View
214
Download
0
Category
Tags:
Preview:
Citation preview
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
20
25
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
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
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
20
40
60
80
100
120
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
Recommended