EP2046960A1 - Procédé de fabrication d'acide arachidonique et/ou d'acide eicosapentaénoïque dans des plantes - Google Patents

Procédé de fabrication d'acide arachidonique et/ou d'acide eicosapentaénoïque dans des plantes

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Publication number
EP2046960A1
EP2046960A1 EP07787358A EP07787358A EP2046960A1 EP 2046960 A1 EP2046960 A1 EP 2046960A1 EP 07787358 A EP07787358 A EP 07787358A EP 07787358 A EP07787358 A EP 07787358A EP 2046960 A1 EP2046960 A1 EP 2046960A1
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EP
European Patent Office
Prior art keywords
acid
desaturase
seq
fatty acids
plant
Prior art date
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EP07787358A
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German (de)
English (en)
Inventor
Michael Geiger
Jörg BAUER
Petra Cirpus
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BASF Plant Science GmbH
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BASF Plant Science GmbH
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Publication of EP2046960A1 publication Critical patent/EP2046960A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • arachidonic acid and eicosapentaenoic acid advantageously in the seed of transgenic plants of the family of
  • further enzymes can be selected from the group of the enzymes ⁇ -3-desaturases, ⁇ -12-desaturases, ⁇ -6-desaturases, ⁇ -6-elongases, ⁇ -5-desaturases, ⁇ -5-elongases and / or ⁇ - 4-desaturases are introduced into the plants.
  • the introduced nucleic acid sequences are the sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.
  • other nucleic acid sequences which code for polypeptides having ⁇ -3-desaturase or ⁇ -12 desaturase activity are preferably introduced into the plants and also expressed simultaneously. These are particularly preferably the nucleic acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 11.
  • these nucleic acid sequences may optionally be expressed in the organism along with other nucleic acid sequences encoding polypeptides of biosynthesis of the fatty acid or lipid metabolism.
  • nucleic acid sequences which code for a ⁇ -4-desaturase and / or ⁇ -5 elongase activity.
  • the oils, lipids or free fatty acids produced in the process which contain ARA and / or EPA, are added to known quantities of feed, food, cosmetics or pharmaceuticals known to the person skilled in the art.
  • the lipid synthesis can be divided into two sections: the synthesis of fatty acids and their attachment to sn-glycerol-3-phosphate and the addition or modification of a polar head group.
  • Common lipids used in membranes include phospholipids, glycolipids, sphingolipids and phosphoglycerides.
  • Fatty acid synthesis begins with the conversion of acetyl-CoA into malonyl-CoA by the acetyl-CoA carboxylase or into acetyl-ACP by the acetyl transacylase. After a condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydration reactions, so that a saturated fatty acid molecule with the desired chain length is obtained.
  • the production of unsaturated fatty acids from these molecules is catalyzed by specific desaturases, either aerobically by molecular oxygen or anaerobically (for fatty acid synthesis in microorganisms, see FC Neidhardt et al., (1996) E.
  • fatty acids must subsequently be transported to various modification sites and incorporated into the triacylglycerol storage lipid.
  • Another important step in lipid synthesis is the transfer of fatty acids to the polar head groups, for example by glycerol-fatty acid acyltransferase (see Frentzen, 1998, Lipid, 100 (4-5): 161-166).
  • polyunsaturated fatty acids are referred to as PUFAs, PUFAs, LCPUFAs or LCPUFAs
  • PUFA polyunsaturated fatty acids
  • PUFA polyunsaturated fatty acids
  • LCPUFA long-chain polyunsaturated fatty acids
  • PUFA polyunsaturated fatty acids
  • PUFAs polyunsaturated fatty acids
  • LCPUFA long-chain polyunsaturated fatty acids
  • Fatty acids and triacylglycerides have a variety of uses in the food, animal nutrition, cosmetics and pharmaceutical industries. Depending on whether they are free saturated and unsaturated fatty acids or triacylglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a wide variety of applications. Multiple- Unsaturated fatty acids such as linoleic and linolenic acid are essential for mammals because they can not be produced by them. Therefore, polyunsaturated ⁇ -3 fatty acids and ⁇ -6 fatty acids represent an important component of animal and human food. Thus, for example, in the human diet lipids with unsaturated fatty acids, especially polyunsaturated fatty acids are preferred.
  • the polyunsaturated ⁇ -3 fatty acids thereby a positive effect on the cholesterol level in the blood and thus on the prevention of heart disease is attributed.
  • the risk of heart disease, stroke, or hypertension can be significantly reduced (Shimikawa 2001, World Rev. Nutr., Diet, 88, 100-108).
  • inflammatory, especially chronic inflammatory, processes in the context of immunological diseases can be positively influenced by ⁇ -3 fatty acids (Calder 2002, Proc Nutr Soc 61, 345-358, Cleland and James 2000, J. Rheumatol. 27, 2305-2307). They are therefore added to foods, especially dietary foods, or are used in medicaments, omega-6 fatty acids such as arachidonic acid have a negative effect in these rheumatic diseases.
  • ARA is beneficial and important for the development of newborns.
  • ⁇ -3 and ⁇ -6 fatty acids are precursors of tissue hormones, called eicosanoids such as the prostaglandins, derived from dihomo- ⁇ -linolenic acid, arachidonic acid and eicosapentaenoic acid and the thromboxanes and leukotrienes derived from arachidonic acid and eicosapentaenoic acid.
  • Eicosanoids (so-called PG 2 -SeMe), which are formed from ⁇ -6 fatty acids, usually promote inflammatory reactions, while eicosanoids (so-called PG 3 -SeMe) from ⁇ -3 fatty acids have little or no pro-inflammatory effect.
  • polyunsaturated long-chain fatty acids such as those mentioned above.
  • polyunsaturated ⁇ -3 fatty acids which are preferred in fish oils, is particularly important for food.
  • the unsaturated fatty acid DHA is thereby attributed a positive effect on the development and maintenance of brain functions. It For this reason there is a need for the production of polyunsaturated long-chain fatty acids.
  • the free fatty acids are advantageously prepared by saponification.
  • Common natural sources of these fatty acids are fish such as herring, salmon, sardine, perch, eel, carp, trout, halibut, mackerel, zander or tuna or algae.
  • Fatty acids from genera of the Brassicaceae family such as Brassica napus or Brassica rapa are often used in the food, feed, cosmetic and / or pharmaceutical industries.
  • a disadvantage of the oils of this family is that they contain some fatty acids such as ⁇ -linolenic acid, icosenoic acid or erucic acid, which are rather undesirable, so that the oils can not be used in any amounts.
  • Other fatty acids contained in the oils, such as oleic acid have a lower value as food additives.
  • the long-chain fatty acids such as icosenoic acid 20: 1 and erucic acid 22: 1 were detected in the family Brassicaceae.
  • the levels of erucic acid are, for example, 35-48% for Brassica carinata, 18-49% for Brassica juncea, 45-54% Brassica napus, 55-60% Crambe abyssinica, 34-47% Eruca sativa, and 33-51 Sinapis alba %, Camelina sativa 3-5%, Raphanus sativa> 22%.
  • Erucic acid should only be present in very small amounts or not at all in oils used in human nutrition.
  • Advantageous oils, lipids and / or fatty acid compositions should have the lowest possible content of fatty acids such as oleic acid, ⁇ -linolenic acid, icosenic acid and / or erucic acid.
  • the highest possible levels of fatty acids such as arachidonic acid and / or eicosapentaenoic acid should be included.
  • the plants used for the production should be as easy to grow as possible and it should be established processing processes for the oils contained, lipids and / or
  • Fatty acid compositions may be present. Externally, the manufacturing process should be simple and inexpensive. Furthermore, the oils, lipids and / or fatty acid compositions of these plants should have been used for an extended period of time for the production of feed, food, cosmetics and / or pharmaceuticals in the industry. It is an object of the present invention to develop a process for the production of large quantities of polyunsaturated fatty acids, especially ARA, EPA and / or DHA, in the seed of transgenic plants and at the same time to reduce the levels of undesired fatty acids.
  • polyunsaturated fatty acids especially ARA, EPA and / or DHA
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • % based on the total lipid content of the transgenic plant characterized in that it comprises the following process steps: a) introduction of at least one nucleic acid sequence into the crop which codes for a ⁇ 6-desaturase, and b) introduction of at least one nucleic acid sequence into the crop c) introducing at least one nucleic acid sequence into the crop which encodes a ⁇ 5-desaturase, and d) harvesting the crop, wherein the enzymatic activity of the enzyme obtained by the steps (a) to (a) (c) enzymes introduced a fatty acid selected from the group consisting of the fatty acids oleic acid [C18: 1 ⁇ 9 ] , Linoleic acid [C18: 2 ⁇ 9 12 ], ⁇ -linolenic acid [C18: 3 ⁇ 6 9 12 ], icosenoic acid [C20: 1 ⁇ 11 ] and erucic acid [C22: 1 ⁇ 13 ] by at least 10%, 11%, 12%,
  • the wild type plant is understood as meaning plants which carry the non-mutated (unmodified) form of a gene or allele and are found, for the most part, in a population living under natural conditions. Under wild-type also so-called zero-zygotes are to be understood. These were transformed with a gene, but lost it again. All of the above data are percentages by weight and refer to the content of the fatty acids in the corresponding wild-type plant.
  • saturated fatty acids are little or not reacted with the nucleic acids used in the process. Little is to be understood, compared to multiple unsatisfied saturated fatty acids have less than 5% of the saturated fatty acids, advantageously less than 3%, more preferably less than 2%, most preferably less than 1%; 0.5; 0.25 or 0.125% are implemented.
  • These produced fatty acids can be produced as the only product in the process or present in a fatty acid mixture.
  • nucleic acid sequences used in the method according to the invention are isolated nucleic acid sequences which code for polypeptides having ⁇ -6-desaturase, ⁇ -6-elongase and / or ⁇ -5-desaturase activity.
  • Nucleic acid sequences coding for polypeptides having ⁇ 6-desaturase, ⁇ 6-elongase, or ⁇ 5-desaturase activity are advantageously used in the method according to the invention selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or b) nucleic acid sequences which, as a result of the degenerate genetic code, differ from those shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, which code for polypeptides which have at least 40% identity at the amino acid level with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and a ⁇ -6-desaturase, ⁇ -6 Have
  • nucleic acid sequences are advantageously introduced into the crop plants which code for an ⁇ -3-desaturase or ⁇ -12-desaturase or ⁇ -3-desaturase and ⁇ -12-desaturase.
  • a preferred embodiment of the method is characterized in that a nucleic acid sequence is additionally introduced into the transgenic plant which codes for polypeptides having ⁇ -3-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO : 9, or b) nucleic acid sequences that result as a result of the degenerate genetic
  • the method is characterized in that a nucleic acid sequence is additionally introduced into the transgenic plant which codes for polypeptides having ⁇ 12-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO B) nucleic acid sequences which can be deduced as a result of the degenerate genetic code from the amino acid sequence shown in SEQ ID NO: 12, or c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1 1, which polypeptides encode at least 60% identity with SEQ ID NO: 10 at the amino acid level and have a ⁇ -12 desaturase activity.
  • ⁇ -12-desaturase sequences can be used alone or in combination with the ⁇ 3-desaturase sequences with the nucleic acid sequences used in the method which code for ⁇ -6 desaturases, ⁇ 6-elongases and / or ⁇ 5-desaturases become.
  • the nucleic acids are expressed in vegetative or reproductive tissue.
  • the nucleic acid sequences used in the method result in addition to a lowering of unwanted fatty acids to increase the ARA or EPA or ARA and EPA content in the plants.
  • the process can thereby increase the ARA or EPA or ARA and EPA content to more than 8%, advantageously up to more than 10%, 11%, 12%, 13%, 14%. , 15%, 16%, 17%, 18%, 19% or 20%, particularly advantageous to more than 21%, 22%, 23%, 24% or 25%, based on the total lipid content of the plant to be achieved
  • the above percentages are by weight.
  • oils and / or triglycerides with an advantageous over oils and / or triglycerides from wild-type plants increased content of polyunsaturated fatty acids, especially of ARA, EPA or mixtures thereof, it may be advantageous to increase the amount of starting material for fatty acid synthesis. This can be achieved, for example, by introducing a nucleic acid encoding a polypeptide having the activity of a ⁇ 12-desaturase and co-expressing it in the organism.
  • a nucleic acid sequence is additionally introduced into the transgenic plant which codes for a polypeptide having ⁇ 12-desaturase activity.
  • the ⁇ -12-desaturases used bind fatty acids bound to phospholipids or CoA fatty acid esters, advantageously bound to CoA fatty acid esters. This, if an elongation step has previously taken place, advantageously leads to higher yields of synthesis products, since the elongation in the
  • nucleic acid sequences are introduced into the plants on a common recombinant nucleic acid molecule, wherein each nucleic acid sequence can be under the control of its own promoter and this own promoter can be a seed-specific promoter.
  • nucleic acids which have a certain degree of identity or homology to the sequences specified in the sequence listing.
  • substantially identical enzymatic activity is to be understood as meaning proteins which are at least 20%, 30%, 40%, 50% or 60%, advantageously at least 70%, 80%, 90% or 95%, particularly advantageously at least 96%, 97%. , 98% or 99% of the enzymatic activity of the wild-type enzymes.
  • the sequences are written among themselves (eg, gaps can be inserted into the sequence of one protein or one nucleic acid to create an optimal alignment with the other protein or nucleic acid).
  • the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding site in the other sequence, then the molecules at that position are identical. If different amino acids are at the same position but have the same properties, eg two hydrophobic amino acids, then they are homologous or similar.
  • Polypeptides cause can occur. These natural variants usually cause a variance of 1 to 5% in the nucleotide sequence of the ⁇ 6-desaturase, ⁇ 5-desaturase and / or ⁇ 6-elongase gene. All and all of these nucleotide variations and resulting amino acid polymorphisms in the ⁇ 6-desaturase, ⁇ 5-desaturase and / or ⁇ 6-elongase, which are the result of natural variation and which do not substantially alter enzymatic activity, are said to be in the art Scope of the invention may be included.
  • Essential enzymatic activity of the ⁇ 6-desaturase ⁇ 6-elongase or ⁇ 5-desaturase used in the process according to the invention is to be understood as meaning that it still has an enzymatic activity of the proteins / enzymes encoded by the sequence and its derivatives at least 10%, preferably at least 20%, more preferably at least 30%, 40%, 50% or at least 60% and most preferably at least 70%, 80%, 90%, 95%, 96%, 97% , 98% or 99%, and thus to the metabolism of compounds that are required to build up fatty acids, fatty acid esters such as diacylglycerides and / or triacylglycerides in a plant or plant cell or participate in the transport of molecules across membranes.
  • fatty acid esters such as diacylglycerides and / or triacylglycerides in a plant or plant cell or participate in the transport of molecules across membranes.
  • nucleic acid molecules which bind under stringent conditions to the complementary strand of ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ -5-desaturase, ⁇ -3-desaturase and / or ⁇ -12-desaturase Hybridize ⁇ 6-elongase nucleic acids.
  • hybridizes under stringent Conditions are meant to describe hybridization and washing conditions under which nucleotide sequences at least 60% homologous to one another usually remain hybridized to each other.
  • the conditions are preferably such that sequences that are at least about 65%, 70%, 80% % or 90%, preferably at least about 91%, 92%, 93%, 94% or 95% and more preferably at least about 96%, 97%, 98%, 99% or more are homologous to each other, usually remaining hybridized to each other stringent conditions are known to those of skill in the art and are described, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6
  • a preferred, non-limiting example of stringent hybridization conditions are hybridizations in Figure 6 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 50-65 ° C It is known that these hybridization conditions differ
  • the hybridization temperature is, for example, under "standard hybridization conditions" depending on the type of nucleic acid between 42 ° C and 58 ° C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2). If organic solvent, for example 50% formamide, is present in the abovementioned buffer, the temperature under standard conditions is about 42 ° C.
  • the hybridization conditions for DNA: DNA hybrids are, for example, 0.1 x SSC and 20 ° C to 45 ° C, preferably 30 ° C to 45 ° C.
  • the hybridization conditions for DNA: RNA hybrids are, for example, 0.1 x SSC and 30 ° C to 55 ° C, preferably 45 ° C to 55 ° C.
  • hybridization conditions required for a particular nucleic acid can be determined by textbooks such as Sambrook et al., "Molecular Cloning", ColD Spring Harbor Laboratory, 1989; Harnes and Higgins (Eds.) 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed.) 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.
  • an isolated nucleic acid molecule By introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence, an isolated nucleic acid molecule can be generated which is suitable for a ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ -5-desaturase, ⁇ -3-desaturase and / or ⁇ 6-elongase encoded with one or more amino acid substitutions, additions or deletions. Mutations can be introduced into one of the sequences by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made on one or more of the predicted nonessential amino acid residues.
  • amino acid residue is raised against an amino acid residue with a similar side chain. exchanges.
  • families of amino acid residues have been defined with similar side chains. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg Alanine, VaNn, Leucine,
  • a predicted non-essential amino acid residue in a ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ -3-desaturase and / or ⁇ 6-elongase thus becomes preferably a different amino acid residue from the same side-chain family replaced.
  • the mutations may be randomized over all or part of those encoding ⁇ -12 desaturase, ⁇ -6 desaturase, ⁇ -5 desaturase, ⁇ -3 desaturase, and / or ⁇ -6 elongase Sequence are introduced, eg by saturation mutagenesis, and the resulting mutants can be prepared by recombinant expression according to the herein described ⁇ -12-desaturase, ⁇ -6-desaturase, ⁇ -5-desaturase, ⁇ -3-desaturase and / or ⁇ -6 -Longase activity to identify mutants carrying the ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ -3-desaturase and / or ⁇ 6-elongase Maintained activity.
  • the polyunsaturated fatty acids ARA or EPA or ARA and EPA produced in the process are advantageously bound in as esters such as membrane lipids such as phospholipids or glycolipids and / or triacylglycerides, but may also occur as free fatty acids or bound in the form of other fatty acid esters in the organisms. They may be present as "pure products" or else advantageously in the form of mixtures of different fatty acids or mixtures of different glycerides.
  • the different fatty acids bound in the triacylglycerides can thereby be derived from short-chain fatty acids having 4 to 6 C atoms, medium-chain fatty acids having 8 to 12 C atoms or long-chain fatty acids having 14 to 24 C atoms, preferably the long-chain fatty acids are particularly preferred the long-chain fatty acids LCPUFAs of DE, C 2 O and / or C 22 fatty acids, very particular preference is given to the long-chain fatty acids LCPUFAs of C 20 and / or C 22 fatty acids such as ARA, EPA or their combination.
  • glycolide is understood to mean a glycerol esterified with one, two or three carboxylic acid residues (mono-, di- or triglyceride).
  • glycolide is also meant a mixture of different glycerides.
  • the glyceride or glyceride mixture may contain other additives, e.g. contain free fatty acids, antioxidants, proteins, carbohydrates, vitamins and / or other substances.
  • a "glyceride” in the sense of the method according to the invention is also understood to mean derivatives derived from glycerol.
  • these also include glycerophospholipids and glyceroglycolipids.
  • Preference is given here to glycerophospholipids such as lecithin (phosphatidylcholine), Cardiolipin, phosphatidylglycerol, phosphatidylserine and Alkylacylglycerophospholipide exemplified.
  • phospholipids are to be understood as meaning phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and / or phosphatidylinositol, advantageously phosphatidylcholine.
  • the fatty acid esters produced in the process may be prepared from the plants used to produce the fatty acid esters in the form of an oil or lipid, for example in the form of compounds such as sphingolipids, phosphoglycerides, lipids, glycolipids such as glycosphingolipids, phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol , Phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters such as the acetyl coenzymeA esters.
  • compounds such as sphingolipids, phosphoglycerides, lipids, glycolipids such as glycosphingolipids, phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatid
  • they are in the form of their diacylglycerides, triacylglycerides and / or in the form of the phospholipids, e.g. the phosphatidylcholine isolated, more preferably in the form of triacylglycerols.
  • the fatty acids produced in the process are also included as free fatty acids or bound to other compounds in the plants.
  • the various compounds mentioned above are present in the organisms in an approximate distribution of 80 to 90% by weight of triglycerides, 2 to 5% by weight of diglycerides, 5 to 10% by weight of monoglycerides, 1 to 5 wt .-% of free fatty acids, 2 to 8 wt .-% phospholipids ago, wherein the sum of the various compounds to 100 wt .-% complements.
  • the LCPUFAs produced have a content of at least 3, 5, 6, 7 or 8% by weight. , preferably of at least 9, 10, 11, 12, 13, 14 or 15 wt .-%, preferably of at least 16, 17, 18, 19 or 20 wt .-%, particularly preferably of at least 21, 22, 23, 24 or 25% by weight, very particularly preferably at least 26, 27, 28, 29 or 30% by weight, based on the total fatty acids in the transgenic organisms, of advantage in the seed of the transgenic plants.
  • Ci ⁇ - and / or C 2 o fatty acids present in the host organisms at least 10%, advantageously at least 20%, more preferably at least 30%, most preferably at least 40% in the corresponding products implemented as ARA, EPA or mixtures thereof.
  • the fatty acids are prepared in bound form.
  • polyunsaturated C 2 o-fatty acids with four or five double bonds in the molecule having a content of all such fatty acids of at least 15, 16, 17, 18, 19 or 20% by weight, advantageously at least 21, are advantageous in the process.
  • ARA is used with a content of at least 3, 5, 6, 7, 8, 9 or 10 wt .-%, advantageously of at least 1 1, 12, 13, 14 or 15 wt .-%, preferably of at least 16 , 17, 18, 19 or 20% by weight, more preferably at least 21, 22, 23, 24 or 25% by weight, most preferably at least 26% by weight, based on the total lipid content in the seeds transgenic plants.
  • EPA is used in the process according to the invention with a content of at least 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9 or 1 wt .-%, advantageously of at least 2, 3, 4 or 5 wt .-%, preferably of at least 6, 7, 8, 9 or 10 wt .-%, particularly preferably of at least 1 1, 12th , 13, 14 or 15% by weight, and most preferably at least 16% by weight, based on the total lipid content in the seeds of the transgenic plants.
  • these unsaturated fatty acids can be brought to the sn1, sn2 and / or sn3 position of the advantageously prepared triglycerides. Since in the process of the invention from the starting compounds linoleic acid (C18: 2) or linolenic acid (C18: 3) are passed through several reaction steps, the end products of the process such as arachidonic acid (ARA) or eicosapentaenoic acid (EPA) are not as absolute pure products, it is always contain small traces of the precursors in the final product.
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • both linoleic acid and linolenic acid are present in the starting organism or in the starting plant, for example, the end products such as ARA or EPA are generally present as mixtures.
  • the end products such as ARA or EPA are generally present as mixtures.
  • only small amounts of the other end products should be present in the final products ARA or EPA. Therefore, in an EPA-containing lipid and / or oil should be less than 15, 14, 13, 12 or 11 wt .-%, advantageously less than 10, 9, 8, 7, 6 or 5 wt .-%, more preferably less than 4 Be contained 3, 2 or 1 wt .-% ARA.
  • ARA-containing lipid and / or oil should therefore less than 15, 14, 13, 12 or 1 1 wt .-%, advantageously less than 10, 9, 8, 7, 6 or 5 wt .-%, particularly advantageously less be contained as 4, 3, 2 or 1 wt .-% EPA.
  • the precursors should advantageously not more than 20 wt .-%, preferably not more than 15 wt .-%, more preferably not more than 10 wt .-%, most preferably not more than 5 wt .-% based on the amount of the respective Final product.
  • ARA or EPA are bound in the process according to the invention or produced as free acids in a transgenic plant as end products. If the compounds ARA and EPA are produced simultaneously, they are advantageously used in a ratio of at least 1: 6 (EPA: ARA), preferably of at least 1: 8, preferably of at least 1:10, more preferably of at least 1: 12 in the plant produced.
  • Fatty acid esters or fatty acid mixtures which have been prepared by the process according to the invention advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid; 7 - 85% oleic acid; 0.5 to 8% vaccenic acid, 0.1 to 1% arachidic acid, 7 to 25% saturated fatty acids, 8 to 85% monounsaturated fatty acids and 60 to 85% polyunsaturated fatty acids in each case based on 100% and on the total fatty acid content of the organisms.
  • the fatty acid esters or fatty acid mixtures prepared by the process according to the invention advantageously contain fatty acids selected from the group of the fatty acids erucic acid (13-docosaic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8,9 Methylene heptadec-8-enoic acid), chaulmo-gruoic acid (cyclopentenodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,1-dienanoic acid), vernoic acid (9,10-epoxyoctadec-12-enoic acid), tartric acid (6-octadecynoic acid), 6-nonadecynoic acid, santalbinic acid (t11-
  • Octadecen-9-ynoic acid 6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynonic acid), crepenynic acid (9-octadecen-12-ynonic acid), 13,14-dihydrooropheic acid, octadecene-13-ene-9, 11-diynoic acid, petroselenoic acid (cis-6-octadecenoic acid), 9c, 12t-octadecadienoic acid, calendulic acid (8t10t12c octadecatrienoic acid), catalpinic acid (9t1 1t13c-octadecatrienoic acid), elicolinic acid (9c11t13t octadecatrienoic acid), jacric acid (8c10t12c octadecatrienoic acid), punicin acid (9c11t13c-octadecatrien
  • fatty acids are generally advantageously present only in traces in the fatty acid esters or fatty acid mixtures prepared by the process according to the invention, that is to say they are less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids. , 22% or 21%, more preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, most preferably less than 4%, 3%, 2% or 1% ago.
  • these aforementioned fatty acids come to less than 0.9% based on the total fatty acids; 0.8%; 0.7%; 0.6%; or 0.5%, more preferably less than 0.4%; 0.3%; 0.2%; 0.1% before.
  • the nucleic acid sequences according to the invention or the nucleic acid sequences used in the method according to the invention can increase the yield of polyunsaturated fatty acids, especially ARA and EPA, by at least 50, 80 or 100%, advantageously at least 150, 200 or 250%, particularly advantageously at least 300, 400, 500, 600, 700, 800 or 900%, most preferably at least 1000, 1100, 1200, 1300, 1400 or 1500% relative to the non-transgenic parent plant of for example a plant such as Brassica juncea or Brassica napus as compared to See the GC analysis for examples.
  • polyunsaturated fatty acids especially ARA and EPA
  • the lipids and / or oils produced in the process according to the invention should advantageously have a high proportion of unsaturated fatty acids of polyunsaturated fatty acids of at least 30, 40 or 50% by weight, advantageously of at least 60, 70 or 80% by weight, based on the Total fatty acid content in the seeds of the transgenic plants amount.
  • All saturated fatty acids together should advantageously account for only a small proportion in the plants preferably used for the process according to the invention.
  • a small proportion in this context is a fraction in GC area units of less than 15%, 14%, 13%, 12%, 11% or 10%, preferably less than 9%, 8%, 7% or 6%. to understand.
  • the advantageous in the process as host plants containing the introduced via different methods used in the process genes for the synthesis of polyunsaturated fatty acids advantageously have a higher oil content than protein content in the seed, advantageous plants have an oil / protein content ratio of 5 to 1, 4 to 1, 3 to 1, 2 to 1 or 1 to 1.
  • Host plants which are advantageous for the method are those which have a high proportion of oleic acid, ie of at least 40, 50, 60 or 70% by weight, based on the total fatty acid content of the plant, in comparison with linoleic acid and / or linolenic acid in the lipids and / or oils especially in triglyceride such as Brassica napus, Brassica alba, Brassica hirta, Brassica nigra, Brassica juncea or Brassica carinata.
  • oleic acid ie of at least 40, 50, 60 or 70% by weight, based on the total fatty acid content of the plant, in comparison with linoleic acid and / or linolenic acid in the lipids and / or oils especially in triglyceride such as Brassica napus, Brassica alba, Brassica hirta, Brassica nigra, Brassica juncea or Brassica carinata.
  • Plants used for the method should advantageously have an erucic acid content of less than 2% by weight based on the total fatty acid content of the plant.
  • the content of saturated fatty acids C16: 0 and / or C18: 0 should advantageously be less than 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10% by weight, advantageously less than 9, 8, 7, 6 or 5 wt .-% based on the total fatty acid content of the plant.
  • longer fatty acids such as C20: 0 or C22: 1 should not at all or in only small amounts advantageously less than 4, 3, 2 or 1 wt .-%, advantageously less than 0.9; 0.8; 0.7; 0.6; 0.5; 0.4; 0.3; 0.2 or 0.1% by weight, based on the total fatty acid content of the plant, in the plants used in the process.
  • no or only small amounts of C16: 1 are present as fatty acid in the plants used for the method according to the invention.
  • Small amounts are to be understood as meaning contents of fatty acids which are less than 4, 3, 2 or 1% by weight, advantageously less than 0.9; 0.8; 0.7; 0.6; 0.5; 0.4; 0.3; 0.2 or 0.1 wt .-% based on the total fatty acid content of the plant.
  • chemically pure polyunsaturated fatty acids or fatty acid compositions can be prepared by the methods described above.
  • the fatty acids or the fatty acid compositions from the plants advantageously the plant seeds in a known manner, for example on breaking up the seeds like Grinding and subsequent extraction, distillation, crystallization, chromatography or combinations of these methods isolated.
  • These chemically pure fatty acids or fatty acid compositions are advantageous for applications in the food industry, the cosmetics industry and especially the pharmaceutical industry.
  • Foliosa Brassica nigra, Brassica sinapioides, Camelina sativa, Melanosinapis communis [mustard], Brassica oleracea [fodder beet] or Arabidopsis thaliana ,
  • nucleic acids and the optionally introduced nucleic acid sequences that for the ⁇ -3-desaturases and / or for the ⁇ -12 Desaturases encode to additionally introduce further nucleic acids encoding enzymes of the fatty acid or lipid metabolism.
  • Elongases or ⁇ -9 elongases are used in combination with the aforementioned genes for the ⁇ -5 elongase, ⁇ -6 elongase and / or ⁇ -3 desaturase, wherein single genes or multiple genes can be used in combination.
  • nucleic acid sequences used according to the invention or their derivative or homologues coding for polypeptides which still possess the enzymatic activity of the proteins encoded by nucleic acid sequences are cloned individually or in combination into expression constructs and used for introduction and expression in plants.
  • these expression constructs enable a favorable optimal synthesis of the polyunsaturated fatty acids produced in the process according to the invention.
  • the method further comprises the step of obtaining a transgenic plant containing the nucleic acid sequences used in the method, the plant having a nucleic acid sequence encoding ⁇ -12 desaturase, ⁇ 5-desaturase, ⁇ - 6-desaturase, ⁇ 6-elongase and / or ⁇ -3-desaturase, a gene construct or a vector as described below, alone or in combination with other nucleic acid sequences coding for proteins of the fatty acid or lipid metabolism is transformed.
  • this method further comprises the step of recovering the oils, lipids or free fatty acids from the seed of the plant. Cultivation is, for example, culturing in the case of plant cells, tissue or organs on or in a nutrient medium or the whole plant on or in a substrate, for example in hydroponics, potting soil or on arable land.
  • a further subject of the invention are gene constructs which contain the nucleic acid sequences used in the method according to the invention which code for a ⁇ 5-desaturase, ⁇ 6-desaturase or ⁇ 6-elongase, wherein the nucleic acid is operably linked to one or more regulatory signals.
  • biosynthesis genes of the fatty acid or lipid metabolism selected from the group consist
  • There may be more than one nucleic acid sequence of an enzymatic activity e.g. a ⁇ -5-desaturase, ⁇ -6-desaturase, ⁇ -12-desaturase, ⁇ -3-desaturase or ⁇ -6 elongase.
  • the nucleic acids used in the method are advantageously subjected to amplification and ligation in a known manner.
  • the procedure is based on the protocol of the Pfu DNA polymerase or of a Pfu / Taq DNA polymerase mixture.
  • the primers are selected taking into account the sequence to be amplified. Conveniently, the primers should be chosen so that the amplificate covers the entire includes codogenic sequence from start to stop codon.
  • the amplificate is conveniently analyzed. For example, a quantitative and qualitative analysis can be carried out after gel electrophoresis separation.
  • the amplificate can be purified according to a standard protocol (eg Qiagen). An aliquot of the purified amplificate is then available for subsequent cloning.
  • Suitable cloning vectors are well known to those skilled in the art. These include, in particular, vectors which can be replicated in microbial systems, ie in particular vectors which ensure efficient cloning in yeasts or fungi, and which enable the stable transformation of plants.
  • various suitable for T-DNA-mediated transformation binary and co-integrated vector systems.
  • Such vector systems are usually characterized in that they contain at least the vir genes required for the Agrobacterium-mediated transformation as well as the T-DNA limiting sequences (T-DNA border).
  • these vector systems also include other cis-regulatory regions such as promoters and terminator sequences and / or selection markers, with which correspondingly transformed organisms can be identified.
  • binary systems are based on at least two vectors, one of them vir genes, but no T-DNA and a second T-DNA, but no carries vir gene.
  • these binary vectors include vectors of the series pBIB-HYG, pPZP, pBecks, pGreen. Bin19, pBI101, pBinAR, pGPTV and pCAMBIA are preferably used according to the invention.
  • the vectors can first be linearized with restriction endonuclease (s) and then enzymatically modified in a suitable manner. The vector is then purified and an aliquot used for cloning. In cloning, the enzymatically cut and, if necessary, purified amplicon is linked to similarly prepared vector fragments using ligase.
  • a particular nucleic acid construct or vector or plasmid construct can have one or more codogenic gene segments.
  • the codogenic gene segments in these constructs are functionally linked to regulatory sequences.
  • the regulatory sequences include in particular plant sequences such as promoters and terminator sequences.
  • the constructs can advantageously be stably propagated in microorganisms, in particular in E.
  • nucleic acids used in the method can be introduced into plants and thus used in the transformation of plants, such as those published and there cited: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-119 (1993); FF White, Vectors for Gene Transfer to Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, 15-38; Genes Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds.
  • nucleic acids and / or vectors used in the process can thus be used for the genetic engineering modification of a broad spectrum of plants so that they become better and / or more efficient producers of PUFAs.
  • a de novo synthesis in a plant lacking the activity and ability to biosynthesize the compounds prior to introduction of the corresponding gene (s) is possible.
  • the use of various divergent, i. At the DNA sequence level of different sequences may be advantageous or the use of promoters, the other temporal gene expression, e.g. depending on the maturity level of a seed or oil-storing tissue.
  • the nucleic acid sequences used in the method are advantageously introduced into an expression cassette which enables expression of the nucleic acids in plants.
  • Desaturase, ⁇ -12-desaturase, ⁇ -3-desaturase or ⁇ -6-elongase encode, with one or more regulatory signals advantageously functionally linked to increase gene expression.
  • These regulatory sequences are intended to allow the targeted expression of genes and proteins. Depending on the host organism, this may mean, for example, that the gene is expressed and / or overexpressed only after induction, or that it is immediately expressed and / or overexpressed.
  • these regulatory sequences are sequences to which inducers or repressors bind and thereby regulate the expression of the nucleic acid. In addition to these new regulatory sequences or instead of these sequences, the natural regulatory elements of these
  • the gene construct may also contain one or more so-called enhancer sequences operably linked to the promoter, which allow for increased expression of the nucleic acid sequence
  • Additional advantageous sequences may also be inserted at the 3 'end of the DNA sequences, such as additional regulatory elements terminator sequences.
  • gene construct gene construct
  • only one copy of the genes is present in the expression cassette.
  • This gene construct or gene constructs can be expressed together in the host plant.
  • the gene construct or the gene constructs can be inserted in one or more vectors and be present freely in the cell or else be inserted in the genome. It is advantageous for the insertion of additional genes in the host genome when the genes to be expressed are present together in a gene construct.
  • the regulatory sequences or factors can, as described above, preferably positively influence the gene expression of the introduced genes and thereby increase them.
  • enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers.
  • an enhancement of the translation is possible by, for example, the stability of the mRNA is improved. It is possible in principle to use all natural promoters with their regulatory sequences, such as those mentioned above, for the new method. It is also possible and advantageous to use synthetic promoters in addition or alone, especially if they mediate seed-specific expression, such as those described in WO 99/16890.
  • the PUFA biosynthesis genes should advantageously be seed-specifically expressed in oilseeds.
  • seed-specific promoters can be used, or such promoters that are active in the embryo and / or in the endosperm.
  • Bce4 [WO 91/13980], legumes B4 (LegB4 promoter) [Bäumlein et al., Plant J., 2,2, 1992], Lpt2 and lpt1 (barley) [WO 95/15389 and US Pat , WO95 / 23230], seed-specific promoters from rice, maize and the like.
  • Plant gene expression can also be facilitated by a chemically inducible promoter (see review in Gatz 1997, Annu Rev. Plant Physiol Plant Mol. Biol., 48: 89-108).
  • Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter
  • each of the nucleic acids used in the process should be selected for ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 12-desaturase, ⁇ -3-desaturase or ⁇ 1-desaturase ⁇ 6-elongase encode under the control of its own, preferably one of the other promoters different promoter can be expressed as repetitive sequence motifs can lead to instability of the T-DNA or to recombination events.
  • the expression cassette is advantageously designed such that a promoter follows a suitable interface, advantageously in a polylinker, for insertion of the nucleic acid to be expressed, and optionally a terminator sequence lies behind the polylinker.
  • This sequence is repeated several times, preferably three, four, five, six or seven times, so that up to seven genes can be brought together in one construct and introduced into the transgenic plant for expression.
  • the sequence is repeated up to four times.
  • the nucleic acid sequences are inserted for expression via a suitable interface, for example in the polylinker downstream of the promoter.
  • each nucleic acid sequence has its own promoter and optionally its own terminator sequence.
  • Such advantageous constructs are disclosed for example in DE 101 02 337 or DE 101 02 338.
  • nucleic acid sequences behind a common promoter and possibly in front of a common terminator sequence.
  • the insertion site or the sequence of the inserted nucleic acids in the expression cassette is not of crucial importance, that is, a nucleic acid sequence may be inserted at the first or last position in the cassette, without thereby significantly affecting its expression.
  • different promoters such as the USP, LegB4 or DC3 promoter and different terminator sequences can be used in the expression cassette.
  • the transcription of the genes introduced should advantageously be effected by means of suitable terminator sequences at the 3 'end of the introduced biosynthesis gene. ne (behind the stop codon) are aborted.
  • suitable terminator sequences at the 3 'end of the introduced biosynthesis gene are aborted.
  • the OCS1 terminator sequence can be used here.
  • different terminator sequences should be used for each gene.
  • the gene construct can, as described above, also comprise other genes which are to be introduced into the plants. It is possible and beneficial in the
  • Host plants regulate genes such as genes for inducers, repressors or enzymes, which intervene by their enzyme activity in the regulation of one or more genes of a biosynthetic pathway to introduce and express.
  • genes may be of heterologous or homologous origin.
  • further biosynthesis genes of the fatty acid or lipid metabolism can advantageously be contained in the nucleic acid construct or gene construct, but these genes can also be located on one or more further nucleic acid constructs.
  • a gene selected from the group consisting of acyl-CoA dehydrogenase (s), acyl-ACP [ acyl carrier protein] desaturase (s), acyl-ACP thioesterase (s), as biosynthesis gene of the fatty acid or lipid metabolism, Fatty acid acyltransferase (s), acyl-CoA: lysophospholipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (n), fatty acid desaturase (s), fatty acid acetylenase (s), lipoxygenase (s), triacylglycerol lipase (s), allene oxide synthase (s), hydroperoxide lyase (s) or fatty acid elongase (s) or combinations thereof.
  • nucleic acid sequences are biosynthesis genes of the fatty acid or lipid metabolism selected from the group of acyl-CoA: lysophospholipid acyltransferase, ⁇ -8-desaturase, ⁇ -4-desaturase, ⁇ -9-desaturase, ⁇ -5 elongase and / or ⁇ -9 -Elongase.
  • acyl-CoA lysophospholipid acyltransferase
  • ⁇ -8-desaturase ⁇ -4-desaturase
  • ⁇ -9-desaturase ⁇ -5 elongase and / or ⁇ -9 -Elongase.
  • the abovementioned nucleic acids or genes can be cloned in combination with other elongases and desaturases in expression cassettes, such as those mentioned above, and used for the transformation of plants with the aid of Agrobacterium.
  • the regulatory sequences or factors can, as described above, preferably positively influence the gene expression of the introduced genes and thereby increase them.
  • enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers.
  • an enhancement of the translation is possible by, for example, the stability of the mRNA is improved.
  • the expression cassettes can be used in principle directly for introduction into the plant or else be introduced into a vector.
  • These advantageous vectors contain the nucleic acids used in the method which code for ⁇ -5-desaturase, ⁇ -6-desaturase, ⁇ -12-desaturase, ⁇ -3-desaturase or ⁇ -6 elongase
  • Nucleic acid construct containing the nucleic acid used alone or in combination with other biosynthesis genes of fatty acid or lipid metabolism such as the acyl-CoA: l_ysophospholipid acyltransferases, ⁇ -8-desaturases, ⁇ -9-desaturases, ⁇ -4-desaturases, ⁇ -5-elongases and / or ⁇ -9-elongases ,
  • vector refers to a nucleic acid molecule that can transport another nucleic acid that is bound to it.
  • plasmid a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector a viral vector, where additional DNA segments can be ligated into the viral genome.
  • Certain vectors can replicate autonomously in a host cell into which they have been introduced (e.g., bacterial origin of bacterial vectors). Other vectors are advantageously integrated into the genome of a host cell upon introduction into the host cell and thereby replicated together with the host genome.
  • certain vectors may direct the expression of genes to which they are operably linked.
  • expression vectors suitable for recombinant DNA techniques are in the form of plasmids.
  • plasmid and “vector” can be used interchangeably because the plasmid is the most commonly used vector form.
  • the invention is intended to encompass other forms of expression vectors, such as viral vectors that perform similar functions.
  • vector is intended to include other vectors known to those skilled in the art, such as phages, viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA.
  • the recombinant expression vectors advantageously used in the method comprise the nucleic acids or the gene construct according to the invention in a form suitable for expression of the nucleic acids used in a host cell, which means that the recombinant expression vectors have one or more regulatory sequences selected on the basis the host cells used for expression, operably linked to the nucleic acid sequence to be expressed.
  • operably linked means that the nucleotide sequence of interest is bound to the regulatory sequence (s) such that expression of the nucleotide sequence is possible and they are linked to each other such that both sequences fulfill the predicted function ascribed to the sequence (eg in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnolgy, CRC Press, Boca Raton , Florida, Ed .: Glick and Thompson, Chapter 7, 89-108, including references therein. Regulatory sequences include those which are constitutive Controlling expression of a nucleotide sequence in many types of host cells, and those that direct the direct expression of the nucleotide sequence only in certain host cells under certain conditions. Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed, the desired expression level of the protein, etc.
  • the ⁇ -12-desaturases, ⁇ -6-desaturases, ⁇ -3-Desatu lawns, ⁇ -6-elongases and / or ⁇ -5-desaturases in unicellular plant cells such as algae
  • unicellular plant cells such as algae
  • plant cells from higher plants eg, spermatophytes, such as crops
  • plant expression vectors include those described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left Biol.
  • a plant expression cassette preferably contains regulatory sequences that can direct gene expression in plant cells and that are operatively linked so that each sequence can fulfill its function, such as termination of transcription, for example, polyadenylation signals.
  • Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens T-DNA, such as the gene 3 of the Ti plasmid pTiACH ⁇ known as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835ff.) Or functional equivalents thereof, but all other terminator sequences functionally active in plants are also suitable.
  • a plant expression cassette preferably contains other operably linked sequences, such as translation enhancers, for example the overdrive sequence comprising the tobacco mosaic virus 5 'untranslated leader sequence encoding the protein / RNA ratio increased (GaIMe et al., 1987, Nucl. Acids Research 15: 8693-871 1).
  • translation enhancers for example the overdrive sequence comprising the tobacco mosaic virus 5 'untranslated leader sequence encoding the protein / RNA ratio increased (GaIMe et al., 1987, Nucl. Acids Research 15: 8693-871 1).
  • the gene to be expressed must be operably linked to a suitable promoter that will trigger gene expression in a timely, cell or tissue-specific manner.
  • suitable promoters are constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those derived from plant viruses, such as 35S CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also US 5352605 and WO 84/02913) or constitutive plant promoters, such as the Rubisco small subunit described in US 4,962,028. Plant gene expression can also be achieved via a chemically inducible promoter as described above (see an overview in Gatz 1997, Annu. Rev.
  • Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner.
  • Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.
  • Promoters which respond to biotic or abiotic stress conditions are also suitable, for example the pathogen-induced PRP1 gene promoter (Ward et al., Plant Mol. Biol. 22 (1993) 361-366), the tomato heat-inducible hsp ⁇ O promoter (US 5,187,267), the potato inducible alpha-amylase promoter (WO 96/12814) or the wound inducible pinll promoter (EP-A-0 375 091).
  • promoters which induce gene expression in tissues and organs in which the fatty acid, lipid and oil biosynthesis take place are preferred in sperm cells such as the cells of the endosperm and the developing embryo.
  • Suitable promoters are the rapeseed napin promoter (US Pat. No.
  • Suitable noteworthy promoters are the lpt2 or lpt1 gene promoter from barley (WO 95/15389 and WO 95/23230) or the promoters described in WO 99/16890 from the barley hordein gene, the rice glutelin gene, the rice oryzine gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum casirin gene, the rye -Secalin gene.
  • promoters which induce plastid-specific expression, since plastids are the compartment in which the precursors as well as some end products of lipid biosynthesis are synthesized.
  • Suitable promoters are the viral RNA polymerase promoter described in WO 95/16783 and WO 97/06250, and the Arabidopsis clpP promoter described in WO 99/46394.
  • promoters such as the plant promoters CaMV / 35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Ward et al., Plant. Biol. 22 (1993)], SSU, OCS, Iib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter.
  • inducible promoters such as those in EP-AO 388 186 (benzylsulfonamide-inducible), Plant J.
  • Plant promoters are the promoter of cytosolic FBPase or the ST-LSI Potato promoter (Stockhaus et al., EMBO J. 8, 1989, 2445), the glycine max phosphoribosyl-pyrophosphatamidotransferase promoter (Genbank Accession No. U87999) or the nodular-specific promoter described in EP-A-0 249 676.
  • Particularly advantageous promoters are promoters which enable expression in tissues involved in fatty acid biosynthesis.
  • seed-specific promoters such as the USP promoter according to the invention but also other promoters such as the LeB4, DC3, phaseolin or napin promoter.
  • promoters are seed-specific promoters which can be used for monocotyledonous or dicotyledonous plants and in US Pat. No. 5,608,152 (napin promoter from rapeseed), WO 98/45461 (oleosin promoter from Arobidopsis), US Pat. No. 5,504,200 (Phaseolin promoter from Phaseolus vulgaris ), WO 91/13980 (Bce4 promoter from Brassica), Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (LeB4 promoter from a legume), these promoters being for dicotyledons suitable.
  • promoters are suitable, for example, for barley monocotylone lpt-2 or ipt-1 promoter (WO 95/15389 and WO 95/23230), barley hordein promoter and other suitable promoters described in WO 99/16890.
  • the PUFA biosynthesis genes should advantageously be seed-specifically expressed in oilseeds.
  • seed-specific promoters can be used, or such promoters that are active in the embryo and / or in the endosperm.
  • seed-specific promoters can be isolated from both dicotolydone and monocotolydonous plants.
  • advantageous promoters are listed above, e.g. the USP, vicilin, napin, oleosin, phaseolin, Bce4, LegB4, Lpt2, lpt1, Amy32b, Amy 6-6, Aleurain or Bce4 promoter.
  • chemically inducible promoter can be used advantageously in the method according to the invention.
  • promoters which are advantageously suitable for expression in soya are the promoters of the ⁇ -conglycinin ⁇ subunit, the ⁇ -conglycinin ⁇ subunit, the Kunitz trypsin inhibitor, the annexin, the glysinin, the albumin 2S, the Legumin A1, Legumin A2 and BD30.
  • promoters are the USP, LegB4, Fad3, SBP, DC-3 or Cruciferin820 promoter.
  • Advantageous regulatory sequences used for expression of the nucleic acid sequences used in the method of the invention are terminators for the expression advantageous in soya are the Leg2A3 ', Kti3', Phas3 ', BD30 3' or the AIS3 '.
  • terminators are the A7T, OCS, LeB3T or cat terminator.
  • each of the nucleic acids used in the method should be used for the ⁇ -12-desaturase, ⁇ -3-desaturase, ⁇ -6-desaturase, ⁇ 6-elongase and / or ⁇ -5-desaturase can be expressed under the control of its own preferably a different promoter, since repetitive sequence motifs can lead to instability of the T-DNA or to recombination events.
  • the gene construct can, as described above, also comprise other genes which are to be introduced into the plant.
  • the regulatory sequences or factors used to express the nucleic acids used in the method according to the invention can, as described above, preferably have a positive influence on the gene expression of the introduced genes and thereby increase it.
  • These advantageous vectors contain the nucleic acids used in the method which code for ⁇ 12-desaturase, ⁇ -3-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase and / or ⁇ 5-desaturase, or a nucleic acid construct which contains the nucleic acid used alone or in combination with further biosynthesis genes of the fatty acid or lipid metabolism, such as the acyl-CoA: lysophospholipid acyltransferases, ⁇ -3-desaturases, ⁇ -4-desaturases, ⁇ -5-desaturases , ⁇ -6-desaturases, ⁇ -8-desatuases, ⁇ -9-desaturases, ⁇ -12-desaturases, ⁇ 3-desaturases, ⁇ -5-elongases, ⁇ -6-elongases and / or ⁇ -9-elongases.
  • the term "vector” refers to a nucleic acid molecule that can
  • the recombinant expression vectors used can be designed for the expression of ⁇ -12-desaturases, ⁇ -3-desaturases, ⁇ -6-desaturases, ⁇ -6-elongases and / or ⁇ -5-desaturases in prokaryotic or eukaryotic cells. This is advantageous because intermediate steps of the vector construction are often carried out in microorganisms for the sake of simplicity.
  • the ⁇ 12-desaturase, ⁇ -3-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase and / or ⁇ 5-desaturase genes can be expressed in bacterial cells, insect cells (using baculovirus - expression vectors), yeast and other fungal cells (see Romanos, MA, et al., (1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488; van den Hondel, CAMJJ, et al ) "Heterologous gene expression in filamentous fungi", in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure, eds., Pp.
  • fusion expression vectors include i.a. pGEX (Pharmacia Biotech Inc., Smith, DB, and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), in which glutathione-S Transferase (GST),
  • Maltose E-binding protein or protein A is fused to the recombinant target protein.
  • Target gene expression from the pTrc vector is based on transcription by host RNA polymerase from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 1 1d vector is based on transcription from a T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from a resident ⁇ prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
  • vectors useful in prokaryotic organisms are known to those of skill in the art, for example, in E. coli pLG338, pACYC184, the pBR series such as pBR322, the pUC series such as pUC18 or pUC19, the M1 13mp series, pKC30, pRep4 , pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pN-1111 13-B1, ⁇ gt1 1 or pBdCI, in Streptomyces plJ101, plJ364, plJ702 or plJ361, in Bacillus pUB1 10, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in the yeast S. cerevisiae include pYeDesaturased (Baldari et al. (1987) Embo J. 6: 229-234), pMFa (Kurjan and
  • Vectors and methods for constructing vectors suitable for use in other fungi, such as filamentous fungi include those described in detail in: van den Hondel, C.A.M.J.J., & Punt, PJ. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, JF Peberdy et al., Eds., Pp.
  • yeast vectors are, for example, pAG-1, YEp6, YEpI 3 or pEMBLYe23.
  • a plant expression cassette preferably contains regulatory sequences that can direct gene expression in plant cells and are operably linked so that each sequence can perform its function, such as termination of transcription, for example, polyadenylation signals.
  • Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens T-DNA, such as the gene 3 of the Ti plasmid pTiACH ⁇ known as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835ff.) Or functional equivalents thereof, but also all other terminators functionally active in plants are suitable.
  • a plant expression cassette preferably contains other operably linked sequences, such as translation enhancers, e.g., the overdrive sequence containing the 5'-untranslated tobacco mosaic virus leader sequence, which is the protein / RNA ratio increases (GaIMe et al., 1987, Nucl. Acids Research 15: 8693-871 1).
  • translation enhancers e.g., the overdrive sequence containing the 5'-untranslated tobacco mosaic virus leader sequence, which is the protein / RNA ratio increases (GaIMe et al., 1987, Nucl. Acids Research 15: 8693-871 1).
  • 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 available from Plant viruses such as 35S CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also US 5352605 and WO 84/02913) or plant promoters such as the Rubisco small subunit described in US 4,962,028.
  • telomeres are preferred sequences necessary to direct the gene product into its corresponding cell compartment (see review in Kermode, Crit., Plant, 15, 4 (1996) 285) -423 and references cited therein), for example to the vacuole, the nucleus, all types of plastids such as amyloplasts, chloroplasts, chromoplasts, extracellular space, mitochondria, endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
  • plastids such as amyloplasts, chloroplasts, chromoplasts, extracellular space, mitochondria, endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
  • Plant gene expression can also be facilitated by a chemically inducible promoter as described above (see review in Gatz 1997, Annu Rev. Plant Physiol Plant Mol. Biol., 48: 89-108).
  • Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.
  • Promoters which react to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-induced PRP1 gene promoter (Ward et al., Plant Mol. Biol. 22 (1993) 361-366), the heat-inducible hsp ⁇ O promoter Tomato (US 5,187,267), the potato inducible alpha-amylase promoter (WO 96/12814) or the wound inducible pinll promoter (EP-A-0 375 091).
  • those promoters which induce gene expression in tissues and organs in which the fatty acid, lipid and oil biosynthesis take place are preferred in sperm cells such as the cells of the endosperm and the developing embryo.
  • Suitable promoters are the rapeseed napkin promoter (US 5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67), the Arabidopsis oleosin promoter (US Pat.
  • phaseolin promoter from Phaseolus vulgaris (US 5,504,200), the Brassica Bce4 promoter (WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9) as well as promoters which induce seed-specific expression in monocotyledonous plants such as corn, barley, wheat, rye, rice, etc.
  • Suitable noteworthy promoters are the lpt2 or lpt1
  • Barley gene promoter (WO 95/15389 and WO 95/23230) or the promoters described in WO 99/16890 from the barley hordein gene, the rice glutelin gene, the rice oryzine gene, the rice Proline protein, wheat gliadin gene, wheat glutelin gene, maize zein gene, oat glutelin gene, sorghum kasirin gene, rye secalin gene).
  • multiparallel expression of ⁇ -5-desaturase, ⁇ -6-desaturase, ⁇ -12-desaturase, ⁇ -3-desaturase or ⁇ -6 elongase used in the method may be desired.
  • the introduction of such expression cassettes can be carried out via a simultaneous transformation of a plurality of individual expression constructs or preferably by combining a plurality of expression cassettes on a construct. It is also possible to transform a plurality of vectors each having a plurality of expression cassettes and to transfer them to the host cell. For the purposes of the invention it is also possible to introduce genes into different plants and to combine them by crossing.
  • Other preferred sequences for use in the functional compound in plant gene expression cassettes are targeting sequences used to direct the gene product into its corresponding cell compartment, for example into the vacuole, the nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, mitochondria, endoplasmic reticulum, oil bodies, peroxisomes, and other compartments of plant cells are necessary (see review in Kermode, Crit., Rev. Plant Sci., 15, 4 (1996) 285-423 and references cited therein).
  • Expression cassette or vector all such constructions realized by genetic engineering methods in which either a) the nucleic acid sequence according to the invention, or b) a genetic control sequence functionally linked to the nucleic acid sequence according to the invention, for example a promoter, or
  • Natural genetic environment means the natural genomic or chromosomal locus in the source organism or presence in a genomic library.
  • the natural genetic environment of the nucleic acid sequence is preferably at least partially conserved.
  • the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, more preferably at least 1000 bp, most preferably at least 5000 bp.
  • non-natural, synthetic ("artificial") methods such as mutagenesis.
  • transgenic plants are therefore to be understood as meaning that the nucleic acids used in the process are not in their natural position in the genome of the plant, it being possible for the nucleic acids to be expressed homologously or heterologously.
  • transgene also means that the nucleic acids according to the invention are in their natural place in the genome of the plant, but that the sequence has been changed from the natural sequence and / or that the
  • Transgenic is preferably understood to mean the expression of the nucleic acids according to the invention or the nucleic acid sequences used in the method according to the invention at a non-natural site in the genome, that is to say a homologous or preferably heterologous expression of the nucleic acids.
  • Preferred transgenic plants are oilseed or oilseed crops and especially their various plant parts.
  • Plant is derived and / or can be used to produce the transgenic plant.
  • Plants in the process according to the invention include whole plants and all plant parts, plant organs or plant parts such as leaves, stems, seeds, roots, tubers, anthers, fibers, root hairs, stems, embryos, callosis, kotelydons, petioles, crop material, plant tissue, reproductive tissue, Cell cultures that can be derived from the transgenic plant and / or used to produce the transgenic plant.
  • the seed includes all seed parts such as the seed shells, epidermis and sperm cells, endosperm or embryonic tissue.
  • the process according to the invention is also suitable for producing polyunsaturated fatty acids, in particular ARA, EPA and / or their
  • Mixtures in plant cell cultures and subsequent extraction of fatty acids from the cultures may in particular be suspension or callus cultures.
  • the compounds prepared in the process according to the invention can also be advantageously isolated from the plants from the plant seeds in the form of their oils, fat, lipids and / or free fatty acids.
  • Produced by this method Polyunsaturated fatty acids can be harvested by harvesting the plants or plant seeds either from the culture in which they grow or from the field.
  • this method further comprises the step of recovering the oils, lipids or free fatty acids from the plant or from the culture.
  • the culture may be, for example, a greenhouse or field crop of a plant.
  • the isolation of the oils, lipids or free fatty acids can be carried out by pressing or extraction of the plant parts, preferably the plant seeds.
  • the oils, fats, lipids and / or free fatty acids by so-called cold beat or cold pressing can be obtained without supplying heat by pressing.
  • the plant parts, especially the seeds, to be easier to digest they are first crushed, steamed or roasted.
  • the thus pretreated seeds can then be pressed or extracted with solvents such as warm hexane. Subsequently, the solvent is removed again. Thereafter, the products thus obtained, which contain the polyunsaturated fatty acids, further processed, that is refined. First of all, for example, the mucilages and turbid matter are removed.
  • the so-called degumming can be carried out enzymatically or, for example, chemically / physically by adding acid, such as phosphoric acid. Subsequently, the free fatty acids are removed by treatment with a base, for example sodium hydroxide solution. The product obtained is thoroughly washed with water to remove the lye remaining in the product and dried. In order to remove the dyes still contained in the product, the products are subjected to bleaching with, for example, bleaching earth or activated carbon. Finally, the product is deodorized, for example, with steam.
  • the oils, lipids, fatty acids or fatty acid mixtures obtained after pressing according to the invention are referred to as so-called crude oils.
  • Such compounds are the various tocopherols such as ⁇ -tocopherol, ⁇ -tocopherol, ⁇ -tocopherol and / or ⁇ -tocopherol or phytosterols such as brassicasterol, campesterol, stigmasterol, ⁇ -sitosterol, sitostanol, ⁇ 5 -avenasterol,
  • ⁇ 5 24-stigmastadienol, ⁇ 7 -stigmastenol or ⁇ 7 -avenasterol.
  • These compounds are contained in a range of 1 to 1000 mg / 100 g, advantageously from 10 to 800 mg / 100 g of lipid or oil.
  • Triterpenes such as germaniol, amyrin, cycloartanol and others may also be included in these lipids and oils.
  • lipids and / or oils contain in the process polyunsaturated fatty acids such as ARA, EPA and / or DHA bound in polar and nonpolar lipids such as phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, galactolipids, monoglycerides, diglycerides or triglycerides only to name a few. Lysophospholipids may also be present in the lipids and / or oils. These components of the lipids and / or oils may be replaced by suitable Methods are separated from each other. Not included in these crude oils is cholesterol.
  • polyunsaturated fatty acids such as ARA, EPA and / or DHA bound in polar and nonpolar lipids
  • phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinos
  • oils, lipids, fatty acids and / or fatty acid composition in feed, food, cosmetics or pharmaceuticals.
  • the oils, lipids, fatty acids or fatty acid mixtures according to the invention may be mixed with other oils, lipids, fatty acids or fatty acid mixtures of animal origin, such as those described in the art, for example.
  • Fish oils are used. Typical of such fish oils short-chain fatty acids such as C12: 0, C14: 0, C14: 1, branched chain C15: 0, C15: 0, C16: 0 or C16: 1.
  • polyunsaturated C16 fatty acids such as C16: 2, C16: 3 or C16: 4, branched chain C17: 0, C17: 1, branched chain C18: 0 and C19: 0 and C19: 0 and C19: 1 are found in fish oil.
  • Such fatty acids are typical of fish oils and are rarely or not found in vegetable oils.
  • Economically relevant fish oils are e.g. Anchovy oil, menhadne oil, tuna oil, sardine oil, herring oil, marjoram oil, whale oil and salmon oil.
  • oils and / or oils of animal origin may be used for blending with the oils of the invention in the form of crude oils, that is in the form of lipids and / or oils that have not yet been purified, or differentially purified fractions may be used for blending .
  • the oils, lipids, fatty acids or fatty acid mixtures according to the invention may be mixed with other oils, lipids, fatty acids or fatty acid mixtures of animal origin, such as those described in the art, for example. Fish oils are used. These oils, lipids, fatty acids or fatty acid mixtures, which consist of vegetable and animal components, can be used for the production of feed, food, cosmetics or pharmaceuticals.
  • oil is understood as meaning a fatty acid mixture which contains unsaturated, saturated, preferably esterified fatty acid (s). It is preferred that the oil, lipid or fat has a high proportion of polyunsaturated free or advantageously esterified fatty acid (s), in particular linoleic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, ⁇ -linolenic acid, stearidonic acid or eicosatetraenoic acid , Preferably, the proportion of unsaturated esterified fatty acids is about 30%, more preferred is a proportion of 50%, even more preferred is a proportion of 60%, 70%, 80%, 85% or more.
  • the proportion of fatty acid after transfer of the fatty acids into the methyl esters can be determined by gas chromatography by transesterification.
  • the oil, lipid or fat may contain various other saturated or unsaturated fatty acids, e.g. Palmitin, palmitoleic, stearic, oleic acid, etc., included.
  • the proportion of the various fatty acids in the oil or fat may vary depending on the starting plant.
  • the polyunsaturated fatty acids produced in the process are, for example, sphingolipids, phosphoglycerides, lipids, as described above. Glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.
  • the polyunsaturated fatty acids containing, for example, via an alkali treatment, for example, aqueous KOH or NaOH or acid hydrolysis advantageously in the presence of an alcohol such as methanol or ethanol or by enzymatic elimination release and isolate over, for example Phase separation and subsequent acidification over eg H 2 SO 4 .
  • an alkali treatment for example, aqueous KOH or NaOH or acid hydrolysis
  • an alcohol such as methanol or ethanol
  • enzymatic elimination release and isolate over, for example Phase separation and subsequent acidification over eg H 2 SO 4 .
  • the release of the fatty acids can also be carried out directly without the workup described above.
  • the polyunsaturated fatty acids produced in the plants used in the process can in principle be increased in two ways. Either the pool of free polyunsaturated fatty acids and / or the proportion of the esterified polyunsaturated fatty acids produced by the process can be increased.
  • the process according to the invention increases the pool of esterified polyunsaturated fatty acids in the transgenic organisms.
  • nucleic acid sequences used in the method according to the invention are derived from a eukaryotic organism such as a plant, a microorganism such as an alga or an animal.
  • the nucleic acid sequences preferably come from the order Salmoniformes, Xenopus or Ciona, algae such as Mantoniella, Crypthecodinium, Euglena or Ostreococcus, fungi such as the genus Phytophtora or diatoms such as the genera Thalassiosira or Phaeodactylum.
  • Nucleic acids useful in the method are derived from bacteria, fungi, diatoms, animals such as Caenorhabditis or Oncorhynchus or plants such as algae or mosses such as the genera Shewanella, Physcomitrella, Thraustochytrium, Fusarium, Phytophthora, Ceratodon, Mantoniella, Ostreococcus, Isochrysis, Aleurita, Muscarioides, Mortierella , Borago, Phaeodactylum, Crypthecodinium, especially from the genera and species Oncorhynchus mykiss, Xenopus laevis, Ciona intestinalis,
  • Thalassiosira pseudonona Mantoniella squamata, Ostreococcus sp., Ostreococcus tauri, Euglena gracilis, Physcomitrella patens, Phytophthora infestans, Fusarium graminaeum, Cryptocodinium cohnii, Ceratodon purpureus, Isochrysis galbana, Aleurita farinosa, Thraustochytrium sp., Muscarioides viallii, Mortierella alpina, Borago officinalis, Phaeodactylum tricornutum, Caenorhabditis elegans, or more particularly from Oncorhynchus mykiss, Euglena gracilis, Thalassiosira pseudonana or Crypthecodinium cohnii.
  • the cloning methods e.g. Restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of Escherichia coli cells, culture of bacteria and sequence analysis of recombinant DNA were performed as described in Sambrook et al. (1989) (CoId Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
  • Example 2 Sequence Analysis of Recombinant DNA Sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al. (1977) Proc. Natl. Acad., See, USA74, 5463-5467 ). Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.
  • Bin19, pBI101, pBinAR, pGPTV and pCAMBIA are preferably used according to the invention for the following examples.
  • a pGPTV derivative was used as described in DE10205607. This vector differs from pGPTV by an additional inserted / Ascl restriction site.
  • the starting point of the cloning was the cloning vector pUC19 (Maniatis et al.). In the first step, the conlinin promoter fragment was amplified with the following primers:
  • composition of the PCR mixture (50 ⁇ l):
  • the OCS terminator (Genbank Accession V00088; De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Chem.
  • OCS_C 5 ' aggcctccatggcctgctttaatgagatatgcgagacgcc
  • OCS_C 3' cccgggccggacaatcagtaaattgaacggag
  • composition of the PCR mixture (50 ⁇ l):
  • the PCR product was first incubated for 2 h at 37 0 C with the restriction enzyme Stu ⁇ and then for 12 h at 25 0 C with the restriction enzyme Sma ⁇ .
  • the vector pUC19-CnH-C was incubated for 12 h at 25 0 C with the restriction enzyme Sma ⁇ .
  • the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised.
  • the Purification of the DNA was carried out using Qiagen Gel Purification Kit according to the manufacturer.
  • vector and PCR product were ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmid pUC19-Cnl1C_OCS was verified by sequencing.
  • the CnH-B promoter was amplified by PCR using the following primers:
  • the OCS terminator for CnII B was inserted.
  • the PCR was carried out with the following primers:
  • OCS2 5 ' aggcctcctgctttaatgagatatgcgagac
  • OCS2 3' cccgggcggacaatcagtaaattgaacggag
  • composition of the PCR mixture (50 ⁇ l):
  • Annealing temperature 1 min 55 0 C denaturation temperature: 1 min 94 0 C elongation temperature: 2 min 72 0 C number of cycles: 35
  • the PCR product was first incubated for 2 h at 37 0 C with the restriction enzyme Stu ⁇ and then for 12 h at 25 0 C with the restriction enzyme Sma ⁇ .
  • the vector pUC19-Cnl1C_Cnl1 B_OCS was incubated for 12 h at 25 0 C with the restriction enzyme Sma ⁇ .
  • the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised.
  • the purification of the DNA was carried out using the Qiagen Gel Purification Kit according to the manufacturer's instructions.
  • vector and PCR product were ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmid pUC19-Cnl1C_Cnl1 B_OCS2 was verified by sequencing.
  • the CnH-A promoter was amplified by PCR using the following primers:
  • composition of the PCR mixture (50 ⁇ l):
  • Annealing temperature 1 min 55 0 C denaturation temperature: 1 min 94 0 C elongation temperature: 2 min 72 0 C number of cycles: 35
  • the PCR product was incubated for 2 h at 37 0 C with the restriction enzyme Stu ⁇ .
  • the vector pUC19-Cnl1-C was incubated for 12 h at 25 0 C with the restriction enzyme Sma ⁇ .
  • the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised.
  • the purification of the DNA was carried out using the Qiagen Gel Purification Kit according to the manufacturer's instructions.
  • vector and PCR product were ligated. To Roche's Rapid Ligation Kit was used.
  • the resulting plasmid pUC19-Cnl1C_Cnl1 B_Cnl1A_OCS2 was verified by sequencing.
  • the OCS terminator for CnIIA was inserted.
  • the PCR was carried out with the following primers:
  • OCS2 5 ' ggcctcctgctttaatgagatatgcga
  • OCS2 3 ' aagcttggcgcgccgagctcgtcgacggacaatcagtaaattgaacggaga
  • composition of the PCR mixture (50 ⁇ l):
  • Annealing temperature 1 min 55 0 C denaturation temperature: 1 min 94 0 C elongation temperature: 2 min 72 0 C number of cycles: 35
  • the PCR product was first incubated for 2 h at 37 0 C with the restriction enzyme Stu ⁇ and then for 2 h at 37 0 C with the restriction enzyme H / ⁇ dlM.
  • the vector pUC19-Cnl1C_Cnl1 B_Cnl1A_OCS2 was incubated for 2 h at 37 0 C with the restriction enzyme Stu ⁇ and for 2 h at 37 0 C with the restriction enzyme H / ⁇ dlM.
  • the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised.
  • the purification of the DNA was carried out using the Qiagen Gel Purification Kit according to the manufacturer's instructions.
  • the plasmid pUC19-Cnl1C_Cnl1 B_Cnl1A_OCS3 was used in the next step to clone the ⁇ 6, ⁇ 5-desaturase and ⁇ 6-elongase.
  • the ⁇ 6-desaturase was amplified from phytium irregular (WO02 / 26946) with the following PCR primers:
  • the PCR product was first incubated for 2 h at 37 0 C with the restriction enzyme BgIW and then for 2 h at 37 0 C with the restriction enzyme ⁇ / col.
  • the vector pUC19-Cnl1C_Cnl1 B_Cnl1A_OCS3 was incubated for 2 h at 37 0 C with the restriction enzyme BgIW and for 2 h at 37 0 C with the restriction enzyme ⁇ / col.
  • the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised.
  • the purification of the DNA was carried out by means of Qiagen Gel Purification Kit according to
  • the plasmid pUC19-Cnl1_d6Des (Pir) was in the next step for cloning the ⁇ 5-desaturase from Thraustochytrium ssp. (WO02 / 26946).
  • the ⁇ 5-desaturase from Thraustochytrium ssp. amplified with the following PCR primers:
  • the purification of the DNA was carried out using the Qiagen Gel Purification Kit according to the manufacturer's instructions. Subsequently, vector and PCR product were ligated. The Rapid Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1_d6Des (Pir) _d5Des (Tc) was verified by sequencing.
  • the plasmid pUC19-Cnl1_d6Des (Pir) _d5Des (Tc) was used in the next step for cloning the ⁇ 6 elongase from Physcomitrella patens (WO01 / 59128) for which it was amplified with the following PCR primers:
  • composition of the PCR mixture (50 ⁇ l):
  • the PCR product was first incubated for 2 h at 37 0 C with the restriction enzyme Not ⁇ and then for 2 h at 37 0 C with the restriction enzyme Xba ⁇ .
  • the vector pUC19-Cnl1_d6Des (Pir) _d5Des (Tc) was incubated for 2 h at 37 0 C with the restriction enzyme Not ⁇ and for 2 h at 37 0 C with the restriction enzyme Xba ⁇ .
  • the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised.
  • the purification of the DNA was carried out by means of Qiagen Gel Purification Kit according to
  • the binary vector for plant transformation was prepared.
  • the pUC19 Cnl1_d6Des (Pir) _d5Des (Tc) _D6Elo (Pp) was incubated for 2 h at 37 0 C with the restriction enzyme Asc ⁇ .
  • the vector pGPTV was treated in the same way.
  • composition of the PCR mixture (50 ⁇ l):
  • Annealing temperature 1 min 55 0 C denaturation temperature: 1 min 94 0 C elongation temperature: 2 min 72 0 C number of cycles: 35
  • the PCR product was incubated for 2 h at 37 0 C with the restriction enzyme Sa / l.
  • the vector pUC19 was incubated for 2 h at 37 0 C with the restriction enzyme Sa / l.
  • the PCR product and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised.
  • the purification of the DNA was carried out using the Qiagen Gel Purification Kit according to the manufacturer's instructions.
  • vector and PCR product were ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmid pUC19-Cnl1_OCS was verified by sequencing.
  • ⁇ 12-desaturase gene from Calendula officinalis (WO01 / 85968) was cloned into pUC19-Cn17_OCS.
  • d12Des (Co) was amplified with the following primers:
  • composition of the PCR mixture 50 ⁇ l: 5.00 ⁇ l template cDNA
  • Annealing temperature 1 min 55 0 C denaturation temperature: 1 min 94 0 C elongation temperature: 2 min 72 0 C number of cycles: 35
  • the PCR product was incubated for 2 h at 37 0 C with the restriction enzyme BgIW and then for 2 h at the same temperature with ⁇ / col.
  • the vector pUC19-Cnl1_OCS was incubated in the same way. Subsequently, the PCR fragment and the cut vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The purification of the DNA was carried out using the Qiagen Gel Purification Kit according to the manufacturer's instructions. Subsequently, vector and PCR product were ligated. The Rapid Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1_D12Des (Co) was verified by sequencing.
  • the plasmid pUC19-Cnl1_D12Des (Co), as well as the plasmid pUC19-Cnl1_d6Des (Pir) _d5Des (Tc) _D6Elo (Pp) were incubated for 2 h at 37 0 C with the restriction enzyme Sa / l. Subsequently, the vector fragment and the vector were separated by agarose gel electrophoresis and the corresponding DNA fragments were excised. The purification of the DNA was carried out using the Qiagen Gel Purification Kit according to the manufacturer's instructions. Subsequently, vector and vector fragment were ligated. The Rapid Ligation Kit from Roche was used for this purpose. The resulting plasmid pUC19-Cnl1_d6Des (Pir) _d5Des (Tc) _D6Elo (Pp) _D12Des (Co) was verified by sequencing.
  • the binary vector for plant transformation was prepared.
  • the pUC19 Cnl1_d6Des was incubated with the restriction enzyme Asc ⁇ (Pir) _d5Des (Tc) _D6Elo (Pp) _D12Des (Co) for 2 h at 37 0 C.
  • the vector pGPTV was treated in the same way.
  • pGPTV-Cnl1_d6Des (Pir) _d5Des (Tc) _D6Elo (Pp) _D12Des (Co) was verified by sequencing.
  • Another vector suitable for plant transformation is pSUN2.
  • this vector was used in combination with the Gateway system (Invitrogen, Düsseldorf).
  • the gateway cassette A was inserted into the vector pSUN2 according to the manufacturer's instructions as follows:
  • the pSUN2 vector (1 ⁇ g) was incubated for 1 h with the restriction enzyme EcoRV at 37 °. Subsequently, the Gateway cassette A (Invitrogen, Düsseldorf) was ligated into the cut vector by means of the Rapid Ligation Kit of Roche, Mannheim. The resulting plasmid was transformed into E. coli DB3.1 cells (Invitrogen). The isolated plasmid pSUN-GW was subsequently verified by sequencing.
  • the expression cassette from pUC19-Cn11_d6Des (Pir) _d5Des (Tc) _D6Elo (Pp) _D12Des (Co) was excised by Ascl and ligated into the similarly treated vector pSUN-GW.
  • Binary vectors in Agrobacterium tumefaciens C58C1: pGV2260 or Escherichia coli can be used to generate transgenic rape plants (Deblaere et al., 1984, Nucl. Acids. Res. 13, 4777-4788).
  • oilseed rape plants Var Drakkar, NPZ Nor Weg plant breeding, Hohenlieth, Germany
  • Petioles or hypocotyls of freshly germinated sterile rape plants are incubated in a Petri dish with a 1:50 agrobacterial dilution for 5-10 minutes. This is followed by a 3-day colncubation in darkness at 25 ° C on 3MS medium with 0.8% Bacto agar. Cultivation is continued after 3 days with 16 hours light / 8 hours darkness and weekly on MS medium with 500 mg / L claforan (Cefotaxime sodium), 50 mg / L kanamycin, 20 microM benzylaminopurine (BAP) and 1, 6 g / l glucose continued. Growing shoots are transferred to MS medium with 2% sucrose, 250 mg / L claforan and 0.8% Bacto agar. If roots do not form after three weeks, then 2-indolebutyric acid was added to the medium as growth hormone for rooting.
  • 2-indolebutyric acid was added to the medium as growth hormone for rooting.
  • Regenerated shoots are obtained on 2MS medium with kanamycin and claforan, transferred into soil after rooting and grown in a climatic chamber or greenhouse after cultivation for two weeks, flowered, harvested mature seeds and for elongase expression such as ⁇ 6-elongase activity or ⁇ -3-desaturase activity by means of lipid analyzes. Lines with elevated levels of C20 and C22 polyunsaturated fatty acids can thus be identified. b) Preparation of transgenic Camelina plants
  • Agrobacterium tumefaciens strain C58 was transformed by electroporation with PUFA vector 81, 191 and 192. Inoculation of explants from Camelina seedlings (age> 1 week) grown on MS medium with agrobacteria was performed. After two weeks of coculture, the plants were washed to remove agrobacteria and then transferred to regeneration medium with optimized BaP and NAA. After a further two days of regeneration, optimized amounts of kanamycin were added. This selection pressure was maintained for 12 days. The shoot regeneration was initiated by transfer to kanamycin-free BaP-containing medium. Sprouting was completed> 3 weeks after inoculation and rooting was induced on medium with NAA.
  • Sprouts were transferred to soil after rooting and grown in a climatic chamber or greenhouse, flowered, harvested mature seeds and examined for elongase expression such as ⁇ 6-elongase activity or ⁇ -3-desaturase activity by lipid analysis. Lines with elevated levels of polyunsaturated fatty acids can be identified.
  • the effect of genetic modification in plants on the production of a desired compound can be determined by cultivating the modified plant under suitable conditions (such as those described above) and increasing the medium and / or cellular components to increased production of the desired product (ie the lipids or a fatty acid).
  • suitable conditions such as those described above
  • suitable conditions such as those described above
  • suitable conditions such as those described above
  • medium and / or cellular components to increased production of the desired product (ie the lipids or a fatty acid).
  • analytical techniques are well known to those skilled in the art and include spectroscopy, thin layer chromatography, staining methods of various types, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and p.
  • Analyzes include measurements of nutrient levels in the medium (eg, sugars, hydrocarbons, nitrogen sources, phosphate, and other ions), measurements, etc., used to produce the desired compound, such as by-products and by-products biomass composition and growth, analysis of production of common metabolites of biosynthetic pathways and measurements of gases generated during fermentation Standard methods for these measurements are in Applied Microbial Physiology, A Practical Approach, PM Rhodes and PF Stanbury, Hrsgb., IRL Press , Pp. 103-129, 131-163 and 165-192 (ISBN: 0199635773) and L mentioned therein Iteraturstellen described.
  • fatty acids abbreviations: FAME, fatty acid methyl ester, GC-MS, gas-liquid chromatography-mass spectrometry, TAG, triacylglycerol, TLC, thin-layer chromatography.
  • FAME fatty acid methyl ester
  • GC-MS gas-liquid chromatography-mass spectrometry
  • TAG triacylglycerol
  • TLC thin-layer chromatography
  • the material to be analyzed may be broken up by sonication, milling in the glass mill, liquid nitrogen and milling or other applicable methods.
  • the material must be centrifuged after rupture.
  • the sediment is distilled in aqua. re-suspended, heated at 100 ° C for 10 min, cooled on ice and recentrifuged, followed by extraction into 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 h at 90 ° C resulting in hydrolyzed oil and lipid compounds. which give transmethylated lipids.
  • fatty acid methyl esters are extracted into petroleum ether and finally subjected to GC analysis using a capillary column (Chrompack, WCOT Fused silica, CP-Wax-52 CB, 25 microm, 0.32 mm) at a temperature gradient between 170 ° C and 240 ° C 20 min and 5 min at 240 ° C subjected.
  • Chropack Chrompack, WCOT Fused silica, CP-Wax-52 CB, 25 microm, 0.32 mm
  • the identity of the resulting fatty acid methyl esters must be defined using standards available from commercial sources (i.e., Sigma).
  • Plant material is first mechanically homogenized by mortars to make it more accessible to extraction. The mixture is then heated for 10 min at 100 ° C and sedimented again after cooling on ice. The cell sediment is hydrolyzed with 1 M methanolic sulfuric acid and 2% dimethoxypropane for 1 h at 90 ° C. and the lipids are transmethylated. The resulting fatty acid methyl esters (FAME) are extracted into petroleum ether.
  • FAME fatty acid methyl esters
  • the extracted FAME are purified by gas chromatography using a capillary column (Chrompack, WCOT Fused silica, CP-Wax-52CB, 25 m, 0.32 mm) and a temperature gradient from 170 ° C to 240 ° C in 20 min and 5 min at 240 ° C analyzed.
  • the identity of the fatty acid methyl esters is confirmed by comparison with corresponding FAME standards (Sigma).
  • the identity and the position of the double bond can be further analyzed by GC-MS by suitable chemical derivatization of the FAME mixtures, for example to give 4,4-dimethoxoxazoline derivatives (Christie, 1998).
  • Example 5 the seeds of the plants transformed with the constructs pGPTV-Cnl1_d6Des (Pir) _d5Des (Tc) _D6Elo (Pp) _D12Des (Co), pSUN-5G and pSUN-8G were analyzed. Compared to control plants that were not transformed (wild-type control, WT), a clear change in the fatty acid spectrum was observed. This showed that the transformed genes are functional. Table 1 summarizes the results.
  • Tab. 2 Fatty acid analysis of transgenic seeds which were transformed with the construct pSUN-5G.
  • Table 3 Gas chromatographic analysis of seed material from Camelina sativa, L. The individual fatty acids are given in area percent.

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Abstract

La présente invention concerne un procédé de fabrication d'acide arachidonique (= ARA) ou d'acide eicosapentaénoïque (= EPA) ou d'acide arachidonique et d'acide eicosapentaénoïque de manière avantageuse dans des semences de plantes transgéniques de la famille des Brassicaceae avec une teneur en ARA ou EPA ou ARA et EPA supérieure ou égale à 3 % en poids par rapport à la teneur totale en lipide de la plante transgénique, selon lequel des acides nucléiques sont introduits dans l'organisme, lesdits acides nucléiques codant pour des polypeptides présentant une activité ?-6-désaturase-, ?-6-élongase- et ?-5-désaturase. Selon l'invention, l'activité enzymatique de l'enzyme introduite permet de réduire un acide gras choisi dans le groupe constitué des acides gras acide oléique [C18:1<SUP>?9</SUP>], acide linoléique [C18:2<SUP>?9, 12</SUP>], acide a-linoléique [C18:3<SUP>?6, 9, 12</SUP>], acide icosénique (20:1<SUP>?11</SUP>) et acide érucique [C22:1<SUP>?13</SUP>]<SUP/>d'au moins 10 % par rapport à la plante de type sauvage non transgénique. De manière avantageuse, des enzymes supplémentaires, choisies dans le groupe comprenant les enzymes ?-3-désaturase, ?-12-désaturase, ?-6-désaturase, ?-6-élongase, ?-5-désaturase, ?-5-élongase et/ou ?-4-désaturase, peuvent être introduites dans les plantes.
EP07787358A 2006-07-21 2007-07-11 Procédé de fabrication d'acide arachidonique et/ou d'acide eicosapentaénoïque dans des plantes Withdrawn EP2046960A1 (fr)

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DE102006034313A DE102006034313A1 (de) 2006-07-21 2006-07-21 Verfahren zur Herstellung von Arachidonsäure und/oder Eicosapentaensäure
PCT/EP2007/057084 WO2008009600A1 (fr) 2006-07-21 2007-07-11 Procédé de fabrication d'acide arachidonique et/ou d'acide eicosapentaénoïque dans des plantes

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CA2563875C (fr) 2004-04-22 2015-06-30 Commonwealth Scientific And Industrial Research Organisation Synthese d'acides gras polyinsatures a chaine longue par des cellules de recombinaison
CN102559364B (zh) 2004-04-22 2016-08-17 联邦科学技术研究组织 用重组细胞合成长链多不饱和脂肪酸
AU2013202498B2 (en) * 2006-08-24 2015-04-30 Basf Plant Science Gmbh Isolation and characterization of a novel Pythium omega 3 desaturase with specificity to all omega 6 fatty acids longer than 18 carbon chains
RU2009111266A (ru) 2006-08-29 2010-10-10 Коммонвелт Сайентифик энд Индастриал Рисерч Организейшн (AU) Синтез жирных кислот
AU2009239949A1 (en) 2008-04-25 2009-10-29 Basf Plant Science Gmbh Plant seed oil
EP2337791B1 (fr) 2008-10-14 2013-07-24 Monsanto Technology LLC Utilisation d'acide gras désaturases de hemiselmis spp.
EP2358882B1 (fr) 2008-11-18 2017-07-26 Commonwealth Scientific and Industrial Research Organisation Enzymes et méthodes de production d'acides gras oméga-3
KR20120092104A (ko) 2009-08-31 2012-08-20 바스프 플랜트 사이언스 컴퍼니 게엠베하 식물에서 구성적 유전자 발현을 향상시키기 위한 조절 핵산 분자
CN112011566B (zh) 2009-08-31 2023-12-05 巴斯夫植物科学有限公司 用于在植物中增强种子特异性基因表达而促进增强的多不饱和脂肪酸合成的调节性核酸分子
WO2011023539A1 (fr) 2009-08-31 2011-03-03 Basf Plant Science Company Gmbh Molécules d'acide nucléique régulatrices pour l'amplification de l'expression génétique spécifique des graines et/ou préférentielle dans les graines dans des plantes
AU2013201740A1 (en) * 2009-08-31 2013-04-11 Basf Plant Science Company Gmbh Regulatory nucleic acid molecules for enhancing seed-specific gene expression in plants promoting enhanced polyunsaturated fatty acid synthesis
US20130129902A1 (en) * 2009-11-03 2013-05-23 Dsm Ip Assets B.V. Composition comprising cells and a polyunsaturated fatty acid having at least 20 carbon atoms (lc-pufa)
AU2010317140B2 (en) * 2009-11-03 2016-04-21 Dsm Ip Assets B.V. Vegatable oil comprising a polyunsaturaded fatty acid having at least 20 carbon atoms
KR20140116445A (ko) * 2011-12-30 2014-10-02 다우 아그로사이언시즈 엘엘씨 카놀라 가공 동안의 ｄｈａ 유지
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EA201991467A1 (ru) 2016-12-15 2019-11-29 Состав смеси, содержащий силикат и микробные и/или растительные клетки и полиненасыщенную жирную кислоту, имеющую по меньшей мере 20 атомов углерода (lc-pufa)

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