EP1963481A2 - Docosahexaenoic acid (dha) producing thraustochytrid strain - sc1 - Google Patents

Docosahexaenoic acid (dha) producing thraustochytrid strain - sc1

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Publication number
EP1963481A2
EP1963481A2 EP05818740A EP05818740A EP1963481A2 EP 1963481 A2 EP1963481 A2 EP 1963481A2 EP 05818740 A EP05818740 A EP 05818740A EP 05818740 A EP05818740 A EP 05818740A EP 1963481 A2 EP1963481 A2 EP 1963481A2
Authority
EP
European Patent Office
Prior art keywords
dha
acid
producing
strains
fatty acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP05818740A
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German (de)
English (en)
French (fr)
Inventor
Villo Morawala Patell
K. R. Rajyashri
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Avesthagen Ltd
Original Assignee
Avestha Gengraine Technologies Pvt Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Avestha Gengraine Technologies Pvt Ltd filed Critical Avestha Gengraine Technologies Pvt Ltd
Publication of EP1963481A2 publication Critical patent/EP1963481A2/en
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/10—Protozoa; Culture media therefor
    • C12N1/105—Protozoal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C12N1/125—Unicellular algae isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6434—Docosahexenoic acids [DHA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/90—Protozoa ; Processes using protozoa

Definitions

  • DHA Docosahexaenoic acid
  • the invention relates to the screening of a novel unreported strain of Thraustochytrid - SCl producing comparable amounts of Docosahexaenoic acid isolated from the backwaters of Goa by the Pollen baiting method.
  • the fatty acid profiles of the isolated organism indicating the production of the omega-3 fatty acids in significant amounts are disclosed. Also revealed is the 18S rRNA sequence of the isolated organisms indicative of their molecular phylogeny.
  • PUFAs polyunsaturated fatty acids
  • Polyunsaturated fatty acids are long chain fatty acids containing two or more double bonds. Interest in them arises from their potential in therapeutic, food and nutritional applications. They are produced commercially from the selected seed plants and some marine sources. But the production of the purified PUFAs from the current sources will become inadequate for supplying the expanding market ( Gill and Valivety 1997). In order to meet the expected rise in demand and to circumvent the drawbacks of fish oils, alternative production processes for the PUFAs are currently being developed.
  • PUFAs are grouped in 2 series on the basis of the position of the terminal bond being 3 C or 6C from the terminal carbon atom of the fatty acid chain. They are generally classified into 2 main groups, the omega-6 (c ⁇ 6 or n-6) and omega-3 ( ⁇ 3 or n-3 series). Of the omega-6 fatty acids, arachidonic acid is gaining particular importance as it is the major precursor of many prostaglandins and eicosanoids and among the omega-3 fatty acids, Docosahexaenoic acid is currently receiving much attention and have been termed as "Essential Fatty Acids". Docosahexaenoic acid (DHA), the unsaturated, 22-carbon long, omega-3 fatty acid is commonly found in fish and marine plants.
  • DHA Docosahexaenoic acid
  • PUFAs can be obtained from microorganisms.
  • the marine algae are thought to be the primary producers of the omega-3 polyunsaturated fatty acids in the marine food chain.
  • These marine microorganisms represent the greatest percentage of the undescribed marine species (Colwell 1997) of which the marine microalgae form a large potential source of Docosahexaenoic acid.
  • the potential source of n-3 oils is the group of micro-heterotrophs called Thraustochytrids. These are a group of non-photosynthetic, heterotrophic organisms presently classified under the Stramenophila kingdom, together with oomycetes and labyrinthulids.
  • Thraustochytrids are the common name of the microheterotrophs that feed as saprobes or occasionally as parasites. Thraustochytrids have a wide geographical distribution with strains isolated from Antarctica, North Sea, India, Japan and Australia. (Reviewed by Lewis et al., 1999).They are rarely found on living plants and appear to be inhibhited by plant anti-microbial agents. Members of these groups are often abound on dead autochthonous as well as allochthonous plant material such as macro algae and submerged Mangrove leaves. They are common in the neritic and the oceanic water column and the sediments including the deep sea.
  • microbes can provide a stable supply of fatty acids that can easily mass produced, have a less fishy smell and highly purified DHA and other PUFAs.
  • DHA and other PUFAs Several recent studies have catalogued the ability of thraustochytrids strains to produce
  • Schizochytrium aggregatum produced a biomass of 0.9g per litre after IOdays (Vazhappilly and Chen,1998), while a biomass of 48g per litre after 4 days was achieved using Schizochytrium sp. SR21 (yaguchi et al., 1997). It is very clear that development of a microbial PUFA production process requires the selection of the proper microorganism and the optimized culture techniques.
  • PUFA production The types and the amounts of the PUFAs produced by individual strains of thraustochytrids are susceptible to manipulation by varying culture conditions. Enhancement of the PUFA profiles using molecular techniques may also be considered. Different markets will provide demand for strains that produce high levels of PUFAs measured either in terms of the biomass (i.e., PUFA production w/w cell mass) or volume (i.e., Production w/v fermentation medium).
  • Schizochytrium sp. is used to produce DHA by heterotrophic cultivation (OmegaTech, Boulder, Colorado, USA).
  • a major drawback of the Schizochytrium strains is the production of the omega-6 docosapentaenoic acid (DPA) in the microbial oils, in addition to DHA (Nakhara et al.1996: Yokochi et al.1998: Ratledge 2001).
  • DPA omega-6 docosapentaenoic acid
  • the nutraceutical properties of DPA are currently not well known and therefore its presence in oils for food and pharmaceutical applications is undesirable. Separation of DPA from DHA is difficult and expensive.
  • the present invention relates to the isolation of several strains of thraustochytrids, a few strains of which produce significant amounts of Docosahexaenoic acid (DHA; C22:6, n- 3) that were screened from the dead leaves and dendrites of marine vascular plants called Mangroves collected from the backwaters of Goa. These strains will have major significance for the commercial production of Docosahexaenoic Acid.
  • DHA Docosahexaenoic acid
  • Huang et al. have described the grouping of newly isolated docosahexaenoic acid producing thraustochytrids based on their polyunsaturated fatty acid profiles and the comparative analysis of the 18S rRNA genes. Seven strains of marine microbes producing a significant amount of DHA were screened from the seawater collected in coastal areas of Japan and Fiji. They accumulate their intermediate respiratory intermediate fatty acids in addition to DHA. These isolates were proved to be new thraustochytrids by their specific insertion sequences in the 18S rRNA genes.
  • the phylogenetic tree constructed by the molecular analysis of the 18S rRNA genes from the isolates and the typical thraustochytrids shows that the strains with the same PUFA profile form each monophyletic cluster. These results suggest that the C20-22 PUFA profile may be appreciable as an eefective characteristic for grouping thraustochytrids.
  • the medium producing 28.1- 41.1% of the total fatty acids, for all strains, with the exception of the Ulkenia sp. KF13.
  • the DHA yield of the Schizochytrium mangrovie ranged from 747.7 to 2778.9 mg/L after 52 hours of fermentation at 25C.
  • the publication titled "Profiles of polyunsaturated fatty acids produced by Thraustochytrium sp KK 17-3 " relates to the isolation of more than 300 strains of microorganisms producing polyunsaturated fatty acids (PUFA) were newly isolated from the coastal seawater in the Seto Inland Sea and around the Iromote Island, Japan, by the baiting method.
  • the profiles of PUFA from the DHA producing strains were classified into four types.
  • KKl 7-3 was then chosen for further study owing to its high DHA content.(52.1% of the total fatty acid.) and a wide range of other PUFAs including arachidonic, eicosapentaenoic acid and docosapentaenoic acid as well as DHA.
  • PUFAs including arachidonic, eicosapentaenoic acid and docosapentaenoic acid as well as DHA.
  • Molecular phylogenetic analysis of the 18S rRNA gene sequences showed KK17-3 to be a thraustochytrids.
  • the publication titled " Fatty acid composition and the Squalene content of the Marine micro algae Schizochytrium mangrovie” relates to the identification of the fatty acid composition and the squalene content in the thraustochytrids S. mangrovie that was newly isolated from the decaying Kandelia candel leaves in the Hong Kong mangrove habitat.
  • the major fatty acid constituents in all the three mangrovie strains were tetradecanoic acid, hexadecanoic acid, docosapentanoic acid, and docosahexaenoic acid.
  • DHA was the most predominant polyunsaturated fatty acid and the percentage of DHA (of total fatty acids) in all these strains varied from 32.29 to 39.14%.
  • the patent No WO9801536 describes the microorganisms belonging to the genus Shewanella or pseudoalteromonas which can produce docosahexaenoic acid within a short culture period.
  • Our invention relates to identification of 10 unreported strains of Thraustochytrids inhabiting in the dead leaves and the dendrites of marine vascular plants called Mangroves from the backwaters of Goa. Subsequent to isolation, GC-MS analysis for the organisms were carried out to determine their total fatty acid content. The 18S rRNA sequences of these organisms indicate their phylogeny. On futher study, these organisms can be efficient large scale producers of Docosahexaenoic acid.
  • Seq ID 1 represents the 18S rRNA sequence of SCl Seq ID 2: represents the 18S ribosomal sequence of AVEl Seq ID 3: represents the 18S ribosomal sequence of AVE2 Seq ID 4: represents the 18S ribosomal sequence of AVE3 Seq ID 5: represents the 18S ribosomal DNA sequence of AVE4 Seq ID 6: represents the 18S ribosomal DNA sequence of AVE5 Seq ID 7: represents the 18S ribosomal sequence of AVE6 Seq ID 8: represents the 18S ribosomal sequence of AVE7 Seq ID 9: represents the 18S ribosomal DNA sequence of AVE8.
  • Seq ID 10 represents the 18S ribosomal sequence of AVE9. Detailed description of the Invention:
  • Thraustochytrium strains are inhabited in the dead leaves and dendrites of marine vascular plants called Mangroves. Dead leaves from these plants were collected from the backwaters of Goa and isolated by pollen baiting method. The leaves were cultured in sterile seawater with antibiotics (streptomycin and penicillin) and pine pollen grains for three days. These pollens were re-inoculated into fresh sterile seawater with antibiotics. After 3 days of incubation the pollens were examined under the microscope.
  • antibiotics streptomycin and penicillin
  • thraustochytrids 10 different strains of thraustochytrids were found and these were plated on MV (Rock salt -3.4%, Peptone - 0.15%, Yeast Extract- 0.1%, Glucose-2%, KH 2 PO 4 - 0.025%, pH 7) agar plate for 5 days with antibiotics. The thraustochytrids form colony/ layer on the plate. These Thraustochytrids were inoculated in fresh MV media with antibiotics. After 3 days of growth the pure culture were obtained. Thraustochytrids were cultured for a period of 5 days and the cells subjected to GC-MS analysis for determining the total fatty acid content.
  • MV Rock salt -3.4%, Peptone - 0.15%, Yeast Extract- 0.1%, Glucose-2%, KH 2 PO 4 - 0.025%, pH 7
  • the thraustochytrids form colony/ layer on the plate.
  • Total lipid was extracted from the cultures using Folch method of extracting with 2:1 v/v of chloroform: methanol.
  • an internal standard heptadecanoic or pentadecanoic acid
  • Fatty acids were extracted, esterified and GC-MS analysis was carried out with Agilent 6890 N gas chromatograph connected to Agilent 5973 mass spectrometer and GC on HP 6850 Series gas chromatograph equipped with a FID detector.
  • the GC-MS detection was performed at 70 eV (m/z 50-550; source" at 230 0 C and quadruple at 150 0 C) in the EI mode with a capillary column (30 m, HP-5ms, WCOT, i.d. 0.25 mm, film thickness 0.25 ⁇ m, oven 2 min at 150 °C, 6 0 C min "1 to 300 °C, 20 min at 300 °C, Helium carrier gas flow, 1.0 ml/min, split ratio 50:1).
  • the capillary column DB-23 (30m, WCOT, i.d. 0.25 mm, film thickness 0.5 ⁇ m) was used.
  • the oven temperature was programmed as 2 min at 160 0 C, 6 0 C min '1 to 180 0 C, 2 min at 180 °C, 4 0 C min "1 to 230 °C and 10 min at 230 °C, with N 2 carrier gas flow, 1.5 ml/min, and injector temp at 230 °C and detector temp at 250 °C; split ratio 50:1.
  • the fatty acid profile of the 10 strains isolated is represented in the Fig.l

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
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  • Biotechnology (AREA)
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  • Oil, Petroleum & Natural Gas (AREA)
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EP05818740A 2005-12-16 2005-12-16 Docosahexaenoic acid (dha) producing thraustochytrid strain - sc1 Pending EP1963481A2 (en)

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EP (1) EP1963481A2 (zh)
JP (1) JP2009519032A (zh)
CN (1) CN101370929A (zh)
AU (1) AU2005339110A1 (zh)
BR (1) BRPI0520823A2 (zh)
CA (1) CA2639071A1 (zh)
IL (1) IL192162A0 (zh)
MX (1) MX2008007860A (zh)
WO (1) WO2007068997A2 (zh)

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JP5886511B2 (ja) 2005-06-07 2016-03-16 ディーエスエム ニュートリショナル プロダクツ アーゲーＤＳＭ Ｎｕｔｒｉｔｉｏｎａｌ Ｐｒｏｄｕｃｔｓ ＡＧ 脂質および抗酸化剤の製造のための真核微生物
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EP2390343A1 (en) 2010-05-31 2011-11-30 InterMed Discovery GmbH Production of fatty acids by heterologous expression of gene clusters from myxobacteria
CA2842272C (en) 2011-07-21 2020-04-28 Dsm Ip Assets B.V. Fatty acid compositions
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AU2015263881B2 (en) * 2014-05-22 2020-06-18 Synthetic Genomics, Inc. Labyrinthulomycete strains for producing docosahexaenoic acid
US9890402B2 (en) 2015-01-24 2018-02-13 Indian Oil Corporation Limited Thraustochytrid based process for treating waste effluents
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US10851395B2 (en) 2016-06-10 2020-12-01 MARA Renewables Corporation Method of making lipids with improved cold flow properties
BR112019012421A2 (pt) * 2016-12-22 2020-02-27 MARA Renewables Corporation Cultura, microrganismo eucariótico, métodos para fazer uma composição lipídica e para produzir uma biomassa rica em proteínas
KR102100650B1 (ko) * 2018-06-29 2020-04-16 씨제이제일제당 주식회사 신규한 트라우즈토카이트리움 속 균주, 및 이를 이용한 다중불포화지방산 생산방법
EP4180513A1 (en) * 2021-11-15 2023-05-17 Indian Oil Corporation Limited An improved process for production of enriched algal biomass

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WO2007068997A2 (en) 2007-06-21
CA2639071A1 (en) 2007-06-21
IL192162A0 (en) 2008-12-29
CN101370929A (zh) 2009-02-18
JP2009519032A (ja) 2009-05-14
AU2005339110A1 (en) 2007-06-21
MX2008007860A (es) 2009-02-11
BRPI0520823A2 (pt) 2011-04-12
WO2007068997A3 (en) 2007-08-16

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