Enzyme Engineering: a key technology for innovation in

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Enzyme Engineering: a key technology for innovation in
Enzyme Engineering: a key technology
for innovation in bio-economy
CLIB International Conference 2013
April 18, 2013
Dr. Mathias Bell
The bio-based economy
Increasing demand for products made from renewable raw materials
by biotechnological production processes
Technology Push
Economic Value
Market Pull
Industrial biotechnology
resources / cost
sustainability
Enzyme uses in consumer-near applications
Enzymes have found their way into processes and
products in several application areas
Bioprocessed
chemicals
- Renewable chemicals /
biopolymers
- Food & Feed Additives
Industrial
enzymes
- Food enzymes
- Feed enzymes
- Small molecule APIs
- Enzymes for cleaning and
washing
- Other fine & specialty
chemicals
- Other industrial
applications
Increasing value by enzyme engineering
Adaptation of catalysts to cost-efficient reaction profiles
Directed evolution of de novo enzyme activities for addressing cheap
and sustainable raw materials or non-natural molecules
Developing new products by artificial enzymes with previously nonexisting catalytic properties, replacing costly product alternatives
c-LEcta as enzyme specialist contributes to innovative, efficient and
market-oriented opportunities of its large industry partners through a
broad technology platform and expertise in commercial, IP and regulatory
aspects at all project stages
Company overview
c-LEcta is a technology company in the field of White / Industrial
Biotechnology located in Leipzig, Germany.
We are specialized on the development and implementation of
biotechnological solutions for industrial applications based on
customized Enzymes and microbial Strains.
Processes for the production of bioprocessed chemicals and
industrial enzymes developed by c-LEcta are implemented at
industry partners and licensees.
The company’s proprietary technologies and products are
protected by currently about 40 patents and patent applications.
Under an ISO9001-certified quality management system c-LEcta
runs a pilot scale Enzyme production and scale-up facility and
cooperates with contract manufacturers for industrial scale enzyme
production.
Company overview
c-LEcta is a spin-off from University of Leipzig and
currently employs 50 highly qualified employees
We recently moved into a new, state-of-the-art
research and production facilities with 2,300 m2
(BioCube) in order to set the basis for a further
dynamic company growth.
c-LEcta was founded about 8 years ago and could establish itself as a technology
partner for the industry in the market place. We successfully cooperate with a number
of well known companies like BASF, DSM, Uhde ThyssenKrupp or Evonik.
Technology platform
Biodiversity
New enzymes and
microbial strains from
natural sources
Protein Engineering
Optimized enzymes
obtained by directed
evolution
Strain Development
Bioprocess Development
Efficient microbial
productions strains
obtained by strain
engineering
Production processes for
enzymes and chemicals
based on microbial
fermentation &
biotransformation
Innovation and economic value of a process are achieved
on every technological level
Biodiversity – Source for new enzymes
Microorganisms
>7.000 strains from various habitats
(prequalified actinomycetes, bacteria
from tree canopy, marine bacteria)
Genomic
Libraries
Genomic DNA of individual high
potential strains
Microbial
Libraries
Genomic DNA of approx. 80-100
pooled strains
Metagenomic
Libraries
Pooled genomic DNA of
environmental probes
Recombinant expression libraries (overall > 55 Mio. clones)
High-Throughput Cluster-Screening
 Highly efficient identification of new enzymes in activity-based screens
Biodiversity – Source for new enzymes
Identity to database enzymes
Microbial
Libraries
40
35
occurence (%)
35
30
25
Cluster
Screening
Genomic
Libraries
20
21
15
16
10
12
5
0
Genomic DNA of individual high
potential strains
Metagenomic
Libraries
Cluster
Screening
0
2
2
7
5
0
c-LEcta’s biodiversity collection
provides a reliable source for new
and protectable enzyme entities
Engineering - Optimized enzymes for non-natural processes
* patented / patent pending
c-LEctaMutagenesis*
Gene
mutagenesis
Enzyme variant with
improved properties
Gene
Library
ClusterScreening*
Activity-based Screening
according industry process
requirements in E.coli, Bacillus,
Pichia hosts
Enzyme Library
Mutagenesis
combining randomized, focussed,
homology-based and recombinatorial
approaches
Overview c-LEcta enzyme engineering
Application
areas
Enzyme
classes
- Alcohol dehydrogenases
- Aldolases
- Transaminases
- Nitrilase
- Lipases
Optimization
targets
- Food & Feed Enzymes
and Additives
- Substrate scope
- Small molecule APIs
- Regio-selectivity
- fine & specialty chemicals
- Temperature and pH
activity/stability
- …
- Stereo-selectivity
- Solvent stability
- Dismutases
- Process stability
- Phosphorylases
- Productivity
- …
Improved processes are
routinely developed in
many areas through
efficient enzyme
engineering by c-LEcta
- High substrate concentration
performance
- Expression yield
- …
Examples
De novo enzyme design
Improved Fructose-6-phosphate aldolase
Nitrilase for API synthesis
Enzyme engineering: de novo enzyme design
Developing an ammonia-lyase for b-amino acid synthesis
Target application
Providing building blocks for bioactive
compounds and fine chemicals
Structure-based approach with
randomization at 4 sites
Cluster Screening (10.000-50.000
variants per plate)
HPLC analysis of product formation with
crotonic acid as model substrate
COOH
HOOC
R
+
O
NH3
COOH
HOOC
mutant
aspartase
+
NH2
aspartase
Aspartase
NH 4+
R
O
O
NH 2
a,b-unsaturated acid
O
b-amino acid
After screening of 300.000 clones:
350000
~10.000 clones/plate
325000
300000
275000
>3 mutations necessary for initial activity
- Hit cluster C6
- other cluster
250000
225000
200000
175000
Identification of active variant possible
only by efficient screening in a
combinatorial library
150000
125000
100000
75000
50000
25000
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
min
Enzyme engineering: Aldolase
Improved Fructose-6-phosphate Aldolase (FSA) for fagomine synthesis
FSA-Variant
Dihydroxyacetone
DHA
Structure-based approach with directed
library design at 7 sites
Cluster Screening (~65.000 variants)
HPLC screening for product formation at
high substrate concentrations
120
100
product concentration [mM]
Optimization demands
Increased activity on non-natural
aldehyde substrate
Process performance (substrate
inhibition, process stability)
80
PA4-23-P2A2 OD5
60
FSA WT
OD5
40
20
0
0
Simultaneous multi-parameter optimization in one screening
100
time [min]
200
Enzyme Engineering: Nitrilase
Engineering of a nitrilase for the synthesis of an API intermediate
Optimization demands:
Activity  activity screen
process stability  thermostability screen
high substrate concentration  process condition screen
Increase of activity
100%
1,2
4,0
90%
1
80%
70%
0,8
wildtype
60%
0,6
50%
plus 10°C
40%
0,4
mutant
30%
0,2
20%
10%
0
wildtype
wild-type
mutant
evolved mutant
3,5
Productivity
activity
Specific activity
higher process
productivity
Increase of stability
3,0
2,5
2,0
1,5
1,0
0,5
0%
40
45
50
55
60
T in °C65
0,0
wildtype
1
mutant
2
Process with optimized enzyme is currently in scale-up for +100 ton synthesis
enzymes and strains
Summary
c-LEcta’s biodiversity collection provides a broad access to novel
enzyme candidates and activities, allowing setup of a strong IP
position
Our enzyme optimization expertise allows quick and processoriented development of enzyme candidates towards cost-efficient
industry processes
c-LEcta is open for collaborations
with industry partners in guiding
biotechnological innovation to the
market
Big Corp.
Thank you for your attention!
Dr. Mathias Bell
Perlickstrasse 5
04103 Leipzig
Germany
E-Mail: [email protected]
Web:
www.c-LEcta.de
Phone: +49 341 355 21 4 0
Fax:
+49 341 355 21 4 33

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