EP1501932A2 - Procede pour produire des acides gras polyinsatures dans des plantes - Google Patents

Procede pour produire des acides gras polyinsatures dans des plantes

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Publication number
EP1501932A2
EP1501932A2 EP03747357A EP03747357A EP1501932A2 EP 1501932 A2 EP1501932 A2 EP 1501932A2 EP 03747357 A EP03747357 A EP 03747357A EP 03747357 A EP03747357 A EP 03747357A EP 1501932 A2 EP1501932 A2 EP 1501932A2
Authority
EP
European Patent Office
Prior art keywords
seq
nucleic acid
fatty acids
plant
plants
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP03747357A
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German (de)
English (en)
Inventor
Petra Cirpus
Andreas Renz
Jens Lerchl
Anne-Marie Kuijpers
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BASF Plant Science GmbH
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BASF Plant Science GmbH
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Filing date
Publication date
Application filed by BASF Plant Science GmbH filed Critical BASF Plant Science GmbH
Publication of EP1501932A2 publication Critical patent/EP1501932A2/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6432—Eicosapentaenoic acids [EPA]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6481—Phosphoglycerides

Definitions

  • the present invention relates to a process for the production of fatty acid esters which contain unsaturated fatty acids with at least three double bonds and free unsaturated fatty acids with a content of at least 1% by weight, based on the total fatty acids contained in the plants, by expression of at least one nucleic acid sequence which codes for a polypeptide with ⁇ -6-desaturase activity, and at least one nucleic acid sequence which codes for a polypeptide with ⁇ -6-elongase activity. These nucleic acid sequences can optionally be expressed in the transgenic plant together with a third nucleic acid sequence which codes for a polypeptide with ⁇ -5 desaturase activity.
  • the invention further relates to the use of defined nucleic acid sequences which code for polypeptides with a ⁇ -6-desaturase activity, ⁇ -6-elongase activity or ⁇ -5-desaturase activity selected from a group of nucleic acid sequences or the use of nucleic acid constructs containing the aforementioned nucleic acid sequences.
  • Fine chemicals also include lipids and fatty acids, among which an exemplary class is the polyunsaturated fatty acids.
  • polyunsaturated fatty acids PUFAs
  • PUFAs polyunsaturated fatty acids
  • PUFAs have a positive influence on the cholesterol level in the blood of humans and are therefore suitable for protection against heart diseases.
  • Fine chemicals such as polyunsaturated fatty acids
  • polyunsaturated fatty acids can be isolated from animal sources, such as fish, or with microorganisms by cultivating microorganisms which have been developed in such a way that they produce and accumulate or secrete large amounts of one or more desired molecules produce large scale.
  • Fatty acids and triglycerides have a multitude of applications in the food industry, animal nutrition, cosmetics and pharmaceuticals. Depending on whether it is free saturated or unsaturated fatty acids or triglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a wide variety of applications.
  • Polyunsaturated ⁇ -3 fatty acids and ⁇ -6 fatty acids are an important part of animal and human food.
  • polyunsaturated ⁇ -3 fatty acids which are preferred in fish oils , particularly important to food.
  • DHA docosahexaenoic acid
  • C22 ß ⁇ 4 ' 7 - 10 ' 13 ' 16 ' 19
  • the free fatty acids are advantageously produced by saponification.
  • Common natural sources of these fatty acids are fish such as herring, salmon, sardine, goldfish, eel, carp, trout, halibut, mackerel, pikeperch or tuna or algae.
  • oils with saturated or unsaturated fatty acids are preferred, for example lipids with unsaturated fatty acids, especially polyunsaturated fatty acids, are preferred in human nutrition.
  • the polyunsaturated ⁇ -3 fatty acids are said to have a positive effect on the cholesterol level in the blood and thus on the possibility of preventing heart disease.
  • Adding these ⁇ -3 fatty acids to food can significantly reduce the risk of heart disease, stroke or high blood pressure.
  • Inflammatory, especially chronic, inflammatory processes in the context of immunological diseases such as rheumatoid arthritis can also be positively influenced by ⁇ -3 fatty acids. They are therefore added to foods, especially dietary foods, or are used in medicines.
  • ⁇ -6 fatty acids such as arachidonic acid tend to have a negative effect on these diseases due to our usual food composition.
  • ⁇ -3 and ⁇ -6 fatty acids are precursors of tissue hormones, the so-called eicosanoids such as prostaglandins, which are derived from dihomo- ⁇ -linolenic acid, arachidonic acid and eicosapentaenoic acid, the thromoxanes and leukotrienes, which are derived from the Derive arachidonic acid and eicosapentaenoic acid.
  • Eicosanoids (so-called PG series) which are formed from ⁇ -6 fatty acids generally promote inflammatory reactions, while eicosanoids (so-called PG 3 series) from ⁇ -3 fatty acids have little or no inflammation-promoting effect ,
  • ⁇ -6-desaturases are described in WO 93/06712, US 5,614,393, US5614393, WO 96/21022, WO00 / 21557 and WO 99/27111 and also the use for production in transgenic organisms as described in W098 / 46763 W098 / 46764, W09846765.
  • microorganisms for the production of PUFAs are microorganisms such as Thraustochytria or Schizochytria strains, algae such as Phaeodactylum tricornutum or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor.
  • strain selection a number of mutant strains of the corresponding microorganisms have been developed which produce a number of desirable compounds, including PUFAs.
  • mutating and selecting strains with improved production of a particular molecule, such as the polyunsaturated fatty acids is a time-consuming and difficult process.
  • the production of fine chemicals can suitably be carried out on a large scale through production in plants designed to produce the above-mentioned PUFAs.
  • Plants that are particularly suitable for this purpose are oil-fruit plants which contain large amounts of lipid compounds, such as rapeseed, canola, flax, soybeans, sunflowers, borage and evening primrose.
  • lipid compounds such as rapeseed, canola, flax, soybeans, sunflowers, borage and evening primrose.
  • other crop plants containing oils or lipids and fatty acids are also well suited, as mentioned in the detailed description of this invention.
  • Using conventional breeding a number of mutant plants have been developed that produce a spectrum of desirable lipids and fatty acids, cofactors and enzymes.
  • the object was therefore to develop a process for the preparation of polyunsaturated fatty acid esters and / or free polyunsaturated fatty acids with at least three double bonds in the fatty acid molecule.
  • This object was achieved by the process according to the invention for the preparation of compounds of the general formula I:
  • transgenic plants with a content of at least 1% by weight, based on the total fatty acids, characterized in that the method comprises the following steps:
  • R 1 -OH, coenzyme A- (thioester), phosphatidylcholine, phosphatidylyl ethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylserine, phosphatidylinositol, sphingolipid, glycoshingolipid or a radical of the following general formula II
  • R 2 H, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylserine, phosphatidylinositol, shingolipid, glycoshingolipid, glycoshingolipid or saturated or unsaturated C 2 -C 4 -alkyl
  • R 3 H, saturated or unsaturated C 2 -C 24 alkylcarbonyl, or
  • R 2 and R 3 independently of one another are a radical of the general formula Ia
  • R i denotes in the compounds of the formula I -OH (hydroxyl), acetyl-coenzyme A, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylserine, phosphatidylinositol, sphingolipid, glycoshingolipid, or a residue of glycoshingol general formula II
  • R 2 in the compounds of formula II denotes hydrogen, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylserine, phosphatidylinositol, shingolipid, glycoshingolipid, glycoshingolipid or saturated or unsaturated C 2 -C 24 carbonyl.
  • C 2 -C 2 alkylcarbonyl radicals 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-tetradecylcarbon- yl-, n-pentadecylcarbonyl-, n-hexadecylcarbonyl-, n-heptadecyl- carbonyl- , n-octadecylcarbonyl-, n-nonadecylcarbonyl-,
  • C 1 -C 2 -alkylcarbonyl radicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadyl are preferred -, n-Octadecylcarbon- yl-, n-nonadecylcarbonyl-, n-eicosylcarbonyl-, n-docosanylcarbon- yl- or n-tetracosanylcarbonyl-, which contain one or more double bonds.
  • C 1 -C 2 -alkylcarbonyl radicals such as Cio-alkylcarbonyl, Cn ⁇ alkylcarbonyl, C ⁇ 2 alkylcarbonyl, C ⁇ 3 alkylcarbonyl, Ci 4 alkylcarbonyl, C ⁇ 6 alkylcarbonyl, C ⁇ s are particularly preferred -Alkylcarbonyl-, C 2 o-Alkylcarbon- yl-, C 2 -Alkylcarbonyl- or C 24 -Alkylcarbonyl residues, which contain one or more double bonds.
  • Saturated or unsaturated Ci are very particularly preferably 6 -C 22 -alkylcarbonyl radicals such as C ß alkylcarbonyl, cis-alkylcarbonyl, C 20 alkylcarbonyl or C22-alkylcarbonyl radicals which hold one or more double bonds corresponds.
  • the radicals mentioned preferably contain two, three, four or five double bonds.
  • R 3 in the compounds of the formula II denotes hydrogen, saturated or unsaturated CC 4 alkylcarbonyl.
  • C 2 -C 22 alkylcarbonyl radicals 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-heptadecylcarbonyl, n-octadecylyl -, n-Nonadecylcarbonyl-, n-Eicosylcarbonyl-, n-
  • C 1 -C 2 -alkylcarbonyl radicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-Hexadecylcarbonyl-, n-heptadecylcarbonyl-, n-octadecylcarbonyl-, n-nonadecylcarbonyl-, n-eicosylcarbonyl-, n-docosanylcarbonyl- or n-tetracosanylcarbonyl-, which contain one or more double bonds.
  • C-o-C 22 alkylcarbonyl radicals such as Cio-alkylcarbonyl, C ⁇ ⁇ alkylcarbonyl, C ⁇ 2 alkylcarbonyl, C ⁇ 3 alkylcarbonyl, C ⁇ 4 ⁇ alkylcarbonyl, C ⁇ 6 alkylcarbonyl are particularly preferred , C ⁇ s-Alkylcarbonyl-, C 2 o-Alkylcarbon- yl- C 22 -alkylcarbonyl- or C 24 -alkylcarbonyl radicals containing one or more double bonds.
  • C ⁇ 6 -C 22 alkylcarbonyl radicals such as C ⁇ 6 ⁇ alkylcarbonyl, C ⁇ s alkylcarbonyl, C 2 o-alkylcarbonyl or C 22 alkylcarbonyl radicals which contain one or more double bonds are very particularly preferred.
  • the radicals mentioned preferably contain two, three, four or five double bonds.
  • C ⁇ -Alkylcarbonylreste which contain one, two, three or four double bonds
  • C 2 o-Alkylcarbonylreste which contain three, four or five double bonds are very particularly preferred. All of the residues mentioned are derived from the corresponding fatty acids.
  • R 2 and R 3 furthermore independently denote a radical of the general formula Ia in the compounds of the formula II
  • radicals R 1 , R 2 and R 3 can also carry substituents such as hydroxyl or epoxy groups or can also contain triple bonds.
  • nucleic acid sequences used in the method according to the invention are isolated nucleic acid sequences which code for polypeptides with ⁇ -5, ⁇ -6 desaturase or ⁇ -6 elongase activity.
  • the compounds of formula I prepared in the process advantageously contain a mixture of different radicals R 1 , R 2 or R 3 , which can be derived from different glycerides. Furthermore, the abovementioned residues of various fatty acids such as short-chain fatty acids with 4 to 6 carbon atoms, medium-chain fatty acids with 8 to 12 carbon atoms or derive long-chain fatty acids with 14 to 24 carbon atoms, long-chain fatty acids are preferred.
  • GLA GLA
  • C18 3 ⁇ 6 ' ° ' 12
  • the fatty acid esters with polyunsaturated C 8 ⁇ , C 2 o and / or C 22 fatty acid molecules can be obtained from the organisms used for the production 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 glycoshingolipid, phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides, diacylglycerides or triacylacid fatty acids such as triacylacid fatty acids such as triacylacid fatty acids such as triacylacid fatty acids Contain double bonds can be isolated.
  • compounds such as sphingolipids, Phosphoglycerides, lipids, glycolipids such as glyco
  • the polyunsaturated fatty acids are also present in the plants as free fatty acids or bound in other compounds.
  • the various aforementioned compounds (fatty acid esters and free fatty acids) in the plant have 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% by weight of free fatty acids, 2 to 8% by weight of phospholipids, the sum of the various compounds adding up to 100% by weight.
  • the compounds of the general formula I with a content of at least 1% by weight, advantageously at least 2% by weight, preferably at least 3% by weight, particularly preferably at least 5% by weight, are whole particularly preferably produced from at least 10% by weight, based on the total fatty acids, in the transgenic plant.
  • the starting compounds linoleic acid (C18: 2) or linolenic acid (C18: 3) go through several reaction steps, the end products of the process, such as arachidonic acid (ERA) or eicosapentaenoic acid (EPA), do not occur as pure products, it there are always slight traces of the precursors in the end product.
  • ERA and EPA are available as mixtures.
  • the precursors should advantageously not more than 20% by weight, preferably not more than 15% by weight, particularly preferably not more than 10% by weight, very particularly preferably not more than 5% by weight, based on the amount of the particular Final product.
  • ERA or only EPA are bound as end products in a transgenic plant in the process according to the invention or prepared as free acids (see compounds of general formula I). If both compounds (ERA + EPA) are produced simultaneously, they are advantageously produced in a ratio of at least 1: 2 (EPA: ERA), advantageously at least 1: 3, preferably 1: 4, particularly preferably 1: 5.
  • Organisms belonging to the oil-producing organisms that is to say those which are used for the production of oils, such as algae such as Crypthecodinium, Phaeodactylum or plants, in particular plants, preferably oil-fruit plants which contain large amounts of lipid compounds, are advantageously used in the process according to the invention , such as peanut, rapeseed, canola, sunflower, safflower (safflower), poppy, mustard, hemp, castor oil, olive, sesame, calendula, punica, evening primrose, mullein, thistle, wild rose, hazelnut, almond, macadamia, avocado, laurel, pumpkin , Flax, soy, pistachio, borage, trees (oil palm, coconut or walnut) or field crops, such as corn, wheat, rye, oat
  • Preferred plants according to the invention are oil fruit plants, such as peanut, rapeseed, canola, sunflower, safflower (safflower), poppy, mustard, hemp, castor oil, calendula, punica, evening primrose, pumpkin, flax, soybean, borage, trees (oil palm, coconut) , Plants rich in C18: 2 and / or C18: 3 fatty acids such as sunflower, safflower, tobacco, mullein, sesame, cotton, pumpkin, poppy, evening primrose, walnut, flax, hemp, thistle or safflower are particularly preferred. Plants such as safflower, sunflower, poppy, evening primrose, walnut, flax or hemp are particularly preferred.
  • Different compounds of the formula I can be prepared by the enzymatic activity of the nucleic acids used in the process according to the invention, which code for polypeptides with ⁇ -5, ⁇ -6 desaturase or ⁇ -6 elongase activity.
  • mixtures of the various compounds of the general formula I or individual compounds such as EPA or ERA can be prepared in free or bound form.
  • compounds of the general formula I are derived which are derived from C18: 2 fatty acids, such as compounds of the formula containing GLA, DGLA or ERA I or which are derived from C18: 3-fatty acids are derived, such as SDA, ETA or EPA compounds of the formula I.
  • GLA, DGLA and ERA can be created as products of the process, which can be present as free fatty acids or bound.
  • SDA, ETA and EPA can be produced as products of the process which are as described above can be present as free fatty acids or bound.
  • ⁇ -5, ⁇ -6-desaturase and ⁇ -6-elongase or by introducing only the first two genes ( ⁇ -6-desaturase and ⁇ -6-elongase) of the synthesis chain, only individual products can be produced specifically in the aforementioned plants (see FIG. I).
  • the activity of the ⁇ -6-desaturase and ⁇ -6-elongase creates GLA and DGLA or SDA and ETA, depending on the starting plant and unsaturated fatty acid.
  • DGLA or ETA or mixtures thereof are preferably formed.
  • additional ERA or EPA arise. Only ERA or EPA or their mixtures are advantageously synthesized, depending on the fatty acid present 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 plants. There are always small amounts of the precursor compounds in the end product.
  • transgenic plants also include plant cells, tissues, organs or whole plants which are grown for the preparation of compounds of the general formula I.
  • Culturing includes, for example, the cultivation of the transgenic plant cells, tissues or organs on a nutrient medium or the whole plant to be understood on or in a substrate, for example in hydroponics or on a field soil.
  • nucleic acids which code for polypeptides with ⁇ -5, ⁇ -6 desaturase or ⁇ -6 elongase activity can be used in the method according to the invention.
  • These nucleic acids are advantageously derived from plants such as algae such as isochrysis or Crypthecodinium, diatoms such as Phaeodactylum, mosses such as Physco itrella, Ceratodon or higher plants such as the Primulaceae such as Aleuritia, Calendula stellata, Osteospermu spinescens or Osteospermum hyoseroids such as microorganisms such as microorganisms such as microorganisms such as microorganisms , Phytophore, entomophthora, Mucor or Mortierella, yeasts or animals such as nematodes such as Caenorhabditis, insects or humans.
  • a nucleic acid sequence is advantageously selected from the group of those in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 or their derivative or homologs which code for polypeptides which still have the enzymatic activity. These sequences are cloned individually or in combination into expression constructs; these expression constructs are reproduced in the sequences SEQ ID NO: 33-37. These expression constructs enable optimal synthesis of the compounds of the general formula I produced in the process according to the invention.
  • the method further comprises the step of obtaining a cell which contains the nucleic acid sequences used in the method which code for a ⁇ -5 or ⁇ -6 desaturase and a ⁇ -6 elongase, a cell with the nucleic acid sequence, a gene construct or a vector which induce the expression of the ⁇ -5, ⁇ -6-desaturase or ⁇ -6-elongase nucleic acid, alone or in combination, is transformed.
  • this method further comprises the step of extracting the fine chemical from the culture.
  • the cell produced in this way is advantageously a cell of an oil fruit plant such as, for example, peanut, rapeseed, canola, flax, soybean, safflower, hemp, sunflower or borage.
  • a transgenic plant in the sense of the invention means that the nucleic acids used in the method are not in their natural place in the genome of an organism, and the nucleic acids can be expressed homologously or heterologously.
  • Tansgen also means that the nucleic acids according to the invention are in their natural place in the genome of an organism, but that the sequence has been changed compared to the natural sequence and / or that the regulatory sequences of the naturally occurring sequences have been changed.
  • Transgenic is preferably to be understood as meaning the expression of the nucleic acids according to the invention at a non-natural location in the genome, that is to say that the nucleic acids are homologous or preferably heterologous.
  • Preferred transgenic plants are the oil fruit plants.
  • Transgenic plants which contain the compounds of the formula I synthesized in the process according to the invention can be marketed directly without isolating the synthesized compounds.
  • Plants in the process according to the invention are to be understood as all parts of plants, plant organs such as leaves, stems, roots, tubers or seeds or the entire plant.
  • the semen comprises all parts of the semen such as the seminal casing, epidermal and sperm cells, endosperm or embyro tissue.
  • the compounds produced in the process according to the invention can also be isolated from the plants in the form of their oils, fats, lipids and / or free fatty acids.
  • Compounds of formula I prepared by this method can be harvested by harvesting the organisms either from the culture in which they grow or from the field.
  • the oils, fats, lipids and / or free fatty acids can be obtained by cold pressing or cold pressing without the addition of heat by pressing.
  • the seeds pretreated in this way can then be pressed or extracted with solvents such as warm hexane. The solvent is then removed again. In this way, more than 96% of the compounds produced in the process can be isolated.
  • the products thus obtained are then processed further, that is to say refined. First, the plant mucilages and turbidities.
  • degumming can be carried out enzymatically or, for example, chemically / physically by adding acid such as phosphoric acid.
  • the free fatty acids are then removed by treatment with a base, for example sodium hydroxide solution.
  • the product obtained is washed thoroughly with water to remove the lye remaining in the product and dried.
  • the products are subjected to bleaching with, for example, bleaching earth or activated carbon. Finally the product is still deodorized, for example with steam.
  • the PUFAs produced by this process are preferably C ⁇ e ⁇ or C 2 o- 22 fatty acid molecules with at least two double bonds in the fatty acid molecule, preferably three, four, or five or six double bonds when combined with a further elongase and a ⁇ -4 desaturase.
  • These C_ ⁇ or C 20 - 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.
  • An embodiment according to the invention are oils, lipids or fatty acids or fractions thereof which have been produced by the process described above, particularly preferably oil, lipid or a fatty acid composition which comprise PUFAs and originate from transgenic plants.
  • a further embodiment according to the invention is the use of the oil, lipid or the fatty acid composition in feed, food, cosmetics or pharmaceuticals.
  • oil or fat is understood to mean a fatty acid mixture which contains unsaturated, saturated, preferably esterified fatty acid (s). It is preferred that the oil or fat has a high proportion of unsaturated, non-conjugated esterified fatty acid (s), in particular linoleic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, ⁇ -linolenic acid, stearidonic acid, eicotetraenoic acid or eicosapentaenoic acid , Preferably the proportion of unsaturated esterified fatty acids is about 30%, more preferred is 50%, more preferred is 60%, 70%, 80% or more.
  • the proportion of fatty acid after conversion of the fatty acids into the methyl esters can be determined by gas chromatography by transesterification.
  • the oil or fat can be various other saturated or unsaturated fatty acids, e.g. Calendulic acid, palmitic, stearic, oleic acid etc. contain.
  • the proportion of the various fatty acids in the oil or fat can fluctuate depending on the starting plant.
  • the compounds of the formula I prepared in the process and containing polyunsaturated fatty acids with at least two double bonds are sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.
  • the polyunsaturated fatty acids containing can be liberated, for example via an alkali treatment, for example aqueous KOH or NaOH or acid hydrolysis, advantageously in the presence of an alcohol such as methanol or ethanol, or via enzymatic cleavage and isolated via, for example Phase separation and subsequent acidification using, for example, H 2 S0 4 .
  • the fatty acids can also be released directly without the workup described above.
  • the nucleic acids used in the method can either lie on a separate plasmid or be integrated into the genome of the host cell.
  • the integration can be random or by recombination such that the native gene is replaced by the inserted copy, thereby modulating the production of the desired compound by the cell, or by using a gene in trans so that the The gene is functionally linked to a functional expression unit which contains at least one sequence ensuring expression of a gene and at least one sequence ensuring polyadenylation of a functionally transcribed gene.
  • the nucleic acids are advantageously introduced into the plants via multi-expression cassettes or constructs for the multiparallel seed-specific expression of genes.
  • Mosses and algae are the only known plant systems that produce significant amounts of polyunsaturated fatty acids such as arachidonic acid (ERA) and / or eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).
  • ERA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • Mosses contain PUFAs in membrane lipids while algae, algae-related organisms and some fungi also accumulate significant amounts of PUFAs in the triacylglycerol fraction.
  • nucleic acid molecules which are isolated from those strains which also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for the process according to the invention and thus for modifying the lipid and PUFA production system in a host, in particular plants such as oil crop plants, for example Rapeseed, canola, flax, hemp, soy, sunflowers, borage. They can therefore be used advantageously in the process according to the invention.
  • the polyunsaturated C ⁇ s fatty acids must first be desaturated by the enzymatic activity of a desaturase and then extended by at least two carbon atoms using an elongase. After one round of elongation, this enzyme activity leads to C 2 o fatty acids, and after two or three rounds of elongation to C 22 or C 24 fatty acids.
  • the activity of the desaturases and elongases used according to the invention preferably leads to C 8 ⁇ , Co ⁇ and / or C 22 fatty acids with at least two double bonds in the fatty acid molecule, preferably with three, four or five double bonds, particularly preferably to C ⁇ 8 ⁇ and / or C 2 o-fatty acids with at least two double bonds in the fatty acid molecule, preferably with three, four or five double bonds in the molecule.
  • further desaturation steps such as one in the ⁇ -5 position can take place.
  • Arachidonic acid and eicosapentaenoic acid are particularly preferred as products of the process according to the invention.
  • the C ⁇ s fatty acids with at least two double bonds in the fatty acid can be extended by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.
  • esters such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.
  • the nucleic acids can be used for the genetic modification of a broad spectrum of plants, so that they become a better or more efficient producer of one or more lipid-derived products, such as PUFAs.
  • This improved production or efficiency in the production of a product derived from lipids, such as PUFAs can be brought about by the direct effect of the manipulation or an indirect effect of this manipulation.
  • the number or activity of the desaturase protein or gene and of gene combinations of desaturases and elongases can be increased, so that larger amounts of these compounds are produced de novo because the organisms lacked this activity and ability to biosynthesize before the introduction of the corresponding gene.
  • the use of different divergent, ie different sequences at the DNA sequence level, can also be advantageous, or the use of promoters for gene expression, which enables a different temporal gene expression, for example depending on the maturity level of a seed or oil-storing tissue.
  • Fatty acids and lipids are desirable even as fine chemicals; by optimizing the activity or increasing the number of one or more desaturases and / or elongases that are involved in the biosynthesis of these compounds, or by destroying the activity of one or more desaturases that are involved in the degradation of these compounds, it may be possible to use the To increase the yield, production and / or efficiency of the production of fatty acid and lipid molecules from plants.
  • the isolated nucleic acid molecules used in the method according to the invention code for proteins or parts thereof, the proteins or the individual protein or parts thereof containing an amino acid sequence which is sufficiently homologous to an amino acid sequence of the sequence SEQ ID NO: 2, 4, 6, 8, 10 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32, so that the protein or part thereof maintains desaturase or elongase activity.
  • the protein or the part thereof which is encoded by the nucleic acid molecule has its essential enzymatic activity and the ability to participate in the metabolism of compounds necessary for the construction of cell membranes of plants or in the transport of molecules across these membranes.
  • the protein encoded by the nucleic acid molecules is at least about 50%, preferably at least about 60% and more preferably at least about 70%, 80% or
  • the protein is preferably a full-length protein which is essentially homologous in parts to an entire
  • the essential enzymatic activity of the desaturases and the elongase used is to be understood as compared to the sequences by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,
  • 25 17, 19, 21, 23, 25, 27, 29 or 31 encoded proteins / enzymes in comparison still have at least one enzymatic activity of at least 10%, preferably 20%, particularly preferably 30% and very particularly 40% and thus on the metabolism necessary to build up fatty acids in a plant cell
  • Nucleic acids which can advantageously be used in the process originate from fungi or plants such as algae or mosses such as the genera Physcomitrella, Thraustochytrium, Phytophtora, Ceratodon, Isochysis, Aleurita, Muscarioides, Mortierella, Borago, Phaeodactylum, Crypthecodinium or from nematitis, such as Ceanorhabod
  • Phytophtora infestans Ceratodon purpureus, Isochrysis galbana, Aleurita farinosa, Muscarioides viallii, Mortierella alpina, Borago officinalis, Phaeodactylum tricormutum or Ceanorhabditis elegans.
  • the isolated nucleotide sequences used can code for desaturases or elongases which bind to a nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 , 29 or 31 hybridize, eg hybridize under stringent conditions.
  • the nucleic acid sequences used in the method are advantageously introduced in an expression cassette which enables the expression of the nucleic acids in plants.
  • Advantageous expression cassettes are reproduced in SEQ ID NO: 33 to 37.
  • the nucleic acid sequences coding for the desaturases and / or the elongases are advantageously functionally linked with one or more regulation signals to increase gene expression.
  • These regulatory sequences are intended to enable targeted expression of the genes and protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed and / or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences may still be present in front of the actual structural genes and may have been genetically modified so that the natural regulation has been switched off and the expression of the genes has been increased.
  • the gene construct can also advantageously contain one or more so-called "enhancer sequences" functionally linked to the promoter, which enable increased expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the DNA sequences.
  • This gene construct or the gene constructs can be expressed together in the host organism.
  • the gene construct or the gene constructs can be inserted in one or more vectors and freely present in the cell or else inserted in the genome. It is advantageous for the insertion of further genes in the host genome if the genes to be expressed are present together in one gene construct.
  • the regulatory sequences or factors can preferably have a positive influence on the gene expression of the introduced genes and thereby increase it.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • Another embodiment of the invention are one or more gene constructs which contain one or more sequences which are represented by Seq ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 are defined and acc.
  • SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 encode polypeptides.
  • the desaturases mentioned introduce a double bond in the ⁇ -5 or ⁇ -6 position, the substrate having one, two, three or four double bonds.
  • the elongase ( ⁇ -6 elongase) has an enzyme activity that extends a fatty acid by at least two carbon atoms. The same applies to their homologs, derivatives or analogs which are functionally linked to one or more regulation signals, advantageously to increase gene expression.
  • Advantageous regulatory sequences for the new method are present, for example, in promoters, such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacis, T7 and T5. , T3-, gal-, trc-, ara-, SP6-, -P R - or ⁇ -Pi, -promotor and are advantageously used in Gram-negative bacteria.
  • promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacis, T7 and T5.
  • promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacis, T7 and T5.
  • promoters such as the cos, tac, trp, tet, trp-tet,
  • Further advantageous regulatory sequences are, for example, in the Gram-positive promoters amy and SP02, in the yeast or fungal promoters ADC1, MF ⁇ , AC, P-60, CYCl, GAPDH, TEF, rp28, ADH or in the plant promoters CaJ_V / 35S [ Franck et al., 1980, Cell 21: 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter.
  • inducible promoters are also advantageous, such as those in EP-A-0 388 186 (benzylsulfonamide inducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracycline inducible), EP-A-0 335 528 (induction of abzisic acid) or WO 93/21334 (ethanol or cyclohexenol inducible) described promoters.
  • Further suitable plant promoters are the cytosolic FBPase promoter or the potato ST-LSI promoter (Stockhaus et al., EMBO J.
  • promoters which enable expression in tissues which are involved in fatty acid biosynthesis.
  • seed-specific promoters such as the USP promoter according to the embodiment, but also other promoters such as the LeB4, DC3, phaseolin or napin promoter are very particularly advantageous.
  • promoters are seed-specific promoters that can be used for monocotyledonous or dicotyledonous plants and in US 5,608,152 (napin promoter from rapeseed), WO 98/45461 (oleosin promoter from Arobidopsis), US 5,504,200 (phaseolin promoter from Phaseolus vulgaris) ), WO 91/13980 (Bce4 promoter from Brassica), by Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (LeB4 promoter from a legume), these promoters being suitable for dicotyledons suitable.
  • the following are seed-specific promoters that can be used for monocotyledonous or dicotyledonous plants and in US 5,608,152 (napin promoter from rapeseed), WO 98/45461 (oleosin promoter from Arobidopsis), US 5,504,200 (phaseolin promoter from Phaseolus vulgaris) ), WO 91/13980
  • Promoters are suitable, for example, for monocotyledons lpt-2 or lpt-1 promoter from barley (WO 95/15389 and WO 95/23230), hordein promoter from barley and other suitable promoters described in WO 99/16890.
  • the PUFA biosynthesis genes should advantageously be expressed seed-specifically in oil seeds.
  • seed-specific promoters can be used, or those promoters that are active in the embryo and / or in the endosperm.
  • seed-specific promoters can be isolated from both dicotolydonous and monocotolydonous plants.
  • Legumes B4 (LegB4 promoter) [Bäumlein et al., Plant J., 2,2, 1992], Lpt2 and Ipt1 (barley) [WO 95/15389 u. WO95 / 23230], seed specific promoters from rice, maize u.
  • Plant gene expression can also be facilitated via a chemically inducible promoter (see an overview in Gatz
  • Chemically inducible promoters are particularly suitable if it is desired that the gene expression be carried out in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible
  • ⁇ -6-desaturase which encode ⁇ -5-desaturase or the ⁇ -6-elongase, are expressed under the control of one's own, preferably a different promoter, since repeating sequence motifs for instability of the T-DNA or for recombination
  • the expression cassette is advantageously constructed in such a way that a promoter is followed by a suitable interface for inserting the nucleic acid to be expressed, advantageously in a polylinker, and optionally a terminator behind the polylinker. This sequence again
  • nucleic acid sequences are inserted for expression via the suitable interface, for example in the polylinker behind the promoter.
  • Each nucleic acid sequence advantageously has its own promoter and possibly its own terminator. However, it is also possible to insert several nucleic acid sequences behind a promoter and possibly 0 in front of a terminator.
  • the insertion point or the sequence of the inserted nucleic acids in the expression cassette is not of crucial importance, that is, a nucleic acid sequence can be inserted in the first or last position in the cassette without the expression 5 being significantly influenced thereby.
  • Different promoters such as the USP, LegB4 or DC3 promoter and different terminators can advantageously be used in the expression cassette be used.
  • the transcription of the introduced genes should advantageously be terminated by suitable terminators at the 3 'end of the introduced biosynthetic genes (behind the stop codon).
  • suitable terminators at the 3 'end of the introduced biosynthetic genes (behind the stop codon).
  • different terminator sequences should be used for each gene.
  • the gene construct can also comprise further genes which are to be introduced into the organisms. It is possible and advantageous to introduce and express regulatory genes, such as genes for inducers, repressors or enzymes, which intervene in the regulation of one or more genes of a biosynthetic pathway due to their enzyme activity. These genes can 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 these genes can be located on a further or more further nucleic acid constructs.
  • regulatory genes such as genes for inducers, repressors or enzymes, which intervene in the regulation of one or more genes of a biosynthetic pathway due to their enzyme activity. These genes can be of heterologous or homologous origin.
  • further biosynthesis genes of the fatty acid or lipid metabolism can advantageously be contained in the nucleic acid construct or gene construct or these genes can be located on a further or more further nucleic acid constructs.
  • the regulatory sequences or factors can preferably have a positive influence on the gene expression of the introduced genes and thereby increase it.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • the expression cassettes can be inserted directly can be used in the plant or introduced into a vector.
  • vectors contain the nucleic acid used in the process, which code for ⁇ -5 or ⁇ -6 desatures or ⁇ -6 elonagases, or a nucleic acid construct which, alone or in combination with other biosynthetic genes, of the nucleic acid used Fatty acid or lipid metabolism.
  • vector refers to a nucleic acid molecule that can transport another nucleic acid to which it is attached.
  • plasmid which stands for a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, whereby additional DNA segments can be ligated into the viral genome.
  • vectors can replicate autonomously in a host cell into which they have been introduced (e.g. bacterial vectors with a bacterial origin of replication). Other vectors are advantageously integrated into the genome of a host cell when they are introduced into the host cell and thereby replicated together with the host genome. In addition, certain vectors can control the expression of genes to which they are operably linked. These vectors are referred to here as "expression vectors". Expression vectors that are suitable for recombinant DNA techniques are usually in the form of plasmids. In the present description, "plasmid” and “vector” can be used interchangeably because the plasmid is the most commonly used vector form. However, the invention is intended to encompass these other expression vector forms, such as viral vectors, which perform similar functions.
  • vector is also intended to include 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 nucleic acids described below or the gene construct described above in a form which is suitable for expression of the nucleic acids used in a host cell, which means that the recombinant expression vectors selected one or more regulatory sequences, based on of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • operably linked means that the nucleotide sequence of interest is bound to the regulatory sequence (s) in such a way that the expression of the nucleotide sequence is possible and they are linked to one another so that both sequences fulfill the predicted function ascribed to the sequence (for example 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 encompass promoters, enhancers and other expression control elements (for example polyadenylation signals).
  • Regulatory sequences include those that control the constitutive expression of a nucleotide sequence in many host cell types and those that control the direct expression of the nucleotide sequence only in certain host cells under certain conditions.
  • the person skilled in the art knows that the design of the expression vector can depend on factors such as the selection of the host cell to be transformed, the extent of expression of the desired protein, etc.
  • the recombinant expression vectors used can be designed to express desaturases and elongases in prokaryotic or eukaryotic cells. This is advantageous since intermediate steps of vector construction are often carried out in microorganisms for the sake of simplicity.
  • desaturase and / or elongase genes 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.
  • Suitable host cells are also 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, for example using T7 promoter regulatory sequences and T7 polymerase.
  • fusion expression vectors include pGEX (Pharmacia Biotech Ine; 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 llD (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.
  • the target gene expression from the pETIld vector is based on the transcription from a T7-gnl0-lac fusion promoter, which is mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is provided by BL21 (DE3) or HMS174 (DE3) host strains from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • vectors suitable in prokaryotic organisms are known to the person skilled in the art, these vectors are, for example, in E. coli PLG338, pACYC184, the pBR series, such as pBR322, the pUC series, such as PUC18 or pUC19, the Mll3mp series, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 113 -Bl, ⁇ gtll or pBdCI, in Streptomyces pIJlOl, pIJ364, pIJ702 or pIJ361, in Bacillus pUBllO, pC194 or pBDneba14, in PBDneba14.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in the yeast S. cerevisiae include pYeDesaturasecl (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 of constructing vectors suitable for use in other fungi include those described in detail in: van den Hondel, CAMJJ, & Punt, PJ (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, JF Peberdy et al., eds., pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi [JW Bennet & LL Lasure , Eds., Pp. 396-428: Academic Press: San Diego]
  • yeast vectors are, for example, pAG-1, YEp6, YEpl3 or pEMBLYe23.
  • the desaturases and / or elongases can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol .. 3: 2156-2165) and the pVL- Series (Lucklow and Summers (1989) Virology 170: 31-39).
  • the desaturases and / or elongases can be used in single-cell plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251 and literature 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. (1992) "New plant binary vectors with selectable markers located proximal to the left border ", Plant Mol. Biol. 20: 1195-1197; and Bevan, MW (1984) "Binary Agrobacterium vectors for plant transformation ", Nucl. Acids Res. 12: 8711-8721; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, ed .: Kung and R. Wu, Academic Press, 1993, p 15-38.
  • a plant expression cassette preferably contains regulatory sequences which can control gene expression in plant cells and are operably linked so that each sequence can fulfill its function, such as termination of transcription, for example polyadenylation signals.
  • Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens-t-DNA, such as gene 3 of the Ti plasmid pTiACH5 known as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835ff.) Or functional equivalents thereof, but all other terminators which are functionally active in plants are also suitable.
  • a plant expression cassette preferably contains other functionally linked sequences, such as translation enhancers, for example the overdrive sequence, which is the 5 'untranslated leader sequence from tobacco mosaic virus, which contains the protein / RNA ratio increased (Gallie et al., 1987, Nucl. Acids Research 15: 8693-8711).
  • translation enhancers for example the overdrive sequence, which is the 5 'untranslated leader sequence from tobacco mosaic virus, which contains the protein / RNA ratio increased (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 that of the small subunit of the Rubisco described in US 4,962,028.
  • Chemically inducible promoters are particularly suitable if it is desired that the gene expression be carried out 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 that react to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-induced PRPl gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), the heat-inducible hsp80 promoter Tomato (US 5,187,267), the cold-inducible alpha amylase promoter from potato (WO 96/12814) or the wound-inducible pin III promoter (EP-A-0 375 091).
  • Suitable promoters are the Napingen promoter from rapeseed (US 5,608,152), the USP promoter from Vicia faba (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67), the oleosin
  • Arabidopsis promoter (WO 98/45461), the Phaseolin promoter from Phaseolus vulgaris (US 5,504,200), the Bce4 promoter from Brassica (WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al., 1992 , Plant Journal, 2 (2): 233-9) and promoters which bring about the seed-specific expression in monocot plants, such as maize, barley, wheat, rye, rice etc.
  • Suitable noteworthy promoters are the lpt2 or lptl 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, wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the Rye secalin gene).
  • the multiparallel expression of the desaturases and / elongases used in the method may be desired alone or in combination with other desaturases or elongases.
  • Such expression cassettes can be introduced via a simultaneous transformation of a plurality of individual expression constructs or preferably by combining a plurality of expression cassettes on one construct. You can also use multiple vectors with each several expression cassettes are transformed and transferred to the host cell.
  • Promoters which bring about plastid-specific expression are also particularly suitable, since plastids are the compartment in which the precursors and 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 clpP promoter from Arabidopsis, described in WO 99/46394.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells using conventional transformation or transfection techniques.
  • transformation and “transfection”, conjugation and transduction, as used here, are intended to mean a large number of methods known in the prior 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.
  • Host cells which are suitable in principle for taking up 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 organisms, such as bacteria, fungi, yeasts or plant cells, preferably plants or parts thereof.
  • Mushrooms, yeasts or plants are preferably used, particularly preferably plants, very particularly preferably plants, such as oil fruit plants, which contain large amounts of lipid compounds, such as rapeseed, evening primrose, hemp, diesel, peanut, canola, flax, soy, safflower, sunflower, borage , or plants, such as corn, wheat, rye, oats, triticale, rice, barley, cotton, manioc, pepper, tagetes, Solanaceae plants, such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, bush plants (coffee, cocoa, tea), salix species, trees (oil plant, coconut) as well as perennial grasses and forage crops.
  • Particularly preferred plants according to the invention are oil fruit plants such as soybean, peanut, rapeseed, canola, flax, hemp, evening primrose, sunflower, safflower, trees (oil palm, coconut).
  • nucleic acid sequences are advantageously used which code for the polypeptides with a ⁇ -6-desaturase activity, ⁇ -6-elongase activity or ⁇ -5-desaturase activity, selected from the group:
  • 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, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
  • SEQ ID NO: 15 SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 or SEQ ID NO: 31 shown nucleic acid sequence, which for polypeptides with the 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 encodes the amino acid sequences shown and have at least 50% homology at the amino acid level without the enzymatic action of the polypeptides being significantly reduced.
  • the above-mentioned nucleic acid according to the invention comes from organisms such as animals, ciliates, fungi, plants such as algae or dinoflagellates, which can synthesize PUFAs.
  • nucleic acid (molecule) also encompasses the untranslated sequence located at the 3 'and 5' ends of the coding gene region: at least 500, preferably 200, particularly preferably 100 nucleotides of the sequence upstream of the 5 ' End of the coding region and at least 100, preferably 50, particularly preferably 20 nucleotides of the sequence downstream the 3 'end of the coding gene region.
  • An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid preferably has no sequences 5 which naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (eg sequences which are located at the 5 'and 3' ends of the nucleic acid).
  • the isolated desaturase or elongase nucleic acid molecule may, for example, contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences that are natural flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid originates.
  • nucleic acid molecules used in the process e.g. a nucleic acid molecule with a nucleotide sequence of SEQ ID N0: 1 or a part thereof can be isolated using standard molecular biological techniques and the sequence information provided here. Also with the help of
  • nucleic acid molecule comprising a complete sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,
  • mRNA can be isolated from cells (e.g. by the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18: 5294-5299)
  • Reverse transcriptase available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St.Petersburg, FL). Leave synthetic oligonucleotide primers for the amplification by means of the polymerase chain reaction
  • a nucleic acid according to the invention can be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to 5 standard PCR amplification techniques.
  • the nucleic acid amplified in this way can be cloned into a suitable vector and characterized by means of DNA sequence analysis.
  • Oligonucleotides which correspond to a desaturase nucleotide sequence can be produced by standard synthesis methods, for example using an automatic DNA synthesizer.
  • 15 15, 17, 19, 21, 23, 25, 27, 29 or 31 means, for example, allelic variants with at least about 50 to 60%, preferably at least about 60 to 70%, more preferably at least about 70 to 80%, 80 to 90 % or 90 to 95% and more preferably at least about 95%, 96%, 97%, 98%, 99% or
  • isolated nucleic acid molecules of a nucleotide sequence which are attached to one of the sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,
  • allelic variants include, in particular, functional variants which are deletion, insertion or substitution of nucleotides from / in the
  • enzymatic activity of desaturase or elongase means proteins with at least 10%, preferably 20%, particularly preferably 30%, very particularly preferably 40% of the original enzyme activity, compared with that by
  • Homologues of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 also mean, for example, bacterial, 45 fungal and plant homologues, shortened sequences , single-stranded DNA or RNA of the coding and non-coding DNA sequence.
  • Homologs of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 also means derivatives, such as promoter variants.
  • the promoters upstream of the specified nucleotide sequences can be modified by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), without however impairing the functionality or activity of the promoters. It is also possible that the activity of the promoters is increased by modifying their sequence or that they are completely replaced by more active promoters, even from heterologous organisms.
  • nucleic acids and protein molecules with desaturase or elongase activity which are involved in the metabolism of lipids and fatty acids, PUFA cofactors and enzymes or in the transport of lipophilic compounds across membranes, are described in Process for modulating the production of compounds of the general formula I in transgenic plants, such as maize, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, linu species such as oil or fiber flax, Brassica species such as rape , Canola and turnip, pepper, sunflower, borage, evening primrose and tagetes, Solanacaen plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, cassava, alfalfa, bush plants (coffee, cocoa, tea), Salix species , Trees (oil palm, coconut) and perennial grasses and forage crops, either directly (eg if the overexpression or optimization of a fatty acid biosynthesis protein has a direct influence
  • PUFAs polyunsaturated fatty acids
  • PUFAs polyunsaturated fatty acids
  • PUFAs polyunsaturated fatty acids
  • Lipid synthesis can be divided into two sections: the synthesis of fatty acids and their binding 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 in acetyl-ACP by the acetyl transacylase. After a condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydration reactions, so that a saturated fatty acid molecule with the desired chain length is obtained.
  • the production of the unsaturated fatty acids from these molecules is catalysed by specific desaturases, either aerobically using molecular oxygen or anaerobically (for fatty acid synthesis in microorganisms see FC Neidhardt et al. (1996) E. coli and Salmonella.
  • Precursors for PUFA biosynthesis are, for example, oleic acid, linoleic and linolenic acid. These C ⁇ s-carbon fatty acids must be extended to C 20 and C 22 in order to obtain fatty acids of the Eicosa and Docosa chain type.
  • desaturases used in the process such as the ⁇ -5 and ⁇ -6 desaturase and the ⁇ -6 elongase, arachidonic acid and eicosapentaenoic acid as well as various other long-chain PUFAs can be obtained, extracted and used for various purposes in food, feed, cosmetics or pharmaceuticals Applications are used.
  • C ⁇ 8 + C o fatty acids with at least two, three, four or five double bonds in the fatty acid molecule preferably to C 2 o ⁇ fatty acids with advantageously three, four or five double bonds in the fatty acid molecule, can be produced.
  • Desaturation can take place before or after elongation of the corresponding fatty acid.
  • Substrates in the process according to the invention are, 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.
  • the C ⁇ s or C 20 ⁇ fatty acids with at least two double bonds in the fatty acid are obtained in the process according to the invention in the form of the free fatty acid or in the form of its esters (see formula I), for example in the form of their glycerides.
  • glycolide means a glycerol esterified with one, two or three carboxylic acid residues (mono-,
  • Di- or triglyceride Di- or triglyceride.
  • Glyceride is also understood to mean a mixture of different glycerides.
  • the glyceride or the glyceride mixture can contain further 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 further understood to mean derivatives derived from glycerol.
  • this also includes glycerophospholipids and glyceroglycolipids.
  • Glycerophospholipids such as lecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol, phosphatidylserine and alkyl acylglycerophospholipids may be mentioned by way of example.
  • fatty acids then have to be transported to different modification sites and incorporated into the triacylglycerol storage lipid.
  • Another important step in lipid synthesis is the transfer of fatty acids to the polar head groups, for example by glycerol fatty acid acyl transferase (see Frentzen, 1998, Lipid, 100 (4-5): 161-166).
  • the PUFAs produced in the process comprise a group of molecules which higher animals can no longer synthesize and therefore have to absorb or which higher animals are no longer able to produce themselves sufficiently and therefore have to absorb, although they are easily synthesized by other organisms, such as bacteria For example, cats can no longer synthesize arachidonic acid.
  • desaturase or elongase or “desaturase or elongase polypeptide” in the sense of the invention encompasses proteins which participate in the desaturation and elongation of fatty acids, as well as their homologs, derivatives or analogs.
  • desaturase or elongase nucleic acid sequence encompass nucleic acid sequences which encode a desaturase or elongase and in which a part can be a coding region and also corresponding 5 'and 3' untranslated sequence regions.
  • production or productivity are known in the art and include the concentration of the fermentation product (compounds of the formula I) which is formed in a certain time period and a certain fermentation volume (for example kg product per hour per liter).
  • efficiency of production encompasses the time it takes to achieve a certain production volume (e.g. how long it takes the cell to set up a certain throughput rate of a fine chemical).
  • yield or product / carbon yield is known in the art and encompasses 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.
  • Increasing the yield or production of the compound increases the amount of molecules or suitable molecules of this compound obtained in a given amount of culture over a predetermined period of time.
  • biosynthesis or biosynthetic pathway are known in the art and encompass the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds, for example in a multi-step and highly regulated process.
  • degradation or degradation pathway are known in the art and include 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 specialist field and encompasses all of the biochemical reactions that take place in an organism. The metabolism of a particular compound (eg the metabolism of a fatty acid) then encompasses all of the biosynthetic, modification and degradation pathways of this compound in the cell that relate to this compound. 5
  • derivatives of the nucleic acid molecule according to the invention encode proteins with at least 50%, advantageously about 50 to 60%, preferably at least about 60 to 70% and more preferably at least about 70 to 80%,
  • the invention also encompasses nucleic acid molecules which differ from one of the nucleotide sequences shown in SEQ ID NO: 1, 3, 5 or 11 (and parts thereof) because of the degenerate genetic code and thus the same desaturase
  • 25 encode as that encoded by the nucleotide sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of the desaturases or elongases can exist within a population.
  • These genetic polymorphisms in the desaturase or elongase gene 35 can 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 desaturase or elongase gene.
  • Nucleic acid molecules which are advantageous for the method according to the invention can be used on the basis of their homology to the desaturase or elongase nucleic acids disclosed here the sequences or a part thereof are isolated as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • isolated nucleic acid molecules can be used that are at least 15 nucleotides long and under stringent conditions with the nucleic acid molecules that have a nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31, hybridize.
  • Nucleic acids of at least 25, 50, 100, 250 or more nucleotides can also be used.
  • hybridizes under stringent conditions is intended to describe hybridization and washing conditions under which nucleotide sequences which are at least 60% homologous to one another usually remain hybridized to one another.
  • the conditions are preferably such that sequences which are at least about 65%, more preferably at least about 70% and even more preferably at least about 75% or more homologous to one another usually remain hybridized to one another.
  • the temperature is about 42 ° C under standard conditions.
  • the hybridization conditions for DNA: DNA hybrids are preferably, for example, 0.1 ⁇ SSC and 20 ° C. to 45 ° C., preferably between 30 ° C. and 45 ° C.
  • the hybridization conditions for DNA: RNA hybrids are preferably, for example, 0.1 ⁇ SSC and 30 ° C. to 55 ° C., preferably between 45 ° C. and 55 ° C.
  • the sequences are written for the purpose of optimal comparison with one another (for example, gaps can be inserted in the sequence of a protein or a nucleic acid in order to produce an optimal alignment with the other protein or the other nucleic acid).
  • the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity”).
  • the terms homology and identity can thus be regarded as synonymous.
  • An isolated nucleic acid molecule that encodes a desaturase or elongase that results in a protein sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 is homologous, by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 are generated so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be made into one of the sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31 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 non-essential amino acid residues.
  • conservative amino acid substitution the amino acid residue is exchanged for an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g.
  • 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 (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, 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, va
  • a predicted non-essential amino acid residue in a desaturase or elongase is thus preferably replaced by another amino acid residue from the same side chain family.
  • the mutations can be randomly introduced over all or part of the desaturase coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for the desaturase activity described herein to identify mutants that desaturase or maintain elongase activity. After mutagenesis of one of the sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined, for example, using the tests described here.
  • Cloning methods such as restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, connection of DNA fragments, transformation of Escherichia coli and yeast cells, cultivation of bacteria and sequence analysis of recombinant DNA were carried out as described in Sairibrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994) "Methods in Yeast Genetics” (Cold Spring Harbor Laboratory Press: ISBN 0-87969-451-3) , b) chemicals
  • RNA Protonema tissue can be obtained from moss by the GTC method (Reski et al., 1994, Mol. Gen. Genet., 244: 352-359).
  • Agrobacterium -mediated plant transformation can be performed, for example, using GV3101- (pMP90-) (Koncz and Schell, Mol. Gen. Genet. 204 (1986) 383-396) or LBA4404- (Clontech) or C58C1 pGV2260 (Deblaere et al 1984 , Nucl. Acids Res. 13, 35 4777-4788) Agrobacterium tumefaeiens strain.
  • the transformation can be carried out using standard transformation techniques (also Deblaere et al. 1984).
  • the Agrobacterium-mediated plant transformation can be carried out using standard transformation and regeneration techniques (Gelvin, Stanton B., Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd ed., Dordrecht: 45 Kluwer Academic Publ., 1995, in Sect., Ringbuc Central signature: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boea Raton: CRC Press, 1993, 360 pages, ISBN 0-8493-5164-2).
  • rapeseed can be transformed using cotyledon or hypocotyl transformation (Moloney et al., Plant
  • Agrobacterium -mediated gene transfer in linseed can be carried out using, for example, one of Mlynarova et al. (1994) Plant Cell Report 13: 282-285 perform the technique described.
  • soybeans can be carried out using, for example, a technique described in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 0397 687, US 5,376,543, US 5,169,770 (University Toledo).
  • Binary vectors such as pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230) or pGPTV (Becker et al 1992, Plant Mol. Biol. 20: 1195-1197) can be used for plant transformation.
  • the binary vectors can be constructed by ligating the cDNA in sense or antisense orientation in T-DNA. 5 'of the cDNA, a plant promoter activates the transcription of the cDNA. A polyadenylation sequence is located 3 'from the cDNA.
  • the binary vectors can carry different marker genes.
  • nptll marker gene coding for kanamycin resistance mediated by neomycin phosphotransferase can be exchanged for the herbicide-resistant form of an acetolactate synthase gene (AHAS or ALS).
  • the ALS gene is described in Ott et al., J. Mol. Biol. 1996, 263: 359-360.
  • the v-ATPase-cl promoter can be cloned into the plasmid pBin19 or pGPTV and used for the marker gene expression by cloning in front of the ALS coding region.
  • the promoter mentioned corresponds to a 1153 base pair question ent from beta-Vulgaris (Plant Mol Biol, 1999, 39: 463-475). You can Both sulphonylureas and imidazolinones such as imazethapyr or sulphonylureas can be used as antimetabolites for selection.
  • Tissue-specific expression can be achieved using a tissue-specific promoter.
  • seed-specific expression can be achieved by cloning the DC3 or the LeB4 or the USP promoter or the phaseolin promoter 5 'of the cDNA. Any other seed-specific promoter element such as the Napin or Arcelin promoter
  • the CaMV-35S promoter or a v-ATPase Cl promoter can be used for constitutive expression in the whole plant.
  • genes coding for desaturases and elongases can be cloned in succession into a binary vector by constructing several expression cassettes in order to simulate the metabolic pathway in plants.
  • the protein to be expressed can be directed into a cellular compartment using a signal peptide, for example for plastids, mitochondria or the endoplasmic reticulum (Kermode, Crit. Rev. Plant Sei. 15, 4 (1996) 285-423).
  • the signal peptide is cloned 5 'in frame with the cDNA in order to achieve the subcellular localization of the fusion protein.
  • Expression cassettes consist of at least two functional units such as a promoter and a terminator. Further desired gene sequences such as targeting sequences, coding regions of genes or parts thereof, etc. can be inserted between the promoter and terminator.
  • promoters and terminators are used (USP promoter: Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67); OCS terminator: Gielen et al. EMBO J. 3 (1984) 835ff.) Isolated using the polymerase chain reaction and tailored with flanking sequences of your choice based on synthetic oligonucleotides.
  • the following oligonucleotides can be used, for example:
  • USP3 rear TCCCCCGGGATCGATGCCGGCAGATCTGCTGGCTATGAAGAAATT OCS1 front: AAAACTGCAGTCTAGAAGGCCTCCTGCTTTAATGAGATAT 0CS2 front: CGCGGATCCGATATCGGGCCCATCTAGCGTTAACCCCCTGCTTGATGAGAGCGTTAACCCCCTGCTTGATGAGAGAGGGTCCAT
  • OCS1 rear CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTAAATTGA
  • OCS2 rear CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTAAATTGA
  • OCS3 rear CCCAAGCTTGGCTCGGGGGAGCGCGTCGAGA
  • a promoter and a terminator are amplified via PCR. Then the terminator is cloned into a recipient plasmid and in a second step the promoter is inserted in front of the terminator. An expression cassette on a carrier plasmid is thus obtained.
  • the plasmids pUT1, 2 and 3 are created on the basis of the plasmid pUC19.
  • the constructs are defined in SEQ ID NO: 33, 34 to 42. They contain the USP promoter and the OCS terminator.
  • the construct pUT12 is created on the basis of these plasmids by cutting pUTl using Sall / Scal and cutting pUT2 using Xhol / Scal.
  • the fragments containing the expression cassettes are ligated and transformed into E. coli XLI blue MRF. After isolating ampicillin-resistant colonies, DNA is prepared and those clones which contain two expression cassettes are identified by restriction analysis.
  • the Xhol / Sall ligation of compatible ends has eliminated the two interfaces Xhol and Sall between the expression cassettes.
  • plasmid pUT12 which is defined in SEQ ID NO: 36.
  • pUTl2 is cut again using Sal / Scal and pUT3 is cut using Xhol / Scal.
  • the fragments containing the expression cassettes are ligated and transformed into E. coli XLI blue MRF.
  • DNA is prepared and those clones which contain three expression cassettes are identified by restriction analysis. In this way, a set of multi-expression cassettes is created that for insertion desired DNA can be used and is described in Table 1 and can also accommodate other expression cassettes.
  • the DC3 promoter is described in Thomas, Plant Cell 1996, 263: 359-368 and consists only of the region -117 to +26, B which is why it is one of the smallest known seed-specific promoters.
  • the expression cassettes can contain the same promoter several times or can be constructed using three different promoters.
  • Vectors used for plant transformation as well as the sequences of the inserted genes / proteins.
  • Polylinker or polylinker terminator 45 polylinkers which are advantageously used can be found in the sequences SEQ ID NO: 50 to 52.
  • Table 2 Multiple expression cassettes
  • seed-specific multi-expression cassettes such as e.g. to use the Napin promoter or the Arcelin-5 promoter.
  • the ⁇ -6 elongase Pp_PSEl is first inserted into the first cassette via BstXI and Xbal. Then the ⁇ -6-desaturase from moss (Pp_des6) is inserted into the 5 second cassette via BamHI / Nael and finally the ⁇ -5-desaturase from Phaeodactylum (Pt_des5) is inserted into the third cassette via BglII / Ncol.
  • the triple construct is given the name pARAl. Taking into account sequence-specific restriction interfaces, further expression cassettes according to Table 3 with the names pARA2, pARA3 and pARA4 can be created.
  • Pp Physcomitrella patens
  • Pt Phaeodactylum tricornutum 15
  • Pp_PSEl corresponds to the sequence from SEQ ID NO: 9.
  • PSE PUFA-specific ⁇ -6-elongase
  • Ce_des5 ⁇ -5 desaturase from Caenorhabditis elegans (Genbank Acc. No. AF078796)
  • Ce_des6 ⁇ -6 desaturase from Caenorhabditis elegans elegans
  • Ce_PSEl ⁇ -6-elongase from Caenorhabditis elegans (Genbank Acc. No. AF244356, bases 1-867)
  • the polylinker is newly synthesized as two double-stranded oligonucleotides, with an additional Ascl DNA sequence being inserted.
  • the oligonucleotide is inserted into the vector pGPTV using EcoRI and HindIII.
  • the cloning techniques required - 0 are known in the art and can just as described in Example 1 to be read.
  • Example 6 Examination of the expression of a recombinant gene product in a transformed organism
  • the activity of a recombinant gene product in the transformed host organism can be measured at the transcription and / or translation level.
  • a suitable method for determining the amount of transcription of the gene is to carry out a Northern blot as outlined below (for reference see Ausubel et al. (1988 ) Current Protocols in Molecular Biology, Wiley, New York, or the sample portion mentioned above), wherein a primer that is designed to bind to the gene of interest with a detectable
  • RNA label usually radioactive or chemiluminescent
  • RNA of a culture of the organism is extracted, separated on a gel, transferred to a stable matrix and incubated with this probe, the binding and extent of binding of the probe indicates the presence and also the amount of mRNA for this gene. This information indicates the level of transcription of the transformed gene.
  • Total cellular RNA can be derived from cells, tissues, or organs by several methods, all of which are known in the art, such as that of Bormann, E.R., et al. (1992) Mol. Microbiol. 6: 317-326.
  • RNA hybridization 20 ⁇ g of total RNA or 1 ⁇ g of poly (A) + RNA were analyzed by gel electrophoresis in agarose gels with a strength of 1.25% using formaldehyde, as described in Amasino (1986, Anal. Biochem. 152 , 304) separated by capillary attraction using 10 x SSC on positively charged nylon membranes (Hybond N +, Amersham, Braunschweig), immobilized by UV light and 3 hours at 68 ° C using hybridization buffer (10% dextran sulfate wt. / Vol., 1 M NaCl, 1% SDS, 100 mg herring sperm DNA) prehybridized.
  • hybridization buffer 10% dextran sulfate wt. / Vol., 1 M NaCl, 1% SDS, 100 mg herring sperm DNA
  • the DNA probe was labeled with the Highprime DNA labeling kit (Röche, Mannheim, Germany) during the prehybridization using alpha- 32 P-dCTP (Amersham, Braunschweig, Germany).
  • the hybridization was carried out after adding the labeled DNA probe in the same buffer at 68 ° C. overnight.
  • the washing steps were carried out twice for 15 min using 2 X SSC and twice for 30 min using 1 X SSC, 1% SDS at 68 ° C. guided.
  • the exposure of the closed filter was carried out at -70 ° C for a period of 1 to 14 T.
  • Standard techniques such as a Western blot can be used to examine the presence or relative amount of protein translated from this mRNA (see, e.g., Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York).
  • the total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix, such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein.
  • This probe is usually provided with a chemiluminescent or colorimetric label that is easy to detect. The presence and amount of the label observed indicates the presence and amount of the desired mutant protein present in the cell.
  • Example 7 Analysis of the effect of the recombinant proteins on the production of the desired product
  • the effect of genetic modification in plants, fungi, algae, ciliates or on the production of a desired compound can be determined by growing the modified microorganisms or the modified plant under suitable conditions (such as those described above) and that Medium and / or the cellular components for the increased production of the desired product (ie lipids or a fatty acid) is examined.
  • suitable conditions such as those described above
  • These analysis techniques are known to the person skilled in the art and include spectroscopy, thin-layer chromatography, staining methods of various types, enzymatic and microbiological methods and analytical chromatography, such as high-performance liquid chromatography (see for example Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and p.
  • the analytical methods include measurements of the amount of nutrients in the medium (e.g. sugar, hydrocarbons, nitrogen sources, phosphate and others
  • fatty acids abbreviations: FAME, fatty acid methyl ester; GC-MS, gas-liquid chromatography-mass spectrometry; TAG, triacylglycerol; TLC, thin-layer chromatography.
  • the unambiguous detection of the presence of fatty acid products can be obtained by analysis of recombinant organisms according to standard analysis methods: GC, GC-MS or TLC, as described variously by Christie and the references therein (1997, in: Advances on Lipid Methodology, Fourth Ed. : Christie, Oily Press, Dundee, 119-169; 1998, gas chromatography-mass spectrometry method, lipids 33: 343-353).
  • the material to be analyzed can be broken up by ultrasound treatment, glass mill grinding, liquid nitrogen and grinding, or other applicable methods.
  • the material must be centrifuged after breaking up.
  • the sediment is in aqua dest. resuspended, 10 min at 100 ° C heated, cooled on ice, and centrifuged again, followed by extraction in 0.5 M sulfuric acid in methanol with 2% dirthhoxypropane for 1 hour at 90 ° C, resulting in hydrolyzed oil and lipid compounds that give transmethylated lipids.
  • fatty acid methyl esters are extracted into petroleum ether and finally subjected to GC analysis using a capillary column (chrome pack, WCOT fused silica, CP-Wax-52 CB, 25 microm, 0.32 mm) at a temperature gradient between 170 ° C and 240 ° C for Subjected for 20 min and 5 min at 240 ° C.
  • the identity of the fatty acid methyl esters obtained must be defined using standards available from commercial sources (ie Sigma).
  • the Escherichia coli strain XLI Blue MRF 'kan (Stratagene) was used for subcloning the new desaturase pPDesaturasel from Physcomitrella patens.
  • the Saccharomyces cerevisiae strain INVSc 1 (Invitrogen Co.) for the functional expression of this gene.
  • E. coli was cultivated in Luria Bertini broth (LB, Duchefa, Haarlem, the Netherlands) at 37 ° C. If necessary, ampicillin (100 mg / liter) was added and 1.5% agar (w / v) was added for LB solid media. S.
  • cDNA clones from SeQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 were modified so that only the coding region can be amplified by means of a polymerase chain reaction with the aid of two oligonucleotides. Care was taken to ensure that a consensus sequence was adhered to before the start codon for efficient translation. Either the base sequence ATA or AAA was chosen and inserted into the sequence before the ATG (Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes, Cell 44, 283-292). Before this consensus triplet, a restriction interface was also introduced, which must be compatible with the interface of the target vector into which the fragment is to be cloned and with the aid of which gene expression in microorganisms or plants is to take place.
  • the PCR reaction was carried out using plasmid DNA as a template in a thermal cycler (Biometra) with the Pfu-DNA (Stratagene) polymerase and the following temperature program: 3 min at 96 ° C, followed by 30 cycles at 30 ° at 96 ° C C, 30 s at 55 ° C and 2 min at 72 ° C, 1 cycle with 10 min at 72 ° C and stop at 4 ° C.
  • the annealing temperature was varied depending on the oligonucleotides chosen. A synthesis time of about one minute can be assumed for each kilobase pair of DNA.
  • Other parameters that influence the PCR e.g. Mg ions, salt, DNA polymerase etc. are known to the person skilled in the art and can be varied as required.
  • the correct size of the amplified DNA fragment was confirmed by agarose TBE gel electrophoresis.
  • the amplified DNA was extracted from the gel with the QIAquick gel extraction kit (QIAGEN) and ligated into the Smal restriction site of the dephosphorylated vector pUC18 using the Sure Clone Ligation Kit (Pharmacia), whereby the pUC derivatives were obtained.
  • QIAquick gel extraction kit QIAGEN
  • ligated into the Smal restriction site of the dephosphorylated vector pUC18 using the Sure Clone Ligation Kit (Pharmacia), whereby the pUC derivatives were obtained.
  • a DNA mini-preparation Ros, MG, & McLachlan, A. (1986) A simplified screening procedure for large numbers of plasmid mini-preparation.
  • BioTechniques 4, 310-313) performed on ampicillin-resistant transformants, and positive clones identified by means of BamHI restriction analysis.
  • the sequence of the cloned PCR product was determined by resequencing using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Rothstadt) confirmed.
  • the total fatty acids were extracted from plant seeds and analyzed by gas chromatography.
  • the seeds were taken up with 1% sodium methoxide in methanol and incubated at RT for 20 min. Then with NaCl
  • Figure 2 Fatty acid profile of transgenic tobacco seeds.
  • the plants were transformed with a triple expression cassette, which expresses the delta-6, the delta-5 and the Physcomitrella patens PpPSEl (pARA2) under the control of the USP promoter.
  • 100 transgenic tobacco and linseed plants were produced, of which approximately 20% synthesized arachidonic acid in the seed.
  • the desired product can be obtained from plant material or fungi, algae, ciliates, animal cells or from the supernatant of the cultures described above by various methods known in the art. If the desired product is not secreted from the cells, the cells can start
  • Cells can be lysed using standard techniques such as mechanical force or ultrasound. Organs of plants can mechanically separated from other tissues or other organs. After homogenization, the cell debris is removed by centrifugation and the supernatant fraction containing the soluble proteins is saved for further purification of the desired compound. If the product is secreted from desired cells, the cells are removed from the culture by slow centrifugation and the supernatant fraction is saved for further purification.
  • the supernatant fraction from each purification process is subjected to chromatography with an appropriate resin, either with the desired molecule retained on the chromatography resin but not many contaminants in the sample, or the contaminants remaining on the resin but not leaving the sample. These chromatography steps can be repeated if necessary using the same or different chromatography resins.
  • the person skilled in the art is skilled in the selection of suitable chromatography resins and their most effective application for a particular molecule to be purified.
  • the purified product can be concentrated by filtration or ultrafiltration and kept at a temperature at which the stability of the product is maximum.
  • the identity and purity of the isolated compounds can be determined by standard techniques in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, in particular thin-layer chromatography and flame ionization detection (IATROSCAN, Iatron, Tokyo, Japan), NIRS, enzyme test or microbiological.
  • HPLC high-performance liquid chromatography
  • spectroscopic methods staining methods
  • thin-layer chromatography in particular thin-layer chromatography and flame ionization detection
  • IATROSCAN Iatron, Tokyo, Japan
  • NIRS enzyme test or microbiological.
  • An overview of these analysis methods can be found in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry (

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Abstract

L'invention concerne un procédé pour produire des esters d'acides gras qui contiennent des acides gras insaturés comportant au moins trois liaisons doubles, ainsi que des acides gras insaturés libres dans une concentration d'au moins 1 % en poids par rapport à la totalité des acides gras contenus dans la plante. Ledit procédé consiste à exprimer au moins une séquence d'acides nucléiques codant pour un polypeptide présentant l'activité de la ?-6-désaturase, et au moins une séquence d'acides nucléiques qui code pour un polypeptide présentant l'activité de la ?-6-élongase. De préférence, ces séquences d'acides nucléiques peuvent éventuellement être exprimées dans la plante transgénique avec une troisième séquence d'acides nucléiques qui codent pour un polypeptide présentant l'activité de la ?-5-désaturase. L'invention concerne en outre l'utilisation de séquences d'acides nucléiques définies qui codent pour des polypeptides présentant l'activité de la ?-6-désaturase, l'activité de la ?-6-élongase ou l'activité de la ?-5-désaturase, sélectionnées dans un groupe de séquences d'acides nucléiques, ou l'utilisation de constructions d'acides nucléiques contenant les séquences d'acides nucléiques mentionnées ci-dessus.
EP03747357A 2002-04-29 2003-04-25 Procede pour produire des acides gras polyinsatures dans des plantes Ceased EP1501932A2 (fr)

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DE10219203 2002-04-29
DE10219203A DE10219203A1 (de) 2002-04-29 2002-04-29 Verfahren zur Herstellung mehrfach ungesättigter Fettsäuren in Pflanzen
PCT/EP2003/004297 WO2003093482A2 (fr) 2002-04-29 2003-04-25 Procede pour produire des acides gras polyinsatures dans des plantes

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CA2485060A1 (fr) 2003-11-13
AU2003232512B2 (en) 2009-08-27
US20070028326A1 (en) 2007-02-01
CA2870809A1 (fr) 2003-11-13
US7893320B2 (en) 2011-02-22
WO2003093482A2 (fr) 2003-11-13
DE10219203A1 (de) 2003-11-13
CA2977570A1 (fr) 2003-11-13

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