Bioprospecting for microorganisms in the sea surface ... · to brown algae found worldwide in...

1Materials and Chemistry

Bioprospecting for microorganisms in the sea surface microlayer

Trond E. EllingsenSINTEF Materials and Chemistry,

Trondheim, Norway


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)


Content

Why bioprospecting at SINTEF/NTNUWhere are we looking?Our focus and strategyConclusions


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

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module 4module 5

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module 8



Robotic liquid handling workstation

Robotic colony picking


One of our two fermentation laboratories, each with 16 3-L fermentors


Our LCMS laboratory has one LC MSD TOF, and two LC MSD SQ


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


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


Surface sample compared with seawater sample from the same location collected approx. 10 cm below the surface.

Lygnenfjorden, Namdalseid


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)


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


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


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?


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





Sea surface microlayer Microbial culture collection




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.



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)





Sea surface microlayer

Microbial culture collection





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


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.


Production of DHA-rich lipid by fermentation of a thraustochytrid isolated from the sea

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Time [hours]

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ght [

g/L]

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al li

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], G

luta

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e [g

/L]

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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


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


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


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


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.


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


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.


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.


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.


Project participants

SINTEFKjell D. JosefsenGeir KlinkenbergKristin F. DegnesPer BruheimHåvard SlettaNina Øino

NTNU Svein VallaArne StrømSergey ZotchevSigrid HakvågTrine AakvikEspen FjærvikHarald BredholtKolbjørn Zahlsen

Bioprospecting for microorganisms in the sea surface ... · to brown algae found worldwide in...

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