Download Rob Speight Selection and Testing of New Bacteria.pps

Selection and Testing of New Microbes for
Green and White Biotechnology
Applications
Industrial Uses of Bacteria
19 May 2010, IOM3, London, UK
Robert Speight
Ingenza Ltd
Roslin, UK
[email protected]
Introduction
• Ingenza - Who we are and what we do
• Finding catalysts from microbes
• Using microbes to make industrial products
Background to Ingenza
INGENZA = “INdustrial GENetics and ENZymes
Innovation at Ingenza is Product Driven
Microbe Engineering
Enzymes
Industrial Products
Background to Ingenza
Bioprocess Improvement in Industrial Biotechnology
Business Model:
• Proprietary bioprocess technologies
– Chemical manufacturing processes (e.g. amino acids, fuels)
– Production organisms, engineering, gene expression
– Biopharma manufacturing systems
• Custom biotechnology services
– Enabling technologies
•
•
•
•
Enzyme discovery and improvement
Strain construction
Gene expression, fermentation
Bioprocess development
Background to Ingenza
Bioprocess Improvement in Industrial Biotechnology
• 19 scientists (12 Ph.D) with integrated skill set:
– Molecular Biology, Biochemistry, Strain Engineering
– Fermentation, Formulation
– Bioprocess/chemical process development and analytical chemistry
• Biotechnology (GMP Compatible), Fermentation and
Process Development Laboratories
• Ingenza History
–
–
–
–
–
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–
Founded in 2002
Spin-out from Edinburgh University
Initial focus on bioprocesses
Customers in pharma, food, agrochem, biofuels
Relocated to Roslin in 2006
Merged with Richmond Chemical Corp. in 2007
Economically Sustainable, Still Growing
Background to Ingenza
Bioprocess Improvement in Industrial Biotechnology
Richmond Chemical Corporation
Oak Brook, Chicago, US
RV Labs
Ingenza
RC Fuel
Hyderabad, India
Roslin, UK
Chicago and Roslin
Fully integrated company with key commercial and scientific
expertise
55 People world-wide
Extensive customer and manufacturing alliances
Strong Portfolio of Enabling Technologies
Cost-effective
processes
Adapting enzymes
to new targets
Bioprocess Development
Biocatalyst Formulation
High-throughput
Screening
Enzyme immobilization
Lyophilization
Enabling Technologies
Fermentation
Directed Evolution
High cell density fermentation
4 x 5 L in house, Scaled to 40,000 L
Enzyme improvement
by mutation/screening
Strain Engineering
Bio-production of
natural products
Biochemistry and
Genomics
Enzyme
Characterization
Enantiopure amino acids and
amines by deracemisation
NH
Enantioselective
Oxidase
biocatalyst
R1
COOH
NH2
R1
COOH
NH2
R1
COOH
Chemical
Reductant
• Platform technology
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–
–
–
Cheap starting materials to high value products
D/L-amino acid oxidases, R/S-amine oxidases
Genes from microbes - enzymes made in other microbes
Needs wide variety of enzymes with wide substrate
specificity and/or adaptability
Speeding Up Enzyme Discovery
New oxidases from diverse sources
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•
Bioinformatics - Genome Sequencing Projects
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1138 microbial sequenced genomes completed and submitted to NCBI
•
Genome sequencing becoming much cheaper
•
Data mining, BLAST searching
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Gene cloning, enzyme expression, assay
Gene synthesis and custom cloning
• In-house expression
systems
• E. coli - diverse plasmid
collections
• Yeast - IP free integration
and expression systems
Graph reproduced from: Nature 458, 719-724 (2009).
SEASCREEN: Organism Screening
Direct identification of oxidase activity
TSB, EPSRC and BBSRC Funding Project
Ingenza Limited
Aquapharm Biodiscovery Limited
Amine/Amino Acid + H2O + O2
Heriot-Watt University
Plymouth Marine Laboratory
Novel Oxidase
Ketone/Keto Acid + H2O2 + NH3
Colorimetric assay
Cell Survival
SEASCREEN: Organism Screening
Cell Survival - Direct selection of oxidase activity
Very Low
Nitrogen
High
Nitrogen
Selective growth on amines
Very Low Nitrogen
+ amine substrate
Screening of >600 strains yielded new broad spectrum L-amino acid and
amine oxidases
Methods developed to control induction, lysis, heterologous expression
and assay/screening
Changing Existing Enzymes
Mutation and Selection
• Generation of large libraries of
variants
• Random mutagenesis
•
e.g. error prone PCR
• Targeted mutagenesis
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•
from structure and mechanism
information
hot-spots selected from random
mutagenesis
• High efficiency plasmid library
construction
• Ligation-free approaches
• Libraries containing up to 2 million
independent variants built
Directed Evolution Strategy
Random
Mutagenesis
Parent Gene
Targeted
Mutagenesis
High Throughput
Screening
Oxidation
Reaction
Analysis
Deracemisation of DL-pipecolic acid using transfer hydrogenation and additional
ammonium formate
100
6.0E+06
90
5.0E+06
80
70
50
3.0E+06
40
2.0E+06
L-pip 1
30
D-pip 1
L-pip 2
D-pip 2
20
L-pip 3
1.0E+06
D-pip 3
Imp-1
10
Imp-2
Imp-3
0
0.0E+00
0
5
10
15
time / h
20
25
Impurity/AU
Yield pipecolic
acid /%
4.0E+06
60
Hit confirmation
Activity Quantification
Validation
Fermentation
Mutation and Screening
• Variety of oxidases (e.g. ScDAO, TvDAO)
• Commercial substrate
• Variable conditions (temperature, pH, inhibitors)
1st screen
Re-assay
ScDAO Mutation and Selection
• Streptomyces coelicolor enzyme previously uncharacterised.
• Initial poor activity towards target compound, low stability and
low expression improved through directed evolution
Activity towards target compound
V0 (mmoles/hr/g)
30
Native Activity
50 deg, 1 hour
25
X 294
20
15
10
5
0
Wild-Type
Thr218Ile
His141Tyr
Round 1
His141Tyr
Thr218Ile
Round 2
His141Tyr
Thr218Ile
Gln68Arg
His141Tyr
Thr218Ile
Glu99Gly
Round 3
His141Tyr
Thr218Ile
Val50Ala
His141Tyr
Thr218Ile
Val50Ala
Asp158Tyr
His141Tyr
Thr218Ile
Val50Ala
Ala111Gly
Round 4
Biocatalyst development
Biocatalyst stability and process robustness
Variant WT1 TvDAAO heat treatment assay
Wild-type TvDAAO heat treatment assay
250
250
mOD/min
150
100
50
100
0
0
10
20
30
40
50
60
70
80
90
100
0
Time (mins)
Not stable under process conditions
High risk for scale-up
10
20
30
40
50
60
70
80
90
100
Time (mins)
•
Not only more thermally stable but
– More resistant to chemical
denaturation
– More resistant to physical
denaturation
– Susceptible to chemical
denaturation
– Susceptible to physical denaturation
•
150
50
0
•
45ûC
50.4ûC
55.8ûC
61ûC
65.6ûC
200
mOD/min
45ûC
50.4ûC
55.8ûC
61ûC
65.6ûC
200
•
Applied in process scale-up
Fine Chemical Manufacture
• For example: 2-Aminobutyric Acid, Norvaline
• Methodology, scalability and economics all validated by
Ingenza process and RC Corp commercial groups
• Engineered microbes and enzymes
• High yield fermentations
– Defined media, fed batch
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High volumetric productivity
High enantiomeric purity
Rapid adaptation
Platform process
H2N
CO2H
H2N
CO2H
Biopharmaceuticals
E.coli expression
HCD fermentation
Protease knockouts
Protein refolding
Active products
H.T. Screens
Biopharma
production
Yeast expression
HCD fermentation
Enabling technology in gene expression and strain improvement
Screening for Improved
Biopharma Production
Test Protein is Soluble
Linker
R is functional
Test Protein
Reporter Protein R
Test Protein is Insoluble
X
Colour
Growth
R is non-functional
No Colour
No Growth
Waldo, G. S. (2003), Current Opinion in Chemical Biology 7, 33-38
The Ingenza System
R.E 1
R.E 2
Constant
TestDomain
Gene
Fusion
Reporter
DAAO fusion
Polylinker
Oxidase
P
R
R
Fusion vector
+ O2 + H2O
H2N
Ori
Ab
COOH
+ H2O2 + NH3
O
COOH
Horse Radish Peroxidase
Substrate
Test gene mutated
The oxidase reporter protein is constant
Assay response proportional to fusion protein concentration
Key Advantages
120
100
80
60
40
20
-S
er
in
e
D
-P
ro
lin
e
D
-L
eu
ci
D
ne
-G
lu
ta
m
D
at
-M
e
et
hi
D
on
-P
in
he
e
ny
la
la
ni
ne
D
-A
rg
in
D
in
-T
e
ry
pt
op
ha
n
D
D
ne
0
an
i
Choice of promoter
Inducer strength
Choice of DAO substrate
Concentration of substrate
Growth and assay time
Growth temperature
-A
l
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Relative Activity (%)
• The screening system is highly tunable
• Total assay response is dependent on each test protein
• Subtle improvements can be seen by finding the right assay ‘window’
• Sensitive, cheap and rapid
Biofuels (RC Fuel)
• Project founded December 2008 to improve biofuel
production process efficiencies through biotechnology
• Commercially Driven
• Strain Engineering
– Molecular Biology, Directed Evolution
• Biochemistry
– High throughput screening for improved strains
– Strain and process characterization
– Analytical method development for process characterization
• Fermentation
– Process modeling and validation
Acknowledgements
Heriot-Watt: Prof. Mark Keane and Alec Foster
Plymouth Marine Laboratory: Sohail Ali, Mike Allen
Aquapharm Biodiscovery: Kim McKendrick, Andrew Mearns-Spragg
Università degli Studi dell'Insubria: Prof. Loredano Pollegioni and Group
ACIB, Graz: Prof. Toni Glieder and Group
CoE Bio3, Manchester: Prof. Nick Turner, Paul Goddard
Everyone at Ingenza and RC Corp.
Our customers
TSB, EPSRC, BBSRC, Scottish Enterprise, Scottish Executive