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SINTEF Materials and Chemistry
• SINTEF Materials and Chemistry
• The Department of Biotechnology
• Infrastructure
• Main research areas
1
Industrial biotechnology at SINTEF
N
O
OHOH 25 23 21 19
24 22 201 3 5 7
2 4 6
18 16 14 12 10
8
9
17 15 13 11
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: [email protected] , +47 93059489
Kolbjørn Zahlsen: [email protected], + 47 98693199
Trygve Brautaset: [email protected], +47 98283977
Håvard Sletta: [email protected], +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
12
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)
19
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