CONFIDENTIAL 2012

Ingenza

NewProt Kick-off meeting25 Jan 2012

Nijmegen

Ian ArcherIngenza Ltd

Roslin, Edinburgh UK

CONFIDENTIAL 2012

Ingenza – what we doIngenza – what we do

Synthetic Biology Technologies Biocatalysis/Bioprocess development Protein expression/enzyme evolution Screening for improved biocatalysts/processes Novel enabling technologies e.g. gENABLE

Business areas Fine chemicals Biologics Biofuels Synthetic biology / petrochemical replacement Biopolymers Diagnostics

CONFIDENTIAL 2012

• Platform technologies– e.g. oxidase and aminotransferase biocatalysts

– Unnatural amino acids and chiral amines

• Large scale processes, compounds >99% e.e.– Highly expressed enzymes– High cell density fermentation– Efficient process chemistry

• Enzymes adapted by mutation/screening– New specificities– Improved reaction properties

• Process optimisation– Stabilised biocatalysts– Diverse enzyme production systems– Biocatalyst formulation– Statistical design of experiments

Initial company focus: biosynthesis of chiral compoundsInitial company focus: biosynthesis of chiral compoundsEngineered microbial biocatalystsEngineered microbial biocatalysts

CONFIDENTIAL 2012

• 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

Expression systemsExpression systemsContinuing to expand and diversifyContinuing to expand and diversify

CONFIDENTIAL 2012

• Adapted to solid phase• Proprietary• Very high throughput

– Millions of variants

HRPSubstrate

oxidase

Screening to adapt enzyme specificity or improve performance Screening to adapt enzyme specificity or improve performance Colorimetric, qualitative solid phase/quantitative liquid phaseColorimetric, qualitative solid phase/quantitative liquid phase

• Micro-titre plate assay

• Straight forward visual read-out

• Kinetic characterisation of variants

CONFIDENTIAL 2012

• The screen detects– improved activityAND/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

Colorimetric screeningColorimetric screeningImproved enzyme activity or protein expressionImproved enzyme activity or protein expression

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• Stable in processStable in process

– ResistantResistant to chemical denaturation to chemical denaturation

– ResistantResistant to physical denaturation to physical denaturation

• Not stable in processNot stable in process

– SusceptibleSusceptible to chemical denaturation to chemical denaturation

– SusceptibleSusceptible to physical denaturation to physical denaturation

Enzyme adaptation:Enzyme adaptation:Increased enzyme thermostability/robustness

• Three rounds of laboratory evolutionThree rounds of laboratory evolution

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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

Screening for efficient protein production Screening for efficient protein production Applied to biologic target to identify top 50 from 50,000Applied to biologic target to identify top 50 from 50,000

Over-expressing clone

CONFIDENTIAL 2012

Synthetic biologySynthetic biologyReplacement of petrochemical and other starting materialsReplacement of petrochemical and other starting materials

• Partnership with Lucite International– 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– 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 assemblygENABLE – Genome segment assembly

• 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

gENABLEgENABLEWhy? Expressing proteins is easy - isn’t it?Why? Expressing proteins is easy - isn’t it?

Welch. M. Journal of the Royal Society. Interface 11 th March (2009)

CONFIDENTIAL 2012

Assemblies of up to 10 parts have been demonstrated

gENABLEgENABLESpecific linker based genetic pathway constructionSpecific linker based genetic pathway construction

CONFIDENTIAL 2012

Pr. 1

2 µ origin

CEN4 origin

Vector backbones

Position A Position B Position C Position D Position E

Gene 1 Ter.

Pr. 2 Gene 1 Ter.

Pr. 3 Gene 1 Ter.

Pr. 1 Gene 2 Ter.

Pr. 2 Gene 2 Ter.

Pr. 3 Gene 2 Ter.

Pr. 1 Gene 3 Ter.

Pr. 2 Gene 3 Ter.

Pr. 3 Gene 3 Ter.

Marker

3 variants 3 variants 3 variants2 origins

of replication1 marker

Assembly of 54 different genetic constructs in a single reactionOvercomes limits of empirical bioprocess optimisation

Faster route to optimal bioprocess

gENABLEgENABLE5 part combinatorial assembly for co-ordinated enzyme expression5 part combinatorial assembly for co-ordinated enzyme expression

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• 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

gENABLEgENABLEExample of resultsExample of results

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NewProtNewProtWorkpackage 6 @ IngenzaWorkpackage 6 @ IngenzaExperimental validationExperimental validation

Tasks1.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