1Materials and Chemistry

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

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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