Effective strain development based on HTS-systems fileMaterials and Chemistry 3 SINTEF SINTEF is the...
Transcript of Effective strain development based on HTS-systems fileMaterials and Chemistry 3 SINTEF SINTEF is the...
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Effective strain development based on HTS-systemsHåvard Sletta
SINTEF Materials and Chemistry,Trondheim, Norway
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Content
SINTEFDefinitionsWhy are we working with HTS-systemsProjects where we use HTS-systemsOur technology platform for strain developmentExamplesConclusions
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SINTEFSINTEF is the largest independent research organisation in Scandinavia (1700 employees). Our fields of research are technology, natural science, medicine and social science. Contract research for industry and the public sector represent > 90 % of our income
NTNU (Norwegian University of Science and Technology)
NTNU staff workon SINTEF projects
SINTEF staff works and teaches at NTNU
Joint laboratories, projectsand equipment
• Established 1978
• 17 employees
• dr. ing.- students, post doc
SINTEF Materials and ChemistryDepartment of biotechnology
Research director: Trond E. Ellingsen
Co-operators• Department of biotechnology (NTNU)
• Matforsk
• SINTEF units
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Definition of HTS (High-throughput screening)
“A combination of sciences and technologies whose simple and powerful aim is to identify candidate lead compounds by testing as large and diverse a set of compounds as possible”Plamen Petrov (AstraZeneca)
HTS-systems in strain development
“A combination of sciences and technologies whose simple and powerful aim is to identify candidate mutants/strains with desired properties by testing as large and diverse a set of mutants/strains as possible”
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Why are we working with HTS-systems at SINTEF/NTNU?
20 years of experience in strain improvement and industrial process development (lysine, polysaccharides, antibiotics, astaxanthin, etc)Bottleneck: The possibility to screen a large number of mutants for improved propertiesEquipment for high throughput screening and analyzes has rapidly improved possibilities for more efficient bio-process development
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Projects where we use HTS-systems in strain development / evaluation (2004)
Antibiotics (Streptomyces)Microbial production of biopolymer and biopolymer modifying enzymes (Pseudomonas, yeast, E. coli)Microbial production of Astaxanthin (yeast)Microbial production of Lysine from methanol (Bacillus)Enzyme engineering (directed evolution) (E. coli)Bioprospecting (antimicrobials, carotenoids and polyunsaturated fatty acids (DHA))
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Our technology platform for HTS- strain developmentWild type
Fermentor studies
Introduction of diversity(mutagenesis, r-DNA technology)
Mutant population
Cultivation(primary screening)
Sample preparation
Analyzes and data processing
Cultivation(secondary screening)
Analyzes and data processing
Cultivation
HTS-protocols
Selection
2 Infors Multitron (capacity 20 000 wells)• 3 mm orbital movement (max 1000 rpm)• 10 – 60 oC, humidity control
Several shaking incubators (>200 flasks)• 25 mm orbital movement (max 400 rpm)• 10 – 60 oC, humidity control
32 Applicon fermentors (3-l)• Advanced control and logging
Our technology platform for HTS- strain developmentWild type
Fermentor studies
Introduction of diversity(mutagenesis, r-DNA technology)
Mutant population
Cultivation(primary screening)
Sample preparation
Analyzes and data processing
Cultivation(secondary screening)
Analyzes and data processing
Robots and analytical equipment
Selection
Tecan Genesis 200 RSP (liquid handling)• 96 and 8 canal pipettes• Washing and filling • Filtration and solid phase extraction• Incubation and reading of well plates• 8 hours 30 000 pipetting cycles
HPLC / LC-MS• 7 HPLC - systems• 4 LC/MS systems (Agilent)
• 2 Single Quadrupoles(up to 700 samples / day)
• TOF• Ion-trap
• Preparative HPLC
Genetics Q-pix colony picker• Inoculation and replication of well plates• Capacity: 2000 colonies / hour
HTS-protocols
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One of our two fermentation laboratories, each with 16 3-L fermentors
Well plate incubator
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Robotic liquid handling workstation
Robotic colony picking
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Our LCMS laboratory (LC MSD TOF, and two LC MSD SQ)
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Streptomyces: strategy(Low producing mutant commercial production process)
Project goal
WT Process withimprovedmutant
Prod
uctiv
ityScreening
Evaluation of mutantsOptimization of process
Adjustments of screening conditions for each loop
A B C
Pro
duct
ion
pote
ntia
l
Candidates
A B C
Pro
duct
ion
pote
ntia
l
Candidates
Good results obtained by running the screening program and the process optimization as parallel activities
Intr
oduc
e di
vers
ity
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Cultivation of Streptomyces in well plates
• Fermentation time 4-5 days• Evaporation position effect
• Morphology in well plates• Oxygen transfer
• Quantitative analyses
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Cultivation of Streptomyces in well plates
• High quality incubators
• Morphology control• Inoculum preparation• Medium• Type of plate
• HTS-Quantitative analyses• Use of filter plates
Optimization of the cultivation conditions is crucial for the quality of the screen
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HTS-analyses : Bioassay of Streptomyces antibiotics
• Bioassays well suited for robotic high throughput analyses
• Protocols for bioassays with the possibility of screening more than 10000 samples / day
• Limitation: more than one active compound in the sample
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Rapid LC-MS analyses of Streptomyces antibiotics
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Sample #
Am
ount
LCMS (1.2 min run)
HPLC (30 min run)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (minutes)
EIC
Comp. A
Internal standard
Comp. BComp. C
Capacity: more than 3500 samples / instrument, week
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Screen for reduced enzyme activityIn vivo screening for mutant enzyme with reduced activity
40
50
60
70
80
90
100
110
120
0 10 20 30 40 50 60 70 80 90
Sample #
Enzy
me
activ
ity
1/100 of screen
• 10 000 mutants screened within a week using robotic protocols • The same work manually performed by an engineer would
take 6 – 8 months
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Main conclusions:
• Effective strain development based on HTS-systems depend on awell equipped technology platform
• HTS-screening and process development / optimization should berun as parallel activities
Molecular biology tools
Equipment for roboticcolony picking and
liquid handling
Equipment forcultivation in micro wells
Equipment forrapid analyses
Fermentors forstrain evaluation
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People involved in projects
NTNUEspen FjærvikEliasbeth RognumSvein VallaSergey ZotchevArne StrømHarald BreholdtSigrid HakvågTrine AakvikKolbjørn Zahlsen
SINTEFTrond E. EllingsenGeir KlinkenbergPer BruheimHåvard SlettaRandi AuneAsgeir WinnbergKjell D JosefsenKristin DegnesNina Øyno