Ingenza NewProt Kick-off meeting 25 Jan 2012 Nijmegen

1. IngenzaNewProt Kick-off meeting25 Jan 2012Nijmegen Ian Archer Ingenza Ltd Roslin, Edinburgh UK

2. Ingenza – 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

3. Initial company focus: biosynthesis of chiral compounds Engineered microbial biocatalysts • 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

4. 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

5. Screening to adapt enzyme specificity or improve performance Colorimetric, qualitative solid phase/quantitative liquid phase oxidase • Adapted to solid phase • Proprietary • Very high throughput • Millions of variants HRP Substrate • Micro-titre plate assay • Straight forward visual read-out • Kinetic characterisation of variants

6. 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

7. Enzyme adaptation:Increased enzyme thermostability/robustness • Three rounds of laboratory evolution • Stable in process • Resistant to chemical denaturation • Resistant to physical denaturation • Not stable in process • Susceptible to chemical denaturation • Susceptible to physical denaturation

8. 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

9. Synthetic biology Replacement 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

10. gENABLE – 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

11. gENABLE Why? Expressing proteins is easy - isn’t it? Welch. M. Journal of the Royal Society. Interface 11th March (2009)

12. gENABLE Specific linker based genetic pathway construction Assemblies of up to 10 parts have been demonstrated

13. gENABLE 5 part combinatorial assembly for co-ordinated enzyme expression Position A Position B Position C Position D Position E Gene 1 Gene 2 Gene 3 Pr. 1 Ter. Pr. 1 Ter. Pr. 1 Ter. 2 µ origin Gene 1 Gene 2 Gene 3 Marker Pr. 2 Ter. Pr. 2 Ter. Pr. 2 Ter. CEN4 origin Gene 1 Gene 2 Gene 3 Pr. 3 Ter. Pr. 3 Ter. Pr. 3 Ter. 2 origins of replication 3 variants 3 variants 3 variants 1 marker Vector backbones Assembly of 54 different genetic constructs in a single reaction Overcomes limits of empirical bioprocess optimisation Faster route to optimal bioprocess

14. 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

15. NewProt Workpackage 6 @ Ingenza Experimental validation • Tasks • Bioinformatics to identify aminotransferases (plus as many as possible of at least another 11 enzyme superfamilies) with diverse, novel activities towards commercially specific targets • Pathway engineering to incorporate activities into hosts • Develop screens to identify best production constructs • Screening of libraries to identify desired activities • Focus areas? • Enzyme promiscuity to identify novel activities • Use of bioinformatics to demystify expression / activity etc