbrueckii wild strains thrive in natural rye sour dough, where maltose is the main
sugar for fermentation. Karyotyping and hybridization to S. cerevisiae SUC2 gene probe or to C.
milleri SUC gene probe revealed several SUC genes on different chromosomes of C. milleri.
Rye sour dough back slopping baking is practiced by half a dozen major and by numerous minor
commercial bakeries as well as by private households. Because dough raising is less eminent using
only natural C. milleri or T. delbrueckii, sometimes maltose-positive commercial baker´s yeast
S. cerevisiae is added to enhance dough raising. We have earlier demonstrated that introducing
Saccharomyces MAL6S gene into T. delbrueckii maltose-negative strain improved dough raising.
254
The Industrial Yeasts Collection DBVPG (Italy): a one hundred years
long history
Benedetta Turchetti, Ciro Sannino, Simone Di Mauro, Sara Filippucci, Pietro Buzzini
Department of Agricultural, Food and Environmental Science & Industrial Yeasts Collection DBVPG (www.dbvpg.
unipg.it), University of Perugia, Italy
[email protected]
The Industrial Yeast Collection DBVPG of the University of Perugia is a biological research Centre
specialized in yeast taxonomy and biodiversity and ex-situ conservation of yeasts and yeast-like
microorganisms. It was founded as “in-house collection” at the beginning of 1900 by Gino de’ Rossi
and rationalized and transformed into a service institution in 1970s by Alessandro Martini and Ann
Vaughan-Martini. The acronym DBVPG was derived by the Italian name of hosting Institution:
Dipartimento di Biologia Vegetale of Perugia - PG.
The Collection DBVPG conserves at present almost 6,000 strains in four distinct sections:
Section 1: yeasts associated with the alcoholic fermentation. About 1,300 fermenting strains for wine,
brewery, bakery industry, including about 1,100 cultures of Saccharomyces cerevisiae (and related
species) thoroughly screened for their technological features at laboratory and pilot plant scale.
Section 2: yeasts isolated from different habitats. About 2,800 strains, belonging to over 400 species of
about 90 genera, isolated during 60 years of ecological surveys in Europe, in tropical and sub-tropical
Countries (Africa and South America) and in worldwide cold areas (mountain glaciers, Antarctica,
Arctic). These strains are currently screened for their ability to produce novel molecules for industry
(e.g. food & beverage, biofuels, bulk and fine chemicals, etc.).
Section 3: type strains and certified yeast strains. Over 1,600 certified cultures obtained through
exchanges with other worldwide culture Collections.
Section 4: like-yeast microorganisms. Over 300 cultures of the genus Prototheca isolated in different
European countries.
The collection is affiliated to World Federation of Culture Collection (WFCC), European Culture
Collection Organization (ECCO), Global Catalogue of Microorganisms (GCM).
Since 1997 the Collection DBVPG has been accredited by the Italian Government as an International
Depositary Authority for the deposit of patented yeast strains under the regulations of Budapest
Convention and the requirements of the European Patent Office (EPO) and the World Intellectual
Property Organization (WIPO).
255
INDEX OF AUTHORS
Abagnale Maria ...................................................... 47
Abarca Valentina .................................................... 60
Abbas Charles A ..................................................... 40
Abdel-Moati Mohamed A ...................................... 10
Adeboye Peter ........................................................ 42
Agirman Bilal ......................................................... 29
Aguilar Cristóbal N .............................................. 126
Aguilar-Jiménez Alejandro .................................. 126
Ahmad Khadija Mohamed ............................. 66, 218
Ait-Kaki Amel ...................................................... 207
Al Malaki Amina .................................................... 10
Al Marri Masoud .................................................... 10
Alamäe Tiina ................................................ 131, 215
Albers Eva ............................................................ 204
Albertin Warren .............................................. 62, 167
Albu Madalina G .................................................. 184
Alexandre Hervé .................................................. 122
Alfonzo Antonio ..................................................... 95
Alves Nuno .......................................................... 138
Amaretti Alberto .................................................. 101
Amaya-Delgado Lorena ............................... 216, 217
Andlid Thomas ..................................................... 147
Aquilanti Lucia .................................................... 145
Arellano-Plaza Melchor ............................... 144, 181
Arena Giovanni .................................................... 200
Arendt Elke ............................................................ 38
Arfelli Giuseppe ..................................................... 26
Ariño Joaquín ....................................................... 102
Arreola Silvana .................................................... 133
Arriola Enrique .................................................... 143
Arrizon Javier ................................................216, 217
Arzanlou Mahdi ..................................................... 85
Assunção Mónica ................................................. 139
Avramova Marta .................................................. 167
Azeredo Joana ........................................................ 17
Babai-Ahari Asadollah ........................................... 85
Backović Ana ....................................................... 129
Bai Feng-Yan ..........................................................56
Bakeeva Albina .....................................................159
Bakhshandeh Maryam ........................................... 66
Baleiras-Couto Margarida .....................136, 138, 139
Balıkcı Eren Kemal ...............................................240
256
Ballario Paola ....................................................... 101
Balzarini Tom ......................................................... 27
Banilas Georgios .......................................... 160, 227
Barbosa Catarina .......................................... 127, 168
Barbosa Piló Fernanda ......................................... 156
Bărbulescu Iuliana-D ............................162, 182, 184
Baronian Keith ....................................................... 70
Bartosz Grzegorz ................................................. 110
Basaglia Marina ......................................49, 109, 205
Baselga Ignacio .................................................... 172
Batista Nadia N .................................................... 132
Bavouzet Jean Michel ............................................ 43
Beckett Michael ..................................................... 38
Begea Mihaela ..................................... 162, 182, 184
Bellasio Martina ..................................................... 41
Bello Cristiano ..................................................... 101
Beltran Gemma ............................................103, 104
Beney Laurent ................................................ 15, 100
Benito Santiago .............................................. 22, 116
Bennamoun Leila ......................................... 152, 207
Beopoulos Thanos................................................... 71
Berardi Enrico ...................................................... 161
Bergström Anders .................................................. 57
Berná Luisa............................................................... 6
Bernardo Ruben T .................................................. 17
Bettiga Maurizio .................................................... 42
Beukes Louisa ........................................................ 25
Biagiotti Claudia .................................................. 118
Białkowska Aneta .......................................... 91, 202
Bianchi Michele M .......................................101, 235
Biernacki Mateuzs ................................................. 70
Bigey Frédéric .......................................... 35, 55, 224
Bing Jian ................................................................ 56
Bischoff Felix ................................................... 51, 70
Bischoff Sonja ........................................................ 51
Bisson Linda .......................................................... 62
Bizzarri Melissa ........................... 178, 179, 225, 253
Blackwell Meredith ................................................63
Bleve Gianluca ..................................... 150, 151, 180
Bleykasten-Grosshans Claudine .......................... 228
Blomqvist Johanna ................................. 72, 244, 245
Boekhout Teun ....................................................... 10
Boido Eduardo ...............................................30, 130
Bojanovič Klara ................................................... 218
Boles Eckhard ................................................64, 189
Bonciani Tommaso ...................................... 178, 253
Bond Ursula ........................................................... 38
Börlin Marine ....................................................... 146
Borneman Anthony ................................................62
257
Borovikova Diana .................................................. 16
Bottagisi Samuele ................................................234
Boundy-Mills Kyria ..................................... 7, 63, 77
Boussaid Abdellatif .............................................. 128
Boyaci Gunduz C Pelin .......................................... 29
Brambilla Luca ......................................................101
Brandenburg Jule ........................................... 72, 245
Braschi Giacomo .................................................. 149
Breiner Hans-Werner ............................................. 10
Breunig Karin ....................................................... 241
Brial Pascale ......................................................... 123
Brice Claire .......................................................... 137
Brion Christian ...............................................61, 218
Brunke Sascha ...................................................... 218
Budroni Marilena ................................... 55, 175, 224
Bukhari Sayed J ..................................................... 10
Buscioni Giacomo ................................................153
Butler Geraldine ...............................................17, 66
Buzzini Pietro ..... 16, 78, 85, 152, 207, 211, 239, 255
Cabral Anderson ..................................................... 97
Čadež Neža ................................................9, 94, 166
Cadiere Axelle ........................................................ 35
Cagnin Lorenzo ...................................... 49, 109, 205
Cakar Petek .......................................................... 106
Caldeira Ilda ......................................................... 125
Callari Roberta ..................................................... 206
Calzada Javier ...................................................... 172
Camacho Rosa ..................................................... 143
Camarasa Carole ............................35, 123, 137, 229
Campos Amador ................................................... 143
Cankorur-Cetinkaya Ayca ............................112, 249
Canonico Laura ...................................... 28, 119, 120
Cantele Giovanni ................................................. 135
Capece Angela ....................................... 24, 169, 176
Capozzi Maria A .................................................. 201
Caputo Gerardo ...................................................... 47
Cardinali Federica ................................................145
Cardinali Gianluigi ................................... 89, 96, 109
Caridi Andrea ...............................................163, 164
Carrau Francisco ............................................ 30, 130
Carrillo Martina ............................................. 45, 197
Cartenì Fabrizio ................................................... 209
Carvalho Bruna T.................................................... 36
Carvalho Cláudia ................................................... 95
Carvalho de Figueiredo Bruna I ........................... 155
Casaregola Serge .................................................. 223
Casella Sergio ........................................ 49, 109, 205
Cassanelli Stefano ................................................225
Cavalieri Duccio .............................................. 6, 147
258
Cayot Philippe ................................................ 15, 100
Cecchi Teresa ....................................................... 161
Cervantes Jesús .................................................... 181
Ceugniez Alexandre ............................................... 23
Chakravarthy Praveen ............................................ 66
Chamas Alexandre ................................................. 70
Chessa Rossella .................................................... 175
Chi Zhenming ........................................................ 92
Chibana Hiroji ........................................................ 17
Chou Hsiu-Chuan .................................................. 92
Chou Yii-Cheng ..................................................... 92
Ciani Maurizio ........................28, 118, 119, 120, 175
Cifuentes Victor ................................................... 111
Cimaglia Fabio ..................................................... 180
Cinquepalmi Vito ................................................. 180
Cirvilleri Gabriella ...............................................107
Clementi Francesca .............................................. 145
Coetsee Elizabeth ......................................... 124, 141
Coi Anna Lisa ........................................ 55, 106, 224
Coimbra Luis ....................................................... 136
Colabella Claudia ..................................... 89, 96, 109
Colavizza Didier .................................................... 43
Colón-González Maritrini .................................... 214
Comitini Francesca .........................28, 118, 119, 120
Compagno Concetta ............................................. 234
Contreras-Esquivel Juan Carlos ........................... 126
Copat Chiara ........................................................ 200
Corallo Daniela .................................................... 135
Corazzi Lanfranco ................................................152
Cordes Arno ........................................................... 51
Corich Viviana ..................................................... 248
Corona Rosa.......................................................... 143
Corsetti Aldo .................................................. 26, 121
Corte Laura .............................................. 89, 96, 109
Costantini Antonella ...............................................13
Coton Emmanuel ................................................. 167
Coton Monika ...................................................... 167
Coucheney Françoise ............................................. 23
Couloux Arnaud ..................................................... 55
Coutrim Maurício Xavier...................................... 156
Cramarossa Maria R ............................................ 239
Cripwell Rosemary ................................................49
Cristaldi Antonio .................................................. 200
Crutz-Le Coq Anne M ........................................... 71
Cubillos Francisco A ..............................60, 137, 173
Cuevas Mara .......................................................... 60
Cunha Diana V ....................................................... 17
Curtin Chris .................................................... 62, 167
D’Achille Fabio ................................................... 239
259
Daisuke Watanabe .................................................. 12
Dakhmouche Scheherazad ........................... 152, 207
Daniel Heide-Marie ...............................................27
Dapporto Leonardo .................................................. 6
Daran Jean-Marc .................................................... 59
Daran-Lapujade Pascale ......................................... 59
Dashko Sofia .................................................. 66, 117
Davey Hazel Marie ................................................14
Davison Steffi A.................................................... 192
de Alteriis Elisabetta ............................................ 209
De Angelis Lorenzo ..............................................101
de Assis Ribeiro Jose R .......................................... 97
De Bari Isabella .................................................... 201
de Boer Carl G ....................................................... 60
de Cássia Franco Afonso Robson J ...................... 156
de Filippo Carlotta ...............................................147
de Garcia Virginia ............................................ 8, 111
De Graeve Marilyn .............................................. 236
Deinhard Pia.......................................................... 189
de la Rosa M Mayela ........................................... 157
De Maeseneire Sofie L ......................................... 247
de Miranda Castro Ieso ................................ 155, 156
De Vero Luciana .......................................... 178, 253
De Vuyst Luc ......................................................... 27
Dellacassa Eduardo ........................................ 30, 130
Delneri Daniela .................................................... 183
Demeke Mekonnen .............................................. 188
den Dulk Ben........................................................ 195
Den Haan Riaan ........................................... 192, 194
Dequin Sylvie ........... 35, 55, 123, 137, 185, 224, 229
Deroma Mario ...................................................... 106
Desfougeres Thomas .............................................. 43
Di Fidio Nicola .................................................... 201
Di Gianvito Paola ................................................... 26
Di Mauro Simone ..................... 78, 85, 211, 239, 255
Dias Disney R ...................................................... 193
Dicks Jo ..................................................................79
Dikicioglu Duygu.................................. 112, 230, 249
Dincă Oana-Romina ............................................. 182
Dithebe Khumisho ....................................... 124, 141
Dlauchy Dénes ....................................................... 94
Dmytruk Kostyantyn .............................................. 68
Doria Francesca ..................................................... 13
Dourou Dimitra .................................................... 160
Drab Vladimir....................................................... 252
Drider Djamel ........................................................ 23
Du Chenyu ............................................................. 50
Duarte Filomena L................................ 125, 136, 138
Dulermo Rémi ........................................................ 71
260
Dulermo Thierry..................................................... 71
Dumont Jennifer ..................................................... 14
Dupont Sébastien ........................................... 15, 100
Durante Miriana.................................................... 151
Duta Denisa .......................................................... 184
Eder Matthias........................................................ 229
Egermeier Michael ...............................................243
Elliston Adam ........................................................ 79
Emisal Selimi ....................................................... 248
Erten Huseyin ................................................. 29, 240
Escalante-García Zazil...........................143, 216, 217
Fagnano Massimo .................................................. 47
Farcasanu Ileana C ............................................... 191
Fariña Laura ......................................................... 130
Fasoli Giuseppe..................................................... 121
Fass Bulelwa........................................................... 25
Fatiha Ben Mellouk .............................................. 128
Favaro Lorenzo ...................................... 49, 109, 205
Favaron Francesco ...............................................248
Fay Justin ............................................................. 117
Febbo Eric .............................................................. 10
Felis Giovanna E .................................................. 147
Fell Jack W ............................................................. 10
Fernandes Lemos Junior Wilson J........................ 248
Fernández-Niño Miguel ....................................... 102
Ferrante Margherita ............................................. 200
Ferrara Massimo .................................................. 180
Ferreira David ...................................................... 185
Filippucci Sara ............................... 78, 211, 239, 255
Filker Sabine .......................................................... 10
Fiorentino Nunzio................................................... 47
Fleet Graham H ...................................................... 20
Florczak Tomasz .................................................. 202
Flores-Villegas Mirelle Citlali ..................... 214, 222
Florio Ciro............................................................... 47
Foligni Roberta..................................................... 145
Folly Christophe .................................................. 206
Fontes Saraiva Margarete A ................................. 155
Forti Luca ............................................................. 239
Fotedar Rashmi ...................................................... 10
Foulquié-Moreno Maria ................... 36, 58, 174, 188
Francesca Nicola .................................................... 95
Francisco-Álvarez Raquel .................................... 172
Franco-Duarte Ricardo ........................................... 55
Franzén Carl J .............................................. 108, 208
Frazão Claudio ..................................................... 190
Freel Kelle ............................................................ 218
Friedrich Anne ...............................................61, 234
Fritsch Emilie ......................................................... 43
261
Fujiyama Kazuhito ................................................. 48
Fülöp László ........................................................... 94
Gabaldon Toni ...................................................... 223
Gaglio Raimondo ................................................... 95
Galeote Virginie................................ 35, 55, 185, 224
Gallardo JCM........................................................ 210
Galli Viola ............................................................ 148
Gallo Antonia ....................................................... 180
Gamboa-Meléndez Heber....................................... 71
Ganucci Donatella ................................................153
García Verónica ...................................... 60, 173, 226
Garcia-Moruno Emilia ........................................... 13
García-Ríos Estéfani ............................................ 220
Gardner Jennie ..................................................... 113
Garofalo Cristiana ................................................145
Gasser Brigitte ....................................................... 69
Geijer Cecilia ...............................................203, 231
Gerhards Daniel ................................................... 122
Gervais Patrick ......................................... 14, 15, 100
Geurts Theo .......................................................... 195
Ghica Mihaela Violeta ......................................... 184
Ghorbal Akram Ben ...............................................29
Giacomini Alessio ................................................ 248
Giannino Francesco ............................................. 209
Gielesen Bianca ................................................... 199
Giersberg Martin..................................................... 70
Giorello Facundo ................................................... 30
Giudici Paolo ............................... 178, 179, 225, 253
Giuffrè Angelo Maria ........................................... 163
Glantschnig Ewald ................................................. 76
Gojkovic Zoran .................................................... 234
Göker Markus ........................................................ 63
Gómez-Angulo Jorge.................................... 216, 217
Goncerzewicz Anna ............................................. 134
González Alicia ...................................... 65, 214, 235
González Beatriz .................................................. 104
González David .................................................... 226
González-Flores James .......................... 65, 214, 235
González-Manjarrez Alicia .................................. 222
Goovaerts Annelies ................................................58
Goral Ceren .......................................................... 106
Gottardi Davide..................................................... 246
Gozzi Giorgia ....................................................... 246
Granchi Lisa ................................................. 153, 154
Grande Ricardo ............................................ 216, 217
Grangeteau Cédric ...............................................122
Grasso Alfina ........................................................ 200
Grazia Luigi ......................................................... 121
Grbin Paul ............................................................ 237
262
Grieco Francesco............................ 89, 135, 150, 151
Grigorică Liviu ..................................................... 162
Grigoriev Igor ........................................................ 63
Grubješić Goran ................................................... 129
Gschaedler-Mathis Anne .............. 144, 181, 216, 217
Guaragnella Nicoletta ............................................ 24
Guatemala Guadalupe .......................................... 143
Guerreiro Marco Alexandre ................................... 95
Guerrini Simona ........................................... 148, 154
Guevara Mirely..................................................... 133
Guézenec Stéphane .................................................35
Guillamón José Manuel ....................................... 220
Guilloux-Benatier Michèle .................................. 122
Guy Julie................................................................. 55
Guyot Stéphane ...................................................... 14
Guzzon Raffaele ................................................... 165
Hagler Allen N.................................................... 4, 97
Hähnel Urs.............................................................. 70
Hamberger Björn ................................................. 206
Han Pei-Jie ............................................................. 56
Harashima Satoshi ...............................................196
Haridas Sajeet ........................................................ 63
Harold Grant........................................................... 25
Hayakawa Takashi ...............................................238
Heavens Darren ...................................................... 79
Heider Harald ....................................................... 206
Henriques Silvia F ................................................190
Herbage Felipe ....................................................... 60
Hernández M Eduardo ......................................... 157
Hernández-Almanza Ayerim ................................ 126
Herrera Enrique .................................................... 181
Herrera S Teófilo .................................................. 158
Hinks Roberts Alexander J.................................... 221
Hittinger Chris ....................................................... 63
Ho Ping-Wei.................................................... 45, 198
Hoff Justin .............................................................. 25
Howell Kate S......................................................... 21
Hranilovic Ana ..................................................... 237
Huang Sing-Yi ....................................................... 92
Hube Bernhard ..................................................... 218
Hürlimann Hans C ............................................... 241
Iñiguez Laura ....................................................... 144
Ionete Roxana-Elena ............................................ 182
Iradukunda Clarisse ............................................. 128
Ishchuk Olena P.............................................. 66, 218
Islam Zia Ul .................................................... 45,197
Iwasaki Wataru ....................................................... 87
Jacques Noémie ................................................... 223
Jacques Philippe ..................................................... 23
263
Jaibangyang Sopin ................................................... 5
James Steve ............................................................ 79
Jankowska Dagmara .............................................. 70
Jansen Mickel ....................................................... 195
Jarboe Laura R ..................................................... 190
Jeffries Thomas ...................................................... 63
Jiménez S Valeria .................................................158
Jiménez-Benítez Ángel ........................................ 214
Jiranek Vladimir ........................................... 113, 237
Jõgi Eerik ............................................................. 131
Jolly Neil ................................................................ 25
Joseph Lucy ........................................................... 62
Jouffrey Cécile...................................................... 100
Kanai Keiko ........................................................... 37
Karklina Daina ..................................................... 140
Karlsson Hanna ...................................................... 72
Kasalica Anka ...................................................... 129
Kasper Lydia ........................................................ 218
Kasprzak Jakub ...................................................... 70
Katz Michael ........................................................ 234
Kavkova Miloslava ........................................ 93, 252
Kgotle Evodia ...................................................... 141
Kibilds Juris ......................................................... 242
Kirchmayr Manuel ...............................................144
Klein Mathias ......................................... 45, 197, 198
Klenk Hans-Peter ................................................... 63
Knecht Wolfgang ................................... 66, 218, 234
Kobayashi Osamu .................................................. 37
Kodedov Marie .................................................... 105
Kokina Agnese ..................................................... 242
Kolecka Anna ......................................................... 10
Korhola Matti ....................................................... 254
Koruza Katarina ................................................... 218
Košmerl Tatjana ................................................... 166
Kozdraś Agnieszka.................................................110
Kraszewska Joanna .................................................38
Krüger Klaus .......................................................... 51
Krysiak Joanna ...............................................91, 202
Kuijpers Niels GA ..................................................59
Kunze Gotthard .................................................51, 70
Kurtzman Clete ...................................................... 63
Kurylenko Olena .................................................... 68
Labagnara Tilde ................................................... 165
Labbani Fatima-Zohra K............................... 152, 207
Lachance Marc-André ............................................. 2
Lafarge Céline ................................................ 15, 100
Lage Patrícia ................................................127, 168
Laier Marcus ........................................................ 177
Laluce Cecilia .............................................. 176, 210
264
Lam Samuel STH.................................................... 21
Lanciotti Rosalba ......................................... 121, 149
Landi Carmine ..................................................... 209
Landolfo Sara ...............................................106, 175
Lappe-Oliveras Patricia ....................... 133, 157, 158
Larrondo Luis F ..................................................... 60
Laureau Raphaëlle ................................................. 57
Lazar Zbigniew ...................................................... 71
Ledesma-Amaro Rodrigo........................................ 71
Lee Ching-Fu ................................................... 90, 92
LeGall Gwenaelle .................................................. 79
Legras Jean-Luc .............................55, 146, 224, 229
Lemaire Marc ....................................................... 235
Lemos Ana ........................................................... 168
León C Concepción .............................................. 158
Leong Su-lin L ..................................................... 159
Lertwattanasakul Noppon ...................................... 48
Leventdurur Sezgi .................................................. 29
Li Yingying .......................................................... 188
Libkind Diego .................................................. 8, 111
Lidums Ivo ........................................................... 140
Liepins Janis ......................................................... 242
Limtong Savitree .................................... 5, 48, 84, 86
Lindahl Lina ........................................................... 42
Lisi Silvia ............................................................... 16
Liti Gianni....................................................... 57, 220
Litwińska Katarzyna ...............................................51
Liu Wan-Qiu .......................................................... 56
Liuzzi Federico .................................................... 201
Lizalova Marketa.................................................... 93
Lodens Sofie......................................................... 247
Lodolo Elizabeth .................................................. 124
Loeillet Sophie ....................................................... 57
Logrieco Antonio Francesco ................................ 151
Lopes Brandão Rogelio ................................ 155, 156
Lopes Mariana ....................................................... 63
López Geovani ....................................................... 65
Lopičić-Vasić Tijana ............................................ 129
Los Alrik............................................................... 195
Louis Edward J ............................................ 183, 221
Lukjanenko Jekaterina ......................................... 242
Luttik Marijke AH ..................................................59
Luzhetskyi Taras .................................................. 244
Lyzak Oleksiy ........................................................ 68
Macedo Araújo Thalita ................................ 155, 156
Maguire Sarah ........................................................ 66
Manabe Ri-ichiroh ................................................. 87
Manara Rich ......................................................... 108
Mandl Neža .......................................................... 166
265
Mannazzu Ilaria ........................................... 106, 175
Mapelli Valeria ..................................................... 108
Marcet-Houben Marina ........................................ 223
Mari Eleonora ...................................................... 154
Marinescu Simona-I .............................162, 182, 184
Markova Jaroslava ................................................. 93
Marques Patrícia .................................................. 138
Marquina Maribel ................................................ 102
Marsit Souhir ......................................................... 55
Martin Valentina ..................................................... 30
Martínez Claudio............................ 60, 137, 173, 226
Martins Luísa L..................................................... 139
Martí-Raga Maria ................................................. 103
Martynenko Nikolay N ........................................ 219
Marullo Philippe .................................................. 103
Marx Christian ....................................................... 42
Marx Hans ...................................................... 41, 243
Mas Albert .............................................. 30, 103, 104
Masiero Maria O C .............................................. 176
Maslowska Agnieszka .................................. 183, 221
Masneuf- Pomarede Isabelle .................. 62, 146, 167
Mastrolitti Silvio .................................................. 201
Mattanovich Diethard .............................. 41, 69, 243
Mattarelli Paola .................................................... 147
Maura Yurelkys Fernandez .................................... 27
Mazzoleni Stefano ...............................................209
McDowall Mark D ............................................... 230
Medina Karina ..................................................... 130
Meier-Kolthoff Jan P ...............................................63
Meijrink Ben ........................................................ 199
Mendes-Faia Arlete ...................................... 127, 168
Mendes-Ferreira Ana ................................... 127, 168
Mendonça-Hagler Leda Cristina ........................4, 97
Meraihi Zahia ...............................................152, 207
Mervič Mateja .......................................................... 9
Metsla Kati ........................................................... 131
Meyer Wieland ....................................................... 96
Mezzetti Francesco .............................................. 253
Mignat Cora ......................................................... 189
Mihai Constanţa ................................................... 162
Mihai Doru ........................................................... 162
Mikkelsen Michael Dalgaard ............................... 206
Milanović Vesna ................................................... 145
Mills David A ......................................................... 31
Milović Boris ....................................................... 129
Mira Nuno P ........................................... 17, 127, 190
Misiewicz Anna ................................................... 134
Mita Giovanni ...................................... 135, 150, 151
Modesto Monica .................................................. 147
266
Mokhtarnejad Lachin ............................................. 85
Moliné Martín .................................................. 8, 111
Molnárová Jana ...................................................... 88
Montalvo-Arredondo Javier ................................. 214
Montañez-Sáenz Julio .......................................... 126
Morel Guillaume .................................................. 223
Moreno Antonio D ....................................... 203, 231
Moreno-Terrazas Rubén ....................... 133, 157, 158
Morrissey John ....................................................... 64
Moschetti Giancarlo ...............................................95
Moudi Koullè Esther Lauree................................. 246
Mourato Miguel P ................................................139
Mozzon Massimo ................................................. 145
Mudura Radu ....................................................... 162
Müller Ulrike ....................................................... 199
Munzel Lena ........................................................ 241
Nakagawa Tomoyuki ........................................... 238
Nasanit Rujikan................................................... 5, 86
Naseeb Samina...................................................... 183
Naumov Gennadi I ..................................... 7, 90, 219
Naumova Elena S ....................................... 7, 90, 219
Neagoe Aurora D ................................................. 191
Neuvéglise Cécile .......................................... 71, 228
Nevoigt Elke .................................. 45, 102, 197, 198
Niba Aziwo T ....................................................... 159
Nicaud Jean M ....................................................... 71
Nicolas Alain .......................................................... 57
Nicolau Ioana ....................................................... 191
Nidelet Thibault ........................................... 123, 229
Nishimura Hisami .................................................. 37
Nisiotou Aspasia .......................................... 160, 227
Noti Olta ................................................................. 13
Nouadri Tahar ...................................................... 207
Novy Vera ............................................................ 208
Nyman Jonas ........................................................ 108
Oda Saori ............................................................... 73
Odorizzi Silvana ................................................... 248
Ohashi Takao .......................................................... 48
Ohkum Moriya ....................................................... 87
Ohyama Akira ........................................................ 87
Oliver Stephen G .......................... 112, 113, 230, 249
Oliveri Conti Gea ................................................. 200
Olsson Joakim ...................................................... 204
Olsson Lisbeth ............................... 42, 203, 208, 231
Olstorpe Matilda .................................................. 159
Oreb Mislav ......................................................... 189
Oro Lucia ...............................................28, 119, 120
Ortega-Granillo Augusto ...................................... 222
Ortiz Raul ............................................................... 64
267
Ortiz-Julien Anne ................................................. 185
Osimani Andrea ................................................... 145
Ota Taku ................................................................. 37
Ottaviano Daniela ................................................235
Pais Thiago ............................................................. 58
Pajarola Ana Lucia ............................................... 111
Papović-Vranješ Anka .......................................... 129
Parachin Nádia S .................................................... 46
Parafati Lucia ....................................................... 107
Parascandola Palma ............................................. 209
Parpinello Giuseppina P ....................................... 149
Parts Leopold ....................................................... 220
Passoth Volkmar ..................................... 72, 244, 245
Patrick de Souza Pimenta Paloma ........................ 155
Patrignani Francesca .................................... 121, 149
Patz Claus-Dieter ................................................. 177
Pedraza Lorena ............................................. 133, 157
Pedrosa Fernando ................................................. 138
Pelayo Carlos ....................................................... 143
Pellicanò Teresa ................................................... 163
Peltier Emilien ..................................................... 167
Perez Lago Estela ................................................. 172
Perpetuini Giorgia .......................................... 26, 121
Perrone Giancarlo ................................................180
Perrotta Carla ......................................................... 89
Péter Gábor ........................................................ 9, 94
Petrovic Uros ....................................................... 117
Pfeffer Martin ......................................................... 69
Pflieger David ........................................................ 61
Pickova Jana ........................................................... 72
Pietrangeli Biancamaria.......................................... 47
Pignede Georges .................................................... 43
Pinheiro Ana CM ................................................. 132.
Pirozzi Domenico ................................................... 47
Piskur Jure .............................. 66, 117, 218, 222, 234
Pohl Carolina ....................................... 124, 141, 142
Poiana Marco ....................................................... 163
Polakova Silvia ...................................................... 66
Polburee Pirapan .................................................... 48
Poldermans Rolf ................................................... 195
Polsinelli Mario ........................................................ 6
Powell Chris ......................................................... 183
Pretorius Isak S ...................................................... 34
Pronk Jack T ........................................................... 59
Qian Michael ........................................................ 172
Qvirist Linnea ...................................................... 147
Ramires Francesca Anna....................................... 151
Ramos Cíntia L .................................................... 132
Rampino Patrizia .................................................... 89
268
Rapoport Alexander ...............................................16
Raquel Moita Maria ............................................. 190
Rauhut Doris ........................................................ 177
Rauter Marion ........................................................ 70
Regenberg Birgitte ...............................................218
Restuccia Cristina ........................................ 107, 200
Reverberi Massimo ..............................................101
Ribeiro Disney D .................................................132
Riechen Jan ............................................................ 70
Riego-Ruiz Lina ................................................... 214
Rigou Peggy ......................................................... 229
Riley Robert ........................................................... 63
Rinaldi Teresa ...................................................... 101
Ripari Valery ........................................................ 161
Robert Vincent ....................................................... 96
Roberti Rita .......................................................... 152
Roberts Ian ............................................................. 79
Robles Edson ....................................................... 222
Roceanu Gabriel ................................................... 162
Rodrigues Nicole M ............................................. 190
Rodríguez-Porrata Boris ...................................... 102
Rodríguez-Tarduchy Gemma ............................... 172
Roelants Sophie ................................................... 247
Rokas Antonis ........................................................ 63
Romaniello Rossana ............................................. 169
Romano Patrizia ..................................... 24, 169, 176
Romo Rebeca ....................................................... 133
Rosa Carlos A.......................................................... 63
Roscini Luca ............................................ 89, 96, 109
Rose Shaunita H ............................................. 49, 205
Rossignol Tristan ................................................... 71
Roubal Petr ........................................................... 252
Roubos Hans ........................................................ 199
Rousseaux Sandrine ............................................. 122
Rowswell Dudley.................................................... 25
Rozenfelde Linda ................................................... 16
Ruchala Justyna ..................................................... 68
Russmayer Hannes ...............................................243
Ruta Lavinia L ..................................................... 191
Rutherford Kim M ............................................... 230
Rydengård Victoria ................................................66
Saaiman Susanna ................................................. 142
Sá-Correia Isabel ............................................ 17, 190
Sadowska-Bartosz Izabela ................................... 110
Sakai Yasuyoshi ..................................................... 73
Salamov Asaf ......................................................... 63
Salin Franck ................................................. 146, 167
Salinas Francisco ............................................. 57, 60
Säll Torbjörn ........................................................ 218
269
Sampaio José Paulo .............................. 54, 55, 80, 95
Sánchez Allan ....................................................... 181
Sanchez Isabelle ........................................... 123, 229
Sánchez M Juan M ............................................... 157
Sanchez-Flores Alejandro ............................216, 217
Sandgren Mats ............................................. 244, 245
Sannino Ciro .................................. 78, 211, 239, 255
Sannino Filomena .................................................. 47
Santomartino Rosa .......................................101, 235
Santos Cruz .......................................................... 172
Sarilar Véronique ................................................. 228
Sarli Margherita ................................................... 169
Sasano Yu ....................................................... 73, 196
Sauer Michael ................................................41, 243
Scalabrelli Giancarlo ............................................ 165
Schacherer Joseph .................................. 61, 218, 234
Scheuner Carmen ................................................... 63
Schirone Maria............................................... 26, 121
Schmidtchen Artur ................................................. 66
Schuller Dorit ......................................................... 55
Schwan Rosane F ......................................... 132, 193
Scovacricchi Eugenio ........................................... 173
Scully Damhan ....................................................... 64
Settanni Luca ......................................................... 95
Shalamitskiy Maxim Yu ....................................... 219
Sibirny Andrei A............................................. 68, 244
Sicari Vincenzo .................................................... 163
Sidari Rossana ...................................................... 163
Silva Campos Anna C................................... 155, 156
Silva Sónia ............................................................. 17
Siroli Lorenzo ...................................................... 149
Sirri Ambe C ........................................................ 159
Soares Géssyca P A .............................................. 193
Soares Marco ......................................................... 63
Soetaert Wim ................................................236, 247
Solieri Lisa ................................... 178, 179, 225, 253
Solis-Escalante Daniel ........................................... 59
Søndergaard Leif .................................................... 66
Souciet Jean-Luc .................................................. 223
Souffriau Ben ................................................. 36, 174
Soulard Alexandre ................................................235
Souza Karla S T ................................................... 193
Spano Giuseppe ................................................... 135
Spyropoulos Apostolos ........................................ 160
Srisuk Nantana ....................................................... 84
Stefanini Irene .......................................................... 6
Sterck Lieven ....................................................... 223
Stielow Benjamin ................................................... 63
Stoeck Thorsten ..................................................... 10
270
Stojiljkovic Marija ...............................................174
Strati Francesco .................................................... 147
Strauss Joseph ........................................................ 65
Suchanova Eva ..................................................... 252
Sugita Takashi ........................................................ 87
Sugiyama Minetaka ............................................. 196
Sundstrom Joanna ................................................ 113
Surussawadee Janjira ............................................. 84
Susca Antonia ....................................................... 180
Suzzi Giovanna .............................................. 26, 121
Swamy Krishna BS .............................................. 234
Swart Chantel ....................................... 124, 141, 142
Swart Hendrik .............................................. 124, 141
Swennen Dominique ............................................ 223
Swinnen Steve ..........................45, 58, 102, 197, 198
Sychrová Hana ..................................................... 105
Synnott John .......................................................... 66
Szulczewska Katarzyna M .............................91, 202
Taccari Manuela ................................................... 145
Tadlaoui Ouafi Ahmed ......................................... 128
Taj-Aldeen Saad J................................................... 10
Takagi Hiroshi ........................................................ 12
Takashima Masako ................................................. 87
Takeki Shiga ........................................................... 12
Tamaian Radu ...................................... 162, 182, 184
Tanguler Hasan .................................................... 240
Tantirungkij Manee ............................................ 5, 86
Teixeira Andreia ................................................... 125
Tek Ee Lin ............................................................ 113
Teodorescu Răzvan .............................................. 162
Tharappel James C .................................................38
Thery Thibaut ......................................................... 38
Thevelein Johan M ............................ 36, 58,174, 188
Thomik Thomas ................................................... 189
Thonart Philippe ................................................... 207
Tiecco Matteo ................................................96, 109
Tilloy Valentin ........................................................ 35
Tinsley Colin R .................................................... 223
Tiukova Ievgeniia ................................................244
Tofalo Rosanna .............................................. 26, 121
Toffanin Annita .................................................... 165
Tommasi Luca ...................................................... 151
Tondini Federico .................................................. 237
Torija Maria Jesús ................................................ 104
Toscano Giuseppe .................................................. 47
Tosetto Niccolò .................................................... 204
Trautwein Anke ...................................................... 70
Tripp Joanna ......................................................... 189
Tristezza Mariana ................................... 89, 135, 150
271
Tucker Gregory ...................................................... 50
Tufariello Maria ................................... 135, 150, 151
Turchetti Benedetta ... 78, 85,152, 207, 211, 239, 255
Turillazzi Stefano ..................................................... 6
Turkiewicz Marianna ..................................... 91, 202
Vadkertiová Renáta ................................................88
Valentin Ionescu ................................................... 184
Van Bogaert Inge .......................................... 236, 247
Van de Peer Yves .................................................. 223
van der Ploeg Ralph ...............................................64
van Maris Antonius JA ........................................... 44
van Wyk Pieter ..................................... 124, 141, 142
Van Zyl John Henry D ......................................... 194
Van Zyl Willem H .......................... 49, 192, 194, 205
Vannini Lucia ....................................................... 246
Varela Javier ........................................................... 64
Vasconcelos Isabel ...............................................127
Vaudano Enrico ...................................................... 13
Vázquez B Tania .................................................. 158
Vázquez Jennifer .................................................. 104
Vega-Alvarado Leticia ................................. 216, 217
Veide Vilg Jenny .................................................. 204
Venturi Manuel ..................................................... 148
Verspohl Alexandra .............................. 179, 225, 253
Verwaal Rene ....................................................... 195
Vetrano Cosimo .................................................... 150
Vigants Armands .................................................. 242
Viigand Katrin .............................................. 131, 215
Vija Heiki ............................................................. 131
Viktor Marko J ....................................................... 49
Vila Santa Ana ...................................................... 190
Viljoen-Bloom Marinda ......................................... 49
Vincenzini Massimo .............................148, 153, 154
Vinga Susana ........................................................ 190
Visca Andrea ........................................................ 235
Visnapuu Triinu ............................................ 131, 215
von Wallbrunn Christian ...................................... 122
Votta Sonia ............................................................. 24
Vu Duong ............................................................... 96
Wakayama Keishi ................................................238
Waldron Keith ........................................................ 79
Wang Can ......................................................... 17, 66
Wang Qi-Ming ....................................................... 56
Wang Ruifei ......................................................... 208
Westman Johan O ........................................ 108, 208
Wincker Patrick .................................................... 223
Winckler Pascale .................................................... 14
Wisecaver Jennifer H ............................................. 63
Wisén Sofia Mebrahtu ............................................ 66
272
Wisniewski Michael ............................................. 107
Wolfe Kenneth ................................................. 63, 64
Wood Valerie ........................................................ 230
Worch Sebastian ...............................................51, 70
Wu Junyuan .......................................................... 196
Wu Liang .............................................................. 199
Yongmanitchai Wichien ......................................... 48
Yoshida Satoshi ...................................................... 37
Yoshimoto Hiroyuki ...............................................37
Young Michael ....................................................... 14
Yurimoto Hiroya .................................................... 73
Yurkiv Mariana ...................................................... 68
Yurkov Andrey ....................................................... 80
Zahrl Richard ......................................................... 69
Zaky Abdelrahman S .............................................. 50
Zambuto Marianna ................................................. 24
Zappia Clotilde ..................................................... 163
Zara Giacomo ....................................................... 224
Zara Severino ....................................................... 175
Zeppel Ryan ........................................................... 62
Zeyara Aisha .......................................................... 10
Zhao Zheng .................................................. 195, 199
Zhenyu Zhai ........................................................... 73
Zhou Nerve .......................................................... 234
Zitti Silvia ............................................................ 145
Zuccaro Gaetano .................................................... 47
273
Acknowledgements
Editors would like to thank the staff of the Industrial Yeasts Collection DBVPG of the Dipartimento di
Scienze Agrarie, Alimentari e Ambientali, University of Perugia: Simone Di Mauro, Sara Filippucci
and Ciro Sannino, for their remarkable support in editing this Book of Abstracts.
A special thank also goes to the talented staff of the Agency Consulta Umbria, Perugia, Italy: Simona
Sarti, Giuseppina Meniconi, Laura Scarfò and Daniela Brunori, for their logistic and administrative
support in organizing ISSY32
274
This book of abstracts was designed, assembled and realized for the
32nd INTERNATIONAL SPECIALIZED SYMPOSIUM ON YEASTS
Perugia (Italy), 13-17 September 2015
by the staff of the Industrial Yeasts Collection DBVPG
www.dbvpg.unipg.it
Department of Agricultural, Food and Environmental Science
University of Perugia, Italy
ISBN 978-88-99407-00-1

Book of Abstracts

Transcription

Similar documents

Modelling respiro-fermentative dynamics of Saccharomyces

FLEISCHMANN`S YEAST IS ON THE RISE

Points to Remember

Red Yeast Rice - Biotics Research Corporation

GIGARO

Coopers Original Pale Ale

Rye Whiskey - White Labs

Garden Wine Making - Organic Growers School

Recommended by Autism Specialists to Address