Bioprospecting for microorganisms in the sea surface microlayer

Transcription

1 Bioprospecting for microorganisms in the sea surface microlayer Trond E. Ellingsen SINTEF, Trondheim, Norway 1

2 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 Research director/prof. Trond E. Ellingsen (sampling, cultivation, analyses, and activities above) 2

3 Content Why bioprospecting at SINTEF/NTNU Where are we looking? Our focus and strategy Conclusions 3

4 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 4

5 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 ( base pairs) and deduced the biosynthetic pathway (patent pending). New possibility: new agents by genetic engineering (ca variants possible theoretically). H 3 C O OH OH OH OH CH 3 O OH OH OH OH O CH Nystatin O O COOH CH 3 OH NysC OH NH 2 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 4 module 55 module 6 module 7 module 8 5

6 Robotic colony picking Robotic liquid handling workstation 6

7 One of our two fermentation laboratories, each with 16 3-L fermentors 7

8 Our LCMS laboratory has one LC MSD TOF, and two LC MSD SQ 8

9 The sea surface microlayer has some unique properties Strong UV-radiation Accumulation of hydrophobic compounds Accumulation of floating particles ~10 µm High density of microorganisms 9

10 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 10

11 Surface sample compared with seawater sample from the same location collected approx. 10 cm below the surface. Lygnenfjorden, Namdalseid 11

12 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) 12

13 The culturable isolates are screened for several products Sea surface microlayer Microbial culture collection Antimicrobial compounds Screening strategy: - Designed growth media - Identification of bioactivity - LC-fractioning - LCMS analysis Carotenoids Screening strategy: - Growth on agar media - Visual inspections - LCMS analysis Polyunsaturated fatty acids (DHA) Screening strategy: - Pollen baiting - Selective growth media - GC & LCMS analysis 13

14 Screening for antimicrobial compounds: Our strategy Step 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 14

15 Screening for antimicrobial compounds: Our strategy Step 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 = Preliminary identification: - LC (DAD)-MS TOF - LC-MS TRAP (Fragmentation Ms n ) - Extended bioassay Production of larger amounts for: - NMR studies - Application testing -Novel compound? -New organism? 15

16 Screening of actinomycetes isolated from the surface microlayer for antimicrobial activity against selected microorganisms. Results from an inhibiting zone assay on solid media Total no. of isolates tested Fraction of isolates showing antimicrobial effect against the organisms below (%) Candida albicans Micrococcus luteus Escherichia coli K

17 The culturable isolates are screened for several products Sea surface microlayer Microbial culture collection Antimicrobial compounds Screening strategy: - Designed growth media - Identification of bioactivity - LC-fractioning - LCMS analysis Carotenoids Screening strategy: - Growth on agar media - Visual inspections - LCMS analysis Polyunsaturated fatty acids (DHA) Screening strategy: - Pollen baiting - Selective growth media - GC & LCMS analysis 17

18 Carotenoids Group of yellow to red compounds that include Astaxanthin the red color in salmon Lycopene the red color in tomato, strong antioxidant β-carotene the red color in carrots, pro-vitamin A Yellow to red colonies constitute a significant fraction of the colonies isolated from the sea surface microlayer. High price ( 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. SINTEF/NTNU collection: currently a few hundred colored strains. 18

19 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? C 40 H 56 O 2, M+H + : (2.8 ppm error from measured mass ) 19

20 The culturable isolates are screened for several products Sea surface microlayer Microbial culture collection Antimicrobial compounds Screening strategy: - Designed growth media - Identification of bioactivity - LC-fractioning - LCMS analysis Carotenoids Screening strategy: - Growth on agar media - Visual inspections - LCMS analysis Polyunsaturated fatty acids (DHA) Screening strategy: - Pollen baiting - Selective growth media - GC & LCMS analysis 20

21 DHA and Thraustochytrids Thraustochytrids are single-cell microorganisms distantly related to brown algae found worldwide in marine environments easy to cultivate industrially DHA-rich fat (up to 50 %) accounts for up to % of cell dry weight the DHA-rich fat is approved for human consumption the DHA-rich fat is suited for fish feed, but currently more expensive than marine oils SINTEF/NTNU collection currently contains around 100 strains, some of which are high DHA-producers 21

22 Isolation of thraustochytrids from the sea surface microlayer Sea surface sample ~40 ml Filtration (0.45 µm) Pine pollen baiting Filter w. sample Pine pollen Sterile seawater with antibiotics and small amounts of glutamate and vitamins With this technique a sea surface microlayer sample (70-90 ml) on average yields 1-2 (assumed) different potential thraustos. Plating at intervals of pollen samples on rich agar medium with antibiotics Isolates are inoculated into shake flasks and the culture frozen at -80 ºC. Microscopic examination of colonies. Potential thraustos are replated on a new agar plate. 22

23 Production of DHA-rich lipid by fermentation of a thraustochytrid isolated from the sea 100 2,0 Dry weight [g/l], Total lipid [g/l], Glutamate [g/l] ,5 1,0 0,5 CER [L/h] Total cellmass (d.w) Total lipid [Glutamate] CO2 (CER) 0 0, Time [hours] Volumetric yield: 12.2 g DHA per liter Volumetric productivity: 77 mg DHA per liter and hour 23

24 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 24

25 Construction of a bacterial artificial chromosome (BAC) library of 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 bacteria that can be cultivated. Hopefully, some genes will code for new products of industrial interest. Goal: clones with an average insert size of kb Where: Trondheimsfjorden 25

26 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 clones 100 x 26

27 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 to transfer the library to different host cells. This implies greater screening potential. 27

28 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 to make it broad-host-range and stable The existing vector is known to function well for metagenome library construction 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 28

29 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. 29

30 DNA library aspects It 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. 30

31 Conclusions The sea surface microlayer contains significant amounts of culturable microorganisms 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. 31

32 Project participants SINTEF Kjell D. Josefsen Geir Klinkenberg Kristin F. Degnes Per Bruheim Håvard Sletta Nina Øino NTNU Svein Valla Arne Strøm Sergey Zotchev Sigrid Hakvåg Trine Aakvik Espen Fjærvik Harald Bredholt Kolbjørn Zahlsen 32