WO2006069710A1 - Verfahren zur herstellung mehrfach ungesättigter fettsäuren in transgenen organismen - Google Patents
Verfahren zur herstellung mehrfach ungesättigter fettsäuren in transgenen organismen Download PDFInfo
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- WO2006069710A1 WO2006069710A1 PCT/EP2005/013803 EP2005013803W WO2006069710A1 WO 2006069710 A1 WO2006069710 A1 WO 2006069710A1 EP 2005013803 W EP2005013803 W EP 2005013803W WO 2006069710 A1 WO2006069710 A1 WO 2006069710A1
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- 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)
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- 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
Definitions
- the present invention relates to a process for the production of polyunsaturated fatty acids in an organism by introducing into the organism nucleic acids which are polypeptides having ⁇ 5 -enongase, ⁇ 6-desaturase, a ⁇ 5-desaturase Encode ⁇ -4-desaturase, ⁇ -12-desaturase and / or ⁇ -6 elongase activity.
- nucleic acids which are polypeptides having ⁇ 5 -enongase, ⁇ 6-desaturase, a ⁇ 5-desaturase Encode ⁇ -4-desaturase, ⁇ -12-desaturase and / or ⁇ -6 elongase activity.
- desaturases and elongases are advantageously derived from Ostreococcus.
- the invention relates to a process for the preparation of oils and / or triacylglycerides having an increased content of long-chain polyunsaturated fatty acids.
- the invention further relates to the nucleic acid sequences, nucleic acid constructs, vectors and organisms containing the nucleic acid sequences of the invention, vectors comprising the nucleic acid sequences and / or the nucleic acid constructs and transgenic organisms contain the aforementioned nucleic acid sequences, nucleic acid constructs and / or vectors.
- Another part of the invention relates to oils, lipids and / or fatty acids prepared by the process according to the invention and their use.
- the invention relates to unsaturated fatty acids and triglycerides having an increased content of unsaturated fatty acids and their use.
- 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 oleic and linolenic acids are essential for mammals as they can not be produced by them. Therefore multiple polyunsaturated ⁇ -3 fatty acids and ⁇ -6 fatty acids an important part of human and animal food are unsaturated long-chain ⁇ -3 fatty acids such as eicosapentaenoic acid.
- polyunsaturated long-chain fatty acids Due to the customary composition of human food today, addition of polyunsaturated ⁇ -3 fatty acids, which are preferred in fish oils, is particularly important for food.
- the unsaturated fatty acid DHA is thereby attributed a positive effect on the development and maintenance of brain functions.
- polyunsaturated fatty acids are referred to as PUFA, PUFAs, LCPUFA or LCPUFAs (poly unsaturated fatty acids, PUFA, polyunsaturated fatty acids, long chain poly unsaturated fatty acids, LCPUFA, long-chain polyunsaturated fatty acids).
- the free fatty acids are advantageously prepared by saponification.
- 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 especially polyunsaturated fatty acids
- 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.
- ⁇ -3 fatty acids By adding these ⁇ -3 fatty acids to the diet, the risk of heart disease, stroke or hypertension can be significantly reduced.
- 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 found in medicines application.
- ⁇ -6 fatty acids such as arachidonic acid tend to have a negative effect on these diseases in these rheumatic diseases due to our usual food composition.
- ⁇ -3- and ⁇ -6 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, the thromoxanes and leukotrienes derived from arachidonic acid and Derive the eicosapentaenoic acid.
- Eicosanoids which are formed from ⁇ -6 fatty acids usually promote inflammatory reactions, while eicosanoids (so-called PG 3 series) of ⁇ -3 fatty acids have little or no pro-inflammatory effect.
- ⁇ 6-desaturases are described in WO 93/06712, US 5,614,393, US 5614393, WO 96/21022, WO00 / 21557 and WO 99/27111 and also the application for production in transgenic organisms as described in WO98 / 46763 WO98 / 46764, WO9846765.
- the expression of various desaturases as described in WO99 / 64616 or WO98 / 46776 and formation of polyunsaturated fatty acids is also described and claimed. Concerning.
- microorganisms for the production of PUFAs are microorganisms such as microalgae such as Phaeodactylum tricomutum, Porphiridium species, Thraustochytrien species, Schizochytria species or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor and / or mosses such as Physcomitrella, Ceratodon and Marchantia (R. Vazhappilly & F. Chen (1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Lipids 22: 1060-1062; Akimoto, M.
- microalgae such as Phaeodactylum tricomutum, Porphiridium species, Thraustochytrien species, Schizochytria species or Crypthecodinium species
- ciliates such as Stylonychia or Colpid
- the starting material for the ⁇ -6 pathway is the fatty acid linoleic acid (18: 2 ⁇ 9 12 ), while the ⁇ -3 pathway is via linolenic acid (18: 3 ⁇ 9 ' 12 ' 15 ).
- Linolenic acid is formed by the activity of an ⁇ -3-desaturase (Tocher et al., 1998, Prog. Lipid Res., 37, 73-117, Domergue et al., 2002, Eur. J. Biochem., 269, 4105-4113).
- the elongation of fatty acids by elongases of 2 and 4 C atoms, respectively, is of crucial importance for the production of C 20 and C 22 PUFAs, respectively.
- This process runs over 4 stages.
- the first step is the condensation of malonyl-CoA the fatty acid-acyl-CoA by ketoacyl-CoA synthase (KCS, hereinafter referred to as elongase).
- KCS ketoacyl-CoA synthase
- KCR ketoacyl-CoA reductase
- dehydratase dehydratase
- enoyl-CoA reductase enoyl-CoA reductase
- LCPUFAs in higher plants, preferably in oilseeds such as oilseed rape, linseed, sunflower and soybean, since in this way large quantities of high-quality LCPUFAs can be obtained inexpensively for the food industry, animal nutrition and for pharmaceutical purposes.
- gene coding for enzymes of the biosynthesis of LCPUFAs in oilseeds must be advantageously introduced and expressed via genetic engineering methods. These are 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 the membranes or triacylglycerides. For example, ⁇ 6-desaturase genes from the moss Physcomitrella patens and ⁇ 6 elongase genes from P. patens and the nematode C. elegans have been isolated.
- transgenic organisms with a content of at least 1 wt .-% of these compounds based on the total lipid content of the transgenic organism, characterized in that it comprises the following steps: a) introducing at least one nucleic acid sequence into the organism, which for a ⁇ -6-desaturase And b) introduction of at least one nucleic acid sequence into the organism which codes for a ⁇ 6-elongase activity, and c) introduction of at least one nucleic acid sequence into the organism which codes for a ⁇ 5-desaturase activity, and d) introducing into the organism at least one nucleic acid sequence encoding ⁇ 5-elongase activity, and e) introducing into the organism at least one nucleic acid sequence encoding a ⁇ 4-desaturase activity, and wherein the variables and substituents in the formula I have the following meaning:
- R 2 hydrogen, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine, lysophosphatidylglycerol, lyso-diphosphatidylglycerol, lyso-phosphatidylserine, lyso-phosphatidylinositol or saturated or unsaturated C 2 -C 24 -alkylcarbonyl-,
- R 3 J _ hydrogen, saturated or unsaturated C 2 -C 24 -alkylcarbonyl-, or R 2 or R 3 independently of one another a radical of general formula Ia:
- R 1 in general formula I denotes hydroxyl, coenzyme A (thioester), lysophosphatidylcholine, lyso-phosphatidylethanolamine, lyso-phosphatidylglycerol, lyso-diphosphatidylglycerol, lyso-phosphatidylserine, lyso-phosphatidylinositol, sphingobase, or a radical of the general formula II
- R 1 are always bonded in the form of their thioesters to the compounds of general formula I.
- R 2 in the general formula II denotes hydrogen, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine, lyso-phosphatidylglycerol, lyso-diphosphatidylglycerol, lyso-phosphatidylserine, lyso-phosphatidylinositol or saturated or unsaturated C 2 -C 24 - alkylcarbonyl,
- alkyl radicals are substituted or unsubstituted, saturated or unsaturated C 2 -C 24 -alkylcarbonyl chains such as ethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, n-pentyl carbo ⁇ yl, n-hexylcarbonyl, n-heptylcarbonyl, n-octylcarbonyl, n-nonylcarbonyl, n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n- Pentadecylcarbonyl, n-hexadecylcarbonyl, n-hepta-decylcarbonyl, n-octadecylcarbonyl, n-nonadecyl
- C 10 -C 22 -alkylcarbonyl radicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-hepta decylcarbonyl, n-octadecylcarbonyl, n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl or n-tetracosanylcarbonyl.
- C 10 -C 22 -alkylcarbonyl radicals such as C 10 -alkylcarbonyl, C 1-4 -alkylcarbonyl, C 12 -alkylcarbonyl, C 1-3 -alkylcarbonyl, C 14 -alkylcarbonyl, C 16 -alkylcarbonyl, , C 18 - alkylcarbonyl, C 20 alkylcarbonyl or C 22 alkylcarbonyl radicals which contain one or more double bonds.
- C 16 -C 22 -alkylcarbonyl radicals such as C 6 alkylcarbonyl, C 18 alkylcarbonyl, C 20 are especially preferred - alkylcarbonyl or C 22 -alkylcarbonyl radicals which contain one or more double bonds.
- These advantageous radicals may contain two, three, four, five or six double bonds.
- the particularly advantageous radicals having 20 or 22 carbon atoms in the fatty acid chain contain up to six double bonds, advantageously three, four, five or six double bonds, more preferably five or six double bonds. All these radicals are derived from the corresponding fatty acids.
- R 3 in the general formula II is hydrogen, saturated or unsaturated C 2 -C 24 -alkylcarbonyl.
- Alkyl radicals which may be substituted or unsubstituted, saturated or unsaturated C 2 -C 24 -alkylcarbonyl chains, such as ethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, n-pentylcarbonyl, n-hexylcarbonyl, n-heptylcarbonyl, n-octylcarbonyl, n-nonylcarbonyl, n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-hepta-
- C 1 -C 22 -alkylcarbonyl radicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-hepta decylcarbonyl, n-octadecylcarbonyl, n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl or n-tetracosanylcarbonyl containing one or more double bonds are preferred.
- saturated and / or unsaturated C 10 -C 22 -alkylcarbonyl radicals such as C 10 -alkylcarbonyl, C 1-4 -alkylcarbonyl, C 12 -alkylcarbonyl, C 13 -alkylcarbonyl, C 14 -alkylcarbonyl, C 16 -alkylcarbonyl, , C 18 - alkylcarbonyl, C 20 alkylcarbonyl or C 22 alkylcarbonyl radicals which contain one or more double bonds.
- C 16 -C 22 -alkylcarbonyl radicals such as C 16 -alkylcarbonyl, C 18 -alkylcarbonyl, C 20 -alkylcarbonyl or C 22 -alkylcarbonyl radicals, having one or more double bonds contain.
- These advantageous radicals may contain two, three, four, five or six double bonds.
- the particularly advantageous radicals having 20 or 22 carbon atoms in the fatty acid chain contain up to six double bonds, advantageously three, four, five or six double bonds, more preferably five or six double bonds. All these radicals are derived from the corresponding fatty acids.
- R 1 , R 2 and R 3 may be substituted by hydroxyl and / or epoxy groups and / or may contain triple bonds.
- the polyunsaturated fatty acids prepared in the process according to the invention advantageously contain at least two, preferably three, four, five or six double bonds. Particularly advantageously, the fatty acids contain four five or six double bonds.
- Fatty acids produced in the process advantageously have 18, 20 or 22 C atoms in the fatty acid chain, preferably the fatty acids contain 20 or 22 carbon atoms in the fatty acid chain.
- saturated fatty acids are little or not reacted with the nucleic acids used in the process. Little is understood to mean that compared to polyunsaturated fatty acids, the saturated fatty acids have less than 5% of the activity, advantageously less than 3%, more preferably less than 2%, most preferably less than 1; 0.5; 0.25 or 0.125% are implemented.
- nucleic acid sequences used in the method according to the invention are isolated nucleic acid sequences which are suitable for polypeptides having ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase and / or ⁇ - 4-encoding desaturase activity.
- Nucleic acid sequences which code for polypeptides having ⁇ -6-desaturase, ⁇ -6-elongase, ⁇ -5-desaturase, ⁇ -5-elongase or ⁇ -4-desaturase activity are advantageously used in the process according to the invention
- SEQ ID NO: 9 SEQ ID NO: 11 or SEQ ID NO: 13 shown for polypeptides having at least 40% identity at the amino acid level with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID N0: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 and have a ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ -4-desaturase activity.
- the substituents R 2 or R 3 in the general formulas I and II independently of one another are saturated or unsaturated C 18 -C 22 -alkylcarbo ⁇ yls, particularly advantageously they independently of one another denote unsaturated C 8 -, C 20 - or C 22 -alkylcarbonyl with at least two double bonds.
- the method is characterized in that a nucleic acid sequence is additionally introduced into the organism which codes for polypeptides having ⁇ 12-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 15, or b) nucleic acid sequences which can be derived as a result of the degenerate genetic code from the amino acid sequence shown in SEQ ID NO: 16, or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 15, which polypeptides have at least 50 % Identity at the amino acid level with SEQ ID NO: 16 and have ⁇ -12 desaturase activity.
- a nucleic acid sequence is additionally introduced into the organism which codes for polypeptides having ⁇ 12-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 15, or b) nucleic acid sequences which can be derived as a result of the degenerate genetic code from the
- ⁇ -12-desaturase sequences can be used alone or in combination with the ⁇ 3-desaturase sequences with the nucleic acid sequences used in the method which are useful for ⁇ -6-desaturases, ⁇ -6-elongases, ⁇ -5-desaturases, ⁇ -5-elongases and or ⁇ 4-desaturases are used.
- Table 1 represents the nucleic acid sequences, the organism of origin and the sequence ID number.
- 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 glycerides.
- the different fatty acids bound in the triacylglycerides can thereby be derived from short-chain fatty acids having 4 to 6 C atoms, medium-chain fatty acids having 8 to 12 C atoms or long-chain fatty acids having 14 to 24 C atoms, preferably the long-chain fatty acids are particularly preferred the long-chain fatty acids LCPUFAs of C 18 , C 2 o and / or C 22 fatty acids.
- 9 12 15) 'Dihomo- ⁇ -linolenic acid ( DGLA, 20: 3 ⁇ 8
- 11 M ), ⁇ -3-eicosatetraenoic acid ( ETA, C20: 4 ⁇ W114 ), arachidonic acid (ARA, C20: 4 ⁇ 5 8 11 ' 14 ), Eicosapent
- 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 which contain at least two polyunsaturated fatty acids, preferably, they are isolated in the form of their diacylglycerides, triacylglycerides and / or
- 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 compounds mentioned above are present in the organisms in an approximate distribution of 80 to 90% by weight of triglycerides, 2 to 5% by weight of diglycerides, 5 to 10% by weight of 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 are present in a content of at least 3% by weight, advantageously of at least 5% by weight, preferably of at least 8% by weight, more preferably of at least 10% by weight, very particularly preferably at least 15 wt .-% based on the total fatty acids in the transgenic organisms advantageously produced in a transgenic plant.
- C 18 and / or C 20 fatty acids present in the host organisms become at least 10%, advantageously at least 20%, particularly advantageously at least 30%, very particularly advantageously at least 40% in the corresponding products such as DPA or DHA, to name just two.
- the fatty acids are prepared in bound form.
- these unsaturated fatty acids can be brought to the sn1, sn2 and / or sn3 position of the advantageously prepared triglycerides. Since in the process of the invention from the starting compounds linoleic acid (C18: 2) or linolenic acid (C18: 3) are passed through several reaction steps, the end products of the process such as
- ARA Arachidonic acid
- EPA eicosapentaenoic acid
- DHA DHA
- the precursors should advantageously not 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 Final product amount.
- ARA, EPA or only DHA are bound in the process according to the invention or produced as free acids in a transgenic plant as end products. If the compounds ARA, EPA and DHA are prepared simultaneously, they are advantageously used in a ratio of at least 1: 1: 2 (EPA: ARA: DHA), preferably of at least 1: 1: 3, preferably of 1: 1: 4 preferably prepared from 1: 1: 5.
- 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 in each case based 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 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), chaulmo-gruoic acid (cyclopentenodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,11-dienoic acid), vernon acid (9,10-epoxyoctadec-12-enoic acid), Taric acid (6-octadecynoic acid), 6-nonadecynoic acid, santalbinic acid (t11-octadecen-9-ynoic acid), 6,9- Octadecenynoic acid, pyrulic acid (t10-heptadecen-8-y
- the nucleic acid sequences according to the invention or the nucleic acid sequences used in the method according to the invention can increase the yield of polyunsaturated fatty acids by at least 50%, advantageously by at least 80%, particularly advantageously by at least 100%, very particularly advantageously by 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, see Examples.
- chemically pure polyunsaturated fatty acids or fatty acid compositions can be prepared by the methods described above.
- the fatty acids or fatty acid compositions from the organism such as the microorganisms or plants or the culture medium in which or on which the organisms were grown, or from the organism and the culture medium in a known manner, for example, extraction, distillation, crystallization, chromatography or combinations isolated from 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.
- Advantageous plants are selected from the group of the plant families Adelotheciaceae, Anacardiaceae, Asteraceae, Apiaceae, Betulaceae, Boraginaceae, Brassicadown, Bromeliaceae, Caricaceae, Cannabaceae, Convolvulaceae, Chenopodiaceae, Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae, Elaeagnaceae, Ericaceae, Euphorbiaceae , Fabaceae, Geraniaceae, Gramineae, Juglandaceae, Lauraceae, Leguminose, Linaceae, Prasinophyceae or vegetables or ornamental plants such as Tagetes.
- 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, Carthage, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta, Tagetes, Valeriana e.g.
- Brassicaceae such as the genera Brassica, Camelina, Melanosinapis, Sinapis, Arabopsopsis e.g. the genera and species Brassica napus, Brassica rapa ssp. [Canola], Sinapis arvensis Brassica juncea, Brassica juncea var. Juncea, Brassica juncea var. Crispifolia, Brassica juncea var.
- Bromeliaceae such as the genera Anana, Bromelia (pineapple) eg the genera and species Anana comosus, pineapple pineapple or Bromelia comosa
- Convolvulus panduratus [sweet potato], Chenopodiaceae such as the genus 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 [pumpkin]
- 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
- Physcomitrella Physcomitrium eg the genera and species Aphanorrhegma serrateum, Entosthodon attenuatus, Entosthodon bolanderi, Entosthodon bonplandii, Entosthodon californicus, Entosthodon drummondii, Entosthodon jamesonii, Entosthodon leibergii, Entosthodon neoscoticus, Entosthodon rubrisetus, Entosthodon spathulifolia, Entosthodon tucsoni, Funaria americana Funaria bolanderi, Funaria calcarea, Funaria californica, Funaria calvescens, Funaria convoluta, Funaria flavicans, Funaria groutiana, Funaria hygrometrica, Funaria hygrometrica var.
- Steffensia elongata. (Cayenne pepper], Poaceae such as the genera Hordeum, Seeale, Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea (maize), Triticum eg the genera and species Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon Hordeum aegiceras, Hordeum hexastichon, Hordeum hexastichum, Hordeum irregular, Hordeum sativum, Hordeum secalinum [Barley], Sea ale cereale [Rye], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.
- Sativa Avena hybrida [Oats], Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghum vulgaris, Andropogon drummondii, Holcus bicolor, Holcus Sorghum, Sorghum aethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum, Sorghum bathna, Sorghum drummondii, Sorghum durra, Sorghum guineense, Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghum subglabrescens, Sorghum verticilliflorum, Sorghum vulgaris, Holcus halepensis, Sorghum Miliaceum, Panicum militaceum [millet], Oryza sativa, Oryza latifolia [rice], Zea mays [maize] Triticum aestivum, Triticum durum,
- Verbascum blattaria Verbascum chaixii, Verbascum densiflorum, Verbascum lagurus, Verbascum longifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum, Verbascum phlomoides, Verbascum phenicum, Verbascum pulverulentum or Verbascum thapsus [Mullein], Solanaceae such as the genera Capsicum, Nicotiana , Solanum, Lycopersicon eg the genera and species Capsicum annuum, Capsicum annuum var.
- 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 ⁇ forme, Solanum integrifolium or Solanum lycopersicum [tomato], Sterculiaceae such as the genus Theobroma eg the genus and species Theobroma cacao [cocoa] or Theaceae such as the genus Camellia e.
- Advantageous microorganisms are, for example, fungi selected from the group of the families Chaetomiaceae, Choanephoraceae, Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycesaceae, Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae or Tubercularaceae.
- Examples include the following microorganisms selected from the group: Choanephoraceae as the genera Blakeslea, Choanephora eg the genera and species Blakeslea trispora, Choanephora cueurbitarum, Choanephora infundibulifera var.
- Mortierellaceae as the genus Mortierella eg the genera and species Mortierella isabellina, Mortierella polycephala , Mortierella ramanniana, Mortierella vinacea, Mortierella zonata, Pythiaceae such as the genera Phytium, Phytophthora eg the genera and species Pythium debaryanum, Pythium intermedium, Pythium irregular, Pythium megalacanthum, Pythium paroecandrum, Pythium sylvaticum, Pythium ultimum, Phytophthora cactorum, Phytophthora cinnamomi, Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, Phytophthora erythroseptica, Phytophthora lateralis, Phytophthora megasperma, Phytophthora
- Minuta Pichia minuta var.
- Saccharomyces ellipsoideus Saccharomyces chevalieri, Saccharomyces delbrueckii, Saccharomyces diastaticus, Saccharomyces drosophilarum, Saccharomyces elegans, Saccharomyces ellipsoideus, Saccharomyces fermentati, Saccharomyces florentinus, 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 pretorien
- Thraustochytriaceae such as the Genera Althomia, Aplanochytrium, Japono- chytrium, Schizochytrium, Thraustochytrium eg the species Schizochytrium aggregatum, Schizochytrium limacinum, minutum Schizochytrium mangrovei, Schizochytrium, Schizochytrium octosporum, aggregatum Thraustochytrium, Thraustochytrium amoe- boideum, antacticum Thraustochytrium, Thraustochytrium arudimentale, aureum Thraustochytrium, benthicola Thraustochytrium, globosum Thraustochytrium, Thrausto- chytrium indicum, Thraustochytrium kerguelense, Thraustochytrium kinnei, Thraustochytrium motivum, Thraustochytrium multirudimentale, Thraustochytriacea
- Bacillaceae such as the genus Bacillus eg 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.
- Bacillaceae such as the genus Bacillus eg the genera and species Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus spha
- 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 genomicpecies, 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 caroto
- 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.
- Rhizobiaceae such as the genera Agrobacterium, Carbophilus, Chelatobacter, Ensifer, Rhizobium, Sinorhizobium eg the genera and species Agrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacterium gelatino- vorum, Agrobacterium larrymoorei, Agrobacterium meteori, Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium rubi, Agrobacterium stellulatum , Agrobacterium tumefaciens, Agrobacterium vitis, Carbophilus carboxidus, Chelatobacter heint
- 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 pustulara, Stylonychia putrina, Stylonychia notophora , Stylonychia sp., Colpidium campylum or Colpidium sp.
- 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 pustulara, Stylonychia putrina, Stylonychi
- transgenic organisms such as fungi such as Mortierella or Traustochytrium, yeasts such as Saccharomyces or Schizosaccharomyces, mosses such as Physcomitrella or Ceratodon, nonhuman 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
- nonhuman animals such as Caenorhabditis
- algae such as Nephroselmis, Pseudoscourfield
- Organisms which belong to the oil-producing organisms ie those used for the production of oils, such as fungi such as Mortierella or Thraustochytrium, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, are particularly advantageously used in the process according to the invention.
- oilseed crops containing high levels of lipid compounds 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, bay leaf, pumpkin, flax, soya, pistachios, borage, trees (oil palm, coconut or walnut) or crops such as corn, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa or Bush plants (coffee, cocoa, tea), Salix species and perennial grasses and forage crops
- oilseed crops containing high levels of lipid compounds such as peanut, rapeseed, canola, sunflower, safflower
- Particularly preferred are C 18: 2 and / or C18: 3 fatty acid rich plants such as sunflower, safflower, tobacco, mullein, sesame, cotton, pumpkin, poppy, evening primrose, walnut, flax, hemp, thistle or safflower.
- C 18: 2 and / or C18: 3 fatty acid rich plants such as sunflower, safflower, tobacco, mullein, sesame, cotton, pumpkin, poppy, evening primrose, walnut, flax, hemp, thistle or safflower.
- Plants such as safflower, sunflower, poppy, evening primrose, walnut, flax or hemp.
- genes selected from the group of ⁇ -4-desaturases, ⁇ -5-desaturases, ⁇ -6-desaturases, ⁇ -9-desaturases, ⁇ -12-desaturases, ⁇ -6-elongases or ⁇ -5 are used, wherein individual Genes or multiple genes can be used in combination.
- the ⁇ -5 elongases according to the invention have the advantageous property that they do not elongate C 22 -fatty acids to the corresponding C 24 -fatty acids compared to the human elongases.
- Particularly advantageous ⁇ -5-elongases preferably convert only unsaturated C 2 o-fatty acids.
- only C 2 o-fatty acids are reacted with a double bond in ⁇ 5-position, with ⁇ -3-C 20 fatty acids being preferred (EPA).
- EPA ⁇ -3-C 20 fatty acids
- they have the property that they have no or only a relatively low ⁇ 6-elongase activity in addition to the ⁇ -5 elongase activity.
- a yeast feeding text in which EPA has been added to the yeasts as substrate, at least 15% by weight of the EPA added to docosapentaenoic acid (DPA, c22: 5 ⁇ 7
- DPA docosapentaenoic acid
- C18: 3 ⁇ 5t9 12 is also not elongated.
- GLA is not reacted.
- ⁇ -4-desaturases according to the invention have the advantage over the known ⁇ -4-desaturases, ⁇ -5-desaturases and ⁇ -6-desaturases that they bind to fatty acids or phospholipids CoA fatty acid esters, advantageous to implement CoA fatty acid esters.
- the processes according to the invention used ⁇ -12-desaturases oleic acid (C18: 1 ⁇ 9 ) to give linoleic acid (C18: 2 ⁇ 9 12 ) or C18: 2 ⁇ 6
- 9 to C18: 3 ⁇ 6 9 '12 ( GLA).
- the ⁇ -12-desaturases used bind fatty acids bound to phospholipids or CoA fatty acid esters, advantageously bound to CoA fatty acid esters.
- the desaturases used in the process according to the invention convert their respective substrates in the form of the CoA fatty acid esters. 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.
- An exchange reaction which would make a further possibly limiting enzyme reaction erfoderlich, between the CoA fatty acid esters and the phospholipids or triglycerides is therefore not required.
- nucleic acids used in the method according to the invention for polypeptides with ⁇ -5-desaturase, ⁇ -6-desaturase, ⁇ -4-desaturase, ⁇ -12-desaturase, ⁇ -5-elongase and or ⁇ 6-elongase activity, advantageously in combination with nucleic acid sequences which are suitable for polypeptides of the fatty acid or lipid metabolism, such as further polypeptides having ⁇ -4-, ⁇ -5, ⁇ -6, ⁇ -12-desaturase or ⁇ -5 or ⁇ -6 elongase activity, it is possible to prepare a wide variety of polyunsaturated fatty acids in the process according to the invention.
- fatty acids derived from C18: 2 fatty acids such as GLA, DGLA or ARA, or those derived from C18: 3 fatty acids derive, such as SDA, ETA or EPA.
- GLA 1 DGLA and ARA can be produced as products of the process, which may be present as free fatty acids or bound.
- ⁇ -5-desaturase By modifying the activity of the enzymes involved in the synthesis ⁇ -5-desaturase, ⁇ -6-desaturase, ⁇ -4-desaturase, ⁇ -12-desaturase, ⁇ -5 elongase and / or ⁇ -6 elongase can be targeted in the aforementioned organisms advantageously produce only individual products in the aforementioned plants.
- Due to the activity of ⁇ -6-desaturase and ⁇ -6 elongase for example, GLA and DGLA or SDA and ETA are formed, depending on the starting plant and unsaturated fatty acid. Preference is given to DGLA or ETA or mixtures thereof.
- ARA, EPA and / or DHA are additionally produced.
- ARA, EPA or DHA 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 these are biosynthetic 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.
- 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 to the end product DGLA, ETA or mixtures thereof or ARA, EPA, DHA or mixtures thereof advantageously EPA or DHA or mixtures thereof.
- the fatty acids can also be fed from the outside. For cost reasons, production in the organism is preferred.
- Preferred substrates are the linoleic acid (C18: 2 ⁇ 9 '12 ), the ⁇ -linolenic acid (C18: 3 ⁇ 6 9 12 ), the eicosadienoic acid (C20: 2 ⁇ 11 14 ), the dihomo- ⁇ -linolenic acid (C20: 3 ⁇ 8 ' 11 '14), arachidonic acid (C20: 4 ⁇ 5811'. 14) docosatetraenoic acid (C22 4 ⁇ 7, i o, i3, i6) and DJE Docosapen taenLitere (C22: 5 ⁇ 4 ⁇ 10 '13 15).
- 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,
- algae such as Isochrysis or Crypthecodinium
- algae / diatoms such as Thalassiosira, Phaeodactylum or Thraustochytrium
- mosses such as Physcomitrella or Ceratodon or higher plants such as Primulaceae such as
- the isolated nucleic acid sequences according to the invention are derived from an animal from the
- the nucleic acid sequences are of the vertebrate class; Euteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei, Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus.
- the nucleic acids originate particularly advantageously from fungi, animals or from plants such as algae or mosses, preferably from the order of the 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 Mantonielle or Ostreococcus or from the diatoms such as the genera Thalassiosira or 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 Mantonielle or Ostreococcus
- diatoms such as the genera Thalassiosira or Crypthecodinium.
- nucleic acid sequences or their derivative or homologs which code for polypeptides which still possess the enzymatic activity of the proteins encoded by nucleic acid sequences.
- These sequences are used singly or in combination with the nucleic acid sequences encoding ⁇ 12-desaturase, ⁇ 4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 5-elongase and / or ⁇ 6-elongase cloned into expression constructs and used for introduction and expression in organisms.
- This expression By their construction, constructs enable a favorable optimum 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 containing the nucleic acid sequences used in the method, wherein the cell and / or the organism with a nucleic acid sequence according to the invention, for the ⁇ -12-desaturase , ⁇ -4-desaturase, ⁇ -5-desaturase, ⁇ -6-desaturase, ⁇ -5 elongase and / or ⁇ -6 elongase, a gene construct or a vector as described below, alone or in combination with other nucleic acid sequences which code for proteins of the fatty acid or lipid metabolism is transformed.
- 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 e.g. Mortierella, Thalassiosira, Mantoniella, Ostreococcus, Saccharomyces or Thraustochytrium or to act as 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 preferably at least partially preserved.
- 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.
- non-natural, synthetic such as mutagenization.
- transgenic organism or transgenic plant As mentioned above is to be understood that the nucleic acids used in the method are not in their natural place in the genome of an organism, while the nucleic acids can be expressed homologously or heterologously.
- transgene also means, as mentioned, 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 the regulatory sequences of the natural sequences have been changed.
- Transgenic is preferably understood to mean the expression of the nucleic acids according to the invention at a non-natural site in the genome, that is to say 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 crop plants.
- all organisms which are capable of synthesizing 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 method 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, Thrausto- chytrium, 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.
- organisms which are naturally capable of synthesizing oils in large quantities, such as fungi such as Mortierella alpina, Pythium insidiosum, Phytophtora infestans or plants such as soybean, oilseed rape, coconut, oil palm, dyer's safflower, flax, hemp, castor, calendula, peanut,
- Cocoa bean or sunflower or yeasts such as Saccharomyces cerevisiae, with particular preference being given to soybean, flax, rapeseed, dyer's safflower, sunflower, calendula, mortierella or Saccharomyces cerevisiae.
- Transgenic animals are advantageously also suitable for non-human animals, for example C. elegans, for the abovementioned transgenic organisms.
- Useful host cells are also mentioned in: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
- Useful expression strains e.g. those which have lower protease activity are described in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128.
- transgenic plants include plant cells and certain tissues, organs and parts of plants in all their manifestations, such as anthers, fibers, root hairs, stems, embryos, cilia, kotelydons, 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.
- 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, callosis, kotelydons, petioles, crop material, plant tissue, reproductive tissue, Cell cultures that can be 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 plant parts, especially the seeds, to be easier to digest they are first crushed, steamed or roasted. The thus 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 mucilages and turbid matter are removed. So-called degumming can be enzymatic or ⁇
- the PUFAs or LCPUFAs C 1 8, C 2 O or C 22 fatty acid molecules produced by this process are preferably C 20 - or C 22 -fatty acid molecules having at least two double bonds in the fatty acid molecule, preferably three, four, five or six double bonds ,
- These C 18 , C 20 or C 22 fatty acid molecules can be isolated from the organism in the form of an oil, lipid or a free fatty acid. Suitable organisms are, for example, those mentioned above. Preferred organisms are transgenic plants.
- One embodiment of the invention is therefore oils, lipids or fatty acids or fractions thereof which have been prepared by the method described above, more preferably oil, lipid or fatty acid composition comprising PUFAs derived from transgenic plants.
- oils, lipids or fatty acids advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid as described above; 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 in each case based 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 prepared by the process according to the invention advantageously contain fatty acids selected from the group of the fatty acids erucic acid (13-docosaoic 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-9,11-dienoic acid), vernonic acid (9,10-epoxyoctadec-12-enoic acid), tartric acid ( 6- octadecynoic acid), 6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid), 6,9-octadecenynoic acid, pyrulic acid (t10-heptadec
- 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.
- 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%, especially advantageous 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, rapeseed, coconut, oil palm, safflower, flax, hemp, castor, calendula, peanut , Cocoa bean, sunflower or the above-mentioned other monocotyledonous or dicotyledonous oil crops.
- an oil crop such as soybean, rapeseed, coconut, oil palm, safflower, flax, hemp, castor, calendul
- oils, lipid, fatty acids and / or fatty acid composition in feed, food, cosmetics or pharmaceuticals.
- the oils, lipids, fatty acids or fatty acid mixtures 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.
- the term “oil”, “lipid” or “fat” is understood as meaning a fatty acid mixture which contains unsaturated, saturated, preferably esterified fatty acid (s).
- 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.
- 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 transesterification by gas chromatography.
- the oil, lipid or fat can be various other saturated or unsaturated fatty acids, eg calendulic acid, palmitic, palmitoleic, stearic, oleic acid etc., contain.
- the proportion of the various fatty acids in the oil or fat may vary depending on the starting organism.
- the polyunsaturated fatty acids having advantageously at least two double bonds which are produced in the process are, as described above, for example sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.
- the polyunsaturated fatty acids containing, 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 over a release enzymatic cleavage and isolate via, for example, phase separation and subsequent acidification over, for example, H 2 SO 4 .
- an alkali treatment for example aqueous KOH or NaOH or acid hydrolysis in the presence of an alcohol such as methanol or ethanol or over a release 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 process can after introduction into a
- Organism advantageously a plant cell or plant are either on a separate plasmid or advantageously 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 plant 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 accumulate significant levels of PUFAs in the triacylglycerol fraction.
- nucleic acid molecules isolated from strains which also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for the process of the invention and thus for modification of the lipid and PUFA production system in a host, in particular plants such as oilseed crops, for example oilseed rape. Canola, flax, hemp, soy, sunflower, borage. They are therefore advantageous for use in the process according to the invention.
- substrates of the nucleic acids used in the method according to the invention which are useful for polypeptides having ⁇ -12-desaturase, ⁇ -5-desaturase, ⁇ -4-desaturase, ⁇ -6
- Encode desaturase, ⁇ -5 elongase and / or ⁇ -6 elongase 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-CoA dehydrogenase (s), acyl-ACP [ acyl carrier protein] desaturase (s), acyl-ACP thioesterase (n ), Fatty acid acyltransferase (s), acyl-CoAlysophospholipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl
- Coenzyme A carboxylase s
- acyl coenzyme A oxidase s
- fatty acid desaturase s
- fatty acid acetylenase s
- lipoxygenase s
- triacylglycerol lipase s
- allene oxide synthase s
- Hydroperoxide lyase s
- fatty acid elongase encode advantageously Ci 6 -, C 18 - or C 2 o fatty acids.
- the fatty acids reacted as substrates in the process are preferably reacted in the form of their acyl-CoA esters and / or their phospholipid esters.
- the polyunsaturated C 18 -fatty acids must first be desaturated by the enzymatic activity of a desaturase and then be extended by at least two carbon atoms via an elongase. After one round of elongation, this enzyme activity leads to C 20 -fatty acids, and after two rounds of elongation to C 22 -fatty acids.
- the activity of the method of the desaturases and elongases used in the invention preferably leads to C 8 -, C 20 - and / or C 22 fatty acids advantageously having at least two double bonds in the fatty acid molecule, preferably with three, four, five or six double bonds, especially preferably to C 20 - and / or C 22 fatty acids having at least two double bonds in the fatty acid molecule, preferably having three, four, five or six double bonds, most preferably having five or six double bonds in the molecule.
- further desaturation and elongation steps such as desaturation at ⁇ -5 and ⁇ -4 positions, may occur.
- Particularly preferred products of the process according to the invention are dihomo- ⁇ -linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and / or docosaheic acid.
- the C 20 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.
- 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 seed, so that a seed-specific expression of the nucleic acids used in the process makes sense.
- biosynthesis of fatty acids, oils or lipids need not be limited to the seed tissue, but may also be tissue-specific in all other parts of the plant - for example in epidermal cells or in the tubers.
- organisms microorganism such as 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 prepared in the process can be at least 5%, preferably at least 10%, more preferably at least 20%, very particularly preferably at least 50%. be increased compared to the wild type of organisms that do not contain the nucleic acids recombinantly.
- 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 process according to the invention increases the pool of esterified polyunsaturated fatty acids in the transgenic organisms.
- 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 to 60 0 C attracted under oxygen fumigation.
- the pH of the nutrient fluid can be kept at a fixed value, that is regulated during the cultivation 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 method according to the invention when the host organisms are microorganisms, is particularly advantageous at a temperature between 0 ° C to 95 °, preferably between 10 ° C to 85 ° C, more preferably between 15 ° C to 75 ° C preferably carried out between 15 ° C to 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 can be found in the textbook by Chmiel (Bioreatechnik 1. Introduction to Bioprocessing Methods). technique (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).
- the culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are included in the Manual of Methods for General Bacteriology of the American Society of Bacteriology (Washington D.C. 1 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 which 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, panthogenate 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.
- the exact composition of the media compounds will depend heavily on the particular experiment and will be decided on a case by case basis. Information about the media optimization is available from the textbook "Applied Microbiol Physiology, A Practical Approach" (ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3).
- Growth media may also be obtained from commercial suppliers, such as standard 1 (Merck) or BHI (Brain Heart Infusion, DIFCO) and the like.
- 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 foaming anti-foaming agents such. As fatty acid polyglycol, are used.
- the medium can be suitably selected substances such. As antibiotics, are added.
- oxygen or oxygen-containing gas mixtures such as ambient air, are introduced into the culture.
- the temperature of the culture is normally from 20 0 C to 45 0 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 removed from the fermentation broth or left completely 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 sequences which code for polypeptides with ⁇ -6-desaturase activity selected from the group: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 13, b) nucleic acid sequences resulting from the degenerate genetic code or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 13 which code for polypeptides having at least 40% homology at the amino acid level with SEQ ID NO: 14 and a ⁇ - derivative of the amino acid sequence shown in SEQ ID NO: 14. Having 6-desaturase activity.
- a further subject of the invention are isolated nucleic acid sequences which code for polypeptides with ⁇ 5-desaturase activity, selected from the group: a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, b) nucleic acid sequences which can be derived as the result of the degenerate genetic code from the amino acid sequence shown in SEQ ID NO: 10 or in SEQ ID NO: 12, or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 9 or in SEQ ID NO: 11, which code for polypeptides having at least 40% homology at the amino acid level with SEQ ID NO: 10 or in SEQ ID NO: 12 and have a ⁇ -5-desaturase activity.
- a further subject of the invention are isolated nucleic acid sequences which code for polypeptides with ⁇ -4-desaturase activity selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 7, b) nucleic acid sequences resulting from the degenerate genetic code or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 7, which code for polypeptides having at least 40% homology at the amino acid level with SEQ ID NO: 8 and a ⁇ -4- Have desaturase activity.
- a further subject of the invention are isolated nucleic acid sequences which code for polypeptides with ⁇ -12-desaturase activity, selected from the group: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 15, b) nucleic acid sequences resulting from the degenerate genetic code derive from the amino acid sequence shown in SEQ ID NO: 16, or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 15 which encode polypeptides having at least 50% homology at the amino acid level with SEQ ID NO: 16 and a ⁇ - Have 12-desaturase activity.
- a further subject of the invention are gene constructs which contain the nucleic acid sequences according to the invention SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15, wherein the nucleic acid is functional with one or more regulatory signals connected is.
- biosynthesis genes of the fatty acid or lipid metabolism selected from the group acyl-CoA dehydrogenase (s), acyl-ACP [acyl carrier protein] desaturase (s), acyl-ACP-thioesteraseCn), fatty acid acyl transferase (n), acyl-CoA: lysophospholipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (s) , Fatty acid desaturase (s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allene oxide synthases, hydroperoxide lyases or fatty acid elongase (s) may be present in the gene construct.
- acyl-CoA dehydrogenase s
- acyl-ACP
- biosynthesis genes of the fatty acid or lipid metabolism selected from the group of ⁇ 4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 9-desaturase, ⁇ 12-desaturase or ⁇ 6-elongase are advantageously also contained ,
- nucleic acid sequences used in the method according to the invention are derived from a eukaryotic organism such as a plant, a microorganism or an animal.
- the nucleic acid sequences are 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.
- an expression cassette nucleic acid construct
- There may be more than one nucleic acid sequence of an enzymatic activity e.g. a ⁇ -12-desaturase, ⁇ -4-desaturase, ⁇ -5-desaturase, ⁇ -6-desaturase, ⁇ -5 elongase and / or ⁇ -6 elongase.
- 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 Pfu DNA polymerase or a Pfu / Taq DNA. Polymerasegemisches ago.
- 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. Subsequently, the amplificate can after a
- Suitable cloning vectors are generally known to the person 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 usually 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 appropriately transformed organisms can be identified.
- vir genes and T-DNA sequences are located 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 no 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.
- Bin19, pBI101, 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 become. 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 can have one or more 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.
- the 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 plant transformation, such as those published in and cited therein Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-119 (1993); F. F. 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 the genetic modification of a broad spectrum of organisms to plants, so that they become better and / or more efficient producers of PUFAs.
- the number or activity of ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ 4-desaturase proteins or genes can be increased so that larger amounts of the gene products and thus ultimately larger amounts of the compounds of general formula I are produced. Also, a de novo synthesis in an organism lacking the activity and ability to biosynthesize the compounds before introducing the gene (s) of interest is possible. The same applies to the combination with other desaturases or elongases or other enzymes from the fatty acid and lipid metabolism.
- the use different divergent, ie different sequences on DNA sequence level may be advantageous or the use of promoters for gene expression, which allows a different time gene expression, for example, depending on the degree of ripeness of a seed or oil-speichemden tissue.
- a ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase and / or ⁇ 4-desaturase gene into one Organism alone or in combination with other genes in a cell can not only increase the biosynthetic flux to the final product, but also increase the corresponding TYi acylglycerin composition 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 isolated nucleic acid molecules used in the method of the invention encode proteins or parts thereof, wherein the proteins or the single protein or parts thereof contain an amino acid sequence sufficiently homologous to an amino acid sequence represented in the sequences SEQ ID NO: 2, SEQ ID NO 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16, so that the proteins or parts thereof still one ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ 4-desaturase activity.
- the proteins or portions thereof encoded by the nucleic acid molecule still have their essential enzymatic activity and the ability to metabolically metabolize compounds or to transport molecules necessary to build cell membranes or lipid bodies in organisms in organisms to participate through these membranes.
- the proteins encoded by the nucleic acid molecules are at least about 40%, preferably at least about 50% or 60%, and more preferably at least about 70%, 80% or 90%, and most preferably at least about 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to those shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 shown amino acid sequences.
- the homology was calculated over the entire amino acid or nucleic acid sequence range.
- a number of programs that are based on different algorithms are available to the person skilled in the art.
- the algorithms of Needleman and Wunsch or Smith and Waterman provide particularly reliable results.
- the program PiIeUp was used (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48: 443-453 (1970) and Smith and Waterman (Adv. Appl. Math.
- ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ -6 elongase, ⁇ -5-desaturase, ⁇ -5 elongase or ⁇ -4-desaturase used in the method according to the invention is understood to mean that they are opposite to those represented by the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 and their derivatives encoded proteins / enzymes by comparison still have at least one enzymatic activity of at least 10%, preferably 20%, more preferably 30% and very particularly 40% and thus the metabolism of the structure of fatty acids, fatty acid esters such as diacylglycerides and / or triacylglycerides in an organism can advantageously participate in a plant or plant cell necessary compounds or in the transport of molecules via membranes, wherein C 18 , C 20 or C 22 carbon chains in the fatty
- 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 may be used in the method according to the invention which are suitable for a ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ 4-desaturase and which are linked to a nucleotide sequence as in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 are shown to hybridize advantageously under stringent conditions.
- 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 encoding ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ -6 elongase, ⁇ -5-desaturase, ⁇ -5 elongase or ⁇ -4-desaturase are thereby amplified with one or more regulatory signals advantageously for
- 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 expressed and / or overexpressed immediately.
- these regulatory sequences are sequences that bind to the inducers or repressors and thus regulate the expression of the nucleic acid.
- the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically altered so that natural regulation is eliminated and expression of genes 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.
- gene construct gene construct
- 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. In this case, the gene construct or the gene constructs can be inserted in one or more vectors and be present freely in the cell or else in the
- 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 is one or more gene constructs which contain one or more sequences represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 or its derivatives are defined and for polypeptides according to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.
- ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ 4-desaturase proteins advantageously lead to desaturation or Elong réelle of fatty acids, wherein the substrate advantageously one, two, three, four, five or six double bonds and advantageously has 18, 20 or 22 carbon atoms in the fatty acid molecule.
- the substrate advantageously 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.
- promoters amy and SP02 in the yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV / 35S [ Franck et al., Cell 21 (1980) 285-294], PRP1 [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-A-0 388 186
- 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) described promoters.
- Further suitable plant promoters are the promoter of cytosolic FBPase or the potato ST LSI promoter (Stockhaus et al., EMBO J. 8, 1989, 2445), the glycine max phosphoribosyl pyrophosphatamidotransferase promoter (Genbank Accession No.
- promoters which allow expression in tissues involved in fatty acid biosynthesis.
- seed-specific promoters such as the USP
- promoters such as the LeB4, DC3, phaseolin or napin promoter.
- Further particularly advantageous promoters are 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 (Phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoter from Brassica), von Baeumlein et al , Plant J., 2, 2, 1992: 233-239 (LeB4 promoter from a legume), these promoters being suitable for dicotyledons.
- promoters are suitable, for example, for barley monocotylone 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 that 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 u. WO95 / 23230], seed-specific promoters from rice, maize and the like.
- Promoter facilitate (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.
- each of the nucleic acids used in the method which is responsible for ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ -5 elongase and / or ⁇ -4-desaturase are expressed under the control of its own preferred a different promoter, as Repetitive sequence motifs can lead to instability of the T-DNA or to recombination events.
- the expression cassette is advantageously constructed in such a way that a promoter is followed by a suitable interface for insertion of the nucleic acid to be expressed, advantageously followed by a terminator behind the polylinker in a polylinker.
- each nucleic acid sequence has its own promoter and optionally its own terminator.
- advantageous constructs are disclosed for example in DE 10102337 or DE 10102338. However, it is also possible to insert several 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 to say a nucleic acid sequence can be inserted at the first or last position in the cassette, without this significantly influencing the expression.
- 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). It can be used here e.g. the OCS1 terminator. As for the promoters, 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 beneficial to regulate genes in the host organisms, such as genes for inducers, repressors or
- Enzyme which engage by their enzyme activity in the regulation of one or more genes of a biosynthetic pathway, introduce and express therein. These genes may be of heterologous or 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 to transform plants using Agrobacterium.
- 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.
- the expression cassettes can in principle be used directly for introduction into the plant or else introduced into a vector.
- These advantageous vectors contain the nucleic acids used in the method which are useful for ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturases, ⁇ 5-elongase or ⁇ -4 Or a nucleic acid construct containing the nucleic acid used alone or in combination with other fatty acid or lipid metabolism biosynthesis genes such as the acyl-CoAl-lysophospholipid acyltransferases, ⁇ -4-desaturases, ⁇ -5-desaturases, ⁇ -6-desaturases, ⁇ -9-desaturases, ⁇ -12-desaturases, ⁇ 3-desaturases, ⁇ -5-elongases and / or ⁇ -6-elongases.
- acyl-CoAl-lysophospholipid acyltransferases such as the acyl-CoAl-lysophospholipid acyltransferases, ⁇ -4-desaturases, ⁇ -5-desaturases,
- vector refers to a nucleic acid molecule that can transport another nucleic acid to which it is attached.
- plasmid which is a circular double-stranded DNA loop into which additional DNA segments can be ligated.
- viral vector Another type of vector is a viral 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.
- certain vectors may direct the expression of genes to which they are operably linked.
- 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 these other forms of expression vectors, such as viral vectors that perform similar functions.
- vector is also intended to mean other vectors which are 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 below-described nucleic acids or the above-described gene construct 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 comprises.
- 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 fulfill the predicted function ascribed to the sequence (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 mean promoters
- Enhancers and other expression control elements e.g., polyadenylation signals.
- These regulatory sequences are e.g. in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnolgy, CRC Press, Boca Raton, Florida, Eds .: Glick and Thompson, chapters 7, 89-108, including references therein.
- Regulatory 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 can be used to express ⁇ -12-desaturases, ⁇ -6-desaturases, ⁇ -6 elongases, ⁇ -5-desaturases, ⁇ -5 elongases and / or ⁇ -4-desaturases in prokaryotic or eukaryotic cells be designed. This is advantageous because often intermediate steps of the vector construction are carried out for simplicity in microorganisms.
- the ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase and / or ⁇ -4-desaturase genes 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. (1991) "Heterologous gene expression in filamentous fungi", in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure, eds., Pp.
- Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
- the recombinant expression vector may alternatively 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
- Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al. (1988) Gene 69: 301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60 -89).
- Target gene expression from the pTrc vector is based on transcription by host RNA polymerase from a hybrid trp-lac fusion promoter.
- Target gene expression from the pET 11d vector is based on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1).
- This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from a resident ⁇ prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
- Other suitable vectors in prokaryotic organisms are known to the person skilled in the art, these vectors are, for example, in E.
- the expression vector is a yeast expression vector.
- 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, YEp13 or pEMBLYe23.
- ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ -6 elongase, ⁇ -5-desaturase, ⁇ -5 elongase and / or ⁇ -4-desaturase 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 Bio! 3: 2156-2165) and the pVL Series (Lucklow and Summers (1989) Virology 170: 31-39). The above vectors provide only a brief overview of possible suitable vectors.
- ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ -6 elongase, ⁇ -5-desaturase, ⁇ -5 elongase and / or ⁇ -4-desaturase can be produced in unicellular plant cells (such as Algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251 and references cited therein, and plant cells from higher plants (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.
- a plant expression cassette preferably contains regulatory sequences that can control gene expression in plant cells and are functional 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 ⁇ known as octopine synthase (Gielen et al., EMBO J. 3 (1984) 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 translation enhancers, e.g., the overdrive sequence containing the 5'-untranslated tobacco mosaic virus leader sequence, which is the protein / RNA ratio increases (Gallie et al., 1987, Nucl. Acids Research 15: 8693-8711).
- translation enhancers e.g., the overdrive sequence containing the 5'-untranslated tobacco mosaic virus leader sequence, which is the protein / RNA ratio increases (Gallie et al., 1987, Nucl. Acids Research 15: 8693-8711).
- Plant gene expression must be operably linked to a suitable promoter that performs gene expression in a timely, cell or tissue-specific manner.
- useful 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 5352605 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, in the vacuole, the nucleus, all kinds of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the
- Mitochondria the endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
- Plant gene expression can also be facilitated by a chemically inducible promoter as described above (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. 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 react to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-induced PRP1 gene promoter (Ward et al., Plant Mol. Biol. 22 (1993) 361-366), the heat-inducible hsp80 promoter Tomato (US 5,187,267), the potato inducible alpha-amylase promoter (WO 96/12814) or the wound inducible pinll promoter (EP-A-0 375 091).
- those promoters which induce gene expression in tissues and organs in which the fatty acid, lipid and oil biosynthesis take place are preferred in sperm cells such as the cells of the endosperm and the developing embryo.
- Suitable promoters are the rapeseed napin promoter (US Pat. No. 5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen Genet.
- promoters which induce seed specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice, etc.
- Suitable noteworthy promoters are the lpt2 or Ipt1 gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene , the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene).
- multiparallel expression of ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ -6 elongase, ⁇ -5-desaturase, ⁇ -5 elongase and / or ⁇ -4-desaturase used in the method may be desired.
- the introduction of such expression cassettes can be carried out via a simultaneous transformation of a plurality of individual expression constructs or preferably by combining a plurality of 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 since plastids are the compartment in which the precursors as well as some end products of lipid biosynthesis are synthesized.
- 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. (Molecular Cloning: A Laboratory Manual, 2nd ed., CoId Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColD Spring Harbor, NY, 1989) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol.
- Host cells which are suitable in principle for receiving the nucleic acid according to the invention, the gene product according to the invention or the vector according to the invention are all prokaryotic or eukaryotic organisms.
- the host organisms which are advantageously used are microorganisms, such as fungi or yeasts or plant cells, preferably plants or parts thereof.
- Fungi, yeasts or plants are preferably used, more preferably plants, most preferably plants such as oilseed crops containing high levels of lipid compounds such as rapeseed, evening primrose, hemp, Diestel, peanut, canola, flax, soy, safflower, sunflower, borage , or plants such as corn, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, tagetes, solanaceae plants, such as
- Particularly preferred plants according to the invention are oil crop plants, such as soybean, peanut, rapeseed, canola, flax, hemp, evening primrose, sunflower, safflower, trees (oil palm, coconut).
- nucleic acid sequences enumerated below which code for ⁇ -6-desaturases, ⁇ -5-desaturases, ⁇ -4-desaturases or ⁇ -12-desaturases.
- Isolated nucleic acid sequences encoding polypeptides having ⁇ -6 desaturase activity selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 13, b) nucleic acid sequences resulting from the degenerate genetic code of the sequence shown in SEQ Or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 13, which code for polypeptides having at least 40% homology at the amino acid level with SEQ ID NO: 14 and have a ⁇ 6-desaturase activity.
- Isolated nucleic acid sequences encoding polypeptides having ⁇ 5-desaturase activity selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 11, b) nucleic acid sequences resulting as the result of degenerate genetic code derived from the amino acid sequence shown in SEQ ID NO: 10 or in SEQ ID NO: 12, or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 9 or in SEQ ID NO: 11, which code for polypeptides having at least 40% homology at the amino acid level with SEQ ID NO: 10 or SEQ ID NO: 12 and a ⁇ - 5- have desaturase activity.
- Isolated nucleic acid sequences encoding polypeptides having ⁇ -4-desaturase activity selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 7, b) nucleic acid sequences resulting from the degenerate genetic code of the sequence shown in SEQ Or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 7, which code for polypeptides having at least 40% homology at the amino acid level with SEQ ID NO: 8 and have a ⁇ 6-desaturase activity.
- Isolated nucleic acid sequences encoding polypeptides having ⁇ 12-desaturase activity selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 15, b) nucleic acid sequences resulting from the degenerate genetic code of the sequence shown in SEQ Or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 15, which code for polypeptides having at least 50% homology at the amino acid level with SEQ ID NO: 16 and have a ⁇ -12 desaturase activity.
- nucleic acids of the present invention are derived from organisms such as non-human animals, ciliates, fungi, plants such as algae or dinoflagellates which can synthesize PUFAs.
- the isolated above-mentioned nucleic acid sequences advantageously originate from the order Salmoniformes, the diatom genus Thalassiosira or Crythecodinium or from the family Prasinophyceae such as the genus Ostreococcus or Pythiaceae such as the genus Phytophtora.
- the articles of the invention also include, as described above, isolated nucleic acid sequence encoding polypeptides having ⁇ -12-desaturases, ⁇ -4-desaturases, ⁇ -5-desaturases and ⁇ -6-desaturases, the ⁇ .
- Coding sequences encoded by these nucleic acid sequences -12-desaturases, ⁇ -4-desaturases, ⁇ -5-desaturases or ⁇ -6-desaturases C 18 , C 2 o and C 22 fatty acids having one, two, three, four or five double bonds and advantageously polyunsaturated Ci 8 -fatty acids with one, two or three double bonds such as C18: 1 ⁇ 9 , C18: 2 ⁇ 9 12 or C18: 3 ⁇ 9 12 / l5 , polyunsaturated C 20 -fatty acids having three or four double bonds such as C 2 0: 3 ⁇ 8, ii, i4 or C 2 0: 4 ⁇ 8, ii, i4, i 7 or menf acn unsaturated C 22 fatty acids having four or five double bonds as C22: 4 ⁇ 7 ' 10 ' 13 '16 or C22: 5 ⁇ 7 ' 10 '13 16 ' 19 implement.
- nucleic acid (molecule) also comprises, in an advantageous embodiment, the untranslated sequence located at the 3 'and 5' end of the coding gene region: 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.
- An "isolated" 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 ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ -4-desaturase molecule may be 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 flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.
- nucleic acid molecules used in the method for example a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 or a part thereof can be isolated using standard molecular biology techniques and the sequence information provided herein. Also, by comparison algorithms, for example, a homologous sequence or homologous, conserved sequence regions at the DNA or amino acid level can be identified.
- a nucleic acid molecule comprising a complete sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO 13 or SEQ ID NO: 15 or a part thereof, by polymerase chain reaction, using oligonucleotide primers based on this sequence or parts thereof (eg, a nucleic acid molecule comprising the complete sequence or a part thereof, by polymerase chain reaction isolated 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.
- Reverse transcriptase available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Louis, 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, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 or using the sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO : 12, SEQ ID NO: 14 or SEQ ID NO: 16 create amino acid sequences shown.
- 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 can be prepared by standard synthetic methods, for example, with an automated DNA synthesizer.
- Homologs of the ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ 4-desaturase nucleic acid sequences of the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 means for example allelic variants with at least about 40 or 50%, preferably at least about 60 or 70%, more preferably at least about 70 or 80%, 90% or 95% ⁇ , and even more preferably at least about 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to each other in SEQ ID NO: 1, SEQ ID NO: 3 , SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, S
- isolated nucleic acid molecules of a nucleotide sequence which correspond to one of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 hybridize or a part thereof hybridized, for example, under stringent conditions.
- allelic variants include functional variants which are obtained by deletion, insertion or substitution of nucleotides from / in which is shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15, but the intention is that the enzyme activity of the resulting synthesized proteins is advantageously retained for the insertion of one or more genes , Proteins which still possess the enzymatic activity of ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ 4-desaturase, ie their Activity is substantially not reduced, means proteins with at least 10%, preferably 20%, more preferably 30%, most preferably 40% of the original enzyme activity compared to that represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, S
- Homologs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 for example, also 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, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 also derivatives, such as, for example, promoter variants.
- the promoters upstream of the indicated nucleotide sequences may be modified by one or more nucleotide exchanges, insertions, and / or deletions, without, however, interfering with the functionality or activity of the promoters. 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 having ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase and / or ⁇ -4-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 advantageously used in the method according to the invention for modulating the production of PUFAs in transgenic organisms in plants such as corn, wheat, rye, oats, Triticale, rice, barley, soybean, peanut, cotton, linum species such as oil or fiber kidney, Brassica species such as oilseed rape, canola and turnip rape, pepper, sunflower, borage, evening primrose and Tagetes, Solanacaen plants such as
- Efficiency of the production of the PUFAs or a decrease of undesired compounds leads (eg if the modulation of the metabolism of lipids and fatty acids, cofactors and enzymes leads to changes in the yield, production and / or efficiency of the production or the composition of the desired compounds within the cells in turn, may affect the production of one or more fatty acids).
- PUFAs polyunsaturated fatty acids
- PUFAs 1 for example stearidonic acid, eicosapentaenoic acid and docosahexaenoic acid
- Brasicaceae for example stearidonic acid, eicosapentaenoic acid and docosahexaenoic acid
- Brasicaceae boraginaceous plants, primulas or linaceae.
- Lein Leinum usitatissimum
- the lipid synthesis can be divided into two sections: the synthesis of fatty acids and their attachment to sn-glycerol-3-phosphate and 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-CoA into malonyl-CoA by the acetyl-CoA carboxylase or into acetyl-ACP by the acetyl transacylase.
- Precursors for the PUFA biosynthesis are, for example, oleic acid, linoleic acid and linolenic acid. These C 18 -carbon fatty acids must be extended to C 2 o and C 22 in order to obtain fatty acids of the eicosa- and docosa-chain type.
- Arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid can be used with the aid of the desaturases used in the process, such as ⁇ -12, ⁇ -4-, ⁇ -5- and ⁇ -6-desaturases and / or ⁇ -5-, ⁇ -6-elongases advantageously eicosapentaenoic acid and / or docosahexaenoic acid are prepared and then used for various purposes in food, feed, cosmetic or pharmaceutical applications.
- C 2 - and / or C 22 -fatty acids having at least two, preferably at least three, four, five or six double bonds in the fatty acid molecule, preferably C 20 - or C 22 -fatty acids with advantageously four, five or six double bonds in the Fatty acid molecule can be produced.
- the desaturation can take place 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 degree of desaturation, including a further elongation of C 2 O to C 22 fatty acids, to fatty acids such as ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid , Stearidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.
- fatty acids such as ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid , Stearidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.
- Elongases in the process according to the invention are C 16 , C 18 or C 20 fatty acids, for example linoleic acid, ⁇ -linolenic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, eicosatetraenoic 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.
- 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.
- fatty acids must then be transported to various modification sites and incorporated into the triacylglycerol storage lipid.
- Another important Step in the 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 known in the art and include the concentration of the fermentation product (compounds of formula I) formed in a given period of time and fermentation volume (eg, kg of product per hour per liter). It also includes 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.
- the term efficiency of production includes the time required to reach 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 of product per kg of carbon source.
- biosynthesis or biosynthetic pathway are known in the art and include the synthesis of a compound, preferably an organic compound. bond, through a cell of interconnects, 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 eg the metabolism of a fatty acid
- the percent sequence homology values given above were determined using the BestFit program over the entire sequence range with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10,000 and Average Mismatch: 0.000, which were always used for sequence comparisons unless otherwise stated
- the invention also includes nucleic acid molecules which differ from any of the amino acid molecules shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 shown nucleotide sequences (and parts thereof) due to the degenerate differing genetic codes and thus encode the same ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ -5-desaturase or ⁇ -4-desaturase as that derived from those shown in SEQ ID NO: 7, SEQ ID NO: 9 , SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 is encoded.
- Desaturase, ⁇ -5-desaturase and / or ⁇ -4-desaturase can exist within a population. These genetic polymorphisms in ⁇ 12-desaturase, ⁇ -6 Desaturase, ⁇ -5-desaturase and / or ⁇ -4-desaturase genes may exist between individuals within a population due to natural variation. These natural variants usually cause a variance of 1 to 5% in the nucleotide sequence of the ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase and / or ⁇ 4-desaturase gene.
- Nucleic acid molecules which are advantageous for the process according to the invention can be prepared on the basis of their homology to the ⁇ -12-desaturase, ⁇ -5-elongase, ⁇ -6-desaturase, ⁇ -5-desaturase, ⁇ -4-desaturase and / or ⁇ -6 elongase nucleic acids using the sequences or a portion thereof as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
- isolated nucleic acid molecules which are at least 15 nucleotides long and can be used under stringent conditions with the nucleic acid molecules which have a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 comprise hybridizing.
- Nucleic acids of at least 25, 50, 100, 250 or more nucleotides may also be used.
- hybridized under stringent conditions as used herein 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 6x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 0.2x SSC, 0.1%. SDS at 50 to 65 ° C.
- hybridization conditions differ depending on the type of nucleic acid and, for example, if organic solvents are present, with regard 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). If organic solvent is present in the above buffer, for example 50% formamide, the temperature is about 42 ° C under standard conditions.
- the hybridization conditions for DNA 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.
- sequences are written one below the other for the purpose of optimal comparison (eg, gaps may be inserted into the sequence of a protein or nucleic acid for optimal alignment) the other protein or nucleic acid).
- amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding site in the other sequence, then the molecules are homologous at that position (ie, amino acid or nucleic acid "homology" as used herein corresponds to amino acid
- the programs or algorithms used are described above.
- An isolated nucleic acid molecule which encodes a ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 4-desaturase, ⁇ 5-elongase and / or ⁇ 6-elongase resulting in a protein sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 is homologous can be prepared by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
- Mutations may be included in any of the sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 by standard techniques such as site-specific mutagenesis and PCR-mediated mutagenesis.
- conservative amino acid substitutions are made on one or more of the predicted nonessential amino acid residues.
- the amino acid residue is challenged exchanged an amino acid residue with a similar side chain.
- families 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 non-polar side chains, (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine)
- a predicted nonessential amino acid residue in a ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 4-desaturase, ⁇ 5-elongase or ⁇ 6-elongase is thus preferably replaced by a different amino acid residue exchanged from the same side chain family.
- the mutations may be randomized over all or part of the ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 4-desaturase, ⁇ 5-elongase or ⁇ -6.
- Elongase coding sequence can be introduced, eg by saturation mutagenesis, and the resulting mutants can be prepared according to the herein described ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 4-desaturase, ⁇ 5-elongase or ⁇ 6 elongase activity to identify mutants carrying the ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 4-desaturase, ⁇ 5-elongase or ⁇ 6-elongase activity.
- After mutagenesis one of
- sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 may be the coded protein can be recombinantly expressed, and the activity of the protein can be eg be determined using the tests described herein.
- Further subjects of the invention are transgenic non-human organisms which contain the inventive nucleic acids SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 or a gene construct or a vector, containing 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.
- the cloning methods such as restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of Escherichia coli cells, culture of bacteria and sequence analysis of recombinant DNA were as in Sambrook et al. (1989) (CoId Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
- the sequencing of recombinant DNA molecules was carried out with 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 and checked to avoid polymerase errors in constructs to be expressed.
- Example 3 Lipid Extraction from Yeasts and Seeds: The effect of genetic modification in plants, fungi, algae, ciliates or on the production of a desired compound (such as a fatty acid) can be determined by subjecting the modified microorganisms or modified plant under suitable conditions ( as described above) and the medium and / or cellular components are assayed for increased production of the desired product (ie, lipids or a fatty acid).
- a desired product ie, lipids or a fatty acid.
- These analytical techniques are well known to those 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 p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.
- the analytical methods include measurements of nutrient levels in the medium (eg, sugars, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of biomass composition and growth, analysis of production of common biosynthetic pathway metabolites, and measurements of gases generated during fermentation. Standard methods for these measurements are in Applied Microbial Physiology; A Practical Approach, PM Rhodes and PF 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 unambiguous evidence for 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, Oliver Press, Dundee, 119-169, 1998, Gas Chromatography Mass Spectrometry Method, Lipids 33: 343-353).
- the material to be analyzed can be prepared by ultrasonic treatment, grinding in the
- fatty acid methyl ester are extracted in petroleum ether and finally subjected to GC analysis using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 mikrom, 0.32 mm) at a temperature gradient between 170 0 C and 240 ° C for 20 min and 5 min at 240 ° C subjected.
- Chropack Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 mikrom, 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. The mixture is then heated for 10 min at 100 0 C and sedimented again after cooling on ice. The cell pellet is hydrolyzed with 1 M methanolic sulfuric acid and 2% dimethoxypropane for 1 h at 90 ° C and transmethylated the lipids. The resulting fatty acid methyl esters (FAME) are extracted into petroleum ether.
- FAME fatty acid methyl esters
- the extracted FAME are purified by gas chromatography using a capillary column (Chrompack, WCOT Fused silica, CP-Wax-52CB, 25 m, 0.32 mm) and a temperature gradient from 170 ° C to 240 ° C in 20 min and 5 min at 240 ° C analyzed.
- the identity of Fatty acid methyl ester is confirmed by comparison with corresponding FAME standards (Sigma).
- the identity and the position of the double bond can be further analyzed by GC-MS by suitable chemical derivatization of the FAME mixtures, for example to give 4,4-dimethoxoxazoline derivatives (Christie, 1998).
- Example 4 Cloning of genes from Ostreococcus tauri
- OteloI has the highest similarity to an elongase from Danio rerio (GenBank AAN77156, about 26% identity), while OtElo2 bears the greatest similarity to the Physcomitella EIo (PSE) [ca. 36% identity] (alignments were performed with the tBLASTn algorithm (Altschul et al., J. Mol. Biol. 1990, 215: 403-410).
- the cloning was carried out as follows: 40 ml of a Ostreococcus tauri culture in the stationary phase were collected by centrifugation and resuspended in 100 .mu.l double-distilled Aqua and stored at -20 0 C. Based on the PCR method, the associated genomic DNAs were amplified. The appropriate primer pairs were selected to carry the yeast consensus sequence for high efficiency translation (Kozak, Cell 1986, 44: 283-292) adjacent to the start codon. The amplification of the OtEIo DNAs was carried out in each case with 1 ⁇ l of thawed cells, 200 ⁇ M dNTPs, 2.5 U day polymerase and 100 pmol of each primer in a total volume of 50 ⁇ l.
- the conditions for the PCR were as follows: first denaturation at 95 ° C for 5 minutes, followed by 30 cycles at 94 ° C for 30 seconds, 55 ° C for 1 minute and 72 ° C for 2 minutes and a final extension step at 72 ° C for 10 minutes.
- Saccharomyces cerev / s / ae strain 334 was transformed by electroporation (1500 V) with the vector pOTE1 and pOTE2, respectively.
- a yeast was used, which was transformed with the empty vector pYES2.
- the selection of the transfor- Yeasts were made on complete minimal medium (CMdum) agar plates with 2% glucose but without uracil. After selection, three transformants each were selected for further functional expression.
- CMdum liquid medium For the expression of the Ot elongases, first precultures each of 5 ml of CMdum liquid medium with 2% (w / v) raffinose but without uracil with the selected transformants were inoculated and incubated for 2 days at 30 ° C., 200 rpm. 5 ml of CMdum liquid medium (without uracil) containing 2% raffinose and 300 ⁇ M of various fatty acids were then inoculated with the precultures to an OD 600 of 0.05. Expression was induced by the addition of 2% (w / v) galactose. The cultures were incubated for a further 96 h at 20 ° C.
- the PCR products were incubated for 16 h at 37 0 C with the restriction enzyme Notl.
- the plant expression vector pSUN300-USP was incubated in the same way. Subsequently, the PCR products and the vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The purification of the DNA was carried out using Qiagen Gel Purification Kit according to the manufacturer. Subsequently, vector and PCR products were ligated. The Rapid Ligation Kit from Roche was used for this purpose. The resulting plasmids pSUN-OtELO1 and pSUN-OtELO2 were verified by sequencing.
- pSUN300 is a derivative of the plasmid pPZP (Hajdukiewicz P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25: 989-994).
- pSUN-USP was generated from pSUN300 by inserting into pSUN300 a USP promoter as an EcoRI fragment.
- the polyadenylation signal is that of the Ostreococcus gene from the A.
- tumefaciens Ti plasmid (ocs terminator, Genbank Accession V00088) (De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu M. and Schell J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol.
- the USP promoter corresponds nucleotides 1 to 684 (Genbank Accession X56240), wherein part of the non-coding region of the USP gene is contained in the promoter
- the 684 base pair promoter fragment was transfected via commercially available T7 standard primer (Stratagene) and with the aid of a synthesized primer PCR reaction amplified by standard methods (primer sequence: ⁇ '-GTCGACCCGCGGACTAGTGGCCCTCTAGACCCGGGGGATCCGGATCTGCTGGCTATGAA-S ')
- the PCR fragment was rescored with EcoRI / SalI and inserted into the vector pSUN300 with OCS terminator to give the plasmid designated pSUN-USP .
- the K Onstrukt was used to transform Arabidopsis thaliana, rapeseed, tobacco and flaxseed.
- Example 7 Expression of OtELOI and OtELO2 in Yeasts Yeasts transformed with the plasmids pYES3, pYES3-0tEL01 and pYES3-OtELO2 as in example 5 were analyzed as follows:
- the yeast cells from the main cultures were (100 ⁇ g, 5 min, 20 0 C) harvested by centrifugation and washed with 100 mM NaHCO 3, pH 8.0 to remove residual medium and fatty acids to be removed.
- fatty acid methyl esters FAMEs
- the cell sediments were incubated with 2 ml of 1N methanolic sulfuric acid and 2% (v / v) dimethoxypropane for 1 h at 80 ° C.
- the extraction of the FAMES was carried out by extraction twice with petroleum ether (PE).
- the organic phases were washed once each with 2 ml of 100 mM NaHCO 3 , pH 8.0 and 2 ml of distilled water. washed. Subsequently, the PE phases were dried with Na 2 SO 4 , evaporated under argon and taken up in 100 ⁇ l of PE.
- the samples were separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 ⁇ m, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector.
- the conditions for the GLC analysis were as follows: The oven temperature was programmed from 50 ° C to 250 ° C at a rate of 5 ° C / min and finally 10 min at 250 ° C (hold).
- the substrate specificity of OtEIoI was determined after expression and feeding of various fatty acids (Table 2).
- the lined substrates can be detected in large quantities in all transgenic yeasts.
- the transgenic yeasts showed the synthesis of new fatty acids, the products of the OtEIoI reaction. This means that the gene OteloI could be functionally expressed.
- Table 2 shows that OteloI has a narrow substrate specificity.
- the OteloI was able to elongate only the C20 fatty acids eicosapentaenoic acid (Figure 2) and arachidonic acid (Figure 3), but preferred the ⁇ -3-desaturated eicosapentaenoic acid.
- Figure 2 The OteloI was able to elongate only the C20 fatty acids eicosapentaenoic acid (Figure 2) and arachidonic acid (Figure 3), but preferred the ⁇ -3-desaturated eicosapentaenoic acid.
- Table 2 shows the substrate specificity of the elongase OteloI for C20 polyunsaturated fatty acids with a double bond in ⁇ 5 position towards different fatty acids.
- the yeasts transformed with the vector pOTE1 were cultured in minimal medium in the presence of the indicated fatty acids.
- the substrate specificity of OtElo2 (SEQ ID NO: 1) could be determined after expression and feeding of various fatty acids (Table 3).
- the lined substrates can be detected in large quantities in all transgenic yeasts.
- the transgenic yeasts showed the synthesis of new fatty acids, the products of the OtElo2 reaction. This means that the gene OtElo2 could be expressed functionally.
- Table 3 shows the substrate specificity of the elongase OtElo2 towards different fatty acids.
- the yeasts transformed with the vector pOTE2 were cultured in minimal medium in the presence of the indicated fatty acids.
- the reconstitution of the biosynthesis of DHA (22: 6 ⁇ 3) can be carried out starting from EPA (20: 5 ⁇ 3) or stearidonic acid (18: 4 ⁇ 3) by the coexpression of OtEIoI with the ⁇ -4-desaturase from Euglena gracilis or the ⁇ -5-desaturase from Phaeodactylum tricornutum and the ⁇ -4-desaturase from Euglena gracilis performed.
- the expression vectors pYes2-EgD4 and pESCLeu-PtD5 were further constructed. The o.g.
- Yeast strain already transformed with the pYes3-OtElo1 can then be further transformed with the pYes2-EgD4 or simultaneously with pYes2-EgD4 and pESCLeu-PtD5.
- the selection of the transformed yeasts can be performed on complete minimal medium agar plates with 2% glucose but without tryptophan and uracil in the case of the pYes3-OtElo / pYes2-EgD4 strain and without tryptophan, uracil and leucine in the case of the pYes3-OtElo / pYes2- EgD4 + pESCLeu-PtD5 strain.
- Expression is then induced by the addition of 2% (w / v) galactose.
- the cultures are then incubated for a further 120 h at 15 ° C.
- Example 10 Generation of Transgenic Plants a) Generation of Transgenic Rape Plants (Modified by Moloney et al., 1992, Plant Cell Reports, 8: 238-242) The transgenic rape plants are grown using the binary vectors in Agrobacterium tumefaciens C58C1: pGV2260 or Escherichia coli (Deblaere et al., 1984, Nucl. Acids. Res. 13, 4777-4788).
- Regenerated shoots are obtained on 2MS medium with kanamycin and claforan, transferred into soil after rooting and grown in a climatic chamber or greenhouse after cultivation for two weeks, flowered, harvested mature seeds and for elongase expression such as ⁇ -5 elongase or ⁇ 6-elongase activity by lipid analysis. Lines with elevated levels of C20 and C22 polyunsaturated fatty acids can thus be identified. b) Production of transgenic flax plants
- transgenic flax plants can be carried out, for example, according to the method of Bell et al., 1999, In Vitro Cell. Dev. Biol. Plant. 35 (6): 456-465 by means of particle bombartment.
- Agrobacteria-mediated transformations can be carried out, for example, according to Mlynarova et al. (1994), Plant Cell Report 13: 282-285.
- Example 11 Cloning of desaturase genes from Ostreococcus tauri
- the conditions for the PCR were as follows: first denaturation at 95 ° C for 5 minutes, followed by 30 cycles at 94 0 C for 30 seconds, 55 ° C for 1 minute and 72 ° C for 2 minutes, and a final extension step at 72 0 C for 10 minutes.
- Table 4 List of fatty acid desaturases with the highest sequence homologies to the ⁇ 6-desaturase of Osterococcus.
- the Saccharomyces cerews / ' ae strain 334 was transformed by electroporation (1500 V) with the vector pYES2.1-OtD6.1.
- a yeast was used, which was transformed with the empty vector pYES2.
- the selection of the transformed yeasts was carried out on complete minimal medium (CMdum) agar plates with 2% glucose, but without uracil. After selection, three transformants each were selected for further functional expression.
- CMdum liquid medium containing 2% (w / v) raffinose but no uracil were first inoculated with the selected transformants and incubated for 2 days at 30 ° C., 200 rpm.
- Expression was induced by the addition of 2% (w / v) galactose. The cultures were incubated for a further 96 h at 20 ° C.
- Annealing temperature 1 min 55 0 C denaturation temperature: 1 min 94 0 C elongation temperature: 2 min 72 0 C number of cycles: 35
- the PCR products are incubated for 16 h at 37 0 C with the restriction enzyme Notl.
- the plant expression vector pSUN300-USP is incubated in the same way.
- the PCR products and the vector are subsequently separated by agarose gel electrophoresis and the corresponding DNA fragments are excised.
- the DNA is purified using the Qiagen Gel Purification Kit according to the manufacturer's instructions. Subsequently, vector and PCR products are ligated.
- the Rapid Ligation Kit from Roche was used for this purpose. The resulting plasmids are verified by sequencing.
- pSUN300 is a derivative of the plasmid pPZP (Hajdukiewicz P, Svab, Z 1 Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation, Plant Mol Biol 25: 989-994).
- pSUN-USP was generated from pSUN300 by inserting into pSUN300 a USP promoter as an EcoRI fragment.
- the polyadenylation signal is that of the Ostreococcus gene from the A.
- tumefaciens Ti plasmid (ocs terminator, Genbank Accession V00088) (De Greve.H., Dhaese.P., Seurinck J., Lemmers, M., Van Montagu M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol.
- the USP promoter corresponds nucleotides 1 to 684 (Genbank Accession X56240), wherein part of the non-coding region of the USP gene is contained in the promoter
- the 684 base pair promoter fragment was transfected via commercially available T7 standard primer (Stratagene) and with the aid of a synthesized primer PCR reaction amplified by standard methods (primer sequence: 5'-
- the PCR fragment was rescored with EcoRI / SalI and inserted into the vector pSUN300 with OCS terminator.
- the resulting plasmid was named pSUN-USP.
- the construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and linseed.
- Yeasts transformed with the plasmids pYES2 and pYES2-OtDes6.1 as in Example 11 were analyzed as follows: The yeast cells from the main cultures were (100 ⁇ g, 5 min, 20 0 C) harvested by centrifugation and washed with 100 mM NaHCO 3, pH 8.0 to remove residual medium and fatty acids to be removed. The yeast cell pellets were extracted with chloroform / methanol (1: 1) for 4 hours. The resulting organic phase was extracted with 0.45% NaCl, dried with Na 2 SO 4 and evaporated in vacuo. The lipid extract was purified by thin layer chromatography (horizontal tank,
- Chloroform: methanol: acetic acid 65: 35: 8) are further separated into the lipid classes phosphatidylcholine (PC), phosphatidiylinositol (PI), phosphatidyserine (PS), phosphatidylethanolamine (PE) and neutral lipids (NL).
- PC phosphatidylcholine
- PI phosphatidiylinositol
- PS phosphatidyserine
- PE phosphatidylethanolamine
- NL neutral lipids
- FAMEs Fatty acid methyl ester
- the cell sediments were incubated with 2 ml of 1 N methanolic sulfuric acid and 2% (v / v) dimethoxypropane at 80 ° C. for 1 h.
- the extraction of the FAMES was carried out by extraction twice with petroleum ether (PE).
- PE petroleum ether
- the organic phases were washed once each with 2 ml of 100 mM NaHCO 3 , pH 8.0 and 2 ml of distilled water. washed.
- the PE phases were dried with Na 2 SO 4 , evaporated under argon and taken up in 100 ⁇ l of PE.
- the samples were separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 ⁇ m, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector.
- the conditions for the GLC analysis were as follows: The oven temperature was finally programmed from 50 ° C to 250 0 C at a rate of 5 ° C / min and 10 min at 250 ° C (hold).
- the signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma).
- the methodology is described, for example, in Napier and Michaelson, 2001, Lipids. 36 (8): 761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52 (360): 1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388 (2): 293-298 and Michaelson et al., 1998, FEBS Letters. 439 (3): 215-218.
- the substrate specificity of desaturases can be determined after expression in yeast (see examples cloning of desaturase genes, yeast expression) by feeding using various yeasts. Descriptions for the determination of the individual activities can be found in WO 93/11245 for ⁇ 15 desaturases, WO 94/11516 for ⁇ 12 desaturases, WO 93/06712, US 5,614,393, US 5614393, WO 96/21022, WO0021557 and WO 99/27111 for ⁇ 6-desaturases, Qiu et al. 2001, J. Biol. Chem. 276, 31561-31566 for ⁇ 4-desaturases, Ho ⁇ g et al. 2002, Lipids 37,863-868 for ⁇ 5-desaturase.
- Table 4 shows the substrate specificity of the desaturase OtDes6.1 versus various fatty acids.
- the substrate specificity of OtDes6.1 could be determined after expression and feeding of different fatty acids.
- the lined substrates can be detected in large quantities in all transgenic yeasts.
- the transgenic yeasts showed the synthesis of new fatty acids, the products of the OtDes6.2 reaction ( Figure 4). This means that the gene OtDes6.1 could be expressed functionally.
- Yeasts transformed with the vector pYES2-OtDes6.1 were cultured in minimal medium in the presence of the indicated fatty acids.
- the activity corresponds to the conversion rate calculated according to [substrate / (substrate + product) * 100].
- Table 4 shows that the OtDes6.1 has a substrate specificity for linoleic and linolenic acid (18: 2 and 18: 3), since these fatty acids achieve the highest activities.
- the preferred reaction of linoleic and linolenic acid shows the suitability of this desaturase for the production of polyunsaturated fatty acids.
- FIG. 4 shows the conversion of linoleic acid by OtDes6.2.
- the analysis of the FAMEs was done by gas chromatography.
- the lined substrate (C18: 2) is converted to ⁇ -C18: 3. Both starting material and the resulting product are marked by arrows.
- a culture of the yeast strain INVSd transformed with pYES-Ot6.1 (see Example 12) was incubated for 24 h at 30 0 C in the presence of galactose. Subsequently 250 ⁇ M linoleic acid (18: 2 ⁇ 9,12) was added and yeast cells were removed at various times and analyzed (0 min, 5 min, 1 h, 4 h). The total fatty acid spectrum was analyzed by GC (FIG. 6, left) or the acyl-CoA esters by HPLC (FIG. 6, right).
- isoARA (20: 4 ⁇ 8, 11, 14, 17) was also investigated in the CoA ester pool in a similar experiment as described above.
- a yeast culture transformed with pYES-Ot6.1 and pLEU-PSE1 (described in Domergue et al., 2002, Eur. J. Biochem., 269, 4105-4113) was added and linolenic acid (18: 3 ⁇ 9, 12, 15) added.
- yeast cells were removed and the total lipids were analyzed by GC (FIG. 7, left) or the acyl-CoA esters by HPLC (FIG. 7, right ).
- Annealing temperature 1 min 55 0 C denaturation temperature: 1 min 94 0 C elongation temperature: 2 min 72 0 C number of cycles: 35
- the PCR products are incubated for 16 h at 37 0 C with the restriction enzyme Notl.
- the plant expression vector pSUN300-USP is incubated in the same way. Subsequently, the PCR products and the vector are replaced by agarose
- the DNA is purified using the Qiagen Gel Purification Kit according to the manufacturer's instructions. Subsequently, vector and PCR products are ligated. The Rapid Ligation Kit from Roche was used for this purpose. The resulting plasmids are verified by sequencing.
- pSUN300 is a derivative of the plasmid pPZP (Hajdukiewicz P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation, Plant Mol Biol 25: 989-994).
- pSUN-USP was generated from pSUN300 by inserting into pSUN300 a USP promoter as an EcoRI fragment.
- the polyadenylation signal is the OCS gene from the A. tumefaciens Ti plasmid (ocs terminator, Genbank Accession V00088) (De Greve.H., Dhaese.P., Seurinck J., Lemmers.
- the USP promoter corresponds to the Nucleotides 1 to 684 (Genbank Accession X56240), wherein part of the non-coding region of the USP gene is contained in the promoter
- the 684 base pair promoter fragment was obtained by commercially available T7 standard primer (Stratagene) and by means of a synthesized primer via PCR Reaction amplified by standard methods (primer sequence: 5'-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCCGGATCTGCTGGCTATGAA-S 1 ).
- the PCR fragment was rescored with EcoRI / SalI and inserted into the vector pSUN300 with OCS terminator.
- the resulting plasmid was named pSUN-USP.
- the construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and linseed.
- Yeasts which are transformed as described in Example 11 with the plasmids pYES2 and pYES2-Ostreococcus desaturases are analyzed as follows:
- the yeast cells from the main cultures are (100 ⁇ g, 5 min, 20 0 C) harvested by centrifugation and washed with 100 mM NaHCO 3, pH 8.0 to remove residual medium and fatty acids to be removed.
- fatty acid methyl esters (FAMEs) are produced by acid methanolysis.
- the cell sediments are incubated with 2 ml of 1N methanolic sulfuric acid and 2% (v / v) dimethoxypropane for 1 h at 80 ° C.
- the extraction of the FAMES was carried out by extraction twice with petroleum ether (PE).
- the organic phases are each determined once with 2 ml of 100 mM NaHCO 3 , pH 8.0 and 2 ml of distilled water. washed. Subsequently, the PE phases are dried with Na 2 SO 4 , evaporated under argon and taken up in 100 ⁇ l of PE.
- the samples are separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 ⁇ m, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector.
- the conditions for the GLC analysis are as follows: The oven temperature is eventually programmed from 50 0 C to 25O 0 C at a rate of 5 ° C / min and 10 min at 250 ° C (hold).
- the signals are identified by comparison of the retention times with corresponding fatty acid standards (Sigma).
- the methodology is described, for example, in Napier and Michaelson, 2001, Lipids. 36 (8): 761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52 (360): 1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388 (2): 293-298 and Michaelson et al., 1998, FEBS Letters. 439 (3): 215-218.
- the substrate specificity of desaturases can be determined after expression in yeast (see examples cloning of desaturase genes, yeast expression) by feeding using various yeasts. Descriptions for the determination of the individual activities can be found in WO 93/11245 for ⁇ 15 desaturases, WO 94/11516 for ⁇ 12 desaturases, WO 93/06712, US 5,614,393, US 5614393, WO 96/21022, WO0021557 and WO 99/27111 for ⁇ 6-desaturases, Qiu et al. 2001, J. Biol. Chem. 276, 31561-31566 for ⁇ 4-desaturases, Hong et al. 2002, Lipids 37,863-868 for ⁇ 5-desaturases.
- the activity of the individual desaturases is calculated from the conversion rate according to the formula [substrate / (substrate + product) * 100].
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EP05819684A EP1831377A1 (de) | 2004-12-23 | 2005-12-21 | Verfahren zur herstellung mehrfach ungesättigter fettsäuren in transgenen organismen |
US11/794,006 US20080076166A1 (en) | 2004-12-23 | 2005-12-21 | Method For Producing Polyunsaturated Fatty Acids In Transgenic Organisms |
CA002590329A CA2590329A1 (en) | 2004-12-23 | 2005-12-21 | Method for producing polyunsaturated fatty acids in transgenic organisms |
AU2005321576A AU2005321576A1 (en) | 2004-12-23 | 2005-12-21 | Method for producing polyunsaturated fatty acids in transgenic organisms |
NO20073045A NO20073045L (no) | 2004-12-23 | 2007-06-15 | Fremgangsmate for fremstilling av polyumettede fettsyrer i transgene organismer |
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CA2590329A1 (en) | 2006-06-06 |
AU2005321576A1 (en) | 2006-07-06 |
US20080076166A1 (en) | 2008-03-27 |
EP1831377A1 (de) | 2007-09-12 |
NO20073045L (no) | 2007-09-11 |
DE102004063326A1 (de) | 2006-07-06 |
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