Robert Speight Ingenza Ltd Roslin, UK r.speight@ingenza

1. Selection and Testing of New Microbes for Green and White Biotechnology ApplicationsIndustrial Uses of Bacteria19 May 2010, IOM3, London, UK Robert Speight Ingenza Ltd Roslin, UK r.speight@ingenza.com

2. Introduction • Ingenza - Who we are and what we do • Finding catalysts from microbes • Using microbes to make industrial products

3. Background to Ingenza • INGENZA = “INdustrial GENetics and ENZymes

4. Innovation at Ingenza is Product Driven Microbe Engineering Enzymes Industrial Products

5. 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

6. 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 • 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

7. Background to Ingenza Bioprocess Improvement in Industrial Biotechnology Richmond Chemical Corporation Oak Brook, Chicago, US RV Labs Hyderabad, India Ingenza Roslin, UK RC Fuel 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

8. 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 Biochemistry and Genomics Strain Engineering Bio-production of natural products Enzyme Characterization

9. Enantioselective Oxidase biocatalyst Chemical Reductant Enantiopure amino acids and amines by deracemisation • Platform technology • 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

10. Speeding Up Enzyme Discovery New oxidases from diverse sources • Bioinformatics - Genome Sequencing Projects • 1138 microbial sequenced genomes completed and submitted to NCBI • Genome sequencing becoming much cheaper • Data mining, BLAST searching • 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).

11. SEASCREEN: Organism Screening Direct identification of oxidase activity TSB, EPSRC and BBSRC Funding Project Ingenza Limited Heriot-Watt University Aquapharm Biodiscovery Limited Plymouth Marine Laboratory Novel Oxidase Amine/Amino Acid + H2O + O2 Ketone/Keto Acid + H2O2 + NH3 Colorimetric assay Cell Survival

12. SEASCREEN: Organism Screening Cell Survival - Direct selection of oxidase activity Very Low Nitrogen High Nitrogen Very Low Nitrogen + amine substrate Selective growth on amines 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

13. Changing Existing Enzymes Mutation and Selection • Generation of large libraries of variants • Random mutagenesis • e.g. error prone PCR • Targeted mutagenesis • 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

14. Directed Evolution Strategy Random Mutagenesis Parent Gene Targeted Mutagenesis High Throughput Screening Hit confirmation Activity Quantification Validation Oxidation Reaction Analysis Fermentation

15. Mutation and Screening • Variety of oxidases (e.g. ScDAO, TvDAO) • Commercial substrate • Variable conditions (temperature, pH, inhibitors) 1st screen Re-assay

16. ScDAO Mutation and Selection • Streptomyces coelicolor enzyme previously uncharacterised. • Initial poor activity towards target compound, low stability and low expression improved through directed evolution X 294 Round 1 Round 2 Round 3 Round 4

17. Biocatalyst developmentBiocatalyst stability and process robustness • Not only more thermally stable but • More resistant to chemical denaturation • More resistant to physical denaturation • Applied in process scale-up • Not stable under process conditions • Susceptible to chemical denaturation • Susceptible to physical denaturation • High risk for scale-up

18. 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 • High volumetric productivity • High enantiomeric purity • Rapid adaptation • Platform process

19. 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

20. Screening for Improved Biopharma Production Test Protein is Soluble Linker R is functional Test Protein Reporter Protein R Colour Growth Test Protein is Insoluble X R is non-functional No Colour No Growth Waldo, G. S. (2003), Current Opinion in Chemical Biology 7, 33-38

21. The Ingenza System Test Gene Reporter Test gene mutated The oxidase reporter protein is constant Assay response proportional to fusion protein concentration

22. Key Advantages • The screening system is highly tunable • Choice of promoter • Inducer strength • Choice of DAO substrate • Concentration of substrate • Growth and assay time • Growth temperature • Total assay response is dependent on each test protein • Subtle improvements can be seen by finding the right assay ‘window’ • Sensitive, cheap and rapid

23. 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

24. 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