1

Microbial biotechnology research at SINTEF Department of Biotechnology

By Senior Researcher Trygve Brautaset07.08.2009

2

Research director: Trond E. Ellingsen

Department of BiotechnologySINTEF Materials and Chemistry

Main working areas • Microbial molecular biology• High throughput screening and analysis• Fermentation and enzyme technology• Research-based analyses • Downstream processing• Immobilized biocatalysts• Food technology • Environmental and oil microbiology• Technical and economical evaluations

FocusDevelopment and optimization of biotechnological processes for the pharmaceutical, fine chemicals, food and feed industries

Close cooperation with NTNU (Department of Biotechnology)

3

Process optimization

Choice of strainImprovement of process

Modification of strain

Focus: microbial biotechnology

4

Available infrastructure for cost-efficient development of bioprocesses

Strain development

Robotized colony-picking and liquid

handling

Fermentors forprosess

optimizationsCultivations in

well-plates

High-throughputscreening and

analyses

Product isolationand purification

Pilot scaleequipment

5

High throughput screening at SINTEF/NTNU

EquipmentRobotized colony pickingCustom-made incubatorsRobotized liquid handling

Robotized liquid handling

Robotized colony picking

Robotized liquid handling

>240 000 operations / 24 hours

> 30 000 parallel running cultures

6

Cultivation and advanced analyses at SINTEF/NTNU

Cultivations5 fermentors (500 ml)32 fermentors (3-l)2 fermentors (14-l)1 fermentor (300-l)1 fermentor (1500-l)

Pilot scale downstream equipment

Analyses4 LC-MS-SQ1 LC-IonTrap2 LC-QQQ1 LC-TOF1 LC-QTOF3 GC-MS-SQ1 GCxGC-TOF (2009)Prep HPLC

3-l fermentors

Pilot scale equipment

7

Example 1: Bioproduction of amino acids from methanol

Alternative production process for two of the world’s most important biotechnological products:

Microbial production of lysine and glutamate from methanol

Estimated volumes:Lysine 800,000 tons / yearGlutamate 1,500,000 tons / year

Methane

Methanol

LysineGlutamate

met

abol

icen

gine

erin

g

ther

mot

oler

ant

Bac

illus

8

Metabolic engineering for effective microbial conversionof Methanol into the amino acid L-lysine

METHANOL

FORMALDEHYDE

RUMP

FORMATE

CO2

PYRUVATE

OXALOACETATE

-OXOGLUTARATE

GLUTAMATE

L-ASPARTATE

METHIONINE THREONINE

LYSINE

TCA

MESO -DIAMINOPIMELAT

metabolicengineering

METHANOL

FORMALDEHYDE

RUMP

FORMATE

CO2

PYRUVATE

OXALOACETATE

-OXOGLUTARATE

GLUTAMATE

L-ASPARTATE

METHIONINE THREONINE

LYSINE

TCA

MESO -DIAMINOPIMELAT

9

Example 2: Microbial Production of human-medical Proteins

ProteinL

pJB6586762 bps

1000

2000

30004000

5000

6000

NcoI 531

BseRI 922

TatI 2461

FspI 2719

PsiI 3700

PvuII 4038ClaI 4140

SfiI 4533

SexAI 5312HindIII 5384

BamHISmaIXmaIKpnISacI

EcoRINdeI

StuI 6068XbaI 6075

AgeI 6324

xylS

oriT

bla

oriV

neo

trfA

terminator

Pm

Development ofgenetic tools

Strain construction

Production in high celldensity cultivations

(>100g/l DW)Antibody fragments

10

Example 3: Bioprospecting

Lygnenfjorden, NamdalseidPolyunsaturated fatty acids (DHA)

Carotenoides

Metagenome-library Antimicrobial andAnticancer compounds

Sea surface / sediments

New patent in 2008

11

Example 4. Bioproduction and tailoring of alginate

Alginates are extracted from brown seaweed

Market value (2001): 1.3 - 1.5 billion NOK

Volume (2001): 36 000 tons

Perspectives :Expected added value in the fields of pharmaceutical and medical use of alginates

ManA ManAGulAGulAGulA ManA

OOH

HO

HOOCO

OHO

OH

HOOC

O OH

OH

HOOCO

OHOOC

HO

OOH

O

OH

HOOC

O

OOH

O

OHHOOC

HO

O

O

ManA ManAGulAGulAGulA ManA

OOH

HO

HOOCO

OHO

OH

HOOC

O OH

OH

HOOCO

OHOOC

HO

OOH

O

OH

HOOC

O

OOH

O

OHHOOC

HO

O

O

AlginateAlginate overproducing

mutants

Bacterial alginate

substrates

Tailor-made alginates

Engineeredstrains for different alginates

Genetic engineering Fermentation

Fermentation Enzymatic modification

Photo: Håvard SlettaPhoto: Håvard Sletta

12

Example 5: Systems biology

Genes (DNA) –(ca. 3000 - 7000 in bacteria)

Proteins (ca.500 – 1500 different, vary)

Metabolites (>1000 different, vary)

Gene copies (RNA)(ca. 500 - 1500 different, vary)

REGULATION

REGULATION

REGULATION

Physiologicalstudies

Quantitative analyses

Datahandling

Predictive models

13

Systems biology at SINTEF / NTNU

Global metabolic switching in Streptomyces coelicolor(14 partners)

Systems biology of a genetically engineered Pseudomonas fluorescens with inducible exo-polysaccaride production (5 partners, Svein Valla coordinator)

ACT

REDCDA

RED

CPKCDALAC

up

~const or down

14

Example 6: 2nd Generation Bioethanol Production

Lignocellulosic biomass Pre-treatment

Enzymatic hydrolysis

Fermentation Distillation or separation

Wood, grain straw, bagasse

Physical separation of the lignocellulosic fibers Enzymatic hydrolysis of

cellulose and hemicellulose to release C6 and C5 mono-sugars

Fermentation of C6 and C5 mono-sugars to ethanol by microorganisms

Separation and concentration of ethanol

Technology development challenges Lignocellulosic biomass: harvesting logistics Pre-treatment: new technologies for increased sugar polymer yield Enzyme hydrolysis: development of new enzymes and enzyme recycling technologies Fermentation: development of “tailor-made” microorganisms and optimization of fermentation Distillation or separation: more energy efficient technologies

The main process steps for second generation bioethanol production

15

Example 7: Oil related research

Upgrading of heavy oil Viscosity reduction Increased fuel value Environmentally more

friendly products Asphaltenes

Paraffin (alkanes)

PAHs

16

Summarised…

Applied and Basic research Close cooperation with national and international

universities Close cooperation with industry