SINTEF Materials and Chemistry

• SINTEF Materials and Chemistry

• The Department of Biotechnology

• Infrastructure

• Main research areas

1

Industrial biotechnology at SINTEF

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O

OHOH 25 23 21 19

24 22 201 3 5 7

2 4 6

18 16 14 12 10

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BE-14106

Industrial biotech meeting 2012.07.11

Research director : Trond E. Ellingsen

SINTEF Materials and Chemistry 2

Organisation

SINTEF Materials

& Chemistry

SINTEF Materials and Chemistry

SINTEF Materials and Chemistry - 400 employees from 45 countries + Norway.

SINTEF Materials and Chemistry

Organization

Research director: Trond E. Ellingsen

30 employees + PhD students, post docs

3 research units

Focus

Development and optimization of

biotechnological processes for the

pharmaceutical, fine chemicals, food

and feed industries

Close cooperation with NTNU, department of

Biotechnology

The Department of Biotechnology -

SINTEF Materials and Chemistry

Molecular biology Trygve Brautaset

Mass

spectrometry

Kolbjørn Zahlsen

Bioprocess

technology Håvard Sletta

Contact

Trond Ellingsen: Trond.Ellingsen@sintef.no , +47 93059489

Kolbjørn Zahlsen: Kolbjorn.zahlsen@sintef.no, + 47 98693199

Trygve Brautaset: Trygve.brautaset@sintef.no, +47 98283977

Håvard Sletta: Havard.Sletta@sintef.no, +47 93086028

URL: http://www.sintef.no/Home/Materials-and-Chemistry/Biotechnology/

SINTEF Materials and Chemistry

• Microbial bioprocess optimization

• Microbial molecular biology

• High throughput screening and analysis

• Fermentation and enzyme technology

• Immobilized biocatalysts

• Environmental and oil microbiology

• Bioprospecting

• Metabolic engineering

• Synthetic and systems biology

• Technical and economical evaluations

Main working areas and core competence

Bacteria

Yeast

Fungi

ANTIBIOTICS

SUGAR

PRODUCT

ENZYMES

VITAMINS

PIGMENTS

ORGANIC

ACIDS

BIO-POLYMERS

BIO-ETHANOL

LYSINE

GLUTAMIC ACID

Process optimization

Choice

of strain Improvement

of process

Modification

of strain

SINTEF Materials and Chemistry

Customers Internationally R&D network

SINTEF Materials and Chemistry

High throughput screening at SINTEF/NTNU

Equipment

Robotized colony picking Custom-made incubators Robotized liquid handling

Robotized liquid handling

Robotized colony picking

Robotized liquid handling

>240 000 operations / 24 hours

> 30 000 parallel running cultures

SINTEF Materials and Chemistry

Cultivation and advanced analyses at SINTEF/NTNU

Cultivations

8 fermentors (1000 ml) 32 fermentors (3-l) 2 fermentors (14-l) 1 fermentor (300-l) 1 fermentor (1500-l)

Pilot scale downstream equipment

Analyses

4 LC-MS-SQ 1 LC-IonTrap 6 LC-QQQ 1 LC-TOF 1 LC-QTOF 3 GC-MS-SQ 1 GCxQQQ

Prep HPLC

3-l fermentors

Pilot scale equipment

SINTEF Materials and Chemistry 9

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 / year

Glutamate 1,500,000 tons / year

Methane

Methanol

Lysine

Glutamate

me

tab

oli

c

en

gin

ee

rin

g

the

rmo

tole

ran

t

Ba

cil

lus

Heggeset TMB, Krog A, Balzer S, Wentzel A, Ellingsen TE, Brautaset T (2012) Genome sequence

of thermotolerant Bacillus methanolicus: Features and regulation related to methylotrophy and

production of L-lysine and L-glutamate from methanol. Appl Environ Microbiol., In press

Brautaset T, Ellingsen T. E (2011) Lysine: Industrial Uses and Production. Comprehensive

Biotechnology, 2nd edition. : Elsevier B.V.

Heggeset TMB, Krog A, Balzer S, Wentzel A, Ellingsen TE, Brautaset T (2012) Genome sequence of

thermotolerant Bacillus methanolicus: Features and regulation related to methylotrophy and

production of L-lysine and L-glutamate from methanol. Appl Environ Microbiol., In press

SINTEF Materials and Chemistry 10

ESF-EuroSYNBIO Project: A Synthetic Biology Approach for Engineering of Bacterial Methylotrophy FP7-project: proMyse: Products from methanol by synthetic cell factories

IP-1 .....

Brautaset

IP-2 …..

Vorholt

IP-3 ……

Wendisch

IP-4 ……

Quax

AP ……

Portais

Coordination

MethylotrophyGram positives

Geneticengineering

MethylotrophyGram negatives

Enzymology

Metabolomics& Proteomics

Metabolicengineering

Amino acidproduction

Transcript-omics

Metabolicmodeling

NMR

Fluxomics

Directedevolution

Genome reduction

Fermentation

Use of emerging technologies (systems and synthetic biology) in

bioprocess development

Wentzel, A, Bruheim, P, Øverby, A, Jakobsen, ØM, Sletta, H, Omara, WAM, Hodgson, DA, Ellingsen, TE.

(2012). Optimized submerged batch fermentation strategy for systems scale studies of metabolic

switching in Streptomyces coelicolor A3(2). BMC Systems Biology. in press.

Wentzel A, Sletta H, Ellingsen TE, Bruheim P, Stream Consortium (2012) Intracellular

metabolite pool changes in response to nutrient depletion induced metabolic switching

in Streptomyces coelicolor. Metabolites 2:178-194.

Correa E, Sletta H, Ellis DI, Hoel S, Ertesvåg H, Ellingsen TE, Valla S, Goodacre R (2012) Rapid reagentless

quantification of alginate biosynthsesis in Pseudomonas fluorescens bacteria mutants using FT-IR

spectroscopy coupled to multivariate partial least square regression. Analytical and Bioanalytical

Chemistry, In press

STREAM: Global metabolic switching in Streptomyces coelicolor SCaRAB: Systems Biology of a genetically engineered Pseudomonas fluorescens with inducible exo-polysaccharide production: analysis of the dynamics and robustness of metabolic networks

SINTEF Materials and Chemistry 11

Fuel

Chemicals

Feed

Carbohydrate

Heat, electricity

Biorefinery: Bio-mass – the new oil?

Moe ST, Janga KK, Hägg MB, Øyaas K, Dyrset N (2012) Saccharification of lignocellulosic

biomass for biofuel and biorefinery applications – A renaissance for the concentrated acid

hydrolysis, Energy Procedia / The Renewable Energy Research Conference (RERC) 2012,

In press

Current research activities Enzyme pre-treatment

Fermentation optimization

Isolation and screening of microorganisms

for production of biofuel

Development of “tailor-made”

microorganisms, metabolic engineering

SINTEF Materials and Chemistry

Alginate and alginate engineering

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Sources :

• Brown seaweed 120 000 metric

tons harvested annually in Norway.

• Alginate producing microorganisms

(important research area for the

department)

Alginates from brown seaweed:

Alginate based Products

Industrial and pharmaceutical

applications

Microbial alginates

Perspectives: tailored alginates

designed for new industrial and

pharmaceutical and biomedical

applications

NOVEL ALGINATE

MODIFYING ENZYMES

Aarstad OA, Tøndervik A, Sletta H, Skjåk-Bræk G (2012) Alginate sequencing - An

analysis of block distribution in alginates using specific alginate degrading

enzymes. Biomacromolecules 13: 106-116

Tøndervik A, Klinkenberg G, Aarstad OA, Drabløs F, Ertesvåg H, Ellingsen TE, Skjåk-Bræk G, Valla S. Sletta H

(2010) Isolation of mutant alginate lyases with cleavage specificity for di-guluronic acid linkages. J. Biol. Chem.

285: 35284-92

Stenvik J, Sletta H, Grimstad Ø, Pukstad B, Ryan L, Aune R, Strand WI, Tøndervik A, Torp

SH, Skjåk-Bræk G, Espevik T (2012) Alginates induce differentiation and expression of

CXCR7 and CXCL12/SDF-1 in human keratinocytes-the role of calcium. Journal of

Biomedical Materials Research, In press.

SINTEF Materials and Chemistry 13

Enzyme evolution and production

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 of

genetic tools

Strain construction

Production in high cell

density cultivations

(>100g/l DW)

Antibody fragments

Sletta et.al. The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the

Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density

Cultivations of Escherichia coli. Appl Environ Microbiol, 2007. 73(3): p. 906-912.

A-module

R-module

Mannuronate

A-module

R-module

Mannuronate

Alginate epimerase

Berg L, Kucharova V, Bakke I, Valla S, Brautaset T (2012) Exploring the 5'-UTR DNA region

as a target for optimizing recombinant gene expression from the strong and inducible

Pm promoter in Escherichia coli. J. Biotechnol. 158:224-230

Brautaset T, Netzer R, Lale R, Stüttgen F, Valla S, Berg L, Aune TE, Stafsnes M (2011)

Continuous control of the flow in biochemical pathways through 5'-UTR sequence

modifications in mRNA expressed from the broad-host-range Pm promoter. Appl.

Environ.Microbiol., 77: 2648-2655

SINTEF Materials and Chemistry 14

Marine bioprospecting Lygnenfjorden, Namdalseid

Polyunsaturated fatty

acids (DHA) Carotenoids

Metagenome-library

Antimicrobial and

Anticancer compounds

Sea surface / sediments

New patent in 2008

SINTEF Materials and Chemistry

Structures of new molecules from the Trondheim fjord bacteria

TP-1161 Antibacterial thiopeptide

ML-449 Cytotoxic macrolactam

N

O

OHOH

Jørgensen et al. 2010

Appl. Environ. Microbiol. 76: 283-293

Engelhardt et al. 2010

Appl. Environ. Microbiol. Accepted

A large collection of marine bacterial isolates and relevant technology for efficient detection of

novel bioactive compounds are established at SINTEF/NTNU.

SINTEF/NTNU can perform all steps from cultivation and high-throughput screening to

identification/isolation of active compounds, including development of a production process.

Spin of company MarBiLeads established in 2011

SINTEF Materials and Chemistry 16

Module 3

NysC

Module 4 Module 5

Module 6

KS AT DH KR ACP KS AT DH KR ACP KS AT DH ER KR ACP KS AT DH KR ACP KS AT DH KR ACP KS AT DH KR ACP

Module 7

Module 8

O

OOH

CH3

OH OHO OH

OH

OH

OH

O

COOHCH3

CH3

O

OHNH

2

CH3

OH

Nystatin A1

OH

1 3 5 7 9 11 13 15

16

33 31 29 27 25 23 21 19

34 32 30 28 26 24 22 20 18

2 4 6 8 10 12 14

1735

36

37

Genetic engineering – Biosynthesis of nystatin; design of new

and improved antibiotics

Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces

noursei ATCC 11455: analysis of the gene cluster and deduction of the

biosynthetic pathway. (Chemistry & Biology. 2000: 7, 395-403)

Heia S, Borgos SEF, Sletta H, Escudero L, Seco EM, Malpartida F, Ellingsen TE, Zotchev

SB (2011) Initiation of polyene macrolide biosynthesis: Interplay between polyketide

synthase domains and modules as revealed via domain swapping, mutagenesis, and

heterologous complementation. Appl Microbiol Biotechnol. 77: 6982-6990

Brautaset T, Sletta H, Degnes KF, Sekurova ON, Bakke I, Volokhan O, Andreassen T,

Ellingsen TE, Zotchev SB (2011) New nystatin-related antifungal polyene macrolides with

altered polyol region generated via biosynthetic engineering of Streptomyces noursei.

Appl. Microbiol. Biotechnol., 77: 6636-6643

”Longterm cooperation

between Xellia and

SINTEF in strain and

process development for

production of antibiotics

including Nystatin”

Biosergen AS er

Company established

with basis in

technology patented

by SINTEF /NTNU

SINTEF Materials and Chemistry 17 17

• Strain collection – (>100 strains)

• 90 -100 g/l cell dry weight

• 58 % total lipids

• 26-28 % DHA of fatty acids

0

5

10

15

20

25

30

35

40

45

50

C14:0 C16:0 C16:1 C18:0 C18:1 C22:5 n-6

C22:6 n-3

Fatt

y a

cid

co

nte

nt (%

)

87 hours

111 hours

135 hours

159 hours

182 hours

Strain T66 with glycerol as C-source,

glutamate as N-source

Microbial production of DHA - Thraustochytrides

Jakobsen AN, Aasen IM, Josefsen KD, Strøm AR (2008) Accumulation of docosahexaenoic

acid-rich lipid in thraustochytrid Aurantiochytrium sp. strain T66: effects of N and P

starvation and O2 limitation. Appl. Microbiol. Biotechnol., 80: 297-306

SINTEF Materials and Chemistry 18

Oil Reservoir Bioprospecting

Microorganisms for use in

added value generation

Wax control through biocatalysis

Novel enzymes for industrial

applications

• Lipases

• Esterases

• ..........

Kotlar HK, Lewin A, Johansen J, Throne-Holst M, Haverkamp T, Markussen S, Winnberg A, Ringrose P,

Aakvik T, Ryeng E, Jakobsen K, Drabløs F, Valla S (2011) High coverage sequencing of DNA from

microorganisms living in an oil reservoir 2.5 kilometres subsurface. Environmental Microbiology Reports, 3:

674-681.

SINTEF Materials and Chemistry

Efficient fish vaccines – a prerequisite for aquaculture (Production farmed salmon in Norway 2011 – 1005 metric tons)

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Main focus -

vaccine development:

Bacterial infections: • New vaccine targets based on

understanding of host – pathogen

interaction

• Cost efficient production processes

Viral infections: • Vaccine design and efficacy

• Cost efficient production

SINTEF Materials and Chemistry 20

Concluding remarks

Our department is involved in both strain improvement and

process development resulting in commercial feasible

bioprocesses for the industry

We publish in high-ranking scientific journals

We have cost-effective laboratories specially designed for high-

through-put operations

We are now using Systems- and Synthetic Biology approaches in

our research