Using Streptomyces Bacteria to Produce Renewable

Transcription

Towards Sustainable Living: Using Streptomyces Bacteria to Produce Renewable Energy and Commodity Chemicals from Plant Biomass Prof. Jason K. Sello Department of Chemistry Brown U niversity [email protected] Sources of Renewable Energy
WIND SOLAR BIOMASS HYDRO GEOTHERMAL Increasing World Biofuels Production
•  15.9 billion gallons of
biofuels were produced
domestically in 2010
–  13.2 billion gallons of ethanol
–  2.7 billion gallons of biodiesel
•  138.6 billion gallons of
gasoline was consumed in
the US during 2010
BP Statistical Review of Energy June 2011. bp.com/statisticalreview
Biotechnology for Conversion of Plant Biomass to
Biofuels
Rubin E. Genomics of cellulosic biofuels. Nature 454: 841-845, 2008.
Energy Crops (switch grass) Agricultural Residue Plant Biomass Feedstocks Organic Trash Forestry Waste Biotechnology for Conversion of Plant Biomass to
Biofuels
Structural Components of Plant Biomass
Lignin
Hemicellulose
Cellulose
Lignin
Hemicellulose
Cellulose
Using Microorganisms for Biofuel Production
Fermentation of yeast on
plant sugars is currently
used to produce
bioethanol
Engineered bacteria are
being developed for the
production of biodiesel by
fermentation of plant
sugars (Steen, Nature,
2010)
Image by Marcin Zemla and Manfred Auer, JBEI. http://newscenter.lbl.gov
Synthetic Biology in Production of Biofuels
Keasling and co-workers have engineered E. coli to convert
hemicellulose into biofuels.
Steen. Nature 463, 559-564, 2010.
Lignin
Hemicellulose
Cellulose
Lignin Component of Plant Biomass
•  Lignin constitute up to
30% of plant biomass
•  Highly stable and
heterogeneous polymer
consisting of aromatic
building blocks
•  Lignin interferes with
utilization of cellulose
for the production of
biofuels
•  Lignin can be
enzymatically
depolymerized by some
bacteria and fungi
Bugg TD, Ahmad M, Hardiman EM & R Singh. Current Opinion in Biotechnology. 22:394–400, 2011.
Phanerochaete chrysosporium diark.org P. chryosporium (white rot fungus) can consume lignin. Lignin Depolymerization
What is the fate of depolymerized lignin?
Catabolism of Depolymerized Lignin (e.g., Sphingomonas)
Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71(1) 1-15, 2007.
Catabolism of Depolymerized Lignin (e.g., Sphingomonas)
Commodity Chemicals from TCA Cycle
Triglycerides
K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, 2010
Biodiesel O
R
O
Alkyl ester R is methyl, ethyl, or propyl. Conversion of Triglycerides into Biodiesel 01.-‐ 0.5% Sodium or Potassium Hydroxide Or Sodium Methoxide O
O
O
O
+ 3 MeOH
O
O
Triglyceride (Triacylglycerols) Methanol Me
80° Celsius OH
O
O
Biodiesel (FaLy Acid Methyl Ester) Chemical reacVon is a “trans-‐esterificaVon”. +
OH
OH
Glycerin (Glycerol) Bioconversion of Lignin to Biofuels
Lignin AromaPc Compounds Acetyl-‐CoA Succinyl-‐CoA Triacyglycerols And FaLy Acids An organism that can convert all the
components of plant biomass into biofuels
would be an efficient “biorefinery”.
Complete Conversion of Lignocellulose to
Biofuels
Cellulose Lignin Hemicellulose AromaPc Compounds Acetyl-‐CoA Succinyl-‐CoA Triacyglycerols And FaLy Acids Prospecting for Plant Biomass Degraders
“An antibiotic is a chemical substance produced by microbes that inhibits the growth of or even destroys other microbes” Selman Waksman (1888-1973)
Timeline of Antibiotic Discovery
Antibiotics in use as Anti-Bacterial Agents
Antibiotics in use as Anti-Tumor Agents
Antibiotics in Use as Immunosuppresants
Diverse Morphologies and Colors of
Streptomyces Species
Image courtesy of T. Kieser
Two Evolutionary Oddities
Streptomycetes Duckbill platypus Streptomyces: An Unconventional Genus of Bacteria
Multi-cellular
Hyphal morphology and mode of growth like fungi
Complex life cycle
Linear chromosomes and plasmids
>8 Mb chromosomes are common
Ubiquitous in terrestrial environments, easily cultured
More than 500 species described
Non-pathogenic relative of Mycobacterium tuberculosis
Prodigious producers of antibiotics
The Majority of Antibiotics are Produced
by Streptomycetes
Waksman screened soil samples in search of microorganisms that produce an8bio8cs. How can we iden2fy microorganisms that degrade plant biomass? IdenVficaVon of LigninolyVc Streptomyces Strains S. lividans S. griseus S. coelicolor S. avermi8lis S. natalensis S. cha<anoogensis S. setonii S. badius S. viridosporus LigininolyPc Streptomyces species can decolorize the aromaPc dye, Azure B. Streptomyces viridosporus
S. viridosporus is a bona fide ligninolyVc streptomycete. It also is capable of consuming cellulose and hemicellulose. D.L. Crawford, Appl. Environ. Microbiol, 53: 2754-‐2760, 1987 D.L. Crawford, Appl. Environ. Microbiol, 41: 442-‐448, 1981 R L. Crawford, Appl. Environ. Microbiol, 45: 898-‐904, 1983
Metagenomic-based Enzyme Discovery in
Lignocellulolytic Microbial Communities
DeAngelis, A. Bioengineering Research, 3, 146-158 (2010)
Richness (Number of Taxa
Biodiversity in Tropical Forest Soil from Puerto Rico
DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)
Biodiversity in Tropical Forest Soil from Puerto Rico
DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)
Biodiversity in Lignin-Enriched Compost
Compost Compost + Alkali Lignin DeAngelis,A. Bioeng. Res., 3, 146-158 (2010)
Biodiversity in Lignin-Enriched Compost
Compost Compost + Alkali Lignin Actinobacteria are Populous Soil Bacteria
Mahidul University-‐ Osaka University -  Large group of terrestrial bacteria with high G+C content
genomes (e.g., Streptomyces, Corynebacteria, Nocardia,
Actinoplanes, and Mycobacteria).
- Many are filamentous like fungi
- Play a critical role in the decomposition of organic matter in soil
- Important organisms in biotechnology source of enzymes
and medicinal antibiotics
Actinobacteria Produce Two-Thirds of the
23,000 Known Antibiotics
Streptomyces derived compounds in red boxes
Sir David A. Hopwood
Overview of Research in the Sello Group
O
N
O
O
NH O
O
O
N
N
O
N
H
HN
O
Chemical Synthesis
and Drug Discovery
Renewable Energy
Streptomyces Bacteria
HO
NH2
O
HO
HO
HO
O
O
O
Chemical Ecology
O
O
NH2
N
OH
NH3
OH
O
H
N
H
N
OH
OH
trpRS1
v
cmlR
Antibacterial Drug
Resistance
O
Biosynthesis and
Metabolomics
NH
Overview of Research in the Sello Group
O
N
O
O
O
NH O
O
N
N
O
N
H
HN
O
Chemical Synthesis
and Drug Discovery
Okandeji, JOC, 2008
Okandeji, JOC, 2009
Socha, BMC, 2010
Okandeji, BMC, 2011
Carney, JOC, 2012
Compton, ACS Chem. Biol. 2013
Nelson, mBio. 2013
Carney, JACS, 2014
trpRS1
v
cmlR
Renewable Energy
Streptomyces Bacteria
HO
H
N
H
N
Vecchione, J. Bacteriol., 2008
Vecchione, AAC, 2009
Vecchione, AAC, 2009
Vecchione, J. Bacteriol., 2010
NH3
HO
HO
O
O
O
NH2
O
HO
N
OH
Antibacterial Drug
Resistance
Socha, Energy & Fuels, 2010
Socha, OBC, 2010
Davis, AMB, 2010
Davis, J. Bacteriol., 2012
Davis, NAR, 2013
Davis, Genome Ann. 2013
O
OH
OH
NH2
OH
O
NH
O
O
Biosynthesis and
Metabolomics
Chemical Ecology
Sello, J. Bacteriol., 2008
Badu-Nkansah, FEMS Lett., 2010
Totaro, ChemBioChem, 2012
Davis, Org. Lett., 2009
Morin, Org. Lett., 2010
Morin, OBC, 2012
Actinobacteria are Potential “Lignocellulose
Biorefineries”
•  Gram-positive soil-dwelling
bacteria
•  Degrade all components of
plant biomass
–  Cellulose
–  Hemicellulose
–  Lignin
•  Naturally accumulate
triacylglycerols, the precursors
of biodiesel, and make
commodity chemicals
•  Long history in industrial-scale
fermentation for the production
of antibiotics
E. Wellington
Plant Biomass-Degrading Actinobacteria
Amycolatopsis setonii
Streptomyces viridosporus
A. setonii and S. viridosporus are bona fide ligninolytic bacteria.
They also consume cellulose and hemicellulose.
D.L. Crawford, Appl. Environ. Microbiol, 53: 2754-2760, 1987
D.L. Crawford, Appl. Environ. Microbiol, 41: 442-448, 1981
R L. Crawford, Appl. Environ. Microbiol, 45: 898-904, 1983
The first bacterial lignin peroxidase was
isolated from Streptomyces viridosporus
Ramachandran et al. Appl. Environ. Microbiol. 53(12): 2754-2760, 1987.
Lignin Depolymerization
Genomics Approaches in Bioenergy Technology
In collaboration with the Joint Genome Institute (JGI), the
genomes of A. setonii and S. viridosporus has been sequenced.
http://www.jgi.doe.gov/education/bioenergy/
2012
2013
Global Genome Comparisons of Four Actinomycetes
A. setonii S. viridosporus S. coelicolor A3(2) A. mediterranei U32 8,442,518 8,292,505 9,054,847 10,236,715 % GC 71.9 72.5 72.0 71.3 Total Genes 8,328 7,648 8,325 9,292 Protein Coding Genes 8,264 7,553 8,210 9,228 Proteins with Predicted FuncVons 6,446 5,653 5,226 6,431 Predicted Secreted Enzymes 1,750 1,618 1,949 3,019 Genome Size Data are from JGI (DOE JOINT GENOME INSTITUTE)
https://img.jgi.doe.gov
Numbers of Genes in Certain COG Functional Categories
A. setonii DescripPon S. viridosporus Gene # % of Genome Gene # % of Genome Amino Acid Transport and Metabolism 539 8.4 452 8.5 Carbohydrate Transport and Metabolism 587 9.2 503 9.4 Coenzyme Transport and Metabolism 303 4.7 238 4.5 Energy ProducVon and Conversion 584 9.1 340 6.4 Lipid Metabolism 448 6.9 310 5.82 Secondary Metabolism 397 6.2 288 5.4 Signal TransducVon 1018 15.86 689 12.93 PosdranslaVonal ModificaVon, Protein turnover, chaperones 149 2.32 169 3.17 Global Genome Comparisons of Four Actinomycetes
Number of Genes with (or without) a homolog in: Comparison Organism A. setonii S. viridosporus S. coelicolor A3(2) A. mediterranei U32 -‐ (3,730) (2,300) (3,545) (3,522) -‐ (3,441) (1,719) -‐ 4,534 5,964 4,719 4,030 1,618 1,949 3,019 Comparisons for Unique Genes A. setonii S. viridosprous Comparisons for Common genes A. setonii S. viridosporus Number of genes without a homolog in the organism being compared are indicated in parenthesis.
Predicted Carbohydrate Degrading Genes in A. setonii and
S. viridosporus
A. setonii S. viridosporus Gene # Gene # Glyco_hydro 36 71 Carbohydrate Binding Module 1 18 Polysacc_deac 5 9 α-‐amylase 9 15 Pectate Lyase 0 3 51 116 Pfam DescripPon Total # Predicted Lignin Degrading Genes in A. setonii and S. viridosporus
A. setonii S. viridosporus Gene # Gene # An_Peroxidase 2 1 Catalase 1 4 CMD* 5 6 Cu-‐oxidase 3 2 Dyp_perox 3 1 GSHPx 1 1 Mn_catalase 2 1 peroxidase 1 1 18 17 Pfam DescripPon Total # *(CMD) Carboxymuconolactone decarboxylase
Both species have a comparable number of genes
encoding enzymes with potential activity against lignin.
Pathways for Catabolism of Depolymerized Lignin in
Sphingomonas
Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71(1) 1-‐15, 2007. Homologs of Sphingomonas Lignin Catabolism Pathway
Genes in Amycolatopsis setonii
PCA 3,4- cleavage pathway!
Homologs of Sphingomonas Lignin Catabolism Pathway
Genes in Streptomyces viridosporus
PCA 3,4- cleavage pathway!
Masai E, Katayama Y, Fukuda M. Biosci. Biotechnol. Biochem., 71, 1-15 (2007)
Biofuels
Cellulose Lignin Hemicellulose AromaPc Compounds Acetyl-‐CoA Succinyl-‐CoA Triacyglycerols And FaLy Acids Streptomyces viridosporus as a Model for Catabolism
of Lignin-Derived Aromatic Compounds
Catabolism of a Lignin-Derived Aromatic Compound
via the β-Ketoadipate Pathway in S. viridosporus
O
S-CoA
HOOC
OH
PcaHG
OH
Protocatechuate
HOOC
COOH
COOH
β-hydroxy
muconate
PcaB
COOH
O
HOOC
PcaL
O
β-hydroxy
muconolactone
COOH
O
PcaL
O
β-ketoadipate
enol-lactone
PcaI, J, and F
O
acetyl coenzme A
COOH
COOH
O
HO
β-ketoadipate
S-CoA
O
Succinyl coenzyme A
pcaL
pcaB
pcaG
pcaH
pcaF
pcaJ
pcaI
β-ketoadipate enol-lactone hydrolase/decarboxylase
β-carboxymuconate cycloisomerase
protocatechuate 3,4 dioxygenase, α-subunit
protocatechuate 3,4 dioxygenase, β-subunit
β-ketoadipyl CoA thiolase
β-ketoadipate succinyl-CoA transferase, β-subunit
β-ketoadipate succinyl-CoA transferase, α-subunit
Commodity Chemicals from TCA Cycle
Triglycerides
K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, 2010
Lignin Derived Aromatics to Commodity
Chemicals
O
ACC
Carboxylase
CoA-S
Tet
O
CoA-S
HO
OH N
H
OH
O
NH 2
OH
OH
Malonyl CoA
Acetyl-CoA
H
OH
OH O
O
O
Tetracycline
O
O
HO
MMA
Mutase
S-CoA
-O
O
O
MMA
Epimerase
S-CoA
-O
O
DEBS
O
OH
HO
OH
S-CoA
O
Succinyl-CoA
(S)-methyl
Malonyl CoA
(R)-methyl
Malonyl CoA
HO
O
O
O
O
O
NMe2
O
OMe
OH
Biofuels
Cellulose Lignin Hemicellulose AromaPc Compounds Acetyl-‐CoA Succinyl-‐CoA Triacyglycerols And FaLy Acids

Using Streptomyces Bacteria to Produce Renewable

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