DE10063387A1 - New elongase gene extends 16, 18 and 20 carbon fatty acids, useful to manipulate plants to produce polyunsaturated fatty acids for the foodstuffs, cosmetics and pharmaceutical industries - Google Patents

Abstract

An isolated nucleic acid from a plant or algae which encodes a polypeptide which extends a C16, C18 or C20 fatty acid having at least two double bonds by at least two carbon atoms, is new. Independent claims are also included for the following: (1) an isolated nucleic acid encoding a polypeptide which extends a C16, C18 or C20 fatty acid having at least two double bonds by at least two carbon atoms, and having the 1192, 687, 955 or 708 base pair (bp) sequence fully defined in the specification, a genetic code degenerate or derivative of one of those sequences, or encoding the 290, 195, 297 or 214 amino acid sequence fully defined in the specification, or a sequence with at least 50% homology to one of those sequences where the enzymatic activity is not substantially reduced; (2) a gene construct comprising one of the above nucleic acids linked to one or more regulatory sequences; (3) amino acid sequences encoded by the above nucleic acids or construct; (4) a vector comprising the above nucleic acids or construct; (5) an organism comprising the above nucleic acids, construct or vector; (6) an antibody which specifically binds to the product of the above nucleic acids; (7) an antisense nucleic acid molecule complementary to one of the above nucleic acids; (8) producing polyunsaturated fatty acids (PUFAs), comprising breeding organisms containing the above nucleic acids, construct or vector, encoding a polypeptide which extends a C16, C18 or C20 fatty acid having at least two double bonds by at least two carbon atoms, under PUFA forming conditions; (9) an oil, lipid, fatty acid or its fraction, produced by the above method; (10) an oil, lipid or fatty acid composition containing PUFAs from transgenic plants, preferably where the transgenic plant comprises the above nucleic acids, construct or vector; (11) identifying an elongase (ant)agonist comprising contacting cells expressing the polypeptide of the invention with a candidate compound, testing PSE activity and comparing activity with a control standard.

Description

Field of the Invention

The invention relates to a new elongase gene with the in sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 sequences or their homologues, Derivatives or analogues, a gene construct that contains these genes or theirs Contains homologs, derivatives or analogues, and its use. The invention relates to vectors or transgenic organisms, which is an elongase gene with the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 or contain its homologs, derivatives or analogues. Farther The invention relates to the use of the elongase gene sequences alone or in combination with other elongases and / or other fatty acid biosynthetic genes. This invention relates to a new elongase gene with the sequence SEQ ID NO: 1 or its Homologs, derivatives or analogs.

Furthermore, the invention relates to a method of manufacturing more polyunsaturated fatty acids and a method of incorporation of DNA in organisms that contain large amounts of oils and in particular Produce oils with a high content of unsaturated fatty acids. The invention also relates to an oil and / or a fatty acid Preparation with a higher content of polyunsaturated fat acids with at least two double bonds and / or a triacyl glycerin preparation with a higher content of polyunsaturated Fatty acids with at least two double bonds.

Background of the Invention

Certain products and by-products are more naturally occurring Metabolic processes in cells are relevant to a wide range Industries including feed, food, Cosmetic and pharmaceutical industries, useful. To this molecules commonly referred to as "fine chemicals" also lipids and fatty acids, among which an exemplary class which are polyunsaturated fatty acids. Polyunsaturated Fatty acids (polyunsaturated fatty acids, PUFAs) are examples given to children's nutritional formulations to one to produce higher nutritional value of these formulations. Have PUFAs for example a positive impact on cholesterol levels in the blood of humans and are therefore suitable for protection against heart diseases.  Fine chemicals and polyunsaturated fatty acids (polyunsaturated fatty acids, PUFAs) can be derived from animal Sources such as fish or microorganisms iso lose. By growing these microorganisms, large ones can be grown Produce quantities of one or more desired molecules and isolate.

Particularly suitable microorganisms for the production of PUFAs are microorganisms like Thraustochytria or Schizochytria Strains, algae such as Phaeodactylum tricornutum or Crypthecodinium- Species, ciliates, such as Stylonychia or Colpidium, mushrooms, such as Mortierella, Entomophthora or Mucor. Through stem selection is a number of mutant strains of the corresponding micro organisms have been developed which contain a number of desirable ver produce bonds, including PUFAs. The selection of Strains with improved production of a particular molecule however, a time-consuming and difficult process. A disadvantage is also that with a defined microorganism only certain unsaturated fatty acids or only a certain fat acid spectrum can be produced.

Alternatively, the production of fine chemicals may 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 rape, canola, flax, soybeans, sunflowers, borage and evening primrose. However, other crop plants containing oils or lipids and fatty acids are also well suited, as mentioned in the detailed description of this invention. By means of conventional breeding, a number of mutant plants have been developed which produce a spectrum of desirable lipids and fatty acids, cofactors and enzymes. However, the selection of new plant varieties with improved production of a particular molecule is a time-consuming and difficult process or even impossible if the compound does not naturally occur in the corresponding plant, as in the case of polyunsaturated C ₂₀ fatty acids and C ₂₂ fatty acids and those with longer carbon chains.

Summary of the invention

The invention provides new nucleic acid molecules which are used for Identification and isolation of elongase genes from PUFA-Bio are suitable for synthesis and for the modification of oils, fatty acids, Lipids, compounds derived from lipids and most potent preferably used for the production of polyunsaturated fatty acids  because there is still a great need for new ones Genes coding for enzymes involved in biosynthesis saturated fatty acids are involved and make it possible to manufacture on a technical scale. In particular there is a need for fatty acid biosynthetic enzymes that will Elongation of polyunsaturated fatty acids preferred with enable two or more double bonds in the molecule. The Nucleic acid according to the invention code for enzymes that these Possess ability.

Microorganisms such as Phaeodactylum, Colpidium, Mortierella, Ent omophthora, Mucor, Crypthecodinium and other algae and fungi and plants, especially oil crops, are commonly found in the industry to produce a variety of fine chemicals used on a large scale.

Assuming the availability of cloning vectors and techniques for genetic manipulation of the above mentioned microorganisms and ciliates, as in WO 98/01572 and WO 00/23604 discloses, or algae and related organisms, such as Phaeodactylum tricornutum, described in Falciatore et al. [1999, Marine Biotechnology 1 (3): 239-251]; as well as Dunahay et al. [1995, Genetic transformation of diatoms, J. Phycol. 31: 10004-1012] and the quotations in it, can be inventive appropriate nucleic acid molecules for genetic engineering modification of these organisms are used so that they are better or more efficient producers of one or more fine chemicals especially unsaturated fatty acids. This improved Production or efficiency of the production of a fine chemical can be manipulated by a direct effect of a gene of the invention or by an indirect effect this manipulation.

Mosses and algae are the only known plant systems the significant amounts of polyunsaturated fatty acids, such as arachidonic acid (= ARA) and / or eicosapentaenoic acid (= EPA) and / or produce docosahexaenoic acid (= DHA). Mosses included PUFAs in membrane lipids during algae, algae-related organisms and some mushrooms also have significant amounts of PUFAs in them Accumulate triacylglycerol fraction. Therefore, nucleuses are suitable acid molecules isolated from such strains, the PUFAs also accumulate in the triacylglycerol fraction, especially for Modification of the lipid and PUFA production system in one Host, particularly in microorganisms such as those above mentioned microorganisms, and plants such as oil crops, for example rapeseed, canola, flax, soy, sunflower, borage, Castor oil palm, safflower (Carthamus tinctorius), coconut,  Peanut or cocoa bean. Furthermore, nucleic acids from triacyl glycerol-accumulating microorganisms for identification such DNA sequences and enzymes in other species that are to modify the biosynthesis of precursor molecules of PUFAs can be used in the corresponding organisms. In particular microorganisms such as Crypthecodinium cohnii and Thraustochytrium species are microorganisms that PUFAs like ARA, Accumulate EPA or DHA in triacylglycerols. Thraustochytria are also closely related phylogenetically to schizochytria Strains. Although these organisms don't like closely with mosses Physcomitrella are related to DNA sequence and ins special polypeptide level sequence similarities in such Measurements that DNA molecules in heterologous hybridi sation experiments, sequence comparisons and experiments using the polymerase chain reaction also from evolutionary distant organisms identifiable and isolatable and are functionally characterizable. In particular, Derive consensus sequences that lead to heterologous viewing or for the functional complementation and prediction of Gene functions in three types are suitable. The ability to do this Identify functions, e.g. B. the prediction of the substrate specificity of enzymes, can therefore be of significant importance his. Furthermore, these nucleic acid molecules can be used as a reference sequences for mapping related genomes or for derivation of PCR primers.

The new nucleic acid molecules code for proteins here as PUFA-specific elongases (= PSEs or in the singular PSE) be designated. For example, these PSEs can be a function exercise that is related to metabolism (e.g. biosynthesis or Degradation) of compounds used for lipid or fatty acid synthesis are necessary, such as PUFAs, are involved or in the transmembrane transport one or more lipid / fatty acid compounds ent neither participate in or out of the cell.

In this new application, the isolation of such new ones Elongase gene shown in more detail. We have for the first time Elongas genes isolated, which are used to produce long-chain multiple unsaturated fatty acids, preferably with more than eighteen or twenty carbon atoms in the carbon skeleton of the fatty acid and / or at least two double bonds in the carbon chain, are suitable and come from typical organisms that contain high amounts of PUFAs in the triacylgycerol fraction. Therefore, here is the singular of a PSE gene or protein Speech or in the plural of PSE genes or proteins. Other known ones Patent applications and publications do not disclose or show anything functionally active PSE gene, although there are several known ones  There are patent applications which are the extension of saturated Short or medium chain fatty acids (WO 98/46776 and US 5,475,099) or the extension or production of long chain Fatty acids, but then no more than a double bond or lead to long-chain fatty acid wax esters (see: WO 98/54954, WO 96/13582, WO 95/15387) show. The ones here lying invention describes the isolation of novel elongases with new properties. Starting from the one in SEQ ID NO: 1 said sequence could, further nucleic acids for Encode elongases that extend unsaturated fatty acids, being found.

WO 99/64616, WO 98/46763, WO 98/46764, WO 98/46765 describe the production of PUFAs in transgenic plants and show the cloning and functional expression of corresponding desaturase activities, in particular from fungi, but do not show any PSE-coding gene and no functional PSE activity. The expression of the desaturase activities leads to a shift in the fatty acid composition in the transgenic plants. An increase in the content of unsaturated fatty acids was not observed. The production of a trienoic acid with a C ₁₈ carbon chain has been shown and has been claimed on the basis of gamma-linolenic acid, but it has not hitherto been the production of very long-chain polyunsaturated fatty acids (with a C ₂₀ and longer carbon chain and of trienoic acids and higher unsaturated types) shown.

To produce long-chain PUFAs, the polyunsaturated C ₁₆ or C ₁₈ fatty acids must be extended by at least two carbon atoms by the enzymatic activity of an elongase. The nucleic acid sequence SEQ ID NO: 1 according to the invention encodes the first plant elongase, which can extend C ₁₆ or C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms. After one round of elongation, this enzyme activity leads to C ₂₀ fatty acids, and after two, three and four rounds of elongation leads to C ₂₂ , C ₂₄ or C ₂₆ fatty acids. With the help of the further elongases disclosed (SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11), longer-chain PUFAs can also be synthesized. These can be individually, several or z. B. additive to PUFA elongase from the moss Physcomitrella patens (SEQ ID NO: 1) to increase the PUFA content in a new process for the production of PUFAs. The activity of the elongases according to the invention preferably leads to C ₂₀ fatty acids with at least two double bonds in the fatty acid molecule, preferably with three or four double bonds, particularly preferably three double bonds in the fatty acid molecule and / or C ₂₂ fatty acids with at least two double bonds in the fatty acid molecule, preferably with four, five or six double bonds, especially preferred with five or six double bonds in the molecule. After the extension with the enzyme according to the invention has taken place, further desaturation steps can be carried out in order to obtain the highly desatured fatty acids. Therefore, the products of the elongase activities and the possible further desaturation lead to preferred PUFAs with a higher degree of desaturation as docosadienoic, arachidonic acid, ω6-Eicosatriendihomo-γ-linolenic acid, eicosapentaenoic acid, ω3-eicosatrienoic, ω3-Eicosatetraen acid, docosapentaenoic acid or docosahexaenoic acid. Substrates of the enzyme activity according to the invention are, for example, taxolic acid; 7,10,13-hexadecatrienoic acid, 6,9-octadecadienoic acid, linoleic acid, pinolenic acid, α- or γ ± linolenic acid or stearidonic acid as well as arachidonic acid, eicosatetraenoic acid, docosapentaenoic acid, eicosa pentaenoic acid. Preferred substrates are linoleic acid, γ-linolenic acid and / or α-linolenic acid and arachidonic acid, Eicosa tetraenoic acid, docosapentaenoic acid, eicosapentaenoic acid. Arachidonic acid, docosapentaenoic acid, eicosapentaenic acid are particularly preferred. The C ₁₆ or C ₁₈ fatty acids with at least two double bonds in the fatty acid can be extended by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol become.

Conjugated linoleic acid "CLA" is of particular importance for human nutrition. CLA is understood in particular to mean fatty acids such as C18: 2 ^{9 cis, 11trans} or the isomer C18: 2 ^{10trans, 12 cis} , which can be desaturated or elongated in the body due to human enzyme systems and which can contribute to health-promoting effects. Elongases according to the invention can also be used to elongate such conjugated fatty acids with at least two double bonds in the molecule and thus to supply such health-promoting fatty acids to human nutrition. Another example of conjugated fatty acids are alpha-parinic acid, eleostearic acid and calendulic acid.

Assuming cloning vectors for use in Plants and in plant transformation, such as those that are published in and cited there: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-119 (1993); F. F. White, Vectors for Gene Transfer to higher plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, ed .: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Trans fer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,  Ed .: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225)), the nucleic acids according to the invention for the genetic engineering change of a broad spectrum Use plants to make them a better, more efficient one or modified producer of one or more of lipids managed products such as PUFAs. This improved production or efficiency of producing one derived from lipids Product, such as PUFAs, can be manipulated by direct action or an indirect effect of this manipulation become.

There are a number of mechanisms through which change takes place the yield, production of a PSE protein according to the invention and / or efficiency of producing a fine chemical from a Oil crop or a microorganism due to a ver modified protein can directly affect. The number or Activity of the PSE protein or gene can be increased so that larger quantities of these compounds are produced de novo, because the organisms have this activity and ability to biosynthesize before the corresponding gene was introduced. Also the Using different divergent, d. H. at the DNA sequence level different sequences can be advantageous.

The introduction of one or more PSE genes into one Organism or a cell can not only the biosynthetic flow to increase the final product, but also the corresponding triacyl Increase glycerin composition or create de novo. As well can be the number or activity of other genes involved in the import of Nutrients necessary for the biosynthesis of one or more fines microals (e.g. fatty acids, polar and neutral lipids) necessary are increased so that the concentration of these precursors, Cofactors or interconnections within the cells or is increased within the storage compartment, making the Ability of cells to produce PUFAs as follows described, further increased. Are fatty acids and lipids desirable even as fine chemicals; by optimizing the Activity or increase in the number of one or more PSEs that are involved in the biosynthesis of these compounds, or by Destroy the activity of one or more PSEs that are degrading of these connections are involved, it may be possible that Yield, production and / or efficiency of fat production acid and lipid molecules from plants or microorganisms increase.  

The mutagenesis of the PSE gene according to the invention can also be increased a PSE protein with modified activities that cause the Production of one or more desired fine chemicals directly or indirectly affect. For example, the number or Activity of the PSE genes according to the invention can be increased, so that the normal metabolic waste or by-products of Cell (its amount may be due to overproduction the desired fine chemical is increased) efficiently exported before other molecules or processes within the Cell (which would lower the cell's viability) disrupt or disrupt the biosynthetic pathways of the fine chemical (which increases the yield, production or efficiency of production the desired fine chemical is reduced). Can also the relatively large intracellular amounts of the desired fines chemical itself may be toxic to the cell or enzyme return interfere with coupling mechanisms, such as allosteric regulation, for example, by increasing activity or To number of other downstream enzymes or detoxification enzymes of the PUFA route, the allocation of the PUFA to the triacylglycerol Increase fraction, you could viability of seed cells increase, which in turn leads to better cell development in Culture or leads to seeds that the desired fine chemical to produce. The PSE gene according to the invention can also be mani be pulped that the appropriate amounts of the different Lipid and fatty acid molecules are produced. This can be a incisive effect on the lipid composition of the membrane the cell have and produces new oils in addition to occurrence newly synthesized PUFAs. Because each lipid type is different has physical properties, can change the lipid composition of a membrane significantly increases the membrane fluidity change. Changes in membrane fluidity can affect the Transport of molecules across the membrane and on the Unver affect cell integrity, both of which are crucial Have an effect on the production of fine chemicals. In Plants can also change these changes paint like tolerance to abiotic and biotic stress situations, influence.

Biotic and abiotic stress tolerance is a common feature times that you can think of a wide range of plants, like corn, Wheat, rye, oats, triticale, rice, barley, soybean, earth nut, cotton, rapeseed and canola, cassava, pepper, sunflower and tagetes, solanaceae plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, bush plants (coffee, Cocoa, tea), salix species, trees (oil palm, coconut) and out permanent grasses and fodder crops, would like to inherit. This Field crops are another embodiment of the invention  also preferred target plants for genetic engineering. Especially preferred plants according to the invention are oil fruit plants, such as Soybean, peanut, rapeseed, canola, sunflower, safflower, trees (Oil palm, coconut) or crops such as corn, wheat, rye, Oats, triticale, rice, barley, alfalfa, or bush plants (Coffee, cocoa, tea).

Thus, one aspect of the invention relates to isolated nucleotides acid molecules (e.g. cDNAs) comprising nucleotide sequences, the one PSE or several PSEs or biologically active parts encode it, or nucleic acid fragments that act as primers or hybridization probes for detection or amplification Nucleic acids encoding PSE (e.g. DNA or mRNA) are suitable. at particularly preferred embodiments include the nucleic acid molecule one of those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 Nucleotide sequences or the coding region or a complement one of these nucleotide sequences. Others especially before Preferred embodiments include the isolated invention Nucleic acid molecule is a nucleotide sequence attached to a nucleotide sequence as in the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 provided, or hybridized a part thereof or at least about 50%, preferably at least about 60%, more preferred at least about 70%, 80% or 90% and even more preferred is at least about 95%, 96%, 97%, 98%, 99% or more. In other preferred embodiments, the isolated code Nucleic acid molecule one of those in the sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 amino acid sequences shown. The preferred PSE gene according to the invention preferably also has at least one of the PSE activities described here.

In a further embodiment, the isolated code Nucleic acid molecule is a protein or a part thereof, wherein the protein or part thereof contains an amino acid sequence, which are sufficiently homologous to an amino acid sequence of the sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 is that the protein or part thereof is a Maintains PSE activity. Preferably the protein or the Part of it encoded by the nucleic acid molecule that Ability to build on cell membranes by metabolism Plants necessary connections or at the transport of molecules participate through these membranes. In one embodiment at least the protein encoded by the nucleic acid molecule about 50%, preferably at least about 60% and more before adds at least about 70%, 80% or 90% and most preferred  at least about 95%, 96%, 97%, 98%, 99% or more homologous to an amino acid sequence of the sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. In a further preferred embodiment the protein is a full length protein that is essentially in parts homologous to an entire amino acid sequence of the SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 (those of the one in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 open reading frame) and is in full length with the specialist common methods and experiments can be isolated.

In another preferred embodiment, the isolated one stirs Nucleic acid molecule from Phytophthora infestans, Physcomitrella patens, Crypthecodinium cohnii or Thraustochytrium forth and encodes a protein (e.g. a PSE fusion protein) that contains a bio logically active domain containing at least about 50% or more homologous to an amino acid sequence of the sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and the ability to metabolize from to Building cell membranes of plants necessary connections or participate in the transport of molecules across these membranes take, maintain, or at least one of the elongation activities resulting in PUFAs such as ARA, EPA or DHA or their precursors molecules or one of the activities listed in Table 1 owns, and also includes heterologous nucleic acid sequences which encode a heterologous polypeptide or regulatory proteins.

Table 1

Fatty acid pattern of five transgenic yeast strains in mol%. The proportions of the g-linolenic acid added and taken up are emphasized by numbers in bold, those of the extended products are underlined and those of the extended γ-linolenic acid by numbers in bold (last line).

In another embodiment, this is nucleic acid isolated molecule at least 15 nucleotides long and hybridizes under stringent conditions on a nucleic acid molecule that a Nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11. Preferably the isolated nucleic acid molecule corresponds to a natural one occurring nucleic acid molecule. Coding is more preferred isolated nucleic acid molecule of naturally occurring crypthe codinium, Phytophthora or Thraustochytrium PSE or one biologically active part of it.

Another aspect of the invention relates to vectors, e.g. B. recombinant expression vectors containing at least one fiction contain appropriate nucleic acid molecule, and host cells into which these vectors have been introduced, in particular micro organisms, plant cells, plant tissues, organs or whole Plants. In one embodiment, such a host cell Store fine chemical compounds, especially PUFAs; to Isolation of the desired compound, the cells are harvested. The compound (oils, lipids, triacylglycerides, fatty acids) or the PSE can then be extracted from the medium or the host cell in plants there are cells that contain fine chemicals or store, most preferred cells from storage tissues, such as seed shells, tubers, epidermis and sperm cells become.

Yet another aspect of the invention relates to a genetic one modified plant, preferably an oil crop, as before mentioned above, particularly preferably a rapeseed, flax or Physcomitrella patens plant into which a PSE gene has been introduced has been. In one embodiment, the genome is of Rapeseed, flax or Physcomitrella patens by introducing one Nucleic acid molecule according to the invention, which is a wild-type or mutated PSE sequence encoded as transgene. In another embodiment, an endogenous PSE gene is in the Genome of the donor organism Physcomitrella patens, Phytophthora infestans, Crypthecodinium or Thraustochytrium for example by homologous recombination with an altered PSE gene or Mutagenesis and detection by means of DNA sequences changed that means functionally destroyed. With a preferred off the plant organism belongs to the genus Physco mitrella, Ceratodon, Funaria, rapeseed or flax, whereby Physco mitrella, rapeseed or flax is preferred. In a preferred one The embodiment is also used for Physcomitrella, rape or linseed Production of a desired compound such as lipids and fat acids, with PUFAs being particularly preferred.  

In yet another preferred embodiment, this can Moss Physcomitrella patens to demonstrate the function of a Elongasegens using homologous recombination on the Based on the nucleic acids described in this invention, ver be applied.

Yet another aspect of the invention relates to an isolated one PSE gene or part, e.g. B. a biologically active part, from that. In a preferred embodiment, the isolated one PSE or part of it on the metabolism of to build up Cell membranes in a microorganism or a plant cell necessary connections or on the transport of molecules via whose membranes participate. Another preferred Embodiment is the isolated PSE or part thereof sufficient homologous to an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 that this protein or part thereof is capable speed, on the metabolism of to build up cell membranes in micro organisms or plant cells necessary connections or on To participate in transporting molecules across these membranes reserves.

The invention also provides an isolated preparation of a PSE. In preferred embodiments, the PSE gene comprises an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. In another In a preferred embodiment, the invention relates to an isolated full-length protein which is essentially homologous to an entire amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 (encoded by the open reading frames shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11) , In a further embodiment, the protein is at least about 50%, preferably at least about 60% and more preferably at least about 70%, 80% or 90% and most preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to an amino acid sequence of the sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. Included in other embodiments the isolated PSE is an amino acid sequence that is at least about 50% homologous to one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and can participate in the metabolism of compounds necessary for the formation of fatty acids in a microorganism or in a plant cell or in the transport of molecules across these membranes or has one or more of the PUFA-prolonging activities, the prolongation advantageously being desaturated C ₁₆ or C ₁₈ or C ₂₀ double carbon chains ties are made in at least two places.

Alternatively, the isolated PSE can have an amino acid sequence summarize, which is encoded by a nucleotide sequence attached to a Nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 hybridized e.g. B. hybridized under stringent conditions, or at least about 50%, preferably at least about 60%, more preferred at least about 70%, 80% or 90% and even more preferred at least about 95%, 96%, 97%, 98%, 99% or more homologous is to. It is also preferred that the preferred PSE Form one of the PSE activities described here have.

The PSE polypeptide or a biologically active part thereof can be operably linked to a non-PSE polypeptide so that a fusion protein is formed. With preferred off this fusion protein has an activity that differs from that of the PSE alone. With others preferred embodiment takes this fusion protein on the fabric alternation of compounds used to synthesize lipids and fat acids, cofactors and enzymes in microorganisms or plants are necessary, or for the transport of molecules via them Membranes part. In particularly preferred embodiments modulates the introduction of this fusion protein into a host the production of a desired connection by the cell Cell. In a preferred embodiment, these contain Fusion proteins also Δ4-, Δ5- or Δ6-, Δ8-, Δ15-, Δ17- or Δ19 desaturase activities alone or in combination.

Another aspect of the invention relates to a method for Manufacture of a fine chemical. This procedure includes either the cultivation of a suitable microorganism or the cultivation of plant cells, tissues, organs or whole Plants comprising the nucleotide sequences of the invention SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 or their homologues, derivatives or analogues or a gene construct that has SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 or their homologues, Derivatives or analogs, or a vector comprising them Sequences or the gene construct comprising the expression of a PSE nucleic acid molecule according to the invention, so that a fine chemical is produced. In a preferred one Embodiment, the method further comprises the step of Obtaining a cell which contains such an elongase Contains nucleic acid sequence, where a cell with an elongase  Nucleic acid sequence, a gene construct or a vector, which the expression of a PSE nucleic acid according to the invention lead, is transformed. In another preferred way this method further comprises the step of Extracting the fine chemical from the culture. With one particularly preferred embodiment, the cell belongs to the order of Ciliates, to microorganisms, such as fungi, or to the plant kingdom, especially for oil crops, micro are particularly preferred organisms or oil crops.

Another aspect of the invention relates to methods for Modulation of the production of a molecule by a micro organism. These procedures involve bringing the Cell with a substance that has PSE activity or PSE nucleic acid expression modulated so that a cell-associated Activity relative to the same activity in the absence of Substance is changed. In a preferred embodiment is one or two pathways of the cell for lipids and modulates fatty acids, cofactors and enzymes or the trans port of compounds modulated over these membranes so that the Yield or the rate of production of a desired fine chemical is improved by this microorganism. The Substance that modulates PSE activity can be a substance be the PSE activity or PSE nucleic acid expression stimulated or as an intermediate in the fatty acid bio synthesis can be used. Examples of substances which stimulate PSE activity or PSE nucleic acid expression, are u. a. small molecules, active PSEs and PSEs encoding Nucleic acids that have been introduced into the cell. at play for substances that have PSE activity or expression inhibit, are u. a. small molecules and / or antisense PSE nucleotides acid molecules.

Another aspect of the invention relates to methods for Modulation of the yields of a desired compound from a Cell comprising introducing a wild type or mutant PSE Gene that is either kept on a separate plasmid or in the genome of the host cell is integrated into a cell. at Integration into the genome can be random or by recombination such that the native gene is replaced by the incorporated copy, reducing production the desired compound is modulated by the cell, or by using a gene in trans so that the gene is associated with a functional expression unit, which at least one the Expression of a gene ensuring sequence and at least  a the polyadenylation of a functionally transcribed gene ensuring sequence, is functionally linked.

In a preferred embodiment, the yields are modified. In another preferred embodiment the desired chemical multiplied, with unwanted disruptive Connections can be reduced. With one especially before preferred embodiment is the desired fine chemical Lipid or a fatty acid, a cofactor or an enzyme. at particularly preferred embodiment, this chemical is a polyunsaturated fatty acid. It is more preferred from selected from arachidonic acid (= ARA), eicosapentaenoic acid (= EPA) or docosahexaenoic acid (= DHA).

Detailed description of the invention

The present invention provides PSE nucleic acids and proteins molecules ready, involved in lipid and fatty acid metabolism, PUFA cofactors and enzymes in the moss Physcomitrella patens, Phytophthora infestans, Crypthecodinium or Traustochytrium or involved in the transport of lipophilic compounds across membranes are. The compounds of the invention can be used for Modulation of the production of fine chemicals from organisms, for example microorganisms, such as ciliates, fungi, yeasts, Bacteria, algae, and / or plants, such as corn, wheat, rye, Oats, triticale, rice, barley, soybean, peanut, cotton, Brassica species, such as rape, canola and turnip, pepper, sun flower, borage, evening primrose and tagetes, solanaceae plants, such as potato, tobacco, eggplant and tomato, Vicia species, pea, Cassava, alfalfa, bush plants (coffee, cocoa, tea), salix species, Trees (oil palm, coconut) and perennial grasses and forage field crops, either directly (e.g. when overexpression or Optimization of a direct fatty acid biosynthesis protein Influence on the yield, production and / or efficiency of the Production of the fatty acid from modified organisms has) ver turn or can have an indirect impact, but still have an increase in yield, production and / or efficiency of Production of a desired connection or a decrease in desired connections (e.g. if the modulation of the substance alternating between lipids and fatty acids, cofactors and enzymes Changes in yield, production and / or efficiency of the Production or the composition of the desired compounds inside the cells, which in turn leads to the production of a or several fine chemicals). Aspects of Invention are further explained below.  

I. Fine chemicals and PUFAs

The term "fine chemical" is known in the art and includes molecules produced by an organism and find applications in different industries, like, but not limited to, the pharmaceutical, farmer industry, food and cosmetics industry. This ver Bonds 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, Rehm et al., ed., VCH: Weinheim and the literature contained therein places), lipids, saturated and unsaturated fatty acids (e.g. Arachidonic acid), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, Vitamins, pp. 443-613 (1996) VCH: Weinheim and contained therein References; and Ong, A.S., Niki, E., & Packer, L. (1995) Nutrition, Lipids, Health and Disease Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia and the Society for Free Radical Research - Asia, held on 1-3. Sept. 1994 in Penang, Malaysia, AOCS Press (1995)), Enzyme and all others by Gutcho (1983) in Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086, and literature references described therein Chemicals. The metabolism and uses of certain Fine chemicals are further explained below.

The combination of different precursor molecules and biosynthesis enzyme leads to the production of various fatty acid molecules, what a decisive impact on the composition of the membrane Has. It can be assumed that PUFAs are not just simple in Triacylglycerol, but also be incorporated into membrane lipids.

The synthesis of membranes is a well characterized process on which a number of components, including lipids as Part of the bilayer membrane, are involved. The production of new ones Fatty acids, such as PUFAs, can therefore have new membrane properties create functions within a cell or organism.

Cell membranes serve a variety of functions in one Cell. First and foremost, a membrane limits the content a cell from the environment, thereby giving cell integrity gives. Membranes can also act as barriers to this Influx of dangerous or unwanted connections and also serve against the outflow of desired connections.  

More detailed descriptions and participations of membranes and the mechanisms involved see in: Bamberg, E., et al. (1993) Charge transport of ion pumps on lipid bilayer membranes, Q. Rev. Biophys. 26: 1-25; Gennis, R.B. (1989) Pores, Channels and Transporters, in: Biomembranes, Molecular Structure and Function, Springer: Heidelberg, pp. 270-322; and Nikaido, H., and Saier, H. (1992) Transport proteins in bacteria: common themes in their design, Science 258: 936-942, and that in everyone of these citations.

Lipid synthesis can be divided into two sections: the synthesis of fatty acids and their binding to sn-glycerin-3- Phosphate and the addition or modification of a polar Head group. Common lipids used in membranes include phospholipids, glycolipids, sphingolipids and phospho glycerides. Fatty acid synthesis begins with the conversion of Acetyl-CoA in either malonyl-CoA through the acetyl-CoA-carboxy lase or in acetyl-ACP by acetyl transacylase. To These two product molecules form a condensation reaction together acetoacetyl-ACP, which has a series of condensation, Reduction and dehydration reactions is converted, so that a saturated fatty acid molecule with the desired chains length is obtained. Production of unsaturated fatty acids these molecules are catalyzed by specific desaturases based, either aerobically using molecular oxygen or anaerobic (with regard to the synthesis of fatty acids in microorganisms see F.C. Neidhardt et al. (1996) E. coli and Salmonella. ASM Press: Washington, D.C., pp. 612-636 and contained therein References; Lengeler et al. (Ed.) (1999) Biology of Procaryotes. Thieme: Stuttgart, New York, and the one included References, as well as Magnuson, K., et al. (1993) Micro biological reviews 57: 522-542 and the literature included put).

Precursors for PUFA biosynthesis are, for example, linoleic and linolenic acid. These C ₁₈ carbon fatty acids must be extended to C ₂₀ or C ₂₂ in order to obtain fatty acids of the Eicosa and Docosa chain type. With the help of various desaturases, such as enzymes which have Δ6-desaturase, Δ5 and Δ4-desaturase activity, arachidonic acid, Eicosa pentaenoic acid and docosahexaenoic acid and various other long-chain PUFAs can be obtained, extracted and for various purposes in food, feed and cosmetics - or pharmaceutical applications are used.

To produce long-chain PUFAs, as mentioned above, the polyunsaturated C ₁₆ or C ₁₈ or C ₂₀ fatty acids have to be extended by at least two carbon atoms through the enzyme activity of an elongase. The nucleic acid sequences according to the invention encode first microbial elongases from typical PUFA in the triacylglycerol fraction-containing producers, who can extend C ₁₆ or C ₁₈ or C ₂₀ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms or this can be done e.g. B. sequentially successively by converting a C ₁₆ or C ₁₈ fatty acid into a C ₂₀ fatty acid and then a C ₂₀ - in a C ₂₂ or higher even numbering 2C atomic units ent containing fatty acid. After one round of elongation, this enzyme activity leads to C ₂₀ fatty acids, and after two, three and four rounds of elongation leads to C ₂₂ , C ₂₄ or C ₂₆ fatty acids. Longer PUFAs can also be synthesized with the elongase according to the invention. The activity of the elongases according to the invention preferably leads to C ₂₀ and / or C ₂₂ fatty acids with at least two double bonds in the fatty acid molecule, C ₂₀ fatty acids preferably with three, four or five double bonds, particularly preferably three double bonds in the fatty acid molecule, C ₂₂ fatty acids preferred example with three, four, five or six double bonds, particularly preferably five or six double bonds in the fatty acid molecule. After the elongation with the enzyme according to the invention, further desaturation steps can take place. Therefore, the products of the elongase activities and the possible further desaturation lead to preferred PUFAs with a higher degree of desaturation as docosadienoic, arachidonic acid, ω6-Eicosatriendi homo-γ-linolenic acid, eicosapentaenoic acid, ω3-eicosatrienoic, ω3-eicosatetraenoic, docosapentaenoic acid or docosahexaenoic acid. Substrates of this enzyme activity according to the invention are, for example, taxolic acid, 7,10,13-hexadecatrienic acid, 6,9-octadecadienic acid, linoleic acid, γ-linolenic acid, pinolenic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid or stearidonic acid. Preferred substrates are linoleic acid, γ-linolenic acid and / or α-linolenic acid or arachidonic acid, eicosatetraenoic acid or eicosapentaenoic acid. The C ₁₆ or C ₁₈ - or C ₂₀ -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, monoacylglycerol, diacylglycerol or triacylglycerol , be extended.

Furthermore, fatty acids have to be added to various Modifications transported and into the triacylglycerol Storage lipid can be installed. 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).

Publications on plant fatty acid biosynthesis, Desaturation, lipid metabolism and membrane transport of fatty compounds, beta oxidation, fatty acid modification and cofactors, triacylglycerol storage and -Assembly including the references therein see in the following articles: Kinney, 1997, Genetic Engeneering, Ed .: JK Setlow, 19: 149-166; Ohlrogge and Browse, 1995, Plant Cell 7: 957-970; Shanklin and Cahoon, 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 611-641; Voelker, 1996, Genetic Engineering, Ed .: JK Setlow, 18: 111-13; Gerhardt, 1992, Prog. Lipid R. 31: 397-417; Gühnemann-Schäfer & Kindl, 1995, Biochim. Biophys Acta 1256: 181-186; Kunau et al., 1995, Prog. Lipid Res. 34: 267-342; Stymne et al., 1993, in: Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants, ed .: Murata and Somerville, Rockville, American Society of Plant Physiologists, 150-158, Murphy & Ross 1998, Plant Journal. 13 (1): 1-16.

Vitamins, cofactors and "nutrazeuticals" such as PUFAs include a group of molecules that higher animals no longer synthesize can and must therefore take up or the higher animals can no longer produce enough and therefore additional need to absorb, although they are easily from other organisms, like bacteria, are synthesized. The biosynthesis of these moles cool in organisms that they can produce, such as bacteria, has been broadly characterized (Ullmann's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, Pp. 443-613, VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley &Sons; Ong, A.S., Niki, E., & Packer, L. (1995) "Nutrition, Lipids, Health and Disease" Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia and the Society for Free Radical Research Asia, held on 1-3. Sept. 1994 in Penang, Malaysia, AOCS Press, Champaign, IL X, 374 S).

The molecules mentioned above are either biological themselves active molecules or precursors of biologically active substances that either as an electron carrier or as an intermediate in one Serve a variety of metabolic pathways. Have these connections in addition to their nutritional value, also a significant industrial value as dyes, antioxidants and catalysts or other ver processing aids. (An overview of structure, activity and for industrial applications of these compounds see e.g. B.  in Ullmann's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, pp. 443-613, VCH: Weinheim, 1996). Polyunsaturated Fatty acids have various functions and are beneficial to health Effects, for example in coronary heart disease, ent ignition mechanisms, child nutrition, etc. publications and references, including literature cited therein see, in: Simopoulos, 1999, Am. J. Clin. Nutr. 70 (3rd Suppl.): 560-569, Takahata et al., Biosc. Biotechnol. Biochem. 1998, 62 (11): 2079-2085, Willich and Winther, 1995, German Medical weekly 120 (7): 229ff.

II. Elements and methods of the invention

The present invention is based at least in part on the Discovery of new molecules, here called PSE nucleic acid and -Protein molecules are referred to, which have an effect on the production of cell membranes in Physcomitrella patens, Crypthecodinium cohnii, Phytophthora infestans, Thraustochytrium and / or exercise Ceratodon purpureus and, for example, the Affect movement of molecules across these membranes. at In one embodiment, the PSE molecules take part in the metabolism of building cell membranes in organisms such as micro organisms and plants, necessary connections in part or indirectly affect the transport of molecules through them Membranes. In a preferred embodiment, the Ak activity of the PSE molecules according to the invention for regulating the Production of membrane components and membrane transport one Impact on the production of the desired fine chemical through this organism. With a particularly preferred Aus The management form is the activity of the PSE molecules according to the invention modulated so that the yield, production and / or efficiency the production of the metabolic pathways of microorganisms or Plants that regulate the PSEs according to the invention are modulated are and the efficiency of transporting connections through the Membranes is what is changed either directly or indirectly Yield, production and / or efficiency of producing one desired fine chemical by microorganisms and plants modulated.

The term PSE or PSE polypeptide includes proteins that are on Metabolism of to build cell membranes in organisms, such as microorganisms and plants, necessary compounds or participate in the transport of molecules across these membranes. at games for PSEs are in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 or their homo logen, derivatives or analogs disclosed. The terms PSE or PSE nucleic acid sequence (s) include nucleic acid sequences that  encode a PSE and part of which encodes a coding region and also corresponding 5'- and 3'-untranslated sequence areas can be. Examples of PSE genes are those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 sequences shown. The terms production or productivity are known in the art and include the concentration of the fermentation product (e.g. the desired fine chemical) in a certain period of time and a certain fermentation volume is formed (e.g. kg of product per hour per liter). The concept of efficiency Production includes the time it takes to achieve a particular Production quantity is necessary (e.g. how long the cell needs to open direction of a certain throughput rate of a fine chemical required). The term yield or product / carbon yield is known in the art and encompasses conversion efficiency the carbon source in the product (i.e. the fine chemical). This is usually expressed, for example, as kg of product per kg of carbon source. By increasing the yield or Production of the compound is the amount of molecules obtained or the appropriate recovered molecules of this compound in a certain amount of culture over a fixed period of time elevated. The terms biosynthesis or biosynthetic pathway are in Known in the art and include the synthesis of a compound preferably an organic compound, through a cell Interconnections, for example in a multi-step and highly regulated process. The terms degradation or degradation route are known in the art and include splitting a compound, preferably an organic compound, through a cell in breakdown products (more generally, smaller or fewer complex molecules) for example in a multi-step and strong regulated process. The term metabolism is in the specialty known and encompasses all biochemical reactions, that take place in an organism. The metabolism of one certain compound (e.g. the metabolism of a fatty acid) then comprises the entirety of the biosynthesis, modification and Degradation pathways of this connection in the cell that this connection affect.

In another embodiment, the inventive PSE molecules like the production of a desired molecule a fine chemical, in a microorganism or in plants modulate. There are a number of mechanisms through which the Changing a PSR according to the invention, the yield, production and / or efficiency of production of a fine chemical a microorganism or plant strain that changed it Contain protein, can directly affect. The number or Activity of PSEs involved in the transport of fine chemical molecules  involved inside or out of the cell can be increased so that larger amounts of these compounds across membranes trans be ported from which they are more easily extracted and into each other being transformed. Also fatty acids are triacylglycerols and / or lipids themselves desirable fine chemicals; by Optimizing activity or increasing the number of one or of several PSEs according to the invention which are involved in the biosynthesis of these Connections are involved, or by disrupting the activity one or more PSEs involved in breaking down these connections involved, it may be possible the yield, production and / or efficiency of production of fatty acid and lipid molecules from organisms, such as microorganisms or plants, to increase.

The mutagenesis of the PSE gene according to the invention can also be PSEs with changed activities that spawn production one or more desired micro fine chemicals indirectly affect organisms or plants. For example can PSEs according to the invention, the export of waste products are involved in a greater number or higher activity point so that the normal metabolic waste of the cell (whose Quantity may be due to overproduction of the desired one Fine chemical is increased) can be exported efficiently before they can damage the molecules in the cell (what life capacity of the cell) or the fine chemical Biosynthetic pathways can disrupt (what the yield, production or Reduce the efficiency of the production of a desired fine chemical would). The relatively large intracellular amounts of the desired Fine chemicals themselves can also be toxic to the cell so that by increasing the activity or number of trans porters who can export these connections from the cell what can increase the viability of the cell in culture again leads to a larger number of cells in the culture, which produce the desired fine chemical. The fiction according to PSEs can also be manipulated so that the ent speaking amounts of different lipid and fatty acid molecules to be produced. This can have a significant impact on the Have lipid composition of the cell membrane. Because every lipid type has different physical properties, a Change in the lipid composition of a membrane the membrane change fluidity significantly. Changes in membrane fluidity can transport molecules across the membrane as well Integrity affect the cell, each of which is significant Impact on the production of fine chemicals from micro organisms and plants in fermentation culture on a large scale stab. Plant membranes impart specific properties, such as tolerance to heat, cold, salt, dryness as well  Tolerance to pathogens such as bacteria and fungi. Therefore the modulation of the membrane components has a fundamental effect on the viability of the plants among the above Have stress parameters. This can result in changes in signal cascades or directly via the changed membrane composition (see for example: Chapman, 1998, Trends in Plant Science, 3 (11): 419-426) and signal cascades (see Wang 1999, Plant Physiology, 120: 645-651) or the cold tolerance, such as disclosed in WO 95/18222.

The isolated nucleic acid sequences according to the invention are for example contained in the genome of a Thraustochytrium strain, with the American Type Culture Collection (ATCC collection) the strain number ATCC26185 (Thraustochytrium) is available, resp in the case of Crypthecodinium, for example, by Provasoli-Guillard National Center for Culture of Marine Phytoplancton ((CCMP) West Boothbay Harbor, ME, USA) with the regular culture no. CCMP316 accessible is. In the case of Phytophthora infestans, these are mentioned Nucleic acid molecules isolated from the ATCC 48886 strain.

The nucleotide sequence of the isolated Physcomitrella, Crypthe codinium, Phytophthora infestans or Thraustochytrium cDNA and the deduced amino acid sequences of Physcomitrella patens- PSEs are shown in SEQ ID NO: 1 to SEQ ID NO: 12. There were Computer analysis performed these nucleotide sequences as Classify and / or identify sequences related to the substance Change of cell membrane components involved proteins or Transport of connections across proteins involved in cell membranes or coding the PUFA biosynthesis. EST's with the database reproducing no. PP001019019F, CC001042041R, PI001002014R as well TC002034029R, TC002034029R-11 and TC002014093R in the data bank of the inventor put the sequences according to the invention in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11. The partial Polypeptides as PP001019019F, CC001042041R, PI001002014R as well TC002034029R, TC002034029R-11 and TC002014093R and represent the sequences according to the invention in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 according to Table 2. The complete fragment Insert of the EST TC002034029R was sequenced to give the SEQ ID NO: 3, which is the complete sequence of TC002034029R is. TC002034029R-11 describes a full length sequence of a Elongase from Thraustochytrium. The naming of the other clones is analog. In addition, the various clones were given names assigned speaking. Abbreviations: Tc = Thraustochytrium, Cc = Crypthecodinium, Pp = Physcomitrella patens, Pi = Phytophthora infestans.  

Table 2

The present invention also relates to proteins with an amino acid sequence that is essentially homologous to an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. As used here is a Protein with an amino acid sequence that is essentially homologous to a selected amino acid sequence, at least about 50% homologous to the selected amino acid sequence, e.g. B. the entire selected amino acid sequence. A protein with one Amino acid sequence that is essentially homologous to one amino acid sequence selected 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 his.

The PSE according to the invention or the biologically active part or the fragment thereof can participate in the metabolism of compounds necessary for the construction of cell membranes in microorganisms or plants or in the transport of molecules over these membranes or one or more of those for elongating C ₁₆ or C ₁₈ or C ₂₀ PUFAs have required activities so that C ₂₀ , C ₂₂ or C ₂₄ PUFAs as well as related PUFAs are obtained.

Various aspects of the invention are further discussed in the following subsections.

A. Isolated nucleic acid molecules

One embodiment of the invention is isolated nucleic acids which come from PUFA-producing microorganisms and code for poly peptides which extend C ₁₆ or C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms or C ₂₀ fatty acids with at least two double Extend bonds in the fatty acid by at least two carbon atoms.

A further embodiment according to the invention are isolated nucleic acids, comprising nucleotide sequences which code for polypeptides which extend C ₁₆ , C ₁₈ or C ₂₀ fatty acids with at least two double bonds in the fatty acid and are selected from the group consisting of

• a) those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 nucleic acid synthesis shown frequencies,
• b) a nucleic acid sequence which according to the degenerate genetic code of one of those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 sequences made, or
• c) derivatives of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 Sequence required for polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 code amino acid sequence shown and at least Have 50% homology at the amino acid level without the enzymatic effect of the polypeptides significantly reduced is.

The above-mentioned nucleic acids according to the invention, which act as C ₁₆ , C ₁₈ or C ₂₀ elongase, originate from organisms, such as ciliates, fungi, algae, plants or dinoflagellates, which can synthesize PUFAs, preferably from plants or algae, particularly preferably from the genus Phytophthora, Physcomitrella, Crypthecodinium, Thraustochytrium or Schizo chytrium and most preferably from Phytophthora infestans, Physcomitrella patens, Crypthecodinium cohnii or Thrausto chytrium sp., Schizochytrium sp. or closely related organisms.

One aspect of the invention relates to isolated nucleic acid molecules, the PSE polypeptides or biologically active parts thereof encode, as well as nucleic acid fragments for use as Hybridization probes or primers for identification or ampli fication of a PSE-encoding nucleic acid (e.g. PSE-DNA) pass. The term "nucleic acid molecule" as used herein is said to be DNA molecules (e.g. cDNA or genomic DNA) and RNA moles cool (e.g. mRNA) as well as DNA or RNA analogues that are generated using nucleo tid analogues are generated include. This term also includes that located at the 3 'and 5' ends of the coding gene region untranslated sequence: at least about 100 nucleotides of the Sequence upstream of the 5 'end of the coding region and at least about 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 preferred wise double-stranded DNA. An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are in the natural source of the nucleic acid. An "isolated" Nucleic acid preferably has no sequences that the Nucleic acid in the genomic DNA of the organism from which the Nucleic acid originates, naturally flanking (e.g. sequences, which are located at the 5 'and 3' ends of the nucleic acid). at In various embodiments, the isolated PSE nucleus acid molecule for example less than about 5 kb, 4 kb, 3 kb, Contain 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences, which naturally contains the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid originates (e.g. a Flank Physcomitrella patens cell). An "isolated" Nucleic acid molecule, such as a cDNA molecule, can also be in the essentially free of other cellular material or culture be medium if it is made by recombinant techniques becomes, or free of chemical precursors or other chemicals be when it's chemically synthesized.

A nucleic acid molecule according to the invention, e.g. B. a nucleic acid molecule with a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 or a part thereof using standard molecular biological techniques and the sequence information provided here. With the help of comparison algorithms, for example, a homologous sequence or homologous, conserved sequence regions can be identified at the DNA or amino acid level. For example, a Phytophthora, Physco mitrella, Crypthecodinium or Thraustochytrium cDNA can be isolated from a Phythophthora, Physcomitrella, Crypthecodinium or Thrausto chtrium bank by the complete SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and / or SEQ ID NO: 11 or a part thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sam brook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Furthermore, a nucleic acid molecule comprising a complete sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 or a part thereof isolate by polymerase chain reaction, oligonucleotide primers based on this sequence or parts thereof, in particular regions around His box motifs of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO : 8, SEQ ID NO: 10 and SEQ ID NO: 12 or modifications of the same in individually defined amino acids can be used (e.g. a nucleic acid molecule comprising the complete sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 or a part thereof, by polymerase chain reaction using oligonucleotide primers which are based on this same sequence of SEQ ID NO : 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11). Furthermore, partial sequences such as those shown in FIG. 10 are particularly suitable for this. For example, mRNA can be isolated from cells (e.g. by the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA using reverse transcriptase (e.g. 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 amplification by means of the polymerase chain reaction can be produced on the basis of one of the nucleotide sequences shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 or with the aid of the amino acid sequences shown in FIG. 10. A nucleic acid according to the invention can be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to 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. Oligo nucleotides which correspond to a PSE nucleotide sequence can be produced by standard synthesis methods, for example using an automatic DNA synthesizer.

The SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 cDNA shown comprises sequences encode the PSEs (i.e. the "coding region") and 5'-un translated sequences and 3'-untranslated sequences. age the nucleic acid molecule can only encode the coding region one of the sequences in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 whole genomic fragments isolated from genomic DNA included.

The SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 are replaced by the same EST Entry number identifier identifies as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 so that they can be easily correlated.  

In a further preferred embodiment, a isolated nucleic acid molecule according to the invention a nucleotide acid molecule that is a complement of one of those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 shown nucleotide sequences or a part of it is. A nucleic acid molecule that belongs to one of the in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 is complementary nucleotide sequences shown, is sufficiently complementary if it matches one of the in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 can hybridize specified sequences, which creates a stable duplex.

Homologs of the new elongase nucleic acid sequences with the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 means, for example, allelic variants at least about 50 to 60%, preferably at least about 60 to 70%, more preferably at least about 70 to 80%, 80 to 90% or 90 to 95% and more preferably at least about 95%, 96%, 97%, 98%, 99% or more homology to one in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 shown nucleotide sequences or their homo logen, derivatives or analogs or parts thereof. At a another preferred embodiment comprises an isolated one nucleic acid molecule according to the invention a nucleotide sequence, to one of those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 nucleotide shown sequences or part thereof, e.g. B. under strin conditions hybridized. Include allelic variants in particular functional variants that are distinguished by deletion, Insertion or substitution of nucleotides from in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 sequence obtained, wherein but the intention is for the enzyme activity to be the result leading synthesized proteins for insertion of one or several genes is advantageously retained. proteins, which still have the enzymatic activity of the elongase, means proteins with at least 10%, preferably 20%, particularly preferably 30%, very particularly preferably 40% the original enzyme activity compared to that by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 encoded protein. Elongasen, the pre Activities mentioned, elongases are their enzymatic Activity are not significantly reduced.  

Homologues of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, For example, SEQ ID NO: 9 and SEQ ID NO: 11 also means bacterial, fungal and plant homologues, shortened sequences, single-stranded DNA or RNA of coding and non-coding the DNA sequence.

Homologues of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 also means derivatives, as in for example promoter variants. The promoters upstream of the specified nucleotide sequences can be one or more Nucleotide exchanges, by insertion (s) and / or deletion (s) be modified without, however, the functionality or Activity of the promoters is disturbed. It is still possible that the activity of the promoters by modification of their sequence is increased or that it is completely replaced by more active promoters, even from heterologous organisms.

In addition, the nucleic acid molecule according to the invention can only have one Part of the coding region of one of the sequences in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, for example a fragment used as a probe or primer can be used, or a fragment which encodes a biologically active portion of a PSE. The one from the Cloning of the PSE gene from Physcomitrella patens, Phytophthora infestans, Thraustochytrium and Crypthecodinium determined Nucleotide sequences allow the generation of probes and Primers used to identify and / or clone PSE Homologues in other cell types and organisms as well as PSE homologues are made from other mosses or related species. The Probe / primer usually comprises essentially purified Oligonucleotide. The oligonucleotide usually includes one Nucleotide sequence region that under stringent conditions at least about 12, preferably about 16, more preferably about 25, 40, 50 or 75 consecutive nucleotides of a sense Strands one of those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 specified sequences, of an antisense strand one of those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 sequences or its homologues, derivatives or analogues or naturally occurring mutants hybridized therefrom. primer based on a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 can be used in PCR reactions to clone PSE Homologues are used. Probes based on the PSE Nucleotide sequences can be used for the detection of transcripts or genomic sequences that are the same or homologous proteins encode, are used. In preferred embodiments  the probe also includes a label group attached to it, z. B. a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. These probes can be part of a Test kits for genomic markers to identify cells, that mis-express a PSE, for example by measuring one Amount of a PSE-encoding nucleic acid in a cell sample, z. B. Measure PSE mRNA levels, or to determine whether a genomic PSE gene is mutated or deleted can be used.

In one embodiment, the nucleic acid according to the invention encodes acid molecule a protein or part thereof, the one Amino acid sequence that is sufficiently homologous to an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 is that the protein or part of it the ability to build on your metabolism of cell membranes in microorganisms or plants necessary Connections or the transport of molecules across these membranes to participate, maintains. As used here, the term refers to "sufficiently homologous" proteins or parts thereof, their amino acid sequences a minimal number of identical or equivalent Amino acid residues (e.g. an amino acid residue with a similar one Side chain, like an amino acid residue in one of the sequences of the SEQ ID NO: 2 to 12) to an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 so that the protein or portion of which on the metabolism of to build cell membranes in micro organisms or plants necessary connections or at the trans port of molecules across these membranes can participate. protein components of these metabolic pathways for membrane components or membrane transport systems can, as described here, a Role in the production and secretion of one or more fine play chemicals. Examples of these activities are here also described. Thus, the "function of a PSE" neither directly or indirectly to yield, production and / or Efficiency of the production of one or more fine chemicals at. Examples of PSE substrate specificities of the catalytic Activity is given in Table 1.

In a further embodiment, derivatives of the Nucleic acid molecule according to the invention 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%, 90 to 95% and most preferably at least about 96%, 97%, 98%, 99% or more homology to complete Amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. The homology of Amino acid sequence was with the  PileUp program (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5, 1989: 151-153) or BESTFIT or GAP (Henikoff, S. and Henikoff, J.G. (1992). Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 89: 10915-10919.)

Parts of proteins produced by the PSE nucleic acid according to the invention acid molecules are encoded, are preferably biological active parts of one of the PSEs. As used here, the term is meant to "biologically active part of a PSE", a section, e.g. Legs Domain / motive to include a PSE that is involved in the metabolism of to build up cell membranes in microorganisms or plants necessary connections or on the transport of molecules via these membranes can participate or one in Table 1 has given activity. To determine whether a PSE or a biologically active part of it on the metabolism of to build up Ver. Necessary cell membranes in microorganisms or plants bonds or the transport of molecules across these membranes can take a test of enzymatic activity by be performed. These test procedures, as detailed in Example 8 described in the example part, are familiar to those skilled in the art.

Additional nucleic acid fragments that are biologically active Coding sections of a PSE can be done by isolation part of one of the sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, Express the encoded portion of the PSE or peptide (e.g. by recombinant expression in vitro) and determining the activity of the encoded portion of the PSE or peptide produce.

The invention also includes nucleic acid molecules that are from one of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 nucleotide shown sequences (and parts thereof) due to the degenerate geneti differentiate codes and thus encode the same PSE as the one of those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 nucleotide shown sequences is encoded. In another embodiment, a isolated nucleic acid molecule according to the invention a nucleotide sequence that is a protein with a in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 Amino acid sequence encoded. In another embodiment the nucleic acid molecule according to the invention encodes a full-length PSE protein which forms an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 (that of one in SEQ ID NO: 1, SEQ ID NO: 3,  SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 open reading frame is coded) is essentially homologous and can be identified and isolated using common methods.

In addition to those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 PSE shown Those skilled in the art will recognize nucleotide sequences that DNA sequence poly morphisms that lead to changes in the amino acid sequences of the PSEs lead within a population (e.g. the Physcomitrella Phytophthora, Crypthecodinium or Thraustochytrium population) can exist. These genetic polymorphisms in the PSE gene can occur between individuals within a population of natural variation exist. As used here, be mean the terms "gene" and "recombinant gene" nucleic acid molecules with an open reading frame that prefer a PSE as a Phytophthora, Physcomitrella, Crypthecodinium or Thraustochytrium -PSE, coded. These natural variants be usually a variance of 1 to 5% in the nucleotide sequence of the PSE gene. All and all of this nucleotide variations and resulting amino acid polymorphisms in PSE that are the result of natural variation and that functional activity of PSEs should not change in the Scope of the invention may be included.

Nucleic acid molecules that correspond to the natural variants, and not Physcomitrella, Phytophthora, Crypthecodinium or Thraustochytrium homologues, derivatives and analogues of Phytophthora, Physcomitrella, Crypthecodinium or Thrausto chytrium cDNA can be based on their homology to the Phytophthora, Physcomitrella, Crypthecodinium disclosed here or Thraustochytrium PSE nucleic acid using the Physcomitrella, Phytophthora, Crypthecodinium or Thrausto chytrium cDNA or a part thereof as a hybridization probe according to standard hybridization techniques under stringent Hybridization conditions are isolated. Another one The embodiment is an isolated nucleus according to the invention acid molecule at least 15 nucleotides long and hybridized under stringent conditions with the nucleic acid molecule that a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11. With others Embodiments, the nucleic acid is at least 25, 50, 100, 250 or more nucleotides long. The term "hybridizes under stringent conditions "as used here Describe hybridization and washing conditions under which Nucleotide sequences that are at least 60% homologous to one another, usually remain hybridized to each other. The conditions are preferably such that sequences that are at least about 65%,  more preferably at least about 70% and even more preferred are at least about 75% or more homologous to one another, usually remain hybridized to each other. This 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., Find. A preferred, non-limiting one Hybridi are examples of stringent hybridization conditions in 6 × sodium chloride / sodium citrate (sodium chloride / sodiumcitrate = SSC) at around 45 ° C, followed by one or several washing steps in 0.2 × SSC, 0.1% SDS at 50 to 65 ° C. Those skilled in the art are aware that these hybridization conditions itself depending on the type of nucleic acid and, if for example organic solvents are present in terms of temperature and the concentration of the buffer. The temperature differs for example under "Standard Hybridi sierungsbedingungen "depending on the type of nucleic acid between 42 ° C and 58 ° C in aqueous buffer with a concentration of 0.1 to 5 × SSC (pH 7.2). If organic solvent in the above mentioned buffer is present, for example 50% formamide, is the Temperature under standard conditions about 42 ° C. Are preferred the hybridization conditions for DNA: DNA hybrids 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 0.1x SSC and 30 ° C to 55 ° C, preferably between 45 ° C and 55 ° C. The above hybridization For example, for a nucleic acid, temperatures are about 100 bp (= base pairs) in length and a G + C content of 50% in Absence of formamide determined. The expert knows how to do that required hybridization conditions based on textbooks, such as that mentioned above or from the following textbooks Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1989; Hames and Higgins (eds.) 1985, "Nucleic Acids Hybridization: A Practical Approach, "IRL Press at Oxford University Press, Oxford; Brown (ed.) 1991, "Essential Molecular Biology: A Practical Approach, "IRL Press at Oxford University Press, Oxford.

An isolated one according to the invention preferably corresponds Nucleic acid molecule that is attached to a Sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 hybridized, of course before upcoming nucleic acid molecule. As used here, affects one "naturally occurring" nucleic acid molecule an RNA or DNA Molecule with a nucleotide sequence that occurs in nature (e.g. encoded a natural protein). With an execution the nucleic acid encodes a naturally occurring form  Physcomitrella patens-PSE, Phytophthora infestans-PSE, Crypthe codinium cohnii-PSE or Thraustochytrium-PSE.

In addition to naturally occurring variants of the PSE sequence, those skilled in the art recognize those that may exist in the population further that changes by mutation into a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 can be introduced what leads to changes in the amino acid sequence of the encoded PSE, without affecting the functionality of the PSE protein becomes. For example, nucleotide substitutions that are "non-essential" amino acid residues for amino acid substitutions lead in a sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 put. A "non-essential" amino acid residue is a residue which is in a wild-type sequence of one of the PSEs (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12) can be changed without the activity of the PSE is changed, whereas an "essential" amino acid residue for the PSE activity is required. Other amino acid residues (e.g. those who are not conserved in the domain with PSE activity or are only semi-preserved) but can be used for Activity can not be essential and can therefore be true apparently change without changing PSE activity.

Accordingly, another aspect of the invention relates to nucleic acid Molecules that encode PSEs, the modified amino acid residues ent hold that are not essential for PSE activity. This PSEs differ in amino acid sequence from one Sequence in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and still keep at least one of the PSE activities described here. The isolated In one embodiment, nucleic acid molecule comprises one Nucleotide sequence encoding a protein, the protein being a Amino acid sequence with at least about 50% homology to one Amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 Metabolism to build cell membranes in Phytophthora, Physcomitrella, Crypthecodinium- or Thraustochytrium not agile connections or the transport of molecules via them Membranes can participate. That from the nucleic acid molecule encoded protein is preferably at least about 50 to 60% homologous to one of the sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 stronger preferably at least about 60 to 70% homologous to one of the Sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID N 99999 00070 552 001000280000000200012000285919988800040 0002010063387 00004 99880O: 12 even more preferably at least  about 70 to 80%, 80 to 90%, 90 to 95% homologous to one of the Sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and most preferably min at least about 96%, 97%, 98% or 99% homologous to one of the Sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.

To determine the percentage homology of two amino acids sequences (e.g. one of the sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and one mutated form thereof) or of two nucleic acids Sequences for the purpose of optimal comparison with one another written (e.g., gaps in the sequence of a protein or a nucleic acid to be optimal Alignment with the other protein or nucleic acid to create). The amino acid residues or nucleotides on the ent speaking amino acid positions or nucleotide positions then compared. If a position in a sequence (e.g. one the sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12) by the same Amino acid residue or the same nucleotide as the corresponding one Place in the other sequence (e.g. a mutated form of the SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 selected sequence) is occupied, then the molecules at this position are homologous (i.e. amino acid or Nucleic acid "homology" as used herein corresponds to amino acid or nucleic acid "identity"). The percentage homology between the two sequences is a function of the number identical positions that are common to the sequences (i.e. % Homology = number of identical positions / total number of Positions × 100).

An isolated nucleic acid molecule that encodes a PSE a protein sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 is homologous by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 are generated so that one or more amino acid substitutions, additions or deletions into the coded Protein are introduced. Mutations can occur in one of the Sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 by standard techniques such as site-specific mutagenesis and PCR-mediated mutagenesis, be introduced. Preferably conservative amino acid substitutions on one or more of the predicted non- essential amino acid residues. With a "conservative  Amino acid substitution "is the amino acid residue against an amino acid residue with a similar side chain exchanges. Families of amino acid residues are included in the field similar side chains have been defined. These families include Amino acids with basic side chains (e.g. lysine, arginine, Histidine), acidic side chains (e.g. aspartic acid, glutamine acid), uncharged polar side chains (e.g. glycine, asparagine, Glutamine, serine, threonine, tyrosine, cysteine), non-polar sides chains, (e.g. alanine, valine, leucine, isoleucine, proline, phenyl alanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). One before said non-essential amino acid residue in a PSE thus preferably by another amino acid residue from the same family of side chains. Alternatively, at another embodiment randomly over the mutations all or part of the PSE coding sequence brought, e.g. B. by saturation mutagenesis, and resulting mutants can be according to the one described here PSE activity to be screened to identify mutants maintain PSE activity. After mutagenesis one of the Sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 can recombine the encoded protein nant expressed, and the activity of the protein z. B. using the tests described here (see example part) can be determined.

In addition to the nucleic acid molecules that the above encoding PSEs described relates to a further aspect of Invention isolated nucleic acid molecules, the "antisense" to it are. An "antisense" nucleic acid comprises a nucleotide sequence to a "sense" nucleic acid that encodes a protein, is complementary, e.g. B. complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA Sequence. An antisense nucleic acid can therefore be above water Bind fabric bonds to a sense nucleic acid. The Antisense nucleic acid can encode an entire PSE Strand or only complementary to part of it. At a Embodiment is an antisense nucleic acid molecule "antisense" to a "coding area" of the coding strand Nucleotide sequence encoding a PSE. The term "coding Area "refers to the area of the nucleotide sequence, the codons which are translated into amino acid residues (e.g. the entire coding area starting with the stop codon and ends, d. H. the last codon before the stop codon). At a Another embodiment is the antisense nucleic acid molecule "Antisense" to a "non-coding area" of the coding  Strands of a nucleotide sequence encoding PSE. The term "non-coding region" refers to 5 'and 3' sequences, that flank the coding area and not in amino acids are translated (i.e. which are also called 5'- and 3'-untranslated areas designated).

Assuming the PSE coding disclosed here Sequences of the coding strand (e.g. those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 sequences shown) can be according to the invention Antisense nucleic acids according to the rules of the Watson-Crick bases pairing can be designed. The antisense nucleic acid molecule can be complementary to the entire coding region of PSE mRNA, but is more preferred an oligonucleotide that only one Part of the coding or non-coding region of PSE mRNA "Antisense" is. The antisense oligonucleotide can e.g. B. to the Area surrounding the translation start site of PSE mRNA, to be complementary. An antisense oligonucleotide can e.g. B. about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 and more nucleotides To be long. An antisense oligonucleotide 15 to is advantageous 25 nucleotides long. An antisense nucleic acid according to the invention can be done using chemical synthesis and enzymatic Ligation reactions using methods known in the art be constructed. An antisense nucleic acid (e.g. an anti sense oligonucleotide) can e.g. B. are chemically synthesized, where naturally occurring nucleotides or different modified nucleotides are used, which are designed that they increase the biological stability of the molecules or the physical stability of the between the antisense and increase the duplex formed sense nucleic acid, for example wise phosphorothioate derivatives and acridine substituted Nucleotides are used. Examples of modified nucleo tide used to generate the antisense nucleic acid can be u. a. 5-fluorouracil, 5-bromouracil, 5-chlorine uracil, 5-ioduracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, Beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methyl guanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl 2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyl uracil, 5-methoxyuracil, 2-methylthio-N6-isopentyladenine, Uracil-5-oxyacetic acid (v), wybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5- oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-  carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine. The anti Alternatively, sense nucleic acid can be used biologically an expression vector are prepared in which a Nucleic acid has been subcloned in the antisense direction (i.e. RNA that transcribes from the inserted nucleic acid is a target nucleic acid of interest in antisense Direction oriented, which continues in the subsection below is described).

The antisense nucleic acid molecules according to the invention are usually administered to a cell or generated in situ, so that they are compatible with the cellular mRNA and / or the genomic DNA encoding, hybridizing or binding to a PSE to thereby express the protein, e.g. B. by inhibiting the To inhibit transcription and / or translation. The hybridization can be formed by conventional nucleotide complementarity a stable duplex or z. B. in the case of an antisense Nucleic acid molecule that binds DNA duplexes by specific Interactions occur in the large groove of the double helix. The antisense molecule can be modified to be specific to a receptor or to a selected cell top area expressed antigen binds e.g. B. by binding the anti sense 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 used of the vectors described here are supplied to the cells. To achieve sufficient intracellular concentrations of the antisense molecules are vector constructs in which the Antisense nucleic acid molecule under the control of a strong one prokaryotic, viral or eukaryotic, including vegetable, promoter is preferred.

In a further embodiment, this is the invention Antisense nucleic acid molecule is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific doubles stranded hybrids with complementary RNA, the strands in the Contrary to ordinary β-units ver to run. [Gaultier et al. (1987) Nucleic Acids Res. 15: 6625-6641]. The antisense nucleic acid molecule can also be a 2'-o-methylribo nucleotide [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].

In a further embodiment, one according to the invention Antisense nucleic acid a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are single-stranded Can cleave nucleic acid, such as an mRNA, to which they join  have complementary area. Thus, ribozymes can e.g. B. Hammer head-Ribozyme [described in Haselhoff and Gerlach (1988) Nature 334: 585-591] for the catalytic cleavage of PSE mRNA transcripts used to thereby translate the PSE mRNA inhibit. A ribozyme with specificity for a PSE coding Nucleic acid can be based on the nucleotide sequence of a PSE cDNA disclosed herein (i.e. 38_ck21_g07fwd in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11) or on the basis of one in accordance with this Invention taught method to isolate heterologous sequence be designed. For example, a derivative of a tetra hymena-L-19-IVS-RNA can be constructed using the nucleotide sequence of the active site complementary to the nucleotide is sequence that are cleaved in a PSE-encoding mRNA should. See e.g. B. Cech et al., US 4,987,071 and Cech et al., US 5,116,742. Alternatively, PSE mRNA can be used to select a catalytic RNA with a specific ribonuclease activity from a pool of RNA molecules [see e.g. B. Bartel, D., and Szostak, J.W. (1993) Science 261: 1411-1418].

Alternatively, PSE gene expression can be inhibited by nucleo tid sequences that are complementary to the regulatory area of a PSE nucleotide sequence (e.g. a PSE promoter and / or enhancer) are so directed that triple helix structures are formed which are the transcription of a PSE gene in target cells inhibit [see generally Helene, C. (1991) Anticancer Drug Res. 6 (6) 569-84; Helene, C., et al. (1992) Ann. N. Y. Acad. Sci. 660: 27-36; and Maher. L. J. (1992) Bioassays 14 (12): 807-815].

B. gene construct

Another embodiment of the invention is a new gene constructed which contains an isolated nucleic acid derived from Physcomitrella, Phytophthora, Crypthecodinium or Thrausto chytrium and which encodes a polypeptide which encodes C ₁₆ , C ₁₈ or C ₂₀ fatty acids with at least two double bonds in the fatty acid is extended by at least two carbon atoms, or the gene sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, their Contains homologs, derivatives or analogs that are operably linked to one or more regulatory signals, advantageously to increase gene expression. Examples of these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid. In addition to these new regulatory sequences, the natural regulation of these sequences may still be present before the actual structural genes and, if appropriate, may have been genetically modified so that natural regulation has been switched off and the expression of the genes has been increased. However, the gene construct can also have a simpler structure, ie that no additional regulation signals before the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 or their homologues and the natural promoter with its regulation has not been deleted. Instead, the natural regulatory sequence has been mutated so that regulation no longer takes place and gene expression is increased. The gene construct can also advantageously comprise one or more so-called enhancer sequences which are operably linked to the promoter and which enable increased expression of the nucleic acid sequence. It is also possible to additionally insert advantageous sequences at the 3 'end of the DNA sequences, for example further regulatory elements or terminators. The elongase genes can be present in the gene construct in one or more copies. It is advantageous for the insertion of further genes in organisms if further genes are present in the gene construct.

Advantageous regulatory sequences for the new method are present, for example, in promoters, such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI ^q , T7, T5 -, T3, gal, trc, ara, SP6, λ-P _R or λ-P _L promoter and are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are, for example, in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFcL, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV / 35S [Franck et al., 1980, Cell 21: 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter. Also advantageous in this context are inducible promoters, such as those in EP-A-0 388 186 (benzylsulfonamide inducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracycline inducible), EP-A -0 335 528 (abscisic acid inducible) or WO 93/21334 (ethanol or cyclohexenol inducible) described promoters. Further suitable plant promoters are the cytosolic FBPase promoter or the potato ST-LSI promoter (Stockhaus et al., EMBO J. 8, 1989, 2445), the glycine max phosphoribosylpyrophosphatamidotransferase promoter (Genbank accession number U87999) or the node-specific promoter described in EP-A-0 249 676. Particularly advantageous promoters are promoters which enable expression in tissues which are involved in fatty acid biosynthesis. Seed-specific promoters such as the usp, LEB4, phaseolin or napin promoter are particularly advantageous. Further particularly advantageous promoters are seed-specific promoters that can be used for monocot or dicotyledonous plants and in US 5,608,152 (napin promoter from rapeseed), WO 98/45461 (phaseolin promoter from Arobidopsis), US 5,504,200 (phaseolin promoter from Phaseolus vulgaris) ), WO 91/13980 (Bce4 promoter from Brassica), by Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (LEB4 promoter from a legume), these promoters being suitable for dicotyledons suitable. The following promoters are suitable for example for monocotyledons lpt-2 or lpt-1 promoter made from barley (WO 95/15389 and WO 95/23230), hordein promoter made from barley and other suitable promoters described in WO 99/16890.

In principle it is possible to use all natural promoters with their Regulatory sequences, such as those mentioned above, for the new ver driving to use. It is also possible and advantageous additionally use synthetic promoters.

As described above, the gene construct can also contain other genes include that are to be introduced into the organisms. It is possible and beneficial, in the host organisms regulatory genes, such as genes for inducers, repressors or enzymes, which are caused by their enzyme activity in the regulation of one or more genes intervene in a biosynthetic pathway, introduce it and close it express. These genes can be heterologous or homologous Be of origin. The inserted genes can have their own Have promoter or under the control of the promoter the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 or its homologs, derivatives or analogues.

The gene construct advantageously comprises for the expression of the other present genes further 3'- and / or 5'-terminal Regulatory sequences to increase expression, which in Dependence on the selected host organism and the gene or the genes are selected for optimal expression.

As mentioned above, these regulatory sequences are intended to: specific expression of the genes and protein expression enable. This can, for example, depending on the host organism wise mean that the gene expresses only after induction or is overexpressed or that it expresses immediately and / or is overexpressed.

The regulatory sequences or factors may also be preferred as a beneficial effect on the expression of the brought genes and thus increase them. That way it is possible that the regulatory elements using strong  Transcription signals, such as promoters and / or enhancers, are advantageous will be reinforced at the level of transcription. It is however, translation is still possible, for example by enhancing mRNA stability. The nucleic acid according to the invention are advantageously sequences together with at least one reporter gene Gene construct (= expression cassette, nucleic acid construct) cloned into the organism via a vector or directly is introduced into the genome. This reporter gene should be one easy detectability via a growth, fluorescence, Chemo, bioluminescence or resistance assay or via a photo enable metric measurement. Be exemplary as a reporter antibiotic or herbicide resistance genes, hydrolase genes, Fluorescence protein genes, bioluminescence genes, sugar or Nucleotide metabolic genes or biosynthetic genes such as the Ura3 Gene, the Ilv2 gene, the luciferase gene, the b-galactosidase gene, the gfp gene, the 2-deoxyglucose-6-phosphate phosphatase gene, the b-glucuronidase gene, b-lactamase gene, the neomycin phospho transferase gene, the hygromycin phosphotransferase gene or that BASTA (= gluphosinate resistance) gene called. These genes make it possible easy measurability and quantifiability of the Transcription activity and thus the expression of the genes. In order to genome sites can be identified that make a difference Show productivity.

The nucleic acid sequences according to the invention with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, which can code for elongases in one or several copies in the expression cassette (= gene construct) be included.

In the expression cassette (= gene construct, nucleic acid construct) can contain at least one further nucleic acid encoded for a gene preferably from fatty acid biosynthesis, be contained, which are introduced into the host organisms should. These genes can be under separate regulation or under the same regulatory region as the genes of the invention Elongase lie. These genes are, for example for other biosynthesis genes advantageous in fatty acid biosynthesis, which enable an increased synthesis. For example the genes for the Δ19-, Δ17-, Δ15-, Δ12-, Δ9-, Δ8-, Δ6-, Δ5-, Δ4-desaturase, the various hydroxylases, the Δ12-acetylenase, the acyl-ACP thioesterases, β-ketoacyl-ACP- Synthases or β-ketoacyl-ACP reductases called. Advantageous the desaturase genes are used in the nucleic acid construct.  

These genes can also be found in one or more copies of the gene construct included.

C. Recombinant Expression Vectors and Host Cells

Another aspect of the invention relates to vectors, preferably wise expression vectors, the nucleic acid of the invention or contain a gene construct according to the invention which have a PSE (or part of it) encode. As used here concerns the term "vector" means one nucleic acid molecule, another Can transport nucleic acid to which it is bound. A vector type is a "plasmid", what a circular double stranded DNA loop is in the additional DNA segments can be ligated. Another type of vector is viral Vector, with additional DNA segments in the viral genome can be ligated. Certain vectors can be in a host autonomously replicate the cell into which they have been inserted (e.g. bacterial vectors with bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g. non-episomal Mammalian vectors) are introduced into the host cell integrates the genome of a host cell and thus together with replicated to the host genome. In addition, certain vectors can Expression of genes with which they are operably linked are, control. These vectors are called "expressions vectors ". Usually expression vectors have the are suitable for recombinant DNA techniques, the form of Plasmids. In the present description, "plasmid" and "Vector" can be used interchangeably because the plasmid is the most most common vector form is. However, the invention is intended these other expression vector forms, such as viral vectors (e.g. replication-deficient retroviruses, adenoviruses and adeno-related Viruses) that perform similar functions. Furthermore should the term vector also includes other vectors known to those skilled in the art are, like phages, viruses, like SV40, CMV, baculovirus, adenovirus, Transposons, IS elements, phasmids, phagemids, cosmids, linear or include circular DNA and RNA.

The recombinant expression vectors according to the invention summarize a nucleic acid according to the invention or a appropriate gene construct in a form which is suitable for expression of the Nucleic acid in a host cell is suitable, which means that the recombinant expression vectors one or more regulations sequences selected on the basis of the expression to be ver turning host cells with the nucleotide to be expressed acid sequence is operatively linked. In one recombinant expression vector means "functional ver bound "that the nucleotide sequence of interest to the  Regulatory sequence (s) is bound that the expression of the Nucleotide sequence is possible and they are bound to each other, so that both sequences are the predicted, attributed to the sequence Perform function (e.g. in an in vitro transcription / trans lation system or in a host cell if the vector is in the Host cell is introduced). The term "regulatory sequence" is said to be promoters, enhancers and other expression control elements (e.g., polyadenylation signals). These regulations sequences are e.g. B. described in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnolgy, CRC Press, Boca Raton, Florida, ed .: Glick and Thompson, Chapter 7, 89-108 finally the references in it. regulatory sequences include those that constitute the constitutive expression of a Control nucleotide sequence in many host cell types, and those which the direct expression of the nucleotide sequence only in control certain host cells under certain conditions. Those skilled in the art know that the design of the expression vector of Factors such as the selection of the host cell to be transformed, the extent of expression of the desired protein, etc. can. The expression vectors according to the invention can be found in hosts cells are introduced in order to thereby produce proteins or peptides, including producing fusion proteins or peptides, encoded by the nucleic acids as described here (e.g. PSEs, mutant forms of PSEs, fusion proteins, etc.).

The recombinant expression vectors according to the invention can for the expression of PSEs in prokaryotic or eukaryotic Cells. For example, PSE genes can be found in bacterial cells, such as C. glutamicum, insect cells (under Use of baculovirus expression vectors), yeast and other fungal cells [see Romanos, M.A., et al. (1992) "Foreign gene expression in yeast: a review ", Yeast 8: 423-488; by the Hondel, C.A.M.J., et al. (1991) "Heterologous gene expression in filamentous fungi ", in: More Gene Manipulations in Fungi, J.W. Bennet & L.L. Lasure, eds., Pp. 396-428: Academic Press: San Diego; and von den Hondel, C.A.M.J., & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F., et al., eds., pp. 1-28, Cambridge University Press: Cambridge], Algae [Falciatore et al., 1999, Marine Biotechnology. 1, 3: 239-251)], ciliates of the types: such as Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella and Stylonychia, especially of the genus Stylonychia lemnae, with vectors after a transformation process,  as described in WO 98/01572, as well as cells cellular plants [see Schmidt, R. and Willmitzer, L. (1988) "High efficiency Agrobacterium tumefaciens-mediated trans formation of Arabidopsis thaliana leaf and cotyledon explants " Plant Cell Rep .: 583-586; Plant Molecular Biology and Biotechno logy, C Press, Boca Raton, Florida, Chapter 6/7, pp. 71-119 (1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, ed .: Kung and R. Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225 (and references cited therein) or mammalian cells become. Suitable host cells are also discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). The recombinant expression vector Alternatively, for example using T7 promoter Regulatory sequences and T7 polymerase, transcribed in vitro and be translated.

Proteins are usually expressed in prokaryotes with vectors that are constitutive or inducible promoters contain which express the expression of fusion or non- Control fusion proteins. Fusion vectors add a series from amino acids to a protein encoded therein, usually on Amino terminus of the recombinant protein, but also at the C-terminus or fused within appropriate ranges in the proteins. These fusion vectors usually have three functions: 1) the Enhancing the expression of recombinant protein; 2) the Increasing the solubility of the recombinant protein and 3) the Support the purification of the recombinant protein by Effect as a ligand in affinity purification, for example via so-called His tags. For fusion expression vectors often a proteolytic cleft at the junction of the Fusion unit and the recombinant protein introduced so that the separation of the recombinant protein from the fusion unit after purification of the fusion protein is possible. These enzymes and their corresponding recognition sequences include factor Xa, Thrombin and enterokinase.

Typical fusion expression vectors include a. pGEX [Pharmacia Biotech Inc. Smith, D.B. and Johnson, K.S. (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 to the recombinant target protein is fused. With an execution form is the PSE coding sequence in a pGEX expression vector cloned so that a vector is generated that merges protein encoding that is from the N-terminus to the C-terminus GST thrombin  Cleavage site X protein. The fusion protein can by Affinity Chromatography Using Glutathione Agarose resin can be cleaned. Recombinant PSE that does not participate GST is fused by cleavage of the fusion protein can be obtained with thrombin.

Examples of suitable inducible non-fusion E. coli Expression vectors are u. a. pTrc (Amann et al. (1988) Gene 69: 301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). The target gene expression from the pTrc vector relies on host RNA polymerase transcription from one Hybrid trp-lac fusion promoter. The target gene expression from the pET 11d vector is based on the transcription of a T7-gn10-lac Fusion promoter generated from a coexpressed viral RNA poly merase (T7 gn1) is mediated. This viral polymerase will from the host strains BL21 (DE3) or HMS174 (DE3) from one resident λ prophage provided that a T7 gn1 gene under the transcription control of the lacUV 5 promoter.

Other vectors suitable in prokaryotic organisms are known to the person skilled in the art, these vectors are, for example, in E. coli pLG338, pACYC184, the pBR series, such as pBR322, the pUC series, such as pUC18 or pUC19, the M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III ¹¹³ -B1, pgt11 or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or p77debacterium, in C774 or p77db14A6.

A strategy to maximize the expression of recombinant Protein is the expression of the protein in a host bacterium, its ability to proteolytically cleave the recombinant Protein is disturbed [Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128]. Another strategy is to change the Insert nucleic acid sequence into an expression vector the nucleic acid so that the individual codons for each amino Acid are those that are preferred for expression in one selected bacteria such as C. glutamicum can be used [Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118]. This The nucleic acid sequences according to the invention are changed through standard DNA synthesis techniques.

In another embodiment, the PSE expression vector is a yeast expression vector. Examples of vectors for expression in the yeast S. cerevisiae include pYepSec1 [Baldari et al. (1987) Embo J. 6: 229-234], pMFa [Kurjan and Herskowitz (1982) Cell 30: 933-943], pJRY88 [Schultz et al. (1987) Gene 54: 113-123]  and pYES2 [Invitrogen Corporation, San Diego, CA]. Vectors and Process of constructing vectors that are ready for use in other fungi, such as filamentous fungi those that are described in detail in: from the Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J.F. Peberdy et al., eds., pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi [J. W. Bennet & L.L. Lasure, eds., Pp. 396-428: Academic Press: San Diego]. Other suitable yeast vectors are examples wise pAG-1, YEp6, YEp13 or pEMBLYe23.

Alternatively, the PSEs according to the invention can be used in insect cells expressed using baculovirus expression vectors become. Baculovirus vectors used to express proteins are available in cultured insect cells (e.g. Sf9 cells), include the pAc series [Smith et al. (1983) Mol. Cell Biol .. 3: 2156-2165] and the pVL series [Lucklow and Summers (1989) Virology 170: 31-39].

The above vectors only provide a small overview about possible suitable vectors. Other plasmids are that Known to those skilled in the art and are described, for example, in: Cloning Vectors (ed. Pouwels, P.H., et al., Elsevier, Amsterdam- New York-Oxford, 1985, ISBN 0 444 904018).

In yet another embodiment, an invention appropriate nucleic acid in mammalian cells using a Mammalian expression vector expressed. Examples of mammalian Expression vectors include pCDM8 [Seed, B. (1987) Nature 329: 840] and pMT2PC [Kaufman et al. (1987) EMBO J. 6: 187-195]. When used in mammalian cells, the control functions of the expression vector often from viral regulatory elements provided. Commonly used promoters come from e.g. B. from Polyoma, Adenovirus2, Cytomegalievirus and Simian Virus 40. Other suitable expression systems for prokaryotic and eukaryotic cells see in chapters 16 and 17 of Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989th

In another embodiment, the recombinant mammalian Expression vector the expression of the nucleic acid preferably in control a specific cell type (e.g. tissue-specific Regulatory elements used to express the nucleic acid). Tissue-specific regulatory elements are known in the art.  

Non-limiting examples of suitable tissue-specific Promoters include a. 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. 8: 729-733] and immunoglobulins [Banerji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell 33: 741-748], neuron-specific promoters [e.g. B. Neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477], pancreas specific promoters [Edlund et al., (1985) Science 230: 912-916] and mammary-specific promoters (e.g. milk serum-promoter; US 4,873,316 and EP-A-0 264 166). Also ent winding regulated promoters are included, e.g. B. the hox Mouse promoters [Kessel and Gruss (1990) Science 249: 374-379] and the fetoprotein promoter [Campes and Tilghman (1989) Genes Dev. 3: 537-546].

In a further embodiment, the inventive PSEs in single-cell plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251 and therein cited references, and plant cells from higher Plants (e.g. spermatophytes, such as crops) are expressed become. Examples of plant expression vectors include sol che, which are 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, ed .: Kung and R. Wu, Academic Press, 1993, pp. 15-38. More suitable Vegetable vectors are described in "Methods in Plant Molecular Biology and Biotechnology "(CRC Press), Chap. 6/7, Pp. 71-119. Advantageous vectors are so-called shuttle Vectors or binary vectors found in E. coli and Agrobacterium replicate.

A plant expression cassette preferably contains Regulatory sequences that regulate gene expression in plants can control cells and are operatively connected so that each sequence its function, like termination of transcription, can fulfill, for example polyadenylation signals. before pulled polyadenylation signals are those derived from Agro bacterium tumefaciens-t-DNA originate, like that as octopine synthase known gene 3 of the Ti plasmid pTiACH5 [Gielen et al., EMBO J. 3 (1984) 835ff.] Or functional equivalents thereof, but also  all other terminators are functionally active in plants suitable.

Because plant gene expression very often does not rely on transcription level restricted, contains a plant expression cassette preferably other operably linked sequences, such as Translation enhancers, for example the overdrive sequence, which is the 5'-untranslated tobacco mosaic leader sequence virus that increases the protein / RNA ratio contains [Gallie et al., 1987, Nucl. Acids Research 15: 8693-8711].

Plant gene expression must be functional with a suitable promoter linked to gene expression perform timely, cell or tissue specific ways. Preferred are promoters which have the constitutive expression bring about [Benfey et al., EMBO J. 8 (1989) 2195-2202], like those derived from plant viruses, like 35S CAMV [Franck et al., Cell 21 (1980) 285-294], 19S CaMV (see also US 5,352,605 and WO 84/02913) or plant promoters, such as that in US 4,962,028 described the small subunit of the Rubisco.

Other preferred sequences for use in function capable compound in plant gene expression cassettes Targeting sequences that are used to control the gene product appropriate cell compartments are necessary [see an over view in Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and references cited therein], for example in the vacuole, the cell nucleus, all types of plastids, such as amyloplasts, chloro plastic, chromoplasts, extracellular space, the mitochon three, the endoplasmic reticulum, oil body, peroxisomes and other compartments of plant cells.

Plant gene expression can also be done chemically facilitate inducible promoter [see an overview in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48: 89-108]. Chemically inducible promoters are particularly useful when it is desired that gene expression be time-specific he follows. Examples of such promoters are salicylic acid inducible promoter (WO 95/19443), a tetracycline-induced bar promoter [Gatz et al. (1992) Plant J. 2, 397-404] and a Ethanol inducible promoter.

Also promoters who are on biotic or abiotic stress React conditions are suitable promoters, for example the pathogen-induced PRP1 gene promoter [Ward et al., Plant. Mol. Biol. 22 (1993) 361-366], the heat-inducible hsp80- Promoter from tomato (US 5,187,267), the cold-inducible alpha amylase promoter  from potato (WO 96/12814) or by Wound inducible pinII promoter (EP-A-0 375 091).

In particular, promoters are preferred which the Induce gene expression in tissues and organs in which lipid and oil biosynthesis takes place in sperm cells, such as the cells of the endosperm and the developing embryo. Suitable promoters are the Napingen promoter from rapeseed (US 5,608,152), the USP promoter from Vicia faba [Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67], the oleosin Arabidopsis promoter (WO 98/45461), the phaseolin promoter from Phaseolus vulgaris (US 5,504,200), the Bce4 promoter Brassica (WO 91/13980) or the legumin B4 promoter [LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9] and Promoters showing seed-specific expression in Mono cotyledonous plants such as corn, barley, wheat, rye, rice, etc. cause. Suitable notable promoters are the lpt2- or lpt1 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 corn zein gene, the oat glutelin gene, the sorghum Kasirin gene, the rye secalin gene).

Promoters which are also particularly suitable induce plastid-specific expression because plastids are the compartment in which the precursor as well as some end products of lipid biosynthesis are synthesized. suitable Promoters, such as the viral RNA polymerase promoter described in WO 95/16783 and WO 97/06250, and the clpP- Arabidopsis promoter, described in WO 99/46394.

The invention also provides recombinant expression vector ready, comprising a DNA molecule according to the invention, that clones into the expression vector in the antisense direction is. d. H. the DNA molecule is so regulatory Sequence operably linked to expression (by Transcription of the DNA molecule) of an RNA molecule that mRNA "antisense" is made possible. There can be regulations sequences are selected that work with an in Antisense direction are linked to cloned nucleic acid and the continuous expression of the antisense RNA molecule in control a variety of cell types, for example viral ones Promoters and / or enhancers or regulatory sequences selected which are the constitutive, tissue-specific or cell type Control specific expression of antisense RNA. The antisense Expression vector can be in the form of a recombinant plasmid,  Phagemids or attenuated virus are present in which antisense Nucleic acids under the control of a highly effective regulator toric area are produced, its activity by the cell type into which the vector is introduced can be determined has been. An explanation of the regulation of gene expression using antisense genes, see in Weintraub, H., et al., Anti sense-RNA as a molecular tool for genetic analysis, reviews- Trends in Genetics, Vol. 1 (1) 1986.

Another aspect of the invention relates to host cells in which introduced a recombinant expression vector according to the invention has been. The terms "host cell" and "recombinant host cells "are used interchangeably here. Even understandably, these terms do not only apply to the specific Target cell, but also the offspring or potential offspring come this cell. Because in successive generations certain modifications due to mutation or environmental influences these offspring are not necessarily with the Parental cell are identical, but are still of the size of the Term as used herein.

A host cell can be a prokaryotic or eukaryotic Be a cell. For example, a PSE can be found in bacterial cells such as C. glutamicum, insect cells, fungal cells or mammalian cells (such as Chinese hamster ovary (CHO) or COS cells), algae, Ciliates, plant cells, fungi or other microorganisms, such as C. glutamicum. Other suitable hosts cells are familiar to the person skilled in the art.

Vector DNA can be expressed in prokaryotic or eukaryotic cells using conventional transformation or transfection techniques contribute. The terms "transformation" and "transfection", Conjugation and transduction, as used here, are said to be one A variety of methods known in the art for one bring foreign nucleic acid (e.g. DNA) into a host cell finally calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, natural Competence, chemically mediated transfer, electroporation or Particle bombardment. Suitable procedures for transformation or transfection of host cells, including plants 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, ed.: Gartland and Davey, Humana Press, Totowa, New Jersey.  

It is known about the stable transfection of mammalian cells that depending on the expression vector and trans used only a small part of the cells contain the foreign DNA integrated into their genome. For identification and selection of these Integrants usually become a gene that has a selectable one Markers (e.g. antibiotic resistance) encoded, along with introduced the gene of interest into the host cells. preferred selectable markers include those that are resistant to Give drugs such as G418, hygromycin and methotrexate or in plants those which are resistant to a herbicide, such as Glyphosphate or glufosinate. Other suitable markers are, for example, markers which encode genes which are linked to bio involved in the synthesis of, for example, sugars or amino acids are like β-galactodsidase, ura3 or ilv2. Markers what genes such as encode luciferase, gfp or other fluorescent genes also suitable. These markers can be found in mutants where these genes are not functional because they are for example, have been deleted using conventional methods are. Furthermore, markers which encode a nucleic acid can encoding a selectable marker into a host cell the same vector as the one that PSE encoded, or can be on a separate vector to be brought. Cells with the introduced nucleic acid have been stably transfected, for example by Drug selection can be identified (e.g. survival Cells that have integrated the selectable marker, where die against the other cells).

To produce a homologously recombined microorganism made a vector that has at least a portion of a PSE Contains gene in which a deletion, addition or substitution has been introduced to change the PSE gene, z. B. functionally disrupt. This PSE gene is preferred as a Physcomitrella, Phytophthora, Crypthecodinium or Thraustochytrium PSE gene, however, it can be a homologue or Analogue from other organisms, even from a mammalian fungal or source of insects. With a preferred off the vector is designed so that the endogenous PSE Gene is functionally disrupted in homologous recombination (i.e. no longer encodes a functional protein, also as Knockout vector). Alternatively, the vector can be like this be designed that the endogenous PSE gene with homologous recombi nation is mutated or otherwise changed, but still encodes a functional protein (e.g., upstream located regulatory area so changed that the expression of the endogenous PSE is changed). For generation A point mutation via homologous recombination can also be used as  Chimeraplasty known DNA-RNA hybrids can be used that are made from Cole-Strauss et al., 1999, Nucleic Acids Research 27 (5): 1323-1330 and Kmiec, Gene therapy, 19999, American Scientist, 87 (3): 240-247 are known.

In the vector for homologous recombination is the changed Ab cut the PSE gene at its 5 'and 3' ends from additional ones Flanked nucleic acid of the PSE gene so that homologous recombi nation between the exogenous PSE gene that precedes the vector and an endogenous PSE gene in a microorganism or a plant is possible. The additional flanking PSE Nucleic acid is essential for successful homologous recombination the endogenous gene is long enough. Usually are in vector several hundred base pairs up to kilobases flanking DNA (see above) probably at the 5 'and 3' ends) [a description of For homologous recombination vectors, see e.g. B. in Thomas, K.R., and Capecchi, M.R. (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 in a microorganism or a plant cell (e.g. using poly ethylene glycol-mediated DNA) and cells in which the introduced PSE gene homologous with the endogenous PSE gene recombined are known in the art using Techniques selected.

In another embodiment, recombinant organisms, like microorganisms of plants that are made from selected systems that contain regulated expression allow the introduced gene. The inclusion of a PSE gene in a vector, being under the control of the lac operon brought, enables z. B. the expression of the PSE gene only in Presence of IPTG. These regulation systems are in the field known.

A host cell according to 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 PSE. In plants, an alternative procedure can also be followed direct transfer of DNA into developing flowers Electroporation or gene transfer using Agrobacterium be applied. The invention therefore also provides methods for the production of PSEs using the inventive Host cells ready. In one embodiment, the ver drive the cultivation of the host cell according to the invention (in the a recombinant expression vector encoding a PSE has been brought in, or a gene has been introduced into their genome encoding a wild-type or altered PSE) in  a suitable medium until the PSE has been produced. The In another embodiment, the method comprises isolating the PSEs from the medium or the host cell.

Host cells, in principle for receiving the invention Nucleic acid, the new gene product according to the invention or of the vector according to the invention are suitable, are all pro caryotic or eukaryotic organisms. The more advantageous host organisms used wisely are organisms such as bacteria, Mushrooms, yeasts, animal or plant cells. More beneficial Organisms are animals or preferably plants or parts of. The term "animal" is understood here to mean that humans are not included. Mushrooms, yeasts or plants are preferred used wisely, particularly preferably mushrooms or plants, whole particularly preferred plants, such as oil crops, the large ones Contain amounts of lipid compounds, such as oilseed rape, evening primrose, Castor, canola, peanut, flax, soy, safflower, sunflower, Borage, oil palm, coconut or plants such as corn, wheat, Rye, oats, triticale, rice, barley, cotton, cassava, Pepper, tagetes, solanaceae plants, such as potatoes, tobacco, Eggplant and tomato, Vicia species, pea, alfalfa, bush plants (Coffee, cocoa, tea), salix species, trees (oil plant, coconut) as well as perennial grasses and forage crops. Especially before plants according to the invention are oil-fruit plants, such as soybeans, Peanut, rapeseed, canola, castor, linseed, evening primrose, sunflower, Safflower, trees (oil palm, coconut).

In a particularly preferred aspect, the invention relates to a Plant cell containing the polynucleotide according to the invention or the includes vector according to the invention. Transgenes are further preferred Plants or plant tissues containing the plants according to the invention include cell. In another aspect, the present concerns Invention those parts of the plants according to the invention which one can harvest, and that material that is suitable for propagation the transgenic plants according to the invention and which either plant cells according to the invention, the fiction express appropriate nucleic acid, or contain cells that have a possess increased levels of the protein according to the invention. Erntebar are basically all parts of a plant, especially flowers, Pollen, fruits, seedlings, roots, leaves, seeds, tubers, Stems, etc. Propagation material includes z. B. seeds, fruits, Seedlings, tubers, cuts and rhizomes.  

D. Isolated PSE

Another aspect of the invention relates to isolated PSEs and biologically active parts of it. An "isolated" or purified "protein or a biologically active part thereof essentially free of cellular material when DNA Recombination techniques is produced, or by chemical pre stages or other chemicals if it chemically synthesizes becomes. The term "essentially free of cellular material" includes PSE preparations in which the protein is cellular Components of the cells in which it is natural or recombinant is produced, is separated. In one embodiment the expression "essentially free of cellular material" PSE- Preparations with less than about 30% (based on the dry weight) non-PSE (here also as "contaminating protein" designated), more preferably less than about 20% non-PSE, even more preferably less than about 10% non-PSE and am most preferably less than about 5% non-PSE. If the PSE or a biologically active part thereof is produced recombinantly , he / she is also essentially free of culture medium, d. H. the culture medium makes up less than about 20%, more preferably less than about 10% and most preferred accounts for less than about 5% of the volume of the protein preparation out. The term "essentially free of chemical precursors or other chemicals "includes PSE preparations in which the protein from chemical precursors or other chemicals is separated, which are involved in the synthesis of the protein. In one embodiment, the term "essentially includes free of chemical precursors or other chemicals "PSE- Preparations with less than about 30% (based on the dry weight) chemical precursors or non-PSE chemicals, stronger preferably less than about 20% chemical precursors or non- PSE chemicals, more preferably less than about 10% chemical precursors or non-PSE chemicals and most potent preferably less than about 5% chemical precursors or non- PSE chemicals. In preferred embodiments, iso gated proteins or biologically active parts thereof no ver impure proteins from the same organism from which the PSE comes from. In the case of the protein of the invention, the Sequence shown in SEQ ID NO: 10 or by one Gene encoding SEQ ID NO: 9 is encoded, however notice that the sequence starts with two mead. This can do that result in the translation of a corresponding coding Nucleic acid sequence two derivatives of the protein according to the invention be expressed starting at the first or second mead Expression ratio between the two derivatives may vary Expression type or host organism vary between 0 and 1.  

This includes PSE, the two named derivatives or only contains one of the derivatives. The two derivatives can be found under different activities, locations, half-lives, Have regulatory mechanisms etc. These proteins are usually by recombinant expression for example Physco mitrella, Phytophthora, Crypthecodinium or Thraustochytrium PSE in plants such as Physcomitrella patens or the above. or micro organisms, for example bacteria, such as E. coli, Bacillus subtilis, C. glutamicum, mushrooms such as Mortierella, yeast such as Saccharomyces, or ciliates such as Colpidium or algae such as Phaeo dactylum produced.

An isolated PSE according to the invention or a part thereof can also participate in the metabolism of compounds necessary for the construction of cell membranes in Physcomitrella, Phytophthora, Crypthecodinium or Thrausto chytrium or in the transport of molecules across these membranes. In preferred embodiments, the protein or the part thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 is that the protein or part thereof maintains the ability to participate in the metabolism of compounds necessary for the construction of cell membranes in Physcomitrella, Phytophthora, Crypthecodinium or Thrausto chytrium or in the transport of molecules across these membranes. The part of the protein is preferably a biologically active part, as described here. In a further preferred embodiment, a PSE according to the invention has one of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. In a further preferred embodiment, the PSE has an amino acid sequence which is encoded by a nucleotide sequence which, for example under stringent conditions, is linked to a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 , SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 hybridized. In yet another preferred embodiment, the PSE 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 even more preferably at least about 96%, 97%, 98%, 99% or even more homologous to one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO : 8, SEQ ID NO: 10 and SEQ ID NO: 12. The preferred PSE according to the invention preferably also has at least one of the PSE activities described here. For example, a preferred PSE according to the invention comprises an amino acid sequence which is encoded by a nucleotide sequence which, for example under stringent conditions, is attached to a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 hybridize and participate in the metabolism of compounds necessary for the construction of cell membranes in Physcomitrella, Phytophthora, Crypthecodinium or Thraustochytrium or in the transport of molecules over these membranes or one or more polyunsaturated Can extend fatty acids with at least two double bonds and a chain length of C ₁₆ or C ₁₈ .

In other embodiments, the PSE is essentially homo log to an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and retains the functional activity of one of the protein Sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 at, their amino acid sequence below however differs due to natural variation or Mutagenesis as described in detail in subsection I above. In a further embodiment, the PSE is consequently on 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 the SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 and is at least one of those described here PSE activities. In another embodiment The invention relates to a complete Physcomitrella, Phytophthora, Crypthecodinium or Thraustochytrium protein, which is essentially homologous to a complete amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.

Biologically active parts of a PSE include peptides comprising Amino acid sequences derived from the amino acid sequence of a PSE are derived, e.g. B. one in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 Amino acid sequence or the amino acid sequence of a protein that is homologous to a PSE that has fewer amino acids than that Have full length PSE or the full length protein that results in a PSE is homologous, and at least one activity of a PSE occurs point. Usually include biologically active parts (peptides, z. B. peptides, for example 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids are long) a domain or a motif with at least one activity of a PSE. moreover can have other biologically active parts in which other areas of the protein are deleted by recombinant techniques and related to one or more of those described herein  Activities are examined. The biologically active parts of a PSEs preferably include one or more selected ones Domains / motifs or parts thereof with biological activity.

Such domains and motifs can be z. B. using computer-aided methods.

In the sequences according to the invention, z. B. so-called KK- Methods found.

Kermode 1996, Critical Reviews in Plant Sciences 15 (4): 285-423 describes KK motifs, a double lysine, primarily as KKXX or K X K XXX motif is found and recycling from ER to Golgi apparatus and thus the residence time of the protein and its Enzyme activity at a specific location, especially the ER affected.

Double lysine motifs were e.g. B. also found in Δ12-desaturases (Arondel et al. 1992, Science 258: 1353) and are also present in elongases according to the invention. In particular, such motifs are described as being C-terminally localizable. In the sequences according to the invention there is a striking accumulation of lysines at the C-terminus.
Moss Elongase PSE1: C-terminus KQKGAKTE
SEQ ID NO 2: KTKKA
SEQ ID NO 4: KKSTPAAKKTN
SEQ ID NO 6: KHLK

This could be a possible genetic variation.

There are lys leftovers that targeting and addressing Affect localization on or in the ER. Masking this Sequence, modification or spatial modification, the proximity to the End of the C-terminus e.g. B. by fusion with GFP "green fluorescent protein "can be used to influence the compartmentalization become.

PSEs are preferably made by recombinant DNA techniques manufactured. For example, one encoding the protein Nucleic acid molecule into an expression vector (as above described) cloned, the expression vector is in a Host cell (as described above), and the PSE is expressed in the host cell. The PSE can then through a suitable cleaning scheme using standard protein cleaning techniques are isolated from the cells. alternative for recombinant expression, a PSE, a polypeptide,  or peptide chemically using standard peptide synthesis techniques be synthesized. In addition, native PSE can be derived from cells (e.g. endothelial cells) e.g. B. using an anti-PSE Antibody can be isolated by standard techniques can be produced, wherein a PSE or a fragment of it is used.

The invention also provides chimeric PSE proteins or PSE Fusion proteins ready. As used herein, includes a "Chimeric PSE Protein" or "PSE Fusion Protein" a PSE poly peptide that is operably linked to a non-PSE polypeptide is. A "PSE polypeptide" refers to a polypeptide with one Amino acid sequence corresponding to a PSE whereas a "non-PSE polypeptide" means a polypeptide with an amino acid sequence concerns that corresponds to a protein that essentially is not homologous to the PSE, e.g. B. a protein that differs from the PSE differs and from the same or a different one Organism. The term is intended within the fusion protein "Functionally linked" means that the PSE polypeptide and the non-PSE polypeptide are fused together so that both sequences the predicted, the used sequence fulfill the written function. The non-PSE polypeptide can be on fused the N-terminus or the C-terminus of the PSE polypeptide his. In one embodiment, the fusion protein is included play a GST-PSE fusion protein in which the PSE sequences the C-terminus of the GST sequences are fused. This fusion Proteins can facilitate the purification of the recombinant PSEs. In another embodiment, the fusion protein is one PSE, which has a heterologous signal sequence at its N-terminus has. In certain host cells (e.g. mammalian host cells) the expression and / or secretion of a PSE through use a heterologous signal sequence can be increased.

A chimeric PSE protein or PSE fusion according to the invention protein is produced by standard recombinant DNA techniques provides. For example, DNA fragments are different Encode polypeptide sequences according to conventional techniques ligated together in the reading frame, 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 links, and enzymatic ligation be used. In a further embodiment, this can Fusion gene by conventional techniques, including DNA Automatic synthesizers to be synthesized. Alternatively, one PCR amplification of gene fragments using anchors primers are performed that have complementary overhangs between  successive gene fragments that subsequently generate can 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). Furthermore, there are many expression vectors commercially available that already has a fusion unit (e.g. a GST polypeptide). A nucleic acid encoding PSE can be cloned into such an expression vector so that the Fusion unit is linked in frame with the PSE protein.

Homologs of PSE can by mutagenesis, e.g. B. by specific Point mutation or shortening of the PSE. The The term "homologue" as used here refers to a variant Form of PSE that acts as an agonist or antagonist of PSE activity acts. An PSE agonist can be essentially the same Activity like that or part of biological activities maintain the PSE. An PSE antagonist can have one or several activities of the naturally occurring form of PSE for example competitive binding to a downstream or upstream element in the metabolic cascade for the cell membrane components that comprise the PSE, or by binding to a PSE, which is the transport of compounds across cell membranes mediates, inhibit, whereby the translocation is inhibited.

In an alternative embodiment, homologs of the PSE by screening combinatorial banks of mutants, e.g. B. Shortening mutants, the PSE in terms of PSE agonists or Antagonist activity can be identified. With an off a varied bank of PSE variants is implemented combinatorial mutagenesis at the nucleic acid level and encoded by a varied gene bank. A varied bank of PSE variants can e.g. B. by enzymatic ligation Mixtures of synthetic oligonucleotides in gene sequences be made so that a degenerate sentence of potential PSE sequences as individual polypeptides or alternatively as Set of larger fusion proteins (e.g. for the phage display), that contain this set of PSE sequences. There are a variety of processes used to manufacture Banks of potential PSE homologues from a degenerate Oligonucleotide sequence can be used. The chemical Synthesis of a degenerate gene sequence can be done in a DNA Synthesized machines performed and then the synthetic gene be ligated into a suitable expression vector. The Ver using a degenerate set of genes enables the Provision of all sequences that match the desired sentence encode potential PSE sequences in a mixture. method for the synthesis of degenerate oligonucleotides are in the art  known [see e.g. B. Narang, S.A. (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].

In addition, banks of PSE fragments can be used to produce a varied population of PSE fragments for screening and used for the subsequent selection of homologs of a PSE become. In one embodiment, a bank of fragments the coding sequence by treating a double stranded PCR fragment of a coding PSE sequence with a nuclease under conditions where double strand breaks only about one times per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded DNA, what sense / antisense pairs of different products with Double-strand breaks can include single-strand removal Sections from newly formed duplexes by treatment with S1 nuclease and ligating the resulting fragment library into one Expression vector can be generated. With this procedure a Expression bank can be derived, the N-terminal, C-terminal and encoded PSE internal fragments of various sizes.

There are several techniques for gene screening in the art products in combinatorial banks caused by point mutations or foreshortening, and for sighting cDNA libraries for gene products with a selected property known. These techniques can be seen in the quick sighting of the Adapt gene banks that are generated by combinatorial mutagenesis of PSE Homologues have been generated. The most used Techniques for screening large gene banks that are analyzed using high throughput can usually include cloning the gene bank into replicable expression vectors, Transform suitable cells with the resulting one Vector library and expression of the combinatorial genes below Conditions under which the detection of the desired activity the isolation of the vector encoding the gene, its product was proven, relieved. Recursive ensemble mutagenesis (REM), a new technique that makes the frequency more functional Mutants increased in banks, in combination with the Screening tests used to identify PSE homologues [Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. United States 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331]. Combinations of the above methods are used.

Another known technique for changing catalytic Properties of enzymes or their coding genes the so-called "gene shuffling" (see, for example, WO 97/20078 or  WO 98/13487), which represents a combination of gene fragments, this new combination is additionally due to faulty Polymerase chain reactions can be varied and thus creates a high sequence diversity to be tested. ahead setting for the use of such an approach is, however a suitable screening system for the created genetic diversity to check for functionality.

In particular for the screening of elongase activities is a Screening procedure Prerequisite, the PUFA-dependent enzyme activity (s) recorded. Concerning. Elongase activities with specificity for PUFAs one can in Mucor species, which by well-known Trans Formation process with desired gene constructs transform are the toxicity of arachidonic acid in the presence of a toxic metabolites (here: salicylic acid or salicylic acid derivatives) (Eroshin et al., Mikrobiologiya, Vol. 65, No. 1, 1996, pages 31-36), for a growth-based first sighting perform. Resulting clones can then be analyzed their lipid ingredients using gas chromatography and masses be subjected to spectroscopy to determine educts and products in Art and capture quantity.

In another embodiment, cell-based tests can be performed to analyze a varied PSE bank using other methods known in the art can be used.

In another embodiment, the present relates Invention an antibody that is specific to the polypeptide of present invention or parts, e.g. B. epitopes, such Protein binds. The antibody of the invention can be used to identify other elongases, especially PSEs and isolate. These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies and Frag ment of these antibodies, such as. B. Fab, Fv or scFV fragments etc., be. Monoclonal antibodies can e.g. B. by method, as originally described in Köhler and Milstein, Nature 256 (1975), 485, and Galfrö, Meth. Enzymol. 73 (1981) become. Antibodies and fragments thereof can also be made are after z. B. in Harlow & Lane, "Antibodies, a Laboratory Manual ", CSH, Press, Cold Spring Harbor, 1988. These antibodies can be used to e.g. B. the proteins of the invention to precipitate and localize or to synthesize this Proteins e.g. B. to check in recombinant organisms and for the identification of compounds with the Invention interact according proteins. In many cases the bond is of antibodies with antigens equivalent to the binding of others Ligands and anti-ligands.  

Furthermore, the present invention relates to a method for identifying an agonist or antagonist of elongases, in particular PSEs, comprising

• a) contacting the cells containing the polypeptide before express lying invention with a candidate substance;
• b) testing PSE activity;
• c) comparing the PSE activity with a standard activity in Absence of the candidate substance, with an increase in PSE Activity above the standard indicates that the candidate substance an agonist and a decrease in PSE activity indicates that the candidate substance is an antagonist.

The candidate substance mentioned can be a chemically synthesized or microbiologically produced substance and z. B. in cell extracts of z. B. plants, animals or microorganisms occur. 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 PSEs. The reaction mixture can be a cell-free extract or can comprise a cell or cell culture. Suitable methods are known to the person skilled in the art and are described, for. As generally described in Alberts, Molecular Biology the cell, ^3rd Edition (1994), for example. B. Chapter 17. B. added to the reaction mixture or the culture medium or injected into the cells or sprayed onto a plant.

If a sample, the one according to the method of the invention active substance has been identified, then it is ent neither possible to get the fabric directly from the original sample isolate or you can divide the sample into different groups, z. B. if they consist of a variety of different components consists of the number of different substances per sample to reduce and then the inventive method with a to repeat such "sub-sample" of the original sample. Depending on the complexity of the sample, the above can be described steps are repeated several times, preferably until the only identified sample according to the method of the invention a small number of substances or only one substance includes. Preferably that according to the method of the invention identified substance or derivative thereof further formulated, that he is for use in plant breeding or plants cell or tissue culture is suitable.  

The substances tested according to the method of the invention and identified, can be: expression libraries, z. B. cDNA expression libraries, peptides, proteins, nucleic acid ren, antibodies, small organic substances, hormones, PNAs or the like (Milner, Nature Medicin 1 (1995), 879-880; Hupp, Cell. 1995, 83: 237-245; Gibbs, Cell. 79 (1994), 193-198 and therein zi references). These substances can also be functional derivatives or analogue of the known inhibitors or activators. Process for the preparation of chemical derivatives or analogue are known to the person skilled in the art. The derivatives and analogues mentioned can be tested according to state-of-the-art methods the. Furthermore, computer aided design or peptidomime tics used to prepare suitable derivatives and analogue become. The cell or tissue responsible for the fiction method can be used, it is preferably one host cell according to the invention, plant cell or a plant tissue, as described in the above embodiments.

Accordingly, the present invention also relates to a substance identifi according to the above inventive method was adorned. The substance is e.g. B. a homologue of the invention PSE. Homologs of the PSEs can by mutagenesis, e.g. B. by point mutation or deletion of the PSE. Used here The term "homolog" is used as a variant form of the PSEs that acts as an agonist or antagonist for the activity of the PSEs. On Agonist can be essentially the same or part of the bio logical activity of the PSEs. An antagonist of the PSEs can one or more activities of the naturally occurring forms of Inhibit PSEs, e.g. B. competitive to a downstream or upstream located member of the fatty acid synthesis pathways that include, bind, or bind to PSEs and thereby the Reduce or inhibit activity.

Accordingly, the present invention also relates to an antibody or a fragment thereof as described herein, the inhibits the activity of the PSEs according to the invention.

In one aspect, the present invention relates to an anti body that specifically described above according to the invention Detects or binds agonists or antagonists.

Another aspect relates to a composition that the anti body identified by the method according to the invention Stop or includes the antisense molecule.  

E. Uses and methods according to the invention

The nucleic acid molecules, proteins, protein described here homologous, fusion proteins, antibodies, primers, vectors and Host cells can be used in one or more of the following ver driving: identification Physcomitrella patens, Crypthecodinium, Phytophthora infestans or Thraustochytrium and related organisms, mapping the genomes of organisms, those with Physcomitrella, Phytophthora, Crypthecodinium or Thraustochytrium related, identification and localization from Physcomitrella, Phytophthora, Crypthecodinium or Thraustochytrium sequences of interest, evolution studies, Be tuning PSE protein areas necessary for function are modulating a PSE activity; Modulation of the fabric alternating one or more cell membrane components; modulation the transmembrane transport of one or more compounds and Modulation of the cellular production of a desired ver bond, like a fine chemical. The PSE according to the invention Nucleic acid molecules have a variety of uses. she can first be used to identify an organism as Physco mitrella, Phytophthora, Crypthecodinium or Thraustochytrium or used as a close relative of it. You can also to identify the presence of Physcomitrella, Crypthe codinium, Phytophthora or Thraustochytrium or a relative ten of them used in a mixed population of microorganisms become. The invention provides the nucleic acid sequences one Series of Physcomitrella, Phytophthora, or Crypthecodinium or Thraustochytrium genes ready; by probing the extracted genomic DNA of a culture of a uniform or mixed Population of microorganisms under stringent conditions a probe covering an area of a Physcomitrella, Crypthe codinium, Phytophthora or Thraustochytrium gene or of Parts of it spanned, which is unique to this organism one can determine whether this organism is present. physco mitrella, Crypthecodinium, Phytophthora or Thraustochytrium themselves become polyunsaturated for commercial production Acids used. The nucleic acids according to the invention are suitable in addition to PUFA production in other organisms especially if the resultant PUFAs are to be achieved should also be incorporated into the triacylglycerol fraction.

Furthermore, the nucleic acid and protein according to the invention molecules serve as markers for specific areas of the genome. These are not only for mapping the genome, but also for functional studies of Physcomitrella, Phytophthora, Crypthe codinium or Thraustochytrium proteins suitable. Identi Specification of the genome area to which a specific DNA-binding  Protein from Physcomitrella, Crypthecodinium, or Phytophthora Thraustochytrium binds, could the Physcomitrella, Crypthe Codinium, Phytophthora or Thraustochytrium genome for the accessory be cleaved and the fragments with the DNA binding Protein to be incubated. Those who bind the protein can additionally with the nucleic acid of the invention molecules, preferably with easily detectable markings, to be probed; the binding of such a nucleic acid molecule to the genome fragment enables the fragment to be located on the genome map of Physcomitrella, Phytophthora, Crypthe codinium or Thraustochytrium and relieved if this more sometimes with different enzymes, a quick one Determination of the nucleic acid sequence to which the protein binds. The nucleic acid molecules according to the invention can also consist of be sufficiently homologous to the sequences of related species that these nucleic acid molecules as markers for the construction of a genomic map can be used in related fungi or algae.

The PSE nucleic acid molecules according to the invention are suitable also for evolution and protein structure studies. The Metabolism and transport processes in which the fiction Appropriate molecules are involved by many prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules according to the invention with those which encode similar enzymes from other organisms Evolution degree of kinship of the organisms can be determined. Accordingly, such a comparison enables the determination which sequence areas are conserved and which are not what be helpful in determining areas of the protein can, which are essential for the enzyme function. This type of Determination is valuable and valuable for protein engineering studies can give an indication of how much mutagenesis the protein can tolerate without losing its function.

The manipulation of the PSE nucleic acid molecules according to the invention can be used to produce PSEs with functional differences lead the wild type PSEs. The efficiency or activity of this Proteins can be improved, they can be in larger numbers than usually present in the cell, or its efficiency or activity may be reduced. Improved efficiency or For example, activity means that the enzyme has a higher one Selectivity and / or activity, preferably at least one 10% higher, particularly preferably an at least 20% higher Activity, most preferably at least 30% higher Has activity than the original enzyme.  

There are a number of mechanisms through which change takes place the yield, production and / or a PSE according to the invention Efficiency of the production of a fine chemical, which a contains such modified protein, can directly influence. The extraction of fine chemical compounds from cultures of ciliates, algae or mushrooms on a large scale is signi savory improves when the cell makes the desired connections secreted because these compounds from the culture medium (in the opposite set for extraction from the mass of grown cells) easily can be cleaned. Otherwise the cleaning can be done improve when the cell connects in vivo in a specialty compartment with a kind of concentration mechanism stores. For plants that express PSEs, a ge increased transport for better distribution within the Plant tissue and organs. By enlarging the Number or activity of transporter molecules, which fine exporting chemicals from the cell, it may be possible Amount of fine chemical produced in the extracellular Medium is present to increase, making harvesting and cleaning or in plants a more efficient distribution can be facilitated. to efficient overproduction of one or more fine chemicals are increased amounts of cofactors, precursors and Interconnections required for the appropriate biosynthetic pathways Lich. By increasing the number and / or activity of Transporter proteins involved in the import of nutrients such as coal Substance sources (i.e. sugars), nitrogen sources (i.e. amino acids, Ammonium salts), phosphate and sulfur, can be involved the production of a fine chemical due to disposal all restrictions on the supply of nutrients in biosynthesis improve process. Fatty acids, such as PUFAs, and lipids, the PUFAs contain, are themselves desirable fine chemicals; by Optimize activity or increase the number of one or of several PSEs according to the invention which are involved in the biosynthesis of these Connections are involved, or by disrupting the activity one or more PSEs involved in breaking down these connections are involved, you can thus the yield, production and / or Efficiency of the production of fatty acid and lipid molecules in Ciliates, algae, plants, mushrooms, yeasts or other micro increase organisms.

The manipulation of one or more PSE genes according to the invention can also lead to PSEs with changed activities, which the production of one or more desired fine chemicals from algae, plants, ciliates or fungi indirectly. The normal biochemical metabolic processes lead e.g. B. for Production of a variety of waste products (e.g. hydrogen peroxide and other reactive oxygen species) that affect these metabolic processes  can actively disrupt [z. B. nitrides peroxynitrite famously known tyrosine side chains, whereby some enzymes with Tyrosine to be inactivated in the active center, Groves, J. T. (1999) Curr. Opin. Chem. Biol. 3 (2); 226-235]. This waste products are usually eliminated, but those for cells used for large-scale fermentative production are used for the overproduction of one or more fine chemicals optimized and can therefore produce more waste products than common for a wild type cell. By optimizing the activity one or more PSEs according to the invention which are involved in the export of Waste molecules are involved, the viability of the Improve cell and have an efficient metabolic activity get right. The presence of high intracellular amounts The desired fine chemical can actually be used for the cell be toxic so that by increasing the ability of the cell cell viability for the secretion of these compounds can improve.

The PSEs according to the invention can also be manipulated in such a way that the relative amounts of different lipid and fatty acid molecules to be changed. This can have a critical impact have the lipid composition of the cell membrane. Because every lipid has different physical properties, the Change in the lipid composition of a membrane the membrane change fluidity significantly. Changes in membrane fluidity can affect the transport of molecules across the membrane, which, as explained above, is the export of waste products or the fine chemical produced or the import necessary Can modify nutrients. This changes the membrane Fluidity can also be critical to cell integrity influence; Cells with comparatively weaker membranes are more susceptible to abiotic and biotic stress conditions that can damage or kill the cell. By manipulating PSEs involved in the production of fatty acids and lipids for membrane construction are involved, so that the resulting membrane has a membrane composition that is suitable for in the cultures used to produce fine chemicals prevailing environmental conditions are more receptive, should a larger proportion of the cells survive and ver multiply. Larger amounts of producing cells should appear in larger yields, higher production or efficiency of Manipulate the production of fine chemicals from culture.

The above mutagenesis strategies for PSEs leading to should lead to increased yields of a fine chemical not be restrictive; Variations on these strategies are that Easily seen by a specialist. Using these mechanisms  and with the help of the mechanisms disclosed here, the Nucleic acid and protein molecules according to the invention for generation of algae, ciliates, plants, animals, fungi or other micro organisms such as C. glutamicum can be used, the mutated Express PSE nucleic acid and protein molecules so that the Yield, production and / or efficiency of production of a ge desired connection is improved. This desired connection can be any natural product of algae, ciliates, Plants, animals, fungi or bacteria, which is the end products of biosynthetic pathways and intermediates, of course Coming metabolic pathways includes, as well as molecules in the substance change of these cells does not occur naturally, but that of the cells of the invention are produced.

A further embodiment according to the invention is a method for producing PUFAs, the method comprising growing an organism which contains a nucleic acid according to the invention, a gene construct according to the invention or a vector according to the invention which code for a polypeptide which is C ₁₆ -, C ₁₈ - or C ₂₀ fatty acids with at least two double bonds in the fatty acid molecule 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 containing lipids, phospholipids, sphingolipids, glycolipids, triacylglycerols and / or free fatty acids with a higher PUFA content can be isolated from this solvent. The free fatty acids with a higher PUFA content can be isolated by basic or acidic hydrolysis of the lipids, phospholipids, sphingolipids, glycolipids, triacylglycerols. A higher PUFA content 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 coding for the elongase according to the invention.

The PUFAs produced by this process are preferably C ₁₈ , C ₂₀ or C ₂₂ fatty acid molecules with at least two double bonds in the fatty acid molecule, preferably three, four, five or six double bonds, particularly preferably three or five double bonds. These C ₁₈ , C ₂₀ or C ₂₂ fatty acid molecules can be isolated from the organism in the form of an oil, lipid or a free fatty acid. Suitable organisms are, for example, those mentioned above. Preferred organisms are microorganisms, animals or plants, particularly preferably plants or algae, very particularly preferably transgenic plants.

An embodiment of the invention are oils, lipids or fat acids or fractions thereof by the above described Processes have been produced, particularly preferably oil, lipid or a fatty acid composition comprising PUFAs and from originate from transgenic plants.

One embodiment of the invention is oils, lipids or fat acids produced by the process according to the invention were. Further embodiments of the invention are oil, lipid or fatty acid compositions that are manufactured according to the PUFAs Contain methods of the invention and of transgenic plants originate, which a nucleic acid according to the invention, a gene construct or contain vector.

Another embodiment of the invention is the use of the oil, lipids or fatty acid composition in feed agents, foodstuffs, cosmetics or pharmaceuticals.

In another embodiment, the present relates Invention a kit comprising the nucleic acid of the invention, the gene construct according to the invention, the amino according to the invention acid sequence, the antisense nucleic acid molecule according to the invention, the antibody and / or composition according to the invention, a Antagonists or agonists, the ver drive was produced, and / or oils, lipids according to the invention and / or fatty acids or a fraction thereof. It can also do that Kit the host cells, organisms, plants according to the invention or parts thereof, harvestable parts of the plants according to the invention or propagation material or also the invention Antagonists or agonists include. The components of the kit the present invention can be in suitable containers, at packaged for example with or in buffers or other solutions his. One or more of the components mentioned can be in one and packed in the same container. Additionally or alternatively can one or more of the components mentioned z. B. on one solid surface to be adsorbed, e.g. B. nitrocellulose filter, Glass plates, chips, nylon membranes or microtiter plates. The Kit can be used for any of the methods and methods described herein leadership forms are used, e.g. B. 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 using the Kits for one of the applications mentioned are included.  

This invention is further illustrated by the examples below illustrated, which are not to be taken as limiting should. The content of all in this patent application cited references, patent applications, patents and published patent application 61932 00070 552 001000280000000200012000285916182100040 0002010063387 00004 61813ungen is incorporated herein by reference added.

Examples example 1 General procedures a) General cloning procedures

Cloning methods, such as restriction cleavages, Agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids on nitrocellulose and nylon membranes, Ver binding of DNA fragments, transformation of Escherichia coli and yeast cells, cultivation of bacteria and sequence analysis recombinant DNA were performed as described in Sambrook et al. [(1989), Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6] or Kaiser, Michaelis and Mitchell [(1994), Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press: ISBN 0-87969-451-3]. The transformation and cultivation algae such as Chlorella or Phaeodactylum are leads as described by El-Sheekh [(1999), Biologia Plantarum 42: 209-216] or Apt et al. [(1996) Molecular and General Genetics 252 (5): 872-9].

b) chemicals

The chemicals used were, unless otherwise stated in the text, in p. A. quality from Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Solutions were prepared from a Milli-Q water system water purification plant (Millipore, Eschborn) using pure pyrogen-free water, hereinafter referred to as H ₂ O. Restriction endonucleases, DNA-modifying enzymes and molecular biological kits were obtained from the companies AGS (Heidelberg), Amersham (Braunschweig), Biometra (Göttingen), Boehringer (Mannheim), Genomed (Bad Oeynhausen), New England Biolabs (Schwalbach / Taunus), Novagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden) and Stratagene (Amsterdam, Netherlands). Unless otherwise stated, they were used according to the manufacturer's instructions.

c) Cell material

A Thraustochytrium strain was used for these studies who uses the American Type Culture Collection (ATCC-Samm lung) with the strain number ATCC26185 (Thraustochytrium) is, or in the case of Crypthecodinium from Provasoli-Guillard National Center for Culture of Marine Phytoplancton ((CCMP) West Boothbay Harbor, ME, USA) with the regular culture no. CCMP316 is accessible. The algae were kept at 25 degrees Celsius in the dark cultivated in glass tubes that were aerated from below with air. Alternatively, Thraustochytrium was grown as described in detail by Singh & Ward (1997, Advances in Microbiology, 45, 271-311).

The f / 2 culture medium with 10% organic medium according to Guillard, RRL was used as culture medium for Crypthecodinium [1975; Culture of phytoplankton for feeding marine invertebrates. In: Smith, WL and Chanley, MH (Eds.) Culture of marine Invertebrate animals, NY Plenum Press, pp. 29-60.]: It contains
995.5 ml sea water (artificial)
1 ml NaNO ₃ (75 g / l), 1 ml NaH ₂ PO ₄ (5 g / l), 1 ml Trace metal solution, 1 ml Tris / Cl pH 8.0, 0.5 ml f / 2 vitamin solution
Trace metal solution: Na ₂ EDTA (4.36 g / l), FeCl ₃ (3.15 g / l),
Primary Trace metals: CuSO ₄ (10 g / l), ZnSO ₄ (22 g / l), CoCl ₂ (10 g / l), MnCl ₂ (18 g / l), NaMoO4 (6.3 g / l)
f / 2 vitamin solution: Biotin: 10 mg / l, thiamine 200 mg / l, Vit B12 0.1 mg / l
org medium: Na acetate (1 g / l), glucose (6 g / l), Na succinate (3 g / l), bacto-tryptone (4 g / l), yeast extract (2 g / l )

d) moss material (= plant material)

Plants of the Physcomitrella species were used for this study patens (Hedw.) B. S. G. from the collection of the Department for Genetic studies from the University of Hamburg used. They come from strain 16/14, which was developed by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire (England) collected and used by angels (1968, Am J Bot 55, 438-446) was subcultured from a spore. The plants were proliferated by means of spores and by means of regeneration of the gametophytes. The Protonema developed from the haploid spore to chloroplast-rich chloronema and low-chloroplast caulonema, which results after about 12 days Buds formed. These grew into gametophores with antheridia and archegonia. The diploid emerged after fertilization  Sporophyte with a short Seta and spore capsule, in which the Meio spores mature.

e) Plant cultivation

The cultivation took place in an air-conditioned chamber at one Air temperature of 25 ° C and a light intensity of 55 micro mol-1m-2 (white light; Phillips TL 65 W / 25 fluorescent tube) and a light / dark change of 16/8 hours. The moss was removed neither in liquid culture using Knop medium Reski and Abel (1985, Planta 165, 354-358) modified or on solid button medium using 1% oxoid agar (Unipath, Basingstoke, England).

The protonemata used for RNA and DNA isolation were described in aerated liquid cultures. The protonemata were all Crushed for 9 days and transferred to fresh culture medium.

f) Cultivation of Phytophthora infestans

First, a cDNA library from Phytophthora infestans was created provides. For this purpose the strain ATCC 48886 from the American Type Culture Collection Rockville, USA. deviant described by the cultivation protocol for the strain ATCC 48886 Phytophthora spores are washed with cold bidistilled water and sporulated for 6 hours in the refrigerator. After that the material was transferred to pea medium. For this purpose, 150 g Frozen peas (igloo, available from local supermarkets) in 1 liter of water is sterile autoclaved for 20 minutes. The material is in 100 ml flask at room temperature on a rotary shaker tightened (200 rpm). After two days, 2 flasks and in the next 4 days also filter 2 flasks each and ground in liquid nitrogen.

Example 2 Isolation of total DNA from plants and micro organisms such as Thraustochytrium and Crypthecodinium for hybridization experiments

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 mM Tris-HCl, pH 8.0; 1.4 M NaCl; 20mM EDTA.
N-lauryl sarcosine buffer: 10% (w / v) N-lauryl sarcosine; 100 mM Tris-HCl, pH 8.0; 20mM EDTA.

The plant material or Crypthecodinium or Thraustochytrium cell material was triturated under liquid nitrogen in a mortar so that a fine powder was obtained and transferred to 2 ml Eppendorf tubes. The frozen plant material was then overlaid with a layer of 1 ml decomposition buffer (1 ml CTAB buffer, 100 ml N-lauryl sarcosine buffer, 20 ml β-mercaptoethanol and 10 ml Proteinase K solution, 10 mg / ml) and under for one hour incubated continuous shaking at 60 ° C. The homogenate obtained was divided into two Eppendorf tubes (2 ml) and extracted twice by shaking with the same volume of chloroform / isoamyl alcohol (24: 1). For phase separation, centrifugation was carried out at 8000 × g and RT (= room temperature = ~ 23 ° C.) for 15 min each. The DNA was then precipitated at -70 ° C for 30 minutes using ice cold isopropanol. The precipitated DNA was sedimented at 10,000 g for 30 min at 4 ° C. and resuspended in 180 ml TE buffer (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). For further purification, 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 taken up in 50 ml H ₂ O + RNAse (50 mg / ml final concentration). 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 plants and microorganisms (Crypthecodinium and Thraustochytrium)

The isolation of total RNA from plants such as flax and Rapeseed etc. is made according to one described by Logemann et al Method (1987, Anal. Biochem. 163, 21) isolated. Moss can the total RNA Protonema tissue according to the GTC method (Reski et al., 1994, Mol. Gen. Genet., 244: 352-359).

RNA isolation from Crypthecodinium and Thraustochytrium

Frozen algae samples (-70 ° C) are ground into fine powder in an ice-cold mortar under liquid nitrogen. 2 volumes of homogenization medium (12.024 g sorbitol, 40.0 ml 1 M 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 are made up to 200 ml with H ₂ O and the pH is adjusted to 8.5) and 4 volumes of phenol with 0.2% mercaptoethanol are mixed at 40-50 ° C with good mixing added to frozen cell powder. Then add 2 volumes of chloroform and stir vigorously for 15 min. It is centrifuged at 10,000 g for 10 min and the aqueous phase is extracted with phenol / chloroform (2 vol / 2 vol) and finally with chloroform.

The volume of the aqueous phase obtained is 1/20 vol 4 M Na acetate (pH 6) and 1 volume of isopropanol (ice cold) are added and the nucleic acids precipitate at -20 ° C above sea level. It is 30 min at 10000 g centrifuged and the supernatant aspirated. A wash follows step with 70% EtOH and centrifugation again. The sediment is in Tris-borate buffer (80 mM Tris-borate buffer, 10 mM EDTA, pH 7.0). Then the supernatant is mixed with 1/3 vol 8 M LiCl, mixed incubated at 4 ° C for 30 min. After centrifuging again the sediment is washed with 70% ethanol, centrifuged and the sediment is dissolved in RNAse-free water.

Poly (A) ⁺ RNA is isolated using Dyna Beads® (Dynal, Oslo, Finland) according to the instructions in the manufacturer's protocol.

After determining the RNA or poly (A) ⁺ RNA concentration, the RNA is precipitated by adding 1/10 volumes of 3 M sodium acetate, pH 4.6, and 2 volumes of ethanol and stored at -70 ° C.

For the analysis, 20 µg RNA are in a formaldehyde containing 1.5% agarose gel and separated on nylon membranes (Hybond, Amersham) convicted. Evidence of specific trans scripts are carried out as described in Amasino ((1986) Anal. Biochem. 152, 304)).

Isolation of total RNA and poly- (A) + RNA from Phytophthora infestans:
Total RNA was obtained using the RNeasy Plant Total RNA Kit (Qiagen, Hilden) and the RLT buffer contained therein, according to the manufacturer. The poly- (A) + RNA was isolated from the total RNA obtained using the Poly Attract mRNA Isolation System III from Promega (Heidelberg) according to the manufacturer's instructions.

Example 4 Construction of the cDNA bank

For the construction of the cDNA bank from Physcomitrella, Crypthe codinium or Thraustochytrium was the first strand synthesis under Use of mouse leukemia virus reverse transcriptase (Roche, Mannheim, Germany) and oligo-d (T) primers, the second strand synthesis  by incubation with DNA polymerase I, Klenow Enzyme and RNAse H cleavage at 12 ° C (2 hours), 16 ° C (1 hour) and 22 ° C (1h). The reaction was by incubation stopped at 65 ° C (10 min) and then transferred to ice. Double-stranded DNA molecules were created using T4 DNA polymerase (Roche, Mannheim) at 37 ° C (30 min) with smooth ends. The nucleotides were extracted by phenol / chloroform and Sephadex G50 centrifugation columns removed. EcoRI / XhoI adapters (Pharmacia, Freiburg, Germany) were using T4 DNA ligase (Roche, 12 ° C, overnight) ligated to the cDNA ends with XhoI trimmed and by incubation with polynucleotide kinase (Roche, 37 ° C, 30 min) phosphorylated. This mixture became the Subject to separation on a low-melting agarose gel. DNA Molecules over 300 base pairs were eluted from the gel, phenol extracted on Elutip-D columns (Schleicher and Schüll, Dassel, Germany) concentrated and ligated to vector arms and in lambda ZAPII phage or lambda ZAP express phage under Ver Use of the Gigapack Gold Kit (Stratagene, Amsterdam, Nieder land) packed, using manufacturer 's material and his instructions were followed.

Construction of a cDNA bank for Phytophthora infestans was carried out as described above.

Example 5 DNA sequencing and computer analysis

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-Elmer, Weiterstadt, Germany). The sequencing to mature, isolated clones was then carried out after the preparative plasmid extraction from cDNA banks via in vivo mass excision and retransformation of DH10B on agar plates (details on material and protocol from Stratagene, Amsterdam, the Netherlands). Plasmid DNA was obtained from overnight grown E. coli cultures grown in Luria broth with ampicillin (see Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6)) , prepared on a Qiagen DNA preparation robot (Qiagen, Hilden) according to the manufacturer's protocols. Sequencing primers with the following nucleotide sequences were used:

The sequences were generated using the standard software pa kets EST-MAX, commercially available from Bio-Max (Munich, Germany) is delivered, processed and annotated. By using Ver like algorithms and using a search sequence searched for homologous genes using the BLAST program (Alt Schul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs ", Nucleic Acids Res. 25: 3389-3402.). A sequence of Crypthecodinium and Thrausto chytrium with homologies to the search sequence of the moss long gases Physcomitrella patens were characterized in more detail.

Example 6a Identification of the Tc_PSE1 and Tc_PSE2 gene (Tc = Thraustochytrium) and the Cc_PSE1 and Cc_PSE2 gene (Cc = Crypthecodinium cohnii) by comparison with the Pp_PSE1 gene from Physcomitrella patens

The full length sequence of the moss longase Pp_PSE1 according to the invention (for the designation see also Table 2) was used for the sequence comparison in the TBLASTN search algorithm:

The complete nucleotide sequence of the moss longase Pp_PSE1 cDNA consists of about 1200 bp. It contains an open reading frame of 873 bp, the 290 amino acid with a calculated molecular mass coded from 33.4 Da. The protein sequence has only 38.5% Identity and 48.3% similarity to one, e.g. B. PSE1, Gen product of Saccharomyces cerevisiae, which is used to elongate Medium chain fatty acids required in yeast (Toke & Martin, 1996, Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae. Journal of Biological Chemistry 271, 18413-18422).

The EST sequence CC001042041R as well as TC002034029R and TC002014093R were initially considered as target genes due to weak homologies with the elongase from Physcomitrella patens (see Table 2), the PSE1 gene among other candidate genes. In FIG. 5, the result of the comparison of Pp_PSE1 peptide sequence is presented with the found sequence. It is part of the nucleic acid according to the invention from Seq ID NO: 3 (name: Tc_PSE1, own database number of the inventors TC002034029R). 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 3.

The sequencing of the complete cDNA fragment from clone TC002034029R resulted in a 693 base pair sequence starting with the first base in the open reading frame. The sequence codes for a polypeptide of 195 amino acids shown in Seq ID NO: 4 with a stop codon in base pair position translated from SEQ ID NO: 3 in base pair position 586-588. The clone TC002014093R contains an almost complete elongase polypeptide, as can be seen from the sequence comparison in FIG. 7. Dashes mean identical amino acids while colons and single points represent chemically exchangeable, ie chemically equivalent amino acids. The comparison was carried out with the BLOSUM62 exchange matrix for amino acids according to Henikoff & Henikoff ((1992) Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 89: 10915-10919). Parameters used: Gap Weight: 8; Average Match: 2,912, Length Weight: 2, Average Mismatch: -2,003.

Furthermore, a second est was identified by the sequence comparison. Shown in Fig. 6 is a comparison of the Pp_PSE1 peptide sequence with the found sequence. Although the homology is limited to a few amino acids under selected parameters, this relates to a highly conserved range of PUFA-specific elongases. The sequence of the complete cloned fragment was therefore determined.

Sequencing of the complete cDNA fragment from clone TC002014093R resulted in a 955 base pair sequence starting with first base in the open reading frame. This is according to the invention indicated with SEQ ID NO: 5. The sequence codes for a poly peptide of 297 amino acids with a stop codon in base pair position 892-894 according to the invention shown in SEQ ID NO: 6.

Using the sequence PpPSE1 the est CC001042041R from Crypthecodinium cohnii coding for the gene Cc_PSE1 identified. The isolated est CC001042041R, according to the invention represented as SEQ ID NO: 7 is 708 base pairs long with one coding open reading frames from the first base of 642 base pairs for 214 amino acids and with a stop codon in position 643-645. The amino acid sequence up to the stop codon is shown according to the invention placed in SEQ ID NO: 8.

In addition to the similarity to the PSE1 gene product, the similarity can also Elongase from Saccharomyces cerevisiae (sce elo 1P) be used to elongate fatty acids with medium chain length is required in the yeast (Toke &  Martin, 1996, Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae. Journal of Biological Chemistry 271, 18413-18422). In Table 3 they are Identities and homologies of elongases according to the invention under each other and with the elongases from Physcomitrella patens and out Yeast presented. The information was obtained using the GAP program received as part of the following software: Wisconsin Package Version 10.0 (Genetics Computer Group (GCG), Madison, Wisc., UNITED STATES).

Table 3

In particular, the following sequence motifs can be derived from FIGS. 5 to 10 as areas of high homology and corresponding consensus sequences derived therefrom, which, by back-translating the amino acids in three-base pair codons, lead to oligonucleotides which can be used to isolate new elongases by means of a polymerase chain reaction. These are in particular the sequence motifs shown in FIG. 10. Oligonucleotides can be derived from these motifs and used in combination with two oligonucleotides in PCR experiments to isolate further elongase fragments. It is expedient to construct and synthesize an oligonucleotide suitable for the conventionally defined 5'-3'S strand and a second one with an oligonucleotide downstream for the 3'-5'S strand. This results in a definable number of primer combinations, which is limited by permutation of the possible variants.

Oligo-dT primers and variants thereof can also be used be, e.g. B. by the last base specificity for a pool of Permits transcripts such as B. Oligo dT (12-20) X, where X is a G, Can be C or T. A second base Oligo dT can also be used (12-20) Use XY, where X can be a G, C or A while that Y can be an A, G, C, or T.

Sequences defined above can be 17 to 20mers Derive oligonucleotides by varying the primer combinations and test parameters such as temperature program, Mg ion concentration etc. for the isolation of gene fragments can be used. The fragments thus obtained can be in  appropriate vectors are cloned and the sequence obtained Clones to identify new elongases according to common ver driving can be determined.

Example 6b Isolation of the cDNA clone from Pythophtora infestans

The cDNA clone with the designation PI001002014R from Phytophthora infestans was identified from randomly sequenced cDNAs due to homologies to PUFA elongase from the moss Physcomitrella patens (ATCCC 48886). The clone contains the consensus motif MYxYYF shown in Fig. 8, whereby, unlike previously identified PUFA elongases, a threonine residue was found as a variable amino acid x. This further variation can be used to derive PCR primers.

Example 7 Identification of genes using hybridization (TC002034029R-11 iGenTc-PCE1)

Gene sequences can be used to identify homologous or Use heterologous genes from cDNA or genomic banks.

Homologous genes (ie full-length cDNA clones that are homologous or homologs) can be isolated via nucleic acid hybridization using, for example, cDNA libraries: in particular for the isolation of functionally active full-length genes of the in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 the method can be used. Depending on the frequency of the gene of interest, 100,000 to 1,000,000 recombinant bacteriophages are plated and transferred to a nylon membrane. After denaturing with alkali, the DNA is z. B. immobilized by UV crosslinking. Hybridization takes place under highly stringent conditions. In aqueous solution, the hybridization and the washing steps are carried out at an ionic strength of 1 M NaCl and a temperature of 68 ° C. Hybridization probes were e.g. B. prepared by labeling using radioactive ( ³² P) nick transcription (High Prime, Roche, Mannheim, Germany). The signals are detected by means of autoradiography.

Partially homologous or heterologous genes that are related but not are identical, can be analogous to the Ver drive using low-stringent hybridization and Identify washing conditions. For aqueous hybridization the ionic strength was usually kept at 1 M NaCl, where the temperature was gradually reduced from 68 to 42 ° C.  

Isolation of gene sequences that only lead to a single Domain of, for example, 10 to 20 amino acid homologies can be demonstrated using synthetic, radioactive perform labeled oligonucleotide probes. Radioactively marked Oligonucleotides are phosphorylated at the 5 'end two complementary oligonucleotides with T4 polynucleotide kinase manufactured. The complementary oligonucleotides are joined together hybridizes and ligates so that concatems are formed. The double-stranded concatems are used, for example, by nick radioactive labeled transcription. The hybridization takes place usually using under low-stringent conditions high oligonucleotide concentrations.

Oligonucleotide hybridization solution

6 × SSC
0.01 M sodium phosphate
1 mM EDTA (pH 8)
0.5% SDS
100 microg / ml denatured salmon sperm DNA
0.1% low-fat dry milk

During the hybridization, the temperature gradually increases to 5 up to 10 ° C below the calculated oligonucleotide Tm or up to space temperature (means RT = ~ 23 ° C in all experiments, if not otherwise specified), followed by washing steps and auto radiography. Washing is done with extremely low stringency carried out, for example 3 washing steps using 4 × SSC. Further details are as described by Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, or Ausubel, F.M., et al. (1994) "Current Protocols in Molecular Biology," John Wiley & Sons, described.

The clone TC002034029R-11 with the nickname Tc_PCE1_1 is one Full length sequence of an elongase from Thraustochytrium and thus longer than clone TC002034029R from Seq. ID No. 3 and Seq. ID No. 4th The clone was isolated using hybridization methods as described above (Example 7). It is a 1054 Base pair long DNA sequence coding for 271 amino acids with a start codon in base pair position 43-45 and a stop codon in base pair position 856-858.  

Example 8 Identification of target genes by screening Expression banks with antibodies

There are cDNA sequences for the production of recombinant Protein used for example in E. coli (e.g. Qiagen QIAexpress pQE system). The recombinant proteins then become common via Ni-NTA affinity chromatography (Qiagen) affinity cleaned. The recombinant proteins then become the Her Position specific antibodies using, for example of standard rabbit immunization techniques. The antibodies are then grown using a Ni-NTA column, which is pre-saturated with recombinant antigen, affinity purified as described by Gu et al., (1994) BioTechniques 17: 257-262 described. The antibody can then be used to screen for Expression cDNA libraries using immunological screening can be used (Sambrook, J., et al. (1989), "Molecular Cloning: A Laboratory Manual ", Cold Spring Harbor Laboratory Press, or Ausubel, F.M., et al. (1994) "Current Protocols in Molecular Biology ", John Wiley & Sons).

Example 9 Plasmids for plant transformation

Binary vectors such as pBinAR can be used for plant transformation can be used (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230). The construction of the binary vectors can be done by Ligation of the cDNA in sense or antisense orientation in T-DNA done. 5 'of the cDNA activates a plant promoter Transcription of the cDNA. There is a polyadenylation sequence 3 'away from the cDNA.

Tissue-specific expression can be determined using a achieve tissue-specific promoter. For example, the seed-specific expression can be achieved by the napin or the LeB4 or USP promoter 5 'of the cDNA becomes. Any other seed-specific promoter element can also be used. For constitutive expression in the whole The CaMV-35S promoter can be used in plants.

The expressed protein can be expressed using a signal peptides, for example for plastids, mitochondria or that Endoplasmic reticulum, in a cellular compartment to be conducted (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423). The signal peptide is 5 'in reading frame with the cDNA cloned to the subcellular localization of the fusion protein to reach.  

Example 10 Agrobacterium transformation

The Agrobacterium-mediated plant transformation can Example using the GV3101- (pMP90-) (Koncz and Schell, Mol. Gen. Genet. 204 (1986) 383-396) or LBA4404- (Clontech) Agrobacterium tumefaciens strain. The Transformation can be done through standard transformation techniques (Deblaere et al., Nucl. Acids. Tes. 13 (1984), 4777-4788).

Example 11 plant transformation

The Agrobacterium-mediated plant transformation can be found at Use of standard transformation and regeneration techniques are carried out (Gelvin, Stanton B., Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuc Central signature: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 pages, ISBN 0-8493-5164-2).

For example, rapeseed can be made using cotyledons or hypocotyls transformation can be transformed (Moloney et al., Plant Cell Report 8 (1989) 238-242; De Block et al., Plant Physiol. 91 (1989) 694-701). The use of antibiotics for that Agrobacterium and plant selection depends on that for that Transformation used binary vector and Agrobacterium Stem from. Rapeseed selection is commonly used of kanamycin as a selectable plant marker.

Agrobacterium-mediated gene transfer in linseed (Linum usitatissimum) can be used using, for example one of Mlynarova et al. (1994) Plant Cell Report 13: 282-285 perform the technique described.

The transformation of soy can be done using examples as one 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) described technique can be performed.

Plant transformation using particles bombardment, polyethylene glycol-mediated DNA recording or is about silicon carbon fiber technology, for example described by Freeling and Walbot "The maize handbook" (1993) ISBN 3-540-97826-7, Springer Verlag New York).  

Example 12 Plasmids for plant transformation

Binary vectors such as pBinAR can be used for plant transformation can be used (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230). The construction of the binary vectors can be done by Ligation of the cDNA in sense or antisense orientation in T-DNA done. 5 'of the cDNA activates a plant promoter Transcription of the cDNA. There is a polyadenylation sequence 3 'away from the cDNA.

Tissue-specific expression can be determined using a achieve tissue-specific promoter. For example, the seed-specific expression can be achieved by the napin or the LeB4 or USP promoter 5 'of the cDNA becomes. Any other seed-specific promoter element can also be used. For constitutive expression in the whole The CaMV-355 promoter can be used in plants. In particular, genes coding for elongases and. Desaturases by constructing several expression cassettes Clone one after the other in a binary vector to the substance to reproduce the path of change in the plants.

This can be expressed within an expression cassette Protein using a signal peptide, e.g. for Plastids, mitochondria or the endoplasmic reticulum, be directed into a cellular compartment (Kermode, Crit. Rev. Plant Sci. 15: 4 (1996) 285-423). The signal peptide is 5 ' cloned in frame with the cDNA to the subcellular Achieve localization of the fusion protein.

Example 13 In vivo mutagenesis

The in vivo mutagenesis of microorganisms can be done by passage the plasmid (or other vector) DNA by E. coli or other microorganisms (e.g. Bacillus spp. or yeast, like Saccharomyces cerevisiae), where the skills that Maintain the integrity of their genetic information, is disturbed. Common mutator strains have mutations in the genes for the DNA repair system (e.g. mutHLS, mutD, mutT etc.; see Rupp, W. D. (1996) DNA repair for a reference 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 described, for example, in Greener, A., and Callahan, M. (1994) Strategies 7: 32-34. The transfer mutated DNA molecules in plants preferably follow Selection and test of the microorganisms. Become transgenic plants  after various examples in the example part of this document generated.

Example 14 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 was based on the transcription and / or the Translation plane measured.

A suitable method for determining the amount of Transcription of the gene (an indication of the amount of RNA, available for the translation of the gene product) is a Northern blot as outlined below (For reference, see Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York, or the ones mentioned above Example part), wherein a primer that is designed so that it binds the gene of interest with a detectable label (usually radioactive or chemiluminescent) is marked so that when the total RNA is extracted from a culture of the organism, separated on a gel, transferred to a stable matrix and incubated with this probe, binding and extent the presence of the probe and the amount of mRNA for this gene. This information shows the degree of Transcription of the transformed gene. Total cellular RNA can be made from cells, tissues or organs using several methods, who are all known in the art, such as that by Bormann, E.R., et al. (1992) Mol. Microbiol. 6: 317-326 described, prepared.

Northern hybridization

For the RNA hybridization, 20 ug total RNA or 1 ug poly (A) ⁺ RNA were gel electrophoresis in agarose gels with a strength of 1.25% using formaldehyde, as described in Amasino (1986, Anal. Biochem 152, 304) transferred separately, by capillary attraction using 10 × 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 (10% dextran sulfate W / v, 1 M NaCl, 1% SDS, 100 mg herring sperm DNA) prehybridized. The DNA probe was labeled with the Highprime DNA labeling kit (Roche, Mannheim, Germany) during the prehybridization using alpha- ³² P-dCTP (Amersham, Braunschweig, Germany). The hybridization was carried out after adding the labeled DNA probe in the same buffer at 68 ° C. overnight. The washing steps were carried out twice for 15 min using 2 × SSC and twice for 30 min using 1 × 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.

To examine the presence or relative amount of this mRNA translated protein can use standard techniques such as a Western blot can be used (see e.g. Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York). In this process, the total cellular Proteins extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose and with a Probe, such as an antibody, specific to the desired one Protein binds, incubated. This probe is usually with one chemiluminescent or colorimetric marking, which is easy to prove. The presence and amount of observed mark shows the presence and amount of desired mutant protein present in the cell.

Example 15 Analysis of the impact of the recombinant proteins on the production of the desired product

The impact of genetic modification in plants, fungi, Algae, ciliates or on the production of a desired ver binding (such as a fatty acid) can be determined by the modified microorganisms or the modified plant under suitable conditions (such as those described above) be grown and the medium and / or the cellular compo the increased production of the desired product (i.e. of 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 processes as well as analytical Chromatography, such as high performance liquid chromatography (see for example Ullman, Encyclopedia of Industrial Chemis try, vol. A2, pp. 89-90 and pp. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery and purification", pp. 469-714, VCH: Weinheim; Belter, P.A., et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, J.F., and Cabral, J.M.S. (1992) Recovery processes for biological materi as, John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, pp. 1-27, VCH: Weinheim; and  Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

In addition to the methods mentioned above, plant lipids are made from Plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940, and Browse et al. (1986) Analytic Biochemistry 152: 141-145. The qualitative and quantitative lipid or fatty acid analysis is described with Christie, William W., Advances in Lipid Methodology, Ayr / Scotland: Oily Press (Oily Press Lipid Library; 2); Christie, William W., Gas Chromatography and Lipids. A Practical Guide- Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 S. (Oily Press Lipid Library; 1); "Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952) -16 (1977) u. d. T .: Progress in the Chemistry of Fats and Other Lipids CODEN.

In addition to measuring the end product of the fermentation it is also possible to add other components of the metabolic pathways analyze the ver. to produce the desired connection be applied, like intermediates and by-products, to the total to determine the efficiency of the production of the connection. 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 occur during the Fermentation are generated. Standard procedure for this Measurements are in Applied Microbial Physiology; A practical Approach, P.M. Rhodes and P.F. Stanbury, eds., IRL Press, Pp. 131-163 and 165-192 (ISBN: 0199635773) and specified therein References described.

One example is the analysis of fatty acids (abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid chromatography mass spectrometry; TAG, triacylglycerol; TLC, thin film chromatography).

The unambiguous proof of the presence of fatty acid products can be analyzed using recombinant organisms Standard analysis methods are obtained: GC, GC-MS or TLC, as variously described by Christie and the literature (1997, in: Advances on Lipid Methodology, Fourth Ed .: Christie, Oily Press, Dundee, 119-169; 1998, Gas Chromato graph mass spectrometry method, lipids 33: 343-353).  

The material to be analyzed can be Grinding in a glass mill, liquid nitrogen and grinding or be broken up through other applicable procedures. The Material must be centrifuged after breaking open. The Sediment is in aqua dest. resuspended, 10 min at 100 ° C heated, cooled on ice and centrifuged again, followed by Extraction in 0.5 M sulfuric acid in methanol with 2% dimethoxy propane for 1 h at 90 ° C, resulting in hydrolyzed oil and lipid leads compounds that result in transmethylated lipids. This Fatty acid methyl esters are extracted into petroleum ether and finally a 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 20 min and subjected to 5 min at 240 ° C. The identity of the received Fatty acid methyl esters must be made using standards that are out commercial sources are available (i.e. sigma) become.

For fatty acids for which no standards are available, the Identity via derivatization and subsequent GC-MS analysis to be shown. For example, the localization of fat acids with triple bond via GC-MS after derivatization with 4,4-dimethoxyoxazoline derivatives (Christie, 1998, see above) to be shown.

Example 16 Expression constructs in heterologous microbial systems Strains, growth conditions and plasmids

The Escherichia coli strain XL1 Blue MRF 'kan (Stratagene) will subclone the new elongases like PpPSE1 Physcomitrella patens used. For functional expression of this gene we use the Saccharomyces cerevisiae strain INVSc 1 (Invitrogen Co.). E. coli is in Luria Bertini broth (LB, Duchefa, Haarlem, The Netherlands) cultivated at 37 ° C. If if necessary, ampicillin (100 mg / liter) is added and 1.5% agar (W / V) is added for solid LB media. S. cerevisiae is at 30 ° C either in YPG medium or in complete minimal medium without uracil (CMdum; see in: Ausubel, F. M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K, Albright, L.B., Coen, D.M., 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 ™ agar (Difco) is added.  The plasmids used for cloning and expression are pUC18 (Pharmacia) and pYES2 (Invitrogen Co.).

Example 17 Cloning and expression of PUFA-specific elongases from Physcomitrella, Crypthecodinium and Thrausto chytrium

The full length genes of sequences according to the invention can be as in Example 7 described isolated and carried out as follows to be processed further. Execution will be concrete Expression examples shown.

A) Elongation of fatty acids by the moss elongase Pp_PSE1

The P. patens cDNA clone PpPSE1 was used for expression in yeast (former database sequence name: 08_ck19_b07, new name: pp001019019f), which contains the PUFA-specific elongase (PSE1) gene encoded, first modified so that a BamHI restriction site and the yeast consensus sequence for highly efficient translation (Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes, Cell 44, 283-292) next to the start codon and one BamHI restriction site flanking the stop codon were. A was used to amplify the open reading frame Pair of primers complementary to its 5 'and 3' ends, synthesized.

The gene of the sequence according to the invention shown in SEQ ID NO: 1 was cloned into pYES by means of the polymerase chain reaction and the plasmid pYPp_PSE1 was obtained:
The following oligonucleotides were used for the PCR experiment:

The PCR reaction was carried out with plasmid DNA from clone PP001019019F as a template in a thermal cycler (Biometra) with the Pfu-DNA (Stratagene) polymerase and the following temperature program performed: 3 min at 96 ° C, followed by 25 cycles of 30 s at 96 ° C, 30 s at 55 ° C and 1 min at 72 ° C, 1 cycle at 10 min at 72 ° C.

The correct size of the amplified DNA fragment was determined using Agarose TBE gel electrophoresis confirmed. The amplified DNA was made from the gel with the QIAquick gel extraction kit (QIAGEN)  extracted and using the Sure Clone Ligation Kit (Pharmacia) first cloned in pUC18. The fragment cloned in this way was cut with BamHI, ligated into pYES, pYPp_PSE1 was obtained. The fragment orientation was determined using HindIII checked. After transforming E. coli XL1 Blue MRF ' a mini DNA preparation (Riggs, M.G., & McLachlan, A. (1986) A simplified screening procedure for large numbers of plasmid mini-preparation. BioTechniques 4, 310-313) by Transformants and positive clones using BamHI Restriction analysis identified. The sequence of the cloned PCR product was resequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt) confirmed.

A clone was used for the DNA maxipreparation with the Nucleobond® AX 500 plasmid DNA extraction kit (Macherey-Nagel, Düringen) dressed.

Saccharomyces INVSc1 was identified as pYPp_PSE1 and pYES2 Control using a modified PEG / lithium acetate Protocol transformed (Ausubel et al., 1995). After Selection on CMdum agar plates with 2% glucose were carried out Transformants and a pYES2 transformant for further Cultivation and functional expression as already stated selected and fed with various fatty acids in the medium.

• a) Lipid patterns of yeasts which have been transformed with the pYES plasmid without fragment insertion or which express the Pp-PSE1 gene (data in mol%) after feeding with 250 microm hexadecatrienoic acid (16: 3 ^{Δ7c, 10c, 13c} ).

Table 4

• a) Lipid patterns of yeasts which have been transformed with the pYES plasmid without fragment insertion or which express the Pp-PSE1 gene (data in mol%) after feeding with 500 micromole pinolenic acid (18: 3 ^{Δ 6c, 9c, 12c} ).

Table 5

• a) Lipid patterns of yeasts which have been transformed with the pYES plasmid without fragment insertion or which express the Pp-PSE1 gene (data in mol%) after feeding with 500 microm stearidonic acid (18: 4 ^{Δ6c, 9c, 12c, 15c} ).

Table 6

• a) Lipid patterns of yeasts which have been transformed with the pYES plasmid without fragment insertion or which express the Pp-PSE1 gene (data in mol%) after feeding with 500 microm linoleic acid (18: 2 ^{Δ9c, 12c} ).

Table 7

B) Elongation of fatty acids by an elongase from Thrausto chytrium

Thraustochytrium cDNA is used for expression in yeast. Clone from SEQ ID NO: 3 (Tc_PSE2), which is a PUFA specific Elongase (PSE) gene encoded, first modified to be a represents functionally active polypeptide. For this purpose the N-terminus of the protein at the DNA level by the few missing bases from the Physcomitrella patens elongase 42 base pairs extended. But it can only be a start codon can be added to the sequence in the appropriate reading frame.

The following oligonucleotides are used for the PCR experiment set:

In addition, a BamHI is in both oligonucleotides Restriction site and the yeast consensus sequence for high contain efficient translation (Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes, Cell 44, 283-292).

The PCR reaction is performed using plasmid DNA as a template in one Thermocycler (Biometra) with the Pfu-DNA (Stratagene) polymerase and the following temperature program: 3 min at 96 ° C, followed by 25 cycles of 30 s at 96 ° C, 30 s at 55 ° C and 3 min at 72 ° C, 1 cycle with 10 min at 72 ° C and stop at 4 ° C.

The correct size of the amplified DNA fragment will be confirmed by agarose TBE gel electrophoresis. The ampli Fected DNA is extracted from the gel with the QIAquick gel extraction kit (QIAGEN) extracted and into the SmaI restriction site of dephosphorylated vector pUC18 using the Sure Clone Ligation Kit (Pharmacia) ligated using pUC-hybrid-Tc_PSE2 is obtained. After transforming E. coli XL1 Blue MRF ' a mini DNA preparation (Riggs, M.G., & McLachlan, A. (1986) A simplified screening procedure for large numbers of plasmid mini-preparation. BioTechniques 4, 310-313) 24 ampicillin resistant transformants performed, and positive clones were identified by means of BamHI restriction analysis identified. The sequence of the cloned PCR product was by resequencing using the ABI PRISM Big Dye  Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt) confirmed.

The plasmid DNA of pUC-PSE1 and pUC-hybrid-Tc_PSE2 were also cleaved with BamHI and the fragments obtained were cut into the BamHI restriction site of dephosphorylated yeast E. coli Shuttle vector pYES2 ligated, pY2 receiving hybrid Tc_PSE2 becomes. After the transformation of E. coli and DNA mini-preparation the transformants became the orientation of the DNA fragment checked in the vector by HindIII cleavage. Became a clone each for the DNA maxipreparation with the Nucleobond® AX 500 Plasmid-DNA extraction kit (Macherey-Nagel, Düringen) tightened. Saccharomyces INVSc1 is linked to pY2PSE1, pYES2, pY2-hybrid-Tc_PSE2 and pYES2 using a modified PEG / lithium acetate Protocol transformed (Ausubel et al., 1995). After Selection on CMdum agar plates with 2% glucose are four each pY2PSE1 transformants (pY2PSE1a-d), pY2-hybrid-Tc_PSE2-Trans formants (pY2-hybrid-Tc_PSE2 1a-d) and a pYES2 transformant selected for further cultivation and functional expression.

Functional expression of elongase activity in yeast preculture

20 ml of CMdum liquid medium with 2% (w / v) raffinose were inoculated with the transgenic yeast clones (pY2-hybrid-Tc_PSE2 1a-d, pYES2) and grown 3 T at 30 ° C., 200 rpm until an optical density at 600 nm (OD ₆₀₀ ) of 1.5-2 was reached.

main Culture

For expression, 20 ml of CMdum liquid medium with 2% raffinose and 1% (v / v) Tergitol NP-40 were enriched with fatty acid substrates to a final concentration of 0.003% (w / v). The media are inoculated with the precultures to an OD ₆₀₀ of 0.05. Expression was induced at an OD ₆₀₀ of 0.2 with 2% (w / v) galactose for 16 hours, after which the cultures were harvested an OD ₆₀₀ of 0.8-1.2.

fatty acid analysis

The total fatty acids were extracted from yeast cultures and analyzed by gas chromatography. Of these, cells from 5 ml culture were harvested by centrifugation (1000 × g, 10 min, 4 ° C.) and washed once with 100 mM NaHCO ₃ , pH 8.0, in order to remove residual medium and fatty acids. To produce the fatty acid methyl ester (FAMES), the cell sediments were treated with 1 M methanolic H ₂ SO ₄ and 2% (v / v) dimethoxypropane at 80 ° C. for 1 hour. The FAMES were extracted twice with 2 ml of petroleum ether, once with 100 mM NaHCO ₃ , pH 8.0 and once with distilled water and dried with Na ₂ SO 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 fatty acid patterns of five transgenic yeast strains in mol% are shown in Table 1.

The ratios of the g-linolen added and taken up acid are highlighted in bold numbers that the elongated products by red numbers and those of the elongated ones g-linolenic acid by bold numbers (last line).

The GC analysis of FAMES, which is made from total lipids of the yeasts transformed with pYES2 (i / control) and pY2PSE1 (ii-iv c + d / each transformed with pY2PSE1A, pY2PSE1B, pY2PSE1C, pY2PSE1D), is shown in FIG. 2a-e shown. For the analysis, the transgenic yeasts were grown in the presence of g-18: 3. Tab. 1 shows their fatty acid patterns in mol%. The uptake of g-18: 3 is highlighted in bold numbers, the elongation product dihomo-g-linolenic acid (20: 3 D8, 11, 14) is underlined and the ratio g-18: 3 elongation product (also in mol% ) by numbers in bold (last line). The structure and the mass spectra of the DMOX derivative of cis-D6, 9, 12 C18: 3 are shown in Fig. 3a + b. The structure and the mass spectra of the DMOX derivative of D8,11,14 C20: 3 are shown in Fig. 4a + b.

The results show that g-18: 3 in large amounts in all trans gene yeast has been incorporated. All four transgenic yeast clones that were transformed with pY2PSE1 show an additional one Peak in the gas chromatogram obtained by comparing the retention times when 20: 3 D8,11,14 was identified. A gas chromato graphy / mass spectroscopy can provide additional support for Provide confirmation of this identity. The proportion of elongated g-18: 3 was 23.7 to 40.5% as shown in Table 1. Further  was unable to significantly elongate palmitic acid (16: 0), Palmitoleic acid (16: 1), stearic acid (18: 0) or oleic acid (18: 1 D9) can be observed.

The identified products show that the nucleotide sequence of PpPSE1 a D6-selective fatty acid elongase from the moss Physcomitrella patens codes for the formation of new fatty acids leads in transgenic yeasts.

The ratios of the fatty acid added and absorbed substrates can be determined as above and so Quantity and quality of the elongase reaction are recorded.

The structure and mass spectra of DMOX derivatives result also the respective position of a double bond.

Further feeding experiments with various other fat acids (e.g. arachidonic acid, eicosapentaenoic acid etc.) for more detailed confirmation of the substrate selectivity of this Elongase be carried out.

Example 18 Cleaning the desired product transformed organisms in general

Obtaining the desired product from plant material or Mushrooms, algae, ciliates, animal cells or from the supernatant of the cultures described above can be procedures known in the art. Will be the one you want Product not secreted from the cells, the cells can the culture is harvested by slow centrifugation, the Cells can be made using standard techniques such as mechanical force or Ultrasound treatment to be lysed. Organs of plants can mechanically separated from other tissues or other organs become. After homogenization, the cell debris are removed Centrifugation removed, and the supernatant fraction, which which contains soluble proteins is used for further purification of the desired connection. The product is made desired cells are secreted, the cells are slow Centrifugation removed from the culture, and the supernatant Fraction is kept for further cleaning.

The supernatant fraction from each cleaning procedure becomes one Chromatography with a suitable resin, which desired molecule either back on the chromatography resin is held, but not many impurities in the sample, or the impurities remain on the resin, the sample however not. These chromatography steps can be done if necessary  can be repeated, using the same or different Chromato graphic resins are used. The specialist is in the selection suitable chromatography resins and their most effective application for a specific molecule to be purified. The purified product can by filtration or ultrafiltration concentrated and kept at a temperature at which is the maximum stability of the product.

There is a wide range of cleaning processes in the field known, and the above cleaning process should not be restrictive. These cleaning procedures are included Game described in Bailey, J.E., & Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined by standard techniques of the field. These include high performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography graphic, NIRS, enzyme test or microbiological. An overview about these analysis methods see in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, Pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A., et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.

equivalent

Those skilled in the art will recognize or can understand many equivalents of here described specific embodiments of the invention determine by using only routine experiments. These equivalents are intended to be encompassed by the claims.  

SEQUENCE LISTING

Claims (28)

1. Isolated nucleic acid derived from a plant or algae and encoding a polypeptide that extends C ₁₆ , C ₁₈ or C ₂₀ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms. 2. Isolated nucleic acid, comprising a nucleotide sequence which encodes a polypeptide which extends C ₁₆ , C ₁₈ or C ₂₀ fatty acids with at least two double bonds in the fatty acid molecule, selected from the group consisting of

• a) a nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7,
• b) a nucleic acid sequence which, according to the degenerate genetic code, comes from the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7,
• c) Derivatives of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, for poly peptides of the sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ Encode ID NO: 6 or SEQ ID NO: 8 and have at least 50% homology at the amino acid level without significantly reducing the enzymatic action of the polypeptides.

3. The isolated nucleic acid sequence according to claim 2, wherein the Sequence derived from a plant, algae or fungus. 4. Isolated nucleic acid sequence according to claim 2 or Claim 3, wherein the sequence of Physcomitrella, Thrausto chytrium or Crythecodinium. 5. A gene construct comprising an isolated nucleic acid one of claims 1 to 4, wherein the nucleic acid functional with one or more regulation signals connected is. 6. gene construct according to claim 5, the gene expression by the regulatory signals are amplified.   7. Gene construct contain a nucleic acid sequence according to the Claims 1 to 4 or a gene construct according to claim 5 or 6 and at least one further nucleic acid which are suitable for encodes a gene of fatty acid biosynthesis. 8. gene construct according to claim 7, characterized in that the nucleic acid responsible for a gene of fatty acid biosynthesis coded selected from the group Δ19-, Δ17-, Δ15-, Δ12-, Δ9-, Δ8-, Δ6-, Δ5-, Δ4-desaturase, the different Hydroxylases, the Δ12-acetylenase, the acyl-ACP thio esterases, β-ketoacyl-ACP synthases or β-ketoacyl-ACP- Is reductases. 9. Amino acid sequence by the isolated nucleic acid sequence according to claims 1 to 4 or the gene construct is encoded according to one of claims 5 to 8. 10. Vector comprising a nucleic acid according to one of the Claims 1 to 4 or a gene construct according to one of the An sayings 5 to 8. 11. organism comprising at least one nucleic acid according to one of claims 1 to 4, a gene construct according to one of the An sayings 5 to 8 or a vector according to claim 10. 12. The organism of claim 11, wherein the organism is a micro organism, an animal or a plant. 13. Organism according to claim 11 or 12, wherein the organism is a transgenic plant. 14. Antibody that is specifically a polypeptide derived from one of the Encoding nucleic acids according to one of claims 1 to 4, binds. 15. Antisense nucleic acid molecule, which is the complementary sequence the nucleic acid according to any one of claims 1 to 4. 16. A method for producing PUFAs, the method comprising growing an organism comprising a nucleic acid according to claim 1, a gene construct according to claim 6 or a vector according to claim 10, encoding a polypeptide which is C ₁₆ -, C ₁₈ - or C ₂₀ 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 formed in the organism. 17. The method according to claim 16, wherein the PUFAs produced by the method are C ₁₈ , C ₂₀ or C ₂₂ fatty acid molecules with at least two double bonds in the fatty acid molecule. 18. The method according to claim 16 or 17, wherein the C ₁₉ , C ₂₀ or C ₂₂ fatty acid molecules are isolated from the organism in the form of an oil, lipid or a free fatty acid. 19. The method according to any one of claims 16 to 18, wherein the Organism a microorganism, an animal or a plant is. 20. The method according to any one of claims 16 to 19, wherein the Organism is a transgenic plant. 21. The method according to any one of claims 16 to 20, wherein the C ₁₆ -, C ₁₈ - or C ₂₀ fatty acid is a fatty acid with three double bonds in the molecule. 22. Oil, lipids or fatty acids or a fraction thereof provided by the method according to one of claims 16 until 21. 23. Oil, lipid or fatty acid composition that ent PUFAs keep and come from transgenic plants. 24. oil, lipid or fatty acid composition according to claim 23, the PUFAs originating from transgenic plants which have a Nucleotide sequence according to one of claims 1 to 4, the Gene construct according to one of claims 5 to 8, the antisense Nucleic acid molecule according to claim 15 or the vector according to Claim 10 include. 25. Use of the oil, lipid or fatty acid composition according to Claims 22 to 24 in feed, food, cosmetics or Pharmaceuticals. 26. A composition comprising the antibody of claim 14, the antisense nucleic acid construct according to claim 15 or an antagonist or agonist identified after Claim 25. 27. Kit comprising the nucleic acid according to one of claims 1 to 4, the gene construct according to one of claims 5 to 8, the antisense nucleic acid molecule of claim 15, the Antibody according to claim 14, an antagonist or Agonists identified according to claim 25, the composition  according to claim 26, the amino acid sequence according to Claim 9 and / or oil, lipids and / or fatty acids or a fraction thereof according to any one of claims 22 to 25. 28. A method of identifying an antagonist or agonist of elongases, comprising

• a) contacting the cells expressing the polypeptide of the present invention with a candidate substance;
• b) testing PSE activity;
• c) comparing the PSE activity to a standard activity in the absence of the candidate substance, an increase in PSE activity above the standard indicating that the candidate substance is an agonist and a decrease in PSE activity indicating that the candidate substance is an antagonist.