Selection and Testing of New Microbes for Green and White Biotechnology Applications Industrial Uses of Bacteria 19 May 2010, IOM3, London, UK Target high value products that are difficult to make using traditional chemistry Develop novel and proprietary manufacturing routes High tech mol biol through fermentation process chemistry and scale up Robert Speight Ingenza Ltd Roslin, UK r.speight@ingenza.com

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 Our name gives a clue to where our technology and innovation lies The team compromises a core scientific team with cutting edge skills in chemistry, genetics and biology. We are a team of twenty highly skilled and educated staff and our company is doubling in size every 2 years. We relocated to Roslin Biocentre in Dec 2006 The picture flow on the right shows how we work at Ingenza. We start by generating the right genetic construct to engineer a microbe to do the job we want. Then we take the microbe and ferment it. Then we using the fermentation output to make the products for our customers using process chemistry. 3

Innovation at Ingenza is Product Driven Innovation at Ingenza is product driven. TO SAY IT ANOTHER WAY, WE ENGINEER BUGS TO PRODUCE ENZYMES THAT MAKE INDUSTRIAL PRODUCTS AND THESE PRODUCTS ARE…. We engineer microbes to make products. We aim to reduce the costs and efficiency of these products. We are world leaders in these product areas and have achieve significant successes in each of these areas. We are highly ambitious and eager to exploit our skills and expertise into new markets. With our platform skills and capabilities in genetics, microbiology and chemistry we believe we are unique placed to fulfill and realise these opportunities. Our secret power to enable us to deliver this reality is from our understanding of how we can produce, modify, detect and predict the behaviour of enzyme science. We have three current product areas where we are adding value. These products are fine chemicals, biopharmaceuticals and biofuels. Microbe Engineering Enzymes Industrial Products 4

Background to Ingenza Business Model: 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 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 Multidisciplinary team, stable, grown together and improved. GMP compatible lab just opened, relevant for biopharma

Background to Ingenza Richmond Chemical Corporation RV Labs 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

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

Enantiopure amino acids and amines by deracemisation Enantioselective Oxidase biocatalyst Chemical Reductant 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 Need to be able to access a wide range of substrates as platform technology takes a long time and cost to develop

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).

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 Or look directly for activities Detection of by-products Cell survival if substrate is the only source of nitrogen that it needs to survive Colorimetric assay Cell Survival

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

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 Take the enzymes you have and mutate them to change their properties (such as which substrates they accept)

Directed Evolution Strategy Random Mutagenesis Parent Gene Targeted Mutagenesis High Throughput Screening Hit confirmation Activity Quantification Validation Oxidation Reaction Key aspect is how to select out of 2 million random variants which ones have the characteristics we desire - needle in the haystack Benefits from interdisciplinary team Analysis Fermentation

Mutation and Screening Variety of oxidases (e.g. ScDAO, TvDAO) Commercial substrate Variable conditions (temperature, pH, inhibitors) Stability screening used due to our previous experience with ScDAO H141Y selected in first round of ScDAO screening Watch for diagram flipping over. 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 X 294 Activity at 20 mM racemate.Corrected for total protein concentration. Good/fast way of assaying Vmax AND KM 141 position in all other proteins is Tyr. Is this just a correction? Is 218 therefore the most important for specificity? Round 1 Round 2 Round 3 Round 4

Biocatalyst development Biocatalyst stability and process robustness Not stable under process conditions Susceptible to chemical denaturation Susceptible to physical denaturation High risk for scale-up Not only more thermally stable but More resistant to chemical denaturation More resistant to physical denaturation 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 High volumetric productivity High enantiomeric purity Rapid adaptation Platform process Adapted readily between ABA, norvaline and also an L-amino acid oxidase based process but relies on high enzyme activity IF YOU FEEL LIKE IT AND TIME IS OK YOU CAN SAY THAT MANY BIO BASED METHODS EXIST FOR AMINO ACID MANUFACTURE (FERMENTATION, PATHWAY ENG, BIOTRANS, RESOLUTION ETC...BUT THIS FACT ILLUSTRATES NO SINGLE METHOD HAS DOMINATED) OUR VIEW IS THAT TO MAXIMISE ADAPTABILITY OF OUR ROUTE WILL GIVE GREATEST PAYBACK AND MITIGATE RISKS OF EXPENSIVE BIOPROCESS DEVELOPMENT

Biopharmaceuticals Biopharma production 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 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

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

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 Need to find right window to see subtle improvements

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 At Ingenza we strongly believe that educating and sharing knowledge is key to our future success. Last November we hired 2 modern apprentices on the Scottish government backed 2 for 1 scheme for modern apprentices in life sciences. Ross and Kirsty are study one day a week at Forth Valley College for a HNC in life sciences. We also engage with the local community the world-leading science we are exploiting. Last month we opened our doors to a local high school to enthuse our next generation of scientific innovators to become excited about the opportunities they have in science. 24

To summarise Ingenza is an exciting world leading bioprocessing company which is gaining a international reputation to deliver high quality products in various markets. Our unique selling point is our ability to use our multi skills set in synthetic chemistry, fermentation, and molecular biology to the customer to exploit the power of enzyme technology. 25