Selection and Testing of New Microbes for Green and White Biotechnology

Applications

Industrial Uses of Bacteria19 May 2010, IOM3, London, UK

Robert Speight

Ingenza LtdRoslin, UK

r.speight@ingenza.com

Introduction

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

INGENZA = “INdustrial GENetics and ENZymes

Background to IngenzaBackground to Ingenza

Innovation at Ingenza is Product DrivenInnovation at Ingenza is Product Driven

Microbe EngineeringMicrobe EngineeringEnzymesEnzymes

Industrial ProductsIndustrial Products

Background to IngenzaBackground to IngenzaBioprocess Improvement in Industrial BiotechnologyBioprocess 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 IngenzaBackground to IngenzaBioprocess Improvement in Industrial BiotechnologyBioprocess 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 IngenzaBackground to IngenzaBioprocess Improvement in Industrial BiotechnologyBioprocess Improvement in Industrial Biotechnology

Richmond Chemical CorporationOak Brook, Chicago, US

RC FuelChicago and Roslin

IngenzaRoslin, UK

RV LabsHyderabad, India

Fully integrated company with key commercial and scientific expertise55 People world-wideExtensive customer and manufacturing alliancesStrong Portfolio of Enabling Technologies

Biochemistry and Biochemistry and GenomicsGenomics

Directed EvolutionDirected Evolution

High-throughputHigh-throughputScreeningScreening

Strain EngineeringStrain Engineering

FermentationFermentation

Bioprocess DevelopmentBioprocess DevelopmentBiocatalyst FormulationBiocatalyst Formulation

Cost-effectiveCost-effectiveprocessesprocesses

Adapting enzymesAdapting enzymesto new targetsto new targets

EnzymeEnzymeCharacterizationCharacterization

High cell density fermentationHigh cell density fermentation4 x 5 L in house, Scaled to 40,000 L4 x 5 L in house, Scaled to 40,000 L

Enzyme immobilizationEnzyme immobilizationLyophilizationLyophilization

Bio-production ofBio-production ofnatural productsnatural products

Enzyme improvementEnzyme improvementby mutation/screeningby mutation/screening

Enabling Technologies

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

EnantioselectiveOxidase

biocatalyst

R1 COOH

NH2

R1 COOH

NH2R1 COOH

NH

Chemical Reductant

Speeding Up Enzyme DiscoveryNew oxidases from diverse sources

Graph reproduced from: Nature 458, 719-724 (2009).

• 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

TSB, EPSRC and BBSRC Funding Project

Ingenza Limited Heriot-Watt UniversityAquapharm Biodiscovery Limited Plymouth Marine Laboratory

Amine/Amino Acid + H2O + O2

Novel OxidaseKetone/Keto Acid + H2O2 + NH3

Cell Survival

Colorimetric assay

SEASCREEN: Organism ScreeningSEASCREEN: Organism ScreeningDirect identification of oxidase activityDirect identification of oxidase activity

Selective growth on amines

High Nitrogen

Very Low Nitrogen

Very Low Nitrogen+ amine substrate

Screening of >600 strains yielded new broad spectrum L-amino acid and amine oxidasesMethods developed to control induction, lysis, heterologous expression and assay/screening

SEASCREEN: Organism ScreeningSEASCREEN: Organism ScreeningCell SurvivalCell Survival - Direct selection of oxidase activity - Direct selection of oxidase activity

Changing Existing Enzymes Changing Existing Enzymes Mutation and SelectionMutation 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

Parent Gene

Random Mutagenesis

Targeted Mutagenesis

Hit confirmationActivity Quantification

High Throughput Screening

Validation

FermentationOxidation ReactionAnalysis

Deracemisation of DL-pipecolic acid using transfer hydrogenation and additional ammonium formate

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25

time / h

Yie

ld p

ipe

co

lica

cid

/%

0.0E+00

1.0E+06

2.0E+06

3.0E+06

4.0E+06

5.0E+06

6.0E+06

Imp

uri

ty/A

U

L-pip 1

D-pip 1

L-pip 2

D-pip 2

L-pip 3

D-pip 3

Imp-1

Imp-2

Imp-3

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

0

5

10

15

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30

Wild-Type Thr218Ile His141Tyr His141TyrThr218Ile

His141TyrThr218IleGln68Arg

His141TyrThr218IleGlu99Gly

His141TyrThr218IleVal50Ala

His141TyrThr218IleVal50Ala

Asp158Tyr

His141TyrThr218IleVal50Ala

Ala111Gly

V0

(m

mole

s/h

r/g

)

Native Activity50 deg, 1 hour

Round 1 Round 2 Round 3 Round 4

X 294

• Not only more thermally stable butNot only more thermally stable but– More resistant to chemical More resistant to chemical

denaturationdenaturation

– More resistant to physical More resistant to physical denaturationdenaturation

• Applied in process scale-upApplied in process scale-up

• Not stable under process conditionsNot stable under process conditions– Susceptible to chemical denaturationSusceptible to chemical denaturation

– Susceptible to physical denaturationSusceptible to physical denaturation

• High risk for scale-upHigh risk for scale-up

Biocatalyst developmentBiocatalyst stability and process robustness

Wild-type TvDAAO heat treatment assay

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250

0 10 20 30 40 50 60 70 80 90 100

Time (mins)

mO

D/m

in

45ûC

50.4ûC

55.8ûC

61ûC

65.6ûC

Variant WT1 TvDAAO heat treatment assay

0

50

100

150

200

250

0 10 20 30 40 50 60 70 80 90 100

Time (mins)

mO

D/m

in

45ûC

50.4ûC

55.8ûC

61ûC

65.6ûC

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

E.coliE.coli expression expressionHCD fermentationHCD fermentationProtease knockoutsProtease knockouts

Yeast expressionYeast expressionHCD fermentationHCD fermentation

BiopharmaBiopharmaproductionproduction

Protein refoldingProtein refoldingActive productsActive productsH.T. ScreensH.T. Screens

Enabling technology in gene expression and strain improvementEnabling technology in gene expression and strain improvement

Biopharmaceuticals

Screening for Improved Biopharma Production

Waldo, G. S. (2003), Current Opinion in Chemical Biology 7, 33-38

Test Protein

Linker

Reporter Protein R

R is functional

Test Protein is Soluble

Test Protein is Insoluble

R is non-functionalX

ColourGrowth

No ColourNo Growth

The Ingenza System

OxidaseFusion PolylinkerConstant Domain

P

DAAO fusion

AbOri

R.E 1 R.E 2

Fusion vectorH2N COOH

R

+ O2 + H2O

O COOH

R

+ H2O2 + NH3

Horse Radish PeroxidaseSubstrate

Test Gene Reporter

Test gene mutatedThe oxidase reporter protein is constantAssay 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

0

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D-Alanine

D-Ser

ine

D-Pro

line

D-Leu

cine

D-Gluta

mat

e

D-Met

hion

ine

D-Phe

nylalanine

D-Arg

inine

D-Tryp

toph

an

Rela

tive A

ctiv

ity (

%)

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

AcknowledgementsAcknowledgements

Heriot-Watt: Prof. Mark Keane and Alec FosterPlymouth Marine Laboratory: Sohail Ali, Mike AllenAquapharm Biodiscovery: Kim McKendrick, Andrew Mearns-SpraggUniversità degli Studi dell'Insubria: Prof. Loredano Pollegioni and GroupACIB, Graz: Prof. Toni Glieder and GroupCoE Bio3, Manchester: Prof. Nick Turner, Paul Goddard

Everyone at Ingenza and RC Corp.Our customersTSB, EPSRC, BBSRC, Scottish Enterprise, Scottish Executive