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WO2004005442A1 - Procede de production d'acides gras polyinsatures conjugues comportant au moins deux liaisons doubles dans des plantes - Google Patents

Procede de production d'acides gras polyinsatures conjugues comportant au moins deux liaisons doubles dans des plantes Download PDF

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Publication number
WO2004005442A1
WO2004005442A1 PCT/EP2003/006833 EP0306833W WO2004005442A1 WO 2004005442 A1 WO2004005442 A1 WO 2004005442A1 EP 0306833 W EP0306833 W EP 0306833W WO 2004005442 A1 WO2004005442 A1 WO 2004005442A1
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seq
nucleic acid
acid sequence
fatty acids
organism
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PCT/EP2003/006833
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German (de)
English (en)
Inventor
Andreas Renz
Martijn Gipmans
Ivo Feussner
Claudia Krüger
Ellen Hornung
Andrea Porzel
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BASF Plant Science GmbH
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BASF Plant Science GmbH
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Priority claimed from DE2002129978 external-priority patent/DE10229978A1/de
Priority claimed from DE10308850A external-priority patent/DE10308850A1/de
Application filed by BASF Plant Science GmbH filed Critical BASF Plant Science GmbH
Priority to AU2003258496A priority Critical patent/AU2003258496A1/en
Publication of WO2004005442A1 publication Critical patent/WO2004005442A1/fr
Anticipated expiration legal-status Critical
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
    • C12P7/6431—Linoleic acids [18:2[n-6]]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings or cooking oils
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/14—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by isomerisation
    • 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
    • C12N9/90—Isomerases (5.)
    • 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

Definitions

  • the present invention relates to a process for the production of conjugated polyunsaturated fatty acids with at least two advantageously three double bonds in eukaryotes, especially in plants, and a process for the production of oils and / or triglycerides with an increased content of conjugated and / or unconjugated polyunsaturated fatty acids with at least two advantageously three double bonds.
  • the present invention also relates to a process for the production of conjugated linoleic acid in eurkaryotes, especially in plants, and to a process for the production of oils and / or triglycerides with an increased content of conjugated linoleic acid.
  • the invention further relates to nucleic acid sequences; Nucleic acid constructs, vectors and organisms containing the nucleic acid sequences, nucleic acid constructs and / or vectors.
  • the invention relates to fatty acid mixtures and triglycerides with an increased content of conjugated and / or unconjugated polyunsaturated fatty acids with at least two advantageously three double bonds, in particular conjugated linoleic acid, and their use.
  • Fatty acids and triglycerides have a large number of applications in the food industry, animal nutrition, cosmetics and in the pharmaceutical sector. Depending on whether it is free fatty acids or triglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a wide variety of applications, for example polyunsaturated fatty acids are added to baby food to increase the nutritional value.
  • the various fatty acids and triglycerides are mainly obtained from microorganisms such as Mortierella or Schizochytrium or from oil-producing plants such as soybean, rapeseed, sunflower and others, where they are generally obtained in the form of their triacylglycerides. But they are also advantageously obtained from animals such as fish.
  • the free fatty acids are advantageously produced by saponification.
  • oils with saturated or unsaturated fatty acids are preferred, e.g. In human nutrition, lipids with unsaturated fatty acids, especially polyunsaturated fatty acids, are preferred because they have a positive influence on the cholesterol level in the blood and thus on the possibility of heart disease. They are used in various dietary foods or medications.
  • conjugated unsaturated fatty acids are particularly valuable and sought-after unsaturated fatty acids.
  • Conjugated polyunsaturated fatty acids are rare in nature compared to other polyunsaturated fatty acids. In plants, these conjugated fatty acids only come in individual species such as in the Euphorbiacease such as Aleurites fordii or in Calendula officinalis. There is no natural vegetable source for conjugated linoleic acid (CLA).
  • conjugated fatty acids for example punicic acid or conjugated linoleic acid
  • CLA or other conjugated fatty acids can easily be obtained chemically via an alkaline isomerization from linoleic acid or linolenic acid or oils that contain linoleic acid or linolenic acid.
  • Two reactions are catalyzed by the alkaline isomerization at temperatures of, for example, 180 ° C., on the one hand the hydrolysis of the fatty acid ester bond in the triglycerides and on the other hand the isomerization of the double bonds (WO 99/32604).
  • WO 99/32604 isomerization of the double bonds
  • conjugated linoleic acid by the enzymatic activity of a desaturase is described by Qiu et al., (Plant Physiology, 125, 2001, 847-855).
  • a disadvantage of this activity is that the enzymatic action produces the undesired 8,10-isomer of the conjugated linoleic acid.
  • CLA is presumably a degradation product of linoleic acid on the way to stearidonic acid.
  • WO 99/29886 discloses bacteria for the enrichment of CLA in food and feed.
  • WO 99/32604 describes a linoleate isomerase from Lactobacillus reuteri.
  • the enzyme activity leads to the conversion of linoleic acid into six different isomers of CLA [(cis, trans) -9.11-CLA, (trans, cis) -10.12-CLA, (cis, cis) -9.11-CLA, (cis.cis) - 10,12-CLA, (trans, trans) -9,11-CLA and (trans, trans) -10,12-CLA].
  • the disadvantage of this isomerase is that the yield in the reaction is low and the purity of the CLA is unsatisfactory. This leads to an economically unattractive process.
  • Kepler et al. an isomerase from Butyrivibrio fibrisolvens (Kepler and Tovee, J. Biol. Chem., 1966, 241: 1350) and by Deng et al. one from Propionibacterium acnes (Deng et al., Ist International Conference on CLA, 2001, Alesund, Norway) was described.
  • WO 99/29886 discloses and claims an isomerase from Bifidobacterium.
  • the gene coding for CLA isomerase from Lactobacillus reuteri and Propionibacterium acnes was cloned and could be functionally expressed in prokaryotic microorganisms.
  • Conjugated linoleic acid is an intermediate product of the linoleic acid metabolism in ruminants.
  • CLA is a collective term for positional and structural isomers of linoleic acid, which are characterized by a conjugated double bond system on the carbon atom 8, 9, 10, 11, 12 or 13. Geometric isomers exist for each of these positional isomers, i.e. cis-cis, trans-cis, cis-trans, trans-trans.
  • C18: 2 cis-9, trans-11 and C18: 2 trans-10, cis-12 CLAs which are the most biologically active isomers, are of particular interest because they have been shown to be cancer-preventive in animal experiments, anti-arteriosclerotic act and reduce body fat in humans and animals.
  • CLAs are mainly sold as free fatty acids.
  • CLA CLA-containing animal fats.
  • Fats from ruminant animals such as cattle (Chin, Journal of Food Composition and Ahalysis, 5, 1992: 185-197) and sheep, as well as dairy products, have very high CLA concentrations.
  • Cattle have 2.9 to 8.9 mg / g fat.
  • vegetable oils, margarines and fats from non-ruminant animals have CLA concentrations of only 0.6 to 0.9 mg / g fat.
  • Linoleic acid (18: 2, c9, c12)
  • conjugated linoleic acid reduces body fat in humans and animals or increases the feed turnover per body weight in animals (WO 94/16690, WO 96/06605, WO 97/46230, WO 97/46118).
  • conjugated linoleic acid By administering conjugated linoleic acid, allergies (WO 97/32008), diabetes (WO 99/29317) or cancer can also be positive (Banni, Carcinogenesis, Vol. 20, 1999: 1019-1024, Thompson, Cancer, Res., Vol 57, 1997: 5067-5072).
  • Polyunsaturated fatty acids are also baby food for "increasing the nutritional value" and added as essential building blocks that ensure growth and brain development.
  • CLA has very far-reaching positive nutritional effects.
  • CLA naturally only occurs in significant quantities in ruminants and their products, such as milk, cheese, etc.
  • consumption of meat and dairy products has decreased to reduce the proportion of saturated fatty acids that are considered unhealthy. At the same time, this means a reduction in the intake of "healthy" CLA.
  • conjugated polyenoic acids are not found in significant amounts in animal lipids, whereas in some vegetable oils they mainly occur as cis-trienes or tetraenes.
  • Conjugated octadecatrienoic acids can also be isolated from marine algae such as the green algae Anadyomena stellata. These conjugated polyunsaturated fatty acids have great potential for use in foods, cosmetics and / or pharmaceuticals. For example, calendulic acid is already used as a component in many cosmetics.
  • the task was therefore to provide a corresponding method.
  • This object was achieved by the methods described below, specifically by a method for producing conjugated polyunsaturated fatty acids with at least two double bonds in transgenic eurkaryotic organisms, characterized in that it comprises the following method steps: a) introducing at least one nucleic acid sequence selected from the group consisting of from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 into an organism that produces unsaturated fatty acids; or b) introduction of at least one nucleic acid sequence which is derived by back-translation into a nucleic acid sequence from the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 on the basis of the degenerated genetic code, or c) introduction of at least one derivative the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, which code for polypeptides with the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4 or S
  • the conjugated polyunsaturated fatty acids produced in the process according to the invention advantageously contain at least three double bonds.
  • the fatty acids particularly advantageously contain two or three double bonds.
  • further non-conjugated double bonds can also be contained in the fatty acid molecule.
  • the fatty acids produced can contain up to 6 double bonds in the molecule.
  • Fatty acids produced or reacted in the process advantageously have 18 to 22 carbon atoms in the fatty acid chain. Table 1 shows the substrates and the reaction products. C18 fatty acids are advantageously implemented.
  • a preferred reaction product is the triene fatty acid 18: 3 (11E, 13E, 15Z), which is formed by reaction of the fatty acid 18: 3 (9Z, 12Z, 15Z).
  • the configuration of the double bonds was determined by NMR analysis unless otherwise described.
  • An advantageous embodiment of the aforementioned method is a method for the production of conjugated linoleic acid in transgenic eurkaryotic organisms, characterized in that it comprises the following method steps: a) introduction of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO : 3 or SEQ ID NO: 5 in an organism producing linoleic acid advantageously in an unsaturated fatty acid; or b) introduction of at least one nucleic acid sequence which is derived by back-translation into a nucleic acid sequence from the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 on the basis of the degenerated genetic code, or c) introduction of at least one derivative the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, which code for polypeptides with the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 and at least Have 90% identity
  • Amino acid coding is extended without the enzymatic effect of the polypeptides encoded by the nucleic acids being significantly changed, and f) expression of the nucleic acid in the organism produced according to (a), (b), (c), (d) or (e) , and if applicable g) if appropriate, cultivation and harvesting of the organism produced according to (a), (b), (c), (d) or (e).
  • nucleic acids are advantageously used in the method according to the invention after their so-called codon usage for expression in eukaryotes has preferably been changed for expression in yeasts. Corresponding modifications have been made and can be seen from the examples.
  • CLA conjugated linoleic acid
  • the oils contained in the organism or the triglycerides contained in the organism are isolated from the organism.
  • the oils and triglycerides can be obtained either from the culture in which they grow or from the field.
  • the further processing In the case of plants, for example, the plant seeds can preferably be pressed or extracted from the plant parts.
  • 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 cold or advantageously warm hexane or cyclohexane.
  • 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 turbid substances.
  • 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 and / or potassium 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.
  • the product is deodorized with water vapor, for example.
  • the fatty acids contained in the oil or in the glycerides can subsequently be released by acidic or alkaline hydrolysis using methods known to those skilled in the art.
  • glycolide means a glycerol esterified with one, two or three carboxylic acid residues (mono-, di- or triglyceride). “Glyceride” is also understood to mean a mixture of different glycerides. The glyceride or -
  • Glyceride mixture may contain other additives, e.g. contain free fatty acids, antioxidants, proteins, carbohydrates, vitamins and / or other substances.
  • a “glyceride” in the sense of the method according to the invention is further understood to mean derivatives derived from glycerol.
  • these also include glycerophospholipids and glyceroglycolipids.
  • Glycerophospholipids such as lecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol, phosphatidylserine and alkylacylglycerophospholipids may be mentioned here by way of example.
  • An embodiment of the invention is therefore oils, lipids or fatty acids or fractions thereof, which have been prepared by the process described above, particularly preferred are oils, lipids or fatty acid compositions which have an increased content of conjugated polyunsaturated fatty acids with at least two, advantageously three, double bonds include conjugated linoleic acid and originate from transgenic plants.
  • the lipids advantageously consist essentially of glycerides, which in turn consist essentially of triglycerides.
  • the above-mentioned% data relate to the total amount of the oil, lipids or fatty acid mixtures in the starting organisms and not to other constituents in the organisms.
  • conjugated polyunsaturated fatty acids with at least two advantageously three double bonds can be produced in eukaryotic organisms such as yeasts, fungi or advantageously plants. In principle, production in non-human animals is also possible.
  • conjugated polyunsaturated fatty acids produced in the process other unsaturated or saturated fatty acids can also be found in the oils. Lipids or fatty acid mixtures can be included.
  • the above weight percentages relate to the total amount of the oil, lipids or fatty acid mixtures and not to other constituents.
  • the method is characterized in that it comprises the following method steps: a) introducing at least one nucleic acid sequence selected from the group consisting of from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 into an organism producing unsaturated fatty acids; or b) introduction of at least one nucleic acid sequence which differs from the prokaryotic codon usage of the nucleic acid sequences SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 by at least 0.5% of a eukaryotic codon usage, or c) Introduction of at least one derivative of the nucleic acid sequences claimed under (b), which is extended by at least one codon triplet of the nucleic acid sequences which code for an amino acid, without the enzymatic action of the polypeptides coded by the nucleic acids having been
  • the conjugated polyunsaturated fatty acids with at least two advantageously three double bonds produced in the aforementioned process are advantageously conjugated linoleic acid.
  • a further embodiment according to the invention relates to a method for producing conjugated linoleic acid with or in transgenic eurkaryotic organisms, characterized in that it comprises the following method steps: a) introducing at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 in a linoleic acid producing organism; or b) introducing at least one nucleic acid sequence which differs from the prokaryotic codon usage of the nucleic acid sequences SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 by at least 0.5% of a eukaryotic codon usage, or c ) Introduction of at least one derivative of the nucleic acid sequences claimed under (b), which is extended by at least one codon triplet of the nucleic acid sequences which code for an amino acid, without the enzymatic action of the polypeptides encoded by the nucleic acids having been significantly changed, d) expression the nucleic
  • the nucleic acid sequences mentioned in each of the two aforementioned methods under point (b) have the least possible change in the codon usage, since these few changes already have the inventive effect, that is to say enable the expression of the nucleic acid sequences in the eurkaryotic organisms and there the fewer changes to the codon usage that have to be inserted into the sequences, the easier these sequences can be generated in vitro.
  • the changes should advantageously be in a range from 0.5 to 99%, advantageously between 0.5 to 90%, preferably between 1 to 50%, particularly preferably between 1 to 40%, very particularly preferably between 1 to 20% of the entire sequence.
  • These changes are preferably inserted at the amino terminus or 5 'end of the sequences.
  • a change at another point is also possible, for example at the C terminus or within the sequence or at several points within the sequence.
  • the mRNA-instructed process of translation into a protein is influenced by the codon usage of each special organism.
  • the function f (x) -> y, with x from the set of codons, the genetic code, y from the set of amino acids, is noted here, f is a function that maps each codon exactly to an amino acid. This mapping is not unambiguous in the sense of mathematics, that is, several codons code for the same amino acid.
  • the genetic code is called degenerate because some amino acids are encoded by several (synonymous) codons.
  • a specific tRNA does not exist for each codon, modified bases can be incorporated at position one of the anticodon and mismatches are permitted at the third position of the codon in the codon / anticodon complex (Crick's wobble rule).
  • the information content of the three base positions in the codon is not the same, the highest information content applies in position 2, followed by position 1 followed by position 3. Therefore a mutation of the third base in the codon often does not change the amino acid composition, a mutation in the first Base position often leads to the incorporation of an amino acid with similar properties, a mutation of the middle base often causes the incorporation of an amino acid with different properties.
  • codon frequencies vary within a species between different genes, depending on how strongly these genes are to be expressed. In certain genes, a subset of the codons occurs preferentially, depending on the species. As already mentioned, this distortion of the codon frequencies is positively correlated with the gene expression. Possible causes for this distortion of the codon frequencies are the different concentrations of the tRNAs, the maintenance of the maximum elongation rate, the cost of proofreading and different translation rates of the codons etc. This distortion of the codon frequencies is interpreted as a "strategy", the growth rates to optimize.
  • the sequence is advantageously changed in such a way that the codon usage after Start codon is changed in such a way that it is optimally adapted to the codon usage of the eurkaryotic non-human organism used for expression and is thus optimally expressed.
  • This change can advantageously be carried out with the aid of the following primers, the primers I advantageously being used for changing the bifidobacterium sequence (SEQ ID NO: 9) and the primers II advantageously being used for changing the lactobacillus sequence (SEQ ID NO: 11):
  • BBI-Kpnla GGT ACC ATG GGT TAG TAC TCC TCC GGT AAC TAC GAA GCT
  • LRI-EcoRla GAA TTC ACC ATG GGT TAC TAC TCC AAC GGT AAC TAC GAA GCT TTC GCT AGA CCA AAG AAG CCA GCT GGT G (5'Primer)
  • LRI-Xholb CTC GAG TTA TAG TAA GTG CTG TTG CTC CAT TAA TTC
  • SEQ ID NO: 7 propionibacterium sequence
  • SEQ ID NO: 9 and SEQ ID NO: 11 can be found in the sequences SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.
  • BBI Bactidobacterium sequence: atgggttactactcctccggtaactacgaagctttcgctagaccaaagaagccagctggtgttg (modified)
  • nucleic acid sequences described under point (c) advantageously have only one further codon triplet. This leads to an amino acid sequence of the isomerase being extended by one amino acid. In principle, it is also possible to change the sequence without extending the nucleic acid and amino acid sequence. In the present case, the codon GGT was introduced after the start codon ATG by the extension.
  • the ATGG base sequence which is thereby formed is considered to be a consensus sequence which has an advantageous effect on translation, that is to say an advantageous effect on protein synthesis.
  • the organism produced in this way can then advantageously be grown and harvested as described above after cultivation.
  • the oils, lipids and / or fatty acid mixtures contained in the organism can then be isolated and further treated as described.
  • linoleic acid and / or linolenic acid and / or other polyunsaturated C 8 -, C 20 - and / or C 22 fatty acids can advantageously be fed, so that the content of conjugated polyunsaturated is thereby Fatty acids with at least two advantageously three double bonds can advantageously be increased to CLA in the organism or in the culture medium of the organism.
  • the organism produced according to (a), (b) or (c) can then be grown and harvested.
  • SEQ ID NO: 1 and their derivatives are preferably used.
  • Another embodiment of the invention is the use of the aforementioned oil, lipid or fatty acid composition in animal feed, food, cosmetics or pharmaceuticals.
  • the present invention therefore relates to a method for producing a food, dietary supplement, animal feed or pharmaceutical preparation, comprising the addition of a conjugated and / or non-conjugated trans / cis octadecadienoic acid, octadecatrienoic acid, octadecatetraenoic acid, eicosatrienoic acid , Eicosatetraenoic acid, eicosapentaenoic acid, docosatrienoic acid, docosatetraenoic acid, docosapentaenoic acid and / or docosahexaenoic acid for preparation.
  • said trans / cis octadecatrienoic acid has a c9, t11, t10, c12 or 11E, 13E, 15Z configuration.
  • the present invention relates to a process for the production of individual biologically active isomers of the aforementioned fatty acids in advantageous plants.
  • Individual biologically active CLA isomers can preferably be produced.
  • the isomerase gene is advantageous either in unchanged form or in fusion with others
  • Gene fragments expressed so that the protein is either present in the cytosol, in the chloroplast or associated with membranes or is bound to oleosomes can advantageously take place in all parts of the plant, such as roots, stems, leaves, flowers, buds or seeds, advantageously in the seeds of these plants.
  • Two or more of the double bonds in the fatty acid molecules can be conjugated by the enzymatic reaction, two or three double bonds are advantageously conjugated to one another.
  • Linoleic acid is advantageously converted into the c9, t11 or t10, c12-CLA isomer.
  • other double bonds which are not in conjugation can also be present in the fatty acid molecule. In principle, however, all double bonds of a converted fatty acid molecule can also be in conjugation.
  • the conjugated and / or non-conjugated fatty acids thus produced can enter the membranes of the plant cells and / or in various lipids, for example, be incorporated into the triglycerides and stored in the seed oil.
  • the present invention therefore also relates to a food, dietary supplement, animal feed or pharmaceutical preparation containing conjugated polyunsaturated fatty acids with at least two advantageously three double bonds specifically containing conjugated linoleic acid.
  • Preparations according to the invention are outstandingly suitable as a food or feed additive, e.g. in diets or on animal fattening. They can be used in combination or alone with a reduced calorie intake to support a diet, e.g. to reduce body weight in humans, which also has an advantageous effect on eating habits.
  • the improved utilization of the increased food leads to a reduction in food consumption, which can be particularly advantageous in less developed regions with food shortages or in extreme situations (illnesses, competitive sports).
  • Preparations according to the invention can also be used economically and ecologically advantageously in animal nutrition, in particular for reducing the amount of feed.
  • the conjugated polyunsaturated fatty acids with at least two advantageously three double bonds especially the conjugated linoleic acid
  • various other saturated or unsaturated fatty acids e.g. Linoleic acid, linolenic acid, palmitic acid
  • the proportion of the different fatty acids in the oils, lipids or fatty acid mixtures can vary depending on the production process. Every fatty acid pattern is encompassed by the preparation according to the invention, in particular fatty acid patterns which arise in the production of oil from vegetable material. It is preferred that the oils, lipids or fatty acid mixtures contain as little scatter as possible of different fatty acids or that the number of different fatty acids is small.
  • the preparation mentioned contains further additives.
  • additives is understood to mean further additives which are advantageous for nutrition or health, e.g. "Nutrients” or "active ingredients”.
  • the preparation can contain one or more additives for animal or human nutrition or treatment and can be diluted or mixed therewith. Additives can be administered together with or separately from the feed, food, dietary supplement or pharmaceutical.
  • a food, dietary supplement, animal feed or pharmaceutical preparation contains no additives or no amounts of additives which can be considered harmful to animal or human nutrition.
  • “Nutrients” are understood to mean additives which are advantageous for the nutrition of humans or animals.
  • the accessory according to the invention preferably contains therefore vitamins, for example vitamins A, B1, B2, B6, B12, C, D3, and / or E, folic acid, nicotinic acid, pantothenic acid, taurine, carboxylic acids, eg tricarboxylic acids, citrate, isocitrate, trans / cis aconitate , and / or homo-citrate, enzymes, eg phytases, carotenoids, minerals, eg P, Ca, Mg, Mn and / or Fe, proteins, carbohydrates, fats, amino acids and / or trace elements Sn.
  • the preparation can also
  • Active ingredients are understood to mean those substances which support the use of the inventive preparation as a pharmaceutical or whose effect is used to treat diseases, in particular the treatment of cancer, diabetes, AIDS, allergies and cardiovascular diseases (see also below).
  • the preparation according to the invention can also comprise preservatives, antibiotics, antimicrobial additives, antioxidants, chelating agents, inert gases, physiologically acceptable salts, etc.
  • preservatives antibiotics, antimicrobial additives, antioxidants, chelating agents, inert gases, physiologically acceptable salts, etc.
  • the person skilled in the art knows how to add the additives suitable for the particular use as a pharmaceutical, animal feed, food supplement or food additive to the preparation or to determine them by simple tests known in the prior art.
  • antioxidants are also understood to mean antioxidants. Antioxidants are e.g. advantageous to protect the double bonds of the fatty acids from oxidation. However, the general health-promoting effects of antioxidants are also known. For example, ethoxyquin is used as an antioxidant in animal nutrition, otherwise gamma and alpha tocopherols, tocotrienol, rosemary extract, naturally occurring polyphenols, e.g. Flavonoids, isoflavones and carotenoids are used. In a further embodiment, the preparation mentioned contains further polyunsaturated fatty acids (PUFAs).
  • PUFAs polyunsaturated fatty acids
  • fatty acid is understood to mean an unbranched carboxylic acid with an even number of carbon atoms and 12 to 22 advantageously 16 to 22 carbon atoms.
  • unsaturated fatty acid means a fatty acid with at least two double bonds.
  • Conjugated unsaturated fatty acid is understood here to mean an unsaturated fatty acid with at least two advantageously three double bonds which are conjugated to one another.
  • the preparation contains omega-3 fatty acids, e.g.
  • Flavorings can also be added to the preparations mentioned.
  • the preparation can be combined with common food components.
  • common food components include vegetable but also animal products, in particular sugar, optionally in the form of syrups, fruit preparations, such as fruit juices, nectar, fruit pulps, purees or dried fruits; Cereal products and starches of the mentioned cereals; Dairy products such as milk protein, whey, yogurt, lecithin and milk sugar.
  • the preparation according to the invention is suitable for use in animal nutrition and contains e.g. Aggregates.
  • Aggregates are understood to mean substances which serve to improve the product properties, such as dust behavior, flow properties, water absorption and storage stability. Examples of such additives and / or mixtures thereof can be based on sugars e.g. Lactose or maltodextrin, based on cereal or legume products e.g. Corn spindle flour, wheat bran and soybean meal, based on mineral salts, etc. Calcium, magnesium, sodium, potassium salts or based on silicas such as silica gel.
  • 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, conjugated and / or non-conjugated esterified fatty acid (s), in particular conjugated linoleic acid but also other unsaturated fatty acids such as ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, ⁇ - Has linolenic acid, stearidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.
  • the proportion of unsaturated conjugated and / or non-conjugated polyunsaturated esterified fatty acids with at least two advantageously three double bonds is approximately 30% by weight, more preferred is a proportion of 50% by weight, even more preferred is a proportion of 60 % By weight, 70% by weight, 80% by weight 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 also contain various other saturated or unsaturated fatty acids, e.g. CLA, palmitic, stearic, linoleic, linolenic, oleic acid etc. included. In particular, depending on the starting organism, especially the starting plant, the proportion of the different fatty acids in the oil or fat can fluctuate.
  • the conjugated and / or non-conjugated polyunsaturated fatty acids with at least two advantageously three double bonds produced in the process can be found in the eurkaryotic transgenic organisms in the form of their sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid glycerol or in the form of the free fatty acid.
  • the conjugated and / or non-conjugated polyunsaturated fatty acids containing at least two advantageously three double bonds and / or further fatty acids can advantageously be obtained from the compounds prepared in the process according to the invention in the presence of an alcohol such as Release and isolate methanol or ethanol or via an enzymatic cleavage, for example by phase separation and subsequent acidification using, for example, H 2 SO 4 .
  • the fatty acids can also be released directly without the workup described above.
  • conjugated and / or non-conjugated polyunsaturated fatty acids produced in the process according to the invention are administered with at least two advantageously three double bonds in feed, they can be administered individually or in combination with other substances in the feed, the active compounds e.g. the above-mentioned fatty acids can be administered as pure substance or substance mixtures or liquid or solid extracts together with customary further feed ingredients or other active compounds such as vitamins, carotenoids etc.
  • Examples of common feed ingredients are: maize, barley, wheat, oats, rye, tritale, sorghum, rice and bran, semolina as well as flours of these cereals, soybeans, soy products such as soybean meal, rapeseed, rapeseed meal, cottonseed and extraction meal, sunflower, sunflower extract, sunflower extract , Oilseed expeller, field beans, peas, gluten, gelatin, tapioca, yeast, single cell protein, fish meal, salts, minerals, trace elements, vitamins, amino acids, oils / fats and the like.
  • Advantageous compositions are e.g. in Jeroch, H. et al. Nutrition of farm animals, UTB described.
  • the preparation according to the invention can be provided as a powder, granulate, pellet, extrudate with a coating ("coated") and / or as combinations thereof.
  • the preparation of the animal feed according to the invention is used e.g. to improve product properties such as dust behavior, flow properties, water absorption and storage stability.
  • Such preparations are widely known in the prior art. For example, in animal nutrition Blocks of a solid, cohesive, shape-retaining mass of several kilos are used.
  • Animal nutrition is composed in such a way that the corresponding nutrient requirements for the respective animal species are optimally covered.
  • vegetable feed components such as corn, wheat or barley meal, soybean bean meal, soy extraction meal, linseed meal, rapeseed meal, green meal or pea meal are chosen as raw protein sources. Soybean oil or other animal or vegetable fats are added to ensure an appropriate energy content of the feed. Since the vegetable protein sources only contain an insufficient amount of some essential amino acids, feed is often enriched with amino acids. This is mainly lysine,
  • Methionine and / or threonine are also added.
  • the type and amount of minerals and vitamins added depends on the animal species and is known to the person skilled in the art (see, for example, Jerosch et al., Nutrition of Agricultural Animals, Ulmer, UTB).
  • complete feed can be used that contains all nutrients in a ratio that meets the needs of each other. It is the only animal feed.
  • to one Grain feed from cereals can be given a supplementary feed.
  • the invention further relates to a preparation according to the invention which is a medicament.
  • the medicament produced using the preparation according to the invention can therefore also be used to treat cancer, cardiovascular diseases, e.g. Atherosclerosis (MacDonald, J.J. American College of Nutrition, (2000) 19, 111S-118S), diabetes (WO99 / 29317), allergies and disease-accompanying diets can be used. It is therefore advantageous e.g. the use of the preparation mentioned for accelerated body building, e.g. after an extended illness associated with weight loss, e.g. chemotherapy, and to support or accelerate the recovery process.
  • cardiovascular diseases e.g. Atherosclerosis (MacDonald, J.J. American College of Nutrition, (2000) 19, 111S-118S)
  • diabetes WO99 / 29317)
  • allergies and disease-accompanying diets can be used. It is therefore advantageous e.g. the use of the preparation mentioned for accelerated body building, e.g. after an extended illness associated with weight loss, e.g. chemotherapy, and to support or accelerate the recovery process.
  • the drug may also contain other active ingredients, e.g. the above or others.
  • the active ingredients can be used to treat cancer, cardiovascular diseases, e.g. Atherosclerosis, diabetes, allergies and the support of diets serve or improve the effect of the preparation according to the invention.
  • a drug for the treatment of diabetes can e.g. Contain insulin, sulfonylureas, sulfonamides, lipoic acid, ⁇ -glucosidase inhibitors, thiazolidinediones, metformin and / or acetylsalicylic acid.
  • Cancer diseases are e.g. by adding cytostatics such as vinca alkaloids, alkylating agents such as e.g.
  • immunosuppressants e.g. Cyclophophosphamide and azathioprine, glucocorticoids, such as prednisolone, or cyclosporin treated.
  • HIV infections or AIDS can e.g. can be treated by the administration of reverse transcriptase inhibitors and / or protease inhibitors. Allergies are e.g. treated by stabilizing the mast cells, e.g. by cromoglyxate, by blocking the histamine receptors, e.g.
  • H1 anithistamines or by functional antagonists of the allergy mediators, e.g. with alpha-sympathomimetics, adrenaline, beta2-sympathomimetics, theophylline, ipratropium or glucocorticoids.
  • Cardiovascular diseases are treated with the aid of anticoagulants, ACE inhibitors, cholesterol-lowering agents such as steatins and fibrates, niacin and cholestyramine.
  • the drug may comprise a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include physiologically acceptable salts, for example phosphate-buffered salt solutions. solutions, water, emulsions such as oil / water emulsions, sterile solutions etc. Sterile solutions can be, for example, aqueous or non-aqueous solutions.
  • Aqueous solutions are, for example, water, alcohol / water solutions, emulsions or suspensions and include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride etc.
  • non-aqueous solutions are propylene, glycol, polyethylene glycol, vegetable oils, organic esters, for example ethyl oleate.
  • the medicament can comprise one of the suitable additives mentioned above.
  • Drugs can be administered orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperotoneally) in the usual way. It can also be applied with steam or sprays through the nasopharynx.
  • the dosage depends on the age, condition and weight of the patient as well as the type of application.
  • the daily dose of active substance is between approximately 0.05 and 100 mg / kg body weight when administered orally and between approximately 0.01 and 20 mg / kg body weight when administered parenterally. 0.5 to 50 mg / kg are particularly preferred.
  • the new preparations can be used in the customary pharmaceutical application forms, solid or liquid, e.g. as tablets, film-coated tablets, capsules, powders, granules, dragees, suppositories, solutions, ointments, creams or sprays. These are manufactured in the usual way.
  • the active ingredients can be processed with the usual pharmaceutical auxiliaries such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, retardants, antioxidants and / or propellants (see H. Sucker et al .: Pharmaceuticals Technology, Thieme-Verlag, Stuttgart, 1991).
  • the administration forms obtained in this way contain active substances, including CLA or oil, normally in an amount of 0.1 to 90% by weight.
  • a medicament according to the invention can e.g. are produced by obtaining and formulating raw extracts from plants which contain conjugated polyunsaturated fatty acids such as CLA. Standard manufacturing processes for drugs are well known to those skilled in the art.
  • the amount of conjugated polyunsaturated fatty acids used with at least two advantageously three double bonds, such as trans / cis conjugated linoleic acid must be adjusted.
  • the amount of the aforementioned fatty acids used can e.g. Represent 0.01% or 0.1% of the amount of fat added to the diet. Also preferred are 0.5%, 1%, 2% or 3%, 5% or 10% of the conjugated and / or non-conjugated polyunsaturated fatty acids with at least two advantageously three double bonds. These quantities can also include other fatty acids.
  • all eukaryotic non-human organisms can be considered as the transgenic organism for the method according to the invention.
  • Microorganisms such as fungi or yeasts or plants such as algae, moss, se, monocotyledons or dicotyledons used, these are advantageously oil-producing organisms. If plants are used in the process, they are advantageously so-called oil fruit plants, fruit plants, useful plants or other plants.
  • nucleic acid used in the method according to the invention [the plural is intended to include the singular for the invention described here and vice versa]
  • nucleic acid according to the invention can in principle be applied to all methods known to the person skilled in the art.
  • the person skilled in the art can use the corresponding textbooks from Sambrook, J. et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, by FM Ausubel et al. (1994) Current protocols in molecular biology, John Wiley and Sons, by DM Glover et al., DNA Cloning Vol.
  • transformation The transfer of foreign genes into the genome of a plant is called transformation.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the use of a gene cannon, electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and the gene transfer mediated by Agrobacterium.
  • the methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published by S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol.
  • the construct to be expressed or the nucleic acid to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
  • Agrobacterium tumefaciens for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
  • the transformation of plants with Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877.
  • Agrobacteria transformed with an expression vector according to the invention can likewise be used in a known manner to transform plants such as test plants such as Arabidopsis and tobacco or crop plants, in particular oil-containing crop plants such as soybean, peanut, castor bean, sunflower, corn, cotton, flax, rapeseed, co coconut, oil palm, safflower (Carthamus tinctorius), canola, poppy seeds, mustard, hemp, olive, sesame, calendula, punica, evening primrose, mullein, thistle, wild roses, hazelnut, almond, macadamia, avocado, bay leaves, pumpkin, pistachios, borage, Walnut, wheat, rye, oats, triticale, rice, barley, cassava, pepper, tagetes, potato, tobacco, eggplant, tomato, pea, alfalfa, coffee, tea or cocoa bean are used, For example, by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable
  • the genetically modified plant cells can be regenerated using all methods known to the person skilled in the art. Appropriate methods can be found in the above-mentioned writings by S.D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer can be found.
  • all eukaryotic organisms which are able to synthesize fatty acids, especially unsaturated fatty acids or are suitable for the expression of recombinant genes are suitable as organisms or host organisms for the nucleic acid according to the invention, the nucleic acid construct or the vector.
  • Examples include plants such as Arabidopsis, Asteraceae such as Calendula or crops such as soybean, peanut, castor oil, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean, microorganisms such as fungi, for example the genus Mortierella, Saprolegnia or Pythium, yeasts such as the genus Saccharomyces, algae or protozoa such as dinoflagellates such as crypthecodinium.
  • plants such as Arabidopsis, Asteraceae such as Calendula or crops such as soybean, peanut, castor oil, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean
  • microorganisms such as fungi, for example the genus Mortierella, Saprolegnia or Pythium
  • yeasts such as
  • Organisms which can naturally synthesize oils in large amounts such as fungi such as Mortierella alpina, Pythium insidiosum or plants such as soybean, rapeseed, coconut, oil palm, safflower, castor bean, calendula, peanut, cocoa bean or sunflower or yeasts such as Saccharomyces cerevisiae, are particularly preferred soy, rapeseed, linseed, sunflower, calendula or Saccharomyces cerevisiae.
  • transgenic non-human animals for example C. elegans, are also suitable as host organisms.
  • 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 or another native gene contained in the nucleic acid construct (see below) is replaced by the inserted copy, whereby the production of the desired compound by the cell is modulated, or by using a gene in trans, so that the gene is functionally linked to a functional expression unit which contains at least one sequence ensuring the expression of a gene and at least one sequence ensuring the polyadenylation of a functionally transcribed gene.
  • the nucleic acids are advantageously introduced into the transgenic organisms such as plants via multi-expression cassettes or constructs for the multiparallel expression of genes. In the case of plants, seed-specific expression is advantageous.
  • the nucleic acids can be used for the genetic engineering modification of a broad spectrum of plants, so that this becomes a better or more efficient producer of one or more products derived from lipids, such as PUFAs specially conjugated and / or non-conjugated polyunsaturated fatty acids with at least two advantageously three double bonds.
  • PUFAs specially conjugated and / or non-conjugated polyunsaturated fatty acids with at least two advantageously three double bonds.
  • This improved production or efficiency of the production of a product derived from lipids, such as the aforementioned PUFAs can be brought about by the direct effect of the manipulation or an indirect effect of this manipulation.
  • 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.
  • genes for enzymes such as desaturase such as for a ⁇ -4-desaturase, a ⁇ -5-desaturase, a ⁇ -6-desaturase and / or a ⁇ -8-desaturase can advantageously be used or elongases such as a ⁇ -5 elongase, a ⁇ -6 elongase and / or ⁇ -9 elongase are introduced into the host organisms.
  • conjudia and / or conjutrien fatty acids for example from linoleic acid, linolenic acid and / or dihomo- ⁇ -uenolenic acid, is particularly advantageously possible from oil seeds such as soya, sunflower, safflower or advantageously linseed, which provide inexpensive and easy access to conjudia and / or allow Konutrien due to their high content of linoleic acid and / or linolenic acid.
  • rapeseed which has a high oleic acid content
  • a further enzymatic activity ( ⁇ -12-desaturase) must be introduced into the plant in addition to the isomerase.
  • Another aspect of the invention is an isolated nucleic acid sequence which codes for a polypeptide with isomerase activity, selected from the group: h) a nucleic acid sequence with the one in SEQ ID NO: 1, SEQ ID NO: 3 or
  • SEQ ID NO: 5 sequence or i) a nucleic acid sequence which is derived by back-translation into a nucleic acid sequence from the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 on the basis of the degenerated genetic code, or j) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, for polypeptides with that shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 Encode amino acid sequences and have at least 90% identity at the amino acid level, or k) a nucleic acid sequence which differs from the prokaryotic codon usage of the nucleic acid sequences SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 by at least 0.5%, advantageously at least 1, 2, 3 or 5%, preferably at least 6, 7, 8, 9 or 10%, particularly preferably at least 20, 30, 40 or 50%, very particularly preferably at least 60, 70, 80 or 90% of
  • nucleic acid sequences claimed under (d) which by at least one, advantageously at least 2, preferably at least 3, particularly preferably at least 4, very particularly preferably at least 5-
  • Codon triplets are advantageously extended at the 5 'end of the nucleic acid sequences which code for an amino acid, without the enzymatic action of the polypeptides encoded by the nucleic acids having been significantly changed.
  • derivatives or functional derivatives of the sequence mentioned in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 are to be understood, for example, as allelic variants which have at least 90% homology at the derived amino acid level, preferably at least 95% homology, particularly preferably at least 96, 97 or 98% homology, very particularly preferably 99; 99.5; 99.6; 99.7; 99.8; 99.9 or have 99.95% homology.
  • the homology was calculated over the entire amino acid or nucleic acid sequence range.
  • the PileUp program was used for sequence comparisons (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981)], which are contained in the GCG software package [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)] The sequence homology values given above in percent were determined with the program BestFit over the entire sequence range with the following settings: gap weight: 8, length weight: 2.
  • amino acid sequences derived from the nucleic acids mentioned can be found in sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
  • Allelic variants include, in particular, functional variants which can be deleted, inserted or substituted by Nucleotides from the in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 can be obtained, the enzymatic activity of the derived synthesized proteins and the eukaryotic codon usage being retained.
  • DNA sequences can be derived from the DNA sequences described in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or parts of these sequences, for example using conventional hybridization methods or the PCR technique from other organisms isolate as mentioned above. These DNA sequences hybridize to the sequences mentioned under standard conditions. For the hybridization, short oligonucleotides, for example of the conserved regions, which can be determined by comparison with other isomerase genes known to the person skilled in the art, are advantageously used. However, longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization.
  • RNA hybrids are approx. 10 ° C lower than that of DNA: RNA hybrids of equal length. Standard conditions are understood to mean conditions as disclosed in this description above.
  • the nucleic acid sequences mentioned under point (b) advantageously have the least possible change in the codon usage, since these few changes already have the inventive effect, that is to say the expression of the nucleic acid sequences in the eurkaryotic organisms and the fewer changes in the codon -usage must be inserted into the sequences, the easier it is to generate these sequences in vitro.
  • the changes should advantageously be in a range from 0.5 to 99%, advantageously between 0.5 to 90%, preferably between 1 to 50%, particularly preferably between 1 to 40%, very particularly preferably between 1 to 20% of the entire sequence lie. These changes are preferably inserted at the amino terminal end or 5 'end of the sequences. It is also a change is possible elsewhere, for example at the C-terminus or within the sequence or at several locations within the sequence.
  • the mRNA-instructed process of translation into a protein is influenced by the codon usage of each special organism.
  • the function f (x) -> y, with x from the set of codons, the genetic code, y from the set of amino acids, is noted here, f is a function that maps each codon exactly to one amino acid. This mapping is not unambiguous in the sense of mathematics, that is, several codons code for the same amino acid.
  • the genetic code is called degenerate because some amino acids are encoded by several (synonymous) codons.
  • a specific tRNA does not exist for each codon, modified bases can be incorporated at position one of the anticodon and mismatches are permitted at the third position of the codon in the codon / anticodon complex (Crick's wobble rule).
  • the information content of the three base positions in the codon is not the same, the highest information content applies in position 2, followed by position 1 followed by position 3. Therefore a mutation of the third base in the codon often does not change the amino acid composition, a mutation in the first base position often leads to the incorporation of an amino acid with similar properties, a mutation of the middle base often causes the incorporation of an amino acid with different properties.
  • codon frequencies vary within a species between different genes, depending on how strongly these genes are to be expressed. In certain genes, a subset of the codons occurs preferentially, depending on the species. As already mentioned, this distortion of the codon frequencies is positively correlated with the gene expression. Possible causes for this distortion of the codon frequencies are the different concentrations of the tRNAs, the maintenance of the maximum elongation rate, the costs for proofreading as well as different translation rates of the codons etc. This distortion of the codon frequencies is interpreted as a "strategy" which Optimize growth rates.
  • the sequence is advantageously changed in a transgenic (host) organism in such a way that the codon usage changes the start codon is changed in such a way that it is optimally adapted to the codon usage of the eurkaryotic non-human organism used for expression and is thereby optimally expressed.
  • This change can advantageously be carried out with the aid of the following primers, the primer I advantageously being used for changing the bifidobacterium sequence (SEQ ID NO: 9) and the primer II advantageously for changing the lactobacillus sequence (SEQ ID NO: 11) become:
  • BBI-Xholb CTC GAG CAG ATC ACA TGG TAT TCG CGT AGC AG (3'Primer)
  • LRI-EcoRla GAA TTC ACC ATG GGT TAC TAC TCC AAC GGT AAC TAC GAA GCT TTC GCT AGA CCA AAG AAG CCA GCT GGT G (5'Primer)
  • LRI-Xholb CTC GAG TTA TAG TAA GTG CTG TTG CTC CAT TAA TTC ( 3 'primer)
  • the primer used to change SEQ ID NO: 7 can be found in the examples.
  • the corresponding amino acid sequences of the sequences SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 can be found in the sequences SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.
  • This change in codon usage inserts an additional amino acid into the amino acid sequences (see SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6), but the other amino acid sequence is retained.
  • an interface is inserted into the nucleic acid (s) for a restriction enzyme.
  • BBI Bactedobacterium sequence
  • atgggttactactcctccggtaactacgaagctttcgctagaccaaagaagccagctggtgttg (modified) atg - tactacagcagcggcaactatgaggcgtttgcccgtccgaagaagccagccggcgtag (original)
  • LRI (Lactobacillus sequence): atgggttactactccaacggtaactacgaagctttcgctagaccaaagaagccagctggtg (modified) atg - tattattcaaacgggaattatgaagcctttgctcgaccaaagaagcctgctggcg .. (original).
  • nucleic acid sequences described under point (c) advantageously have only one further codon triplet. This leads to an amino acid sequence of the isomerase which is extended by one amino acid.
  • an extension for a restriction enzyme was inserted into the sequence.
  • this interface can also be inserted outside the coding sequence.
  • Another embodiment of the invention is an amino acid sequence encoded by the aforementioned nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or their aforementioned derivatives. These amino acid sequences can be found in SEQ ID NO: 2, SEQ ID NO: 4 or.
  • Derivatives in the process according to the invention or to the nucleic acid according to the invention include, for example, functional homologs of the enzymes encoded by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 or their enzymatic activity, i.e. To understand enzymes which catalyze the same enzymatic reactions as the enzyme encoded by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. These genes also enable advantageous production of unsaturated conjugated fatty acids.
  • nucleic acid sequences or fragments thereof used in the method according to the invention can advantageously be used to isolate further genomic sequences via homology screening.
  • the derivatives mentioned can be isolated, for example, from other organisms such as rumen rumen microorganisms, the intestines of other animals or from starter cultures of dairy products or silages.
  • Advantageous derivatives or homologs can be microorganisms such as fungi, yeasts, protozoa or bacteria such as gram-negative or gram-positive bacteria, preferably from gram-positive bacteria such as, for example, from the genera Propionibacterium, Lactococcus, Bifidobacterium or Lactobacillus, particularly preferably from the genera Isolate Lactobacillus or Bifidobacterium.
  • microorganisms such as fungi, yeasts, protozoa or bacteria such as gram-negative or gram-positive bacteria, preferably from gram-positive bacteria such as, for example, from the genera Propionibacterium, Lactococcus, Bifidobacterium or Lactobacillus, particularly preferably from the genera Isolate Lactobacillus or Bifidobacterium.
  • derivatives or functional derivatives of the sequence mentioned in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 mean, for example, allelic variants which have at least 90% homology at the derived amino acid level, preferably at least 95% homology, particularly preferably at least 96, 97 or 98% homology, very particularly preferably 99; 99.5; 99.6; 99.7; 99.8; 99.9 or 99.95% homology.
  • the homology was calculated over the entire amino acid or nucleic acid sequence range.
  • the PileUp program was used for sequence comparisons (J. Mol.
  • amino acid sequences derived from the nucleic acids mentioned can be found in sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
  • Allelic variants include in particular functional variants which are characterized by Deletion, insertion or S Substitution of nucleotides from the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 can be obtained, the enzymatic activity of the derived synthesized proteins and the eukaryotic codon usage being retained.
  • DNA sequences can be derived from the DNA sequences described in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or parts of these sequences, for example using conventional hybridization methods or the PCR technique from other organisms isolate as mentioned above. These DNA sequences hybridize to the sequences mentioned under standard conditions. For the hybridization, short oligonucleotides, for example of the conserved regions, which can be determined by comparison with other isomerase genes known to the person skilled in the art, are advantageously used. However, longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization.
  • RNA hybrids are approx. 10 ° C lower than that of DNA: RNA hybrids of equal length.
  • DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 to 45 ° C., preferably between approximately 30 to 45 ° C.
  • DNA: RNA hybrids the hybridization Conditions advantageous at 0.1 x SSC and temperatures between about 30 to 55 ° C, preferably between about 45 to 55 ° C.
  • These specified temperatures for the hybridization are, for example, calculated melting temperature values for a nucleic acid with a length of approx. 100 nucleotides and a G + C content of 50% in the absence of formamide.
  • the experimental conditions for DNA hybridization are described in relevant textbooks of genetics such as Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be made according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G + C content. The person skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al.
  • derivatives include homologs of the sequences SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, for example advantageously prokaryotic homologs, shortened sequences, single-stranded DNA of the coding and non-coding DNA sequence or RNA of the coding and non-coding DNA Understand sequence.
  • homologs of the sequences SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 are to be understood as derivatives such as promoter variants. These variants can be changed by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), but without the functionality or effectiveness of the promoters being impaired.
  • the effectiveness of the promoters can be increased by changing their sequence, or completely replaced by more effective promoters, including organisms of other species.
  • Derivatives are also advantageously to be understood as variants whose nucleotide sequence in the range from -1 to -2000 before the start codon has been changed such that the gene expression and / or the protein expression is changed, preferably increased. Derivatives are also to be understood as variants that have been changed at the 3 'end.
  • the isomerase gene can advantageously be combined in the process according to the invention with other genes of fatty acid biosynthesis.
  • the combination with a ⁇ -12 desaturase is particularly advantageous.
  • Derivatives of the amino acid sequences according to the invention are to be understood as proteins which have an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 or a substitution, inversion, insertion or deletion of one or more amino acid residues Available sequence contain, wherein the enzymatic activity of the proteins shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 is retained or is not changed significantly. These not significantly modified proteins are therefore still enzymatically active, that is, functional nell.
  • Not significantly changed means all enzymes which still have at least 10%, preferably 20%, particularly preferably 30% of the enzymatic activity of the starting enzyme. Or their enzymatic activity is increased by at least 10%, preferably by 50%, particularly preferably by at least 100%, compared to the starting enzyme or the starting amino acid sequence.
  • certain amino acids can be replaced by those with similar physicochemical properties (space filling, basicity, hydrophobicity, etc.).
  • arginine residues are exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues for glutamic acid residues.
  • one or more amino acids can also be interchanged, added or removed in their order, or several of these measures can be combined with one another.
  • nucleic acid constructs or fragments used in the method according to the invention are to be understood as sequences which contain at least one of the nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or which are the result of the genetic code by back-translation can be derived from SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 or contain derivatives of the sequences SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 and which advantageously with one or more regulation signals were functionally linked to increase gene expression.
  • 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 before 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 increased.
  • These modified promoters can also be placed in front of the natural gene to increase activity.
  • 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.
  • the isomerase gene or the isomerase genes can be contained in one or more copies in the gene construct.
  • Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, lacl q " 'T7, T5- , T3-, gal-, trc-, ara-, SP6-, ⁇ -P R - or in the ⁇ -P L promoter, which are advantageously used in gram-negative bacteria and advantageously for the multiplication of nucleic acid in these organisms can be used.
  • promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, lacl q " 'T7, T5- , T3-, gal-, trc-, ara-, SP6-, ⁇ -P R - or in the ⁇ -P L promoter, which are advantageously used in gram-negative bacteria and advantageously for
  • Partial regulatory sequences are, for example, in the gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MF ⁇ , AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters such as CaMV / 35S [Franck et al., 1980, Cell 21: 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, Iib4, STLS1, B33, nos or in the ubiquitin promoter.
  • plant promoters are, for example, one that can be induced by benzenesulfonamide (EP 388186), one that can be induced by tetracycline (Gatz et al., (1992) Plant J. 2, 397-404), one that can be induced by abscisic acid (EP335528) or one by ethanol or cyclohexanone inducible (WO9321334) promoter.
  • Further plant promoters are, for example, the promoter of the cytosolic FBPase from potato, the ST-LSI promoter from potato (Stockhaus et al., EMBO J.
  • the promoter of the phosphoribosyl pyrophosphate amidotransferase from Glycine max can advantageously be used.
  • Plant promoters which ensure expression in tissues or parts of plants in which fat biosynthesis or its precursors take place are particularly advantageous. Promoters which ensure seed-specific expression, such as the usp promoter, the LEB4 promoter, the phaseolin promoter or the napin promoter, should be mentioned in particular. In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the method according to the invention. In addition, synthetic promoters can also be used advantageously.
  • SEQ ID NO: 1 SEQ ID NO: 3 and / or SEQ ID NO: 5
  • These genes can be under separate regulation or under the same regulatory region as the isomerase genes used in the method according to the invention. These genes are, for example, further biosynthesis genes advantageously in fatty acid biosynthesis.
  • the isomerase genes are advantageously used in the same nucleic acid construct as the other genes.
  • nucleic acid constructs according to the invention are advantageously inserted into a vector such as, for example, a plasmid, a phage or other DNA, which optimally expresses the genes in the host allows.
  • a vector such as, for example, a plasmid, a phage or other DNA, which optimally expresses the genes in the host allows.
  • Suitable plasmids are, for example, in E.
  • plasmids mentioned represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985,
  • vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA. These vectors can be replicated autonomously in the host organism or can be replicated chromosomally. Chromosomal replication is preferred.
  • vectors e.g. recombinant expression vectors which contain at least one nucleic acid molecule used in the method according to the invention, and host cells into which these vectors have been introduced, in particular eukaryotic microorganisms, plant cells, plant tissues, plant organs or whole plants.
  • a host cell can store the conjugated and non-conjugated polyunsaturated fatty acids produced with at least two advantageously three double bonds; the cells are harvested to isolate the desired compound.
  • the compound (oils, lipids, triacylglycerides, fatty acids) can then be isolated from the medium or the host cell which contain or store the abovementioned polyunsaturated fatty acids, most preferably cells from storage tissues, such as in plants from cells of seed shells, tubers , Epidermal and sperm cells.
  • Yet another aspect of the invention relates to a genetically modified plant, preferably an oil fruit plant, as mentioned above, particularly preferably a linseed, sunflower, cotton or safflower body, into which an isomerase gene has been introduced.
  • the genome of linseed, rapeseed, soybean, sunflower, cotton or safflower has been changed as a transgene by introducing an isomerase gene which codes for a wild-type or mutated isomerase sequence.
  • flax, sunflower, soybean or safflower is also used to produce a desired compound, such as lipids and fatty acids, with triene and / or dienes being particularly preferred and CLA being particularly preferred.
  • the vector advantageously contains at least one copy of the nucleic acid sequence used in the method according to the invention and / or the nucleic acid construct according to the invention.
  • nucleic acid sequences or homologous genes can be incorporated, for example, into a nucleic acid fragment or into a vector which preferably contains the regulatory gene sequences assigned to the respective genes or promoter activity acting in an analogous manner.
  • those regulatory sequences are used which increase gene expression.
  • nucleic acid fragments for the expression of the further genes contained additionally contain 3 'and / or 5' terminaie regulatory sequences for increasing the expression, which are selected depending on the selected host organism and gene or genes for optimal expression.
  • transgenic organism these regulatory sequences are intended to enable targeted expression of the genes and protein expression.
  • this can mean, for example, "that the gene is only expressed and / or overexpressed after induction, or that it is immediately expressed and / or overexpressed.
  • 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.
  • This linear DNA can consist of a linearized plasmid or only of the nucleic acid fragment as a vector or the nucleic acid sequence used in the method according to the invention.
  • the nucleic acid sequence (the singular should also include the plural for the present description) is advantageously cloned together with at least one reporter gene into a nucleic acid construct which is introduced into the genome.
  • This reporter gene should enable easy detection via a growth, fluorescence, chemo, bioluminescence or resistance assay or via a photometric measurement.
  • These genes enable the transcription activity and thus the expression of the genes to be measured and quantified easily. This enables genome sites to be identified that show different productivity. Furthermore, successful transformations can advantageously be identified with these reporter genes.
  • a nucleic acid construct for plants preferably contains regulatory sequences which can control the gene expression in plant cells and are operably linked so that each sequence can fulfill its function, such as termination of the transcription, for example polyadenylation signals.
  • Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens-t-DNA, such as gene 3 of the Ti plasmid pTiACH ⁇ (Gielen et al., EMBO J. 3 (1984) 835ff.) Known as octopine synthase or functional equivalents thereof, but all other terminators that 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.
  • telomeres are targeting sequences which are necessary for the control of the gene product into its corresponding cell compartment (see an overview in Kermode, Grit. Rev. Plant Sei. 15, 4 (1996) 285 -423 and references cited therein), for example in the vacuole, the cell nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
  • plastids such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
  • Plant gene expression can also be facilitated as described above using a chemically inducible promoter (see an overview in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Bio!., 48: 89-108).
  • 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 one 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 PRP1 gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), the heat-inducible hsp80-
  • 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 promoter from Arabidopsis ( WO 98/45461), the phaeolin promoter from Phaseolus vulgaris (US 5,504,200), the Bce4 promoter from Brassica (WO
  • legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9) and promoters which promote seed-specific expression in monocot plants, such as maize, Bring barley, wheat, rye, rice, etc.
  • Suitable noteworthy promoters are the barley lpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230) or those described in WO 99/16890
  • 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
  • nucleic acid sequences used in the methods according to the invention can also be introduced into an organism alone. If, in addition to the nucleic acid sequence according to the invention, further genes are to be introduced into the organism, they can all be introduced into the organism together with a reporter gene in a single vector or each individual gene with a reporter gene in one vector, the different vectors being carried out simultaneously or successively can be introduced.
  • the host organism advantageously contains at least one copy of the nucleic acid sequence and / or the nucleic acid construct.
  • nucleic acids according to the invention can in principle be carried out by all methods known to the person skilled in the art.
  • transformation The transfer of foreign genes into the genome of a plant is called transformation.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the use of a gene cannon, electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and the gene transfer mediated by Agrobacterium. The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene
  • the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
  • Agrobacterium tumefaciens for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
  • the transformation of plants with Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877.
  • Agrobacteria transformed with an expression vector according to the invention can also be used in a known manner to transform plants such as test plants such as Arabidopsis or crop plants, in particular oil-containing crop plants such as soybean, peanut, castor bean, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, Dyer safflower (Carthamus tinctorius) or cocoa bean can be used, for example by bathing wounded leaves, leaf pieces, hypocotyl pieces or roots in an agrobacterial solution and then cultivating them in suitable media. Using the Invitrogen GATE-WAY system for cloning, no more suitable interfaces in the vector are necessary.
  • the genetically modified plant cells can be regenerated using all methods known to the person skilled in the art. Appropriate methods can be found in the above-mentioned writings by S.D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer can be found.
  • each of the nucleic acids used in the method which code for the isomerase and / or the advantageous ⁇ -12-desaturase, should be expressed under the control of its own, preferably a different promoter , since repeating sequence motifs can lead to instability of the T-DNA or to recombination events earlier.
  • 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 then optionally a terminator behind the polylinker.
  • This sequence is repeated several times, preferably three, four or five times, so that up to five genes are brought together in one construct and can thus be introduced into the transgenic plant for expression.
  • the sequence is advantageously repeated up to two or three times.
  • the 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 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, i.e.
  • a nucleic acid sequence can be inserted at the first or last position in the cassette without the expression being significantly influenced thereby.
  • Different promoters such as the USP, LegB4, DC3 or the ubiquitin promoter and different terminators can advantageously be used in the expression cassette.
  • the transcription of the introduced genes should advantageously be terminated by suitable terminators at the 3 'end of the introduced biosynthesis genes (behind the stop codon).
  • suitable terminators at the 3 'end of the introduced biosynthesis genes can be used e.g. the OCS1 terminator.
  • different terminator sequences should be used for each gene.
  • Suitable organisms or host organisms for the nucleic acids, nucleic acid constructs or vectors used in the method according to the invention are, in principle, all eukaryotic organisms, as have already been mentioned, which are able to synthesize fatty acids, especially unsaturated fatty acids or are suitable for the expression of recombinant genes.
  • Examples include plants such as Arabidopsis, branches raceae such as Calendula, Punicaceae such as Punica granatum or crops such as soybean, peanut, castor oil, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean, microorganisms such as fungi, for example the genus Mortierella, Saprolegnia or Pythium Yeasts such as the genus Saccharomyces, algae or protozoa such as dinoflagellates such as Crypthecodinium.
  • plants such as Arabidopsis, branches raceae such as Calendula, Punicaceae such as Punica granatum or crops such as soybean, peanut, castor oil, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean
  • microorganisms such as fungi,
  • Organisms which can naturally synthesize oils in large quantities such as fungi such as Mortierella alpina, Pythium insidiosum, or plants such as soybean, rapeseed, coconut, oil palm, safflower, castor bean, flax, calendula, peanut, cocoa bean or sunflower or yeasts such as Saccharomyces cerevisiae are preferred. Soybeans, rapeseed, sunflower, safflower, flax, calendula or Saccharomyces cerevisiae are particularly preferred.
  • transgenic animals for example C. elegans, can also be used as host organisms.
  • Transgenic in the sense of the invention means that the nucleic acids or nucleic acid constructs used in the method are not in their natural position in the genome of an organism, and the nucleic acids can be expressed homologously or heterologously.
  • transgene also means that the nucleic acids or expression cassettes 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, the natural sequences, have been changed.
  • Transgenic is preferably understood to mean the expression of the nucleic acids according to the invention at a non-natural location in the genome, i.e. homologous or preferably heterologous expression of the nucleic acids is present.
  • transgenic organisms are the above-mentioned transgenic plants, preferably oil crop plants which contain large amounts of lipid compounds, such as oilseed rape, evening primrose, hemp, diesel, peanut, canola, flax, soybean, safflower, sunflower, borage, or plants such as
  • 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 used in the process according to the invention or the nucleic acid construct for the production of transgenic eukaryotic organisms or transgenic plants is therefore also part of the subject matter of the invention.
  • a nucleic acid sequence selected from the group is advantageously used for the production of transgenic eukaryotic organisms or transgenic plants: a) nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or b) nucleic acid sequence which is derived by back-translation into a nucleic acid sequence from the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 on the basis of the degenerate genetic code, or c) derivatives of those in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, which encode polypeptides with the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 and have at least 90% identity at the amino acid level , without the enzymatic action of the polypeptides being significantly changed, or d) a nucleic acid sequence which differs from the prokaryotic codon usage of the nucleic acid sequences SEQ
  • Another object of the invention is a process for the production of fatty acid mixtures with an increased unsaturated fatty acid content, characterized in that at least one nucleic acid sequence described above or at least one nucleic acid construct or vector is brought into a preferably oil-producing organism, attracts this organism and isolates the oil and / or triglyceride contained in the organism and releases the fatty acids contained in the oil and / or triglyceride.
  • a process for the preparation of oils and / or triglycerides with an increased content of conjugated and / or non-conjugated polyunsaturated fatty acids with at least two advantageously three double bonds characterized in that at least one nucleic acid sequence described above or at least one nucleic acid construct or brings a vector into an oil producing organism, attracts this organism and isolates the oil contained in the organism is one of the objects of the invention.
  • Both methods advantageously enable the synthesis of fatty acid mixtures or triglycerides with an increased content of conjugated and / or non-conjugated polyunsaturated fatty acids with at least two advantageously three double bonds. Since this does not occur naturally in plants, they are from the Plants resulting oils, lipids and / or fatty acid mixtures new. The same applies to other eukaryotic organisms.
  • organisms for the processes mentioned are plants such as arabidopsis, soybean, peanut, castor bean, sunflower, maize, cotton, flax, rapeseed, coconut, oil palm, dyed savannah (Carthamus tinctorius) or cocoa bean, microorganisms such as fungi Mortierella, Saprolegnia or Pythium Yeasts such as the genus Saccharomyces, algae or protozoa such as dinoflagellates such as Crypthecodinium.
  • plants such as arabidopsis, soybean, peanut, castor bean, sunflower, maize, cotton, flax, rapeseed, coconut, oil palm, dyed savannah (Carthamus tinctorius) or cocoa bean
  • microorganisms such as fungi Mortierella, Saprolegnia or Pythium Yeasts such as the genus Saccharomyces, algae or protozoa such as dinoflagellates such as Crypthecodin
  • fungi such as Mortierella alpina, Pythium insidiosum or plants such as soybean, rapeseed, coconut, oil palm, savory, castor bean, calendula, punica, peanut, cocoa bean or sunflower or yeasts such as Saccharomyces cerevisiae, soya, rape, sunflower, calendula, punica or Saccharomyces cerevisiae are particularly preferred.
  • Microorganisms are usually in a liquid medium that contains a carbon source mostly in the form of sugars, a nitrogen source mostly in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and possibly vitamins, at temperatures between 0 and 100 ° C, preferably between 10 to 60 ° C attracted with oxygen.
  • the pH of the nutrient liquid can be kept at a fixed value, i.e. regulated or not during cultivation.
  • the cultivation can take place batchwise, semi batchwise or continuously. Nutrients can be introduced at the beginning of the fermentation or fed in semi-continuously or continuously. After transformation, plants are first regenerated as described above and then grown or cultivated as usual.
  • the lipids are usually obtained from the organisms.
  • the organisms can first be digested after harvesting or used directly.
  • the lipids are advantageously extracted with suitable solvents such as apolar solvents such as hexane or ethanol, isopropanol or mixtures such as hexane / isopropanol, phenol / chloroform / isoamyl alcohol at temperatures between 0 to 80 ° C., preferably between 20 to 50 ° C.
  • suitable solvents such as apolar solvents such as hexane or ethanol, isopropanol or mixtures such as hexane / isopropanol, phenol / chloroform / isoamyl alcohol at temperatures between 0 to 80 ° C., preferably between 20 to 50 ° C.
  • the biomass is usually extracted with an excess of solvent, for example an excess of solvent to biomass of 1: 4.
  • the solvent is then removed, for example by distillation.
  • the extraction can also
  • the crude oil obtained in this way can then be further purified, for example by removing turbidity by adding polar solvents such as acetone or chloroform and then filtering or centrifuging. Further cleaning via columns is also possible.
  • polar solvents such as acetone or chloroform
  • Further cleaning via columns is also possible.
  • free fatty acids from the triglycerides they are usually saponified.
  • the cDNA of the CLA isomerase from Propionibacterium acnes which converts linoleic acid into the t10, c12-CLA isomer, was known from WO 01/00846 and was - using the following primers - via PCR with Pfu polymerase (Röche - Diagnostics) amplified and cloned into the pSE380 vector (Invitrogen):
  • Primer A 5 '- ATC TGC AGA TGT CCA TCT CGA AGG AT - 3 "forward primer (with Pstl interface)
  • Primer B 5 '- GCG AGC TCA CAC GAA GAA CCG CGT C - 3' Reverse Primer (with Sacl interface)
  • the PCR reaction mixture was composed as follows: dNTP mix (IO mM) 1.0 ⁇ l
  • the amplified DNA fragment was cut from a preparative agarose gel and eluted with the QiaQuick Gel Elution Kit (Qiagen). Both the PCR fragment and the pSE380 were cut with Pstl and Sacl and the open vector was additionally dephosphorylated with an alkaline phosphatase. Both DNA fragments were ligated to one another with the T4 ligase.
  • the pSE-PAI construct produced in this way was amplified in E. coli and sequenced as a double-stranded control.
  • Example 2 Expression of a codon-optimized CLA isomerase from Propionibacterium acnes in yeast
  • the N-terminus of the isomerase was changed so that the first 60 base pairs of the coding sequence correspond to the optimal codon composition of the yeast.
  • the modified cDNA was then cloned into a yeast expression vector and the functionality of the modified isomerase in yeast was checked.
  • a forward primer with a codon-optimized sequence was used to generate a codon-optimized Propionibacterium isomerase by means of PCR.
  • the PCR was carried out using the Expand High Fidelity PCR system (Röche Diagnostics) using the following primers:
  • Primer C 5 ' - GAA TTC CAC CAT GGG TTC CAT TTC CAA GGA CTC CAG AAT TGC TAT TAT TGG TGC TGG CCC GGC CGG GCT GGC - 3 ' Forward Primer (with EcoRI interface)
  • Primer D 5 '- GCG GCC GCT CAC ACG AAG AAC CGC GTC ACC AG - 3 '
  • SEQ ID NO: 1 shows the optimized, modified overall sequence of the Propionibacterium acnes nucleic acid sequence.
  • SEQ ID NO: 7 the original sequence can be found.
  • the PCR reaction mixture was composed as follows: dNTP mix (10 mM) 1.0 ⁇ l Forward primer (10 ⁇ M) 4.0 ⁇ l reverse primer (10 ⁇ M) 4.0 ⁇ l template (1/50 diluted pSE-PAl plasmid DNA) 1.0 ⁇ l 10-fold buffer 5.0 ⁇ l polymerase (3.5 U / ⁇ l) 0.5 ⁇ l water 34.5 ⁇ l total volume 50.0 ⁇ l
  • the amplified DNA fragment was cut from a preparative agarose gel, eluted with GFXTM PCR DNA and Gel Band Purification Kit (Amersham Bioscience) and cloned into the pGEM-T vector (Promega) according to the manufacturer's instructions.
  • the codon-optimized isomerase cDNA was ligated into the likewise cut vector pYES2 via the EcoRI and NotI interfaces.
  • the pY2-coPAI construct thus produced was amplified in E. coli and for yeast expression in the yeast strain INVSd (Invitrogen) with the LiAc method - (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1995).
  • the main culture was grown in 25 ml SD medium with 2% (w / v) galactose, amino acid / base solution without uracil for selection, 0.02% linoleic acid (2% stock solution in 5% Tergitol NP40), 10% Tergitol NP40 for 72 hours at 30 ° C.
  • the main culture was harvested by centrifugation (10 min, 4000 rpm, 4 ° C.).
  • the cell pellet was frozen at -20 ° C and then lyophilized for at least 18 hours.
  • the lyophilized yeast pellets were extracted in 1.35 ml of methanol / toluene (2: 1) and 0.5 ml of sodium methoxide solution.
  • the cell material was ground up as finely as possible with a glass rod and then incubated for 1 hour at room temperature with shaking. Then 1.8 ml of 1 M NaCl solution and 3 ml of n-heptane were added and incubated for 10 min at RT with shaking. After phase separation by centrifugation (10 min, 4000 rpm, 4 ° C) the heptane supernatant was transferred to a test tube and evaporated under nitrogen.
  • the P. acnes isomerase takes free linoleic acid as the substrate.
  • the free fatty acids were extracted using the HIP method, methylated with methanol and then detected by GC or GC / MS.
  • the yeast pellet was placed in 15 ml HIP buffer (hexane / isopropanol 3: 2 (v / v) with 0.0025% BHT (2,6-di-tert-butyl-4-methylphenol) ) recorded and homogenized as finely as possible with a glass rod. After addition of 75 ⁇ l of 1 M HCl, the mixture was incubated for 5 min at RT with shaking.
  • HIP buffer hexane / isopropanol 3: 2 (v / v) with 0.0025% BHT (2,6-di-tert-butyl-4-methylphenol)
  • the extracted sample in 400 ⁇ l of methanol was mixed with 10 ⁇ l of EDAC solution (N- (3-dimethylaminoproyl) -N " -ethylcarbodiimide hydrochloride, 1 mg in 10 ml of methanol) and shaken for 2 h at RT
  • EDAC solution N- (3-dimethylaminoproyl) -N " -ethylcarbodiimide hydrochloride, 1 mg in 10 ml of methanol
  • the mixture was thoroughly mixed by vortexing and centrifuged (5 min, 12000 rpm, RT) the upper phase was transferred to a new microcentrifuge tube.
  • the mixture was extracted again with 1 ml of hexane, both hexane phases combined and evaporated under nitrogen. The residue was taken up in 40 ⁇ l acetonitrile.
  • GC analysis N- (3-dimethylaminoproyl) -N " -ethylcar
  • fatty acid methyl esters For GC analysis of the fatty acid methyl esters (FAME), 7 ⁇ l of the sample (in acetonitrile) was transferred to a sample tube and 1 ⁇ l was injected. The GC analysis was carried out using an HP-DB23 (cross-linked PEG; 30 m ⁇ 0.32 mm ⁇ 0.5 ⁇ m coating thickness) at a flow rate of 1.5 ml / min. Helium served as the carrier gas. The injection temperature was 220 ° C. The following temperature gradient was applied: 1 min 150 ° C, 150 ° C to 200 ° C (with 15 ° C / min), 200 ° C to 250 ° C (with 2 ° C / min), 5 min 250 ° C.
  • HP-DB23 cross-linked PEG; 30 m ⁇ 0.32 mm ⁇ 0.5 ⁇ m coating thickness
  • the FAME was detected using a flame ionization detector (FID) at 275 ° C.
  • FID flame ionization detector
  • the retention times for conjugated linoleic acid 10c, 12-CLA were 14.3 min.
  • the retention times were 12.68 min for linoleic acid and 11.86 min for oleic acid.
  • 1A shows the gas chromatogram of the lipid extracts from yeast cells which have been transformed with the empty vector pYES2. The cells were grown for 72 hours at 30 ° C with 0.02% linoleic acid. The gas chromatogram shows no FAME with a retention time of t10, c12-CLA.
  • 1B shows the gas chromatogram of the lipid extracts from yeast cells transformed with pY2-coPAI.
  • the cells were again grown for 72 hours at 30 ° C with 0.02% linoleic acid.
  • the gas chromatogram has a clear peak with a retention time of 14.37 min, which does not occur in the control batch (see FIG. 1A) and has the same retention time as t10, c12-CLA (see FIG. 1C).
  • the expression was carried out in the INVSd strain, which was grown on linoleic acid at 30 ° C. for 3 days, and then the fatty acids were transmethylated.
  • Figure 2 shows the presence of the t10, c12-CLA isomer in the free fatty acid pool.
  • 2A shows the gas chromatogram of the methylated fatty acids from yeast cells which were transformed with the empty vector pYES2. The cells were grown for 72 hours at 30 ° C with 0.02% linoleic acid. The gas chromatogram shows no FAME with a retention time of t10, c12-CLA.
  • 2B shows the gas chromatogram of the methylated fatty acids from yeast cells transformed with pY2-coPAl. The cells were again grown for 72 hours at 30 ° C with 0.02% linoleic acid.
  • the gas chromatogram has a clear peak with a retention time of 14.29 min, which does not occur in the control batch (see FIG. 2A) and has the same retention time as the t10, c12-CLA isomer (see FIG 2C).
  • the expression took place in the INVSd strain, which was grown on linoleic acid at 30 ° C. for 3 days, and then the fatty acids were methylated.
  • Table 1 shows the results of the GC analyzes.
  • the empty control shows the fatty acid composition of transgenic yeast cells, which with the empty pYES Vector were transformed.
  • PAI 4.3.1, PAI 4.3.2, PAI 6.1.1 and PAI 6.1.2 correspond to independent repeats of coPAI expression in transgenic yeast cells.
  • Experiment A shows the FAME analyzes of the lipids after incubation for 72 h at 30 ° C with 0.02% linoleic acid.
  • Experiment B shows the FAME analyzes of the free fatty acids after incubation for 72 h at 30 ° C with 0.02% Linoleic acid.
  • Experiment C shows the FAME analyzes of the lipids after incubation for 10 days at 16 ° C. with 0.02% linoleic acid.
  • FIG. 1, FIG. 2 and Table 1 show that the codon-optimized isomerase from P. acnes leads to the formation of t10, c12-CLA in yeast by the conversion of linoleic acid.
  • the detection of t10, c12-CLA from transformed yeast cells was achieved after hydrolysis of the lipids. Since yeast contains hardly any triacylglycerides, it must be assumed that the majority of the t10, c12-CLA detected in this way was bound in the yeast phopholipids. Detection of t10, c12-CLA from transformed yeast cells was also successful after extraction and methylation of the free fatty acids. This indicates that the coPAl in transgenic yeast cells also use the free fatty acids as a preferential substrate.
  • PAI Propionibacterium acnes isomerase
  • Primer C 5 ' - CAG ACA TAT GTC CAT CTC GAA GGA TTC - 3 Forward Primer (with Ndel interface)
  • Primer D 5' - CTA TCT CGA GTC ACA CGA AGA ACC GCG TC - 3 ' Reverse Primer (with Xhol interface)
  • the PCR reaction mixture was composed as follows: dNTP mix (10 mM) 1.0 ⁇ l
  • the amplified DNA fragment was cut from a preparative agarose gel, eluted with GFX TM PCR DNA and Gel Band Purification Kit (Amersham Bioscience) and cloned into the pGEM-T vector (Promega) according to the manufacturer's instructions. After amplification of the plasmid DNA in E. coli, the isomerase cDNA was cut out over the Ndel and Xhol interfaces and the cDNA ends with the T4-
  • the pYES2 vector was opened with EcoRI, the cDNA ends - smoothed with the T4 polymerase and dephosphorylated with an alkaline phosphatase. Both DNA fragments were alloyed with each other using the T4 ligase and the direction of insertion was checked by a BamHI digest.
  • the pY2-PAI construct thus produced was amplified in E. coli and for yeast expression in the yeast strain
  • example 2 it was shown that the codon-optimized isomerase from Propionibacterium acnes (coPAl), when expressed in yeast cells, can convert the added linoleic acid into the t10, c12-CLA isomer.
  • coPAl Propionibacterium acnes
  • the codon optimization can significantly increase the expression of an active isomerase protein, which can convert linoleic acid into t10, c12 CLA, in a eukaryotic cell such as S. cerevisiae.
  • 4A shows the gas chromatogram of the lipid extracts from yeast cells which were transformed with the empty vector pYES2. The cells were grown for 72 hours at 30 ° C with 0.02% linoleic acid. The gas chromatogram shows no FAME with a retention time of t10, c12-CLA.
  • 4B shows the gas chromatogram of the lipid extracts from yeast cells transformed with pY2-bakt.PAI.
  • the cells were again grown for 72 hours at 30 ° C with 0.02% linoleic acid.
  • the gas chromatogram has a very small peak with a retention time of 14.37 min, which does not occur in the control batch (cf. FIG. 4A).
  • the gas chromatogram of the lipid extracts from yeast cells, which were transformed with pY2-PAI and cultivated under the same conditions as in FIGS. 4A and b, is shown in FIG. 4C.
  • the chromatogram shows a clear peak with a retention time of 14.37 min, the retention time of t10, c12-CLA (see FIG. 1C).
  • the expression was carried out in the INVSd strain, which was grown on linoleic acid at 30 ° C. for 3 days, and then the fatty acids were transmethylated.
  • Table 1 shows the results of the GC analyzes.
  • the empty control shows the fatty acid composition of transgenic yeast cells, which with the empty pYES
  • PAI 4.3.1, PAI 4.3.2, PAI 6.1.1 and PAI 6.1.2 correspond to independent repeats of coPAI expression in transgenic yeast cells.
  • Bakt.PAl 4.1, 4.2, 9.1, 9.2 correspond to independent repetitions of the bakt.PAI-
  • Experiment E shows the FAME analyzes of the lipids after incubation for 72 h at 30 ° C. with 0.02% linoleic acid.
  • Experiment F shows the
  • FAME analyzes of free fatty acids after incubation for 72 h at 30 ° C with 0.02%
  • a main culture with transgenic yeasts carrying either the pY2-PAI or pY2-coPAI construct was grown as described above. After the OD600 had been determined, the main culture was harvested by centrifugation (7 min, 3800 rpm, 4 ° C.) in sterile centrifuge tubes. The cell pellets were washed with 5 ml of sterile H 2 O and harvested again (7 min, 3800 rpm, 4 ° C.). The cell pellet was washed with 5 ml of 0.1 M sodium phosphate buffer, pH 7.0 and harvested again by centrifugation (7 min, 3800 rpm, 4 ° C.). The supernatant was removed as completely as possible and the yeast pellets were snap-frozen at -80 ° C.
  • FIG. 3B shows the gas chromatogram of the methylated fatty acids from yeast cell lysates transformed with pY2-coPAI.
  • the cell lysates were again incubated for 30 min at RT with 200 ng linoleic acid.
  • the gas chromatogram has a clear peak with a retention time of 14.34 min, which does not occur in the control batch (cf. FIG. 3A) and has the same retention time as the t10, c12-CLA isomer (cf. FIG. 3C ).
  • the expression was carried out in the INVSd strain, which was grown for 3 days at 30 ° C., followed by mechanical digestion and incubation in the presence of linoleic acid with subsequent methylation of the fatty acids.
  • Table 1 shows the results of the GC analyzes.
  • the empty control shows the fatty acid composition of transgenic yeast cells which have been transformed with the empty pYES vector.
  • PAI 4.3.1, PAI 4.3.2, PAI 6.1.1 and PAI 6.1.2 correspond to independent repeats of coPAI expression in transgenic yeast cells.
  • Experiment D shows the results of the FAME analyzes of methylated free fatty acids after incubation of yeast cell lysates for 30 min at RT with 200 ng linoleic acid.
  • Experiment E is an independent repetition of Experiment D.
  • Example 5 Expression in E. coli BL21 (DE3) pLysS cells a) cell cultivation
  • the PAI-carrying DNA fragment isolated in accordance with Example 3 was cloned into the vector pET24a (Novagen) with the aid of the T4 ligase (MBI fermentas) and then first transformed into E. coli XL1 blue cells. After confirmation of a successful cloning, the resulting plasmid pET24a-PAI (0.2 ⁇ g) was used to transform E. coli BL21 (DE3) pLysS cells.
  • a single colony of the resulting E.coli BL21 (DE3) pLysS cells transformed with pET24a-PAI was used to inoculate 3 ml LB medium preculture, which additionally contained the antibiotics kanamycin and chloramphenicol for selection.
  • the cells were grown overnight at 37 ° C, 200 rpm.
  • Two 5 ml portions of this culture were transferred to a centrifuge tube, centrifuged (3800 rpm, 15 min, 4 ° C.), the supernatant was discarded and the pellet was frozen at -80 ° C.
  • the cells were disrupted for the implementation of various fatty acids and incubated with the fatty acids.
  • lysis buffer 1.5 ml was added to the Zeil pellets harvested before induction and 5 ml to the Zeil pellets harvested after induction.
  • the cells were resuspended by vortexing and snap-frozen in liquid N 2 for cell disruption and then thawed at 37 ° C. in a water bath. This process was repeated again. After the Zeil pellets were completely resuspended, the digestion and shearing of the genomic DNA was finally carried out by ultrasound: 45 seconds at 50% power in ice. Subsequently, 1.5 ml of lysate were incubated with the fatty acids to be reacted at 37 ° C. with shaking at 180 rpm for 1 to 3 h.
  • the extraction of the fatty acids takes place according to the Bligh and Dyer method by adding 100 ⁇ l glacial acetic acid, 900 ⁇ l 0.1M sodium phosphate buffer pH 7.0; 2.5 ml of methanol and 2.5 ml chloroform. The entire batch was shaken for 1 min, centrifuged off (4000 rpm, 10 min, RT ie at approx. 23 ° C.). The organic lower phase was transferred to a test tube and blown to dryness under N 2 and transferred to another test tube with 2 ⁇ 500 ⁇ l chloroform and dried again with N 2 . The residues were dissolved with 400 ⁇ l.
  • the sample extracted in this way was mixed with 10 ⁇ l EDAC solution [EDAC: N- (3-dimethyiaminopropyl) -N'-ethylcarodiimide hydrochloride, 1 mg / 10 ⁇ l methanol) and shaken at RT for 2 h. Then 200 ⁇ l of 0.1 M Tris / HCl solution pH 7.5 and 1 ml of hexane were added, shaken vigorously (vortexing) and centrifuged off (5 min, 12000 rpm, RT). The top phase was transferred to a new microcentrifuge tube and the extraction repeated with 1 ml of hexane.
  • EDAC solution EDAC: N- (3-dimethyiaminopropyl) -N'-ethylcarodiimide hydrochloride, 1 mg / 10 ⁇ l methanol
  • BBI-3 'primer: CTC GAG CAG ATC ACA TGG TAT TCG CGT AGC AG Primer contains an Xhol interface
  • the original Bifidobacterium breve isomerase sequence (SEQ ID NO: 9) is as follows: atg - tac tac agc agc ggc aac tat gag gcg ttt gcc cgt ccg aag aag cca gcc ggc gta g
  • the codon usage optimized modified sequence (SEQ ID NO : 3) then results as follows: atg ggt tac tac tcc tcc ggt aac tac gaa gct ttc gct aga cca aag ag cca gct ggt gtt g
  • the modification of the LRI isomerase was carried out with the following primers:
  • LRI-5 'primer GAA TTC ACC ATG GGT TAC TAC TCC AAC GGT AAC TAC GAA GCT TTC GCT AGA CCA AAG AAG CCA GCT GGT G
  • Primer contains an EcoRI interface
  • the original Lactobacillus reuteri isomerase sequence can be found in SEQ ID NO: 11.
  • the first base pairs of the nucleic acid sequence are as follows: atg - tat tat tca aac ggg aat tat gaa gcc ttt gct cga cca aag aag cct gct ggc g
  • the corresponding modified sequence (SEQ ID NO: 5) is as follows: atg ggt tac tac tcc aac ggt aac tac gaa gct ttc gct aga cca aag aag cca gct ggt g
  • the PCR reaction was carried out with Expand Fidelity PCR system from Röche Diagnostics and was composed as follows: dNTP mix (10 mM) 1.0 ⁇ l 5'-forward primer (10 ⁇ M) 4.0 ⁇ l 3'- Reverse primer (10 ⁇ M) 4.0 ⁇ l template (1/50 diluted PSE380-PAI plasmid DNA) 1.0 ⁇ l 10-fold buffer 5.0 ⁇ l polymerase (3.5 U / ⁇ l) 0.5 ⁇ l water 34 , 5 ul
  • the amplified DNA fragment was cut from a preparative agarose gel, eluted with GFX TM PCR DNA and Gel Band Purification Kit (Amersham Bioscience) and cloned into the pGEM-T vector (Promega) according to the manufacturer's instructions.
  • the isomerase cDNA was cut out via the Kpnl and Xhol interfaces or EcoRI and Xhol interfaces and the cDNA ends were smoothed with the T4 polymerase.
  • pYES2 vector was opened with EcoRI, the cDNA ends were smoothed with the T4 polymerase and dephosphorylated with an alkaline phosphatase.
  • Both DNA fragments were alloyed with each other using the T4 ligase and the direction of insertion was checked by a BamHI digest.
  • the pY2-PAI construct thus produced was amplified in E. coli and used for yeast expression in the yeast strain INVSd (Invitrogen) using the LiAc method (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York , 1995) transformed. If a cloning was carried out in the pET24a vector, the vector was cut with the same enzymes for cloning and the isomerase genes were cloned into the vector via these interfaces using the T4 ligase.
  • the expression of the isomerase from Bifidobacterium breve or Lactobacillus reuteri in transgenic plants is advantageous in order to increase the CLA content in these plants.
  • the cDNA coding for the isomerases was cloned into binary vectors and transferred via Agrobacterium-mediated DNA transfer into Arabidopsis thaliana, Nicotiana tabacum, Brassica napus and Linum usitatissimum.
  • the expression of the isomerase cDNA was under the control of the cohesive CaMV 35 S promoter or the seed-specific USP promoter.
  • Arabidopsis is particularly suitable as a model plant because it has a short generation time and sufficient amounts of linoleic acid, the substrate of the isomerase for the production of CLA.
  • Tobacco and high linoleic acid varieties of linseed are particularly suitable as oilseeds with a high content of linoleic acid for the heterologous expression of isomerase genes, since linoleic acid is the substrate of the isomerases for the formation of conjugated linoleic acid.
  • the expression vectors were the vector pBinAR (Höfgen and Willmitzer, Plant Science, 66, 1990: 221-230) and the pBinAR derivative pBinAR-USP, in which the CaMV 35 S promoter was exchanged for the USP promoter from V. faba , used.
  • the vectors pGPTV and pGPTV-USP were also used.
  • the isomerase cDNA had to be cut out from the vector pGEM-T and cloned into pBinAR or pBinAR-USP.
  • the resulting plasmids were transformed into Agrobacterium tumefaciens (Höfgen and Willmitzer, Nucl. Acids Res., 16, 1988: 9877).
  • the transformation of A. thaliana was carried out by means of a "floral dip" (Clough and Bent, Plant Journal, 16, 1998: 735-743), that of N. tabacum via cocultivation of tobacco leaf fragments with transformed A. tumefaciens cells, that of linseed and rapeseed by coculturing hypocotyl pieces with transformed A. tumefaciens cells.
  • the expression of the isomerase genes in transgenic Arabidopsis, tobacco, rapeseed and linseed plants was investigated using Northem blot analysis. Selected plants were examined for their CLA content in the seed oil.
  • the napin promoter can also be used to achieve seed-specific expression of the isomerase.
  • Table 3 shows the results in plants. Table 3: Compilation of the experiments in transgenic tobacco plants

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Abstract

L'invention se rapporte à un procédé de production d'acides gras polyinsaturés conjugués comportant au moins deux, de préférence trois liaisons doubles, dans des eucaryotes, en particulier dans des plantes, ainsi qu'à un procédé de production d'huiles et/ou de triglycérides présentant une teneur élevée en acides gras polyinsaturés conjugués et/ou non conjugués comportant au moins deux, de préférence trois liaisons doubles. Cette invention concerne également un procédé de production d'acide linoléique conjugué dans des eucaryotes, en particulier dans des plantes, ainsi qu'un procédé de production d'huiles et/ou de triglycérides présentant une teneur élevée en acide linoléique conjugué. La présente invention se rapporte en outre à des séquences d'acides nucléiques, à des constructions d'acides nucléiques, à des vecteurs ainsi qu'à des organismes contenant ces séquences d'acides nucléiques, constructions d'acides nucléiques et vecteurs. L'invention concerne par ailleurs des mélanges d'acides gras et des triglycérides présentant une teneur élevée en acides gras polyinsaturés conjugués et/ou non conjugués comportant au moins deux, de préférence trois liaisons doubles, en particulier une teneur élevée en acide linoléique conjugué, ainsi que leur utilisation.
PCT/EP2003/006833 2002-07-03 2003-06-27 Procede de production d'acides gras polyinsatures conjugues comportant au moins deux liaisons doubles dans des plantes Ceased WO2004005442A1 (fr)

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