Ingenza
NewProt Kick-off meeting
25 Jan 2012
Nijmegen
Ian Archer
Ingenza Ltd
Roslin, Edinburgh UK
CONFIDENTIAL 2012
Ingenza – what we do
 Synthetic Biology Technologies
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Biocatalysis/Bioprocess development
Protein expression/enzyme evolution
Screening for improved biocatalysts/processes
Novel enabling technologies e.g. gENABLE
 Business areas
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Fine chemicals
Biologics
Biofuels
Synthetic biology / petrochemical replacement
Biopolymers
Diagnostics
CONFIDENTIAL 2012
Initial company focus: biosynthesis of chiral compounds
Engineered microbial biocatalysts
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Platform technologies
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e.g. oxidase and aminotransferase biocatalysts
Unnatural amino acids and chiral amines
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Large scale processes, compounds >99% e.e.
– Highly expressed enzymes
– High cell density fermentation
– Efficient process chemistry
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Enzymes adapted by mutation/screening
– New specificities
– Improved reaction properties
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Process optimisation
– Stabilised biocatalysts
– Diverse enzyme production systems
– Biocatalyst formulation
– Statistical design of experiments
CONFIDENTIAL 2012
Expression systems
Continuing to expand and diversify
• Bacteria: E.coli
– Large range of host/vector systems
– Inducible, constitutive, synthetic
• C.glutamicum / C.acetobutylicum
– Customising host/vector systems
• Yeast: Saccharomyces, Pichia
– Range of host/vector systems
– Cytosolic/Partitioned/Secretion
– Integration vectors/copy number control
– Fusions to identify novel regulatory regions
• Fungi: A.niger, A. terreus
– Customising host/vector systems
– Inducible/Constitutive
• Insect cell/mammalian
– Production of biologics
CONFIDENTIAL 2012
Screening to adapt enzyme specificity or improve performance
Colorimetric, qualitative solid phase/quantitative liquid phase
oxidase
HRP
Substrate
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Micro-titre plate assay
Straight forward visual read-out
Kinetic characterisation of variants
CONFIDENTIAL 2012
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Adapted to solid phase
Proprietary
Very high throughput
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Millions of variants
Colorimetric screening
Improved enzyme activity or protein expression
• The screen detects
– improved activity
AND/OR
– improved expression
Can be used in conjunction with:
• Gene synthesis, now standard in molecular biology
• Gene assembly methods to rapidly generate expression libraries
• Assists selection of the optimal expression system
• Assists selection of the optimal gene sequence
• Delivers the highest quantity and quality of expressed protein
– Screening libraries is a powerful tool
CONFIDENTIAL 2012
Enzyme adaptation:
Increased enzyme thermostability/robustness
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Three rounds of laboratory evolution
Wild-type TvDAAO heat treatment assay
Variant WT1 TvDAAO heat treatment assay
250
250
45˚C
50.4˚C
55.8˚C
61˚C
65.6˚C
150
100
200
mOD/min
mOD/min
200
50
45˚C
50.4˚C
55.8˚C
61˚C
65.6˚C
150
100
50
0
0
0
10
20
30
40
50
60
70
80
90
100
0
Time (mins)
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10
20
30
40
50
60
70
80
90
100
Time (mins)
Not stable in process
– Susceptible to chemical denaturation
– Susceptible to physical denaturation
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CONFIDENTIAL 2012
Stable in process
– Resistant to chemical denaturation
– Resistant to physical denaturation
Screening for efficient protein production
Applied to biologic target to identify top 50 from 50,000
Library built (50,000 variants)
Oversampled (500,000 clones)
Hits identified visually
500 initial positive hits
- re-assayed
- PCR screen to confirm
- liquid phase assay to quantify
50 Best hits identified
- sub-cloned
- SDS-PAGE/Western assay
- Provided to customer
Over-expressing clone
CONFIDENTIAL 2012
Synthetic biology
Replacement of petrochemical and other starting materials
• Partnership with Lucite International
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Global producer of industrial polymers
Engaged in multi year contract
Microbial strain construction
Synthetic Biology – pathway engineering
Screening: Crossfeeding, Zone clearing, pH based
Fermentation development
Management of strategic academic collaboration
• Additional contracts now initiated
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Biomass as replacement for petrochemical feedstock
Fermentation route to natural food additive
Multi-target
Synthetic Biology to develop efficient production microbes
Bacteria/Yeast/Fungi
• Proprietary genetic platforms accelerate strain improvement
CONFIDENTIAL 2012
gENABLE – Genome segment assembly
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Co-developed by Ingenza and Scottish Government
Ingenza applying broadly in Industrial Biotechnology
Assembly of genes, variants, reporters, markers, regulatory elements
High-throughput, one-pot combinatorial assemblies
Combines Bioinformatics, Microfluidics, Novel bio-reactions
Accelerates:
– Optimisation of gene expression
– Pathway construction/engineering
– Efficient synthesis of target products
• Applicable in all areas of protein expression
• Now central to all Ingenza enabling technologies and business areas
• Synergistic with screening
• Colorimetric, pH, crossfeeding, zone clearing, protein fusions
CONFIDENTIAL 2012
gENABLE
Why? Expressing proteins is easy - isn’t it?
Welch. M. Journal of the Royal Society. Interface 11th March (2009)
CONFIDENTIAL 2012
gENABLE
Specific linker based genetic pathway construction
Assemblies of up to 10 parts have been demonstrated
CONFIDENTIAL 2012
gENABLE
5 part combinatorial assembly for co-ordinated enzyme expression
Position A
Pr. 1
Gene 1
Position B
Ter.
Pr. 1
Position C
Ter.
Gene 2
Pr. 1
Gene 3
Position D
Position E
Ter.
2 µ origin
Pr. 2
Gene 1
Ter.
Pr. 2
Ter.
Gene 2
Pr. 2
Gene 3
Ter.
Marker
CEN4 origin
Pr. 3
Gene 1
3 variants
Ter.
Pr. 3
Gene 2
Ter.
Gene 3
Pr. 3
3 variants
3 variants
Ter.
1 marker
2 origins
of replication
Vector backbones
Assembly of 54 different genetic constructs in a single reaction
Overcomes limits of empirical bioprocess optimisation
Faster route to optimal bioprocess
CONFIDENTIAL 2012
gENABLE
Example of results
• Combination of 5 independent DNA fragments
• Assembly of synthetic enzyme pathway
• Typically a problematic empirical process
• Results in 95% success in correct pathway assembly (dark clones)
> 95 % positive clones
CONFIDENTIAL 2012
NewProt
Workpackage 6 @ Ingenza
Experimental validation
Tasks
1. Bioinformatics to identify aminotransferases (plus as many as
possible of at least another 11 enzyme superfamilies) with
diverse, novel activities towards commercially specific targets
2. Pathway engineering to incorporate activities into hosts
3. Develop screens to identify best production constructs
4. Screening of libraries to identify desired activities
Focus areas?
1. Enzyme promiscuity to identify novel activities
2. Use of bioinformatics to demystify expression / activity etc
CONFIDENTIAL 2012