WO2008104559A1 - Procédé de production d'acides gras polyinsaturés dans des organismes transgéniques - Google Patents

Procédé de production d'acides gras polyinsaturés dans des organismes transgéniques Download PDF

Info

Publication number
WO2008104559A1
WO2008104559A1 PCT/EP2008/052358 EP2008052358W WO2008104559A1 WO 2008104559 A1 WO2008104559 A1 WO 2008104559A1 EP 2008052358 W EP2008052358 W EP 2008052358W WO 2008104559 A1 WO2008104559 A1 WO 2008104559A1
Authority
WO
WIPO (PCT)
Prior art keywords
acid
desaturase
nucleic acid
fatty acids
seq
Prior art date
Application number
PCT/EP2008/052358
Other languages
German (de)
English (en)
Inventor
Amine Abbadi
Ivo Feussner
Mareike Hoffmann
Original Assignee
Norddeutsche Pflanzenzucht
Georg-August-Universität Göttingen
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 Norddeutsche Pflanzenzucht, Georg-August-Universität Göttingen filed Critical Norddeutsche Pflanzenzucht
Publication of WO2008104559A1 publication Critical patent/WO2008104559A1/fr

Links

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a process for the preparation of polyunsaturated fatty acids, in particular long-chain polyunsaturated fatty acids such as arachidonic acid and / or eicosapentaenoic acid, in a transgenic organism by introducing into the organism nucleic acids which are suitable for polypeptides having ⁇ 6-desaturase, ⁇ 6-elongase and / or ⁇ 5-desaturase activity.
  • the ⁇ 6-desaturase and ⁇ 5-desaturase are from Mantoniella squamata and the ⁇ 6 elongase is from Physcomitrella patens.
  • a gene which codes for a ⁇ 3-desaturase is also expressed in the organism.
  • further nucleic acid sequences coding for polypeptides of the biosynthesis of the fatty acid and lipid metabolism can be expressed in the organism.
  • Particularly advantageous for this purpose are the nucleic acid sequences which code for a ⁇ 8-desaturase, ⁇ 12-desaturase, ⁇ 15-desaturase, ⁇ 4-desaturase, ⁇ 9-elongase and / or ⁇ 5-elongase activity.
  • the invention further relates to the nucleic acid sequences according to the invention, vectors comprising the nucleic acid sequences and / or the nucleic acid constructs and transgenic organisms containing the abovementioned nucleic acid sequences, nucleic acid constructs and / or vectors. Furthermore, the invention relates to a process for the preparation of oils, lipids and / or triacylglycerides having an increased content of long-chain polyunsaturated fatty acids. Another part of the invention relates to oils, lipids and / or fatty acids prepared by the process according to the invention and their use, in particular the use in feed or food, cosmetics or pharmaceuticals.
  • Fatty acids and triacylglycerides have a variety of uses in the food, animal nutrition, cosmetics and pharmaceutical industries. Depending on whether they are free saturated and unsaturated fatty acids or triacylglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a wide variety of applications.
  • Polyunsaturated fatty acids such as linoleic and linolenic acid are essential for - J -
  • polyunsaturated ⁇ 3 fatty acids and 006 fatty acids are an important component of animal and human food.
  • C20 5 ⁇ 5 ' 8 ' n ' 14 ' 17
  • Aspects such as the development of the child's brain, the functionality of the eye, the synthesis of hormones and other signaling substances, as well as the prevention of cardiovascular complaints, cancer and diabetes include. There is therefore a need for the production of polyunsaturated long-chain fatty acids.
  • polyunsaturated fatty acids which are preferred in fish oils
  • DHA polyunsaturated fatty acids
  • EPA baby food are added to increase the nutritional value.
  • the unsaturated fatty acid DHA is thereby attributed a positive effect on the development and maintenance of brain functions.
  • polyunsaturated fatty acids as PUFA, PUFAs, LCPUFA or
  • LCPUFAs poly unsaturated fatty acids, PUFA, polyunsaturated fatty acids, LCPUFA, long-chain polyunsaturated fatty acids).
  • the free fatty acids are advantageously prepared by saponification.
  • Docosapentaenoic acid (DPA, Q22: 5 ⁇ 7 ' 10 ' 13 ' 16 ' 19 ) is not synthesized in oilseed crops such as rapeseed, soybean, sunflower, safflower. Common natural sources of these fatty acids are fish such as herring, salmon, sardine, perch, eel, carp, trout, halibut, mackerel, zander or tuna or algae.
  • oils with saturated or unsaturated fatty acids are preferred.
  • lipids with unsaturated fatty acids in particular polyunsaturated fatty acids are preferred.
  • the polyunsaturated ⁇ 3-fatty acids thereby a positive effect on the cholesterol level in the blood and thus the possibility of preventing heart disease is attributed.
  • Food can significantly reduce the risk of heart disease, stroke or high blood pressure. Also, inflammatory, especially chronic inflammatory processes in the context of immunological diseases such as rheumatoid arthritis can be positively influenced by ⁇ 3 fatty acids. They are therefore added to foods, especially dietary foods, or are used in medicines.
  • ⁇ 3 and oo6 fatty acids are precursors of tissue hormones, the so-called eicosanoids such as the prostaglandins derived from dihomo- ⁇ -linolenic acid, arachidonic acid and eicosapentaenoic acid, and thromboxanes and leukotrienes derived from arachidonic acid and eicosapentaenoic acid , Eicosanoids (so-called PG 2 -SeHe), which are made of ⁇ 6- Fatty acids are formed, usually promote inflammatory reactions, while eicosanoids (so-called PG3 series) of ⁇ 3 fatty acids have little or no pro-inflammatory effect.
  • eicosanoids such as the prostaglandins derived from dihomo- ⁇ -linolenic acid, arachidonic acid and eicosapentaenoic acid, and thromboxanes and leukotrienes derived from arachidonic acid
  • WO 91/13972 describes a ⁇ 9-desaturase, in WO 93/11245 a ⁇ 15-desaturase and in WO 94/11516 a ⁇ 12-desaturase. Further desaturases are described, for example, in EP-AO 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol.
  • the polyunsaturated fatty acids can be classified according to their desaturation pattern into two broad classes, the ⁇ 6 or ⁇ 3 fatty acids, which have metabolically and functionally different activities (Figure 1).
  • the fatty acid linoleic acid (18: 2 ') acts as the starting material for the co-pathway, while the ⁇ 3 pathway proceeds via linolenic acid (18: 3 ⁇ 9 ' 12 '15 ).
  • Linolenic acid is formed by the activity of ⁇ 15- or ⁇ 3-desaturase (Tocher et al., 1998, Prog. Lipid Res., 37, 73-117, Domergue et al., 2002, Eur. J. Biochem., 269, 4105-4113 ).
  • the first step is the condensation of malonyl-CoA on the fatty acid acyl-CoA by ketoacyl-CoA synthase (KCS, hereinafter referred to as elongase).
  • KCS ketoacyl-CoA synthase
  • elongase ketoacyl-CoA synthase
  • KCR ketoacyl-CoA reductase
  • dehydratase dehydration step
  • enoyl-CoA reductase enoyl-CoA reductase
  • Higher plants contain polyunsaturated fatty acids such as linoleic acid (Cl 8: 2) and linolenic acid (C18: 3).
  • ARA, EPA and / or DHA are not present in the seed oil of higher plants or only in traces.
  • LCPUFAs in higher plants, preferably in oilseeds such as oilseed rape, linseed, sunflower and soybeans, as this will enable large quantities of high quality LCPUFAs to be obtained inexpensively for the food, animal and pharmaceutical industries.
  • gene coding for enzymes of the biosynthesis of LCPUFAs in oilseeds must be advantageously introduced and expressed via genetic engineering methods.
  • genes which encode, for example, ⁇ 6-desaturases, ⁇ 6-elongases, ⁇ 5-desaturases or ⁇ 4-desaturases These genes can be advantageously isolated from microorganisms and lower plants that produce LCPUFAs and incorporate them into membranes or triacylglycerides.
  • ⁇ 6-desaturase genes from the moss Physcomitrella patens and ⁇ 6 elongase genes from P. patens and the nematode C. elegans have already been isolated.
  • a further object was to provide further genes or enzymes which are suitable for the synthesis of LCPUFAs, in particular genes which encode a ⁇ 5-desaturase or ⁇ 6-desaturase activity, for the production of polyunsaturated fatty acids.
  • Another object of this invention has been to provide genes and enzymes, respectively, which permit a shift from the ⁇ 6 fatty acids to the ⁇ 3 fatty acids.
  • the objects of the invention were achieved, inter alia, by the process according to the invention for the production of arachidonic acid or eicosapentaenoic acid in transgenic organisms, characterized in that the content of arachidonic acid and / or eicosapentaenoic acid in the transgenic organism is at least 1% by weight, based on the total lipid content of the transgenic organism and the method comprises the following method step (s): a) introduction of a nucleic acid sequence into the organism which codes for a polypeptide having the activity of a ⁇ 6-desaturase, selected from the group consisting of a nucleic acid sequence having the sequence shown in SEQ ID NO: 1, nucleic acid sequences resulting as a result of degenerate genetic codes from the amino acid sequence shown in SEQ ID NO: 2, and derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, which code for polypeptides having at least 40% identity with SEQ ID NO: 2 at amino acid level and a ⁇
  • the present invention also relates to a process for producing ⁇ 3-fatty acids in transgenic organisms, characterized in that it comprises the following process step (s): a) introduction of a nucleic acid sequence into the organism which represents a polypeptide with the activity of a ⁇ -6 Desaturase encoded selected from the group consisting of a nucleic acid sequence having the sequence shown in SEQ ID NO: 1, nucleic acid sequences which can be derived as the result of the degenerate genetic code of the amino acid sequence shown in SEQ ID NO: 2, and derivatives of in SEQ ID NO: 1 shown Nucleic acid sequence encoding polypeptides having at least 40% identity with SEQ ID NO: 2 at amino acid level and having a ⁇ 6-desaturase activity, and / or b) introducing into the organism a nucleic acid sequence encoding a polypeptide having the activity of a ⁇ 5-desaturase selected from the group consisting of a
  • a ⁇ 6-elongase activity is introduced into the organism.
  • the organism is preferably a microorganism, a yeast or a plant, with useful plants being particularly preferred.
  • Nucleic acid sequences which code for polypeptides having the activity of a ⁇ 6-elongase and which can be used in the context of the invention are described, for example, in WO 2007/017419, WO 2006/069710, WO 2006/100241, WO 2005/083053, WO2006 / 069936, WO2005 / 12316, Domergue et al. (2002) Eur J Biochem 269: 4105-4113; Girke et al. (1998) Plant J 15: 39-48.
  • the ⁇ 6-elongase is a ⁇ 6-elongase from the moss Physcomitrella patens, as described, for example, in Zank et al.
  • the ⁇ -6 elongase has the sequence shown in SEQ ID NO. 14 indicated amino acid sequence.
  • a nucleic acid sequence which codes for an ⁇ -3-desaturase is additionally introduced into the organisms, especially the plants. Suitable nucleic acid sequences coding for an ⁇ -3-desaturase are, for example. described in WO 2007/017419, WO 2006/069710, WO 2006/100241, WO 2005/083053, Michaelson et al. (1998) J Biol Chem 273: 19055-19059; Kaewsuwan et al. (2006) J Biol Chem. 281: 21988-97.
  • the polyunsaturated fatty acids ARA and / or EPA produced in the process according to the invention and further LCPUFAs of the ⁇ -3 or ⁇ -6 fatty acid series contain at least two, advantageously three, four, five or six double bonds.
  • the fatty acids contain four, five or six double bonds.
  • the fatty acids produced in the process advantageously have 18, 20 or 22 carbon atoms in the fatty acid chain, preferably the fatty acids contain 20 or 22 carbon atoms in the fatty acid chain.
  • it is ARA and / or EPA.
  • saturated fatty acids with the nucleic acids used in the process little or not at all, advantageously not reacted. Little is to be understood that, compared to polyunsaturated fatty acids, the saturated fatty acids having less than 5% of the activity, advantageously less than 3%, particularly advantageously less than 2%, very particularly preferably less than 1, 0.5, 0.25 or 0.125% of the activity are reacted.
  • These fatty acids prepared in addition to the fatty acids ARA and / or EPA produced in the process can additionally be prepared as individual fatty acids in the process or be present in a fatty acid mixture.
  • ⁇ 3-fatty acids are understood as meaning those unsaturated fatty acids in which the last double bond in the carbon chain is present at the third-last carbon bond from the carboxyl end.
  • ⁇ 3 fatty acids are ⁇ -linolenic acid (18: 3 ⁇ 9 ' 12 ' 15 ), eicosapentaenoic acid (EPA; 20: 5 ⁇ 5 ' 8 ' n ' 14 ' 17 ) and docosahexaenoic acid (DHA; 22: 6 ⁇ 4 ' 7 ' 10 ' 13 ' 16 '19 ).
  • DHA docosahexaenoic acid
  • ⁇ 3-fatty acids In the process according to the invention, predominantly ⁇ 3-fatty acids are produced, i. the ratio of ⁇ 3 fatty acids produced to ⁇ 6 fatty acids produced is at least 5: 1, 6: 1 or 7: 1, preferably at least 8: 1, 9: 1 or 10: 1, more preferably at least 12: 1, 15: 1 , 18: 1 or 20: 1. Most preferably, no ⁇ 6 fatty acids are produced by the process of the present invention.
  • the ⁇ -6-desaturases and ⁇ 5-desaturases of Mantoniella squamata have the advantage of ⁇ 3 specificity over the desaturases of the prior art, such as, for example, the desaturases from Ostreococcus tauri and Phaeodactylum tricornutum (see, for example, Figures 15 and 16).
  • nucleic acid sequences which code for ⁇ -6-desaturases, ⁇ -5-desaturases and / or ⁇ -6-elongases and / or ⁇ -desaturases are expressed in combination with further genes of the fatty acid and / or lipid metabolism, such as eg Nucleic acid sequences corresponding to polypeptides having ⁇ -8-desaturase, ⁇ -12-desaturase, ⁇ -15-desaturase, ⁇ -4-desaturase, ⁇ -9 elongase and / or ⁇ -5 elongase activity encode.
  • Suitable nucleic acid sequences which code for polypeptides having ⁇ -5-elongase, ⁇ -12-desaturase or ⁇ -4-desaturase activity are described in WO 2006/069710, WO 2006/100241, WO 2005/083053, WO2006 / 069936, WO2005 / 012316, Meyer et al. (2004) J. Lipid Res. 45: 1899-1909, Meyer et al. (2003) Biochemistry 42: 9779-88.
  • the amino acid sequence of a suitable ⁇ -5 elongase is also shown in SEQ ID no. 16 indicated.
  • vegetative tissue is to be understood as meaning that the tissue is characterized by multiplying by mitotic divisions.
  • tissues leaf, flower, root, stem, aboveground or subterranean shoots (lateral shoots, stolons), rhizomes, buds, tubers such as tubers or tubers, onion, broodstock, brood buds, bulbils, or turions.
  • Such tissues can also be caused by spurious, real or man-made viviparous.
  • seeds which are caused by agamospermia, as they are typical for Asteraceae, Poaceae or Rosaceae, belong to the vegetative tissues, in which the expression takes place favorably.
  • the nucleic acids used in the method according to the invention are expressed in the generative tissue (germline tissue).
  • generative tissue is to be understood as meaning that the tissue is formed by meiotic division.
  • tissues which are damaged by sex.
  • Reproduction that is, meiotic cell divisions arise, such as seeds, which have arisen through sexual processes.
  • the expression measured at the RNA and / or protein level is less than 5%, advantageously less than 3%, particularly advantageously less than 2%, very particularly preferably less than 1, 0.5, 0.25 or 0.125%.
  • the polyunsaturated fatty acids produced in the process are advantageously bound in membrane lipids and / or triacylglycerides, but may also be present as free fatty acids or bound in the form of other fatty acid esters in the organisms. They may be present as "pure products" or advantageously in the form of mixtures of different fatty acids or mixtures of different phospholipids such as phosphatidylglycol, phosphatidylcholine, phosphatidylethanolamine and / or phosphatidylserine and / or triacylglycerides, monoacylglycerides and / or diacylglycerides.
  • the LCPUFAs ARA and / or EPA produced in the process are advantageously present in phosphatidylcholine and / or phosphatidylethanolamine and / or in the triacylglycerides.
  • the triacylglycerides may also contain other fatty acids such as short-chain fatty acids having 4 to 6 carbon atoms, medium-chain fatty acids having 8 to 12 carbon atoms or long-chain fatty acids having 14 to 24 carbon atoms, preferably containing long-chain fatty acids, particularly preferably the long-chain Fatty acids LCPUFAs of C 18, C 20 or C 22 fatty acids.
  • the fatty acid esters with polyunsaturated C 18, C 20 and / or C 22 fatty acid molecules can be prepared from the organisms used for the preparation of the fatty acid esters in the form of an oil or lipid, for example in the form of compounds such as sphingolipids, phosphoglycerides, lipids, glycolipids such as glycosphingolipids, phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters such as the acetyl-coenzymeA esters containing the polyunsaturated fatty acids having at least two, three, four, five or six preferably five or more six double bonds are isolated, advantageously they are isolated in the form of their diacyl
  • the polyunsaturated fatty acids are also included as free fatty acids or bound in other compounds in the organisms beneficial to the plants.
  • the various aforementioned compounds (fatty acid esters and free fatty acids) in the organisms in an approximate distribution of 80 to 90 wt .-% triglycerides, 2 to 5 wt .-% diglycerides, 5 to 10 wt .-% monoglycerides, 1 to 5 wt .-% of free fatty acids, 2 to 8 wt .-% phospholipids ago, wherein the sum of the various compounds to 100 wt .-% complements.
  • the LCPUFAs produced have a content of at least 1, 2, 3 or 4% by weight, advantageously of at least 5, 6, 7, 8, 9 or 10% by weight, preferably of at least 11.12, 13, 14 or 15 wt .-%, particularly preferably of at least 16, 17, 18, 19 or 20 wt .-%, most preferably of at least 25, 30, 35 or 40 wt .-% based on the total fatty acids in the transgenic organisms, advantageously produced in a transgenic plant.
  • the fatty acids produced in the process according to the invention are ARA and / or EPA with a content of at least 10% by weight. preferably at least 11, 12, 13, 14 or 15% by weight, more preferably at least 16, 17, 18, 19 or 20% by weight, most preferably at least 25, 26, 27, 28, 29 , 30 or 31 wt .-%, most preferably of at least 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 wt .-% based on the total fatty acids in the triacylglycerols and / or phosphatidylglycerides advantageously contained in the phosphatidylcholine.
  • the fatty acids are prepared in bound form.
  • these unsaturated fatty acids can be brought to the snl, sn2 and / or sn3 position of the advantageously prepared triglycerides.
  • at least 11% of the triacylglycerols are doubly substituted, that is substituted at snl and sn2 or sn2 and sn3 positions.
  • Trisubstituted triacylglycerides are also detectable.
  • the end products of the process such as, for example, arachidonic acid (ARA) or eicosapentaenoic acid (EPA), are not produced as absolute pure products. There are always traces or larger amounts of precursors in the final product. If, for example, both linoleic acid and linolenic acid are present in the starting plant, the end products such as ARA or EPA are present as mixtures.
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • the precursors should not be beneficial more than 20 wt .-%, preferably not more than 15 wt .-%, more preferably not more than 10 wt .-%, most preferably not more than 5 wt .-%, based on the amount of the respective end product.
  • ARA or EPA are bound in the process of the invention in a transgenic plant as end products or prepared as free acids.
  • Fatty acid esters or fatty acid mixtures which have been prepared by the process according to the invention advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid; 7 - 85% oleic acid; 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachidic acid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acids and 60 to 85% of polyunsaturated fatty acids, based in each case on 100% and on the total fatty acid content of the organisms ,
  • polyunsaturated fatty acid in the fatty acid esters or fatty acid mixtures are preferably at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1% based on the total fatty acid content of arachidonic acid.
  • the fatty acid esters or fatty acid mixtures which have been prepared by the process according to the invention advantageously contain fatty acids selected from the group of the fatty acids erucic acid (13-docosaic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8 , 9-methylene heptadec-8-enoic acid), chaulmoogric acid (cyclopentendodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,1-dienanoic acid), vernoic acid (9,10-epoxyoctadec-12-enoic acid), taranic acid (6-octadecynonic acid), 6-nonadecynoic acid, santalbinic acid (tl 1-octadecen-9-ynoic acid), 6,9-octadecenynonic acid, pyrulic acid (tl0-
  • the abovementioned fatty acids are generally advantageously present only in traces in the fatty acid esters or fatty acid mixtures prepared by the process according to the invention, that is to say they come to less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids. , 22% or 21%, more preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, most preferably less than 4%, 3%, 2% or 1% ago.
  • these abovementioned fatty acids come to less than 0.9%, 0.8%, 0.7%, 0.6% or 0.5%, more preferably less than 0, relative to the total fatty acids. 4%, 0.3%, 0.2%, 0.1% ago.
  • nucleic acid sequences according to the invention or in the method according to the invention used nucleic acid sequences can increase the yield of polyunsaturated fatty acids of at least 50%, preferably at least 80%, more preferably at least 100%, most preferably at least 150% compared to the non-transgenic starting organism
  • a yeast, an alga, a fungus, or a plant such as Arabidopsis or flax can be obtained by comparison in GC analysis.
  • chemically pure polyunsaturated fatty acids or fatty acid compositions can be prepared by the methods described above.
  • the fatty acids or the fatty acid compositions from the organism such as the microorganisms or the plants or the culture medium in which or on which the organisms were grown, or isolated from the organism and the culture medium in a known manner, for example via extraction, distillation, crystallization, chromatography or combinations of these methods.
  • These chemically pure fatty acids or fatty acid compositions are advantageous for applications in the food industry, the cosmetics industry and especially the pharmaceutical industry.
  • Agricultural crops are plants that are used for food production for humans and animals, the production of luxury foods, fibers and pharmaceuticals such as cereals such as corn, rice, wheat, barley, millet, oats, rye, buckwheat; such as tubers such as potatoes, cassava, potatoes, yams, etc .; such as sugar beets such as sugar cane or sugar beet; such as legumes such as beans, peas, broad bean, etc .; such as oil and fatty fruits such as soybean, rapeseed, sunflower, safflower, flax, camelina, etc., to name but a few.
  • cereals such as corn, rice, wheat, barley, millet, oats, rye, buckwheat
  • tubers such as potatoes, cassava, potatoes, yams, etc .
  • sugar beets such as sugar cane or sugar beet
  • legumes such as beans, peas, broad bean, etc .
  • Advantageous plants are selected from the group of plant families consisting of the families of Aceraceae, Actinidiaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Arecaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Cannaceae, Caprifoliaceae, Chenopodiaceae, Convolvulaceae, Cucurbitaceae, Dioscoreaceae, Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Fagaceae, Geraniaceae, Gramineae, Grossulariaceae, Juglandaceae, Lauraceae, Leguminosae, Liliaceae, Linaceae, Malvaceae, Moraceae, Musaceae, Oleaceae, Oxalidaceae, Papaveraceae, Poaceae, Polygonaceae, Prasinophycea
  • the following plants may be selected from the group Adelotheciaceae, such as the genera Physcomitrella, e.g. the genus and species Physcomitrella patens, Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium e.g. the genus and species Pistacia vera [pistachio], Mangifer indica [Mango] or Anacardium occidentale [cashew], Asteraceae such as the genera Calendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta, Tagetes, Valeriana e.g.
  • Brassica rapa ssp. Sinapis arvensis, Brassica juncea, Brassica juncea var. Juncea, Brassica juncea var. Crispifolia, Brassica juncea var. Foliosa, Brassica nigra, Brassica sinapioides, Camelina sativa, Melanosinapis communis [mustard], Brassica oleracea [feeder] or Arabidopsis thaliana , Bromeliaceae such as the genera Anana, Bromelia (pineapple) eg the genera and species Anana comosus, pineapple pineapple or Bromelia comosa [pineapple], Caricaceae as the genus Carica as the genus and species Carica papaya [Papaya], Cannabaceae as the genus Cannabis as the genus and species Cannabis sative [hemp], Convolvulaceae as the genera Ipomea, Convolvulus eg the genera
  • Beta such as the genera and species Beta Vulgaris, Beta vulgaris var. Altissima, Beta vulgaris var. Vulgaris, Beta maritima, Beta vulgaris var. Perennis, Beta vulgaris var. Conditiva or Beta vulgaris var.
  • Esculenta [sugar beet], Crypthecodiniaceae such as the genus Crypthecodinium eg the genus and species Cryptecodinium cohnii, Cucurbitaceae such as the genus Cucubita eg the genera and species Cucurbita maxima, Cucurbita mixta, Cucurbita pepo or Cucurbita moschata [squash], Cymbellaceae such as the genera Amphora, Cymbella, Okedenia, Phaeodactylum, Reimeria eg the genus and species Phaeodactylum tricornutum, Ditrichaceae such as the genera Ditrichaceae, Astomiopsis, Ceratodon, Chrysoblastella, Ditrichum, Distichium, Eccremidium, Lophidion, Philibertiella, Pleuridium, Saelania, Trichodon, Skottsbergi
  • Elaeagnaceae such as the genus Elaeagnus eg the genus and species Olea europaea [Olive]
  • Ericaceae like the genus Kalmia eg the genera and species Kalmia latifolia, Kalmia angustifolia, Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistus chamaerhodendros or Kalmia lucida [Berglorbeer]
  • Euphorbiaceae such as the genera Manihot, Janipha, Jatropha, Ricinus eg the genera and species Manihot utilissima, Janipha manihot, Manathot manihot, Manihot aipil, Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot esculenta [Manihot] or Ricinus communis [Castor]
  • Juglandaceae such as the genera Juglans, Wallia e.g. the genera and species of Juglans regia, Juglans ailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea, Juglans bixbyi, Juglans californica, Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa, Juglans nigra or Wallia nigra [Walnut], Lauraceae Like the genera Persea, Laura eg the genera Persea, Laura eg the genera Persea, Laura eg the genera Persea, Laura eg the genera Persea, Laura eg the genera Persea, Laura eg the genera Persea, Laura eg the genera Persea, Laura eg the genera Persea, Laura e
  • Capsicum frutescens [pepper], Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata, Nicotiana attenuata, Nicotiana glauca, Nicotiana slowdorifii, Nicotiana obtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotiana rustica , Nicotiana sylvestris [tobacco], Solanum tuberosum [Potato], Solanum melongena [eggplant], Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon py [eta] forme, Solanum integrifolium or Solanum lycopersicum [tomato], Sterculiaceae such as the genus Theobroma eg the genus and species Theobroma cacao [cocoa] or Theaceae like the genus Camellia
  • Examples of advantageous microorganisms are fungi selected from the families of the families Chaetomiaceae, Choanephoraceae, Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae, Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae or Tuberculariaceae.
  • Choanephoraceae such as the genera Blakeslea, Choanephora e.g.
  • Mortierellaceae such as the genus Mortierella e.g. the genera and species Mortierella isabellina, Mortierella polycephala,
  • Saccharomyces fermentati Saccharomyces florentine, Saccharomyces fragilis, Saccharomyces heterogenicus, Saccharomyces hienipiensis, Saccharomyces inusitatus, Saccharomyces italicus, Saccharomyces kluyveri, Saccharomyces krusei, Saccharomyces lactis, Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomyces montanus, Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomyces paradoxus, Saccharomyces pastorianus , Saccharomyces pretoriensis, Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces uvarum, Saccharomyces ludwigii, Yarrowia lipolytica, Schizosacharomycetaceae such as the generic Schizosaccharomyces
  • Schizosaccharomyces pombe var. Pombe, Thraustochytriaceae such as the genera Althomia, limacinum Aplanochytrium, Japonochytrium, Schizochytrium, Thraustochytrium eg the species Schizochytrium aggregatum, Schizochytrium, Schizochytrium minutum mangrovei, Schizochytrium, Schizochytrium octosporum, aggregatum Thraustochytrium, amoeboideum Thraustochytrium, Thraustochytrium antacticum, arudimentale Thraustochytrium, Thraustochytrium aureum, Thraustochytrium benthicola, Thraustochytrium globosum, Thraustochytrium indicum, Thraustochytrium kerguelense, Thraustochytrium kinnei, Thraustochytrium motivum, Thraustochytrium multi
  • Examples include the following microorganisms selected from the group: Bacillaceae such as the genus Bacillus z.Beg the genera and species Bacillus acidocaldarius,
  • Bacillus acidoterrestris Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus sphaericus subsp. fusiformis, Bacillus galactophilus, Bacillus globisporus, Bacillus globisporus subsp.
  • Bacillus subtilis subsp. marinus Bacillus halophilus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis subsp. spizizenii, Bacillus subtilis subsp.
  • Enterobacteriacae such as the genera Citrobacter, Edwardsieila, Enterobacter, Erwinia, Escherichia, Klebsiella, Salmonella or Serratia eg the genera and species Citrobacter amalonaticus, Citrobacter diversus, Citrobacter freundii, Citrobacter genomospecies, Citrobacter gillenii, Citrobacter intermedium, Citrobacter koseri, Citrobacter murliniae, Citrobacter sp , Edwardsiella hoshinae, Edwardsieila ictaluri, Edwardsiella tarda, Erwinia alni, Erwinia amylovora, Erwiniaananatis, Erwinia aphidicola, Erwinia billingiae, Erwinia cacticida, Erwinia carcinogena, Erwinia carnegieana, Erwinia caro
  • Salmonella daressalaam Salmonella enterica subsp. houtenae, Salmonella enterica subsp. salamae, Salmonella enteritidis, Salmonella gallinarum, Salmonella heidelberg, Salmonella panama, Salmonella senftenberg, Salmonella typhimurium, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia liquefaciens, Serratia marcescens, Serratia marcescens subsp.
  • marcescens Serratia marinorubra, Serratia odorifera, Serratia plymouthensis, Serratia plymuthica, Serratia proteamaculans, Serratia proteamaculans subsp. quinovora, Serratia quinivorans or Serratia rubidaea; Rhizobiaceae such as the genera Agrobacterium, Carbophilus, Chelatobacter, Ensifer, Rhizobium, Sinorhizobium eg the
  • Species and Species Agrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacterium gelatinovorum, Agrobacterium larrymoorei, Agrobacterium meteori, Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium rubi, Agrobacterium stellulatum, Agrobacterium tumefaciens, Agrobacterium vitis, Carbophilus carboxidus, Chelatobacter heintzii, Ensifer adhaerens, Ensifer arboris, Ensifer fredii , Ensifer kostiensis, Ensifer kummerowiae, Ensifer Rhizobium eti, Rhizobium fredii, Rhizobium galegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobium hainanense, Rhizobium huakuii, Rhizo
  • microorganisms for the method according to the invention are, for example, protists or diatoms selected from the group of the families Dinophyceae, Turaniellidae or Oxytrichidae such as the genera and species: Crypthecodinium cohnii, Phaeodactylum tricornutum, Stylonychia mytilus, Stylonychia pustulata, Stylonychia putrina, Stylonychia notophora, Stylonychia sp. , Colpidium campylum or Colpidium sp.
  • transgenic organisms such as fungi such as Mortierella or Traustochytrium, yeasts such as Saccharomyces or Schizosaccharomyces, mosses such as Physcomitrella or Ceratodon, non-human animals such as Caenorhabditis, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium or Phaeodactylum or plants such as dicotyledonous or monocotyledonous plants.
  • fungi such as Mortierella or Traustochytrium
  • yeasts such as Saccharomyces or Schizosaccharomyces
  • mosses such as Physcomitrella or Ceratodon
  • non-human animals such as Caenorhabditis
  • algae such as Nephroselmis, Pseudoscour
  • oils such as fungi such as Mortierella or Thraustochytrium, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium, Phaeodactylum or plants, especially plants, preferably oil crops containing large amounts of lipid compounds contain, such as peanut, rapeseed, canola, sunflower, safflower (Carthamus tinctoria), poppy, mustard, hemp, castor, olive, sesame, calendula, punica, evening primrose, mullein, thistle, wild roses, hazelnut, almond, macadamia, avocado, laurel , Pumpkin, flax, soy, pistachio, borage, trees (
  • Preferred plants according to the invention are oil crop plants, such as peanut, rapeseed, canola, sunflower, safflower, poppy, mustard, hemp, castor, olive, calendula, punica, evening primrose, pumpkin, flax, soy, borage, trees (oil palm, coconut).
  • Particularly preferred are C 18: 2 and / or C 18: 3 fatty acid-rich plants such as sunflower, safflower, tobacco, mullein, sesame seed,
  • nucleic acids and optionally introduced nucleic acid sequences encoding the ⁇ -6 elongase and / or the ⁇ -3-desaturases to additionally introduce further nucleic acids which code for enzymes of the fatty acid or lipid metabolism.
  • genes of the fatty acid or lipid metabolism can advantageously be used in combination with the nucleic acid sequences used in the inventive method, which are suitable for ⁇ 6-desaturase (s), ⁇ 5-desaturase (s), ⁇ 6-elongase (s) and / or ⁇ -3-desaturase (s) [in the context of this application, the plural shall encode the singular and vice versa];
  • genes selected from the group of ⁇ -4-desaturases, ⁇ -8-desatuases, ⁇ -9-desaturases, ⁇ -12-desaturases, ⁇ -5-elongases or ⁇ -9-elongases in combination with the abovementioned genes for the ⁇ -6 elongase, ⁇ -6-desaturase, ⁇ -5-desaturase and / or ⁇ -3-desaturase, wherein single genes or multiple genes can be used in combination.
  • the ⁇ -3-desaturase used in the method according to the invention should advantageously allow a shift from the ⁇ -6 biosynthetic pathway to the ⁇ -3 biosynthetic pathway, which advantageously leads to a shift of Cl 8: 2 to C18: 3 fatty acids.
  • omega-3-desaturase it is advantageously possible to shift the fatty acid spectrum within an organism, advantageously within a plant or a fungus, from the omega-6 fatty acids to the omega-3 fatty acids.
  • ⁇ 5-desaturases and ⁇ 6-desaturases according to the invention from Mantoniella squamata have the advantage over the known ⁇ 5-desaturases and ⁇ 6-desaturases, for example from Phaeodactylum tricornutum, that they bind fatty acids to phospholipids or CoA-
  • Fatty acid esters advantageously CoA-F ettklareester implement.
  • the desaturases used in the process according to the invention convert their respective substrates in the form of the Co A-fatty acid esters (see Examples 10 and 11). This leads, if previously a Elongations Marin has taken place, advantageously to an increased product yield.
  • the respective desaturation products are thereby synthesized in higher amounts, since the elongation step usually takes place on the CoA fatty acid esters, while the desaturation step takes place predominantly on the phospholipids or on the triglycerides.
  • nucleic acids used in the method according to the invention which code for polypeptides with ⁇ -6-elongase, ⁇ -6-desaturase, ⁇ -5-desaturase and / or ⁇ -3-desaturase activity, advantageously in combination with nucleic acid sequences which are polypeptides of fatty acid or lipid metabolism such as other polypeptides with ⁇ -4, ⁇ -5, ⁇ -6, ⁇ -8, ⁇ -12-desaturase or ⁇ -5, ⁇ -6 or ⁇ -9-Elongasecretmaschine encode, a variety of polyunsaturated fatty acids can be prepared in the process according to the invention.
  • mixtures of the various polyunsaturated fatty acids or individual polyunsaturated fatty acids such as EPA or ARA can be prepared in free or bound form.
  • fatty acids derived from C18: 2 fatty acids such as GLA, DGLA or ARA or those derived from C18: 3 Derived fatty acids, such as SDA, ETA or EPA.
  • linoleic acid LA, C18: 2 ⁇ 9 '12
  • GLA, DGLA and ARA can arise as products of the process which may be present as free fatty acids or bound.
  • ⁇ -linolenic acid ALA, C18: 3 ⁇ 9,12,15
  • the products of the process can only be SDA, ETA and / or EPA, which as described above may be present as free fatty acids or bound.
  • ⁇ 6-desaturase By modifying the activity of the enzymes used in the process and involved in the synthesis ⁇ 6-elongase, ⁇ 6-desaturase, ⁇ 5-desaturase and / or ⁇ -3-desaturase advantageous in combination with other genes of lipid or fatty acid metabolism can be specifically produced in the plants only individual products.
  • ARA or EPA or their mixtures are synthesized, depending on the fatty acid present in the organism or in the plant, which serves as the starting substance for the synthesis. Since it is about
  • Biosynthesis chains the respective end products are not present as pure substances in the organisms. There are always small amounts of precursor compounds in the final product. These small amounts are less than 20 wt .-%, advantageously less than 15 wt .-%, more preferably less than 10 wt .-%, most preferably less than 5, 4, 3, 2 or 1 wt .-% based on the end product EPA or ARA or mixtures thereof.
  • the fatty acids can in principle also be fed from the outside.
  • Preferred substrates are linoleic acid (C18: 2 ⁇ 9,12), ⁇ -linolenic acid (C18: 3 ⁇ 6,9,12), Eicosadienoic acid (C20: 2 ⁇ 11,14), dihomo- ⁇ -linolenic acid (C20: 3 ⁇ 8, 11,14), arachidonic acid (C20: 4 ⁇ 5,8, 11,14), docosatetraenoic acid (C22: 4 ⁇ 7, 10,13, 16) and docosapentaenoic acid (C22: 5 ⁇ 4,7,10,13,15).
  • the genus and species Olea europaea or the family Fabaceae such as the genus Glycine e.g. the genus and species Glycine max, which have a high oleic acid content. Since these organisms have only a low content of linoleic acid, the use of said ⁇ -12-desaturases for the preparation of the starting product linoleic acid is advantageous.
  • Nucleic acids used in the method according to the invention are advantageously derived from plants such as algae, for example algae of the family Prasinophyceae as from the genera Heteromastix, Mammella, Mantoniella, Micromonas, Nephroselmis, Ostreococcus, Prasinocladus, Prasinococcus, Pseudoscourfielda, Pycnococcus, Pyramimonas, Scherffelia or Tetraselmis such as the genera and Heteromastix longifillis, Mamiella gilva, Mantoniella squamata, Micromonas pusilla, Nephroselmis olivacea, Nephroselmis pyriformis, Nephroselmis rotunda, Ostreococcus tauri, Ostreococcus sp.
  • algae for example algae of the family Prasinophyceae as from the genera Heteromastix,
  • the nucleic acids used are derived from algae of the genera Euglena, Mantoniella or Ostreococcus.
  • algae such as Isochrysis or Crypthecodinium
  • algae / diatoms such as Thalassiosira or Phaeodactylum
  • mosses such as Physcomitrella or Ceratodon or higher plants such as Primulaceae such as Aleuritia, Calendula stellata, Osteospermum spinescens or Osteospermum hyoseroides
  • microorganisms such as fungi such as Aspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor or Mortierella
  • bacteria such as Shewanella
  • yeast or animals such as nematodes such as Caenorhabditis, insects, frogs, sea cucumbers or fish.
  • the nucleic acid sequences are of the vertebrate class; Euteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei, Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus or Vertebrata, Amphibia, Anura, Pipidae, Xenopus or Evertebrata such as Protochordata, Tunicata, Holothuroidea, Cionidae such as Amaroucium constellatum, Botryllus schlössen, Ciona intestinalis, Molgula citrina, Molgula manhattensis, Perophora viridis or Styela partita.
  • Nucleic acids from fungi, animals or from plants such as algae or mosses preferably from the order of Salmoniformes such as the family Salmonidae such as the genus Salmo, for example, from the genera and species Oncorhynchus mykiss, Trutta trutta or Salmo trutta fario, from algae such as the genera Mantoniella or Ostreococcus or from the diatoms such as the genera Thalassiosira or Phaeodactylum or from algae such as Crypthecodinium.
  • Salmoniformes such as the family Salmonidae such as the genus Salmo
  • Oncorhynchus mykiss Trutta trutta or Salmo trutta fario
  • algae such as the genera Mantoniella or Ostreococcus
  • diatoms such as the genera Thalassiosira or Phaeodactylum
  • algae such
  • the abovementioned nucleic acid sequences or their derivatives or homologs which code for polypeptides which still have the enzymatic activity of the proteins encoded by the nucleic acid sequences are expressed in the organism.
  • These sequences are used alone or in combination with those for ⁇ 12-desaturase, ⁇ 4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 5-elongase, ⁇ 6-elongase and / or ⁇ 3-desaturase-encoding nucleic acid sequences are cloned into expression constructs and used for introduction and expression in organisms. By their construction, these expression constructs enable a favorable optimal synthesis of the polyunsaturated fatty acids produced in the process according to the invention.
  • the method further comprises the step of obtaining a cell or a whole organism, especially a whole plant, which comprises the nucleic acid sequences used in the method which are responsible for a ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ -5 Desaturase and / or ⁇ -3-desaturase encoded, wherein the cell and / or the organism may contain other nucleic acid sequences of the lipid or fatty acid metabolism.
  • this method further comprises the step of recovering the oils, lipids or free fatty acids from the organism or from the culture.
  • the culture may be, for example, a fermentation culture, for example, in the case of culturing microorganisms such as Mortierella, Thalassiosira, Mantoniella, Ostreococcus, Saccharomyces or Thraustochytrium, or a greenhouse or field crop of a plant.
  • the cell or organism thus produced is advantageously a cell of an oil-producing organism such as an oil crop such as peanut, canola, canola, flax, hemp, peanut, soybean, safflower, hemp, sunflower or borage.
  • Cultivation is, for example, culturing in the case of plant cells, tissue or organs on or in a nutrient medium or the whole plant on or in a substrate, for example in hydroponics, potting soil or on arable land.
  • Natural genetic environment means the natural genomic or chromosomal locus in the source organism or presence in a genomic library.
  • the natural genetic environment of the nucleic acid sequence is preferably at least partially conserved.
  • the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, more preferably at least 1000 bp, most preferably at least 5000 bp.
  • a naturally occurring expression cassette for example, the naturally occurring combination of the natural promoter of the nucleic acid sequence used in the method according to the invention, which is suitable for proteins with appropriate ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase and / or ⁇ -3 Desaturase activity, advantageously in combination with nucleic acid sequences which are suitable for proteins having ⁇ -12-desaturase, ⁇ -4-desaturase, ⁇ -8-desaturase, ⁇ -9 elongase, and / or ⁇ -5.
  • Elongase activity becomes a transgenic expression cassette when it is changed by non-natural, synthetic ("artificial") methods such as a mutagenization. Corresponding methods are described, for example, in US Pat. No. 5,565,350 or WO 00/15815.
  • transgenic organism or “transgenic plant” within the meaning of the invention means that the nucleic acids used in the method are not in their natural position in the genome of an organism, in which case the nucleic acids can be expressed homologously or heterologously.
  • transgene also means that the nucleic acids according to the invention are in their natural place in the genome of an organism, but that the sequence has been changed compared to the natural sequence and / or that the regulatory sequences of the natural sequences have been altered.
  • the transgenic expression of the nucleic acids according to the invention at non-natural sites in the genome is preferred understand, that is, a homologous or preferably heterologous expression of the nucleic acids is present.
  • Preferred transgenic organisms are fungi such as Mortierella or Phytophthora, mosses such as Physcomitrella, algae such as Mantoniella or Ostreococcus, diatoms such as Thalassiosira or Crypthecodinium or plants such as the oil crops and oil-producing plants, vegetable, salad or ornamental plants, which are advantageously selected from the A group of plant families consisting of the families of Aceraceae, Actinidiaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Arecaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Cannaceae, Caprifoliaceae, Chenopodiaceae, Convolvulaceae, Cucurbitaceae, Dioscoreaceae, Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Fagaceae, Grossul
  • all organisms which are able to synthesize fatty acids, especially unsaturated fatty acids, or which are suitable for the expression of recombinant genes are suitable in principle as organisms or host organisms for the nucleic acids, the expression cassette or the vector used in the process according to the invention.
  • Examples include plants such as Arabidopsis, Asteraceae such as calendula or crops such as soybean, peanut, castor, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, dyer safflower (Carthamus tinctorius) or cocoa bean, microorganisms such as fungi, for example, the genus Mortierella, Thraustochytrium, Saprolegnia, Phytophthora or Pythium, bacteria such as the genus Escherichia or Shewanella, yeasts such as the genus Saccharomyces, cyanobacteria, ciliates, algae such as Mantoniella or Ostreococcus or protozoans such as dinoflagellates such as Thalassiosira or Crypthecodinium called.
  • fungi for example, the genus Mortierella, Thraustochytrium, Saprolegnia, Phytophthora or Pythium, bacteria
  • transgenic animals advantageously non-human animals, such as, for example, C. elegans, are suitable as host organisms in addition to the abovementioned transgenic organisms.
  • transgenic plants include plant cells and certain tissues, organs and parts of plants in all their forms, such as anthers, fibers, root hairs, stems, embryos, calli, cotyledons, petioles, crops, plant tissue, reproductive tissue and cell cultures, that of the actual transgenic plant is derived and / or can be used to produce the transgenic plant.
  • Transgenic plants which contain the polyunsaturated fatty acids synthesized in the process according to the invention can advantageously be marketed directly, without the synthesized oils, lipids or fatty acids having to be isolated. This form of marketing is particularly beneficial.
  • Plants in the process according to the invention include whole plants and all plant parts, plant organs or plant parts such as leaves, stems, seeds, roots, tubers, anthers, fibers, root hairs, stems, embryos, calli, cotyledons, petioles, crop material, plant tissue, reproductive tissue, Cell cultures derived from the transgenic plant and / or used to produce the transgenic plant.
  • the seed includes all seed parts such as the seed shells, epidermis and sperm cells, endosperm or embryonic tissue.
  • the compounds prepared in the process according to the invention can also be isolated from the organisms advantageously plants in the form of their oils, fat, lipids and / or free fatty acids.
  • Polyunsaturated fatty acids produced by this process can be harvested by harvesting the organisms either from the culture in which they grow or from the field. This can be done by pressing or extraction of the plant parts, preferably the plant seeds.
  • the oils, fats, lipids and / or free fatty acids by so-called cold beat or cold pressing can be obtained without supplying heat by pressing.
  • the seeds are first crushed, steamed or roasted. The pretreated seeds can then be pressed or extracted with solvents such as warm hexane.
  • the solvent is removed again.
  • these are harvested after harvesting, for example, directly without further working steps, or else extracted after digestion by various methods known to the person skilled in the art. In this way, more than 96% of the compounds prepared in the process can be isolated.
  • the products thus obtained are further processed, that is refined.
  • the Pfieszenschleime and turbidity are removed.
  • the so-called degumming can be carried out enzymatically or, for example, chemically / physically by adding acid, such as phosphoric acid.
  • the free fatty acids are removed by treatment with a base, for example sodium hydroxide solution.
  • the product obtained is for removal
  • the lye remaining in the product is thoroughly washed with water and dried.
  • the products are subjected to bleaching with, for example, bleaching earth or activated carbon.
  • the product is deodorized, for example, with steam.
  • the PUFAs or LCPUFAs produced by this process are preferably C 18, C 20 and / or C 22 fatty acid molecules, advantageously C 20 -fatty acid molecules having at least two double bonds in the fatty acid molecule, preferably three, four, five or six double bonds.
  • These C18, C20 or C22 fatty acid molecules can be isolated from the organism in the form of an oil, lipid or free fatty acid. Suitable organisms are, for example, those mentioned above. Preferred organisms are transgenic plants.
  • oils, lipids or fatty acids or fractions thereof which have been prepared by the method described above, more preferably oil, lipid or a fatty acid composition comprising PUFAs and transgenic
  • Plants come from. Another embodiment of the invention is the use of these oils, lipids or fatty acids or fractions thereof, which have been prepared by the process described above, for the production of feed, food, cosmetics, dietary supplements or pharmaceuticals.
  • oils, lipids or fatty acids advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid as described above; 7 to 85% oleic acid; 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachidic acid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acids and 60 to 85% of polyunsaturated fatty acids, based in each case on 100% and on the total fat acid content of the organism.
  • fatty acid esters or fatty acid mixtures such as phosphatidyl fatty acid esters or triacylglyceride esters preferably at least 1, 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 wt .-% based on the total fatty acid content of arachidonic acid and / or at least 20, 22, 24 or 25, advantageously at least 26, 28 or 30, more preferably at least 32, 34, 36, 38 or 40, most preferably at least 42, 44, 45 wt .-% or more based on the total fatty acid content of eicosapentaenoic acid.
  • phosphatidyl fatty acid esters or triacylglyceride esters preferably at least 1, 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 wt .-% based on the total fatty acid content of arachidonic acid and / or at least 20, 22, 24 or 25, advantageously at least 26, 28 or 30, more preferably at least 32, 34, 36, 38 or 40, most preferably at least 42, 44, 45
  • the fatty acid esters or fatty acid mixtures prepared by the process according to the invention advantageously contain fatty acids selected from the group of the fatty acids erucic acid (13-docosaic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8,9 -Methylene heptadec-8-enoic acid), chaulmoogric acid (cyclopentenodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,1-l-dienoic acid), vernonic acid (9,10-epoxyoctadec-12-enoic acid), tartric acid (6-octadecynoic acid), 6-nonadecynoic acid, santalbic acid (tl 1-octadecen-9-ynoic acid), 6,9-octadecenynoic acid, pyrulic acid (tlO-heptade
  • fatty acids are generally advantageously present only in traces in the fatty acid esters or fatty acid mixtures prepared by the process according to the invention, that is to say they are less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids.
  • these abovementioned fatty acids come to less than 0.9%, 0.8%, 0.7%, 0.6% or 0.5%, more preferably less than 0, relative to the total fatty acids, 4%, 0.3%, 0.2%, 0.1% ago.
  • the oils, lipids or fatty acids according to the invention advantageously contain at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least 6%, 7%, 8%, 9% or 10%, particularly advantageously at least 11%, 12%, 13%, 14% or 15% ARA or at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least 6%, or 7%, more preferably at least 8 %, 9% or 10% EPA and / or DHA based on the total fatty acid content of the production organism advantageously a plant, particularly advantageous an oil crop such as soybean oilseed rape, coconut, oil palm, Desirbersafflor, flax, hemp, castor, calendula, peanut, cocoa bean, sunflower or the above other monocotyledonous or dicotyledonous oil crops.
  • an oil crop such as soybean oilseed rape, coconut, oil palm, Desirbersafflor, flax, hemp, castor, calendula, peanut,
  • Another embodiment of the invention is the use of the oils, lipids, the
  • oils, lipids, fatty acids or fatty acid mixtures obtained in the process according to the invention can be used in the manner known to those skilled in the art for blending with other oils, lipids, fatty acids or fatty acid mixtures of animal origin, such as fish oils.
  • oils, lipids, fatty acids or fatty acid mixtures which consist of vegetable and animal components, can be used for the production of feed, food, cosmetics or pharmaceuticals.
  • oil is understood as meaning a fatty acid mixture which contains unsaturated, saturated, preferably esterified fatty acid (s). It is preferred that the oil, lipid or fat contains a high proportion of polyunsaturated free or advantageously esterified fatty acid (s), in particular linoleic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, ⁇ -linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, Docosapentaenoic acid or docosahexaenoic acid has.
  • s polyunsaturated free or advantageously esterified fatty acid
  • the proportion of unsaturated esterified fatty acids is about 30%, more preferred is a proportion of 50%, even more preferred is a proportion of 60%, 70%, 80% or more.
  • the proportion of fatty acid after conversion of the fatty acids into the methyl esters can be determined by gas chromatography by transesterification.
  • the oil, lipid or fat may contain various other saturated or unsaturated fatty acids, e.g. Calendulic acid, palmitic, palmitoleic, stearic, oleic acid, etc. included. In particular, depending on the starting organism, the proportion of the various fatty acids in the oil or fat may vary.
  • the polyunsaturated fatty acids prepared in the process and advantageously having at least two, three, four or five, particularly preferably four or five double bonds are, as described above, advantageously fatty acid esters, for example sphingolipid esters, phosphoglyceride esters, lipid esters, glycolipid esters, phospholipid esters, monoacylglycerol esters, diacylglycerol esters , Triacylglycerinester or other fatty acid esters, it is preferably phospholipid esters and / or Triacylglycerinester.
  • advantageously fatty acid esters for example sphingolipid esters, phosphoglyceride esters, lipid esters, glycolipid esters, phospholipid esters, monoacylglycerol esters, diacylglycerol esters , Triacylglycerinester or other fatty acid esters, it is preferably phospholipid esters and / or Triacylglycerinester.
  • the polyunsaturated fatty acids present can be advantageously with at least five or six double bonds, for example via an alkali treatment, for example, aqueous KOH or NaOH or acid hydrolysis in the presence of an alcohol such as methanol or ethanol or release via an enzymatic cleavage and isolate via, for example, phase separation and subsequent acidification over, for example, H 2 SO 4 .
  • the release of the fatty acids can also be carried out directly without the workup described above.
  • the nucleic acids used in the method can either lie on a separate plasmid or can advantageously be integrated into the genome of the host cell.
  • integration may be at random or by such recombination as to replace the native gene with the incorporated copy, thereby modulating the production of the desired compound by the cell, or by using a gene in trans such that Gene having a functional expression unit, which contains at least one expression of a gene ensuring sequence and at least one polyadenylation of a functionally transcribed gene ensuring sequence is operably linked.
  • the nucleic acids are brought into the plants via multi-expression cassettes or constructs for multiparallel expression in the organisms, advantageously for multiparallel seed-specific expression of genes.
  • Moose and algae are the only known pesticide systems that produce significant amounts of polyunsaturated fatty acids, such as arachidonic acid (ARA) and / or eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • Moose contain PUFAs in membrane lipids, while algae, algae-related organisms and some fungi also contain significant amounts Accumulate levels of PUFAs in the triacylglycerol fraction.
  • nucleic acid molecules isolated from strains that also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for the method of the invention and thus for modification of the lipid and PUFA production system in a host, especially plants such as oil crop plants, for example Rapeseed, canola, flax, hemp, soy, sunflower, borage. They are therefore advantageous for use in the process according to the invention.
  • nucleic acids used in the method according to the invention which code for polypeptides with ⁇ -6-desaturase, ⁇ -5-desaturase, ⁇ -6-elongase and / or ⁇ -3-desaturase activity, and / or the other used nucleic acids such as the nucleic acids selected for polypeptides of fatty acid or lipid metabolism from the group acyl CoADehydrogenase (s), acyl-ACP acyl carrier protein desaturase (s), acyl-ACP thioesterase (s), fatty acid Acyl-transferase (s), acyl-CoA: lysophospho lipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (n), fatty acid desaturase (s), fatty acid acety
  • the saturated, monounsaturated C 16 -fatty acids and / or polyunsaturated C 18 -fatty acids must first be desaturated and / or elongated or only desaturated, depending on the substrate, by the enzymatic activity of a desaturase and / or elongase, and then be further desaturated Elongase be extended by at least two carbon atoms. After a round of elongation this leads Enzyme activity either from C16 fatty acids to C18 fatty acids or from C18 fatty acids to C20 fatty acids, and after two elongation cycles from C16 fatty acids to C20 fatty acids.
  • the activity of the desaturases and elongases used in the process according to the invention preferably leads to C 18- and / or C 20 -fatty acids advantageously having at least two double bonds in the fatty acid molecule, preferably having three, four or five double bonds, more preferably C 20 -fatty acids having at least four double bonds in the fatty acid molecule.
  • Particularly preferred as products of the method according to the invention are dihomo- ⁇ -linolenic acid, arachidonic acid and / or eicosapentaenoic acid.
  • the C18 fatty acids having at least two double bonds in the fatty acid can be extended by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.
  • esters such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.
  • the preferred biosynthesis site of fatty acids, oils, lipids or fats in the advantageously used plants is, for example, generally the seed or cell layers of the
  • microorganism such as yeasts such as Saccharomyces or Schizosaccharomyces
  • fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Thraustochytrium or algae such as Isochrysis, Mantonielia, Ostreococcus, Phaeodactylum or Crypthecodinium used
  • yeasts such as Saccharomyces or Schizosaccharomyces
  • fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Thraustochytrium
  • algae such as Isochrysis, Mantonielia, Ostreococcus, Phaeodactylum or Crypthecodinium used
  • these organisms are advantageously attracted to fermentation.
  • the polyunsaturated fatty acids produced in the process can be at least 5%, preferably at least 10%, particularly preferably at least 20%. , most preferably by at least 50% over the wild-type of organisms which do not recombinantly contain the nucleic acids.
  • the polyunsaturated fatty acids produced in the organisms used in the process can in principle be increased in two ways.
  • the pool of free polyunsaturated fatty acids and / or the proportion of esterified polyunsaturated fatty acids produced by the process can be increased.
  • the inventive method increases the pool of esterified polyunsaturated fatty acids in the transgenic organisms, advantageously in the form of the phosphatidyl esters and / or triacyl esters.
  • microorganisms are used as organisms in the process according to the invention, they are grown or grown, depending on the host organism, in a manner known to the person skilled in the art.
  • Microorganisms are usually in a liquid medium containing a carbon source usually in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally vitamins, at temperatures between 0 0 C and 100 0 C, preferably between 10 0 C and 60 0 C attracted under oxygen fumigation.
  • the pH of the nutrient fluid can be kept at a fixed value be regulated during breeding or not.
  • the cultivation can be batchwise, semi-batch wise or continuous.
  • Nutrients can be presented at the beginning of the fermentation or fed in semi-continuously or continuously.
  • the polyunsaturated fatty acids prepared can be isolated from the organisms by methods known to those skilled in the art as described above. For example, extraction, distillation, crystallization, optionally salt precipitation and / or chromatography. The organisms can be opened up for this purpose yet advantageous.
  • the erfmdungs proper method when it is in the host organisms are microorganisms, advantageously carried out at a temperature between 0 0 C and 95 ° C, preferably between 10 0 C and 85 ° C, more preferably between 15 ° C and 75 ° C, most preferably carried out between 15 ° C and 45 ° C.
  • the pH is advantageously maintained between pH 4 and 12, preferably between pH 6 and 9, more preferably between pH 7 and 8.
  • the process according to the invention can be operated batchwise, semi-batchwise or continuously.
  • a summary of known cultivation methods is in the textbook by Chmiel (bioprocess 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994) ) to find.
  • the culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are given in the Manual of Methods for General Bacteriology of the Merican Society for Bacteriology (Washington D.C, USA, 1981).
  • these media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
  • sugars such as mono-, di- or polysaccharides.
  • very good carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • Sugar can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It may also be advantageous to add mixtures of different carbon sources.
  • Other possible sources of carbon are oils and fats, e.g. Soybean oil, sunflower oil, peanut oil and / or coconut fat, fatty acids such as e.g.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds.
  • Exemplary nitrogen sources include ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soy protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used singly or as a mixture.
  • Inorganic salt compounds that may be included in the media include the Chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • sulfur-containing fine chemicals in particular methionine
  • inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols can be used.
  • Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
  • Chelating agents can be added to the medium to keep the metal ions in solution.
  • Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
  • the fermentation media used according to the invention for the cultivation of microorganisms usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthotenate and pyridoxine.
  • growth factors and salts are often derived from complex media components, such as yeast extract, molasses, corn steep liquor, and the like.
  • suitable precursors can be added to the culture medium.
  • All media components are sterilized either by heat (20 min at 1.5 bar and 121 0 C) or by sterile filtration.
  • the components can either be sterilized together or, if necessary, sterilized separately. All media components may be present at the beginning of the culture or added randomly or batchwise, as desired.
  • the temperature of the culture is usually between 15 ° C and 45 ° C, preferably at 25 ° C to 40 0 C and can be kept constant or changed during the experiment.
  • the pH of the medium should be in the range of 5 to 8.5, preferably 7.0.
  • the pH for cultivation can be controlled during cultivation by addition of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid. To control the foam development antifoams such.
  • B. fatty acid polyglycol esters are used.
  • the medium can be selected selectively acting substances such. As antibiotics, are added.
  • oxygen or oxygen-containing gas mixtures such as ambient air
  • the temperature of the culture is normally from 20 0 C to 45 ° C and preferably at 25 ° C to 40 0 C.
  • the culture is continued until a maximum of the desired product has formed. This goal is usually reached within 10 hours to 160 hours.
  • the fermentation broths thus obtained in particular containing polyunsaturated fatty acids, usually have a dry matter content of 7.5 to 25% by weight.
  • the fermentation broth can then be further processed.
  • the biomass can be wholly or partly by separation methods, such. As centrifugation, filtration, decantation or a combination of these methods are removed from the fermentation broth or completely left in it.
  • the biomass is worked up after separation.
  • the fermentation broth can also without cell separation with known methods such. B. with the aid of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, or by nanofiltration, thickened or concentrated. This concentrated fermentation broth may eventually be worked up to recover the fatty acids contained therein.
  • the fatty acids obtained in the process are also suitable as starting material for the chemical synthesis of further products of value. They may be used, for example, in combination with each other or solely for the manufacture of pharmaceuticals, foods, animal feed or cosmetics.
  • a further subject of the invention are isolated nucleic acid molecules comprising a nucleic acid sequence which codes for polypeptides having ⁇ -6-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 1, b) nucleic acid sequences which are known as Deriving the result of the degenerate genetic code from the amino acid sequence shown in SEQ ID NO: 2, and c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1 which encode polypeptides having at least 68% identity at the amino acid level with SEQ ID NO: 2 and a ⁇ -6 Have desaturase activity.
  • nucleic acid sequences found in the context of this invention which code for a polypeptide with ⁇ -6-desaturase activity, encode a polypeptide which is distinguished from known lipid-dependent desaturases by a very high substrate turnover (35% desaturation).
  • substrate turnover 35% desaturation
  • the ⁇ -6-desaturase according to the invention from M. squamata shows a significantly higher substrate specificity.
  • the ⁇ -6-desaturase according to the invention converts only 18: 3 '' to 18: 4 ''', whereas the ⁇ 6-desaturase from Ostreococccus tauri also accepts 18: 2 ⁇ 9 ' 12 as substrate.
  • the advantage is that one can achieve a more targeted production of .omega.3-fatty acids by the ⁇ -6-desaturase according to the invention from M. squamata (see FIG. 1).
  • High substrate specificity of the ⁇ 6-desaturase of the present invention means in this invention that the substrate 18: 3 ⁇ 9 '12 '15 is reacted significantly more than 18: 2 ⁇ 9' 12th Preference is given to 18: 3 ⁇ 9 ' 12 ' 15 1, 5-fold, 2-fold, 4-fold, 6-fold, 8-fold or 10-fold, more preferably 12-fold, 15-fold or 20-fold stronger accepted and implemented as 18: 2 ⁇ 9 '12 .
  • a further subject of the invention are isolated nucleic acid molecules comprising a nucleic acid sequence which codes for polypeptides with ⁇ -5-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 3, b) nucleic acid sequences which are known as Derive the result of the degenerate genetic code from the amino acid sequence shown in SEQ ID NO: 4, and c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 3, which code for polypeptides having at least 67% identity at the amino acid level with SEQ ID NO: 4 and have a ⁇ -5 desaturase activity.
  • the ⁇ 5-desaturase activity found in the context of this invention is acyl-CoA-dependent. It is the first ⁇ 5-desaturase activity found in a microalgae.
  • nucleic acid sequences By “derivatives" of the nucleic acid sequences according to the invention are meant in particular nucleic acid sequences which under stringent conditions with a
  • Nucleic acid sequence according to SEQ ID no. 1 or SEQ ID NO. 3 hybridize, as well as fragments of the nucleic acid sequences according to SEQ ID NO. 1 or SEQ ID NO. Third
  • a further subject of the invention are gene constructs which contain the nucleic acid sequences according to the invention SEQ ID NO: 1 and / or SEQ ID NO: 3, wherein the nucleic acid is in each case operably linked to one or more regulatory signals.
  • biosynthesis genes of the fatty acid or lipid metabolism selected from the group of ⁇ -4-desaturase, ⁇ 8-desaturase, ⁇ -9-desaturase, ⁇ -12-desaturase, ⁇ -5-elongase, ⁇ - 6-elongase, ⁇ -9 elongase, ⁇ 3-desaturase and / or ⁇ 15-desaturase.
  • ⁇ -4-desaturase ⁇ 8-desaturase
  • ⁇ -9-desaturase ⁇ -12-desaturase
  • ⁇ -5-elongase ⁇ - 6-elongase
  • ⁇ -9 elongase ⁇ 3-desaturase
  • ⁇ 15-desaturase preferably, at least one ⁇ 6-elongase is contained in the gene construct.
  • nucleic acid sequences used in the method of the invention are derived from a eukaryotic organism such as a plant, a microorganism or an animal.
  • the nucleic acid sequences are preferably derived from the order Salmoniformes, algae such as Mantoniella or Ostreococcus, fungi such as the genus Phytophtora or diatoms such as the genera Thalassiosira or Crypthecodinium.
  • Activity such as a ⁇ 5 -desaturase, ⁇ 6-desaturase and / or ⁇ 6-elongase may be included.
  • the nucleic acids used in the method are advantageously subjected to amplification and ligation in a known manner.
  • the procedure is based on the protocol of the Pfu DNA polymerase or of a Pfu / Taq DNA polymerase mixture.
  • the primers are selected on the basis of the sequence to be amplified. Conveniently, the primers should be chosen so that the amplificate comprises the entire codogenic sequence from the start to the stop codon.
  • the amplificate is conveniently analyzed. For example, the analysis can be carried out after gel electrophoretic separation in terms of quality and quantity.
  • the amplificate can be purified according to a standard protocol (eg Qiagen).
  • Suitable cloning vectors are well known to those skilled in the art. These include, in particular, vectors which can be replicated in microbial systems, ie in particular vectors which ensure efficient cloning in yeasts or fungi, and which enable stable transformation of plants. In particular, various suitable for T-DNA-mediated transformation, binary and co-integrated vector systems. Such vector systems are generally characterized in that they contain at least the vir genes required for the Agrobacterium-mediated transformation as well as the T-DNA limiting sequences (T-DNA border).
  • these vector systems also include other cis-regulatory regions such as promoters and terminators and / or selection markers, with which correspondingly transformed organisms can be identified.
  • vir genes and T-DNA sequences are arranged on the same vector
  • binary systems are based on at least two vectors, one of them vir genes, but no T-DNA and a second T-DNA, but none carries vir gene.
  • the latter vectors are relatively small, easy to manipulate and replicate in both E.coli and Agrobacterium.
  • These binary vectors include vectors of the series pBIB-HYG, pPZP, pBecks, pGreen.
  • Binl9, pBHO1, pBinAR, pGPTV and pCAMBIA are preferably used according to the invention.
  • the vectors can first be linearized with restriction endonuclease (s) and then enzymatically modified in a suitable manner. The vector is then purified and an aliquot used for cloning. In cloning, the enzymatically cut and, if necessary, purified amplicon is cloned with similarly prepared vector fragments using ligase.
  • a particular nucleic acid construct or vector or plasmid construct a or also have several codogenic gene segments.
  • the codogenic gene segments in these constructs are functionally linked to regulatory sequences.
  • the regulatory sequences include, in particular, plant sequences such as the promoters and terminators described above.
  • Constructs can advantageously be stably propagated in microorganisms, in particular Escherichia coli and Agrobacterium tumefaciens, under selective conditions and enable a transfer of heterologous DNA into plants or microorganisms.
  • nucleic acids used in the method can be introduced into organisms such as microorganisms or advantageously plants and thus used in pitch transformation, such as those published in and cited herein: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-119 (1993); FF White, Vectors for Gene Transfer to Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, 15-38; Genes Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung and R.
  • nucleic acids used in the method, the inventive nucleic acids and nucleic acid constructs and / or vectors can thus be used advantageously for genetically modifying a broad spectrum of organisms to plants so that they become better and / or more efficient producers of PUFAs.
  • ⁇ 3-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase and / or ⁇ 5-desaturase gene By introducing an ⁇ 3-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase and / or ⁇ 5-desaturase gene into an organism alone or in combination with other genes, not only the biosynthesis flux to the final product can be increased, but also the corresponding triacyl- glycerol and / or phosphatidyl ester composition increased or created de novo.
  • the number or activity of other genes necessary for the import of nutrients necessary for the biosynthesis of one or more fatty acids, oils, polar and / or neutral lipids may be increased, such that the concentration of these precursors, cofactors or intermediates within the cells or within the storage compartment, thereby further increasing the ability of the cells to produce PUFAs, as described below.
  • the nucleic acid molecules used in the method according to the invention encode proteins or parts thereof, the proteins or the individual protein or parts thereof containing an amino acid sequence which is sufficiently homologous to an amino acid sequence which is present in the sequences SEQ ID NO: 2 or SEQ ID NO: 4, so that the proteins or parts thereof still have ⁇ 6-desaturase and / or ⁇ 5-desaturase activity.
  • the proteins or portions thereof encoded by the nucleic acid molecule (s) still retain their essential enzymatic activity and the ability to catabolize cell membranes or lipid bodies in organisms advantageously participate in plants necessary compounds or in the transport of molecules through these membranes.
  • the enzymatic activity of the ⁇ -6-desaturase according to the invention and ⁇ -5-desaturase or derivatives thereof can be determined, for example, by expressing the enzyme to be tested in a suitable host organism such as yeast, exogenous substrates (18: 3 ''). in the case of ⁇ -6-desaturase and 20: 3 "in the case of ⁇ -5-desaturase) and, after a certain incubation time, analyzes the fatty acid pattern.
  • the appearance of the products 18: 4 ⁇ 6 ' 9 ' 12 '15 or 20: 4 ⁇ 5 ' 8 ' ⁇ ' 14 indicates a ⁇ -6-desaturase or ⁇ -5-desaturase activity in the yeast cultures. Details on the experimental procedure can be found in the exemplary embodiments of the present application.
  • a ⁇ 6-desaturase or ⁇ 5-desaturase in the sense of the present invention has an enzymatic activity of at least 10%, 15%, 20% or 25%, preferably 30%, 35%, 40% or 45%, especially preferably 50%, 55%, 60%, 65%, 70% or 75%, most preferably 80%, 82%, 84%, 86% or 88%, and most preferably 90%, 92%, 94%, 96% , 98%, 100%, 110%, 120%, 130%, 150%, 180% or 200% of the enzymatic activity of the enzymes encoded by SEQ ID NO: 1 and 3, respectively.
  • the proteins encoded by the nucleic acid molecules are at least 67%, 68% or 69% and preferably at least about 70%, 74%, 78%, 80% or 84%, and more preferably at least about 85%, 86%, 87%, 88%, 89% or 90%, and most preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to those shown in SEQ ID NO: 2 or SEQ ID NO: 4 amino acid sequences shown.
  • enzymatic activity of the ⁇ -6-desaturase or ⁇ -5-desaturase used in the process according to the invention is to be understood as meaning that it has opposite the proteins / enzymes coded by the sequence with SEQ ID NO: 1 or SEQ ID NO: 3 and their derivatives in comparison, at least one enzymatic activity of at least 10%, preferably 20%, more preferably 30% and very particularly 40% and thus the metabolism of fatty acids for the construction of fatty acid esters such as diacylglycerols and / or triacylglycerides in an organism advantageously a plant or Plant cell necessary compounds or participate in the transport of molecules via membranes, wherein Cl 8, C20 or C22 carbon chains in the fatty acid molecule with double bonds at least two, preferably three, four, five or six digits are meant.
  • Nucleic acids useful in the method are derived from bacteria, fungi, diatoms, animals such as Caenorhabditis or Oncorhynchus or plants such as algae or mosses such as the genera Shewanella, Physcomitrella, Thraustochytrium, Fusarium, Phytophthora, Ceratodon, Mantoniella, Ostreococcus, Isochrysis, Aleurita, Muscarioides, Mortierella , Borago, Phaeodactylum, Crypthecodinium, especially of the genera and species Oncorhynchus mykiss, Thalassiosira pseudonona, Mantoniella squamata, Ostreococcus sp., Ostreococcus tauri, Euglena gracilis, Physcomitrella patens, Phytophthora infestans, Fusarium graminaeum, Cryptocodinium cohn
  • nucleotide sequences encoding a ⁇ 6-desaturase or ⁇ 5-desaturase and which hybridize to a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3, advantageously under stringent conditions, may be used in the method of the invention .
  • Nucleic acid molecules advantageous for the method according to the invention can be isolated on the basis of their homology to the desaturase nucleic acids disclosed herein using the sequences or a part thereof as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • isolated nucleic acid molecules can be used that are at least 15 nucleotides long and hybridize under stringent conditions with the nucleic acid molecules comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
  • Nucleic acids of at least 25, 50, 100, 250 or more nucleotides may also be used.
  • hybridized under stringent conditions is intended to describe hybridization and washing conditions under which nucleotide sequences that are at least 60% homologous to one another usually remain hybridized to one another.
  • the conditions are preferably such that sequences that are at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous, are usually hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridizations in 6 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 0.2 x SSC, 0.1%. SDS at 50 to 65 ° C. It is known to those skilled in the art that these hybridization conditions differ with respect to the type of nucleic acid and, for example, when organic solvents are present, with respect to the temperature and the concentration of the buffer. The temperature differs, for example under "standard hybridization conditions" depending on the type of nucleic acid between 42 ° C and 58 ° C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2).
  • the temperature is about 42 ° C under standard conditions.
  • the hybridization conditions for DNA: DNA hybrids are, for example, 0.1 x SSC and 20 0 C to 45 ° C, preferably between 30 0 C and 45 ° C.
  • the hybridization conditions for DNA: RNA hybrids are, for example, 0.1 x SSC and 30 0 C to 55 ° C, preferably between 45 ° C and 55 ° C.
  • nucleotide sequences coding for an ⁇ -3-desaturase, ⁇ 12-desaturase, ⁇ 9-elongase, ⁇ 8-desaturase or ⁇ -4-desaturase can additionally be used in the method according to the invention.
  • the nucleic acid sequences used in the method are advantageously introduced into an expression cassette which enables expression of the nucleic acids in organisms such as microorganisms or plants.
  • nucleic acid sequences which are responsible for one of the abovementioned enzyme activities advantageously an ⁇ -3-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 6-elongase, ⁇ 12-desaturase, ⁇ 5-elongase, ⁇ -9 elongase, ⁇ -8-desaturase or ⁇ -4-desaturase, with one or more regulatory signals advantageously functionally linked to increase gene expression.
  • These regulatory sequences are intended to allow the targeted expression of genes and protein expression. Depending on the host organism, this may mean, for example, that the gene is expressed and / or overexpressed only after induction, or that it is immediately expressed and / or overexpressed.
  • these regulatory sequences are sequences to which inducers or repressors bind, and so
  • the gene construct may advantageously also contain one or more so-called enhancer sequences functionally linked to the promoter, which allow increased expression of the nucleic acid sequence. Additional advantageous sequences can also be inserted at the 3 'end of the DNA sequences, such as further regulatory elements or terminators.
  • only one copy of the genes is present in the expression cassette.
  • This gene construct or gene constructs can be expressed together in the host organism.
  • the gene construct or the gene constructs can be inserted in one or more vectors and be present freely in the cell or else be inserted in the genome. It is advantageous for the insertion of additional genes in the host genome when the genes to be expressed are present together in a gene construct.
  • the regulatory sequences or factors can, as described above, preferably positively influence the gene expression of the introduced genes and thereby increase them.
  • enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers.
  • an enhancement of the translation is possible by, for example the stability of the mRNA is improved.
  • a further embodiment of the invention are one or more gene constructs which contain one or more sequences which are defined by SEQ ID NO: 1 or SEQ ID NO: 3 or its derivatives and for polypeptides according to SEQ ID NO: 2 or SEQ ID NO: 4 code.
  • the abovementioned ⁇ 6-desaturase and ⁇ 5-desaturase proteins advantageously result in the desaturation or elongation of fatty acids in interaction with other enzymes of fatty acid and lipid biosynthesis, the substrate advantageously having one, two, three, four, five or six Double bonds and advantageously has 18, 20 or 22 carbon atoms in the fatty acid molecule.
  • Advantageous regulatory sequences for the novel process are, for example, in promoters, such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5 , T3, gal, trc, ara, SP6, ⁇ -PR or ⁇ -PL promoter and are advantageously used in Gram-negative bacteria.
  • promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5 , T3, gal, trc, ara, SP6, ⁇ -PR or ⁇ -PL promoter and are advantageously used in Gram-negative bacteria.
  • Further advantageous regulatory sequences are, for example, in the gram-positive promoters amy and SP02, in the yeast or fungal promoters ADC1, MFa, AC, P-60, CYCl, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV / 35S [Franck et al, Cell 21 (1980) 285-294], PRPl [Ward et al., Plant. Biol. 22 (1993)], SSU, OCS, Iib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter.
  • inducible promoters such as those described in EP-AO 388 186 (benzylsulfonamide-inducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracycline Inducible), EP-AO 335 528 ( Abzisinic inducible) or WO 93/21334 (ethanol or cyclohexenol inducible) promoters.
  • Other suitable plant promoters are Promoter of cytosolic FBPase or potato ST-LSIPromotor (Stockhaus et al., EMBO J.
  • promoters which allow expression in tissues involved in fatty acid biosynthesis.
  • seed-specific promoters such as the USP promoter according to the embodiment, but also other promoters such as the LeB4, DC3, phaseolin or napin promoter.
  • seed-specific promoters which can be used for monocotyledonous or dicotyledonous plants and in US Pat. No. 5,608,152 (rapeseed napin promoter), WO 98/45461 (oleosin promoter from Arobidopsis), US Pat. No. 5,504,200
  • WO 91/13980 Phaseolus vulgaris
  • Boce4 promoter from Brassica Phaseolus vulgaris
  • Van Baeumlein et al. Plant J., 2, 2, 1992: 233-239
  • the following promoters are suitable, for example, for monocots: barley lpt-2 or lpt-1 promoter (WO 95/15389 and WO 95/23230), barley hordein promoter and other suitable promoters described in WO 99/16890.
  • the PUFA biosynthesis genes should advantageously be seed-specifically expressed in oilseeds.
  • seed-specific promoters can be used, or such promoters, the are active in the embryo and / or in the endosperm.
  • seed-specific promoters can be isolated from both dicotolydone and monocotolydonous plants.
  • acyl- Carrier protein [US 5,315,001 and WO 92/18634], oleosin (Arabidopsis thaliana) [WO 98/45461 and WO 93/20216], phaseolin (Phaseolus vulgaris) [US 5,504,200], Bce4 [WO 91/13980], Legumes B4 (LegB4 promoter) [Bäumlein et al., Plant J., 2,2, 1992], Lpt2 and lpt1 (barley) [WO 95/15389 and others].
  • WO95 / 23230 seed-specific promoters from rice, maize and the like.
  • Plant gene expression can also be facilitated by a chemically inducible promoter (see review in Gatz 1997, Annu Rev. Plant Physiol Plant Mol. Biol., 48: 89-108). Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner.
  • promoters examples include a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.
  • sequences are advantageously used for the expression that allow constitutive expression in as many tissues of the plant as the CaMV35S, CaMV36S, CaMV35Smas, nos, mas, ubi, stpt, lea or super promoter.
  • the expression is carried out in the vegetative tissue as described above.
  • the regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences before the actual structural genes may still be present and possibly genetically altered, so that the natural regulation was switched off and the expression of genes was increased.
  • the gene construct may advantageously also contain one or more so-called enhancer sequences functionally linked to the promoter, which allow increased expression of the nucleic acid sequence. Additional advantageous sequences can also be inserted at the 3 'end of the DNA sequences, such as further regulatory elements or terminators. Advantageous terminators are, for example, viral terminators such as the 35S terminator or others.
  • the gene construct or the gene constructs can be inserted in one or more vectors and be present freely in the cell or else be inserted in the genome. It is advantageous for the insertion of further genes into the plant if the genes to be expressed are present together in a gene construct.
  • nucleic acid sequences which code for the ⁇ 6-desaturase or ⁇ 5-desaturase according to the invention and optionally further enzymes of the lipid metabolism can also be present individually in a gene construct and introduced into a suitable host plant. These transformed host plants can then be crossed with each other to obtain the desired combination of enzymes in the offspring.
  • each of the nucleic acids used in the process should be expressed under the control of its own, preferably a different promoter, since repeating sequence motifs lead to instability of the T-DNA or to recombination events can.
  • the expression cassette is advantageously constructed so that a promoter follows a suitable interface for insertion of the nucleic acid to be expressed, advantageously in a polylinker.
  • a terminator may be behind the polylinker.
  • This sequence is repeated several times, preferably three, four or five times, so that up to five genes are combined in one construct and thus can be introduced into the transgenic plant for expression.
  • the sequence is repeated up to three times.
  • the nucleic acid sequences are inserted for expression via the appropriate interface, for example in the polylinker downstream of the promoter.
  • each nucleic acid sequence has its own promoter and optionally its own terminator.
  • Such advantageous constructs are disclosed for example in DE 10102337, DE 10102338 or WO 2007/017419.
  • nucleic acid sequences behind a promoter and possibly in front of a terminator.
  • the insertion site or the sequence of the inserted nucleic acids in the expression cassette is not of decisive importance, that is, a nucleic acid sequence may be inserted at the first or last position in the cassette, without this significantly affecting the expression.
  • different promoters can be used in the expression cassette, for example the USP, LegB4 or DC3 promoter and different ones
  • Terminators are used. But it is also possible to use only one type of promoter in the cassette.
  • the CaMV35S promoter can also be used several times.
  • transcription of the introduced genes should be advantageously aborted by suitable terminators at the 3 'end of the introduced biosynthetic genes (beyond the stop codon).
  • the OCSl terminator can be used here.
  • different terminator sequences should be used for each gene.
  • the gene construct may, as described above, also include other genes to be introduced into the organisms. It is possible and advantageous to introduce into the host organisms regulatory genes, such as genes for inducers, repressors or enzymes, which intervene by their enzyme activity in the regulation of one or more genes of a biosynthetic pathway, and to express therein. These genes may be heterologous or of homologous origin. Furthermore, further biosynthesis genes of the fatty acid or lipid metabolism can advantageously be contained in the nucleic acid construct or gene construct, or else these genes can be located on a further or several further nucleic acid constructs.
  • nucleic acid sequences are biosynthesis genes of the fatty acid or lipid metabolism selected from the group of acyl-CoA: lysophospholipid acyltransferase, ⁇ -4-desaturase, ⁇ -5-desaturase, ⁇ -6-desaturase, ⁇ -9-desaturase, ⁇ -12 -Desaturase, ⁇ -5- Elongase and / or ⁇ -6 elongase.
  • nucleic acids or genes can be cloned in combination with other elongases and desaturases in expression cassettes, such as those mentioned above, and used for the transformation of plants with the aid of Agrobacterium.
  • the expression cassettes can be used in principle directly for introduction into the plant or else be introduced into a vector.
  • the term "vector” refers to a nucleic acid molecule that can transport another nucleic acid to which it is attached.
  • a vector is a "plasmid,” which is a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • a viral vector is another type of vector, where additional DNA segments can be ligated into the viral genome.
  • Certain vectors may autonomously replicate in a host cell into which they have been introduced (eg bacterial vectors of bacterial origin of replication). Other vectors are advantageously integrated into the genome of a host cell upon introduction into the host cell and thereby replicated together with the host genome.
  • vectors may direct the expression of genes to which they are operably linked. These vectors are referred to herein as "expression vectors".
  • expression vectors suitable for recombinant DNA techniques are in the form of plasmids.
  • plasmid and “vector” can be used interchangeably because the plasmid is the most commonly used vector form.
  • the invention is intended to encompass other forms of expression vectors, such as viral vectors that perform similar functions.
  • vector is also intended to mean other vectors known to the person skilled in the art, such as phages, viruses, such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, Cosmids, linear or circular DNA.
  • the recombinant expression vectors advantageously used in the method comprise the nucleic acid sequences or the described gene construct used in the method in a form suitable for expression of the nucleic acids used in a host cell, which means that the recombinant expression vectors comprise one or more regulatory sequences selected on the basis of For expression to be used host cells, which is operably linked to the nucleic acid sequence to be expressed include.
  • operably linked means that the nucleotide sequence of interest is bound to the regulatory sequence (s) such that expression of the nucleotide sequence is possible and they are linked to each other such that both sequences correspond to the predicted sequences attributed to the sequence Function (eg in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals).
  • Regulation sequences include those that direct the constitutive expression of a nucleotide sequence in many types of host cells and those that direct the direct expression of the nucleotide sequence only in certain host cells under certain conditions.
  • the design of the expression vector may depend on factors such as the selection of the host cell to be transformed, the level of expression of the desired protein, etc.
  • the recombinant expression vectors used may be designed to express the nucleic acid sequences used in the method in prokaryotic or eukaryotic cells. This is advantageous since intermediate steps of the vector construction are often carried out in microorganisms for the sake of simplicity.
  • the genes of the invention and other genes of fatty acid and lipid metabolism can be expressed in bacterial cells, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, MA, et al., 1992, Foreign gene expression in yeast : a review ", Yeast 8: 423-488; van den Hondel, CAMJJ, et al.
  • recombinant expression vector may be transcribed and translated in vitro using, for example, T7 promoter regulatory sequences and T7 polymerase.
  • Typical fusion expression vectors include i.a. pGEX (Pharmacia Biotech Inc., Smith, DB, and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), in which glutathione-S Transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein.
  • GST glutathione-S Transferase
  • pTrc amann et al., (1988) Gene 69: 301-315
  • pET Id a coexpressed viral RNA polymerase
  • T7 gnl a coexpressed viral RNA polymerase
  • Prophagen provided a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • Other suitable vectors in prokaryotic organisms are known to those skilled in the art, these vectors are, for example, in E. coli pLG338, pACYC184, the pBR series, such as pBR322, the pUC series, such as pUC18 or pUC19, the Ml 13mp series, pKC30, pRep4, pHSl , pHS2, P PLc236, pMBL24, pLG200, pUR290, pN-111113-Bl, ⁇ gtl 1 or pBdCI, in Streptomyces pIJ101, pI364, pIJ702 or pIJ361, in Bacillus pUB10, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in the yeast S. cerevisiae include pYeDesaturased (Baldari et al. (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Vectors and methods for constructing vectors suitable for use in other fungi, such as filamentous fungi include those described in detail in: van den Hondel, CAMJJ, & Punt, PJ.
  • yeast vectors are, for example, pAG-1, YEp6, YEpI 3 or P EMBLYe23.
  • nucleic acid sequences used in the method of the invention can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
  • genes used in the method can be found in unicellular plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251 and references cited therein, and higher plant pesticides (eg, spermatophytes, such as crops). are expressed.
  • plant expression vectors include those described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left Border ", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 8711-8721; Vectors for Gene Transfer to Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, pp. 15-38.
  • a plant expression cassette preferably contains regulatory sequences that can control gene expression in clones and are operably linked so that each sequence can fulfill its function, such as termination of transcription, for example, polyadenylation signals.
  • Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens T-DNA, such as the gene 3 of the Ti plasmid pTiACH [delta] known as octopine synthase (Gielen et al. (1984) EMBO J. 3: 835ff.) Or functional equivalents thereof , but also all other terminators functionally active in plants are suitable.
  • a plant expression cassette preferably contains other operably linked sequences, such as Translational enhancers, such as the overdrive sequence, which contains the 5'-untranslated tobacco mosaic virus leader sequence which increases the protein / RNA ratio (Gallie et al., (1987) Nucl. Acids Research 15: 8693-8711).
  • the inserted gene must be operably linked to a suitable promoter that performs gene expression in an upright, cell or tissue-specific manner.
  • suitable promoters are constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those derived from plant viruses, such as 35S CaMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also US 5,352,605 and WO 84/02913) or plant promoters, such as the Rubisco small subunit described in US 4,962,028.
  • telomeres are preferred sequences necessary to direct the gene product into its corresponding cell compartment (see review in Kermode, Crit., Plant, 15, 4 (1996) 285) -423 and references cited therein), for example to the vacuole, the nucleus, all types of plastids such as amyloplasts, chloroplasts, chromoplasts, extracellular space, mitochondria, endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
  • plastids such as amyloplasts, chloroplasts, chromoplasts, extracellular space, mitochondria, endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
  • the plant gene expression can also be facilitated as described above via a chemically inducible promoter (see a review in Gatz 1997, Annu Rev. Plant Physiol Plant Mol. Biol., 48: 89-108).
  • Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline Inducible promoter (Gatz et al. (1992) Plant J. 2: 397-404) and an ethanol-inducible promoter.
  • Promoters which respond to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-induced PRPl gene promoter (Ward et al. (1993) Plant Mol. Biol. 22: 361-366), the heat-inducible hsp80 promoter from tomato (US Pat. No. 5,187,267), the potato-alpha-amylase-inducible promoter (WO 96/12814) or the wound-inducible pinII promoter (EP-A-0 375 091).
  • the pathogen-induced PRPl gene promoter Ward et al. (1993) Plant Mol. Biol. 22: 361-366
  • the heat-inducible hsp80 promoter from tomato US Pat. No. 5,187,267
  • the potato-alpha-amylase-inducible promoter WO 96/1281
  • the wound-inducible pinII promoter EP-A-0 375 091
  • those promoters which induce gene expression in tissues and organs in which fatty acid, lipid and oil biosynthesis take place are preferred in seed cells such as the cells of the endosperm and the developing embryo.
  • Promoters are the rapeseed napkin promoter (US 5,608,152), the Vicia faba USP promoter (Baeumlein et al (1991) Mol Gen Genet, 225 (3): 459-67), the Arabidopsis oleosin promoter (WO 98/45461), the phaseolin promoter from Phaseolus vulgaris (US 5,504,200), the Brassica Bce4 promoter (WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al. (1992) Plant Journal 2 (1992); 2): 233-9) as well as promoters which induce seed-specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice and the like.
  • Suitable noteworthy promoters are the lpt2 or lpt1 gene promoter from barley (WO 95/15389 and WO 95/23230) or the promoters described in WO 99/16890 from the barley hordein gene, the rice glutelinase. Gene, rice oryzine gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, corn zein gene, oat glutelin gene, sorghum kasirin gene, rye secalin -Gene).
  • the multiparallel expression of the nucleic acid sequences used in the method may be desired.
  • the introduction of such expression cassettes can be carried out or preferred via a simultaneous transformation of a plurality of individual expression constructs by combining several expression cassettes on a construct. It is also possible to transform a plurality of vectors each having a plurality of expression cassettes and to transfer them to the host cell.
  • promoters which induce plastid-specific expression are particularly suitable.
  • Suitable promoters such as the viral RNA polymerase promoter, are described in WO 95/16783 and WO 97/06250, and the Arabidopsis clpP promoter described in WO 99/46394.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection”, conjugation and transduction are intended to encompass a variety of methods known in the art for introducing foreign nucleic acid (eg DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE- Dextran-mediated transfection, lipofection, natural competence, chemically mediated transfer, electroporation or particle bombardment.
  • Suitable methods for transforming or transfecting host cells, including plant cells can be found in Sambrook et al.
  • nucleic acid (molecule) as used herein also includes, in an advantageous embodiment, those located at the 3 'and 5' ends of the coding gene region untranslated sequence: at least 500, preferably 200, more preferably 100 nucleotides of the sequence upstream of the 5 'end of the coding region and at least 100, preferably 50, more preferably 20 nucleotides of the sequence downstream of the 3' end of the coding gene region.
  • An “isolated” nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid.
  • Nucleic acid preferably does not have sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (e.g., sequences located at the 5 'and 3' ends of the nucleic acid).
  • the isolated nucleic acid molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences that naturally comprise the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived flank.
  • nucleic acid molecules used in the method can be isolated using standard molecular biology techniques and the sequence information provided herein. It is also possible with the aid of comparative algorithms to identify, for example, a homologous sequence or homologous, conserved sequence regions at the DNA or amino acid level. These can be used as hybridization probes in standard hybridization techniques (such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CoId Spring Harbor Laboratory, Col.
  • nucleic acid molecule comprising a complete sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof can be isolated by polymerase chain reaction, wherein oligonucleotide primers based on this sequence or parts thereof (eg, a nucleic acid molecule comprising the complete sequence or a portion thereof can be isolated by polymerase chain reaction using oligonucleotide primers prepared on the basis of this same sequence).
  • mRNA can be isolated from cells (eg, by the guanidinium thiocyanate extraction method of Chirgwin et al., (1979) Biochemistry 18: 5294-5299) and cDNA by reverse transcriptase (eg, Moloney MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV Reverse Transcriptase, available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase eg, Moloney MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV Reverse Transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for polymerase chain reaction amplification can be prepared on the basis of one of the sequences shown in SEQ ID NO: 1 or SEQ ID NO: 3 or with the aid of the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4.
  • a nucleic acid of the invention may be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques. The thus amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • Oligonucleotides corresponding to a desaturase nucleotide sequence may be prepared by standard synthetic methods, for example, with an automated DNA synthesizer.
  • Homologs of the desaturase nucleic acid sequences used having the sequence SEQ ID NO: 1 or SEQ ID NO: 3 means for example allelic variants with at least 67%, 68% or 69%, preferably at least about 70%, 74%, 78%, 80% or 84%, more preferably at least about 85%, 86%, 87%, 88%, 89% or 90%, and most preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity or homology to a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or their homologs, derivatives or analogs or parts thereof.
  • isolated nucleic acid molecules of a nucleotide sequence which hybridize to one of the nucleotide sequences shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof, eg hybridized under stringent conditions.
  • allelic variants include functional variants which can be obtained by deletion, insertion or substitution of nucleotides from / in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, but the intention is that the enzyme activity should be that of the one resulting therefrom synthesized proteins for the insertion of one or more genes is advantageously retained.
  • Proteins which still have the enzymatic activity of ⁇ -6-desaturase or ⁇ -5-desaturase, ie whose activity is essentially not reduced means proteins with at least 10%, preferably 20%, particularly preferably 30%, very particularly preferably 40%, 50%, 60%, 70%, 80% of the original enzyme activity compared to the protein encoded by SEQ ID NO: 1 or SEQ ID NO: 3.
  • homologues of SEQ ID NO: 1 or SEQ ID NO: 3 also mean bacterial, fungal and plant homologs, truncated sequences, single-stranded DNA or RNA of the coding and non-coding DNA sequence.
  • Homologs of SEQ ID NO: 1 or SEQ ID NO: 3 also mean derivatives, such as promoter variants.
  • the promoters upstream of the indicated nucleotide sequences may be modified by one or more nucleotide substitutions, insertion (s) and / or deletion (s) without, however, interfering with the functionality or activity of the promoters becomes. It is also possible that the activity of the promoters is increased by modification of their sequence or that they are completely replaced by more active promoters, even from heterologous organisms.
  • nucleic acids and protein molecules with ⁇ -6-desaturase or ⁇ -5-desaturase activity which are involved in the metabolism of lipids and fatty acids, PUFA cofactors and enzymes or in the transport of lipophilic compounds via membranes, are used in the method according to the invention for modulation the production of PUFAs in transgenic organisms advantageous in plants such as maize, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, Linum species such as oil or fiber kidney, Brassica species such as rapeseed, canola and Turnip rape, pepper, sunflower, borage, evening primrose and tagetes, solanacea plants such as potato, tobacco, aubergine and tomato, vicia species, pea, manioc, alfalfa, shrimp (coffee, cocoa, tea), salix species, trees ( Oil palm, coconut) and perennial grasses and forage crops, either directly (eg, if overexpression or optimization of
  • Production and / or production efficiency of the fatty acid from modified organisms and / or may have an indirect effect which nevertheless results in an increase in the yield, production and / or efficiency of the production of the PUFAs or a decrease in undesired compounds (eg modulation of the metabolism of lipids and fatty acids, cofactors and enzymes results in changes in the yield, production and / or efficiency of production or composition of the desired compounds within the cells, which in turn may affect the production of one or more fatty acids).
  • the combination of different precursor molecules and biosynthetic enzymes leads to the production of various fatty acid molecules, which has a decisive effect on the composition of the lipids.
  • PUFAs for example stearidonic acid, eicosapentaenoic acid, arachidonic acid and docosahexaenoic acid, are Brassicaceae, Boraginaceae, Primulaceae, or Linaceae.
  • Lein Linum usitatissimum is particularly advantageously suitable for the production of PUFAS with the nucleic acid sequences according to the invention, preferably as described, in combination with other desaturases and elongases.
  • the lipid synthesis can be divided into two sections: the synthesis of fatty acids and their binding to sn-glycerol-3-phosphate as well as the addition or modification of a polar head group.
  • Common lipids used in membranes include phospholipids, glycolipids, sphingolipids and phosphoglycerides.
  • Fatty acid synthesis begins with the conversion of acetyl-Co A into malonyl-CoA by the acetyl-Co A carboxylase or in
  • Acetyl-ACP by acetyl transacylase. After a condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydration reactions to give a saturated fatty acid molecule of the desired chain length.
  • the production of unsaturated fatty acids from these molecules is catalyzed by specific desaturases, either aerobically by molecular oxygen or anaerobically (for fatty acid synthesis in microorganisms see FC Neidhardt et al., (1996) E.
  • Precursors for the PUFA biosynthesis are, for example, oleic acid, linoleic acid and linolenic acid. These C18 carbon fatty acids must be extended to C20 and C22 to obtain Eicosa and Docosa chain type fatty acids, especially ARA and EPA.
  • Arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid advantageously eicosapentaenoic acid, arachidonic acid and / or docosahexaenoic acid, can be prepared with the aid of the desaturases and / or elongases used in the process and subsequently used for various purposes in food, feed, cosmetic or pharmaceutical applications become.
  • C20 and / or C22 fatty acids having at least two, advantageously at least three, four, five or six double bonds in the fatty acid molecule, preferably C20 or C22 fatty acids with advantageously four, five or six double bonds in the fatty acid molecule, can be produced with the abovementioned enzymes.
  • the desaturation can be carried out before or after elongation of the corresponding fatty acid.
  • the products of desaturase activities and possible further desaturation and elongation result in preferred PUFAs having a higher desaturation level, including a further elongation of C20 to C22 fatty acids, to fatty acids such as ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, Stearidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.
  • Substrates of the desaturases and elongases used in the process according to the invention are C16, C18 or C20 fatty acids such as, for example, linoleic acid, ⁇ -linolenic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, eicosatetrape acid or stearidonic acid.
  • Preferred substrates are linoleic acid, ⁇ -linolenic acid and / or ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid or arachidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.
  • the synthesized C20 or C22 fatty acids having at least two, three, four, five or six double bonds in the fatty acid are obtained in the novel process in the form of the free fatty acid or in the form of their esters, for example in the form of their glycerides.
  • glycolide is understood to mean a glycerol esterified with one, two or three carboxylic acid residues (mono-, di- or triglyceride).
  • glycolide is also meant a mixture of different glycerides.
  • the glyceride or glyceride mixture may contain other additives, e.g. contain free fatty acids, antioxidants, proteins, carbohydrates, vitamins and / or other substances.
  • a "glyceride” in the sense of the method according to the invention is also understood to mean derivatives derived from glycerol.
  • these also include glycerophospholipids and glyceroglycolipids.
  • the glycerophospholipids such as lecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol, phosphatidylserine and alkylacylglycerophospholipids, may be mentioned by way of example here.
  • Lipid synthesis is the transfer of fatty acids to the polar head groups, for example, by glycerol-fatty acid acyltransferase (see Frentzen (1998) Lipid, 100 (4-5): 161-166).
  • the PUFAs produced in the process comprise a group of molecules that are no longer able to synthesize, and therefore need to take up, higher animals, or that can no longer sufficiently produce higher animals themselves, and thus have to additionally take up, even though they are readily synthesized by other organisms, such as bacteria For example, cats can no longer synthesize arachidonic acid.
  • phospholipids are to be understood as meaning phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and / or phosphatidylinositol, advantageously phosphatidylcholine.
  • production or productivity are in the
  • the concentration of the fermentation product formed in a given period of time and fermentation volume eg, kg of product per hour per liter. It also includes the productivity within a plant cell or plant, that is the content of the desired fatty acids produced in the process based on the content of all fatty acids in that cell or plant.
  • Efficiency of production includes the time it takes to produce a certain amount of production (eg how long the cell needs to set up a specific throughput rate of a fine chemical).
  • yield or product / carbon yield is known in the art and includes the efficiency of converting the carbon source into the product (ie, the fine chemical). This is usually expressed, for example, as kg
  • biosynthesis or biosynthetic pathway are known in the art and involve the synthesis of a compound, preferably an organic compound, by a cell from intermediates, for example in a multi-step and highly regulated process.
  • degradation or degradation pathway are well known in the art and involve the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), for example in a multi-step and highly regulated process.
  • metabolism is known in the art and includes the entirety of the biochemical reactions that take place in an organism. The metabolism of a particular compound (e.g., the metabolism of a fatty acid) then comprises all of the biosynthetic, modification, and degradation pathways of that compound in the cell that affect that compound.
  • the desaturation efficiency in the context of the invention can be calculated by the formula (product x 100) / (educt + product).
  • the sequences are written one below the other for the purpose of optimal comparison (eg, gaps may be inserted into the sequence of one protein or one nucleic acid for optimal alignment with the other protein or nucleic acid produce).
  • the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared.
  • An isolated nucleic acid molecule encoding a ⁇ 6-desaturase or ⁇ 5-desaturase homologous to a protein sequence of SEQ ID NO: 2 or SEQ ID NO: 4 may be prepared by introducing one or more nucleotide substitutions, additions, or deletions are generated in a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into any of the sequences of SEQ ID NO: 1 or SEQ ID NO: 3 by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made on one or more of the predicted nonessential amino acid residues.
  • conservative amino acid substitution the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Familie of Amino acid residues have been defined with similar side chains.
  • amino acids with basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains eg Alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains eg, threonine, valine, isoleucine
  • aromatic side chains eg, tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in a ⁇ 6-desaturase or ⁇ 5-desaturase is thus preferably exchanged for another amino acid residue from the same side-chain family.
  • the mutations may be introduced randomly over all or part of the ⁇ 6-desaturase or ⁇ 5-desaturase coding sequence, for example, by saturation mutagenesis, and the resulting mutants may be prepared according to the ⁇ -6 described herein - Desaturase or ⁇ -5-desaturase activity are screened to identify mutants that have retained the ⁇ -6-desaturase or ⁇ -5-desaturase activity.
  • the encoded protein can be recombinantly expressed, and the activity of the protein can be determined using, for example, the assays described herein. Thereafter, as a rule, the activity is detected by expression of the corresponding cDNA in an organism such as yeast and subsequent fatty acid analysis.
  • transgenic non-human organisms which contain the nucleic acids according to the invention according to SEQ ID NO: 1 or SEQ ID NO: 3 or a gene construct or a vector which contain these nucleic acid sequences according to the invention.
  • the non-human organism is a microorganism, a non-human animal or a plant, more preferably a plant. Examples for suitable plants have already been mentioned.
  • the cloning methods e.g. Restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, attachment of DNA fragments, transformation of Escherichia coli cells, culture of bacteria and sequence analysis of recombinant DNA were performed as described in Sambrook et al , (1989) (CoId Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
  • the sequencing of recombinant DNA molecules was carried out using a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al. (1977) Proc. Natl. Acad., See, USA74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced to avoid polymerase defects in constructs to be expressed and checked.
  • the effect of genetic modification in plants, fungi, algae or ciliates on the production of a desired compound may be determined by culturing the modified microorganism or modified plant under suitable conditions (such as those described above) and the medium and / or the cellular components are assayed for increased production of the desired product (ie, lipids or a fatty acid).
  • suitable conditions such as those described above
  • the desired product ie, lipids or a fatty acid.
  • analytical techniques are known to the person skilled in the art and include spectroscopy, thin-layer chromatography, staining methods of various types, enzymatic and microbiological methods and analytical chromatography, such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and Pp.
  • Nutrient levels in the medium e.g., sugars, hydrocarbons, nitrogen sources, phosphate and other ions
  • measurements of biomass composition and growth analysis of the production of common metabolites of biosynthetic pathways, and measurements of gases produced during fermentation. Standard methods for these measurements are in Applied Microbial Physiology; A Practical Approach, P.M. Rhodes and P.F. Stanbury, eds., IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and references cited therein.
  • FAME fatty acid methyl ester
  • GC-MS gas-liquid chromatography-mass spectrometry
  • TAG triacylglycerol
  • TLC thin-layer chromatography
  • the unequivocal evidence of the presence of fatty acid products can be obtained by analysis of recombinant organisms by standard analytical methods: GC, GC-MS or TLC as variously described by Christie and the references therein (1997, in: Advances on Lipid Methodology, Fourth Edition: Christie, OiIy Press, Dundee, 119-169; 1998, gas chromatography-mass spectrometry method, Lipids 33: 343-353).
  • the material to be analyzed may be broken up by sonication, milling in the glass mill, liquid nitrogen and milling or other applicable methods. The material must be centrifuged after rupture. The sediment is distilled in aqua. re-suspended, heated at 100 ° C.
  • fatty acid methyl ester are extracted in petroleum ether and finally subjected to GC analysis (mikrom Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25, 0.32 mm) using a capillary column with a temperature gradient of between 170 0 C and 240 0 C for 20 min and 5 min at 240 0 C subjected.
  • GC analysis mikrom Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25, 0.32 mm
  • the identity of the resulting fatty acid methyl esters must be defined using standards available from commercial sources (ie Sigma).
  • Plant material is first mechanically homogenized by mortars to make it more accessible to extraction. Then it is heated for 10 min at 100 0 C and sedimented again after cooling on ice. The cell sediment is hydrolyzed for 1 h at 90 ° C. with 1 M methanolic sulfuric acid and 2% dimethoxypropane, and the lipids are trans methylated. The resulting fatty acid methyl esters (FAME) are extracted into petroleum ether. The extracted FAME are purified by gas chromatography using a capillary column (Chrompack, WCOT fused silica, CP-Wax-52 CB, 25 m, 0.32 mm) and a temperature gradient of 170 ° C. to 240 ° C.
  • Mantoniella squamata Manton et Parke
  • Desikachary was purchased from the algae culture collection Göttingen (SAG, Germany).
  • plasmid DNA was prepared on a Qiagen DNA purification robot (Qiagen, Hilden, Germany) according to the manufacturer's instructions and subjected to random sequencing by means of the chain termination method Use of the ABI
  • RNA isolated from M. squamata was transcribed and ligated to adapter-ligated double-stranded cDNA (ds-cDNA) using the Marathon cDNA amplification kit (BD Bioscience) according to the manufacturer's instructions.
  • the adapter-ligated ds cDNA was diluted (1: 250) with sterile dd H 2 O and used as template for 5 'and 3' RACE PCR reactions to eliminate the missing 5 'prime and 3' prime To obtain ends of the coding sequences for different desaturases.
  • a Marathon cDNA adapter primer 1 (API) and a gene-specific primer were designed using the EST sequence information.
  • the primers used in 5 'and 3' RACE reactions were as follows: for Ms ⁇ 6 (MsI, ⁇ -6-desaturase from M. squamata) as 5'-RACE primer 5'-CATCCGGGCGGCAGCGTCATCTTCTAC-S 'and as
  • a 50 ⁇ l standard reaction mixture contained 1 ⁇ Ex Taq DNA polymerase buffer, 1 ⁇ Ex Taq DNA polymerase (TaKaRa Bio), 0.2 mM of each dNTP, 0.5 ⁇ M 5 'primer or 3' primer, 0, 5 ⁇ M API and 5 ⁇ l of the diluted adapter-ligated ds cDNA.
  • RACE PCR amplification was performed as follows: 30 sec. At 94 ° C; 5 cycles of 5 s at 94 0 C, 3 min at 72 0 C; 5 cycles of 5 s at 94 0 C, 3 min at 70 0 C; 20 cycles of 5 s at 94 0 C, 3 min at 68 0 C.
  • the amplif ⁇ fashionen products were isolated from agarose gels by means of a kit (GE Healthcare Bioscience) and then purified in the plasmid pGEM-T (Promega). From the 5 'and / or 3' cDNA sequence data obtained by the RACE-PCR, the putative translation start codons and stop codons were identified, and this sequence information was used to obtain full-length cDNA clones of the putative desaturases from M. squamata receive.
  • the tracked EST sequencing strategy generated more than 3,500 non-redundant sequences.
  • the sequences of the two isolated full length cDNA clones for desaturases from M. squamata are given in SEQ ID NO: 1 and 3.
  • the MsI cDNA represents a ⁇ 6-desaturase, shown in SEQ ID NO: 1, which was annotated as Ms ⁇ 6 (M squamata ⁇ 6-desaturase).
  • the amino acid sequence shown in SEQ ID NO: 2 shows an ORF of 450 amino acids.
  • the MsII cDNA shown in SEQ ID NO: 3 encodes a ⁇ 5-desaturase whose amino acid sequence shown in SEQ ID NO: 4 has an ORF of 483 amino acids.
  • the MsII clone was annotated as Ms ⁇ 5 (M squamata ⁇ 5-desaturase).
  • Gene-specific primers were designed to the 5 'and 3' ends of the coding regions of the corresponding nucleotide sequences introducing restriction sites for subsequent cloning into the various yeast expression vectors.
  • the ORF (open reading frame) of Ms ⁇ 6 was cloned with the primers given in Table 1 in pYES2.
  • the forward primers of Ms ⁇ 6 and Ms ⁇ 5 were designed such that the nucleotide sequence ACATA was included before the ATG start codon (Table 1) to enhance translation initiation in eukaryotic cells.
  • the ORFs of Ms ⁇ 6 and Ms ⁇ 5 were amplified by PCR with the described primers (see Table 1) using the Expand High Fidelity 1 "1118 PCR system (Roche Diagnostics), cDNA template and a standard PCR protocol (2 min at 94 0 C, 10 cycles of 10 s at 94 0 C, 30 s at 70 0 C, 80 s at 72 0 C, followed by 20 cycles of 10 s at 94 0 C and 3 min at 72 0 C, and a final elongation step of 7 min at 72 0 C) modified.
  • amplified cDNAs were cloned into the pGEM-T vector (Promega) before they cut again and cloned into a yeast expression vector (pYES2, Invitrogen, pESC-LEU or pESC-TRP, Stratagene) which resulted in the vectors pYES2-Ms ⁇ 6, pESC-LEU-Ms ⁇ 6 and pESC-TRP-Ms ⁇ 5.
  • the ⁇ 6 elongase from Physcomitrella patens, PSE1 was cloned into the yeast expression vector pESC-LEU to add the vector pESC-LEU-PSE1 -Ms ⁇ 6 before the ORF of Ms ⁇ 6 was inserted as explained above.
  • the P. tricornutum cDNA clones were cloned in yeast expression vectors as described above for M.
  • S. cerevisiae cells of the INVScI strain from Invitrogen were prepared by the method of Dohmen et al. (Dohmen et al. (1991) Yeast 7: 691-692). For purposes of inducing expression cultures for 72 h were at 21-23 0 C in the presence of 2% (w / v) galactose, supplemented with 350 uM of a suitable fatty acid substrate and in the presence of 1% Igepal CA 630 ( 'NP 40') of Attracted Sigma-Aldrich.
  • the cells were harvested by centrifugation at 1200 g for 5 min and the pellets washed twice with sterile dd H 2 O before being used for further analysis.
  • the host strain transformed with the empty vector (s) was used as a negative control in all experiments.
  • Fatty acid methyl esters were obtained by transmethylation of the yeast cell pellets with 0.5 M sulfuric acid in methanol containing 2% (v / v) dimethoxypropane at 80 ° C. for 1 h. FAMEs were extracted in hexane and analyzed by gas chromatography (GC). GC analysis was performed on an Agilent GC 6890 system coupled with a FID detector equipped with a 122-2332 DB-23 capillary column (30 mx 0.32 mm, 0.5 ⁇ m coating thickness, Agilent).
  • Helium was "used samples were injected at 220 0 C The temperature gradient was as follows: 150 0 C for 1 min, 150 0 C - 200 0 C at 15 0 C min., As the carrier gas (1 mL min)" 1 200 0 C - 250 0 C at 2 0 C min "1 , and 250 0 C for 10 min. The data were evaluated using HP ChemStation Rev. A09.03. FAMEs were identified by comparison with appropriate reference substances of FAMEs.
  • the filling length cDNAs of Ms ⁇ 6 and Ms ⁇ 5 had been cloned into pYES2, pESC-LEU and pESC-TRP.
  • the desaturase sequences are under the control of the galactose-inducible promoter GAL1 or GAL10.
  • the plasmids obtained were designated Ms ⁇ 6-pYES2, Ms ⁇ 6-pESC-LEU and Ms ⁇ 5-pESC-TRP.
  • the clones were individually expressed in S. cerevisiae strain INVScI.
  • Ms ⁇ 6 enzyme showed very high specificity, only the fed ⁇ -linolenic acid was used as substrate and in stearidonic acid with 17% desaturation efficiency (pYES2-Ms ⁇ 6) and 35% desaturation efficiency (pESC-LEU-Ms ⁇ 6, which is the translation initiation sequence ACATA) ( Figure 4A).
  • Ms ⁇ 5 appears to be less specific and converts both 20: 3 ⁇ 8 ' n ' 14 and 20: 4 ⁇ 8 ' n ' 14 '17 as a substrate for desaturation.
  • lipid analysis For lipid analysis, expression was carried out in 120 ml cultures. Harvested cell pellets were homogenized in 5 ml chloroform / methanol (1: 2) and the lipids extracted on a shaker for 4 h and then for 20 h with 5 ml chloroform / methanol (2: 1) at 4 ° C. The resulting organic phases were combined and evaporated under nitrogen. The remaining lipids were dissolved in 1 ml of chloroform. Separation of the lipid classes (neutral lipids and phospholipids) was achieved using a silica gel column (Bond Elut SI, 100 mg / ml, Varian, Darmstadt).
  • the lipid extracts were loaded onto the silica gel column previously pre-equilibrated with chloroform and then fractionated into the lipid classes by elution as follows: neutral fats with chloroform and phospholipids with methanol / glacial acetic acid (9: 1).
  • the isolation of the individual components from the phospholipid class was achieved by means of thin-layer chromatography (TLC) with methanol / chloroform / glacial acetic acid (65: 25: 8) as the eluent and with suitable standards.
  • the proportions of phospholipids vary from 14% in phosphatidylethanolamine (PE) and phosphatidylinositol / phosphatidylserine (PI / PS) to 22% of total lipid-associated stearidonic acid in phosphatidylcholine (Figure 5A).
  • PE phosphatidylethanolamine
  • PI / PS phosphatidylinositol / phosphatidylserine
  • patens ⁇ 6 elongase resulted from the efficient ⁇ 6 desaturation of the 18: 3 ⁇ 9 ' 12 ' 15 substrate (16% conversion) by Ms ⁇ 6 , the efficient elongation (97% conversion) of the ⁇ 6 desaturation product 18: 4 '''to the corresponding C20 fatty acid as well as the efficient ⁇ 5 desaturation (20% conversion).
  • EPA accumulation was not as significant as the fatty acid intermediates are intermediates between the PC pool and the acyl-CoA pool because of the additional transacylation step required to move back and forth, probably less efficiently presented to the affected enzymes.
  • the indicative fatty acid intermediate 18: 4 ⁇ 6 ' 9 ' 12 '15 accumulated to a greater extent than could be observed with expression of M. squamata enzyme Q, indicating less efficient conversion for ⁇ -6 desaturation (6% conversion) ⁇ 6 elongation (61% conversion) and consequently lower ⁇ 5 desaturation (14% conversion).
  • nucleic acid sequences used in the method according to the invention which code for a ⁇ 6-desaturase and a ⁇ 5-desaturase activity are expressed in an expression cassette alone or in a preferred manner in combination with other nucleic acid sequences, for a ⁇ 6 elongase, a ⁇ 5 elongase, a ⁇ 4-desaturase, an ⁇ 3-desaturase, a ⁇ 12-desaturase, a ⁇ 15-desaturase, a bifunctional ⁇ 12 and ⁇ 15-desaturase, a lysophospholipid acyltransferase, a diacylglycerol acyltransferase or a phospholipid diacylglycerol acyltransferase, are introduced into and expressed in a suitable plant.
  • nucleic acid sequence of an enzymatic activity e.g. a ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 6-elongase, ⁇ 5-elongase, ⁇ 4-desaturase, ⁇ -3-desaturase, ⁇ 12-desaturase, ⁇ 15-desaturase, bifunctional ⁇ 12 and ⁇ 15 desaturase, lysophospholipid acyltransferase, diacylglycerol acyltransferase and / or phospholipid
  • Diacylglycerol acyltransferase be included.
  • plants such as Brassica species such as oilseed rape, sunflower, soybean, linum species such as oillein, corn, rye, wheat, oats, crambe, triticale, rice, greens, potatoes, field beans such as Vicia faba, pea, oil palm, coconut, peanut, borage are promoters that are active in plant cells and the expression of the
  • nucleic acid sequence (s) in plant cells are preferably promoters with a constant and / or seed-specific and / or tissue-specific activity.
  • the nucleic acids used in the context of this invention are preferably amplification and ligation by means of polymerase chain reaction (for example, following the protocol of the Pfu-DNA reaction). Polymerase or Taq polymerase) or T4 DNA ligase or other DNA-ligating enzymes.
  • the primers should be chosen to allow amplification of the entire coding region of the nucleic acids described in this method.
  • the nucleic acids can then be introduced into suitable for expression in microorganisms or plant cells Verktorsysteme.
  • vectors which are replicable in microbial systems include, in particular, vectors which are replicable in microbial systems and, above all, vectors which ensure efficient cloning in yeasts, microorganisms or fungi, and the stable transformation of plants, such as Agrobacterium-mediated transformation.
  • Suitable transformation methods for plants are known in the art and are based on T-DNA mediated transformation suitable binary and co-integrated vector systems.
  • Vector systems include additional cis-regulatory elements and selection markers.
  • the transformation of the plants is based on standard protocols and the selection of trans formants is allowed by means of the selection marker.
  • the fatty acid analysis of the transgenic plants is suitably carried out in the form of a
  • transgenic seeds e.g. transgenic oilseed rape seeds (Brassica napus).
  • transgenic oilseed rape seeds Brassica napus
  • the reaction mixture (200 ⁇ l) containing 100 mM Tris-HCl, pH 8.1, 10 mM MgCl 2 , 5 mM ATP, 5 mM CoASH, 2 mM DTT, 25 ⁇ M free fatty acid, and 2.5 units of the acyl-CoA Synthase was incubated for two hours at 37 ° C.
  • the reaction was stopped with 50 ⁇ l of glacial acetic acid / ethanol 1: 1 (v / v), the desired aqueous phases were washed with petrol to remove residual free fatty acids.
  • acyl-CoA analysis of yeast cells 20 ml of the liquid cultures were harvested at an OD0000 of 1.5 to 2.0 and the acyl-CoA species were extracted as described in Domergue et al. (2005) Biochem. J. 389 (2): 483-490. Conversion of the acyl-CoA esters to their etheno derivatives and acyl-CoA analysis were performed as described by Larson and Graham (2001) Plant J. 25 (1): 115-125.
  • Example 10 Distribution of Ms ⁇ 6 and Ms ⁇ 5 desaturation products in different lipid classes in yeast
  • yeast cultures expressing Ms ⁇ 6 and Ms ⁇ 5 were treated with exogenous 18: 3 ⁇ 9 ' 12 ' 15 and 20: 3 ⁇ 8 ' n, respectively '14 and the fatty acid distribution in the individual lipid classes was analyzed.
  • the same experiments were performed with the acyl-CoA-dependent Ot ⁇ 6 and the lipid-dependent Pt ⁇ 6 and Pt ⁇ 5 desaturase.
  • 18: 4 ⁇ 6 ' 9 ' 12 '15 which was produced by ⁇ 6 desaturation of the 18: 3 ⁇ 9 ' 12 '15 substrate, was predominantly in the neutral
  • the 18: 4 "' 5 product was approximately evenly distributed in all ⁇ 6-desaturase expression cultures in phosphatidylinositol with phosphatidylserine (PI / PS), phosphatidylethanolamine (PE) and diphosphatidylglycerol (CL).
  • PI / PS phosphatidylinositol with phosphatidylserine
  • PE phosphatidylethanolamine
  • CL diphosphatidylglycerol
  • acyl-CoA profiles were determined for the respective yeast expression cultures. Control cultures expressing Ot ⁇ 6, Pt ⁇ 6 or Pt ⁇ 5 were tested in parallel. The data presented were obtained by the method described in Domergue et al. (2005) Biochem. J. 389 (2): 483-490. Exogenous substrates (18: 3 ⁇ 9 ' 12 ' 15 for ⁇ 6-desaturases or 20: 3 ⁇ 8 ' n ' 14 for ⁇ 5-desaturases) were added to the induced yeast cultures at an optical density of 1.5 to 2.0.
  • 18: 3 ⁇ 9 ' 12 ' 15 to 18: 4 ⁇ 6 ' 9 ' 12 '15 in an acyl-CoA dependent manner which is consistent with the data for Ot ⁇ 6 described by Domergue et al. (2005) Biochem. J. 389 (2): 483-490.
  • the delayed appearance of the 18: 4 ⁇ 6 ' 9 ' 12 '15 product in the acyl-CoA pool in the expression of Pt ⁇ 6 indicates that desaturation by Pt ⁇ 6 occurs on fatty acids attached to phospholipids rather than those associated with CoA.
  • Desaturases the desaturases were individually coexpressed with acyl-CoA elongases.
  • the background of this experiment is that fatty acid extension takes place in the acyl-CoA pool (Domergue et al (2003) J. Biol. Chem. 278: 35115-35126) and that fatty acids desaturated in the acyl-CoA pool be extended more efficiently than those that have been denatured by lipid-dependent enzymes and thereby require additional acyl-transferase activities.
  • the efficiency of the combined desaturations / extensions was compared in the same way to that resulting from the expression of the known desaturases Ot ⁇ 6, Pt ⁇ 6 or Pt ⁇ 5.
  • ⁇ 6-desaturases were isolated in yeast with the ⁇ 6-elongase PSE1 from the moss
  • the ⁇ 5-desaturases Ms ⁇ 5 and Pt ⁇ 5 were coexpressed in yeast with the OtELO5 ⁇ 5 elongase from O. tauri (Meyer et al. (2004) J Lipid Res. 45 (10): 1899-1909). 20: 4 ⁇ 8 ' n ' 14 '17 , the ⁇ 3 substrate of the ⁇ 5-desaturases, was added to the expression cultures, since the O. tauri ⁇ 5 elongase omega3 fatty acid substrates is preferred over ⁇ 6 substrates (Meyer et al ) J Lipid Res. 45 (10): 1 899-1909). As shown in Fig.
  • plasmids were constructed based on the pUC 19 vector.
  • a triple cassette containing three seed-specific USP promoter copies (Bäumlein et al. (1991) Mol. Gen. Gent. 225 (3): 459-67), three OCS terminator copies (MacDonald et al., (1991 ) Nucleic Acids Res. 19 (20): 5575-5581) and three different polylinkers between each promoter and terminator were first introduced into the vector pUC19 (Pharmacia) to give the plasmid USP123OCS.
  • the open reading frames of the various desaturases and elongases were modified by PCR to provide appropriate restriction sites adjacent to the start and stop codons, cloned into the pGEM-T vector (Promega) and sequenced to confirm the correctness.
  • the primers used were (restriction sites shown in bold): Ms ⁇ 6, forward 5'-ATGCGCGGCCGCACATAATGTGTCCTCCCAAGGAAT-S 'reverse 5'-GCATTCTAGACTAGTGAGCGTGCCTTC-3'; Ms ⁇ 5, forward 5'-ATGCCCATGGACATAATGCCCCCGCGCGAGACCACCAC-S 'reverse 5'-GCATACCGGTTCACCCGATGGTTTGAAGGC-S'; Pt ⁇ 6, forward 5'-ATGCGCGGCCGCACATAATGGGCAAAGGAGGGGACGC-S 'reverse 5'-GCATTCTAGATTACATGGCGGGTCCATCGCGTA-S'; Pt ⁇ 5, forward 5'-ATGCCCATGGACATAATGGCTCCGGATGCGGATAAGC-S 'reverse 5'-GCATGCGATCGCTTACGCCCGTCCGGTCAA-S', PSE1, forward 5'-AGTCGGATCCTATGGAGGTCGTGGAGAGAT-S
  • the open reading frames were then released using the restriction sites generated by the PCR and then inserted into the same sites of the polylinker of the USP123OCS plasmid.
  • the resulting cassette containing the three genes, each under the control of the USP promoter, was released by digesting the USP123OCS plasmid with Sbfi or SacI and cloned into the appropriate sites of the pCAMBIA3300 binary vector.
  • the binary vector pCAMBIA3300 (CAMBIA, Canberra, Australia) uses the bar gene together with the CaMV 35S promoter as a selection marker in plants.
  • the resulting binary plasmid constructs (Figure 12) were transformed into chemically competent Agrobacterium tumefaciens' cells (strain EH105).
  • Plants of the Arabidopsis thaliana ecotype Columbia were transformed by floral dipping (Clough and Bent (1998) Plant J. 16 (6): 735-43.).
  • T2 seeds were collected from single ammonium glufosinate-resistant Tl plants and analyzed individually by GC. For lipid analysis, 10 mg of seed were homogenized in 4 ml of chloroform / methanol / glacial acetic acid (2: 1: 0, v / v / v) and incubated for 24 h at 4 ° C. Seed residues were pelleted (2 min, 3000 xg).
  • the supernatant was collected and the pelleted seed residues were incubated with 2 ml of hexane for 30 min at room temperature. The resulting organic phases were combined and dried under nitrogen. The dried lipids were dissolved in 200 ⁇ l of chloroform.
  • the separation of the lipid classes (TAG and various phospholipids) was achieved by thin layer chromatography (TLC) with methanol / chloroform / glacial acetic acid (65: 25: 8) as eluent.
  • TLC thin layer chromatography
  • methanol / chloroform / glacial acetic acid 65: 25: 8
  • the lipids (TAG, PC, PI / PS and PE) were identified according to authentic standards (xy), scraped from the TLC plates and reextracted with the eluent for subsequent fatty acid analysis.
  • FAMEs Fatty acid methyl esters
  • TMSH trimethylsulfonium hydroxide
  • FAMEs of DC-separated individual lipids were prepared by transmethylation with 333 ul of toluene / methanol (1: 2 v / v) and 167 ul 0.5 M NaOCH 3 in
  • the enzymes selected should be specific to ⁇ 3 substrates. Another limitation to avoid is the limiting acyl chain shuttling between PC and CoA pools described by Abbadi et al. (2004) Plant Cell 16 (10): 2734-2748 was observed.
  • the acyl-CoA dependent ⁇ 6 and ⁇ 5 desaturases from the M. squamata algae were coexpressed with a ⁇ 6 elongase from the moss P. patens in Arabidopsis plants (referred to as triple Ms).
  • triple Ms a ⁇ 6 elongase from the moss P. patens in Arabidopsis plants
  • the ⁇ 6- and ⁇ 5-desaturases from the diatom P. tricornutum were labeled with the ⁇ 6 elongase from the moss P. patens in Arabidopsis plants (referred to as triple-Pt). according to Abbadi et al. (2004) Plant Cell 16 (10): 2734-2748 coexpressed.
  • the plant transformation constructs encoded each gene under the control of the seed-specific USP promoter (Bäumlein et al (1991) Mol Gen Genet 225 (3): 459-67).
  • the fatty acid analysis of the individual T2 seeds revealed different new fatty acids, which were classified as ⁇ 3-18: 4 ⁇ 6 ' 9 ' 12 '15 , ⁇ 3-20: 3 ⁇ 11 ' 14 '17 , ⁇ 3- 20: 4 ⁇ 8 ' 1 U4 ' 17 and ⁇ 3-20: 5 ⁇ 5 ' 8 ' n ' 14 ' 17 (for triple Ms, see Figure 13A and B) and for ⁇ 6-18: 3 ⁇ 6 ' 9 ' 12 , ⁇ 6- for triple Pt.
  • the ⁇ 3 biosynthesis pathway could be established in the transgenic triple M plants, since the ⁇ 6-desaturase from M. squamata specifically desaturates the oo3 fatty acid 18: 3 ⁇ 9 ' 12 ' 15 and thus does not produce ⁇ 6 fatty acids.
  • both the ⁇ 3 and the ⁇ 6 biosynthetic pathway could be established in the Triple-Ot and Triple-Pt plants, as the ⁇ 6-desaturases from O. tauri and P. tricornutum both the oo3 fatty acid 18: 3 '' and use the ⁇ 6 fatty acid 18: 2 ⁇ 9 '12 as substrate.
  • the percentage of endogenous fatty acids 18: 2 ⁇ 9 '12 and 18: 3 ⁇ 9 ' 12 '15 and the newly formed ⁇ 3 and 006 VLCPUFA in the total fatty acid content of the transgenic Arabidopsis seeds are shown in Figures 15 and 16.
  • the endogenous fatty acids 18: 2 ⁇ 9 '12 and 18: 3 ⁇ 9 ' 12 '15 served as starting substrates of the newly introduced VLCPUF A biosynthesis.
  • the percentages of the two fatty acids were the same in all three transgenic approaches (Fig. 15 A and 16 A).
  • the most accumulating newly formed fatty acids in the Triple-Ms and Triple-Ot plants were the elongation products of the ⁇ 6 elongase, ⁇ 3-20: 4 ⁇ 8 ' n ' 14 '17 and ⁇ 3- in the Triple-Ot plants, respectively. 20: 4 ⁇ 8 ' n ' 14 '17 and ⁇ 6-20: 3 ⁇ 8 ' n '14 (Fig. 15 C and Fig. 16 C).
  • the ⁇ 6-desaturated fatty acids ⁇ 3-l 8: 4 ''' 5 and 006-18: 3 ⁇ 6 ' 9 '12 were the most abundant in the triple-Pt plants ( Figure 15B and Figure 16B).
  • Figure 2 Fatty acid profile of yeast expressing Ms ⁇ 6 with the translation initiation sequence ACATA before the ATG start codon.
  • Figure 3 Fatty acid profile of yeast expressing Ms ⁇ 5.
  • Figure 5 Distribution of fatty acids produced in yeast cultures expressing Ms ⁇ 6 or Ms ⁇ 5 on lipid classes.
  • Yeast cultures expressing the M. squamata DQsaturasm were each supplied with exogenous 18: 3 ⁇ 9 ' 12 ' 15 or 20: 3 ⁇ 8 ' n ' 14 and the distribution of the fatty acids to individual lipid classes was analyzed.
  • A) and B) show the results of Ms ⁇ 6 expression in yeast.
  • C) and D) show the results of Ms ⁇ 5 expression in yeast.
  • the fatty acid produced is expressed in terms of mole percent of total fatty acids in each lipid class (A and C) and in mole percent of total lipid-associated produced fatty acids (B and D).
  • PC Phosphatidylcholine
  • PS Phosphatidylserine
  • PI Phosphatidylinositol
  • PE Phosphatidylethanolamine
  • neutral lipids NL
  • FIG. 6 The establishment of an EPA biosynthesis pathway in yeast.
  • the yeast strain INVScI was either transformed with the empty vector (A) or the various co-expression constructs were supplemented with the fatty acid 18: 3 ⁇ 9 ' 12 ' 15 (B and C). Cell pellet FAMEs were analyzed by GC.
  • FIG. 7 Enrichment of EPA in yeast.
  • Figure 8 Distribution of stearidonic acid and arachidonic acid in different
  • Arachidonic acid the product of ⁇ 5-desaturase, in the various lipid classes was detected in yeast cultures containing Ms ⁇ 6 ( ⁇ 6-desaturase from M. squ ⁇ m ⁇ t ⁇ ), Ot ⁇ 6 ( ⁇ 6-
  • NL neutral lipids
  • PC phosphatidylcholine
  • PI / PS phosphatidylinositol with phosphatidylserine
  • CL diphosphatidylglycerol
  • FIG. 9 Substrate specificity of Ms ⁇ 6 and Ot ⁇ 6
  • Yeast cultures were transformed with Ms ⁇ 6, Ot ⁇ 6 and the corresponding empty vector pYES2 and the FAMEs from the cultures analyzed by GC.
  • Yeast expression cultures expressing Ms ⁇ 6 and control cultures expressing Pt ⁇ 6 and Ot ⁇ 6 were, respectively, after 5 and 60 minutes after addition of exogenous 18: 3 ⁇ 9 ' 12 ' 15 substrate with respect to total fatty acids (left) and CoA-bound fatty acids (right) analyzed by GC (A).
  • Yeast expression cultures expressing Ms ⁇ 5 (on the left) and control cultures expressing Pt ⁇ 5 (on the right, respectively) were observed after 1, 4, 8 and 24 hours, respectively, after addition of exogenous 20: 3 ⁇ 8 ' n ' 14 substrate with respect to total fatty acids (left) and the CoA-bound fatty acids (right) analyzed by GC (B).
  • FIG. 11 Coexpression of ⁇ 6- and ⁇ 5-desaturases with acyl-Co A-elongases
  • the desaturases Ms ⁇ 6, Ot ⁇ 6 and Pt ⁇ 6 were in each case coexpressed with the ⁇ 6 elongase PSE1 from the moss P. patens in yeast, the fatty acid 18: 3 ⁇ 9 ' 12 '15 fed and analyzed the fatty acid composition by GC (A).
  • A a control served a culture that had been transformed with the empty vector pESC-LEU.
  • the desaturases Ms ⁇ 5 and Pt ⁇ 5 were each coexpressed with the ⁇ 5 elongase OtELO5 from O.
  • FIG. 12 Binary pCAMBIA vectors for the transformation of plants
  • LB and RB left and right T-DNA boundaries
  • USP Viciafaba USP promoter
  • Ms ⁇ 6 and Pt ⁇ 6 ⁇ 6-desaturases from M. squamata and P. tricornutum, respectively
  • Ms ⁇ 5 and Pt ⁇ 5 ⁇ 5-desaturases from M. squamata and P. tricornutum, respectively
  • PSE1 ⁇ 6 elongase from P. patens
  • OCS terminator region of the octopine synthase gene of A. tumefaciens
  • 35S-Prom 35S promoter of CaMV
  • 35S term CaMV 35S terminator
  • bar glufosinate resistance gene
  • Kan kanamycin resistance gene
  • FIG. 13 Fatty acid analysis of Arabidopsis seeds
  • FIG. 14 Fatty acid analysis of triple Ms, triple Ot and triple Pt Arabidopsis seeds Fatty Acid Profiles of Single Seed Analyzes of Triple-Ms, Triple-Ot and Triple-Pt Plants.
  • Representative GC fatty acid profiles of single seeds of the transgenic triple Ms (A), triple-Ot (B) and triple-Pt (C) Arabidopsis plants are shown from three to eight independent measurements. The fatty acids were compared and identified by comparison with an authentic standard mixture (D).
  • FIG. 15 ⁇ 3 fatty acid content of transgenic Ar ⁇ bidopsis plants
  • Figure 16 ⁇ 6-F acid content of transgenic Ar ⁇ bidopsis plants Single seed analysis of two or three independent Triple-Ot and Triple-Pt Ar ⁇ bidopsis lines was performed.
  • A averages and individual values of seed analyzes of ⁇ 6-18: 2 ⁇ 9 '12
  • B the ⁇ 6 desaturation product ⁇ 6-18: 3 ⁇ 6 ' 9 '12
  • C the elongation product of the ⁇ 6 elongase, ⁇ 6-20: 3 ⁇ 8 ' ⁇ ' 14
  • D the final product of the ⁇ 6-VLCPUFA biosynthetic pathway AA, ⁇ 6-20: 4 ⁇ 5 ' 8 ' n '14 .

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Polymers & Plastics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Food Science & Technology (AREA)
  • Plant Pathology (AREA)
  • Nutrition Science (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Animal Husbandry (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

La présente invention concerne un procédé de production d'acides gras polyinsaturés, en particulier d'acides gras polyinsaturés à chaîne longue, tels que l'acide arachidonique et/ou l'acide eicosapentanoïque, dans un organisme transgénique. Selon ce procédé, des acides nucléiques codant pour des polypeptides à activité Δ-6-désaturase, Δ-6-élongase et/ou Δ-5-désaturase sont introduits dans l'organisme. De façon avantageuse, la Δ6-désaturase et la Δ5-désaturase proviennent de Mantoniella squamata et la Δ6-élongase de Physcomitrella patens. De façon avantageuse, un gène codant pour une ω-3-désaturase est en outre exprimé dans l'organisme. Dans un autre mode de réalisation avantageux du procédé, d'autres séquences d'acides nucléiques codant pour des polypeptides de la biosynthèse du métabolisme des acides gras et des lipides sont exprimées dans l'organisme. De façon particulièrement avantageuse, les séquences d'acides nucléiques codant pour une activité Δ-8-désaturase, Δ-12-désaturase, Δ-15-désaturase, Δ-4-désaturase, Δ-9-élongase et/ou Δ-5-élongase sont codées à cet effet.
PCT/EP2008/052358 2007-02-27 2008-02-27 Procédé de production d'acides gras polyinsaturés dans des organismes transgéniques WO2008104559A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP07103183.5 2007-02-27
EP07103183 2007-02-27

Publications (1)

Publication Number Publication Date
WO2008104559A1 true WO2008104559A1 (fr) 2008-09-04

Family

ID=39616489

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2008/052358 WO2008104559A1 (fr) 2007-02-27 2008-02-27 Procédé de production d'acides gras polyinsaturés dans des organismes transgéniques

Country Status (1)

Country Link
WO (1) WO2008104559A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010066703A3 (fr) * 2008-12-12 2010-09-23 Basf Plant Science Gmbh Désaturases et procédé pour la production d'acides gras polyinsaturés dans des organismes transgéniques
WO2011023800A1 (fr) * 2009-08-31 2011-03-03 Basf Plant Science Company Gmbh Molécules d'acides nucléiques régulatrices pour amplifier l'expression des gènes spécifiques aux semences dans des plantes, favorisant une synthèse accrue d'acides gras polyinsaturés
WO2011048119A2 (fr) 2009-10-20 2011-04-28 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts Procédés et moyens pour altérer la biosynthèse des lipides par ciblage de multiples enzymes sur des domaines d'organelles sub-cellulaires
WO2011079005A1 (fr) * 2009-12-24 2011-06-30 E.I. Dupont De Nemours And Company Séquences de protéines de la famille des o-acyl transférases liées à la membrane végétale et leurs utilisations pour modifier les compositions en acides gras
US8168858B2 (en) 2008-06-20 2012-05-01 E. I. Du Pont De Nemours And Company Delta-9 fatty acid elongase genes and their use in making polyunsaturated fatty acids
WO2011154947A3 (fr) * 2010-06-10 2013-02-28 Ben-Gurion University Of The Negev Research And Development Authority Gène mutant défectueux pauvre en désaturase delta 5 et utilisation de ce dernier
US9150871B2 (en) 2009-08-31 2015-10-06 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific and/or seed-preferential gene expression in plants
AU2013201738B2 (en) * 2009-08-31 2016-01-28 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific gene expression in plants promoting enhanced polyunsaturated fatty acid synthesis
US9828607B2 (en) 2009-08-31 2017-11-28 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing constitutive gene expression in plants
EP3260544A1 (fr) * 2008-11-18 2017-12-27 Commonwealth Scientific and Industrial Research Organisation Enzymes et procédés de production d'acides gras omega -3
US10005713B2 (en) 2014-06-27 2018-06-26 Commonwealth Scientific And Industrial Research Organisation Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the sn-2 position
US10125084B2 (en) 2013-12-18 2018-11-13 Commonwealth Scientific And Industrial Research Organisation Lipid comprising docosapentaenoic acid
CN109563517A (zh) * 2016-06-01 2019-04-02 加拿大国家研究委员会 菟葵脂肪酸延伸酶及其在脂肪酸生产中的用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005083093A2 (fr) * 2004-02-27 2005-09-09 Basf Plant Science Gmbh Procede de production d'acides gras polyinsatures chez des plantes transgeniques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005083093A2 (fr) * 2004-02-27 2005-09-09 Basf Plant Science Gmbh Procede de production d'acides gras polyinsatures chez des plantes transgeniques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ABBADI A ET AL: "Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: Constraints on their accumulation", PLANT CELL, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 16, no. 10, 1 October 2004 (2004-10-01), pages 2734 - 2748, XP002334261, ISSN: 1040-4651 *

Cited By (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8168858B2 (en) 2008-06-20 2012-05-01 E. I. Du Pont De Nemours And Company Delta-9 fatty acid elongase genes and their use in making polyunsaturated fatty acids
US8465787B2 (en) 2008-06-20 2013-06-18 E. I. Du Pont De Nemours And Company Delta-9 fatty acid elongase genes and their use in making polyunsaturated fatty acids
US11976287B2 (en) 2008-11-18 2024-05-07 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing ω-3 fatty acids
US10648046B2 (en) 2008-11-18 2020-05-12 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing omega-3 fatty acids
US9994792B2 (en) 2008-11-18 2018-06-12 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing omega-3 fatty acids
US9976107B2 (en) 2008-11-18 2018-05-22 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing ω-3 fatty acids
US9938486B2 (en) 2008-11-18 2018-04-10 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing omega-3 fatty acids
EP3260544A1 (fr) * 2008-11-18 2017-12-27 Commonwealth Scientific and Industrial Research Organisation Enzymes et procédés de production d'acides gras omega -3
US9139854B2 (en) 2008-12-12 2015-09-22 Basf Plant Science Company Gmbh Desaturases and process for the production of polyunsaturated fatty acids in transgenic organisms
WO2010066703A3 (fr) * 2008-12-12 2010-09-23 Basf Plant Science Gmbh Désaturases et procédé pour la production d'acides gras polyinsaturés dans des organismes transgéniques
US8822662B2 (en) 2008-12-12 2014-09-02 Basf Plant Science Company Gmbh Desaturases and process for the production of polyunsaturated fatty acids in transgenic organisms
US9828607B2 (en) 2009-08-31 2017-11-28 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing constitutive gene expression in plants
WO2011023800A1 (fr) * 2009-08-31 2011-03-03 Basf Plant Science Company Gmbh Molécules d'acides nucléiques régulatrices pour amplifier l'expression des gènes spécifiques aux semences dans des plantes, favorisant une synthèse accrue d'acides gras polyinsaturés
US9150871B2 (en) 2009-08-31 2015-10-06 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific and/or seed-preferential gene expression in plants
AU2013201738B2 (en) * 2009-08-31 2016-01-28 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific gene expression in plants promoting enhanced polyunsaturated fatty acid synthesis
JP2016028583A (ja) * 2009-08-31 2016-03-03 ビーエーエスエフ プラント サイエンス カンパニー ゲーエムベーハー 多価不飽和脂肪酸合成の増強を促進する植物の種子特異的遺伝子発現を増強する調節核酸分子
US9428757B2 (en) 2009-08-31 2016-08-30 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific gene expression in plants promoting enhanced polyunsaturated fatty acid synthesis
CN106222166A (zh) * 2009-08-31 2016-12-14 巴斯夫植物科学有限公司 用于在植物中增强种子特异性基因表达而促进增强的多不饱和脂肪酸合成的调节性核酸分子
EP3121283A1 (fr) * 2009-08-31 2017-01-25 BASF Plant Science Company GmbH Molécules d'acide nucléique régulatrices pour l'amélioration de l'expression de gènes spécifiques des semences dans des plantes de promotion de la synthèse d'acides gras polyinsaturés améliorée
EP3156492A1 (fr) * 2009-08-31 2017-04-19 BASF Plant Science Company GmbH Molécules d'acide nucléique régulatrices pour renforcer l'expression génique spécifique et/ou préférentielle de semences dans des plantes
EP3156491A1 (fr) * 2009-08-31 2017-04-19 BASF Plant Science Company GmbH Molécules d'acide nucléique régulatrices pour l'amélioration de l'expression génique spécifique et/ou préférentielle à la semence dans des plantes
EP3178937A1 (fr) * 2009-08-31 2017-06-14 BASF Plant Science Company GmbH Molécules d'acide nucléique régulatrices pour l'amélioration de l'expression de gènes spécifiques des semences dans des plantes pour faciliter la synthèse améliorée d'acides gras polyinsaturés
EP3156490A3 (fr) * 2009-08-31 2017-07-05 BASF Plant Science Company GmbH Molécules d'acide nucléique régulatrices pour l'amélioration de l'expression génique spécifique et/ou préférentielle à la semence dans des plantes
EP3153584A3 (fr) * 2009-08-31 2017-07-12 BASF Plant Science Company GmbH Molécules d'acide nucléique régulatrices pour l'amélioration de l'expression génique spécifique et/ou préférentielle de semences dans des plantes
US10689657B2 (en) 2009-08-31 2020-06-23 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing constitutive gene expression in plants
AU2016202700B2 (en) * 2009-08-31 2017-12-07 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific gene expression in plants promoting enhanced polyunsaturated fatty acid synthesis
CN112011566B (zh) * 2009-08-31 2023-12-05 巴斯夫植物科学有限公司 用于在植物中增强种子特异性基因表达而促进增强的多不饱和脂肪酸合成的调节性核酸分子
JP2013502914A (ja) * 2009-08-31 2013-01-31 ビーエーエスエフ プラント サイエンス カンパニー ゲーエムベーハー 多価不飽和脂肪酸合成の増強を促進する植物の種子特異的遺伝子発現を増強する調節核酸分子
US9970019B2 (en) 2009-08-31 2018-05-15 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific and/or seed-preferential gene expression in plants
CN102597245A (zh) * 2009-08-31 2012-07-18 巴斯夫植物科学有限公司 用于在植物中增强种子特异性基因表达而促进增强的多不饱和脂肪酸合成的调节性核酸分子
US11708578B2 (en) 2009-08-31 2023-07-25 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing constitutive gene expression in plants
CN112011566A (zh) * 2009-08-31 2020-12-01 巴斯夫植物科学有限公司 用于在植物中增强种子特异性基因表达而促进增强的多不饱和脂肪酸合成的调节性核酸分子
US10041082B2 (en) 2009-08-31 2018-08-07 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific and/or seed-preferential gene expression in plants
US10041081B2 (en) 2009-08-31 2018-08-07 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific and/or seed-preferential gene expression in plants
US10041083B2 (en) 2009-08-31 2018-08-07 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific and/or seed-preferential gene expression in plants
CN106222166B (zh) * 2009-08-31 2020-09-08 巴斯夫植物科学有限公司 用于在植物中增强种子特异性基因表达而促进增强的多不饱和脂肪酸合成的调节性核酸分子
EP3418387A1 (fr) * 2009-08-31 2018-12-26 Basf Plant Science Company GmbH Molécules d'acide nucléique régulatrices pour l'amélioration de l'expression de gènes spécifiques des semences dans des plantes pour faciliter la synthèse améliorée d'acides gras polyinsaturés
WO2011048119A2 (fr) 2009-10-20 2011-04-28 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts Procédés et moyens pour altérer la biosynthèse des lipides par ciblage de multiples enzymes sur des domaines d'organelles sub-cellulaires
US9006514B2 (en) 2009-12-24 2015-04-14 E. I. Du Pont De Nemours And Company Plant membrane O-acyl transferase (MBOAT) family protein sequences and their uses for altering fatty acid compositions
WO2011079005A1 (fr) * 2009-12-24 2011-06-30 E.I. Dupont De Nemours And Company Séquences de protéines de la famille des o-acyl transférases liées à la membrane végétale et leurs utilisations pour modifier les compositions en acides gras
US8637733B2 (en) 2009-12-24 2014-01-28 E.I. Du Pont De Nemours And Company Plant membrane bound O-acyl transferase (MBOAT) family protein sequences and their uses for altering fatty acid compositions
WO2011154947A3 (fr) * 2010-06-10 2013-02-28 Ben-Gurion University Of The Negev Research And Development Authority Gène mutant défectueux pauvre en désaturase delta 5 et utilisation de ce dernier
US10190073B2 (en) 2013-12-18 2019-01-29 Commonwealth Scientific And Industrial Research Organisation Lipid comprising long chain polyunsaturated fatty acids
US10125084B2 (en) 2013-12-18 2018-11-13 Commonwealth Scientific And Industrial Research Organisation Lipid comprising docosapentaenoic acid
US10800729B2 (en) 2013-12-18 2020-10-13 Commonwealth Scientific And Industrial Research Organisation Lipid comprising long chain polyunsaturated fatty acids
US11623911B2 (en) 2013-12-18 2023-04-11 Commonwealth Scientific And Industrial Research Organisation Lipid comprising docosapentaenoic acid
US10793507B2 (en) 2014-06-27 2020-10-06 Commonwealth Scientific And Industrial Research Organisation Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the SN-2 position
US10005713B2 (en) 2014-06-27 2018-06-26 Commonwealth Scientific And Industrial Research Organisation Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the sn-2 position
CN109563517A (zh) * 2016-06-01 2019-04-02 加拿大国家研究委员会 菟葵脂肪酸延伸酶及其在脂肪酸生产中的用途

Similar Documents

Publication Publication Date Title
EP2166089B1 (fr) Procédé de fabrication d'acides gras insaturés de manière multiple dans des organismes transgéniques
EP1720988B1 (fr) PROCEDE POUR PRODUIRE DES ACIDES GRAS omega-3 INSATURES DANS DES ORGANISMES TRANSGENIQUES
EP2177605B1 (fr) Delta-5 deasturase et procédé de production d'acides gras polyinstaurés dans des organimes transgéniques non-humains
EP2176433B1 (fr) Désaturases et procédés de production d'acides gras polyinsaturés dans des organismes transgéniques
EP2176416B1 (fr) Élongases et procédés de production d'acides gras polyinsaturés dans des organismes transgéniques
WO2006008099A2 (fr) Procede permettant d'augmenter la teneur d'organismes transgeniques en acides gras a chaine longue polyinsatures
WO2006069710A1 (fr) Procede de production d'acides gras polyinsatures dans des organismes transgeniques
WO2006100241A2 (fr) Procede de production d'acides gras c20 et c22 polyinsatures avec au moins quatre liaisons doubles dans des plantes transgeniques
WO2008104559A1 (fr) Procédé de production d'acides gras polyinsaturés dans des organismes transgéniques
DE112009003708T5 (de) Desaturasen und Verfahren zur Herstellung mehrfach ungesättigter Fettsäuren in transgenenOrganismen

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08717165

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08717165

Country of ref document: EP

Kind code of ref document: A1