EP1356056A2 - Procede pour exprimer des genes de biosynthese dans des graines vegetales a l'aide de nouveaux genes hybrides d'expression - Google Patents

Procede pour exprimer des genes de biosynthese dans des graines vegetales a l'aide de nouveaux genes hybrides d'expression

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Publication number
EP1356056A2
EP1356056A2 EP02710015A EP02710015A EP1356056A2 EP 1356056 A2 EP1356056 A2 EP 1356056A2 EP 02710015 A EP02710015 A EP 02710015A EP 02710015 A EP02710015 A EP 02710015A EP 1356056 A2 EP1356056 A2 EP 1356056A2
Authority
EP
European Patent Office
Prior art keywords
seq
desaturase
gene
expression
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP02710015A
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German (de)
English (en)
Inventor
Jens Lerchl
Elke Duwenig
Friedrich Bischoff
Ernst Heinz
Hjördis DREXLER
Jodi Scheffler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF Plant Science GmbH
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BASF Plant Science GmbH
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Publication date
Application filed by BASF Plant Science GmbH filed Critical BASF Plant Science GmbH
Publication of EP1356056A2 publication Critical patent/EP1356056A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

  • the invention relates to expression cassettes, their combination and vectors containing the expression cassettes which contain plant promoters with an expression specificity for plant seeds, in particular linseed, and the use of these expression cassettes or vectors for the recombinant expression of heterologous genes in plants.
  • the invention further relates to transgenic plants transformed with these expression cassettes or vectors, cultures derived therefrom, parts or transgenic propagation material, and the use thereof as food, feed or seed, pharmaceuticals, fine chemicals or as an industrial base material.
  • the expression cassettes according to the invention are advantageously used in a process for the production of unsaturated fatty acids with at least two double bonds and / or a process for the production of triglycerides with an increased content of polyunsaturated fatty acids with at least two double bonds.
  • the nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or 11 are advantageously used in the method and are expressed using the expression cassettes.
  • These aforementioned nucleic acids are suitable in the process and for the production of a transgenic organism, preferably a transgenic plant or a transgenic microorganism with an increased content of fatty acids, oils or lipids with unsaturated Cig-, C o ⁇ / or C -fatty acids.
  • genes besides the nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 11 or its homologs, derivatives or analogs as well as gene constructs containing these genes or their homologs, derivatives or analogs, and their use alone or in combination with other biosynthetic genes preferably biosynthetic genes for polyunsaturated fatty acids, as advantageously shown in SEQ ID NO: 7 and SEQ ID NO: 9, are preferably expressed in organisms in plants.
  • a number of products and by-products of naturally occurring metabolic processes in microorganisms, animal and plant cells are useful for many industries, including the animal feed, food, cosmetic and pharmaceutical industries.
  • These molecules collectively referred to as "fine chemicals", include, for example Lipids and fatty acids, among which an exemplary class are the polyunsaturated fatty acids.
  • Fatty acids and triglycerides have a large number of applications in the food industry, animal nutrition, cosmetics and in the pharmaceutical sector. Depending on whether it is free saturated or unsaturated fatty acids or triglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a wide variety of applications, for example polyunsaturated fatty acids (PUFAs) are used to increase baby food Added nutritional value.
  • PUFAs also have a positive influence on the cholesterol level in the blood of humans and are therefore suitable for protection against heart diseases. So they are used in various dietary foods or medications.
  • microorganisms for the production of PUFAs are microorganisms such as Thraustochytria or Schizochytria strains, algae such as Phaeodactylum tricornutum or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor.
  • strain selection a number of mutant strains of the corresponding microorganisms have been developed which produce a number of desirable compounds, including PUFAs.
  • the selection of strains with improved production of a particular molecule is a time-consuming and difficult process.
  • the production of fine chemicals can suitably be carried out on a large scale through the production of plants designed to produce the above-mentioned PUFAs.
  • Plants that are particularly suitable for this purpose are oil-fruit plants which contain large amounts of lipid compounds, such as rapeseed, canola, linseed, soya, sunflowers, borage and evening primrose.
  • lipid compounds such as rapeseed, canola, linseed, soya, sunflowers, borage and evening primrose.
  • other crop plants containing oils or lipids and fatty acids are also well suited, as mentioned in the detailed description of this invention.
  • Using conventional breeding a number of mutant plants have been developed that produce a spectrum of desirable lipids and fatty acids, cofactors and enzymes.
  • yeasts nor plants naturally produce polyunsaturated C 2 o ⁇ and / or C 22 fatty acids with at least two double bonds in the fatty acid molecule such as arachidonic acid (ERA) and / or eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA) ,
  • ERA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • the aim is to produce plants with advantageous novel properties, for example for increasing agricultural productivity, 'improving quality of human foods or for producing specific chemicals or pharmaceuticals (Dunwell JM, J. Exp Bot, 2000:.. 51 Spec No: 487 -96).
  • a basic requirement for the transgenic expression of certain genes is the provision of plant-specific promoters. Promoters are important tools in plant biotechnology in order to control the expression of a specific gene in a transgenic plant locally and in time and thus to exploit or achieve certain essential characteristics of the plant. Various promoters for various types of plants, certain plant tissues and stages of development are known.
  • constitutive promoters such as the promoter of nopaline synthase from Agrobacterium, the TR double promoter or the promoter of the 35S transcript of the cauliflower mosaic virus (CaMV) (Odell et al., Nature 1985: 313,810-812), the OCS (octopine Synthase) promoter from Agrobacterium, the Ubiguitin promoter (Holtorf S et al., Plant Mol. Biol.
  • Promoters whose activity is regulated in a tissue-specific or development-dependent manner were isolated. Specificities are described for the anthers, ovaries, flowers, leaves, stems, roots and seeds. The stringency of the specificity as well as the expression activity of these promoters is very different. Promoters that ensure leaf-specific expression should be mentioned, such as the promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (small subunit) of the rubi sco (ribulose-1, 5-bisphosphate carboxylase) or the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989), 2445-245).
  • promoters are, for example, specific promoters for tubers, storage roots or roots, such as, for example, the class I patatin promoter (B33), the potato cathepsin D inhibitor promoter, the starch synthase starch (GBSS1) or the sporamine promoter, fruit-specific Promoters like
  • fruit-specific promoter from tomato EP-A 409625
  • fruit-ripening-specific promoters such as for example the fruit-ripening-specific promoter from tomato
  • flower-specific promoters such as the phytoene-synthetic promoter (WO 92/16635) or the promoter of the P-rr gene (WO
  • Promoters are known which control expression in seeds and plant embryos. For example, the promoters of genes that code for storage proteins of different dicotyledons were identified. Seed-specific promoters are, for example, the promoter of phaseoline (US 5504200, Bustos
  • sucrose binding protein WO 00/26388
  • LeB4 promoter Bacillus subtilis
  • Gene expression in other parts of the plant can restrict the use of a promoter in another plant species. For example, if the expression of the gene interferes with the metabolism of the cell, the composition of the membrane lipids or biosynthesis.
  • the task was therefore to provide further expression cassettes for expression in plants. And to use these biosynthesis genes, which are advantageous for the expression of genes, alone or, if appropriate, in combination with other enzymes in a process, for example for the production of polyunsaturated fatty acids.
  • This object was achieved by the expression cassette according to the invention with a structure selected from the group:
  • Ll, L2, promoter and structural gene has the following meaning:
  • Ll SEQ ID NO: 32 or a sequence containing equivalent restriction cleavage sites
  • L2 independently of one another SEQ ID NO: 33, 34 or 35 or sequences containing equivalent restriction sites
  • Structural gene a nucleic acid sequence that can be expressed in plants.
  • the structural gene is advantageously a biosynthetic gene, more preferably it is a biosynthetic gene of the lipid or fatty acid metabolism, advantageously a vegetable gene.
  • the structural gene is a nucleic acid sequence, those selected for proteins from the group:
  • the structural gene is very particularly preferably a nucleic acid sequence selected from the group:
  • nucleic acid sequences which are obtained on the basis of the degenerate genetic code by back-translation of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12,
  • restriction restriction sites are to be understood as sequences which contain restriction sites which are suitable for the construction of multiple expression cassettes, that is to say are suitably not present in the structural gene or in the binary vector.
  • Restriction interfaces such as, for example, EcoRI, BamHI, Sacl, PstI, Ncol, Ndel, Bgll, Bglll, Xhol, Xba and others are known to the person skilled in the art and can be found in relevant specialist books.
  • the expression cassettes according to the invention can be used for the expression of genes in economically important crop plants such as linseed, for which no endogenous seed-specific promoters were known.
  • flax is particularly problematic for a seed-specific expression of genes, since obviously several promoters which are routinely used by the expert for seed-specific expression in other plants do not function in, for example, other plants such as flax, that is to say not one Transcription or ultimately leads to expression of the mRNA of the structural gene.
  • nucleic acid sequences which are obtained on the basis of the degenerate genetic code by back-translation of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12,
  • nucleic acid sequences used in the method are isolated nucleic acid sequences which code for polypeptides with ⁇ -5, ⁇ -6 or ⁇ -12 desaturase activity.
  • fatty acid esters with polyunsaturated cis, C 2 o ⁇ and / or C 2 fatty acid molecules with at least two double bonds in the fatty acid ester are advantageously produced.
  • These fatty acid molecules preferably contain three, four or five double bonds and advantageously lead to the synthesis of arachidonic acid (ERA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).
  • ERA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • the fatty acid esters with polyunsaturated C 20 and / or C 22 fatty acid molecules can be obtained from the organisms used for the production of the fatty acid esters in the form of an oil or lipid, for example in the form of compounds such as sphingolipids, phosphoglycerides , Lipids, glycolipids, phospholipids, Monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters containing the polyunsaturated fatty acids with at least two double bonds are isolated.
  • prokaryotic or eurkaryotic organisms such as prokaryotic or eurkaryotic microorganisms such as gram-positive or gram-negative bacteria, fungi, yeasts, algae, ciliates, animal or plant cells, animals or plants come as organisms for production in the process Moss, dicotyledonous or monocotyledonous plants in question.
  • Organisms belonging to the oil-producing organisms that is to say used for the production of oils, such as microorganisms such as Crypthecodinium, Thraustochytrium, Phaeodactylum and Mortierella, Ento ophthora, Mucor, Crypthecodinium and other algae, are advantageously used in the process according to the invention or mushrooms as well as animals or plants, in particular plants, preferably oil fruit plants which contain large amounts of lipid compounds, such as soybean, peanut, rapeseed, canola, sunflower, safflower, evening primrose, linseed, soya, borage, trees (oil palm, coconut) or field crops , such as corn, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, tagetes, solanaceae plants, such as potato, tobacco, eggplant and tomato, types of Vicia, peas, alfalfa or bush plants (coffee , Cocoa, tea),
  • the method includes either the cultivation of a suitable transgenic organism or transgenic microorganism or the cultivation of transgenic plant cells, tissues, organs or whole plants, comprising the nucleotide sequences according to the invention of SEQ ID NO: 1, 3, 5 or 11 optionally in Compounds with the sequences shown in SEQ ID NO: 7 and / or SEQ ID NO: 9, alone or in combination with sequences from advantageous expression cassettes according to the invention in advantageous vectors with SEQ ID NO: 13-17, or their homologs, derivatives or analogs or a gene construct which comprises SEQ ID NO: 1, 3, 5 or 11, optionally in conjunction with SEQ ID NO: 7 and / or 9 or their homologues, derivatives or analogs, or a vector which comprises this sequence or the gene construct which brings about the expression of nucleic acid molecules according to the invention, so that a fine chemical is produced.
  • the method further comprises the step of obtaining a cell which contains such a nucleic acid sequence according to the invention. contains zen, wherein a cell is transformed with a desaturase nucleic acid sequence, a gene construct or a vector which bring about the expression of a desaturase nucleic acid according to the invention alone or in combination.
  • this method further comprises the step of extracting the fine chemical from the culture.
  • the cell belongs to the order of the ciliates, to microorganisms, such as fungi, or to the plant kingdom, in particular to oil fruit plants, particularly preferred are microorganisms or oil fruit plants, for example peanut, rapeseed, canola, flax, soy, safflower (thistle), sunflowers or borage.
  • microorganisms or oil fruit plants for example peanut, rapeseed, canola, flax, soy, safflower (thistle), sunflowers or borage.
  • Transgenic in the sense of the invention means that the nucleic acids used in the method or the expression cassettes according to the invention are not in their natural position in the genome of an organism, and the nucleic acids can be expressed homologously or heterologously.
  • transgene also means that the nucleic acids or expression cassettes are in their natural positions in the genome of an organism, but that the sequence has been changed compared to the natural sequence and / or that the regulatory sequences, the natural sequences, have been changed.
  • Transgenic is preferably to be understood as meaning the expression of the nucleic acids according to the invention at a non-natural location in the genome, that is to say that the nucleic acids are homologous or preferably heterologous.
  • Preferred transgenic organisms are the abovementioned transgenic plants, preferably oil fruit plants.
  • the polyunsaturated fatty acids containing can be released, for example via an alkali treatment, such as aqueous KOH or NaOH, advantageously in the presence of an alcohol, such as methanol or ethanol, and isolated via, for example, phase separation and subsequent acidification using, for example, H 2 SO 4 .
  • an alkali treatment such as aqueous KOH or NaOH
  • an alcohol such as methanol or ethanol
  • the fatty acid esters produced in the process are obtained in the form of oils, lipids and / or fatty acids which contain at least two double bonds in the fatty acid molecules, preferably three, four, five or six double bonds.
  • Compositions which contain the oleic, lipid and / or fatty acids mentioned, and the use of the oils, lipids and / or fatty acids or the compositions in feed, foods, cosmetics or pharmaceuticals are a further possible application of the abovementioned substances.
  • Another aspect relates to a method for modulating the production of a molecule by a microorganism.
  • These methods include contacting the cell with a substance which modulates the desaturase activity according to the invention alone or in combination or the desaturase nucleic acid expression so that a cell-associated activity is changed relative to the same activity in the absence of the substance.
  • a substance which modulates the desaturase activity can be a substance that stimulates desaturase activity or desaturase nucleic acid expression or that can be used as an intermediate in fatty acid biosynthesis.
  • substances which stimulate desaturase activity or desaturase nucleic acid expression include small molecules, active desaturases and desaturase-coding nucleic acids which have been introduced into the cell.
  • substances that inhibit desaturase activity or expression include small molecules and antisense desaturase nucleic acid molecules.
  • Another aspect relates to a method for modulating the
  • Yielding a desired compound from a cell comprising introducing into a cell a wild-type or mutant desaturase gene that is either maintained on a separate plasmid or integrated into the genome of the host cell.
  • the integration can be random or by recombination such that the native gene is replaced by the inserted copy, thereby modulating the production of the desired compound by the cell, or by using a gene in trans so that the The gene is functionally linked to a functional expression unit which contains at least one sequence which ensures the expression of a gene and at least one sequence which ensures the polyadenylation of a functionally transcribed gene.
  • the yields are modified.
  • the desired chemical is increased, and undesirable interfering compounds can be reduced.
  • the desired fine chemical is a lipid or a fatty acid, a cofactor or an enzyme.
  • this chemical is a polyunsaturated fatty acid. Before In addition, it is selected from arachidonic acid (ERA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).
  • the present invention provides advantageous multi-expression cassettes and constructs for multiparallel seed-specific expression of gene combinations in plants.
  • genes preferably those described in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 .
  • preferred organisms are used for the process.
  • the process can be used in conjunction with the nucleic acid molecules mentioned above for the genetic engineering of plants, so that they ultimately lead to the production of better or more efficient producers of one or more fine chemicals.
  • This improved production or efficiency of the production of a fine chemical can be brought about by a direct effect of the manipulation of a gene according to the invention or by an indirect effect of this manipulation.
  • fine chemicals include, for example, fatty acid esters which contain polyunsaturated fatty acids with at least two double bonds, such as sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters which contain the polyunsaturated fatty acids with at least contain two double bonds.
  • This also includes compounds such as vitamins, for example vitamin E, vitamin C, vitamin B2, vitamin B6, pantolactone, carotenoids such as astaxanthin, ⁇ -carotene, zeaxanthin and others.
  • Mosses and algae are the only known plant systems that produce significant amounts of polyunsaturated fatty acids such as arachidonic acid (ERA) and / or eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).
  • ERA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • Mosses contain PUFAs in membrane lipids while algae, algae-related organisms and some fungi also accumulate significant amounts of PUFAs in the triacylglycerol fraction. Therefore nucleic acid molecules which are isolated from those strains which also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for modifying the lipid and PUFA production system in a host, in particular in microorganisms, such as the microorganisms mentioned above, and plants. like oil crops, for example rapeseed, canola, flax, soybeans, sunflower
  • nucleic acid sequences used in the process using the expression cassettes according to the invention code for desaturases which are suitable for the production of long-chain polyunsaturated fatty acids, preferably with more than sixteen, eighteen or twenty carbon atoms in the carbon skeleton of the fatty acid and / or at least two double bonds in the carbon chain , wherein a nucleic acid codes for an enzyme which can introduce double bonds in the ⁇ -5 position, in another case in the ⁇ -6 position and in a further case in the ⁇ -12 position.
  • These nucleic acids can be used to obtain large amounts of PUFAs in the triacylgycerol fraction.
  • the polyunsaturated cis fatty acids must first be extended by at least two carbon atoms by the enzymatic activity of an elongase. After one round of elongation this enzyme activity leads to C 2 o fatty acids, and after two, three and four rounds of elongation leads to C, C or C 26 fatty acids.
  • the nucleic acid sequences disclosed in this invention which code for various desaturases, can lead to very long-chain, polyunsaturated in concert with elongases.
  • the activity of the desaturases according to the invention preferably leads to cis, C 0 and / or C fatty acids with at least two double bonds in the fatty acid molecule, preferably with three, four, five or six double bonds, particularly preferably to cis and / or C 0 fat acids with at least two double bonds in the fatty acid molecule, preferably with three, four or five double bonds in the molecule.
  • the fatty acid elongation can be carried out by combining the desaturases according to the invention with an elongase activity, the elongase encoded in SEQ ID NO: 9 advantageously being used can be.
  • the products of the desaturase activities and the possible further desaturation lead to preferred PUFAs with a higher degree of desaturation, such as dihomo-gamma-lonolenic acid, docosadienoic acid, arachidonic acid, ⁇ 6-eicosatrienedihomo- ⁇ -linolenic acid, eicosapentaenoic acid, ü) 3-eicosatrienoic acid, ö) 3-eicosatetraenic acid, docosapentaenoic acid or docosahexaenoic acid.
  • substrates of the enzyme activity according to the invention are taxolic acid;
  • 6, 9-0ctadecadienoic acid linoleic acid, pinolenic acid, ⁇ - or ⁇ -linolenic acid or stearidonic acid as well as arachidonic acid, eicosatetraenoic acid, docosapentaenoic acid, eicosapentaenoic acid.
  • Preferred substrates are linoleic acid, ⁇ -linolenic acid and / or ⁇ -linolenic acid and arachidonic acid, eicosatetraenoic acid, docosapentaenoic acid, eicosapentaenoic acid.
  • Ci 8 ⁇ fatty acids with at least two double bonds in the fatty acid can be extended by the inventive enzymatic activity in the form of the free fatty acid or in the form of the esters such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerides, diacylglycerides or triacylglycerides ,
  • Conjugated linoleic acid "CLA” is of particular importance for human nutrition.
  • the desaturases according to the invention ⁇ -12-desaturase
  • conjugated fatty acids with at least two double bonds in the molecule can also be desaturated and such health-promoting fatty acids can thus be supplied to human nutrition.
  • Another example of conjugated fatty acids are alpha-parinic acid, punicic acid, eleostearic acid and calendulic acid.
  • genes can advantageously use biosynthetic genes such as the nucleic acids described above for the genetic engineering modification of a broad spectrum of plants, so that they are a better or more efficient producer, for example of one or more products derived from lipids, like PUFAs.
  • This improved production or efficiency of the production of a product derived, for example, from lipids, such as PUFAs can be brought about by a direct effect of the manipulation or an indirect effect of this manipulation.
  • a desaturase gene or several desaturase genes under control of the expression cassettes according to the invention into an organism alone or in combination with other genes in a cell, not only can the flow of biosynthesis to the end product be increased, but also the corresponding composition of the products, for example the triacylglycerols, can be increased or de novo can be created.
  • the number or activity of other genes required for the import of nutrients required for the biosynthesis of one or more fine chemicals eg fatty acids, polar and neutral lipids
  • concentration of these precursors, cofactors or intermediate compounds within cells or within the storage compartment is increased, which further increases the ability of the cells to produce PUFAs, as described below.
  • Fatty acids and lipids are desirable even as fine chemicals; by optimizing the activity or increasing the number of one or more desaturases involved in the biosynthesis of these compounds, or by destroying the activity of one or more desaturases involved in the degradation of these compounds, the yield, production and / or to increase the efficiency of the production of fatty acid and lipid molecules from plants or microorganisms.
  • the mutagenesis of the desaturase gene (s) according to the invention can furthermore lead to a desaturase protein with modified activities which directly or indirectly influence the production of one or more desired fine chemicals.
  • the number or activity of the desaturase gene (s) according to the invention can be increased so that the normal metabolic waste or by-products of the cell (the amount of which may be increased due to the overproduction of the desired fine chemical) can be efficiently exported before it uses other molecules or destroy processes within the cell (which would lower the cell's viability) or disrupt the fine chemical's biosynthetic pathways (thereby reducing the yield, production, or efficiency of production of the desired fine chemical).
  • the relatively large intracellular amounts of the desired fine chemical can itself be toxic to the cell or interfere with enzyme feedback mechanisms such as allosteric regulation, for example by increasing the activity or number of other downstream enzymes or detoxification enzymes of the PUFA pathway Allocate PUFA allocation to the triacylgylcerin fraction, one could increase the viability of seed cells, which in turn leads to better development of cells in culture or to seeds that produce the desired fine chemical.
  • the desaturase gene according to the invention can also be manipulated in such a way that the corresponding amounts of the different lipid and fatty acid molecules are produced. This can have a drastic effect on the lipid composition of the cell membrane and generates new oils in addition to the occurrence of newly synthesized PUFAs.
  • isolated nucleic acid molecules eg cDNAs
  • 5 comprising nucleotides, which encode a desaturase or several desaturases or biologically active parts thereof, or nucleic acid fragments, which act as primers or hybridization probes for the detection or amplification of desaturase-encoding nucleic acids (eg DNA or mRNA) are used.
  • the nucleic acid molecule comprises one of the nucleotide sequences shown in sequence ID N0: 1 or 3 and 5 or the coding region or a complement of one of these nucleotide sequences.
  • the isolated nucleic acid molecule comprises
  • nucleotide sequence which hybridizes to a nucleotide sequence as shown in the sequence SEQ ID NO: 1, 3, 5 or 11 or a part thereof or at least about 50%, preferably at least about 60%, more preferably at least about 70% , 80% or 90% and even more preferably at least about 95%,
  • the isolated nucleic acid molecule encodes one of the amino acid sequences shown in the sequence SEQ ID NO: 2, 4, 6 or 12.
  • the preferred desaturase gene preferably also has at least one of those described here
  • the isolated nucleic acid molecule encodes a protein or a part thereof, the protein or the part thereof containing an amino acid sequence
  • the protein or part thereof maintains desaturase activity.
  • the protein or part thereof encoded by the nucleic acid molecule retains the ability to metabolize to build
  • the protein encoded by the nucleic acid molecule is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80% or
  • Protein is a full-length protein that is essentially partially homologous to an entire amino acid sequence of sequence SEQ ID NO: 2, 4, 6 or 12.
  • this is Protein is a full-length protein that is essentially partially homologous to an entire amino acid sequence of sequence SEQ ID NO: 2, 4, 6 or 12.
  • the isolated desaturase comprises an amino acid sequence which is at least about 50% homologous to one of the amino acid sequences of SEQ ID NO: 2, 4, 6 or 12 and in the metabolism of compounds necessary for the formation of fatty acids in a microorganism or a plant cell or can participate in the transport of molecules across these membranes, meaning desatured Ci 8 ⁇ or C 2 o- 22 carbon chains with double bonds at at least two sites.
  • the isolated nucleic acid molecule is derived from Phaeodactylum tricornutum UTEX646 and encodes a protein (eg a desaturase fusion protein) which contains a biologically active domain which is at least about 50% or more homologous to an amino acid sequence of the sequence SEQ ID NO 2, 4, 6 or 12 and maintains the ability to participate in the metabolism of compounds necessary for building cell membranes of plants or in the transport of molecules across these membranes, or at least one of the desaturation activities resulting in PUFAs such as GLA, ALA, Dihomo -gamma linolenic acid, ERA, EPA or DHA or their precursor molecules, and also includes heterologous nucleic acid sequences that encode a heterologous polypeptide or regulatory proteins.
  • PUFAs such as GLA, ALA, Dihomo -gamma linolenic acid, ERA, EPA or DHA or their precursor molecules
  • the isolated desaturase may comprise an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11, e.g. hybridizes under stringent conditions, or at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90% and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to it. It is also preferred that the preferred desaturase forms also have one of the desaturase activities described here.
  • the isolated nucleic acid molecule is at least 15, 25, 50, 100, 250 or more nucleotides long and hybridizes under stringent conditions to a nucleic acid molecule which comprises a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 17.
  • the isolated nucleic acid molecule preferably corresponds to a naturally occurring nucleic acid molecule. More preferably, the isolated nucleic acid molecule encodes naturally occurring Phaeodactylum desaturase or a biologically active part thereof.
  • Another embodiment of the invention are expression cassettes which enable the expression of the nucleic acids according to the invention with the sequences SEQ ID NO: 1, 3, 5 or 11 in the various organisms such as plant cells, tissues, parts of plants or whole plants.
  • nucleic acid construct or fragment are those in SEQ ID NO: 32 as L1 and a promoter, a structural gene selected from the advantageous sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or
  • These regulatory sequences contained in the constructs are intended to enable the targeted expression of the genes and the protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed and / or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified so that the natural regulation has been switched off and the expression of the genes increased.
  • the gene construct can also have a simpler structure, that is to say no additional regulation signals have been inserted in front of the nucleic acid sequence or its derivatives, and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence was mutated so that regulation no longer takes place and / or gene expression is increased.
  • the gene construct can also advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable increased expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the DNA sequences.
  • the regulatory sequences or factors can preferably have a positive influence on the gene expression of the introduced genes and thereby increase it.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • vectors e.g. recombinant expression vectors which contain at least one of the expression cassettes according to the invention, and host cells into which the expression cassettes according to the invention or these vectors have been introduced, in particular microorganisms, plant cells, plant tissue or plant organs or whole plants.
  • a host cell can store fine chemical compounds, in particular PUFAs; the cells are harvested to isolate the desired compound.
  • the compound (oils, lipids, triacylglycerides, fatty acids) or the desaturase can then, from the medium or the host cell, which are cells in plants which contain or store fine chemicals, most preferably cells of storage tissues such as seed shells, tubers, epidermis and Sperm cells, endosperm or embyro tissue can be isolated.
  • Yet another aspect of the invention relates to a genetically modified transgenic plant, preferably an oil crop, as mentioned above, particularly preferably a rape or linseed plant, into which an expression cassette according to the invention, which advantageously contains further genes such as desaturase genes, has been introduced.
  • the genome of oilseed rape or linseed has advantageously been changed as a transgene by introducing an expression cassette according to the invention which advantageously contains further nucleic acid molecules which, for example, encodes a wild-type or mutated desaturase sequence.
  • rapeseed or linseed is also used to produce a desired compound, such as lipids and fatty acids, with PUFAs being particularly preferred.
  • the moss Physcomitrella patens can be used to demonstrate the function of an expression cassette with a desaturase curse homologous recombination based on the nucleic acids described in this invention.
  • the desaturase polypeptide or a biologically active part thereof can advantageously be linked to another polypeptide which has a different enzymatic activity than the desaturases, for example an elongase, acyltransferase or other activity, under the control of the expression cassette according to the invention, so that a fusion protein is formed.
  • This fusion protein advantageously has an activity that differs from that of desaturase alone.
  • this fusion protein participates in the metabolism of compounds which are necessary for the synthesis of lipids and fatty acids, cofactors and enzymes in microorganisms or plants, or in the transport of molecules across these membranes.
  • this fusion protein in a host cell particularly advantageously modulates the production of a desired compound within and by the cell.
  • these fusion proteins also contain ⁇ -4, ⁇ -5 or ⁇ -6, ⁇ -8, ⁇ -15, ⁇ -17 or ⁇ -19 desaturase activities alone or in combination.
  • Such gene combinations in particular are preferred embodiments which are selected from SEQ ID NO: 7 or 9, or parts thereof, derivatives or their homologues.
  • those combinations are preferred which contain the complete protein activity as in SEQ ID NO: 1, 3, 5 or 11 and are inserted in multi-expression cassettes as defined by SEQ ID NO: 13, 14, 15, 16 and 17 for the transformation of plants and expression in Plants are suitable.
  • An object of the invention are also the expression cassettes in connection with isolated nucleic acid sequence (s) which codes for a polypeptide with desaturase activity, selected from the group:
  • nucleic acid sequences which are obtained on the basis of the degenerate genetic code by back-translation of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12, 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: 11, which are suitable for polypeptides with the sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ Encode ID NO: 6 or SEQ ID NO: 12 and have at least 50% homology at the amino acid level without significantly reducing the enzymatic action of the polypeptides.
  • the invention relates to an amino acid sequence which is encoded by the above-mentioned nucleic acid sequence (s) (for the invention, the singular is intended to include the plural and vice versa).
  • the invention relates to amino acid sequences which are encoded by the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11.
  • the present invention provides expression cassettes which are suitable for the expression of nucleic acids and protein molecules with desaturase activity and of nucleic acids coding for proteins which are involved in the metabolism of lipids and fatty acids, PUFA cofactors and enzymes in the moss Physcomitrella patens or in the transport of lipophilic compounds are involved over membranes.
  • the compounds according to the invention can be used to modulate the production of fine chemicals from organisms such as plants, such as maize, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, linum species such as oil or fiber flax, Brassica species , such as rapeseed, canola and turnip, pepper, sunflower, borage, evening primrose and tagetes, solanacaen plants, such as potato, tobacco, eggplant and tomato, Vicia species, peas, cassava, alfalfa, bush plants (coffee, cocoa, tea ), Salix species, trees (oil palm, coconut) and perennial grasses and fodder crops, either directly (eg if the overexpression or optimization of a fatty acid biosynthesis protein has a direct influence on the yield, production and / or efficiency of the production of the fatty acid modified organisms) use or may have an indirect effect, which nevertheless leads to an increase in the yield, production and / or efficiency of production of a desired ver
  • Fine chemicals and PUFAs The term "fine chemical” is known in the art and encompasses molecules that have been produced by an organism and find applications in various industries, such as, but not limited to, the pharmaceutical, agricultural, food, and cosmetic industries. These compounds include lipids, fatty acids, cofactors and enzymes, etc. (as described, for example, in Kuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6, Reh et al., Eds. , VCH: Weinheim and the literature references contained therein), lipids, saturated and unsaturated fatty acids (eg arachidonic acid), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, Vol.
  • PUFAs are not only simply incorporated into triacylglycerol, but also into membrane lipids.
  • Precursors for PUFA biosynthesis are, for example, oleic acid, linoleic and linolenic acid. These cis-carbon fatty acids must be extended to C 20 and C 2 in order to obtain fatty acids of the Eicosa and Docosa chain type.
  • Arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid as well as various others can be used with the aid of various desaturases, such as enzymes which have ⁇ -12-desaturase, ⁇ -15-desaturase, ⁇ -6-desaturase, ⁇ -5 and ⁇ -4-desaturase activity
  • desaturases such as enzymes which have ⁇ -12-desaturase, ⁇ -15-desaturase, ⁇ -6-desaturase, ⁇ -5 and ⁇ -4-desaturase activity
  • Long-chain PUFAs are obtained, extracted and used for various purposes in food, feed, cosmetic or pharmaceutical applications.
  • the polyunsaturated Ci 8 ⁇ or C 20 fatty acids have to be desaturated several times.
  • the nucleic acid sequences according to the invention encode first functionally active desaturases from Phyeodactylum tricornutum, a microorganism which contains PUFAs in the triacyl contains glycerol fraction. With the desaturases according to the invention, double bonds can be introduced into the ⁇ -5, ⁇ -6 or ⁇ -12 position.
  • the activities of the desaturases according to the invention preferably lead to Cis + C 0 fatty acids with at least two, three, four or five double bonds in the fatty acid molecule, preferably to C 2 o fatty acids with advantageously three, four or five double bonds in the fatty acid molecule.
  • Desaturation can take place before or after elongation of the corresponding fatty acid.
  • fatty acids such as linoleic acid, docosadienic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, ⁇ -eicostriendihomo- ⁇ -linolenic acid, eicosapentaen
  • Substrates of this enzyme activity according to the invention are, for example, taxolic acid, 6, 9-0ctadecadienoic acid, oleic acid, linoleic acid, ⁇ -linolenic acid, pinolenic acid, ⁇ -linolenic acid, arachidonic acid, eicosapentaenoic acid or stearidonic acid.
  • Preferred substrates are linoleic acid, ⁇ -linolenic acid and / or ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid or arachidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.
  • the cis or C 0 fatty acids with at least two double bonds in the fatty acid can by the enzyme activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerides, diacylglycerides, triacylglycerides or other esters , be extended. Furthermore, fatty acids then have to be transported to different modification sites and incorporated into the triacylglycerol storage lipid. Another important step in lipid synthesis is the transfer of fatty acids to the polar head groups, for example by glycerol fatty acid acyltransferase (see Frentzen, 1998, Lipid, 100 (4-5): 161-166).
  • Vitamins, cofactors and nutraceuticals such as PUFAs comprise a group of molecules that higher animals can no longer synthesize and therefore have to ingest or which higher animals can no longer produce themselves and therefore additionally
  • the present invention is based, among other things, on the discovery of new molecules, referred to herein as desaturase nucleic acid and protein molecules, which have an effect on exert the production of cell membranes and lipids Phaeodactylum tricornutum and, for example, influence the movement of molecules across these membranes.
  • the desaturase molecules participate in the metabolism of compounds necessary for the construction of cell membranes in organisms, such as microorganisms and plants, or indirectly influence the transport of molecules across these membranes.
  • the activity of the desaturase molecules according to the invention for regulating the production of membrane components and membrane transport has an effect on the production of the desired fine chemical by this organism.
  • the activity of the desaturase molecules according to the invention is modulated, so that the yield, production and / or efficiency of the production of the metabolic pathways of microorganisms or plants which regulate the desaturases according to the invention are modulated and the efficiency of the transport of compounds through the membranes is changed, which directly or indirectly modulates the yield, production and / or efficiency of the production of a desired fine chemical by microorganisms and plants.
  • the term "desaturase” or “desaturase polypeptide” includes proteins that participate in the desaturation of fatty acids. Examples of desaturases are disclosed in SEQ ID NO: 1, 3, 5, 11 or their homologues, derivatives or analogs.
  • the terms desaturase or desaturase nucleic acid sequence (s) encompass nucleic acid sequences which encode a desaturase and in which a part can be a coding region and likewise corresponding 5 'and 3' untranslated sequence regions. Examples of desaturase genes are those shown in SEQ ID NO: 1, 3, 5 or 11.
  • production or productivity are known in the art and include the concentration of the fermentation product (for example the desired fine chemical) that is formed in a certain time period and a certain fermentation volume (for example kg product per hour per liter).
  • concentration of the fermentation product for example the desired fine chemical
  • production efficiency encompasses the time it takes to achieve a certain production volume (eg how long it takes the cell to set up a certain throughput rate of a fine chemical).
  • yield or product / carbon yield is known in the art and encompasses the efficiency of converting the carbon source into the product (ie the fine chemical). This is usually expressed, for example, as kg of product per kg of carbon source.
  • biosynthesis or biosynthetic pathway are known in the art and encompass the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds, for example in a multi-step and highly regulated process.
  • degradation or degradation pathway are known in the art and include the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), for example in a multi-step and highly regulated process.
  • metabolism is known in the specialist field and encompasses all of the biochemical reactions that take place in an organism. The metabolism of a particular compound (eg the metabolism of a fatty acid) then encompasses all of the biosynthetic, modification and degradation pathways of this compound in the cell that relate to this compound.
  • the nucleic acid sequences according to the invention which code for desaturase molecules, can modulate the production of a desired molecule, such as a fine chemical, in a microorganism or in plants.
  • a desired molecule such as a fine chemical
  • the number or activity of desaturases involved in the transport of fine chemical molecules inside or out of the cell can be increased so that larger amounts of these compounds are transported across membranes from which they are more easily obtained and converted into one another.
  • fatty acids, triacylglycerols and / or lipids are themselves desirable fine chemicals; by optimizing the activity or increasing the number of one or more desaturases according to the invention which are involved in the biosynthesis of these compounds, or by disrupting the activity of one or more desaturases which are involved in the degradation of these compounds, it may be possible to increase the yield, production and / or to increase the efficiency of the production of fatty acid and lipid molecules from organisms, such as microorganisms or plants.
  • the mutagenesis of the nucleic acid sequences mentioned can produce desaturases with altered activities which indirectly influence the production of one or more desired fine chemicals from microorganisms or plants.
  • desaturases involved in the export of waste products have a higher number or higher activity so that the cell's normal metabolic waste (the amount of which may be increased due to the overproduction of the desired fine chemical) can be efficiently exported before it can damage the molecules in the cell (reducing the cell's viability) would) or disrupt the fine chemical biosynthetic pathways (which would decrease the yield, production, or efficiency in producing a desired fine chemical).
  • the relatively large intracellular amounts of the desired fine chemical itself can also be toxic to the cell, so increasing the activity or number of transporters that can export these compounds from the cell can increase cell viability in culture, which in turn leads to a larger number of cells in the culture which produce the desired fine chemical.
  • the desaturases can also be manipulated in such a way that the corresponding amounts of different lipid and fatty acid molecules are produced. This can have a significant impact on the lipid composition of the cell membrane. Since each type of lipid has different physical properties, changing the lipid composition of a membrane can significantly change the membrane fluidity.
  • Plant membranes impart specific properties, such as tolerance to heat, cold, salt, dryness, and tolerance to pathogens, such as bacteria and fungi.
  • the isolated nucleic acid sequences mentioned are contained in the genome of a Phaeodactylum tricornutum UTEX646 strain, which is available from the algae collection of the University of Texas, Austin.
  • nucleotide sequence of the Phaeodactylum tricornutum cDNA and the derived amino acid sequences of the desaturases are shown in SEQ ID NO: 1 to 6 and 11 and 12.
  • Computer analyzes were carried out which classify and / or identify these nucleotide sequences as sequences which encode proteins involved in the metabolism of cell membrane components or proteins involved in the transport of compounds across cell membranes or PUFA biosynthesis.
  • ESTs with the database entry NO: PT001070010R and PT001078032R by the inventors represent the sequences according to the invention in SEQ ID NO: 1 and 3.
  • the sequence of the fragment from EST PT001070010R was determined and is as shown in SEQ ID NO: 5.
  • the analog is the Sequence of the clone PT001078032R shown in SEQ ID NO: 1.
  • PT001070010R from SEQ ID NO: 5 codes for a new gene homologous to ⁇ -12-desaturase and PT001078032R codes for a novel ⁇ -5-desaturase.
  • Pt_des6 can be isolated according to Example 5a by means of a polymerase chain reaction with the aid of degenerate oligonucleotides.
  • a fragment thus obtained can be isolated from Phaeodactylum tricornutum for screening a cDNA library and the coding region of a Phaeodactylum tricornutum ⁇ -6-desaturase can be obtained.
  • a gene isolated in this way is referred to in Table 1 as Pt_des6 and is shown in SEQ ID NO: 3.
  • the corresponding amino acid sequences are obtained by translating the genetic code of the sequence ID NO: 1, 3 and 5 and are defined as SEQ ID NO: 2, 4 and 6 (see also Table 1).
  • a further nucleic acid sequence which codes for a ⁇ -12 desaturase can also be found in Table 1. It has the clone number PT001072031R.
  • the present invention also relates to proteins with an amino acid sequence which is essentially homologous to an amino acid sequence of SEQ ID NO: 2, 4, 6 or 12.
  • a protein having an amino acid sequence that is substantially homologous to a selected amino acid sequence is at least about 50% homologous to the selected amino acid sequence, e.g. the entire selected amino acid sequence.
  • a protein having an amino acid sequence that is substantially homologous to a selected amino acid sequence can also be at least about 50 to 60%, preferably at least about 60 to 70%, and more preferably at least about 70 to 80%, 80 to 90% or 90 to 95%, and most preferably at least about 96%, 97%, 98%, 99% or more, homologous to a selected amino acid sequence.
  • the desaturases or the biologically active parts or fragments thereof can participate in the metabolism of lipids to build up cell membranes or storage lipids in organisms and in Combination with other genes, in particular those with elongase activity, contribute to the elongation of cis or Co 2 PUFAs, so that cis, C 0 , C or C 4 PUFAs and related PUFAs are obtained. ⁇ •
  • the desaturases in combination with elongases and other desaturases can be cloned in expression cassettes according to the invention and used for the transformation of plants with the aid of Agrobacterium.
  • One embodiment of the invention is isolated nucleic acids which originate from PUFA-producing microorganisms and code for polypeptides which desaturate cis or C o- 22 fatty acids with at least one, two, three or four double bonds in the fatty acid.
  • a further embodiment according to the invention are isolated nucleic acids, comprising nucleotide sequences which code for polypeptides which desaturate cis or C 0 fatty acids with at least one, two, three or four double bonds in the fatty acid and are from the group consisting of
  • nucleic acid sequences which are obtained on the basis of the degenerate genetic code by back-translation of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 12,
  • the above-mentioned nucleic acid according to the invention comes from organisms, such as ciliates, fungi, algae or dinoflagellates, which can synthesize PUFAs, preferably from Phaeodactylum tricornutum or closely related organisms.
  • nucleic acid molecule as used herein is intended to encompass DNA molecules (e.g. cDNA or genomic DNA) and RNA molecules (e.g. RNA) as well as DNA or RNA analogs which are generated by means of nucleotide analogs.
  • This term also includes the untranslated sequence located at the 3 'and 5' end of the coding region: at least 500, preferably 200, particularly preferably 100 nucleotides of the sequence upstream of the 5 'end of the coding region and at least 100, preferably 50, particularly preferably 20 nucleotides of the sequence downstream of the 3 'end of the coding gene region.
  • the nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid preferably has no sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (e.g. sequences located at the 5 'and 3' ends of the nucleic acid).
  • the isolated desaturase nucleic acid molecule can contain, for example, less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences that naturally contain the nucleic acid molecule in flank the genomic DNA of the cell from which the nucleic acid originates (e.g. a Physcomitrella patens cell).
  • An "isolated" nucleic acid molecule, such as a cDNA molecule can also be substantially free of other cellular material or culture medium when made by recombinant techniques, or free of chemical precursors or other chemicals when chemically synthesized.
  • An expression cassette according to the invention with the structure SEQ ID NO: 32 promoter SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35 nucleic acid molecule for example a nucleic acid molecule with a nucleotide sequence of SEQ ID NO: 1 or a part of these, can be isolated using standard molecular biological techniques and the sequence information provided here. Can too Using comparison algorithms, for example, a homologous sequence or homologous, conserved sequence regions can be identified at the DNA or amino acid level.
  • a Phaeodactylum tricornutum cDNA can be isolated from a Phaeodactylum tricornutum bank by using the complete SEQ ID NO: 1, 3, 5 or 11 or a part thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule comprising a complete sequence of SEQ ID NO: 1, 3, 5 or 11 or a part thereof can be isolated by polymerase chain reaction, oligonucleotide primers based on this sequence or on parts thereof, in particular regions Motifs from example 5a are created or modifications of the same in individually defined amino acids can be used (for example, a nucleic acid molecule comprising the complete sequence of SEQ ID NO: 1, 3, 5 or 11 or a part thereof, by polymerase chain reaction using Oligonucleotide primers are isolated, which have been created on the basis of this same sequence of SEQ ID NO: 1, 3, 5 or 11).
  • mRNA can be isolated from cells (for example by the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA using reverse transcriptase (for example Moloney-MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St.Petersburg, FL).
  • reverse transcriptase for example Moloney-MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St.Petersburg, FL.
  • Synthetic oligonucleotide primers for aplification by means of the polymerase chain reaction can be prepared on the basis of one of the sequences shown in SEQ ID NO: 1, 3, 5 or 11 and the sequence shown in FIG.
  • a nucleic acid of the invention can be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid amplified in this way can be cloned into a suitable vector and characterized by means of DNA sequence analysis.
  • Oligonucleotides which correspond to a desaturase nucleotide sequence can be produced by standard synthesis methods, for example using an automatic DNA synthesizer.
  • the cDNA shown in SEQ ID NO: 1, 3, 5 or 11 comprises sequences encoding desaturases (ie the "coding region") as well as 5'-untranslated sequences and 3 '-untranslated sequences.
  • the nucleic acid molecule can only encode the coding region of one of the sequences in SEQ ID NO: 1, 3, 5 or 11.
  • one or more nucleotide exchanges can be modified by insertion (s) and / or deletion (s) without, however, interfering with the functionality or activity of the promoters. It is also possible that the activity of the promoters is increased by modifying their sequence or that they are completely replaced by more active promoters, even from heterologous organisms.
  • nucleic acid molecule according to the invention can only part of the coding region of one of the sequences in
  • SEQ ID NO: 1, 3, 5 or 11 for example a fragment that can be used as a probe or primer, or a fragment that encodes a biologically active portion of a desaturase.
  • the nucleotide sequences determined from the cloning of the desaturase gene from Phaeodactylum tricornutum enable the generation of probes and primers which are designed for the identification and / or cloning of desaturase homologues in other cell types and organisms as well as desaturase homologues from other microalgae or related species.
  • the probe / primer usually comprises essentially purified oligonucleotide.
  • the oligonucleotide usually comprises a nucleotide sequence region which, under stringent conditions, comprises at least about 12, preferably about 16, more preferably about 25, 40, 50 or 75 successive nucleotides of a sense strand of one of the ones in SEQ ID NO: 1, 3, 5 or 11 specified sequences, an antisense strand of one of the sequences given in SEQ ID NO: 1, 3, 5 or 11 or its homologs, derivatives or analogs or naturally occurring mutants thereof.
  • Primers based on a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11 can be used in PCR reactions for cloning desaturase homologues.
  • Probes based on the desaturase nucleotide sequences can be used for the detection of transcripts or genomic sequences which encode the same or homologous proteins.
  • the probe also includes a label group attached thereto, e.g. a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor.
  • These probes can be used as part of a genomic marker assay kit to identify cells that express a desaturase, for example by measuring an amount of a desaturase-encoding nucleic acid in a cell sample, e.g. Measuring desaturase mRNA levels, or to determine whether a genomic desaturase gene is mutated or deleted can be used.
  • the nucleic acid molecule according to the invention encodes a protein or a part thereof which comprises an amino acid sequence which is sufficiently homologous to one Amino acid sequence of SEQ ID NO: 2, 4, 6 or 12 is that the protein or part thereof retains the ability to participate in the metabolism of compounds necessary for the construction of cell membranes in microorganisms or plants or in the transport of molecules across these membranes ,
  • the term "sufficiently homologous” refers to proteins or portions thereof whose amino acid sequences have a minimal number of identical or equivalent amino acid residues (eg an amino acid residue with a similar side chain as an amino acid residue in
  • derivatives of the nucleic acid molecule according to the invention encode proteins with at least about 50 to 60%, preferably at least about 60 to 70% and more preferably at least about 70 to 80%, 80 to 90%,
  • 40 parts of proteins which are encoded by the desaturase nucleic acid molecules according to the invention are preferably biologically active parts of one of the desaturases.
  • biologically active part of a desaturase is intended to mean a section, e.g. a domain / motif, a desaturase
  • Additional nucleic acid fragments encoding biologically active sections of a desaturase can be isolated by isolating part of one of the sequences in SEQ ID NO: 1, 3, 5 or 11, expressing the encoded section of the desaturase or the peptide (for example by recombinant expression in vitro) and determining the activity of the encoded portion of the desaturase or peptide.
  • the invention also encompasses nucleic acid molecules which differ from one of the nucleotide sequences shown in SEQ ID NO: 1, 3, 5 or 11 (and parts thereof) because of the degenerate genetic code and thus encode the same desaturase as that from those is encoded in SEQ ID NO: 1, 3, 5 or 11 nucleotide sequences shown.
  • an isolated nucleic acid molecule according to the invention has a nucleotide sequence which codes for a protein with an amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 12.
  • nucleic acid molecule according to the invention encodes a full-length desaturase protein which forms an amino acid sequence of SEQ ID NO: 2,4, 6 or 12 (that of an open one shown in SEQ ID NO: 1, 3, 5 or 11
  • Reading frame is coded) is essentially homologous and can be identified and isolated by common methods.
  • the person skilled in the art recognizes that DNA sequence polymorphisms which lead to changes in the amino acid sequences of the desaturases exist within a population (for example the Phaeodactylum tricornutum population) can. These genetic polymorphisms in the desaturase gene can exist between individuals within a population due to natural variation.
  • the terms "gene” and “recombinant gene” mean nucleic acid molecules with an open reading frame that encodes a desaturase, preferably a Phaeodactylum tricornutum desaturase.
  • Nucleic acid molecules that correspond to the natural variants and non-Phaeodactylum tricornutum homologues, derivatives and analogues of the Phaeodactylum tricornutum cDNA can be based on their homology to the Phaeodactylum tricornutum desaturase nucleic acid disclosed here using the Phaeornutactumum tricornutum part of it can be isolated as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule according to the invention is at least 15 nucleotides long and hybridizes under stringent conditions with the nucleic acid molecule which comprises a nucleotide sequence of SEQ ID N0: 1, 3, 5 or 11.
  • the nucleic acid is at least 25, 50, 100, 250 or more nucleotides in length.
  • hybridizes under stringent conditions is intended to describe hybridization and washing conditions under which nucleotide sequences which are at least 60% homologous to one another usually remain hybridized to one another.
  • the conditions are preferably such that sequences which are at least about 65%, more preferably at least about 70% and even more preferably at least about 75% or more homologous to one another usually remain hybridized to one another.
  • stringent conditions are known to the person skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • Examples of stringent hybridization conditions are hybridizations in 6 x sodium chloride / sodium citrate (SSC) at around 45 ° C, followed by one or more washing steps in 0.2 x SSC, 0.1% SDS at 50 to 65 ° C , It is known to the person skilled in the art that these hybridization conditions differ depending on the type of nucleic acid and, for example, if organic solvents are present, with regard to the temperature and the concentration of the buffer. The temperature differs, for example, under "standard hybridization conditions” depending on the type of nucleic acid between 42 ° C and 58 ° C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2).
  • the temperature is about 42 ° C under standard conditions.
  • the hybridization conditions for DNA: DNA hybrids are preferably, for example, 0.1 ⁇ SSC and 20 ° C. to 45 ° C., preferably between 30 ° C. and 45 ° C.
  • the hybridization conditions for DNA: RNA are preferably Hybrids for example 0.1 x SSC and 30 ° C to 55 ° C, preferably between 45 ° C and 55 ° C.
  • nucleic acid molecule which hybridizes under stringent conditions to a sequence of SEQ ID NO: 1, 3, 5 or 11 preferably corresponds to a naturally occurring nucleic acid molecule.
  • a "naturally occurring" nucleic acid molecule refers to an RNA or DNA molecule with a nucleotide sequence that occurs in nature
  • the nucleic acid encodes a naturally occurring Phyaedactylum tricornutum desaturase.
  • nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11 which leads to changes in the Amino acid sequence of the encoded desaturase leads without the functionality of the desaturase protein being impaired.
  • nucleotide substitutions which lead to amino acid substitutions at "non-essential" amino acid residues can be produced in a sequence of SEQ ID NO: 2, 4, 6 or 12.
  • a "non-essential" amino acid residue is a residue that is in one
  • Wild-type desaturase sequence of one of the desaturases can be changed without the activity of the desaturase being changed, that is to say substantially reduced, whereas an "essential" amino acid residue is required for the desaturase activity.
  • other amino acid residues for example those which are not conserved in the domain with desaturase activity or are only semi-preserved
  • An isolated nucleic acid molecule which encodes a desaturase which is homologous to a protein sequence of SEQ ID NO: 2, 4, 6 or 12 can be introduced into a nucleotide sequence of SEQ by introducing one or more nucleotide substitutions, additions or deletions ID NO: 1, 3, 5 or 11 are generated so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into one of the sequences of SEQ ID NO: 1, 3, 5 or 11 by standard techniques such as site-specific mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made on one or more of the predicted non-essential amino acid residues.
  • amino acid residue is exchanged for an amino acid residue with a similar side chain.
  • Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g.
  • a predicted non-essential amino acid residue in a desaturase is thus preferably replaced by another amino acid residue from the same side chain family.
  • the mutations can be introduced randomly over all or part of the desaturase coding sequence, e.g.
  • the encoded protein can be expressed recombinantly and the activity of the protein can e.g. can be determined using the tests described here (see example section).
  • nucleic acid molecules which are "antisense” to the nucleic acid sequences according to the invention.
  • An “antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid which encodes a protein, for example complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • An antisense nucleic acid can consequently bind to a sense nucleic acid via hydrogen bonds.
  • the antisense nucleic acid can be complementary to an entire strand coding for desaturase or only to a part thereof.
  • an antisense nucleic acid molecule is "antisense” to a "coding region" of the coding strand of a nucleotide sequence that encodes a desaturase.
  • coding region refers to the region of the nucleotide sequence which comprises codons which are translated into amino acid residues (for example the entire coding region which begins and ends with the stop codon, ie the last codon before the stop codon).
  • the antisense nucleic acid molecule is "antisense” to a "non-coding region" of the coding strand of a nucleotide sequence which codes for desaturase.
  • non-coding region relates to 5 'and 3' sequences which flank the coding region and are not translated into amino acids (ie which are also referred to as 5 'and 3' untranslated regions).
  • antisense nucleic acids according to the invention can be designed in accordance with the rules of Watson-Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of desaturase mRNA, but is more preferably an oligonucleotide that is only part of the coding or non-coding region of desaturase mRNA "antisense".
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of desaturase mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 and more nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art.
  • An antisense nucleic acid eg an antisense oligonucleotide
  • an antisense oligonucleotide can, for example, be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides which are designed in such a way that they increase the biological stability of the molecules or the physical stability of the difference between the antisense and the duplex formed in the sense nucleic acid, for example phosphorothioate derivatives and acridine-substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chloro-uracil, 5-ioduracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) racil , 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine Methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-me
  • the antisense nucleic acid can alternatively be produced biologically using an expression vector into which a nucleic acid has been subcloned in the antisense direction (ie RNA which is transcribed by the introduced nucleic acid is oriented in the antisense direction to a target nucleic acid of interest , which is further described in the subsection below).
  • the antisense nucleic acid molecules according to the invention are usually administered to a cell or generated in situ so that they hybridize with or bind to the cellular mRNA and / or the genomic DNA which encodes a desaturase, in order thereby to express the protein, for example by inhibition to inhibit transcription and / or translation.
  • Hybridization can be accomplished by conventional nucleotide complementarity to form a stable duplex or, for example in the case of an antisense nucleic acid molecule that binds DNA duplexes, by specific interactions in the major groove of the double helix.
  • the antisense molecule can be modified to specifically bind to a receptor or to an antigen expressed on a selected cell surface, for example by binding the antisense nucleic acid molecule to a peptide or an antibody that binds to a cell surface receptor or an antigen ,
  • the antisense nucleic acid molecule can also be delivered to the cells using the vectors described here.
  • vector constructs in which the antisense nucleic acid molecule is under the control of a strong prokaryotic, viral or eukaryotic, including plant, promoter are preferred.
  • the antisense nucleic acid molecule according to the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA, the strands running parallel to one another in contrast to conventional ⁇ -units.
  • the antisense nucleic acid molecule can also be a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987) FEBS Lett. 215: 327-330).
  • an antisense nucleic acid according to the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that can cleave a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g. hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591)) can be used for the catalytic cleavage of desaturase mRNA transcripts, thereby inhibiting the translation of desaturase mRNA.
  • a ribozyme with specificity for a desaturase-encoding nucleic acid can be isolated based on the nucleotide sequence of one of the desaturase cDNAs disclosed in SEQ ID NO: 1, 3, 5 or 11 (ie or based on a method according to the methods taught in this invention
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed, the nucleotide sequence of the active site being complementary to the nucleotide sequence that is to be cleaved in a desaturase-encoding mRNA, see for example Cech et al ., U.S. Patent No. 4,987,071 and Cech et al., U.S. Patent No.
  • desaturase mRNA can be used to select a catalytic RNA with specific ribonuclease activity from a pool of RNA molecules, see, e.g., Bartel, D ., and Szostak, JW (1993) Science 261: 1411-1418.
  • desaturase gene expression can be inhibited by directing nucleotide sequences that are complementary to the regulatory region of a desaturase nucleotide sequence (for example a desaturase promoter and / or enhancer) in such a way that triple helix structures are formed which form the Inhibit transcription of a desaturase gene in target cells.
  • a desaturase nucleotide sequence for example a desaturase promoter and / or enhancer
  • the expression cassette according to the invention includes the sequences mentioned in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 11, which are the result of the genetic code and / or their functional or non-functional derivatives to understand that have been functionally linked to one or more regulatory signals to increase gene expression and which advantageously control the expression of the coding sequence in the host cell. These regulatory sequences are intended for the targeted expression of the genes and the
  • Enable protein expression Depending on the host organism, this can mean, for example, that the gene is only expressed and / or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified, so that the natural regulation has been switched off and the expression of the genes increased.
  • the gene construct can also have a simpler structure, that is to say no additional regulation signals have been inserted in front of the nucleic acid sequence or its derivatives, and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence was mutated so that regulation no longer takes place and / or gene expression is increased.
  • the gene construct can also advantageously contain one or more so-called “enhancer sequences" functionally linked to the promoter, which enable increased expression of the nucleic acid sequence.
  • the regulatory sequences or factors can preferably have a positive influence on the gene expression of the introduced genes and thereby increase it.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • genes for inducers, repressors or enzymes which intervene in the regulation of one or more genes of a biosynthetic pathway through their enzyme activity, to be introduced and expressed therein.
  • genes for inducers, repressors or enzymes which intervene in the regulation of one or more genes of a biosynthetic pathway through their enzyme activity, to be introduced and expressed therein.
  • genes can be of heterologous or homologous origin.
  • further biosynthesis genes of the fatty acid or lipid metabolism can advantageously be contained in the nucleic acid construct or gene construct, or these genes can lie on another or more further nucleic acid constructs.
  • Acyl transferase s
  • fatty acid synthase s
  • fatty acid hydroxylase s
  • acetyl coenzma A carboxylase (s)
  • acyl coenzyme A oxidase (s)
  • fatty acid desaturase s
  • Fatty acid acetylenases lipoxygenases, triacylglycerol lipases, allen oxide synthases, hydroperoxide lyases or fatty acid elongase (s) or combinations thereof are used.
  • gene constructs advantageously comprise further 3'- and / or 5'-terminal regulatory sequences for increasing expression, which are selected depending on the host organism selected and the gene or the genes for the optimal expression.
  • these regulatory sequences are intended to enable specific expression of the genes and protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction or that it is expressed and / or overexpressed immediately.
  • the regulatory sequences or factors can also preferably have an advantageous effect on the expression of the genes introduced and thus increase them.
  • the regulatory elements can be advantageously amplified at the transcription level using strong transcription signals, such as promoters and / or enhancers.
  • strong transcription signals such as promoters and / or enhancers.
  • vectors preferably expression vectors, which contain a nucleic acid, a desaturase alone (or a part thereof) or a nucleic acid construct described under point b in which the nucleic acid according to the invention alone or in combination with other biosynthesis genes of the fatty acid or lipid metabolism such as desaturases or elongases is contained.
  • vector refers to a nucleic acid molecule that can transport another nucleic acid to which it is attached.
  • plasmid which stands for a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • vectors Another type of vector is a viral vector, whereby 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 (eg bacterial vectors with bacterial origin of replication and episomal mammalian vectors).
  • Other vectors eg non-episomal mammalian vectors
  • certain vectors can control the expression of genes to which they are operably linked. These vectors are referred to here as "expression vectors".
  • expression vectors suitable for recombinant DNA techniques are in the form of plasmids.
  • plasmid and "vector” can be used interchangeably because the plasmid is the most commonly used vector form.
  • the invention is intended to encompass these other expression vector forms, such as viral vectors (eg replication-deficient retroviruses, adenoviruses and adeno-related viruses), which perform similar functions.
  • viral vectors eg replication-deficient retroviruses, adenoviruses and adeno-related viruses
  • vector is also intended to include other vectors which are known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phas ide, phagemids, cosmids, linear or circular DNA.
  • the recombinant expression vectors according to the invention comprise a nucleic acid or a gene construct according to the invention in a form which is suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors have one or more regulatory sequences selected on the basis of those to be used for expression Host cells that are operably linked to the nucleic acid sequence to be expressed.
  • operably linked means that the nucleotide sequence of interest is linked to the regulatory sequence (s) in such a way that the expression of the nucleotide sequence is possible and they are linked to one another, so that both sequences have the predicted sequence assigned to the sequence Perform function (for example in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to be promoters, enhancers and include other expression control elements (eg polyadenylation signals).
  • Regulatory sequences include those which control the constitutive expression of a nucleotide sequence in many host cell types and those which control the direct expression of the nucleotide sequence only in certain host cells under certain conditions.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, which are encoded by the nucleic acids as described here (e.g. desaturases, mutant forms of desaturases, fusion proteins, etc.).
  • the recombinant expression vectors according to the invention can be designed for the expression of desaturases and elongases in prokaryotic or eukaryotic cells.
  • desaturase genes in bacterial cells such as C. glutamicum,
  • Insect cells using baculovirus expression vectors
  • yeast and other fungal cells see Romanos, MA, et al. (1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488; van den Hondel , CAMJJ, et al. (1991) "Heterologous gene expression in filamentous fungi", in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure, ed., Pp.
  • the recombinant expression vector may alternatively, for example using T7 promoter regulatory sequences and
  • T7 polymerase can be transcribed and translated in vitro.
  • Proteins are usually expressed in prokaryotes using vectors which contain constitutive or inducible promoters which control the expression of fusion or non-fusion proteins.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually at the amino terminus of the recombinant protein, but also at the C terminus or fused within suitable ranges in the proteins. These fusion vectors usually have three functions: 1) to increase the expression of recombinant protein; 2) increasing the solubility of the recombinant protein and 3) supporting the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is often introduced at the junction of the fusion unit and the recombinant protein, so that the recombinant protein can be separated from the fusion unit after the fusion protein has been purified.
  • These enzymes and their corresponding recognition sequences include factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Ine; Smith, DB, and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) , in which glutathione-S-transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein.
  • GST glutathione-S-transferase
  • the desaturase coding sequence is cloned into a pGEX expression vector to produce a vector encoding a fusion protein comprising GST-thrombin cleavage site X protein from the N-terminus to the C-terminus.
  • the fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant desaturase that is not fused to GST can be obtained by cleaving the fusion protein
  • yeast vectors are, for example, pAG-1, YEp6, YEpl3 or pEMBLYe23.
  • the desaturases according to the invention can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol .. 3: 2156-2165) and the pVL- Series (Lucklow and Summers (1989) Virology 170: 31-39).
  • a nucleic acid according to the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammals are understood to mean all non-human mammals.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (aufman et al. (1987) EMBO J. 6: 187-195).
  • the control functions of the expression vector are often provided by viral regulatory elements. Promoters commonly used are e.g. from Polyoma, Adenovirus2, Cytomegalie virus and Simian Virus 40.
  • the recombinant mammalian expression vector can preferably control the expression of the nucleic acid in a specific cell type (for example, tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275) , in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
  • mammary specific promoters e.g., milk serum promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166.
  • Development-regulated promoters are also included, e.g. the mouse hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546).
  • the desaturases according to the invention can be found in single-cell plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251 and literature references cited therein, and plant cells from higher plants (for example spermatophytes, such as crops) ) are expressed.
  • plant expression vectors include those described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border ", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 8711-8721; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, pp. 15-38.
  • a plant expression cassette preferably contains
  • polyadenylation signals are those derived from Agrobacterium tumefaciens-t-DNA, such as gene 3 of the Ti plasmid pTiACH5 known as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835ff.) Or functional equivalents thereof, but all other terminators that are functionally active in plants are also suitable.
  • a plant expression cassette preferably contains other functionally linked sequences, such as translation enhancers, for example the overdrive sequence which comprises the 5 'untranslated leader sequence from tobacco mosaic. virus that increases the protein / RNA ratio contains (Gallie et al., 1987, Nucl. Acids Research 15: 8693-8711).
  • Plant gene expression must be operably linked to a suitable promoter that performs gene expression in a timely, cell or tissue specific manner.
  • promoters which bring about constitutive expression such as those derived from plant viruses, such as 35S CAMV (Franck et al., Cell 21 (1980) 285 -294), 19S CaMV (see also US 5352605 and WO 84/02913) or plant promoters, such as that in US 4,962.02? described the small subunit of the Rubisco.
  • Targeting sequences which are necessary for controlling the gene product in its corresponding cell compartment (see an overview in Kermode, Crit. Rev. Plant Sei. 15, 4 (1996) 285-423 and references cited therein), for example in the vacuole, the Cell nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other parts of plant cells.
  • plastids such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other parts of plant cells.
  • Plant gene expression can also be facilitated via a chemically inducible promoter (see an overview in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48: 89-108).
  • Chemically inducible promoters are particularly suitable if it is desired that the gene expression takes place in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.
  • Promoters that react to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-induced PRPl gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), the heat-inducible hsp80 promoter from tomato (US Pat. No. 5,187,267), the cold-inducible alpha amylase promoter from potato (WO 96/12814) or the wound-inducible pin III promoter (EP-A-0 375 091).
  • the pathogen-induced PRPl gene promoter Ward et al., Plant. Mol. Biol. 22 (1993) 361-366
  • the heat-inducible hsp80 promoter from tomato US Pat. No. 5,187,267
  • the cold-inducible alpha amylase promoter from potato WO 96/1281
  • the wound-inducible pin III promoter EP-A-0 375 091
  • Suitable promoters are the Napingen promoter from rapeseed (US 5,608,152), the USP promoter from Vicia faba (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67), the oleosin pro otor from Arabidopsis (WO 98/45461), the phaseolin Promoter from Phaseolus vulgaris (US 5,504,200), the Bce4 promoter from Brassica (WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9 ) and promoters which bring about seed-specific expression in monocotyledonous plants, such as maize, barley, wheat, rye, rice, etc.
  • Suitable noteworthy promoters are the lpt2 or iptl gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene , the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the Rye secalin gene).
  • the multiparallel expression of desaturases according to the invention alone or in combination with other desaturases or elongases may be desired.
  • Such expression cassettes can be introduced via a simultaneous transformation of several individual expression constructs or by combining several expression cassettes on one construct.
  • Several vectors, each with several expression cassettes, can also be transformed and transferred to the host cell.
  • Promoters which bring about plastid-specific expression are also particularly suitable, since plastids are the compartment in which the precursors and some end products of lipid biosynthesis are synthesized.
  • Suitable promoters such as the viral RNA polymerase promoter, are described in WO 95/16783 and WO 97/06250, and the clpP promoter from Arabidopsis, described in WO 99/46394.
  • the invention also provides a recombinant expression vector comprising a DNA molecule according to the invention which is cloned into the expression vector in the antisense direction. ie the DNA molecule is operably linked to a regulatory sequence in such a way that expression (by transcription of the DNA molecule) of an RNA molecule that is "antisense" to the desaturase mRNA is made possible.
  • Regulatory sequences can be selected which are operably linked to a nucleic acid cloned in the antisense direction and which control the continuous expression of the antisense RNA molecule in a multiplicity of cell types, for example viral promoters and / or enhancers or regulatory sequences can be selected , which is the constitutive, tissue-specific or control cell type-specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a highly effective regulatory region, the activity of which can be determined by the type of cell into which the vector has been introduced.
  • a highly effective regulatory region the activity of which can be determined by the type of cell into which the vector has been introduced.
  • host cell and “recombinant host cell” are used interchangeably here. Of course, these terms refer not only to the specific target cell, but also to the descendants or potential descendants of this cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, these offspring are not necessarily identical to the parental cell, but are still within the scope of the term as used herein.
  • Recombinant or transgene for example recombinant expression vector or recombinant host or host cells in the sense of the invention, is to be understood that the nucleic acids according to the invention and / or their natural regulatory sequences at the 5 'and 3' position of the nucleic acids are not in their natural environment, that is to say either the position of the sequences in the original organism was changed or the nucleic acid sequences and / or the regulatory sequences were mutated in this or the nucleic acid sequences according to the invention were moved to an organism other than the original organism or its regulatory sequences. Combinations of these changes are also possible.
  • the natural environment is the position of a nucleic acid sequence in an organism as it occurs in nature.
  • a host cell can be a prokaryotic or eukaryotic cell.
  • a desaturase can be expressed in bacterial cells such as C. glutamicum, insect cells, fungal cells or mammalian cells (such as Chinese urinary ovary cells (CHO) or COS cells), algae, ciliates, plant cells, fungi or other microorganisms such as C. glutamicum become.
  • Other suitable host cells are known to the person skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells using conventional transformation or transfection techniques. The terms "transformation” and “transfection”, conjugation and transduction, as used herein, are intended to mean a variety of methods known in the art for
  • transforming or transfecting host cells including plant cells, can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual., 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44 , Agrobacterium protocols, eds .: Gartland and Davey, Humana Press, Totowa, New Jersey.
  • selectable markers include those which confer resistance to drugs such as G418, hygromycin and methotrexate, or in plants which confer resistance to a herbicide such as glyphosphate or glufosinate.
  • markers which code genes which are involved in biosynthetic pathways for example of sugars or amino acids, such as ⁇ -galactodsidase, ura3 or ilv2.
  • Markers which encode genes such as luciferase, gfp or other fluorescent genes are also suitable. These markers can be used in mutants in which these genes are not functional, since they have been deleted, for example, using conventional methods.
  • markers which encode a nucleic acid which encodes a selectable marker can be introduced into a host cell on the same vector as the one which encodes a desaturase, or can be introduced on a separate vector.
  • Cells that have been stably transfected with the introduced nucleic acid can, for example, be identified by drug selection (for example, cells that have integrated the selectable marker survive, whereas the other cells die).
  • a vector is produced which contains at least a section of a desaturase gene into which a deletion, addition or substitution has been introduced in order to thereby change, for example functionally disrupt, the desaturase gene.
  • This desaturase gene is preferably a Phaeodactylum tricornutum desaturase gene, but a homologue or analogue from other organisms, even from a mammalian, fungal or insect source, can be used.
  • the vector is designed such that the endogenous desaturase gene is functionally disrumpted when homologous recombination occurs (ie no longer encodes a functional protein, also referred to as a knock-out vector).
  • the vector can be designed in such a way that the endogenous desaturase gene is mutated or otherwise changed during homologous recombination, but still encodes a functional protein (for example the upstream regulatory region can be changed in such a way that the expression of the endogenous desaturase is thereby changed)
  • DNA-RNA hybrids known as chimeraplasty which are derived from Cole-Strauss et al. , 1999, Nucleic Acids Research 27 (5): 1323-1330 and Kmiec, Gene therapy, 19999, American Scientist, 87 (3): 240-247.
  • the modified portion of the desaturase gene is flanked at its 5 'and 3' ends by additional nucleic acid of the desaturase gene, so that homologous recombination between the exogenous desaturase gene present on the vector and an endogenous desaturase gene in one Microorganism or a plant is possible.
  • the additional flanking desaturase nucleic acid is long enough for successful homologous recombination with the endogenous gene.
  • the vector contains several hundred base pairs of DNA flanking up to kilobases (both at the 5 'and 3' ends) (for a description of vectors for homologous recombination, see, for example, in Thomas, KR, and Capecchi, MR (1987) Cell 51: 503 or recombination in Physcomitrella patens based on cDNA in Strepp et al., 1998, Proc. Natl. Acad. Sci. USA 95 (8): 4368-4373).
  • the vector is introduced into a microorganism or plant cell (eg, using polyethylene glycol-mediated DNA), and cells in which the introduced desaturase gene is homologously recombined with the endogenous desaturase gene are selected using techniques known in the art.
  • recombinant organisms such as microorganisms, can be produced which contain selected systems which allow regulated expression of the introduced gene.
  • the inclusion of a desaturase gene in a vector, whereby it is brought under the control of the lac operon, enables, for example, the expression of the desaturase gene only in the presence of IPTG.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell, growing in culture or in a field, can be used to produce (i.e., express) a desaturase.
  • an alternative method can also be used by direct transfer of DNA into developing flowers via electroporation or gene transfer using Agrobacterium.
  • the invention thus further provides methods for producing desaturases using the host cells of the invention.
  • the method comprises culturing the host cell according to the invention (into which a recombinant expression vector encoding a desaturase has been introduced, or into whose genome a gene encoding a wild-type or modified desaturase has been introduced) in a suitable medium until the desaturase has been produced.
  • the method comprises isolating the desaturases from the medium or the host cell.
  • Host cells which are suitable in principle for taking up the nucleic acid according to the invention, the gene product according to the invention or the vector according to the invention are all prokaryotic or eukaryotic organisms.
  • the host organisms advantageously used are organisms such as bacteria, fungi, yeasts, animal or plant cells. Other advantageous organisms are animals or preferably plants or parts thereof.
  • Mushrooms, yeasts or plants are preferably used, particularly preferably mushrooms or plants, very particularly preferably plants, such as oil fruit plants, which contain large amounts of lipid compounds, such as oilseed rape, evening primrose, canola, peanut, flax, soya, safflower, sunflower, borage , or plants, such as corn, wheat, rye, oats, triticale, rice, barley, cotton, manioc, pepper, tagetes, solanaceae plants, such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, bush plants ( Coffee, cocoa, tea), types of salix, trees (oil plant, coconut) as well as perennial grasses and fodder crops.
  • Particularly preferred plants according to the invention are oil fruit plants such as soybean, peanut, rapeseed, canola, flax, evening primrose, sunflower, safflower, trees (oil palm, coconut).
  • Another aspect of the invention relates to isolated desaturases and biologically active parts thereof.
  • An "isolated” or “purified” protein or a biologically active portion thereof is essentially free of cellular material when it is produced by recombinant DNA techniques, or of chemical precursors or other chemicals when it is chemically synthesized.
  • the term "essentially free of cellular material” encompasses desaturase preparations in which the protein is separated from cellular components of the cells in which it is produced naturally or recombinantly.
  • the term "substantially free of cellular material” includes desaturase preparations with less than about 30% (by dry weight) non-desaturase (also referred to herein as "contaminating protein"), more preferably less than about 20% non-desaturase, more preferably less than about 10% non-desaturase, and most preferably less than about 5% non-desaturase.
  • contaminating protein also referred to herein as "contaminating protein”
  • the desaturase or a biologically active portion thereof has been produced recombinantly, it is also substantially free of culture medium, ie the culture medium makes up less than about 20%, more preferably less than about 10% and most preferably less than about 5 % of the volume of the protein preparation.
  • substantially free of chemical precursors or other chemicals includes desaturase preparations in which the protein is separated from chemical precursors or other chemicals involved in the synthesis of the protein.
  • the term “substantially free of chemical precursors or other chemicals” includes desaturase preparations with less than about 30% (by dry weight) chemical precursors or non-desaturase chemicals, more preferably less than about 20% chemical precursors or non-desaturase chemicals, more preferably less than about 10% chemical precursors or non-desaturase chemicals, and most preferably less than about 5% chemical precursors or non-desaturase chemicals.
  • isolated proteins or biologically active parts thereof have no contaminating proteins from the same organism from which the desaturase originates.
  • Phaeodactylum tricornutum desaturase in plants such as Physcomitrella patens or og or microorganisms, for example bacteria such as E. coli, Bacillus subtilis, C. glutamicum, fungi such as Mortierella, yeast such as Saccharomyces, or Ciliates like Colpidium or algae like Phaeodactylum.
  • An isolated desaturase according to the invention or a part thereof can also participate in the metabolism of compounds necessary for the construction of cell membranes in Phaeodactylum tricornutum or in the transport of molecules across these membranes.
  • the protein or portion thereof comprises an amino acid sequence sufficiently homologous to an amino acid sequence of SEQ ID NO: 2, 4, 6 or 12 that the protein or portion thereof has the ability to metabolize to build Cell membranes in Phaeodactylum tricornutum not-
  • a desaturase according to the invention has one of the SEQ ID NO: 2, 4, 6 or 12
  • the desaturase has an amino acid sequence which is encoded by a nucleotide sequence which, for example under stringent conditions, hybridizes to a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11. Another one
  • the desaturase has an amino acid sequence encoded by a nucleotide sequence that is at least about 50 to 60%, preferably at least about 60 to 70%, more preferably at least about 70 to 80%, 80 to 90%, 90 to 95% and more preferably at least about 96%,
  • a preferred desaturase according to the invention preferably also has at least one of the desaturase activities described here.
  • a preferred desaturase according to the invention comprises an amino
  • nucleotide sequence which, for example under stringent conditions, hybridizes to a nucleotide sequence of SEQ ID NO: 1, 3, 5 or 11 and on the metabolism of compounds necessary for the construction of cell membranes in Phaeodactylum tricornutum or on transport of molecules
  • 35 can participate through these membranes or introduce a double bond into a fatty acid with one, two, three or four double bonds and a chain length of Cis, C 0 or C 2 .
  • the desaturase is substantially 40 homologous to an amino acid sequence of SEQ ID NO: 2, 4 or 6 and maintains the functional activity of the protein of one of the sequences of SEQ ID NO: 2, 4 or 6, their.
  • amino acid sequence differs due to natural variation or mutagenesis, as described in detail in subsection I above.
  • desaturase is thus a protein comprising an "amino acid sequence that is at least about 50 to 60%, preferably at least about 60 to 70% and more preferably at least about 70 to 80%, 80 to 90%, 90 " to 95% and most preferably at least about 96%, 97%, 98%, 99% or even more homologous to a complete amino acid sequence of SEQ ID NO: 2, Is 4 or 6.
  • the invention relates to a complete Phaeodactylum tricornutum protein which is essentially homologous to a complete amino acid sequence of SEQ ID NO: 2, 4 or 6.
  • Biologically active parts of a desaturase include peptides comprising amino acid sequences derived from the amino acid sequence of a desaturase, e.g. an amino acid sequence shown in SEQ ID NO: 2, 4 or 6, or the amino acid sequence of a protein homologous to a desaturase which has fewer amino acids than the full length desaturase or the full length protein homologous to a desaturase, and at least one activity have a desaturase.
  • Usually biologically active parts (peptides, for example peptides that are for example 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids long) comprise a domain or a motif with at least one activity of a desaturase.
  • biologically active parts in which other regions of the protein have been deleted can be produced by recombinant techniques and examined for one or more of the activities described herein.
  • the biologically active parts of a desaturase preferably comprise one or more selected domains / motifs or parts thereof with biological activity.
  • Desaturases are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above), and the desaturase is expressed in the host cell. The desaturase can then be isolated from the cells using a suitable purification scheme using standard protein purification techniques. As an alternative to recombinant expression, a desaturase, a polypeptide or a peptide can be chemically synthesized using standard peptide synthesis techniques.
  • native desaturase can be isolated from cells (for example endothelial cells), for example using an anti-desaturase antibody which can be produced by standard techniques, using a desaturase according to the invention or a fragment thereof.
  • the invention also provides chimeric desaturase proteins or desaturase fusion proteins.
  • a "chimeric desaturase protein” or “desaturase fusion protein” includes a desaturase polypeptide that is operably linked to a non-desaturase polypeptide.
  • a “desaturase polypeptide” refers to a polypeptide with an amino acid sequence that corresponds to a desaturase
  • a “non-desaturase polypeptide” refers to a polypeptide with an amino acid sequence that corresponds to a protein that is substantially non-homologous to the desaturase, e.g. a protein that differs from desaturase and comes from the same or a different organism.
  • the term "operably linked” is intended to mean that the desaturase polypeptide and the non-desaturase polypeptide are fused to one another in such a way that both sequences fulfill the predicted function ascribed to the sequence used.
  • the non-desaturase polypeptide can be fused to the N-terminus or the C-terminus of the desaturase polypeptide.
  • the fusion protein is, for example, a GST-desaturase fusion protein in which the desaturase
  • fusion proteins are fused to the C-terminus of the GST sequences. These fusion proteins can facilitate the purification of the recombinant desaturases.
  • the fusion protein is a desaturase which has a heterologous signal sequence at its N-terminus.
  • the expression and / or secretion of a desaturase can be increased by using a heterologous signal sequence.
  • a chimeric desaturase protein according to the invention or
  • Desaturase fusion protein is made by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences are ligated together in-frame according to conventional techniques, for example by smooth or overhanging ends for ligation, restriction enzyme cleavage to provide suitable ends, filling in cohesive ends as required, treatment with alkaline phosphatase to avoid unwanted linkages to avoid and enzymatic ligation are used.
  • the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers.
  • a PCR amplification of gene fragments can be carried out using anchor primers which generate complementary overhangs between successive gene fragments which can subsequently be hybridized with one another and reamplified so that a chimeric gene sequence is generated (see for example Current Protocols in Molecular Biology, ed. Ausubel et al. , John Wiley & Sons: 1992).
  • anchor primers which generate complementary overhangs between successive gene fragments which can subsequently be hybridized with one another and reamplified so that a chimeric gene sequence is generated
  • many expression vectors are already commercially available which already encode a fusion unit (eg a GST polypeptide).
  • a desaturase-encoding nucleic acid can be cloned into such an expression vector, so that the fusion unit is linked in frame with the desaturase protein.
  • Desaturase homologs can be isolated by mutagenesis, e.g. by specific point mutation or shortening of the desaturase.
  • the term "homologs" as used herein refers to a variant form of desaturase that acts as an agonist or antagonist of desaturase activity.
  • An agonist of desaturase can maintain essentially the same activity as or part of the biological activities of desaturase.
  • An antagonist of desaturase can carry out one or more activities of the naturally occurring form of desaturase by, for example, competitive binding to a downstream or upstream element of the metabolic cascade for cell membrane components comprising the desaturase or by binding to a desaturase which facilitates the transport of compounds mediated via cell membranes, inhibit, whereby the translocation is inhibited.
  • homologs of desaturase can be identified by viewing combinatorial banks of mutants, eg shortening mutants, of desaturase for desaturase agonist or antagonist activity.
  • a varied bank of desaturase variants is generated by combinatorial mutagenesis at the nucleic acid level and encoded by a varied gene bank.
  • a varied bank of desaturase variants can be produced, for example, by enzymatic ligation of a mixture of synthetic oligonucleotides in gene sequences, so that a degenerate set of potential desaturase sequences can be used as individual polypeptides or alternatively as a set of larger fusion proteins (for example for the phage display ) that contain this set of desaturase sequences.
  • a degenerate set of potential desaturase sequences can be used as individual polypeptides or alternatively as a set of larger fusion proteins (for example for the phage display ) that contain this set of desaturase sequences.
  • the chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer and the synthetic gene can then be ligated into a suitable expression vector.
  • degenerate set of genes allows all of the sequences encoding the desired set of potential desaturase sequences to be provided in a mixture.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 11: 477).
  • banks of desaturase fragments can be used to produce a varied population of desaturase fragments for screening and for the subsequent selection of homologues of a desaturase.
  • a bank of fragments of the coding sequence can be obtained by treating a double-stranded PCR fragment of a coding desaturase sequence with a nuclease under conditions under which double-strand breaks occur only about once per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded DNA, which can include sense / antisense pairs of different double stranded break products, removing single stranded sections from newly formed duplexes by treatment with S1 nuclease and ligating the resulting fragment library into an expression vector.
  • an expression bank can be derived which encodes N-terminal, C-terminal and internal fragments of desaturase of different sizes.
  • REM Recursive ensemble mutagenesis
  • a screening process that detects PUFA-dependent enzyme activity (s) is a prerequisite, especially for screening desaturase activities.
  • Desaturase activities with specificity for PUFAs can be used in Mucor species, which can be transformed by known transformation processes with desired gene constructs, the toxicity of arachidonic acid in the presence of a toxic metabolite (here: salicylic acid or salicylic acid derivatives) (Eroshin et al., Mikrobiologiya, Vol 65, No.l 1996, pages 31-36) in order to carry out a growth-based initial inspection.
  • Resulting clones can then be subjected to an analysis of their lipid constituents by means of gas chromatography and mass spectroscopy in order to determine the type and quantity of starting materials and products.
  • cell-based tests can be used to analyze a varied desaturase library using other methods known in the art.
  • nucleic acid molecules, proteins, protein homologues, fusion proteins, primers, vectors and host cells described here can be used in one or more of the following methods: identification of Phaeodactylum and related organisms, mapping of the genomes of organisms related to Phaeodactylum tricornutum, identification and localization of Phaeodactylum tricornutum sequences from
  • the desaturase nucleic acid molecules according to the invention have a multitude of uses. They can initially be used to identify an organism as Phaeodactylum tricornutum or as a close relative of it. They can also be used to identify the presence of Phaeodactylum tricornutum or a relative thereof in a mixed population be used by microorganisms.
  • the invention provides the nucleic acid sequences of a number of Phaeodactylum tricornutum genes; by probing the extracted genomic DNA of a culture of a uniform or mixed population of microorganisms under stringent conditions with a probe spanning a portion or part of a Phaeodactylum tricornutum gene unique to that organism, one can determine whether that organism is present.
  • Phaeodactylum tricornutum itself is used for the commercial production of polyunsaturated acids and is also suitable for PUFA production in other organisms, particularly if the aim is to incorporate the resulting PUFAs in the triacylglycerol fraction.
  • nucleic acid and protein molecules according to the invention can serve as markers for specific regions of the genome. This is not only suitable for mapping the genome, but also for functional Phaeodactylum tricornutum proteins. To identify the genome region to which a specific DNA-binding protein from Phaeodactylum tricornutum binds, the Phaeodactylum tricornutum genome could, for example, be cleaved and the fragments incubated with the DNA-binding protein.
  • nuclemic acid molecules according to the invention can additionally be probed with the nuclemic acid molecules according to the invention, preferably with easily detectable markings; the binding of such a nucleic acid molecule to the genome fragment enables the localization of the fragment on the genome map of Phaeodactylum tricornutum and, if this is carried out several times with different enzymes, facilitates a rapid determination of the nucleic acid sequence to which the protein binds.
  • the nucleic acid molecules according to the invention can also be sufficiently homologous to the sequences of related species that these nucleic acid molecules can serve as markers for the construction of a genomic map in related fungi or algae.
  • the desaturase nucleic acid molecules according to the invention are also suitable for evolution and study studies.
  • the metabolic and transport processes in which the molecules according to the invention are involved are used by many prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules according to the invention with those which code similar enzymes from other organisms, the degree of evolutionary kinship of the organisms can be determined. Accordingly, such a comparison enables determination of which sequence regions are conserved and which are not, which can be helpful in determining regions of the protein which are essential for the enzyme function. This type of determination is valuable for protein engineering studies and can provide an indication of how much mutagenesis the protein can tolerate without losing function.
  • the manipulation of the desaturase nucleic acid molecules according to the invention can lead to the production of desaturases with functional differences from the wild-type desaturases.
  • the efficiency or activity of these proteins can be improved, they can be present in the cell in larger numbers than usual, or their efficiency or activity can be reduced.
  • Improved efficiency or activity means, for example, that the enzyme has a higher selectivity and / or activity, preferably an at least 10% higher, particularly preferably an at least 20% higher activity, very particularly preferably an at least 30% higher activity than the original enzyme.
  • Fatty acids such as PUFAs and lipids containing PUFAs are themselves desirable fine chemicals;
  • the yield, production and / or increase the efficiency of the production of fatty acid and lipid molecules in ciliates, algae, plants, fungi, yeasts or other microorganisms are themselves desirable fine chemicals;
  • the manipulation of one or more desaturase genes according to the invention can likewise lead to desaturases with changed activities which indirectly influence the production of one or more desired fine chemicals from algae, plants, ciliates or fungi.
  • the normal biochemical metabolic processes lead e.g. for the production of a large number of waste products (e.g. hydrogen peroxide and other reactive oxygen species) which can actively disrupt these metabolic processes (e.g. nitrated peroxynitrite, as is known, tyrosine side chains, whereby some enzymes are inactivated with tyrosine in the active center (Groves, JT (1999) Curr Opin. Chem. Biol. 3 (2), -226-235)).
  • the cells used for large-scale fermentative production are optimized for the overproduction of one or more fine chemicals and can therefore produce more waste products than is usual for a wild-type cell.
  • one or more desaturases according to the invention which are involved in the export of waste molecules, one can improve the viability of the cell and maintain an efficient metabolic activity.
  • the presence of high intracellular amounts of the desired fine chemical can actually be toxic to the cell, so that by increasing the ability of the cell to secrete these compounds, the viability of the cell can be improved.
  • the desaturases according to the invention can also be manipulated in such a way that the relative amounts of different lipid and fatty acid molecules are changed. This can have a decisive impact on the lipid composition of the cell membrane. Because each type of lipid has different physical properties, changing the lipid composition of a membrane can significantly change membrane fluidity. Changes in membrane fluidity can affect the transport of molecules across the membrane, which, as explained above, can modify the export of waste products or the fine chemicals produced or the import of necessary nutrients. These changes membrane fluidity can also have a decisive influence on the integrity of the cell; Cells with comparatively weaker membranes are more susceptible to abiotic and biotic stress conditions, which can damage or kill the cell.
  • nucleic acid and protein molecules according to the invention can be used to generate algae, ciliates, plants, animals, fungi or other microorganisms, such as C. glutamicum, which express mutated desaturase nucleic acid and protein molecules - Mieren, so that the yield, production and / or efficiency of production of a desired compound is improved.
  • This desired compound can be any natural product of algae, ciliates, plants, animals, fungi or bacteria, which includes the end products of biosynthetic pathways and intermediate products of naturally occurring metabolic pathways, as well as molecules that are not naturally occurring in the metabolism of these cells, but which are derived from the cells according to the invention are produced.
  • a further embodiment according to the invention is a method for producing PUFAs, the method comprising culturing an organism which comprises a nucleic acid according to the invention, a gene construct according to the invention or a vector according to the invention which encode a polypeptide, the cis, C o ⁇ or C-fatty acids with at least two double bonds in the fatty acid molecule extended by at least two carbon atoms under conditions under which PUFAs are produced in the organism.
  • PUFAs produced by this method can be isolated by harvesting the organisms either from the culture in which they grow or from the field, breaking up and / or extracting the harvested material with an organic solvent.
  • the oil, lipids, phospholipids, sphingolipids, glyco- contains lipids, triacylglycerme and / or free fatty acids with a higher PUFA content.
  • the free fatty acids with a higher PUFA content can be isolated by basic or acidic hydrolysis of the lipids, phospholipids, sphingolipids, glycolipids, triacylglycermes.
  • a higher content of PUFAs means at least 5%, preferably 10%, particularly preferably 20%, very particularly preferably 40% more PUFAs than the original organism, which has no additional nucleic acid which encodes the desaturase according to the invention.
  • the PUFAs produced by this process are preferably cis or C 2 o- 22 fatty acid molecules with at least two double bonds in the fatty acid molecule, preferably three or four, or five or six double bonds when combined with a further elongase and a ⁇ -4 desaturase.
  • These C 18 or C o 22 fatty acid molecules can be isolated from the organism in the form of an oil, lipid or a free fatty acid. Suitable organisms are, for example, those mentioned above. Preferred organisms are transgenic plants.
  • An embodiment according to the invention are oils, lipids or fatty acids or fractions thereof which have been produced by the process described above, particularly preferably oil, lipid or a fatty acid composition which comprise PUFAs and originate from transgenic plants.
  • a further embodiment according to the invention is the use of the oil, lipid or the fatty acid composition in feed, food, cosmetics or pharmaceuticals.
  • Another object of the invention is a method for identifying an antagonist or agonist of desaturases, comprising
  • the candidate substance mentioned can be a chemically synthesized or microbiologically produced substance and can occur, for example, in cell extracts from, for example, plants, animals or microorganisms. Furthermore, although the substance mentioned may be known in the prior art, it has not hitherto been known to increase or represent the activity of the desaturases.
  • the reaction mixture can be a cell-free extract or can comprise a cell or cell culture.
  • Suitable methods are known in the art and are generally described in Alberts eg, molecular cular Biology the cell, 3rd Edition (1994), for example Chapter 17.
  • the substances mentioned can be injected, for example, be added to the reaction mixture or the culture medium or cells or sprayed on a plant.
  • a sample containing a substance active according to the method according to the invention has been identified, then it is either possible to isolate the substance directly from the original sample or the sample can be divided into different groups, e.g. if it consists of a large number of different components so as to reduce the number of different substances per sample and then to repeat the method according to the invention with such a "sub-sample" of the original sample.
  • the steps described above can be repeated several times, preferably until the sample identified according to the method according to the invention only comprises a small number of substances or only one substance.
  • the substance or derivative thereof identified according to the method according to the invention is preferably further formulated so that it is suitable for use in plant breeding or plant cell or tissue culture.
  • the substances that were tested and identified according to the method according to the invention can be: expression libraries, for example cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic substances, hormones, PNAs or the like (Milner, Nature Medicin 1 (1995 ), 879-880; Hupp, Cell. 83 (1995), 237-245; Gibbs, Cell. 79 (1994), 193-198 and references cited therein). These substances can also be functional derivatives or analogs of the known inhibitors or activators. Processes for the production of chemical derivatives or analogs are known to the person skilled in the art. The derivatives and analogues mentioned can be tested according to methods according to the prior art.
  • the cell or tissue that can be used for the method according to the invention is preferably a host cell, plant cell or a according to the invention Plant tissue as described in the above embodiments.
  • the present invention also relates to a substance which has been identified in accordance with the above methods according to the invention.
  • the substance is e.g. a homologue of the desaturases according to the invention.
  • Homologs of the desaturases can be obtained by mutagenesis, e.g. by point mutation or deletion of the desaturases.
  • the term "homolog" is used herein as a variant form of the desaturases, which acts as an agonist or antagonist for the activity of the desaturases.
  • An agonist can have substantially the same or a part of the biological activity of the desaturases.
  • An antagonist of the desaturases can inhibit one or more activities of the naturally occurring forms of the desaturases, e.g. competitively located on a downstream or upstream member of the fatty acid synthesis pathways which include, bind to, or bind to desaturases and thereby reduce or inhibit activity.
  • the present invention also relates to an antibody or a fragment thereof, as described herein, which inhibits the activity of the desaturases according to the invention.
  • the present invention relates to an antibody which specifically recognizes or binds the agonist or antagonist according to the invention described above.
  • compositions which comprises the antibody, the stop identified by the method according to the invention or the antisense molecule.
  • the present invention relates to a kit comprising the nucleic acid according to the invention, the gene construct according to the invention, the amino acid sequence according to the invention, the antisense nucleic acid molecule according to the invention, the antibody and / or composition according to the invention, an antagonist or agonist which was produced by the method according to the invention , and / or oils, lipids and / or fatty acids according to the invention or a fraction thereof.
  • the kit can also comprise the host cells, organisms, plants or parts thereof according to the invention, harvestable parts of the plants or propagation material according to the invention or else the antagonists or agonists according to the invention.
  • the components of the kit of the present invention can be packaged in suitable containers, for example with or in buffers or other solutions.
  • One or more of the components mentioned can be in one and packed in the same container. Additionally or alternatively, one or more of the components mentioned can be adsorbed, for example, on a solid surface, for example nitrocellulose filters, glass plates, chips, nylon membranes or microtiter plates.
  • the kit can be used for any of the methods and embodiments described herein, eg for the production of host cells, transgenic plants, for the detection of homologous sequences, for the identification of antagonists or agonists etc. Furthermore, the kit can provide instructions for the use of the kit for a of the applications mentioned.
  • Cloning processes such as restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, connection of DNA fragments, transformation of Escherichia coli and yeast cells, cultivation of bacteria and sequence analysis of recombinant DNA were carried out as described in Sambrook et al.
  • the isolated nucleic acid sequences according to the invention are contained in the genome of a Phaeodactylum tricornutum UTEX646 strain, 15 which is available from the algae collection of the University of Texas, Austin.
  • Phaeodactylum tricornutum was cultivated at 25 ° C with a light / dark rhythm of 14:10 hours at 22 ° C and 35 microEinstein (corresponding to 20 micromol photons per square meter and second) in glass tubes which were aerated with air from below.
  • the culture medium for Phaeodactylum tricornutum was the f / 2 culture medium with 10% organic medium according to Guillard, R.R.L. 25 used (1975; Culture of phytoplankton for feeding marine invertebrates. In: Smith, W.L. and Chanley, M.H. (Eds.) Culture of marine Invertebrate animals, NY Plenum Press, pp. 29-60.): It contains
  • Trace element solution Na 2 EDTA (4.36 g / 1), FeCl 3 (3.15 g / 1), 35 Primary trace elements: CuS04 (10 g / 1), ZnS0 4 (22 g / 1), CoCl 2
  • 0.1 mg / 1 org medium Na acetate (1 g / 1), glucose (6 g / 1), Na succinate 40 (3 g / 1), bacto-tryptone (4 g / 1), yeast Extract (2 g / 1)
  • the details of the isolation of total DNA relate to the processing of plant material with a fresh weight of one gram.
  • CTAB buffer 2% (w / v) N-acetyl-N, N, N-trimethylammonium bromide (CTAB); 100 M Tris-HCl, pH 8.0; 1.4 M NaCl; 20mM EDTA. 10
  • N-lauryl sarcosine buffer 10% (w / v) N-lauryl sarcosine; 100 mM Tris-HCl, pH 8.0; 20mM EDTA.
  • Phaeodactylum tricornutum cell material was under liquid
  • the DNA was treated with NaCl (1.2 M final concentration) and precipitated again at -70 ° C. for 30 min using twice the volume of absolute ethanol. After a washing step with 70% ethanol, the DNA was dried and then in 50 ml H 2 0 + RNAse (50 mg / ml
  • the DNA was dissolved overnight at 4 ° C. and the RNAse cleavage was then carried out at 37 ° C. for 1 hour. The DNA was stored at 4 ° C.
  • Example 3 Isolation of total RNA and poly (A) + RNA from 40 plants and Phaeodactylum tricornutum
  • RNA isolation from Phaeodactylum tricornutum is isolated according to a method described by Logemann et al (1987, Anal. Biochem. 163, 21).
  • the total RNA Protonema tissue can be obtained from moss by the GTC method (Reski et al., 1994, Mol. Gen. Genet., 244: 352-359).
  • Frozen algae samples (- 70 ° C) were ground into fine powder in an ice-cold mortar under liquid nitrogen.
  • 2 volumes of homogenization medium (12.024 g sorbitol, 40.0 ml IM Tris-HCl, pH 9 (0.2 M); 12.0 ml 5 M NaCl (0.3 M), 8.0 ml 250 mM EDTA, 761, 0 mg EGTA, 40.0 ml 10% SDS were made up to 200 ml with H 2 0 and the pH was adjusted to 8.5) and 4 volumes of phenol with 0.2% mercaptoethanol were added at 40 to 50 ° C with good mixing frozen cell powder. Then 2 volumes of chloroform were added and the mixture was stirred vigorously for 15 min. It was centrifuged at 10,000 g for 10 min and the aqueous phase was extracted with phenol / chloroform (2 vol) and finally with chloroform.
  • the mixture was then centrifuged at 10,000 g for 30 min and the supernatant was sucked off. A washing step with 70% EtOH followed and centrifugation again.
  • the sediment was taken up in Tris-borate buffer (80 mM Tris-borate buffer, 10 mM EDTA, pH 7.0). Then the supernatant was mixed with 1/3 vol 8 M LiCl, mixed and incubated at 4 ° C. for 30 min. After centrifuging again, the sediment was washed with 70% ethanol, centrifuged and the sediment was dissolved in RNAse-free water.
  • Poly (A) + RNA was isolated using Dyna Beads (Dynal, Oslo, Finland) according to the instructions in the manufacturer's protocol.
  • RNA or poly (A) + RNA concentration After determining the RNA or poly (A) + RNA concentration, the RNA was precipitated by adding 1/10 volumes of 3 M sodium acetate, pH 4.6, and 2 volumes of ethanol and stored at -70 ° C.
  • RNA 20 ⁇ g RNA were separated in a 1.5% agarose gel containing formaldehyde and transferred to nylon membranes (Hybond, Amersham). The detection of specific transcripts was carried out as described in Amasino ((1986) Anal. Biochem. 152, 304)).
  • the first strand synthesis using reverse transcriptase from mouse leukemia virus (Röche, Mannheim, Germany) and oligo-d (T) primers the second strand synthesis obtained by incubation with DNA polymerase I, Klenow enzyme and cleavage with RNAse H at 'l2 ° C (2 hrs.), 16 ° C (1 hr.) and 22 ° C (1 hr.). The reaction was stopped by incubation at 65 ° C (10 min) and then transferred to ice. Double-stranded DNA molecules were blunt-ended with T4 DNA polymerase (Röche, Mannheim) at 37 ° C. (30 min).
  • the nucleotides were removed by phenol / chloroform extraction and Sephadex G50 centrifugation columns.
  • EcoRI / XhoI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends using T4 DNA ligase (Röche, 12 ° C, overnight), cut with Xhol and by incubation with polynucleotide kinase (Röche, 37 ° C , 30 min) phosphorylated. This mixture was subjected to separation on a low-melting agarose gel.
  • DNA molecules over 300 base pairs were eluted from the gel, phenol extracted, concentrated on Elutip-D columns (Schleicher and Schüll, Dassel, Germany) and ligated to vector arms and in lambda-ZAP-Express phages using the Gigapack Gold -Kits (Stratagene, Amsterdam, The Netherlands) packed using manufacturer's material and following its instructions.
  • cDNA banks as described in Example 4, were used for DNA sequencing by standard methods, in particular by the chain termination method using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-El er, Rothstadt, Germany).
  • the sequencing of random, isolated clones was carried out after the preparative plasmid extraction from cDNA libraries via in vivo mass excision and retransformation of DH10B on agar plates (details on material and protocol from Stratagene, Amsterdam, Netherlands). Plasmid DNA was obtained from overnight grown E. coli cultures grown in Luria broth with ampicillin (see Sambrook et al.
  • sequences were processed and annotated using the standard software package EST-MAX, which is commercially available from Bio-Max (Munich, Germany).
  • EST-MAX standard software package
  • SEQ ID NO: 8 The search sequence shown was searched for homologous genes using the BLAST program (Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402.).
  • Two sequences from Phaeodactylum tricornutum with homologies to the search sequence from Physco-mitrella patens were characterized in more detail.
  • Example 5a Isolation of desaturases from Phaeodactylum tricornutum via polymerase chain reaction using degenerate oligonucleotides:
  • Oligonucleotide sequences with possible variations are shown below.
  • the amino acid from which the base combination can be derived is shown in the one-letter code below the oligonucleotide sequence.
  • a / G means that at this position in the synthesis of the building block either an A or a G is incorporated into the oligonucleotide in a statistically uniform distribution, since the base triplet derived from the corresponding amino acid can be either an AAA or an AAG.
  • the DNA sequence can also contain an inosine (i) if the determination of a base at this position allows three or four different bases based on the genetic code.
  • the following sequences and primers can be used:
  • F5a L: CA T / C TA T / C TGG AA A / G AA T / C CA G C
  • F5a_ L HYWKNQH / Q F6a: TTG TTG AAi A / CAA / G AA i CA T / C AA
  • the primers can be used in all combinations for polymerase chain reactions. With the aid of individual combinations, desaturase fragments could be isolated if the following conditions were taken into account: 10 nmol primers and 10 ng of a plasmid bank obtained by in vivo excision 0 were used for PCR reactions.
  • the plasmid bank was isolated from the phage bank according to the manufacturer's protocols (Stratagene).
  • the PCR reaction was carried out in a thermal cycler (Biometra) with the Pfu DNA polymerase (Stratagene) and the following temperature program: 3 min at 96 ° C, followed by 5 of 35 cycles with 30 s at 96 ° C, 30 s at 55 ° C and 1 min at 72 ° C.
  • the temperature of the additive was gradually reduced by 3 ° C after the first step of 55 ° C and after the fifth cycle a • annealing temperature of 40 ° C maintained. Finally, a cycle was carried out at 72 ° C. for 10 min and the batch was terminated by cooling to 4 ° C.
  • the primer combination F6a and R4a2 are underlined in the text and could be successfully used to isolate a desaturase fragment.
  • the resulting fragment could be verified by sequencing and showed homologies to a desaturase with the Genbank Accession No. T36617 from Streptomyces coelicolor. Homology was obtained using the BLASTP program. The comparison is shown in FIG. 4. Identities of 34% and homology of 43% to sequence T36617 were found.
  • the DNA fragment was used according to Example 7 in a hybridization experiment to isolate a full length gene according to standard conditions according to the invention.
  • the coding region of a DNA sequence thus isolated was obtained by translating the genetic code into a polypeptide sequence.
  • SEQ ID NO: 3 it is 1434 base pairs long
  • sequence shown that could be isolated by the described method.
  • the sequence has a start codon in position 1 to 3 and a stop codon in position 1432-1434 and could be translated into a 477 amino acid polypeptide.
  • WO 98 46763 By comparison with a gene sequence described in WO 98 46763, it was found that a non-identical but homologous fragment from Phaeodactylum tricornutum coding for 87 amino acids was previously described.
  • WO 98/46763 does not disclose a complete, functionally active desaturase, position or substrate specificity. This is also clear from the fact that homologies to ⁇ -5 and to ⁇ -6 desaturase from Mortierella alpina are reported without specifying an exact function.
  • the sequence according to the invention codes for a functionally active ⁇ -6-acyl lipid desaturase.
  • the result of the two est sequences found is shown in FIG. 1 and in FIG. 2 and in FIG. 2a.
  • the sequences found are part of the nucleic acids according to the invention from SEQ ID NO: 1 (name: Pt_des5, own database number of the inventors PT001078032R), SEQ ID NO: 5 (name: Pt_desl2, own database number of the inventors PT0010070010R) and SEQ ID NO: 11 (name: Pt_desl2.2, own database of the inventor PT001072031R).
  • Letters indicate identical amino acids, while the plus sign means a chemically similar amino acid.
  • the identities or homologies of all sequences found according to the invention are summarized in Table 2.
  • Desaturases can have cytochrome b5 domains that also occur in other genes that do not code for desaturases. Cytochrome b5 domains therefore show high homologies, even though they are different gene functions. Desaturases of weakly conserved areas can only be identified as putative candidate genes and must be checked for the enzyme activity and position specificity of the enzymatic function. For example, various hydroxylases, acetylenases and epoxygenases similar to desaturases show histidine box motifs, so that a specific function has to be verified experimentally and, in addition, the verification of the double bond enables safe enzyme activity and position specificity of a desaturase.
  • ⁇ -6 and ⁇ -5 desaturase according to the invention have particularly suitable substrate specificities and are particularly suitable for use in combination with a ⁇ -6 elongase from Physcomitrella for the production of polyunsaturated fatty acids such as arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid can.
  • Sequencing of the complete cDNA fragment from clone PT001078032R resulted in a 1652 base pair sequence.
  • the sequence codes for a polypeptide of 469 amino acids shown in SEQ ID NO: 2. This was obtained by translating the genetic code from SEQ ID NO: 1 with a start codon in base pair position ' 115-117 and with a stop codon in base pair position 1522- 1524th
  • the clone contains a complete desaturase polypeptide, as can be seen from the sequence comparison in FIG. 3.
  • Dashes mean identical amino acids, while colons and single points represent chemically interchangeable, ie chemically equivalent, amino acids.
  • FIG. 6 and FIG. 7 show the comparison of the MA_desl2 peptide sequence with the sequences found.
  • Sequencing the complete cDNA fragment from clone PT0010070010R resulted in a 1651 base pair sequence shown in SEQ ID NO: 5 with a start codon in position 67-69 and a stop codon in position 1552-1554.
  • the polypeptide sequence according to the invention is shown in SEQ ID NO: 6.
  • Fragment from clone PT0010072031R resulted in a 1526 base pair sequence shown in SEQ ID NO: 11 with a start codon in position 92-94 and a stop codon in position 1400-1402.
  • the polypeptide sequence according to the invention is shown in SEQ ID NO: 12.
  • Table 2 shows the identities and homologies of desaturases according to the invention with one another and with the desaturase from Physcomitrella patens and Mortierella alpina. The information was obtained using the Bestfit program under given parameters as defined below as a sub-program of the following software: Wisconsin Package Version 10.0 (Genetics Computer Group (GCG), Madison, Wisc, USA). Henikoff, S. and Henikoff, J.G. (1992). Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Be. USA 89: 10915-10919.
  • FIG. 5 also shows the comparison of the ⁇ -6-acyl lipid desaturase from Physcomitrella patens with the polypeptide sequence of the clone Pt_des6.
  • Table 2A Homologs with the highest sequence homologies or identities to polypeptide sequences according to the invention from SEQ ID NO. 2, 4, 6 or 12
  • Gene sequences can be used to identify homologous or heterologous genes from cDNA or genomic banks.
  • Homologous genes ie full-length cDNA clones that are homologous or homologs
  • cDNA libraries in particular for the isolation of functionally active full-length genes which can be shown in SEQ ID NO: 3 the method can be used.
  • 100,000 to 1,000,000 recombinant bacteriophages are plated and transferred to a nylon membrane. After denaturing with alkali, the DNA was immobilized on the membrane, for example by UV crosslinking. Hybridization takes place under highly stringent conditions.
  • Hybridization probes were, for example, labeled by means of radioactive ( 32 P) nick transcription (high prime, Röche, Mannheim, Germany). The signals are detected by means of autoradiography.
  • Partially homologous or heterologous genes which are related but not identical, can be identified analogously to the method described above using low-stringent hybridization and washing conditions.
  • the ionic strength was usually kept at 1 M NaCl, the temperature being gradually lowered from 68 to 42 ° C.
  • Radioactively labeled oligonucleotides are produced by phosphorylating the 5 'end of two complementary oligonucleotides with T4 polynucleotide kinase.
  • the complementary oligonucleotides are hybridized to one another and ligated so that concatemers are formed.
  • the double-stranded concatems are radioactively marked, for example, by nick transcription.
  • Hybridization is usually carried out under low-stringent conditions using high oligonucleotide concentrations.
  • CDNA sequences were used to produce recombinant protein, for example in E. coli (e.g. Qiagen QIAexpress pQE system).
  • the recombinant proteins were then usually affinity purified via Ni-NTA affinity chromatography (Qiagen).
  • the recombinant proteins were then used, for example, to produce specific antibodies
  • 15 expression cDNA libraries can be used by immunological screening (Sambrook, J., et al. (1989), “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, or Ausubel, FM, et al. ( 1994) "Current Protocols in Molecular Biology", John Wiley & Sons).
  • Agrobacterium-mediated plant transformation can be performed, for example, using GV3101- (pMP90-) (Koncz and Schell, 25 Mol. Gen. Genet. 204 (1986) 383-396) or LBA4404- (Clontech) or C58C1 pGV2260 (Deblaere et al 1984, Nucl. Acids Res. 13, 4777-4788) Agrobacterium tumefaciens strain.
  • the transformation can be carried out using standard transformation techniques (also Deblaere et al. 1984).
  • Agrobacterium-mediated plant transformation can be performed using standard transformation and regeneration
  • rape can be transformed using cotyledon or hypocotyl transformation (Moloney et al., Plant Cell 8 (1989) 238-242; De Block et al., Plant Physiol. 91 (1989) 45 694-701).
  • the use of antibiotics for Agrobacterium and plant selection depends on the binary vector and Agrobacterium strain used for the transformation.
  • the Rapeseed selection is usually carried out using kanamycin as a selectable plant marker.
  • Agrobacterium -mediated gene transfer in linseed can be carried out using, for example, one of Mlynarova et al. (1994) Plant Cell Report 13: 282-285 perform the technique described.
  • soy can be carried out using, for example, a technique described in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 0397 687, US 5,376,543, US 5,169,770 (University Toledo) ,
  • Binary vectors such as pBinAR can be used for plant transformation
  • the binary vectors can be constructed by ligating the cDNA in sense or antisense orientation in T-DNA. 5 'of the cDNA, a plant promoter activates the transcription of the cDNA. A polyadenylation sequence is located 3 'from the cDNA.
  • the binary vectors can carry different marker genes. For example, resistance can be expressed by expressing the nptll marker gene under the control of the 35S or the nos promoter.
  • nptll marker gene coding for kanamycin resistance mediated by neomycin phosphotransferase can be exchanged for the herbicide-resistant form of an acetolactate synthase gene (AHAS or ALS).
  • the ALS gene is described in Ott et al., J. Mol. Biol. 1996, 263: 359-360.
  • the use of the hygromycin resistance gene is also suitable for some plants.
  • the v-ATPase-cl promoter can be cloned into the plasmid pBin19 or pGPTV and can be used for the marker gene expression by cloning in front of the coding region of the ALS gene.
  • the v-ATPase-cl promoter mentioned corresponds to a 1153 base pair fragment from Beta vulgaris (Plant Mol Biol, 1999, 39: 463-475).
  • the nos promoter mentioned here both sulfonylureas and imidazolinones such as imazethapyr or sulfonylureas can be used as antimetabolites for selection. the.
  • the nos promoter can also be used for the marker gene expression.
  • Example 12 Determination of suitable promoters for expression in linseed
  • Tissue-specific expression can be achieved using a tissue-specific promoter.
  • seed-specific expression can be achieved by using the DC3-
  • any other seed-specific promoter element such as the Napin or Arcelin promoter (Goosens et al. 1999, Plant Phys. 120 (4): 1095-1103 and Gerhardt et al. 2000, Biochimica et Biophysica Acta 1490 (1-2): 87-98)
  • the CaMV-35S promoter or a v-ATPase-cl promoter can be used for constitutive expression in the whole plant.
  • the bacterial 25 ⁇ -glucuronidase (GUS) may be mentioned as an example of a reporter gene (Jefferson et al., EMBO J. 1987, 6, 3901-3907).
  • the ⁇ -glucuronidase activity can be determined in situ using a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid as part of an activity staining (Jefferson, 1987, Plant Molecular Biology Reporter 5, 30 387 -405).
  • a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid as part of an activity staining (Jefferson, 1987, Plant Molecular Biology Reporter 5, 30 387 -405).
  • a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid as part of an activity staining (Jefferson, 1987, Plant Molecular Biology Reporter 5,
  • MUG 4-methyl-umbelliferyl-beta-D-glucuronide
  • MU methyl-umbelliferone
  • a protein extract of the desired tissue is first produced, to which the substrate of the GUS is then added.
  • a calibration series was prepared which contained concentrations of 0.1 mM to 10 mM MU (4-methyl-umbelliferone). If the sample values were outside these concentrations, less protein extract was used (10 ml, 1 ml, 1 ml from 1:10 dilution) and shorter time intervals were measured (0 h, 30 min, 1 h). The measurement was carried out with an exitation of 365 nm and an emission of 445 nm in a Fluoroscan II device (lab system).
  • the substrate cleavage can be monitored fluorometrically under alkaline conditions (excitation at 365 nm, measurement of the emission at 455 nm; SpectroFluorimeter BMG Polarstar +) as described in Bustos MM et al., 1989 Plant Cell 1: 839-853. All samples were subjected to a protein concentration determination according to Bradford (1976) in order to allow a statement about the promoter activity and strength in different tissues and plants.
  • reporter genes examples which may be the green fluorescent protein (GFP) and its derivatives called (C.Reichel et al. (1996) Proc.Natl.Acad. Sei.USA 93, from 5888 to 5893 and J. Sheen et al., (1995) Plant Journal 8, 777-784) and various luciferases (A.Millar et al. (1992) Plant Mol. Biol. Reporter 10, 324-414).
  • GFP green fluorescent protein
  • C.Reichel et al. (1996) Proc.Natl.Acad. Sei.USA 93, from 5888 to 5893 and J. Sheen et al., (1995) Plant Journal 8, 777-784) and various luciferases (A.Millar et al. (1992) Plant Mol. Biol. Reporter 10, 324-414).
  • the Talking detection methods are known to those skilled in the art and described, for example, the literature mentioned.
  • promoter-reporter gene constructs for the above promoters are given below. Fragments of these promoters can be isolated using the polymerase chain reaction and tailored with flanking sequences of your choice based on synthetic oligonucleotides.
  • oligonucleotides can be used, for example:
  • LeB4 front GAAAGCTTCTCGAGTTATGCATTTCTT
  • LeB4 rear GGGTCTAGATCTGTGACTGTGATAG
  • DC3a front AGTGGATCCCCGAGCTAACCACAACT DC3a rear: ATAAGCTTTTTCTTTGGCAGA napinvorne: GAAATACTTACAAACGGATAChinctacatac
  • the promoter fragments are amplified by PCR, cut with suitable restriction enzymes and cloned into the above cassettes.
  • the LeB4 (700) -PCR fragment is cut with Xbal and Hindlll and cloned into the vector pGPTV in the Hindlll and Xbal sites 5 'in front of the GUS reporter gene.
  • the PCR-amplified DC3 promoter fragment can be cut with BamHI and HindIII, for example subcloned into pBluescript (Stratagene) * and then cloned into suitable interfaces in pGPTV in front of the GUS reporter gene.
  • a nanoprotectant fragment PCR-amplified with the above primers and having a size of 1055 bp after digestion with HindIII and Xbal in pGPTV can be cloned in front of the GUS reporter gene.
  • An equivalent construct could be a napin promoter fragment of 11OObp 5 'cloned in front of a GUS reporter gene with intron with a 3 * • subsequent Nos terminator in a pHL9000 vector (Hausmann & Tmür, 1999).
  • the GUS activity can be measured in transgenic flax embryos of various ages that have been transformed with one of the following constructs: Napin-GUS, 35S-GUS, LeB4- CIS, USP-CIS.
  • the values are mean values from one to five measurements with different a lot of protein. Three embryos per construct were analyzed quantitatively.
  • the Napin promoter from Brassica napus was found to be two to three orders of magnitude less active than the two promoters from Vicia faba (LeB4 and USP).
  • GUS activity increased in the order of Napin, LeB4 and USP.
  • the positive control 35S moved in its activity between LeB4 and USP, whereas the negative control, the non-transformed wild type (Flanders variety), had practically no activity.
  • the following table shows the mean values of the activities in the individual age stages and overall for each construct.
  • Table 3.4 Overview of the mean GUS activities of flax embryos, transformed with different GUS constructs. daf: days after flowering begins.
  • Binary vectors such as pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230) or pGPTV (Becker et al 1992, Plant Mol. Biol. 20: 1195-1197) or derivatives thereof can be used for plant transformation.
  • the binary vectors can be constructed by ligating the cDNA in sense or antisense orientation in T-DNA. 5 'of the cDNA, a plant promoter activates the transcription of the cDNA. A polyadenylation sequence is located 3 'from the cDNA.
  • the binary vectors can carry different marker genes.
  • nptII marker gene coding for kanamycin resistance can be mediated can be replaced by neomycin phosphotransferase for the herbicide-resistant form of an acetolactate synthase gene (abbreviation: AHAS or ALS).
  • AHAS acetolactate synthase gene
  • the ALS gene is described in Ott et al. , J. Mol. Biol. 1996, 263: 359-360.
  • the v-ATPase-cl promoter can be cloned into the plasmid pBin19 or pGPTV and used for the marker gene expression by cloning in front of the ALS coding region.
  • the promoter mentioned corresponds to a 1153 base pair fragment from beta-Vulgaris (Plant Mol Biol, 1999, 39: 463-475). Both sulphonylureas and imidazolinones such as imazethapyr or sulphonylureas can be used as antimetabolites for selection.
  • Tissue-specific expression can be achieved using a tissue-specific promoter.
  • seed-specific expression can be achieved by cloning the DC3 or the LeB4 or the USP promoter or the phaseolin promoter 5 'of the cDNA. Any other seed-specific promoter element such as the Napin or Arcelin promoter Goossens et al. 1999, Plant Phys. 120 (4): 1095-1103 and Gerhardt et al. 2000, Biochimica et Biophysica Acta 1490 (1-2): 87-98) can be used.
  • the CaMV-35S promoter or a v-ATPase Cl promoter can be used for constitutive expression in the whole plant.
  • genes coding for desaturases and elongases can be cloned in succession into a binary vector by constructing several expression cassettes in order to simulate the metabolic pathway in plants.
  • the protein to be expressed can be directed into a cellular compartment using a signal peptide, for example for plastids, mitochondria or the endoplasmic reticulum (Kermode, Crit. Rev. Plant Sei. 15, 4 (1996) 285-423).
  • the signal peptide is cloned 5 'in frame with the cDNA in order to achieve the subcellular localization of the fusion protein.
  • Expression cassettes consist of at least two functional units such as a promoter and a terminator. Further desired gene sequences such as targeting sequences, coding regions of genes or parts thereof, etc. can be inserted between the promoter and terminator.
  • promoters and terminators USP promoter: Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67
  • OCS terminator Gielen et al. EMBO J. 3 (1984) 835ff.
  • oligonucleotides can be used, for example: USP1 front: CCGGAATTCGGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCA USP2 front: CCGGAATTCGGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCA USP3 front: CCGGAATTCGGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCA USP1 back: AAAACTGCAGGCGGCCGCCCACCGCGGTGGGCTGGCTATGAAGAAATT USP2 back: CGCGGATCCGCTGGCTATGAAGAAATT
  • USP3 rear TCCCCCGGGATCGATGCCGGCAGATCTGCTGGCTATGAAGAAATT 0CS1 front: AAAACTGCAGTCTAGAAGGCCTCCTGCTTTAATGAGATAT OCS2 front: CGCGGATCCGATATCGGGCCCATCTAGCGTTAACCCCCTGCTTTAATGAGAGCGTTAACCCTGCTTGATGAGAGAGGGTCCATGAGG
  • 0CS1 rear CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTAAATTGA
  • OCS2 rear CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTAAATTGA
  • 0CS3 rear CCCAAGCTTGGCTCGGGGGAGCGGGTCGAGA
  • a promoter and a terminator are amplified via PCR. Then the terminator is cloned into a recipient plasmid and in a second step the promoter is inserted in front of the terminator. An expression cassette on a carrier plasmid is thus obtained.
  • the plasmids pUT1, pUT2 and pUT3 are created on the basis of the plasmid pUCl9.
  • the constructs are defined in SEQ ID NO: 13, 14 and 15. They contain the USP promoter and the OCS terminator based on pUCl9.
  • the construct PÜT12 is created by cutting pUTl using Sall / Scal and pUT2 using Xhol / Scal. The fragments containing the expression cassettes are ligated and transformed into E. coli XLI blue MRF. After separating ampicillin-resistant colonies, DNA is prepared and those clones which contain two expression cassettes are identified by restriction analysis. The Xhol / Sall ligation of compatible ends has eliminated the two interfaces Xhol ' and Sall between the expression cassettes.
  • plasmid pUTl2 which is defined in SEQ ID NO: 16.
  • pUTl2 is cut again using Sal / Scal and pUT3 is cut using Xhol / Scal.
  • the fragments containing the expression cassettes are ligated and transformed into E. coli XLI blue MRF.
  • DNA is prepared and identified by restriction analysis those clones which contain three expression cassettes. In this way, a set of multi-expression cassettes is created that can be used for the insertion of the desired DNA and is described in Table 3 and can also accommodate further expression cassettes.
  • the DC3 promoter is described in Thomas, Plant Cell 1996, 263: 359-368 and consists only of the region -117 to +27 which is why it is one of the smallest known seed-specific promoters.
  • Fragments of these promoters can be isolated using the polymerase chain reaction and tailored with flanking sequences of your choice based on synthetic oligonucleotides.
  • oligonucleotides can be used, for example: LeB4 front: GAAAGCTTCTCGAGTTATGCATTTCTT LeB4 rear: GGGTCTAGATCTGTGACTGTGATAG DC3a front: CCGGAATTCGGCGCGCCGAGCTCCTCGAG DC3a rear: CGCGGATCCTAGCTTTTTCTTGGCAGATG
  • the promoter fragments are amplified by PCR, cut with suitable restriction enzymes and cloned into the above cassettes.
  • the LeB4 (700) PCR fragment is cut with Xhol and BglII and inserted into the Xhol and BglII sites of the plasmid pUT3 to obtain pLT3.
  • Advantageous expression cassettes contain, based on pUCl9 (Vieira and Messing (1982); Gene 19, 259), the SEQ ID NO: 32, the LeB4 promoter and the sequences SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO : 35.
  • the construct pLTl2 is created on the basis of these plasmids by cutting pUTl using Sall / Scal and pUT2 using Xhol / Scal.
  • the fragments containing the expression cassettes are ligated and transformed into E. coli XLI blue MRF. After separation of ampicillin-resistant colonies, DNA is prepared and those clones which contain two expression cassettes are identified by restriction analysis.
  • the Xhol / Sall ligation of compatible ends has eliminated the two interfaces Xhol and Sall between the expression cassettes.
  • the result is the plasmid pUTl2, which is defined in SEQ ID NO: 16.
  • pUT12 is cut again using Sal / Scal and pUT3 cut using Xhol / Scal.
  • the fragments containing the expression cassettes are ligated and transformed into E. coli XLI blue MRF.
  • DNA is prepared and those clones which contain three expression cassettes are identified by restriction analysis. In this way, a selection of multi-expression cassettes is created which can be used for the insertion of desired DNA and is described in Table 3 and can also accommodate other expression cassettes.
  • the expression cassettes can contain the same promoter several times or can be constructed using three different promoters.
  • Table 4 Multiple expression cassettes
  • Phaseolin promoter (Bustos et al. (1989) Plant Cell 1,839-853) or using the
  • Multi-expression cassettes can be inserted using Ascl directly from pUC19 derivatives from Table 3 into the vector pGPTV + AscI (see iii.)) Via the Ascl interface and are available for inserting target genes.
  • the corresponding gene constructs (pBUTl is in SEQUENCE ID NO: 20, pBUT2 is in SEQUENCE ID NO: 21, pBUT 3 is in SEQUENCE ID NO: 22, pBUTl2 is in SEQUENCE ID NO: 22 and pBUTl23 is shown in SEQUENCE ID NO: 24 ) are available according to the invention as a kit.
  • gene sequences can be inserted into the pUC19-based expression cassettes and used as Ascl fragments in pGPTV + AscI.
  • the D-6 elongase Pp_PSEl is first inserted into the first cassette via BstXI and Xbal. Then the D-6 desaturase from moss (Pp_des6) is inserted into the second cassette via BamHI / Nael.
  • the construct pUT-ED is produced.
  • the Ascl fragment from the plasmid pUT-ED is inserted into the vector pGPTV + AscI cut with Ascl and the orientation of the inserted fragment is determined by means of restriction or sequencing.
  • the plasmid pB-DHGLA is formed, the complete sequence of which is shown in SEQUENCE ID NO. 25 is shown.
  • the coding region of the Physcomitrella delta 6 elongase is in SEQUENCE ID NO. 26 shown that the delta 6 desaturase from Physcomitrella in SEQUENCE ID NO: 27.
  • the ⁇ -6 elongase Pp_PSEl is first inserted into the first cassette via BstXI and Xbal. Then the ⁇ -6-desaturase from moss (Pp_des6) is inserted into the second cassette via BamHI / Nael and finally the ⁇ -5-desaturase from Phaeodactylum (Pt_des5) is inserted into the third cassette via BglII.
  • the triple construct is given the name pARAl. Taking into account sequence-specific restriction interfaces, further expression cassettes according to Table 5 with the designation pARA2, pARA3 and pARA4 can be created.
  • the Ascl fragment from the plasmid pARAl is inserted into the vector pGPTV + AscI cut with Ascl and the orientation of the inserted fragment is determined by means of restriction or sequencing.
  • the complete sequence of the resulting plasmid pBARAl is in SEQUENCE ID NO. 28 shown.
  • the coding region of the Physcomitrella delta 6 elongase is in SEQUENCE ID NO. 29 shown that the delta 6 desaturase from Physcomitrella in SEQUENCE ID NO: 30 and that of the delta-5 desaturase from Phaeodactylum tricornutum in SEQUENCE ID NO: 31.
  • Plasmids 1 to 5 are pUC derivatives, plasmids 6 to 7 are binary plant transformation vectors
  • Pp Physcomitrella patens
  • Pt Phaeodactylum tricornutum
  • Pp_PSEl corresponds to the sequence from SEQ ID NO: 9.
  • PSE PUFA specific ⁇ -6 elongase
  • Ce_des5 ⁇ -5 desaturase from Caenorhabditis elegans (Genbank Acc.
  • Ce_des6 ⁇ -6 desaturase from Caenorhabditis elegans elegans
  • Ce_PSEl ⁇ -6-elongase from Caenorhabditis elegans (Genbank Acc.
  • Chimeric gene constructs based on those described in pUC19 can be inserted into the binary vector pGPTV using Ascl.
  • the multiple cloning sequence is extended by an Ascl interface.
  • the polylinker is newly synthesized as two double-stranded oligonucleotides, with an additional Ascl DNA sequence being inserted.
  • the oligonucleotide is inserted into the vector pGPTV using EcoRI and HindIII.
  • the plasmid pGPTV + AscI is formed.
  • the necessary cloning techniques are known to the person skilled in the art and can simply be read as described in Example 1.
  • Example 14 In vivo mutagenesis
  • the in vivo mutagenesis of microorganisms can be accomplished by passing the plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillus spp. Or yeasts such as Saccharomyces cerevisiae), in which the skills, the integrity of their genetic Maintain information, is disturbed, take place.
  • E. coli or other microorganisms e.g. Bacillus spp. Or yeasts such as Saccharomyces cerevisiae
  • Common mutator strains have mutations in the genes for the DNA repair system (e.g. utHLS, mutD, mutT, etc.; for a literature reference see Rupp, WD (1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington). These strains are known to the person skilled in the art. The use of these strains is explained, for example, in Greener, A. and Callahan, M. (1994
  • Transgenic plants are created according to various examples in the example part of this document.
  • Example 15 Examination of the expression of a recombinant gene product in a transformed organism
  • the activity of a recombinant gene product in the transformed host organism can be measured at the transcription and / or the translation level.
  • a suitable method for determining the amount of transcription of the gene is to carry out a Northern blot as outlined below (for reference see Ausubel et al. (1988 ) Current Protocols in Molecular Biology, Wiley: New York, or the example section mentioned above), wherein a primer which is designed in such a way that it binds to the gene of interest is labeled with a detectable label (usually radioactive or chemilumescence), so that when the total RNA of a culture of the organism is extracted, separated on a gel, transferred to a stable matrix and incubated with this probe, the binding and extent of binding of the probe are the presence and also the amount of mRNA therefor Gene indicates.
  • a detectable label usually radioactive or chemilumescence
  • Total cellular RNA can be derived from cells, tissues, or organs by several methods, all of which are known in the art, such as that of Bormann, ER, et al. (1992) Mol. Microbiol. 6: 317-326.
  • RNA hybridization 20 ⁇ g of total RNA or 1 ⁇ g of poly (A) + RNA were analyzed by gel electrophoresis in agarose gels with a strength of 1.25% using formaldehyde, as described in Amasino (1986, Anal. Biochem. 152 , 304) separated, transferred by capillary attraction using 10 x SSC to positively charged nylon membranes (Hybond N +, Amersham, Braunschweig), immobilized by means of UV light and 3 hours at 68 ° C. using hybridization buffer
  • the DNA probe was labeled with the Highprime DNA labeling kit (Röche, Mannheim, Germany) during the pre-hybridization using alpha- 32 P-dCTP (Amersham, Braunschweig, Germany).
  • the hybridization was carried out after adding the labeled DNA probe in the same buffer at 68 ° C. overnight.
  • the washing steps were carried out twice for 15 min using 2 X SSC and twice for 30 min using 1 X SSC, 1% SDS, at 68 ° C.
  • the exposure of the closed filter was carried out at -70 ° C for a period of 1 to 14 T.
  • Standard techniques such as a Western blot can be used to examine the presence or relative amount of protein translated from this mRNA (see, for example, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York).
  • the total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix, such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein.
  • This probe is usually provided with a chemiluminescent or colorimetric label that is easy to detect. The presence and amount of the label observed indicates the presence and amount of the desired mutant protein present in the cell.
  • Example 16 Analysis of the effect of the recombinant proteins on the production of the desired product
  • the effect of genetic modification in plants, fungi, algae, ciliates or on the production of a desired compound can be determined by growing the modified microorganisms or the modified plant under suitable conditions (such as those described above) and that Medium and / or the cellular components for the increased production of the desired product (ie lipids or a fatty acid) is examined.
  • This analysis Techniques are known to the person skilled in the art and include spectroscopy, thin-layer chromatography, staining methods of various types, enzymatic and microbiological methods and analytical chromatography, such as high-performance liquid chromatography (see for example Ullman, Encyclopedia of Industrial
  • the analysis methods include measurements of the amounts of nutrients in the medium (e.g. sugar, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of the biomass composition and growth, analysis of the production of common metabolites of biosynthetic pathways and measurements of gases that are generated during fermentation. Standard procedures for these measurements are in Applied Microbial Physiology; A Practical Approach, PM Rhodes and PF Stanbury, eds., IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and the references cited therein.
  • nutrients in the medium e.g. sugar, hydrocarbons, nitrogen sources, phosphate and other ions
  • fatty acids abbreviations: FAME, fatty acid methyl ester; GC-MS, gas-liquid chromatography-mass spectrometry; TAG, triacylglycerol; TLC, thin-layer chromatography.
  • the unambiguous evidence for the presence of fatty acid products can be obtained by analysis of recombinant organisms according to standard analysis methods: GC, GC-MS or TLC, as described variously by Christie and the references therein (1997, in: Advances on Lipid Methodology, Fourth Ed .: Christie, Oily Press, Dundee, 119-169; 1998, gas chromatography-mass spectrometry method, lipids 33: 343-353).
  • the material to be analyzed can be broken up by ultrasound treatment, glass mill grinding, liquid nitrogen and grinding, or other applicable methods.
  • the material must be centrifuged after breaking up.
  • the sediment is in aqua dest. resuspended, heated at 100 ° C for 10 min, cooled on ice and centrifuged again, followed by extraction in 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 hour at 90 ° C, which leads to hydrolyzed oil and lipid compounds, which result in transmethylated lipids.
  • fatty acid methyl esters are extracted into petroleum ether and finally subjected to GC analysis using a capillary column (chrome pack, WCOT fused silica, CP-Wax-52 CB, 25 microm, 0.32 mm) at a temperature gradient between 170 ° C and 240 ° C for Subjected for 20 min and 5 min at 240 ° C.
  • the identity of the fatty acid methyl esters obtained must be defined using standards available from commercial sources (i.e. Sigma).
  • the Escherichia coli strain XLI Blue MRF 'kan (Stratagene) was used for subcloning the new desaturase pPDesaturasel from Physcomitrella patens.
  • the Saccharomyces cerevisiae strain INVSc 1 (Invitrogen Co.) for the functional expression of this gene.
  • E. coli was cultivated in Luria Bertini broth (LB, Duchefa, Haarlem, the Netherlands) at 37 ° C. If necessary, ampicillin (100 mg / liter) was added and 1.5% agar (w / v) was added for LB solid media. S.
  • cerevisiae was at 30 ° C either in YPG medium or in complete minimal medium without uracil (CMdum; see in: Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Seidan, JG, Smith, JA, Struhl, K., Albright, LB, Coen, DM, and Varki, A. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York) cultivated with either 2% (w / v) raffinose or glucose. For solid media, 2% (w / v) Bacto TM agar (Difco) was added. The plasmids used for cloning and expression are pUCl8 (Pharmacia) and pYES2 (Invitrogen Co.).
  • the Phaeodactylum tricornutum cDNA clones from Seq ID NO: 1, 3, 5 or 11 or the sequences from SEQ ID NO: 7 or 9 or other desired sequences were first modified so that only the coding region was used Polymerase chain reaction can be amplified with the help of two oligonucleotides. Care was taken to ensure that a consensus sequence was followed before the start odon for efficient translation. Either the base sequence ATA or AAA was chosen and inserted into the sequence before the ATG (Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes, Cell 44, 283-292).
  • a restriction interface was also introduced, which must be compatible with the interface of the target vector into which the fragment is to be cloned and with the aid of which gene expression in microorganisms or plants is to take place.
  • the PCR reaction was carried out using plasmid DNA as a template in a thermal cycler (Biometra) with the Pfu-DNA (Stratagene) polymerase and the following temperature program: 3 min at 6 ° C, followed by 30 cycles at 30 ° at 96 ° C, 30 s at 55 ° C and 2 min at 72 ° C, 1 cycle with 10 min at 72 ° C and stop at 4 ° C.
  • the annealing temperature was varied depending on the oligonucleotides chosen.
  • a synthesis time of about one minute can be assumed for each kilobase pair of DNA.
  • Other parameters which influence the PCR such as Mg ions, salt, DNA polymerase etc., are familiar to the person skilled in the art in the field and can be varied as required.
  • the correct size of the amplified DNA fragment was confirmed by agarose TBE gel electrophoresis.
  • the amplified DNA was extracted from the gel with the QIA uick gel extraction kit (QIAGEN) and ligated into the Smal restriction site of the dephosphorylated vector pUCl ⁇ using the Sure Clone Ligation Kit (Pharmacia), whereby the pUC derivatives were obtained.
  • QIAGEN QIA uick gel extraction kit
  • Sure Clone Ligation Kit Pharmacia
  • BioTechniques 4, 310-313) performed on ampicillin-resistant transformants, and positive clones identified by means of BamHI restriction analysis. The sequence of the cloned PCR product was confirmed by resequencing using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Rothstadt).
  • the PCR fragment (1428bp) was cloned into pUC 18 using the Sure Clone Kit (Pharmacia), the inserted fragment Sacl / Xhol was digested and the fragment was inserted into pYES2 or pYES6 using appropriate restriction sites.
  • the PCR fragment (1505bp) was cloned into pUC 18 using the Sure Clone 10 kit (Pharmacia), the inserted fragment BamHI / XhoI digested and the fragment inserted into pYES2 or pYES6 using appropriate restriction sites.
  • the plasmid DNA was matched with restriction enzyme (s)
  • Saccharomyces cerevisiae INVScl was transformed with the pYES derivatives and pYES empty vector using a PEG / lithium acetate protocol (Ausubel et al., 1995). After selection on CMdum agar plates with 2% glucose, pYES derivatives
  • CMdum liquid medium without uracil but with 2% raffinose and 1% (vol. / Vol.) Tergitol NP-40 with fatty acid substrates were enriched to a final concentration of 0.003% (wt. / Vol.).
  • the media were inoculated with the precultures to an ODgoo of 0.05.
  • Expression was induced at an ODgoo of 0.2 with 2% (w / v) galactose for 16 hours, after which the cultures were harvested to an ODgoo of 0.8-1.2.
  • the total fatty acids were extracted from yeast cultures and analyzed by gas chromatography. Cells from 5 ml of culture were harvested by centrifugation (1000 ⁇ g, 10 min, 4 ° C.) and washed once with 100 mM NaHC0 3 , pH 8.0, in order to remove residual medium and fatty acids. To produce the fatty acid methyl ester (FAMES or Singular FAME), the cell sediments were treated with 1 M methanolic HS0 4 and 2% (vol. / Vol.) Dimethoxypropane for 1 hour at 80 ° C.
  • FAME fatty acid methyl ester
  • the FAMES were extracted twice with 2 ml of petroleum ether, once with 100 mM NaHC0 3 , pH 8.0, and once with distilled water and dried with NaS0 4 .
  • the organic solvent was evaporated under a stream of argon and the FAMES were dissolved in 50 microliters of petroleum ether.
  • the samples were separated on a ZEBRON-ZB-Wax capillary column (30 m, 0.32 mm, 0.25 micron; Phenomenex) in a Hewlett Packard 6850 gas chromatograph with a flame ionization detector.
  • the oven temperature was raised from 70 ° C (hold 1 min) to 200 ° C at a rate of 20 ° C / min, then to 250 ° C (hold 5 min) at a rate of 5 ° C / min and finally to 260 ° C programmed at a rate of 5 ° C / min.
  • Nitrogen was used as the carrier gas (4.5 ml / min at 70 ° C).
  • the fatty acids were identified by comparison with retention times of FAME standards (SIGMA).
  • the figures represent mol% fatty acids of cis fatty acids.
  • linoleic acid 20: 3 25 ⁇ -5, 11, 14-fatty acid, alpha or gamma linolenic acid, stearidonic acid, arachidonic acid, eicosapentaenoic acid etc.
  • fatty acids alone or in combination (e.g. linoleic acid, 20: 3 25 ⁇ -5, 11, 14-fatty acid, alpha or gamma linolenic acid, stearidonic acid, arachidonic acid, eicosapentaenoic acid etc.) can be used to confirm the substrate specificity in more detail and selectivity of these desaturases can be carried out.
  • the desired product can be obtained from plant material or fungi, algae, ciliates, animal cells or from the supernatant of the cultures described above by various methods known in the art. If the desired product is not secreted from the cells, the cells can be harvested from the culture by slow centrifugation, the cells can be lysed by standard techniques such as mechanical force or ultrasound treatment. Organs of plants can be mechanically separated from other tissues or other organs. After homogenization, the cell debris is removed by centrifugation and the supernatant fraction containing the soluble proteins is saved for further purification of the desired compound. If the product is secreted from desired cells, the cells are removed from the culture by slow centrifugation and the supernatant fraction is saved for further purification.
  • the supernatant fraction from each purification process is subjected to chromatography with an appropriate resin, either with the desired molecule retained on the chromatography resin but not many contaminants in the sample, or the contaminants remaining on the resin but not leaving the sample. These chromatography steps can be repeated if necessary using the same or different chromatography resins.
  • the person skilled in the art is skilled in the selection of suitable chromatography resins and their most effective application for a particular molecule to be purified.
  • the purified product can be concentrated by filtration or ultrafiltration and kept at a temperature at which the stability of the product is maximum. A wide range of cleaning methods are known in the art and the above cleaning method is not intended to be limiting. These purification processes are described, for example, in Bailey, JE, & Ollis, DF, Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).
  • the identity and purity of the isolated compounds can be determined by standard techniques in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, in particular thin-layer chromatography and flame ionization detection (IATROSCAN, Iatron, Tokyo, Japan), NIRS, enzyme test or microbiological.
  • HPLC high-performance liquid chromatography
  • spectroscopic methods staining methods
  • thin-layer chromatography in particular thin-layer chromatography and flame ionization detection
  • IATROSCAN flame ionization detection
  • Iatron Iatron, Tokyo, Japan
  • NIRS enzyme test or microbiological.
  • An overview of these analysis methods can be found in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-

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Abstract

L'invention concerne des cassettes d'expression, leur combinaison et des vecteurs contenant lesdites cassettes d'expression, contenant des promoteurs végétaux ayant une spécificité d'expression pour des graines végétales, notamment des graines de lin, ainsi que l'utilisation de ces cassettes d'expression ou de ces vecteurs dans l'expression recombinée de gènes hétérologues dans des plantes. L'invention concerne en outre des plantes transgéniques transformées avec ces cassettes d'expression ou ces vecteurs, des cultures qui en sont dérivées, des parties ou des produits de développement transgéniques, de même que leur utilisation comme substances fourragères ou semences, produits pharmaceutiques, produits chimiques de pointe ou comme matières premières industrielles.
EP02710015A 2001-01-19 2002-01-18 Procede pour exprimer des genes de biosynthese dans des graines vegetales a l'aide de nouveaux genes hybrides d'expression Withdrawn EP1356056A2 (fr)

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DE10102338 2001-01-19
DE10102338A DE10102338A1 (de) 2001-01-19 2001-01-19 Verfahren zur Expression von Biosynthesegenen in pflanzlichen Samen unter Verwendung von neuen multiplen Expressionskonstrukten
PCT/EP2002/000461 WO2002057464A2 (fr) 2001-01-19 2002-01-18 Procede pour exprimer des genes de biosynthese dans des graines vegetales a l'aide de genes hybrides d'expression

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WO2002057464A3 (fr) 2003-02-27
AU2002228053A1 (en) 2002-07-30
US20040049805A1 (en) 2004-03-11
DE10102338A1 (de) 2002-07-25
CA2435091A1 (fr) 2002-07-25

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