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1Materials and Chemistry
Bioprospecting for microorganisms in the sea surface microlayer
Trond E. EllingsenSINTEF Materials and Chemistry,
Trondheim, Norway
2Materials and Chemistry
The Norwegian bioprospecting program
Participants in our project:Institute of Biotechnology, NTNU
Prof. Arne Strøm (thraustochytrids/DHA)Prof. Svein Valla (Gene library and carotenoids)Ass. Prof. Sergey Zotchev (antibiotics)
SINTEF Materials and ChemistryResearch director/Prof. Trond E. Ellingsen (sampling, cultivation, analyses, and activities above)
3Materials and Chemistry
Content
Why bioprospecting at SINTEF/NTNUWhere are we looking?Our focus and strategyConclusions
4Materials and Chemistry
Why bioprospecting at SINTEF/NTNU?
20 years of experience in strain improvement and industrial process development (lysine, polysaccharides, antibiotics, astaxanthin, etc)
Experience in marine bioprospecting (crude oil emulsifiers)
Efficient facilities for high throughput screening and strain development
New bioprocesses and new biomolecules from a wild type isolate of Streptomyces noursei• We have developed a commercial process for production of the polyene antifungal
antibiotic nystatin.
• We have done a complete DNA sequence analysis of the nystatin polyene antibiotic biosynthetic gene cluster (123 580 base pairs) and deduced the biosynthetic pathway (patent pending).
• New possibility: new agents by genetic engineering (ca 1017 variants possible theoretically).
O
OOH
CH3
OH OHO OH
OH
OH
OH
O
COOHCH3
CH3
O
OH NH2
CH3
OH
OHNystatin
33 31 29 27 25 23 21 19
32 30 28 26 24 22 20 18
NysC
module 3module 4
module 5module 6
module 7module 8
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 3
module 4module 5
module 6module 7
module 8
5Materials and Chemistry
9Materials and Chemistry
The sea surface microlayer has some unique properties
~10 µm
Strong UV-radiation
Accumulation of hydrophobic compounds
Accumulation of floating particles
High density of micro-organisms
10Materials and Chemistry
Sampling of the surface microlayer
Teflon plate slowly pulled through the surface
Surface microlayer (10-90 µm) adheres to plate and can be scraped off and collected
11Materials and Chemistry
Surface sample compared with seawater sample from the same location collected approx. 10 cm below the surface.
Lygnenfjorden, Namdalseid
12Materials and Chemistry
Two strategies
Direct isolation of culturable strains on agar media etc.
(Traditional method but 90 to >99 % of the microorganisms in seawater
samples can not be cultured)
Isolation and cloning of DNA from the
unculturable fraction(Gene library, see later)
13Materials and Chemistry
The culturable isolates are screened for several products
Antimicrobial compounds
Carotenoids Polyunsaturated fatty acids (DHA)
Sea surface microlayer Microbial culture collection
Screening strategy:- Pollen baiting- Selective growth media- GC & LCMS analysis
Screening strategy:- Growth on agar media- Visual inspections- LCMS analysis
Screening strategy:- Designed growth media- Identification of bioactivity- LC-fractioning- LCMS analysis
14Materials and Chemistry
Screening for antimicrobial compounds: Our strategyStep 1: Isolation of microorganisms and identification of bioactivity
Plating samples on selective agar media
Colony picking
Storage in microwell plates(Culture collections)
Fermentation in microwell plates using several production media
Extraction and filtration
Identification of biological activity in extracts against target organisms
15Materials and Chemistry
Screening for antimicrobial compounds: Our strategyStep 2: Identification of bioactive components
Choice of interesting extracts from strains
Repeated LC-fractioning and LC-MS-analyses of extracts containing bioactive compounds followed by bioassays
LC-F1 LC-F2 LC-F3 LC-F4
MW = 963.1434
Preliminary identification:- LC (DAD)-MS TOF- LC-MS TRAP (Fragmentation Msn)- Extended bioassay
Production of larger amounts for: - NMR studies- Application testing
-Novel compound?-New organism?
16Materials and Chemistry
Screening of actinomycetes isolated from the surface microlayer for antimicrobial activity against selected microorganisms.
Results from an inhibiting zone assay on solid media
Fraction of isolates showing antimicrobial effect against the organisms below (%)
Total no. of isolates tested
Candida albicans
Micrococcus luteus
Escherichia coli K12
456 50 38 32
17Materials and Chemistry
The culturable isolates are screened for several products
Antimicrobial compounds
Carotenoids Polyunsaturated fatty acids (DHA)
Sea surface microlayer Microbial culture collection
Screening strategy:- Pollen baiting- Selective growth media- GC & LCMS analysis
Screening strategy:- Growth on agar media- Visual inspections- LCMS analysis
Screening strategy:- Designed growth media- Identification of bioactivity- LC-fractioning- LCMS analysis
CarotenoidsGroup of yellow to red compounds that include
Astaxanthin – the red color in salmonLycopene – the red color in tomato, strong antioxidantβ-Carotene – the red color in carrots, pro-vitamin A
High price (10 000-20 000 NOK/kg)Currently mainly produced by chemical synthesis and/or extraction from plants.Synthesized by many microorganisms, and high-producing strains may be commercially competitive.
Yellow to red colonies constitute a significant fraction of the colonies isolated from the sea surface microlayer.
SINTEF/NTNU collection: currently a few hundred colored strains.
18Materials and Chemistry
19Materials and Chemistry
IDENTIFICATION OF CAROTENOIDS FROM THE ISOLATES WITH LC-MS
Freeze-dried bacterial pellets are extracted with DMSO. Individual carotenoids in extract are separated on RP C18 HPLC column and characterized with Diode array detector (DAD)and Time of flight (TOF) mass spectrometer (better than 3 ppm mass accuracy)
DAD Spectrum TOF Mass Spectrum
Is the unknown compound Zeaxanthin?C40H56O2, M+H+: 569.4352 (2.8 ppm error from measured mass 569.4337)
20Materials and Chemistry
The culturable isolates are screened for several products
Antimicrobial compounds
Carotenoids Polyunsaturated fatty acids (DHA)
Sea surface microlayer
Microbial culture collection
Screening strategy:- Pollen baiting- Selective growth media- GC & LCMS analysis
Screening strategy:- Growth on agar media- Visual inspections- LCMS analysis
Screening strategy:- Designed growth media- Identification of bioactivity- LC-fractioning- LCMS analysis
21Materials and Chemistry
DHA and Thraustochytrids
Thraustochytrids are single-cell microorganisms distantly related to brown algae
found worldwide in marine environ-mentseasy to cultivate industriallyDHA-rich fat (up to 50 %) accounts for up to 60-70 % of cell dry weightthe DHA-rich fat is approved for human consumptionthe DHA-rich fat is suited for fish feed, but currently more expensive than marine oilsSINTEF/NTNU collection currently contains around 100 strains, some of which are high DHA-producers
22Materials and Chemistry
Isolation of thraustochytrids from the sea surface microlayer
Pine pollen baitingPine pollen
Sea surface sample
~40 ml
Filter w. sample
Sterile seawater with antibiotics and small amounts of glutamate and vitamins
Plating at intervals of pollen samples on rich agar medium with antibiotics
Filtration (0.45 µm)
With this technique a sea surface microlayer sample (70-90 ml) on average yields 1-2 (assumed) different potential thraustos.
Microscopic examination of colonies. Potential thraustos are replated on a new agar plate.
Isolates are inoculated into shake flasks and the culture frozen at -80 ºC.
23Materials and Chemistry
Production of DHA-rich lipid by fermentation of a thraustochytrid isolated from the sea
0
25
50
75
100
0 50 100 150 200 250
Time [hours]
Dry
wei
ght [
g/L]
, Tot
al li
pid
[g/L
], G
luta
mat
e [g
/L]
0,0
0,5
1,0
1,5
2,0
CE
R [L
/h]
Totalcellmass(d.w)
Total lipid
[Glutamate]
CO2 (CER)
Volumetric yield: 12.2 g DHA per liter
Volumetric productivity: 77 mg DHA per liter and hour
24Materials and Chemistry
Two strategies
Direct isolation of culturable strains on agar media etc.
(Traditional method but 90 to >99 % of the microorganisms in seawater
samples can not be cultured)
Isolation and cloning of DNA from the
unculturable fraction(Metagenome library)
The uncultured organisms have, until now not been studied and may represent a source for new compounds
25Materials and Chemistry
Construction of a bacterial artificial chromosome (BAC) libraryof microorganisms from the sea surface microlayer of
Trondheimsfjorden
A BAC library is a collection of clones that contains DNA inserts from one or more genomes.
Aim: Express genes from unculturable microorganisms in bacteriathat can be cultivated. Hopefully, some genes will code for newproducts of industrial interest.
Goal: 10.000 clones with an average insert size of 50-100 kb
Where: Trondheimsfjorden
26Materials and Chemistry
Preparation of the BAC vector and cloning of the insert
Cutting of vector with restriction enzymes
+difficult
Cloning of DNA-fragments into a shuttle vector.
Transformation with electroporation into host cells.
Robotic picking and screening of clones100 x
27Materials and Chemistry
Predicted properties of the RK2 broad-host-range BAC vector
• Regular BAC-vectors are stable in E. coli but do not replicate in most other species.
• The broad-host-range plasmid vector makes it possible totransfer the library to different host cells. This implies greater screening potential.
28Materials and Chemistry
Strategies for construction of metagenome libraries in broad-host-range plasmid vectors
a) Construction of a reduced size version of the naturally occurring plasmid RK2
• High probability of stable maintenance in a variety of host species • Laborious vector DNA preparation due to low copy number• Large vector size may complicate construction of recombinants
b) Construction of a new vector based on an already existing mini-derivative tomake it broad-host-range and stable
• The existing vector is known to function well for metagenome libraryconstruction in E. coli
• The new vector has to be tested for stability in non E. coli hosts• The new vector has an inducible copy number control
29Materials and Chemistry
C) Construction and analysis of a small fragment library in a standard E. coli vector
Preliminary results:
- About 12 % of the sequences had no match with already sequenced DNA.
- About 40 % of the sequences had only very poor (coincidental?) match with already sequenced DNA.
- About 48 % of the sequences had from poor to good match with already sequenced DNA.
30Materials and Chemistry
DNA library aspectsIt is a major challenge to construct the desired DNA library.
We will clone large DNA fragments to cover many genes in a metabolic pathway.
Production of interesting products from the DNA library is limited by the expression of the genes of interest.
By using broad host range vectors the library may be tested for expression of products in many different organisms.
31Materials and Chemistry
ConclusionsThe sea surface microlayer contains significant amounts of culturablemicroorganisms that produce the compounds we are looking for (antimicrobials, carotenoids, DHA).A major challenge is to design efficient screening techniques that enable us to select strains with an industrial potential, i.e. high production, novel and efficient compounds (antibiotics), rapid growth, robustness, etc.The use of robotic equipment facilitates the handling of the large number of isolates in the initial screening phase.The use of modern analytical equipment such as LC-MS, considerably speeds up the identification of unknown compounds. Much work is still needed before a DNA library can be constructed and utilized.