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Selection and Testing of New Microbes for Green and White Biotechnology Applications Industrial Uses of Bacteria 19 May 2010, IOM3, London, UK Robert Speight Ingenza Ltd Roslin, UK [email protected] 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 Innovation at Ingenza is Product Driven Microbe Engineering Enzymes Industrial Products 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 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 Background to Ingenza Bioprocess Improvement in Industrial Biotechnology Richmond Chemical Corporation Oak Brook, Chicago, US RV Labs Ingenza RC Fuel Hyderabad, India Roslin, UK 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 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 Strain Engineering Bio-production of natural products Biochemistry and Genomics Enzyme Characterization Enantiopure amino acids and amines by deracemisation NH Enantioselective Oxidase biocatalyst R1 COOH NH2 R1 COOH NH2 R1 COOH 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 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 Aquapharm Biodiscovery Limited Amine/Amino Acid + H2O + O2 Heriot-Watt University Plymouth Marine Laboratory Novel Oxidase Ketone/Keto Acid + H2O2 + NH3 Colorimetric assay Cell Survival SEASCREEN: Organism Screening Cell Survival - Direct selection of oxidase activity Very Low Nitrogen High Nitrogen Selective growth on amines Very Low Nitrogen + amine substrate 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 Directed Evolution Strategy Random Mutagenesis Parent Gene Targeted Mutagenesis High Throughput Screening Oxidation Reaction Analysis Deracemisation of DL-pipecolic acid using transfer hydrogenation and additional ammonium formate 100 6.0E+06 90 5.0E+06 80 70 50 3.0E+06 40 2.0E+06 L-pip 1 30 D-pip 1 L-pip 2 D-pip 2 20 L-pip 3 1.0E+06 D-pip 3 Imp-1 10 Imp-2 Imp-3 0 0.0E+00 0 5 10 15 time / h 20 25 Impurity/AU Yield pipecolic acid /% 4.0E+06 60 Hit confirmation Activity Quantification Validation Fermentation Mutation and Screening • Variety of oxidases (e.g. ScDAO, TvDAO) • Commercial substrate • Variable conditions (temperature, pH, inhibitors) 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 Activity towards target compound V0 (mmoles/hr/g) 30 Native Activity 50 deg, 1 hour 25 X 294 20 15 10 5 0 Wild-Type Thr218Ile His141Tyr Round 1 His141Tyr Thr218Ile Round 2 His141Tyr Thr218Ile Gln68Arg His141Tyr Thr218Ile Glu99Gly Round 3 His141Tyr Thr218Ile Val50Ala His141Tyr Thr218Ile Val50Ala Asp158Tyr His141Tyr Thr218Ile Val50Ala Ala111Gly Round 4 Biocatalyst development Biocatalyst stability and process robustness Variant WT1 TvDAAO heat treatment assay Wild-type TvDAAO heat treatment assay 250 250 mOD/min 150 100 50 100 0 0 10 20 30 40 50 60 70 80 90 100 0 Time (mins) Not stable under process conditions High risk for scale-up 10 20 30 40 50 60 70 80 90 100 Time (mins) • Not only more thermally stable but – More resistant to chemical denaturation – More resistant to physical denaturation – Susceptible to chemical denaturation – Susceptible to physical denaturation • 150 50 0 • 45ûC 50.4ûC 55.8ûC 61ûC 65.6ûC 200 mOD/min 45ûC 50.4ûC 55.8ûC 61ûC 65.6ûC 200 • 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 H2N CO2H H2N CO2H 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 Screening for Improved Biopharma Production Test Protein is Soluble Linker R is functional Test Protein Reporter Protein R Test Protein is Insoluble X Colour Growth R is non-functional No Colour No Growth Waldo, G. S. (2003), Current Opinion in Chemical Biology 7, 33-38 The Ingenza System R.E 1 R.E 2 Constant TestDomain Gene Fusion Reporter DAAO fusion Polylinker Oxidase P R R Fusion vector + O2 + H2O H2N Ori Ab COOH + H2O2 + NH3 O COOH Horse Radish Peroxidase Substrate Test gene mutated The oxidase reporter protein is constant Assay response proportional to fusion protein concentration Key Advantages 120 100 80 60 40 20 -S er in e D -P ro lin e D -L eu ci D ne -G lu ta m D at -M e et hi D on -P in he e ny la la ni ne D -A rg in D in -T e ry pt op ha n D D ne 0 an i Choice of promoter Inducer strength Choice of DAO substrate Concentration of substrate Growth and assay time Growth temperature -A l – – – – – – Relative Activity (%) • The screening system is highly tunable • Total assay response is dependent on each test protein • Subtle improvements can be seen by finding the right assay ‘window’ • Sensitive, cheap and rapid 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