DE10205607A1 - New nucleic acid encoding fatty acid elongase, useful for producing polyunsaturated fatty acids as oils, lipids or free fatty acids - Google Patents

Abstract

Isolated nucleic acid (I) that encodes a polypeptide (II) able to extend 16 or 18 carbon fatty acids having at least two double bonds by at least 2 carbon atoms, where the fatty acids C18:4(DELTA5t,9,12); C20:3(DELTA8,11,14); C20:4(DELTA5,8,11,14) and C20:5(DELTA5,8,11,14,17) are not extended, is new. Independent claims are also included for: (1) amino acid sequences encoded by (I); (2) gene construct (GC) containing a 1066 nucleotide sequence (N1), functionally linked to at least one regulatory sequence; (3) vector containing (I) or GC; (4) organism containing (I), GC or the vector of (3); (5) producing (M1) polyunsaturated fatty acids (PUFAs) by culturing an organism that includes (I); (6) oils, lipids, fatty acids and their fractions produced by M1; and (7) oils, lipids and fatty acids that contain PUFAs and are derived from transgenic plants.

Description

This invention relates to a new elongase gene with the sequence SEQ ID NO: 1 or its homologues, derivatives or analogues Gene construct that this gene or its homologues, derivatives or Includes analogs, as well as its use. The invention relates also vectors or organisms that have an elongase gene with the Sequence SEQ ID NO: 1 or its homologues, derivatives or analogs include.

The invention further relates to a method of manufacture 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 an Fatty acid composition with a higher content of polyunsaturated Fatty acids with at least two double bonds and / or one Triacylglycerol composition with a higher content of multiple unsaturated fatty acids with at least two double bonds.

Certain products and by-products are more naturally occurring Metabolic processes in microbial cells or in the cells of animals and beneficial plants are for a wide range to 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) for example, food for children added to a higher one To produce nutritional value of these foods. PUFAs have for Example a positive influence on cholesterol levels in the blood of humans and are therefore suitable for protection against Heart disease. Fine chemicals such as polyunsaturated Fatty acids (polyunsaturated fatty acids, PUFAs) can be isolate from animal sources such as fish or with microorganisms by cultivating microorganisms, that have been developed to have large quantities of one or produce and accumulate several desired molecules or secrete, produce on a large scale.

Particularly suitable microorganisms for the production of PUFAs are microorganisms like the algae Phaeodactylum tricornutum or Crypthecodinium species, ciliates, such as Stylonichia or Colpidium, mushrooms such as Mortierella, Entomophthora, Mucor. By Strain selection is a number of mutant strains of the corresponding microorganisms have been developed that a number of desirable compounds, including PUFAs. The selection of strains with improved production of a However, certain molecule is a time consuming and difficult one Method.

Alternatively, the production of fine chemicals can suitably be carried out on a large scale through the production of plants designed to produce the above-mentioned PUFAs. Plants which are particularly suitable for this purpose are oil-fruit plants which contain large amounts of lipid compounds, such as rapeseed, canola, flax, soybeans, sunflowers, thistles, 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, through the selection of suitable plants, a number of mutant plants have been developed which produce a spectrum of desirable lipids and fatty acids, cofactors and enzymes. The selection of new plant varieties with improved production of a particular molecule is, however, a time-consuming and difficult process or even impossible if the compound does not occur naturally in the corresponding plant, as in the case of polyunsaturated C ₂₀ fatty acids and those with longer carbon chains.

Because of the positive properties of unsaturated fatty acids There has been no lack of approaches in the past, genes, involved in the synthesis of fatty acids or triglycerides are for the production of oils in various organisms available with a modified content of unsaturated fatty acids close. So in WO 91/13972 and its US equivalent described a Δ-9 desaturase. In WO 93/11245 a Δ-15 desaturase in WO 94/11516 becomes a Δ-12 desaturase claimed. Δ-6 desaturases are described in WO 93/06712, US 5,614,393, WO 96/21022 and WO 99/27111. Other desaturases are described, for example, in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347, 1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. In WO 96/13591 a Δ-6-palmitoyl ACP desaturase described and claimed. The biochemical Characterization of the different desaturases is however has so far been insufficient because the enzymes are membrane-bound Proteins are very difficult to isolate and characterize (McKeon et al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988: 777-792).

In yeast both a shift in the fatty acid spectrum could be observed towards unsaturated fatty acids as well as an increase in Productivity can be demonstrated (see Huang et al., Lipids 34, 1999: 649-659, Napier et al., Biochem. J., Vol. 330, 1998: 611-614). The expression of the different desaturases in However, transgenic plants did not show the desired success. A shift in the fatty acid spectrum to unsaturated ones Fatty acids could be shown, but at the same time that the synthetic performance of the transgenic plants decreased significantly, that is, compared to the original plants, only few could Amounts of oils are isolated.

The cloning and expression of elongases, the unsaturated Fatty acids as a substrate for the enzymatic reaction by at least elongate two C atoms, has never been found in yeast or in Plants described.

This means that neither in yeasts nor in crops are naturally polyunsaturated C ₂₀ and / or C ₂₂ fatty acids with at least two double bonds in the fatty acid molecule such as arachidonic acid (ARA) and / or eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA) produced.

Therefore, there is still a great need for new genes, that code for enzymes that are involved in unsaturated biosynthesis Fatty acids are involved and make it possible to combine them in one to produce a technical scale. A particularly high demand exists for elongases, the unsaturated fatty acids by at least elongate two C atoms. None of the previously known biotechnological process for the production of polyunsaturated Fatty acids provides the aforementioned fatty acids in an economical way usable quantities.

When it comes to the expression of genes in plants, there are always Problems, that is, it does not come to expression expected increase in the manufacture of the desired Value product.

The task was therefore to identify, clone and express new elongase genes and thereby use them for the synthesis of unsaturated fatty acids such as polyunsaturated C ₂₀ and / or C ₂₂ fatty acids with at least two double bonds in the fatty acid molecule such as arachidonic acid (ARA) and / or To provide eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).

This object was achieved by the isolated nucleic acid according to the invention, which codes for a polypeptide which extends C ₁₆ or C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms, the fatty acids C _{18: 3} ^{Δ 5t, 9, 12} , C _{20: 3} ^{Δ 8,11,14} , C _{20: 4} ^{Δ 5,8,11,14} and C _{20: 5} ^{Δ 58,11,14,17} cannot be extended.

• a) einer in SEQ ID NO: 1 dargestellten Nukleinsäuresequenz,
• b) einer Nukleinsäuresequenz, die sich gemäß dem degenerierten genetischen Code von der in SEQ ID NO: 2 dargestellten Sequenz ableitet,
• c) Derivate der in SEQ ID NO: 1 dargestellten Sequenz, die Polypeptide mit mindestens 50% Homologie zu der Sequenz kodiert, die die Aminosäuresequenzen in SEQ ID NO: 2 kodiert, wobei die Sequenz als C₁₆- oder C₁₈-Elongase wirkt.

The object is advantageously achieved by an isolated nucleic acid comprising a nucleotide sequence which encodes a polypeptide which extends C ₁₆ or C ₁₈ 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,
• b) a nucleic acid sequence which is derived from the sequence shown in SEQ ID NO: 2 in accordance with the degenerate genetic code,
• c) Derivatives of the sequence shown in SEQ ID NO: 1, which encodes polypeptides with at least 50% homology to the sequence which encodes the amino acid sequences in SEQ ID NO: 2, the sequence acting as a C ₁₆ or C ₁₈ elongase.

The nucleotide sequence selected from the aforementioned group of C ₁₆ or C ₁₈ fatty acids with at least two double bonds in the fatty acid is advantageously extended by at least two carbon atoms, the following fatty acids C _{18: 3} ^{Δ 5t, 9.12} , C _{20: 3} ^{Δ 8,11,14} , C _{20: 4} Δ ^5,8,11,14 and C _{20: 5 Δ 5,8,11,14,17} cannot be extended. C ₁₆ or C ₁₈ fatty acids with two, three or four double bonds in the fatty acid molecule are preferably extended. While fatty acids such as C _{18: 3} ^{Δ 5t, 9.12} , C _{20: 3} ^{Δ 8,11,14} , C _{20: 4} ^{Δ 5,8,11,14} and C _{20: 5 Δ 5,8,11,14 , 17 are} not extended, fatty acids are selected from the group C _{18: 2} ^{Δ 9.12} , C _{18: 3} ^{Δ 4.7.10} , C _{18: 3} ^{Δ 5.8.11} , C _{18: 3} ^{Δ 6, 9.12} , C _{18: 3} ^{Δ 7.10.13} , C _{18: 3} ^{Δ 8.11.14} , C _{18: 3} ^{Δ 9.12.15} , C _{18: 4} ^{Δ 6.9.12.15} , C _{18: 3} ^{Δ 5c, 9.12} or C _16: ^{3 Δ 7.10.13} elongated. The elongases ^{according to the} invention show a preference over C _{18: 3} ^{Δ 6.9.12} -, C _{18: 4} ^{Δ 6.9.12.15} - and C _{16: 3} ^{Δ 7.10.13} fatty acids, which are advantageously at least around is a factor of 1.5, preferably at least by a factor of 1.6, particularly preferably at least by a factor of 1.7 or very particularly preferably at least by a factor of 1.8, compared to unsaturated fatty acids such as C _{18: 2} ^{Δ 9, 12} -, C _{18: 3} ^{Δ 4,7,10} -, C _{18: 3} ^{Δ 5,8,11} -, C _{18: 3} ^{Δ 7,10,13} -, C _{18: 3} ^{Δ 8,11,14} - , C _{18: 3} ^{Δ 9,12,15} - or C _{18: 3} ^{Δ 5, c9,12 -fatty} acids.

The nucleic acid sequences according to the invention which elongate C ₁₆ or C ₁₈ fatty acids originally originate advantageously from fungi, preferably from fungi such as the Oomycetes, for example Oomycetes such as those of the genus Phytophthora, particularly preferably from Oomycetes of the genus and type Phytophthora infestans.

The nucleic acids according to the invention can be used to modify Oils, fatty acids, lipids, compounds derived from lipids and most preferred for the production of polyunsaturated Fatty acids are used.

As host organisms for the nucleic acids according to the invention microorganisms such as Phaeodactylum are advantageous Colpidium, Mortierella, Entomophthora, Mucor, Crypthecodinium as well as other algae and fungi and plants, in particular Oil crops used in the production of a variety of industries Fine chemicals can be used on a large scale.

With those disclosed in, for example, WO 98/01572 Cloning vectors and techniques for genetic manipulation of the microorganisms and ciliates mentioned above or those in Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251, and Dunahay et al., 1995, Genetic transformation of diatoms, J. Phycol. 31: 10004-1012 and the quotes therein for algae and relatives Organisms such as Phaeodactylum tricornutum methods described and vectors, the nucleic acid molecules according to the invention used for the genetic engineering change of these organisms so that they become better or more efficient producers of one or several fine chemicals. This improved Production or efficiency of the production of a fine chemical can through a direct effect of manipulating one nucleic acid according to the invention advantageously in the form of the entire gene or caused by an indirect effect of this manipulation become.

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 Prepare docosahexaenoic acid (DHA). Fungal systems too like oomycetes (Eukaryotes / Stramenopiles / Oomyzeten / Phythialen / Pythiaceen) produce the aforementioned fatty acids. Therefore suitable themselves nucleic acid molecules that an oomycete like Phytophtohora infestans come, especially for the modification of the lipid and PUFA production system in a host, especially in Microorganisms such as the aforementioned microorganisms, and Plants, such as oil crops, for example rapeseed, canola, Flax, soy, sunflower, borage. Can also Nucleic acids from an oomycete such as Phytophtohora infestans Identification of such DNA sequences and enzymes in other species that to modify the biosynthesis of precursor molecules from PUFAs can be used in the corresponding organisms.

The fungus Phytophthora infestans is a member of the oomycetes. It is related to other mushrooms in the absence of light can grow. Fungi, such as Phytophthora, are on DNA and sequence Polypeptide level very homologous to each other, which is the use of heterologous screening of DNA molecules with other fungi probes or organisms, so that at there are other nucleic acid sequences in addition to the sequence according to the invention a consensus sequence can be derived, for heterologous screening or functional Commenting and prediction of gene functions in third ways is suitable. Currently the function is predicted by the sequences encoded proteins or enzymes are not yet possible. The Ability to identify these functions, e.g. B. the Prediction of the substrate specificity of enzymes can therefore by be of significant importance. Furthermore, these Nucleic acid molecules as reference sequences for mapping other fungi or for Derivation of PCR primers are used.

Furthermore, a functionally active PSE gene was developed for the first time isolated from the oomycete Phytophthora infestans (Eucariots / Stramenopiles, Oomycetes / Phythialen / Pythiaceen). The gene is advantageous for the production of long-chain polyunsaturated Suitable fatty acids, preferably with more than sixteen or eighteen carbon atoms in the carbon backbone of the Fatty acid and / or at least two double bonds in the Carbon chain, is suitable, the by the invention Sequence encoded enzymes a preference over the above have fatty acids mentioned in the elongation.

This new nucleic acid molecule encodes a protein this as PUFA-specific elongase (PSEs or in the singular PSE). For example, these PSEs can be one Exercise a function that is related to metabolism (e.g. biosynthesis or on degradation) of compounds leading to lipid or Fatty acid synthesis are necessary, such as PUFAs, are involved in or on Transmembrane transport of one or more lipid / fatty acid compounds either participate in or out of the cell.

In this new application, the function of the sequence shown in more detail. We have a functionally active one for the first time Oomycete gene isolated, which is used to produce long-chain multiple unsaturated fatty acids, preferably with more than sixteen or eighteen carbon atoms in the carbon backbone of the Fatty acid and / or at least two double bonds in the Carbon chain, is suitable, the by the invention Sequences encoded enzymes a preference over the above have fatty acids mentioned in the elongation. thats why referred to herein as a PSE gene or protein. Other Publications and patents do not disclose or show any functionality active PSE gene, although there are several known ones There are patent applications which include the extension of saturated fatty acids short or medium chain (WO 98/46776 and US 5,475,099) or the extension or production of long chain fatty acids that but then have no more than one double bond or too lead long-chain fatty acid wax esters (see: WO 98/54954, WO 96/13582, WO 95/15387) show.

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 an inevitable PSE- coding gene and no functional PSE activity. 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 so far the production of very long-chain polyunsaturated fatty acids (with a C ₂₀ and longer carbon chain as well as of trienoic acids and higher unsaturated types) has not been achieved Taught substrate specificity of elongases.

To produce long-chain PUFAs, the polyunsaturated C ₁₆ and / 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 according to the invention encodes the first fungal elongase which can extend C ₁₆ and / 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. An advantage of the enzymatic activity according to the invention is that not all unsaturated C ₂₀ fatty acids are extended. This enables the specific synthesis of individual desirable unsaturated fatty acids or fatty acid mixtures. 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, preferably with three or four double bonds, fatty acids with a double bond in the Δ6 position are particularly preferred. After the extension with the enzymes according to the invention has taken place, further desaturation steps can take place. Therefore, the products of the elongase activity 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-eicosatetraenoic, docosapentaenoic acid or docosahexaenoic acid. Substrates of the enzyme activity according to the invention are, for example, taxolic acid; 6,9-octadecadienoic acid, linoleic acid, γ-linolenic acid, pinolenic acid, α-linolenic acid or stearidonic acid. Preferred substrates are linoleic acid, γ-linolenic acid and / or α-linolenic acid. The C ₁₆ - and / or C ₁₈ -fatty acids with at least two double bonds in the fatty acid can by the inventive enzymatic activity in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol, be extended.

Using cloning vectors to use are suitable in plants and for plant transformation, such as those in Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-119 (1993); F. F. White, Vectors for Gene Transfer in 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 Transfer, 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)) and those in these publications quotations have been published, the Nucleic acids according to the invention for genetic engineering Change a wide range of plants so use them a better or more efficient producer of one or more products derived from lipids, such as PUFAs. This improved production or efficiency of producing one of Lipid-derived product, such as PUFAs, can be obtained by direct Effect of manipulation or an indirect effect of this Manipulation.

There are a number of mechanisms through which change takes place the yield, production of a PSE protein according to the invention and / or production efficiency of a fine chemical an oil crop or a microorganism due to a modified protein can directly affect. The number or Activity of the PSE protein, nucleic acid and / or gene can be increased so that larger amounts of these compounds de novo are produced because the organisms have this activity and Ability to biosynthesize before introducing the appropriate Nucleic acid and / or the corresponding gene was missing.

The introduction of a nucleic acid according to the invention and / or a PSE gene in an organism or a cell cannot only increase the biosynthesis flow to the end product, but also increase the corresponding triacylglycerol composition or create de novo. Likewise, the number or activity of others Genes involved in the import of nutrients necessary for the biosynthesis of a or several fine chemicals (e.g. fatty acids, polar and neutral lipids) are necessary, so that the Concentration of these precursors, cofactors or intermediates within the cells or within the storage compartment is increased, increasing the ability of the cells to produce PUFAs, as described below, is further increased. Fatty acids and lipids are themselves considered fine chemicals desirable; by optimizing activity or increasing the number one or more PSEs involved in the biosynthesis of this Connections are involved, or by destroying 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 To increase lipid molecules from plants or microorganisms.

The mutagenesis of the PSE gene according to the invention or the Nucleic acid can also become a PSE protein with altered activities lead which the production of one or more desired Influence fine chemicals directly or indirectly. For example can be the number or activity of the PSE gene according to the invention or the nucleic acid can be increased so that the normal Metabolic waste or by-products of the cell (their amount possibly due to the overproduction of the desired one Fine chemical is increased) can be exported efficiently before other molecules or processes within the cell (which are the Cell viability) or destroy it Would disrupt biosynthetic pathways of the fine chemical (whereby the yield, production or efficiency of production of the desired fine chemical is reduced). Can also the relatively large intracellular amounts of the desired Fine chemical itself can be toxic to the cell or Enzyme feedback mechanisms, such as allosteric regulation, for example, by increasing activity or Number of other downstream enzymes or Detoxification enzymes of the PUFA route the allocation of the PUFA in the Increase triacylgylcerin fraction, you could viability of Increase seed cells, which in turn leads to better development of Cells in culture or seeds that produce the desired Produce fine chemicals. The PSE genes according to the invention or Nucleic acids can also be manipulated so that the corresponding amounts of the different lipid and fatty acid molecules getting produced. This can have a drastic effect on the Lipid composition of the cell membrane have and produces new ones Oils in addition to the occurrence of newly synthesized PUFAs. Because everyone Lipid type can have different physical properties a change in the lipid composition of a membrane Change membrane fluidity significantly. Changes in Membrane fluidity can affect the transport of molecules across the Membrane as well as affect the integrity of the cell both have a crucial impact on the production of Own fine chemicals. In plants, these changes can occur also other features, such as tolerance to ablotic and biotic stress situations.

Biotic and ablotic stress tolerance is a common one Trait that is associated with a wide range of plants, such as corn, Wheat, rye, oats, triticale, rice, barley, soybean, Peanut, thistle, 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, Inherit from perennial grasses and forage crops would like to. These crops are another according to the invention Embodiment also preferred target plants for genetic engineering. Plants according to the invention are particularly preferred Oil crops, such as soybean, peanut, rapeseed, canola, sunflower, Diesels, trees (oil palm, coconut) or crops such as corn, Wheat, rye, oats, triticale, rice, barley, alfalfa, or Bush plants (coffee, cocoa, tea).

Accordingly, one aspect of the invention relates to isolated Nucleic acid molecules (e.g. cDNAs), comprising a nucleotide sequence, encoding a PSE or biologically active parts thereof, or Nucleic acid fragments that can be used as primers or Hybridization probes for the detection or amplification of PSE-encoding Nucleic acids (e.g. DNA or mRNA) are suitable. With especially preferred embodiments, the nucleic acid molecule comprises a the nucleotide sequences shown in SEQ ID NO: 1 or the coding region or a complement of any of these Nucleotide sequences. In other particularly preferred embodiments comprises the isolated nucleic acid molecule according to the invention a nucleotide sequence attached to a nucleotide sequence as in of the sequence SEQ ID NO: 1, or a part thereof hybridized or at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90% and more preferably at least about 95%, 96%, 97%, 98%, 99% or more is homologous to it, taking below Homology in the sense of the invention is to be understood as identity. at other preferred embodiments encode the isolated one Nucleic acid molecule one of those in the sequence SEQ ID NO: 2 amino acid sequences shown. The preferred one according to the invention Has PSE gene or the nucleic acid sequence according to the invention preferably also at least one of the PSE described here Activities.

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 is that the protein or part thereof is a PSE Maintains activity, taking homology in the sense of the invention To understand identity. Preferably the protein retains or the part of it encoded by the nucleic acid molecule the ability to metabolize to build cell membranes connections necessary for plants or for the transport of Molecules to participate across these membranes. At a Embodiment is the protein encoded by the nucleic acid molecule 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. bei a further preferred embodiment, the protein is a Full-length Phytophthora infestans protein, which is essentially homologous to an entire amino acid sequence of SEQ ID NO: 2 (from the open reading frame shown in SEQ ID NO: 1 stirs).

In another preferred embodiment, the isolated one stirs Nucleic acid molecule from Phytophthora infestans and encoded a protein (e.g. a PSE fusion protein) that is a biological active domain containing at least about 50% or more homologous (identical) to an amino acid sequence of the sequence SEQ ID NO: 2 is and the ability to metabolize mushrooms to Structure of unsaturated fatty acids necessary compounds or participate in the transport of molecules across membranes or at least one of the activities listed in Table I. owns, and also includes heterologous nucleic acid sequences that encode a heterologous polypeptide or regulatory proteins.

The nucleic acids according to the invention code for proteins, the fatty acids selected from the group C _{18: 2} ^{Δ 9.12} , C _{18: 3} ^{Δ 4.7.10} , C _{18: 3} ^{Δ 5.8.11} , C _{18: 3} ^{Δ 6 , 9.12} , C _{18: 3} ^{Δ 7.10.13} , C _{18: 3} ^{Δ 8.11.14} , C _{18: 3} ^{Δ 9.12.15} , C _{18: 4} ^{Δ 6.9.12.15} , C _{18: 3} ^{Δ 5c, 9.12} or C _16: ^{3 Δ 7.10.13} elongate, while fatty acids such as C _{18: 3} ^{Δ 5t, 9.12} , C _{20: 3} ^{Δ 8.11.14} , C _{20: 5} ^{Δ 5,8,11,14,17} cannot be extended. The PSE gene was heterologously expressed in yeast and various poly (poly) unsaturated fatty acids were fed individually and converted from the transformed yeast. Fatty acid profiles of transgenic yeasts were analyzed using GLC. The percentage of fed fatty acids converted was calculated as follows: mol% (product) × 100 / [mol% (starting material) + mol% (product)].

Table I shows fatty acids which are elongated by the enzyme according to the invention.

In another embodiment, this is isolated Nucleic acid 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 comprises. Preferably corresponds the isolated nucleic acid molecule to a naturally occurring one Nucleic acid molecule. The isolated code is more preferred Nucleic acid molecule of naturally occurring Phytophthora infestans PSE or a biologically active part of it.

Another aspect of the invention relates to vectors, e.g. B. recombinant expression vectors containing at least one contain nucleic acid molecule according to the invention, and host cells into which these vectors have been introduced, in particular Microorganisms, plant cells, plant tissue, plant organs or whole Plants. Also not human animal host cells like for example Caenorhabditis elegans can be used advantageously. 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 are cells that contain fine chemicals or store, most preferred cells from storage tissues, such as seed coats, tubers, epidermis and sperm cells become.

Yet another aspect of the invention relates to a genetic one modified plant (= transgenic plant), preferably a Oil crop, as mentioned above, or a genetically modified one Mushroom (= transgenic mushroom), particularly preferably a mushroom from the Genus Phytophthora or a plant in which a PSE gene preferably from an oomycete such as advantageously Phytophthora infestans has been introduced or in which a PSE gene has been changed is. In one embodiment, the genome is from Phytophthora infestans by introducing an inventive Nucleic acid molecule encoding a wild-type or mutated PSE sequence, been changed as a transgene. In another embodiment is an endogenous PSE gene in the genome of the oomycete Phytophthora infestans by homologous recombination with a modified one PSE gene changed, i. H. functionally destroyed. At a preferred embodiment, the plant organism belongs to Genus Physcomitrella, Ceratodon or Funaria, whereby Physcomitrella is particularly preferred. In a preferred one The embodiment of the Physcomitrella plant is also used for production a desired compound such as lipids and fatty acids, wherein PUFAs are particularly preferred.

In yet another preferred embodiment, the Oomyzet Phytophthora to demonstrate the function of a Moosgens using homologous recombination based on the nucleic acids described in this invention can be used.

Yet another aspect of the invention relates to one isolated nucleic acid sequence according to the invention, a PSE gene or a Part, e.g. B. a biologically active part thereof. At a preferred embodiment, the isolated nucleic acid sequence, the PSE or part of it on the metabolism of building up Cell membranes in a microorganism or a plant cell necessary connections or on the transport of molecules whose membranes participate. Another preferred One embodiment is the isolated nucleic acid sequence, the PSE or part of it sufficiently homologous to one Amino acid sequence of SEQ ID NO: 2 that this protein or part of it the ability to metabolize to build cell membranes compounds necessary in microorganisms or plant cells or the transport of molecules across these membranes participate, maintains.

The invention also provides an isolated preparation of a PSE (PSE protein). In preferred embodiments, the nucleic acid sequence or the PSE gene according to the invention comprises an amino acid sequence of SEQ ID NO: 2. In a further 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 (the is encoded by the open reading frame shown in SEQ ID NO: 1). In another 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 (= identical) to an amino acid sequence of the sequence SEQ ID NO: 2. In other embodiments, the isolated nucleic acid sequence or PSE comprises an amino acid sequence which is at least about 50% homologous to one of the amino acid sequences of SEQ ID NO: 2 and can participate in the metabolism of compounds necessary for the formation of fatty acids in a microorganism or a plant cell or in the transport of molecules across these membranes or has one or more of the PUFA-prolonging activities, the prolongation of desatured C ₁₆ - and / or C ₁₈ -Carbon chains with double bonds in at least two places is meant.

Alternatively, the isolated PSE protein can be an amino acid sequence encoded by a nucleotide sequence attached to hybridizes a nucleotide sequence of SEQ ID NO: 1, e.g. More colorful stringent conditions hybridized, or at least approximately 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 to it is. It is also preferred that the preferred PSE forms also have one of the PSE activities described here.

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 This fusion protein has an activity that embodies differs from that of the PSE alone. With others preferred embodiment adopts this fusion protein Metabolism of compounds beneficial to the synthesis of Lipids and fatty acids, cofactors and enzymes in microorganisms or plants are necessary, or in the transport of molecules across these membranes. With particularly preferred Embodiments modulate the incorporation of this fusion protein into one Host cell producing a desired compound like advantageous for the synthesis of PUFAS by the cell. At a preferred embodiment, these fusion proteins also contain Δ-4-, Δ-5 or Δ-6 desaturase activities alone or in combination.

Another aspect of the invention relates to a method for Production of a fine chemical advantageous from unsaturated Fatty acids and / or lipids, the unsaturated fatty acids contain. This process involves either breeding a suitable microorganism or the cultivation of Plant cells, tissues, organs or whole plants, comprising the Nucleotide sequence according to the invention of SEQ ID NO: 1 or its Homologs, derivatives or analogues or a gene construct that the SEQ ID NO: 1 or their homologs, derivatives or analogs, or a vector that contains this sequence or the gene construct comprises the expression of a PSE according to the invention Nucleic acid molecule brings about, making a fine chemical is produced. In a preferred embodiment the method further includes the step of recovering a cell that has a contains such elongase nucleic acid sequence according to the invention, wherein a cell with an elongase nucleic acid sequence, a Gene construct or a vector which expresses an expression bring about PSE nucleic acid according to the invention, transformed becomes. In a further preferred embodiment comprises this method further includes the step of recovering the fine chemical from culture. In a particularly preferred embodiment the cell belongs to the order of the ciliates, to microorganisms, like mushrooms, or to the plant kingdom, especially to Oilfruit plants, microorganisms or are particularly preferred Oil crops.

Another aspect of the invention relates to methods for Modulation of the production of a molecule by a microorganism. These methods involve bringing the cell into contact with one Substance affecting PSE activity or PSE nucleic acid expression is modulated so that a cell-associated activity relative to the same activity in the absence of the substance is changed. In a preferred embodiment one or two metabolic pathway (s) of the cell for lipids and Fatty acids, cofactors and enzymes are modulated or the transport of Connections modulated across these membranes, so the yield or the rate of production of a desired fine chemical is improved by this microorganism. The substance which modulating PSE activity can be a substance that Stimulates PSE activity or PSE nucleic acid expression or the can be used as an intermediate in fatty acid biosynthesis can. Examples of substances that have the PSE activity or Stimulate PSE nucleic acid expression, u. a. small Molecules, active PSEs and PSEs-encoding nucleic acids that Cell have been introduced. Examples of substances which inhibit PSE activity or expression may include: a. small Molecules and antisense PSE nucleic 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 Gens or a wild-type or Mutant nucleic acid that is either kept on a separate plasmid or is integrated into the genome of the host cell, into a cell. at Integration into the genome can be random or by recombination such that the native Nucleic acid sequence or the native gene introduced by the Copy is replaced, thereby producing the desired one Connection is modulated by the cell, or by use of a gene in trans, so the gene is functional Expression unit, which at least one expression of a gene facilitating sequence and at least one Polyadenylation of a functionally transcribed gene or the Contains nucleic acid sequence facilitating sequence, functional connected is.

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 particularly 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 selected from arachidonic acid (ARA), eicosapentaenoic acid (AEP) or Docosahexaenoic acid (DHA).

Detailed description of the invention

The present invention provides a PSE nucleic acid and Protein molecule ready to participate in lipid and metabolism Fatty acids, PUFA cofactors and enzymes in the Oomyzet Phytophthora infestans or in the transport of lipophilic compounds Membranes are involved. The compounds of the invention can be used to modulate the production of fine chemicals 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, diesels, brassica species, such as rapeseed, diesels, and canola Turnips, pepper, sunflower, borage, evening primrose and tagetes, Solanaceae plants such as potato, tobacco, eggplant and tomato, Vicia species, peas, cassava, alfalfa, bush plants (coffee, Cocoa, tea), salix species, trees (oil palm, coconut) and perennial grasses and forage crops, either directly (e.g. if the overexpression or optimization of a Fatty acid biosynthesis protein has a direct impact on yield, Production and / or efficiency of the production of the fatty acid modified organisms) or can use one indirectly Have an impact that still increases yield, Production and / or production efficiency of a desired one Connection or a decrease in unwanted connections leads (e.g. when modulating the metabolism of lipids and Fatty acids, cofactors and enzymes to change the yield, Production and / or efficiency of production or Composition of the desired compounds within the cells, which in turn means the production of one or more fine chemicals can influence). Aspects of the invention are further below explained.

I. Fine chemicals and PUFAs

The term "fine chemical" is known in the art and includes molecules that have been produced by an organism and find applications in different industries, like, but not limited to, pharmaceutical, agricultural, Food and cosmetics industry. These connections include lipids, fatty acids, cofactors and enzymes, etc. (such as described in Kuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6, Rehm et al., Ed., VCH: Weinheim and literature references contained therein), 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, S. 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 on the 1st-3rd Sept. 1994 in Penang, Malaysia, AOCS Press (1995)), Enzymes and all others by Gutcho (1983) in Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086, and chemicals described therein. The Metabolism and uses of certain fine chemicals are explained further below.

The combination of different precursor molecules and Biosynthetic enzymes lead 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 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 Fatty acids, such as PUFAs, can therefore have new properties Generate membrane 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-glycerol 3-phosphate and the addition or modification of a polar Head group. Common lipids used in membranes include phospholipids, glycolipids, sphingolipids and Phosphoglycerides. Fatty acid synthesis begins with the conversion of Acetyl-CoA in either Malonyl-CoA through the Acetyl-CoA carboxylase or in acetyl-ACP by the 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 one Chain length is obtained. Production of unsaturated fatty acids these molecules become specific desaturases catalyzes, either aerobically using molecular oxygen or anaerobic (regarding fatty acid synthesis in microorganisms see F.C. Neidhardt et al. (1996) E. coli and Salmonella. ASM Press: Washington, D.C., pp. 612-636 and included 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) Microbiological Reviews 57: 522-542 and the references contained therein).

Precursors for PUFA biosynthesis are, for example, palmitoleic acid, linoleic and linolenic acid. These C ₁₆ and / or C ₁₈ carbon fatty acids must be extended to C ₂₀ and C ₂₂ in order to obtain fatty acids of the Eicosa and Docosa chain type. This elongation is advantageously carried out with the nucleic acids according to the invention or with the proteins encoded by these nucleic acids. With the help of various desaturases, such as enzymes which have Δ-6-desaturase, Δ-5 and Δ-4-desaturase activity, arachidonic acid, eicosapentenoic acid and docosahexaenoic acid and various other long-chain PUFAs can be obtained, extracted and used for various purposes in food, Feed, cosmetic or pharmaceutical applications can be used.

To produce long-chain PUFAs, as mentioned above, the polyunsaturated C ₁₆ and / or C ₁₆ 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 plant elongases which can extend C ₁₆ and / or C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms. After an elongation round, this enzyme activity leads to C ₁₈ or C ₂₀ fatty acids, and after two, three and four or five rounds of elongation to C ₂₂ , C ₂₄ or C ₂₆ fatty acids. Longer PUFAs can also be synthesized with the elongases 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, preferably with three or four double bonds, particularly preferably three double bonds in the fatty acid molecule. After elongation with the enzymes according to the invention, further desaturation steps can take place. Therefore, the products of the elongase activity 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-eicosatetraenoic, docosapentaenoic acid or docosahexaenoic acid. Substrates of this enzyme activity according to the invention are, for example, taxolic acid, 6,9-octadecadienoic acid, linoleic acid, γ-linolenic acid, pinolenic acid, α-linolenic acid or stearidonic acid. Preferred substrates are linoleic acid, γ-linolenic acid and / or α-linolenic acid. The C ₁₆ and / or C ₁₈ fatty acids with at least two double bonds in the fatty acid can be extended 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 become.

Furthermore, fatty acids have to be added to various Modifications transported and into the triacylglycerol storage lipid to be built in. Another important step in the 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).

Furthermore, the expression of the invention changes Nucleic acids in different host organisms not just that Total membrane lipid composition, but it changed the composition of all compounds in the host cell, which contain unsaturated fatty acids compared to original host cells that do not or do not contain the nucleic acids contained in these quantities. These changes are in Host organisms, for example, plant cells that are more pronounced proteins or enzymes encoded by the nucleic acids naturally not included. By expressing the This creates new lipid compositions that form a nucleic acid are another aspect of the invention.

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 - For assembly, including references, see in the following articles: Kinney, 1997, Genetic Engeneering, Editor: 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 nutraceuticals, such as PUFAs, include one A group of molecules that higher animals no longer synthesize can and must therefore take up or the higher animals not can produce more adequately and thus additionally need to absorb, although easily from other organisms, such as Bacteria to be synthesized. The biosynthesis of these molecules 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 others 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 different functions and are beneficial to health Effects, for example in coronary heart disease, Inflammation mechanisms, child nutrition, etc. publications and references, including those cited therein References, 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. You are also important starting materials for the synthesis of compounds that control important biological processes within the organism. You can therefore find them, for example, in various dietary ones Food or medication application.

II. Elements and methods of the invention

The present invention is based at least in part on the Discovery of a new molecule, which is called PSE nucleic acid and protein molecules, which have an effect on the production of cell membranes or fatty acids as in Physcomitrella patens, Ceratodon purpureus and / or Phytophthora exercise infestans and, for example, the movement of molecules influence over these membranes. In one embodiment take the PSE molecules from the metabolism to build up Cell membranes and / or fatty acids in organisms, such as microorganisms and plants, necessary connections in part or influence indirectly the transport of molecules across these membranes. In a preferred embodiment, the activity of PSE molecules according to the invention for regulating the production of Membrane components and membrane transport have an impact on the production of the desired fine chemical by this Organism. In a particularly preferred embodiment modulates the activity of the PSE molecules according to the invention, so that the yield, production and / or efficiency of production the metabolic pathways of microorganisms or plants, which regulate the PSEs according to the invention, are modulated and the Efficiency of the transport of connections through the membranes is changed, which is either directly or indirectly the yield, Production and / or production efficiency of a desired one Fine chemical modulated by microorganisms and plants.

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. Examples of PSEs are in SEQ ID NO: 1 or their homologs, 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 is a coding region and also corresponding 5'- and 3'-untranslated Sequence areas is. Examples of PSE genes are those in SEQ ID NO: 1 shown. The terms production or productivity are known in the art and include the concentration of Fermentation product (for example the desired one Fine chemical), which in a certain period of time and one certain fermentation volume is formed (e.g. kg product per hour per liter). The concept of production efficiency includes the time it takes to achieve a particular Production quantity is necessary (e.g. how long the cell takes to erect one certain throughput rate of a fine chemical is required). The The term yield or product / carbon yield is in Known in the art and includes the efficiency of converting the Carbon source in the product (i.e. the fine chemical). This is usually expressed, for example, as kg of product per kg Carbon source. By increasing yield or production the compound is the amount of molecules obtained or the appropriate obtained molecules of this compound in one certain amount of culture increased over a specified period. The terms biosynthesis or biosynthetic pathway are in the specialist field known and involve the synthesis of a compound, preferably an organic compound, through a cell Interconnections, for example in a multi-step and / or strong regulated process. The terms degradation or degradation route are in Known in the art and include splitting a connection, preferably an organic compound, through a cell in breakdown products (more generally, smaller or fewer complex molecules) for example in a multi-step and / or highly regulated process. The term metabolism is in the Known in the field and encompasses the entirety of biochemical Reactions that take place in an organism. The Metabolism of a certain compound (e.g. the metabolism a fatty acid) then encompasses all of the biosynthesis, Modification and degradation routes of this connection in the cell, that concern this connection.

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 production efficiency 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 within or out of the cell can be increased so larger amounts of these compounds across membranes are transported, from which they are more easily extracted and into one another 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 nucleic acids or PSE genes according to the invention can also produce PSEs with changed activities, which the production of one or more desired fine chemicals indirectly from microorganisms or plants. For example, PSEs according to the invention which are involved in the export of Waste products are involved, a larger number or higher Have activity so that the normal metabolic waste of the Cell (its amount may be due to overproduction the desired fine chemical is increased) efficiently exported before they can damage the molecules in the cell (what would reduce cell viability) or Fine chemical biosynthetic pathways (which affects the yield, Production or production efficiency of a desired one Would lower fine chemical). The relatively large intracellular Amounts of the desired fine chemical itself can also be used for the cell will be toxic so you can by increasing the activity or number of transporters that make these connections from the Cell export, cell viability in culture can increase, which in turn leads to a larger number of cells in the culture that carries the desired fine chemical to produce. The PSEs according to the invention can also be manipulated in this way that the corresponding amounts of different lipid and fatty acid molecules are produced. This can be a significant impact on the lipid composition of the cell membrane to have. Because each lipid type has different physical Features can change the Lipid composition of a membrane significantly increases membrane fluidity change. Changes in membrane fluidity can transport of molecules across the membrane as well as the integrity of the cell affect what each has a significant impact on the Production of fine chemicals from microorganisms and plants in fermentation culture on a large scale. plant membranes impart specific properties, such as tolerance towards Heat, cold, salt, dryness and tolerance to pathogens, like bacteria and fungi. Therefore, the modulation of the Membrane components have a fundamental impact on survivability of the plants under the stress parameters mentioned above. This can be done via changes in signal cascades or directly via the modified membrane composition take place (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 as disclosed in WO 95/18222, influence.

The isolated nucleic acid sequences according to the invention are in the Genome of a Phytophthora infestans strain, which for example available through collections such as ATCC or DSM are. The nucleotide sequence of the isolated Physcomitrella patens cDNA and the deduced amino acid sequences of the Phytophthora infestans -PSEs are shown in SEQ ID NO: 1 and 2, respectively. There were Computer analysis performed these nucleotide sequences as Sequences classified and / or identified that on Metabolism of cell membrane components or proteins involved in Transport of compounds across cell membranes or fatty acids encode involved proteins. An EST with database entry No. 08_ck19_b07 of the inventor is part of the sequence shown in FIG SEQ ID NO: 1. Meanwhile, these ESTs have been renamed, resulting in the revised name: pp001019019f led. In an analogous way the partial polypeptide was named pp001019019f. The full fragment insert of EST pp001019019f was sequenced and gave SEQ ID NO: 1, which is the complete Sequence of pp001019019f shows. It contains a complete, functionally active clone, of which is more specific Expression in yeast turned out to be the one he wanted Had substrate specificity, as shown in the example part. Yeasts are also suitable organisms according to the invention, for example as host cells for the genes according to the invention, Gene constructs or vectors.

The present invention also relates to proteins with a Amino acid sequence that is essentially homologous (= identical) to one Amino acid sequence of SEQ ID NO: 2. As used here is a protein with an amino acid sequence that is essentially is homologous to a selected amino acid sequence at least about 50% homologous to the selected amino acid sequence, z. B. the entire selected amino acid sequence. A protein with an amino acid sequence that is essentially homologous to a selected amino acid sequence can also be at least about 50 to 60%, preferably at least about 60 to 70% and more preferably at least about 70 to 80%, 80 to 90% or 90 to 95% and most preferably at least about 96%, 97%, 98%, 99% or more homologous to a selected amino acid sequence 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 across these membranes or can have one or more of the activities required for the elongation of C ₁₈ PUFAs , so that C ₂₂ or C ₂₄ PUFAs and 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 an isolated nucleic acid derived from an oomycete and encoding a polypeptide that extends C ₁₆ and / or C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms.

• a) einer in SEQ ID NO: 1 dargestellten Nukleinsäuresequenz,
• b) einer Nukleinsäuresequenz, die gemäß dem degenerierten genetischen Code von der in SEQ ID NO: 1 dargestellten Sequenz stammt,
• c) Derivate der in SEQ ID NO: 1 dargestellten Sequenz, die Polypeptide mit mindestens 50% Homologie zu der Sequenz kodiert, die die Aminosäuresequenzen in SEQ ID NO: 2 kodiert, wobei die Sequenz als C₁₆- oder C₁₈-Elongase wirkt.

Another embodiment of the invention is an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide that extends C ₁₆ and / or C ₁₈ fatty acids with at least two double bonds in the fatty acid, selected from the group consisting of

• a) a nucleic acid sequence shown in SEQ ID NO: 1,
• b) a nucleic acid sequence which, according to the degenerate genetic code, comes from the sequence shown in SEQ ID NO: 1,
• c) Derivatives of the sequence shown in SEQ ID NO: 1, which encodes polypeptides with at least 50% homology to the sequence which encodes the amino acid sequences in SEQ ID NO: 2, the sequence acting as a C ₁₆ or C ₁₈ elongase.

The above-mentioned nucleic acid according to the invention comes from Organisms such as ciliates, fungi, algae or dinoflagellates, that can synthesize PUFAs, preferably from plants or Mushrooms, particularly preferably from the genus Phytophthora and am most preferred by Phytophthora infestans.

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 Amplification of a PSE-encoding nucleic acid (e.g. PSE-DNA) suffice. The term "nucleic acid molecule" as used herein should be single-stranded or double-stranded DNA molecules (e.g. cDNA or genomic DNA) and / or RNA molecules (e.g. mRNA) as well as DNA or RNA analogs that are generated using nucleotide analogs, or DNA / RNA hybrids. This term also includes the 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 double-stranded DNA. An "isolated" nucleic acid molecule is from other nucleic acid molecules separated in the natural Source of the nucleic acid are present. An "isolated" nucleic acid preferably has no sequences which the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates, flank naturally (e.g. sequences that are located at the 5'- and 3 'ends of the nucleic acid). At different Embodiments can be used to isolate the isolated PSE nucleic acid molecule Example less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or contain 0.1 kb of nucleotide sequences that are natural the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid originates (e.g. a Physcomitrella patens cell) flank. An "isolated" nucleic acid molecule, such as a cDNA Molecule, moreover, can be essentially free of anything else cellular material or culture medium if by recombinant Techniques are manufactured, or free of chemical precursors or other chemicals if it is chemically synthesized becomes.

A nucleic acid molecule according to the invention, e.g. B. a Nucleic acid molecule with a nucleotide sequence of SEQ ID NO: 1 or part of it can be used using molecular biology Standard techniques and the provided here Sequence information can be isolated. For example, a Phytophthora infestans cDNA are isolated from a P. infestans library, by using the full SEQ ID NO: 1 or part of it as Hybridization probe as well as standard hybridization techniques (as described, for example, in Sambrook et al., Molecular Cloning: Laboratory manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) can be used. Moreover, one can Nucleic acid molecule comprising a complete sequence of SEQ ID NO: 1 or isolate part of it by polymerase chain reaction, wherein Oligonucleotide primers based on this sequence or by For parts of it, especially regions around His box motifs, see Shanklin et al. (1994) Biochemistry 33, 12787-12794 have been used (e.g. a nucleic acid molecule, comprising the complete sequence of SEQ ID NO: 1 or one Part of it, by polymerase chain reaction using Oligonucleotide primers are isolated based on this same sequence of SEQ ID NO: 1 have been created). To the For example, mRNA can be isolated from oomycete cells (e.g. by the guanidinium thiocyanate extraction method by Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and reverse cDNA 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) produce. Synthetic oligonucleotide primers for amplification by means of a polymerase chain reaction on the basis of a create the nucleotide sequences shown in SEQ ID NO: 1. A The nucleic acid according to the invention can be used using cDNA or alternatively from genomic DNA as a template and suitable Oligonucleotide primers according to standard PCR amplification techniques be amplified. The nucleic acid amplified in this way can be in a suitable vector can be cloned and by means of DNA sequence analysis can be characterized. Oligonucleotides that a PSE Nucleotide sequence can correspond by Standard synthesis methods, for example with an automatic DNA synthesizer.

The cDNA shown in SEQ ID NO: 1 comprises sequences, the PSEs encode (i.e. the "coding area") and 5'-untranslated sequences and 3'-untranslated sequences. alternative the nucleic acid molecule can only encode the coding region of the sequences in SEQ ID NO: 1 or can be whole genomic Contain fragments isolated from genomic DNA.

In a further preferred embodiment, a comprises isolated nucleic acid molecule according to the invention Nucleic acid molecule that is a complement of one of the SEQ ID NO: 1 shown nucleotide sequences or a part thereof. On Nucleic acid molecule belonging to one of those shown in SEQ ID NO: 1 Nucleotide sequences is complementary, is one that is one of the nucleotide sequences shown in SEQ ID NO: 1 are sufficient is complementary that it is with one of the SEQ ID NO: 1 sequences can hybridize, creating a stable duplex arises.

Homologs of the new elongase nucleic acid sequence with the sequence SEQ ID NO: 1 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 shown nucleotide sequences or their homologues, Derivatives or analogs or parts thereof, where homology in Identity means the meaning of the invention. Another preferred embodiment comprises an isolated according to the invention Nucleic acid molecule a nucleotide sequence that is attached to one of the in SEQ ID NO: 1 shown nucleotide sequences or a part thereof hybridized, e.g. B. hybridized under stringent conditions. Allelic variants include functional ones in particular Variants characterized by deletion, insertion, or substitution of nucleotides from / in the sequence shown in SEQ ID NO: 1 , but the intent is that the Enzyme activity of the resulting synthesized proteins for the Advantageously maintain insertion of one or more genes becomes. Proteins that still have the enzymatic activity of elongase own, 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 encoded protein.

Homologs of SEQ ID NO: 1 also means, for example bacterial, fungal and plant homologs, shortened sequences, single-stranded DNA or RNA of the coding and non- coding DNA sequence.

Homologs of SEQ ID NO: 1 also means derivatives such as 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 modifying their Sequence is increased or that it is completely more active Promoters, even from heterologous organisms, can be replaced.

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 include, for example, a fragment used as a probe or primer can be used, or a fragment which a encoded biologically active section of a PSE. The one from the Cloning of the P. infestans PSE gene determined Nucleotide sequences enable the generation of probes and primers those used to identify and / or clone PSE homologs in other cell types and organisms as well as PSE homologs from others Oomycetes or related species are designed. The probe / the Primer usually comprises essentially purified Oligonucleotide. The oligonucleotide usually includes one Nucleotide sequence region of at least under stringent conditions about 12, preferably about 16, more preferably about 25, 40, 50 or 75 consecutive nucleotides of a sense strand one of the sequences given in SEQ ID NO: 1, an antisense Strands one of the sequences given in SEQ ID NO: 1 or its homologs, derivatives or analogues or of course occurring mutants hybridized therefrom. Primer based on a Nucleotide sequence of SEQ ID NO: 1 can be used in PCR reactions Cloning of PSE homologs can be used. Probes on the The PSE nucleotide sequences can be used to detect Transcripts or genomic sequences that are the same or homologous Encode proteins. With preferred In embodiments, the probe also includes a probe attached thereto Marker group, e.g. B. a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. These probes can be part of a test kit for genomic markers to identify Cells that misexpress a PSE, for example by measuring an amount of a PSE-encoding nucleic acid in one Cell sample, e.g. B. Measure PSE mRNA levels, or to determine if a genomic PSE gene is mutated or deleted become.

In one embodiment, the inventive codes Nucleic acid molecules a protein or a part thereof, the one Amino acid sequence that is sufficiently homologous to one Amino acid sequence of SEQ ID NO: 2 is that the protein or the part of which is the ability to build on the metabolism of Cell membranes necessary in microorganisms or plants Connections or the transport of molecules across these membranes or participate in fatty acid synthesis. Like here used, the term "sufficiently homologous" refers to proteins or parts thereof, the amino acid sequences of which are minimal identical or equivalent amino acid residues (e.g. one Amino acid residue with a side chain similar to an amino acid residue in one of the sequences of SEQ ID NO: 2) to one Have amino acid sequence of SEQ ID NO: 2, so that the protein or Part of it on the metabolism of building cell membranes in Microorganisms or plants necessary compounds or Transport of molecules can take place across these membranes. 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 Play fine chemicals. Examples of these activities are also described here. Thus, the "function of a PSE" either directly or indirectly to yield, production and / or Efficiency in the production of one or more fine chemicals.

Examples of PSE substrate specificities for catalytic Activity is given in Table I.

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. The homology of Amino acid sequence was run across the entire sequence range using the program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5, 1989: 151-153).

Parts of proteins used by the invention PSE nucleic 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 / a motive to include a PSE that is involved in the metabolism of the Structure of cell membranes in microorganisms or plants necessary connections or the transport of molecules via them Membranes can participate or participate in fatty acid synthesis or has an activity shown in Table I. to Determine whether a PSE or a biologically active part of it is on Metabolism to build cell membranes in microorganisms necessary connections or plants or at the transport of A test of molecules can participate across these membranes enzymatic activity can be performed. These test procedures, as described in detail in Example 8 of the example part familiar to the expert.

Additional nucleic acid fragments that are biologically active Coding sections of a PSE can be done by isolation a part of the sequence shown in SEQ ID NO: 2 the encoded portion of the PSE or peptide (e.g., by recombinant expression in vitro) and determination of the activity of the encoded portion of the PSE or peptide.

The invention also includes nucleic acid molecules that differ from one of the nucleotide sequences shown in SEQ ID NO: 1 (and parts of this) due to the degenerate genetic code and thus encode the same PSE as that used by those in SEQ ID NO: 1 nucleotide sequences shown is encoded. At a another embodiment has an insulated according to the invention Nucleic acid molecule is a nucleotide sequence that contains a protein an amino acid sequence shown in SEQ ID NO: 2. at a further embodiment encodes the invention Nucleic acid molecule a full-length Phytophthora infestans Protein that forms an amino acid sequence of SEQ ID NO: 2 (the encoded by an open reading frame shown in SEQ ID NO: 1 is) is essentially homologous.

In addition to the Phytophthora infestans shown in SEQ ID NO: 1 -PSE nucleotide sequences, the skilled worker recognizes that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the PSEs result in a population (e.g. the Phytophthora infestans population) can exist. This genetic Polymorphisms in the PSE gene can occur between individuals within one Population due to natural variation exist. How As used herein, the terms "gene" and "recombinant" mean Gene "nucleic acid molecules with an open reading frame, the one PSE, preferably a Phytophthora infestans PSE, encoded. This natural variants usually result in a variance of 1 up to 5% in the nucleotide sequence of the PSE gene. All and all of these nucleotide variations and resulting ones Amino acid polymorphisms in PSE that are the result of natural variation are and do not change the functional activity of PSEs, are intended to be included within the scope of the invention.

Nucleic acid molecules that correspond to the natural variants, and not Phytophthora infestans homologues, derivatives and analogs the Phytophthora infestans-PSE cDNA according to the invention based on their homology to that disclosed here Phytophthora infestans-PSE nucleic acid using the Phytophthora infestans cDNA or a part thereof as Hybridization probe according to standard hybridization techniques below stringent hybridization conditions are isolated. At a another embodiment is an isolated one according to the invention Nucleic acid molecule at least 15 nucleotides long and hybridizes to the nucleic acid molecule under stringent conditions, which comprises a nucleotide sequence of SEQ ID NO: 1. 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 Examples of stringent hybridization conditions are Hybridizations 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. The skilled worker is 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 hybridization conditions" between depending on the type of nucleic acid 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 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 temperatures are, for example, for a nucleic acid with 100 bp (= base pairs) in length and a G + C content of 50% determined in the absence of formamide. The expert knows how the 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 (ed.) 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. The specialist is known that the nature and time of development of the Hybridization results have an impact on the hybridization result Has. In simple experiments, he is able to do that To optimize development conditions so that the aforementioned hybridization delivers reliable safe results.

An isolated one according to the invention preferably corresponds Nucleic acid molecule attached to a sequence under stringent conditions the SEQ ID NO: 1 hybridizes, a naturally occurring one Nucleic acid molecule. As used here, a "of course concerns occurring "nucleic acid molecule an RNA or DNA molecule with a nucleotide sequence that occurs in nature (e.g. a encodes natural protein). Encoded in one embodiment the nucleic acid is a naturally occurring phytophthora infestans PSE.

In addition to naturally occurring variants of the PSE sequence, those skilled in the art will recognize those that may exist in the population furthermore that changes by mutation into one Nucleotide sequence of SEQ ID NO: 1 can be introduced, resulting in Changes in the amino acid sequence of the encoded PSE results without that the functionality of the PSE protein is impaired. For example, nucleotide substitutions that are "non-essential" amino acid residues for amino acid substitutions lead, in a sequence of SEQ ID NO: 1. A "no- essential "amino acid residue is a residue that is in a Wildtypenzsequenz one of the PSEs (SEQ ID NO: 2) can change, without changing the activity of the PSE, whereas a "Essential" amino acid residue for PSE activity is required. Other amino acid residues (e.g. those in the Domain with PSE activity not conserved or only are semi-preserved) but cannot be used for the activity be essential and can therefore probably be changed without that PSE activity is changed.

Accordingly, in another aspect relates to the invention Nucleic acid molecules that encode PSEs, the modified amino acid residues contain that are not essential for PSE activity. This PSEs differ in amino acid sequence from one Sequence in SEQ ID NO: 2 and still keep at least one of the PSE activities described here. The isolated In one embodiment, the nucleic acid molecule comprises a nucleotide sequence, which encodes a protein, the protein being an amino acid sequence with at least about 50% homology to an amino acid sequence which includes SEQ ID NO: 2 and on the metabolism of to build up Cell membranes in Phytophthora infestans necessary connections or participate in the transport of molecules across these membranes can or is involved in fatty acid metabolism or one or has several of the activities listed in Table I. The protein encoded by the nucleic acid molecule is preferred at least about 50 to 60% homologous to one of the sequences in SEQ ID NO: 2, more preferably at least about 60 to 70% homologous to one of the sequences in SEQ ID NO: 2, even more preferred at least about 70 to 80%, 80 to 90%, 90 to 95% homologous to one of the sequences in SEQ ID NO: 2 and most preferred at least about 96%, 97%, 98% or 99% homologous to one of the Sequences in SEQ ID NO: 2.

To determine the percentage homology of two Amino acid sequences (e.g. one of the sequences of SEQ ID NO: 2 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 can be inserted to an optimal Alignment with the other protein or nucleic acid produce). The amino acid residues or nucleotides on the corresponding amino acid positions or nucleotide positions are then compared. If a position in a sequence (e.g. one of the Sequences of SEQ ID NO: 2) by the same amino acid residue or the same nucleotide as the corresponding site in the other Sequence (e.g. a mutated form of the one from SEQ ID NO: 2 selected sequence), the molecules are on it Position homologous (i.e. amino acid or nucleic acid "homology", as used here corresponds to amino acid or nucleic acid "Identity"). The percentage homology between the two Sequences is a function of the number of identical positions, which 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 is homologous to a protein sequence of SEQ ID NO: 2 Introduction of one or more nucleotide substitutions, additions or deletions generated in a nucleotide sequence of SEQ ID NO: 1 so that one or more amino acid substitutions, additions or deletions introduced into the encoded protein become. Mutations can occur in one of the sequences of SEQ ID NO: 1 through standard techniques, such as site-specific Mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are preferred one or more of the predicted non-essential ones Amino acid residues produced. With a "conservative Amino acid substitution "is the amino acid residue against one Amino acid residue exchanged with a similar side chain. in the The subject is families of amino acid residues with similar ones Side chains have been defined. These families include amino acids basic side chains (e.g. lysine, arginine, histidine), acidic Side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, Threonine, tyrosine, cysteine), non-polar side chains, (e.g. Alanine, valine, leucine, isoleucine, proline, phenylalanine, Methionine, tryptophan), beta-branched side chains (e.g. threonine, Valine, isoleucine) and aromatic side chains (e.g. tyrosine, Phenylalanine, tryptophan, histidine). A predicted non- essential amino acid residue in a PSE is thus preferred through another amino acid residue from the same Side chain family replaced. Alternatively, another one Embodiment the mutations randomly over the whole or part of the PSE coding sequence can be introduced, e.g. B. by saturation mutagenesis, and the resulting mutants can be screened for the PSE activity described here to identify mutants that have PSE activity maintained. After mutagenesis of one of the sequences of SEQ ID NO: 1 the encoded protein can be expressed recombinantly, and the activity of the protein can e.g. B. using the here described tests (see example part) can be determined.

In addition to the nucleic acid molecules that the above encoding PSEs described concerns another aspect of Invention isolated nucleic acid molecules, the "antisense" to it are. An "antisense" nucleic acid includes one Nucleotide sequence resulting in a "sense" nucleic acid, which is a protein is coded, 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 via hydrogen bonds to a sense nucleic acid tie. The antisense nucleic acid can form an entire PSE coding strand or only complementary to part of it his. In one embodiment, is a Antisense nucleic acid molecule "Antisense" to a "coding area" of the coding strand of a nucleotide sequence encoding a PSE. The The term "coding area" refers to the area of Nucleotide sequence comprising codons that translate into amino acid residues (e.g. the entire coding area that corresponds to the Stop codon begins and ends, i. H. the last codon before Stop codon). In a further embodiment, the antisense Nucleic acid molecule "antisense" to a "non-coding Region "of the coding strand of a nucleotide sequence which Encoded PSE. The term "non-coding area" refers to 5 'and 3' sequences flanking the coding area and not be translated into amino acids (i.e. which one too referred to as 5'- and 3'-untranslated areas).

Provided the PSE encoding disclosed here Sequences of the coding strand (e.g. those in SEQ ID NO: 1 sequences shown) antisense according to the invention Nucleic acids according to the rules of Watson-Crick base pairing be designed. The antisense nucleic acid molecule can to be complementary to the entire coding region of PSE mRNA more preferably an oligonucleotide that is only a part of the coding or non-coding region of PSE mRNA "Antisense" is. The antisense oligonucleotide can e.g. B. to that 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 long his. An antisense nucleic acid according to the invention can be found under Use chemical synthesis and enzymatic Ligation reactions constructed using methods known in the art become. An antisense nucleic acid (e.g. a Antisense oligonucleotide) can e.g. B. can be synthesized chemically, wherein naturally occurring nucleotides or variously modified Nucleotides are used that are designed so that they increase the biological stability of the molecules or the physical stability of the between the antisense and the sense Increase nucleic acid formed duplexes, for example Phosphorothioate derivatives and acridine substituted nucleotides be used. Examples of modified nucleotides used for Generation of the antisense nucleic acid can be used are u. a. 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-ioduracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, Beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 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 Antisense nucleic acid can alternatively be used biologically an expression vector are prepared, in which a Nucleic acid has been subcloned in the antisense direction (i.e. RNA, which is transcribed by the inserted nucleic acid, is of interest to a target nucleic acid of interest in 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 the cellular mRNA and / or genomic DNA, that encode, hybridize, or bind to a PSE to thereby expression of the protein, e.g. B. by inhibiting transcription and / or translation. The hybridization can be done by conventional nucleotide complementarity to form a stable duplexes or z. B. in the case of a 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 a receptor or to one on a selected cell surface expressed antigen binds e.g. B. by binding the antisense Nucleic acid molecule to a peptide or an antibody, the binds to a cell surface receptor or an antigen. The Antisense nucleic acid molecule can also be used here described vectors are supplied to the cells. To achieve sufficient intracellular concentrations of Antisense molecules are vector constructs in which the Antisense nucleic acid molecule under the control of a strong prokaryotic, viral or eukaryotic, including plant, Promoter is preferred.

In a further embodiment, this is the invention Antisense nucleic acid molecule is an α-anomeric nucleic acid molecule. On α-anomeric nucleic acid molecule forms specific double-stranded ones Hybrid with complementary RNA, the strands being unlike ordinary β units run parallel to each other. (Gaultier et al. (1987) Nucleic Acids Res. 15: 6625-6641). The antisense Nucleic acid molecule can also be a 2'-o-methyl ribonucleotide (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 (e.g. Hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591)) for the catalytic cleavage of PSE mRNA Transcripts are used to help translate to inhibit PSE mRNA. A ribozyme with specificity for one Nucleic acid encoding PSE can be based on the Nucleotide sequence of a PSE cDNA disclosed here (i.e. 38_ck21_g07fwd in SEQ ID NO: 1) or on the basis of one in accordance with this Invention taught methods to isolate heterologous sequence be designed. For example, a derivative of a Tetrahymena-L-19 IVS RNA are constructed, the Nucleotide sequence of the active site complementary to the nucleotide sequence which is to be cleaved in a PSE-encoding mRNA. See e.g. B. Cech et al., U.S. Patent No. 4,987,071 and Cech et al., U.S. Patent No. 5,116,742. Alternatively, PSE mRNA can be used for selection a catalytic RNA with a specific Ribonuclease activity from a pool of RNA molecules can be used. See e.g. B. Bartel, D., and Szostak, J.W. (1993) Science 261: 1411-1418.

Alternatively, PSE gene expression can be inhibited by Nucleotide sequences complementary to regulatory Region of a PSE nucleotide sequence (e.g. a PSE promoter and / or enhancers) are directed so that Triple helix structures are formed, which are the transcription of a Inhibit PSE genes in target cells. 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.

Another alternative is PSE gene expression inhibit by co-suppression, also simultaneous expression of a combined antisense / sense strand (RNAi technique) advantageously possible.

B. gene construct

Another embodiment of the invention is a new gene construct, which means an isolated nucleic acid that comes from a plant or a fungus and encodes a polypeptide that encodes C ₁₆ and / or C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two Extends carbon atoms, or the gene sequence of SEQ ID NO: 1, their homologs, derivatives or analogs, which 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 have been inserted before the sequence SEQ ID NO: 1 or its homologs 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 allow increased expression of the nucleic acid sequence. It is also possible to additionally insert advantageous sequences, for example further regulatory elements or terminators, at the 3 'end of the DNA sequences. 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 novel process 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, MFα, 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. In this context, inducible promoters are also advantageous, such as those in EP-A-0 388 186 (benzylsulfonamide inducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracycline inducible), EP-A-0 335 528 (abzisic 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 which can be used for monocotyledons or dicotyledons and are described in US Pat. , WO 91/13980 (Bce4 promoter from Brassica), by Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (LEB4 promoter from legumes) are described, these promoters being suitable for dicotyledons. The following promoters are suitable, for example, for monocotyledons lpt-2 or lpt-1 promoter from barley (WO 95/15389 and WO 95/23230), hordein promoter 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 one Procedure to use. It is also possible and advantageous additionally use synthetic promoters.

As described above, the gene construct can also contain other genes include which 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 Intervene, insert and insert genes of a biosynthetic pathway to 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 or its homologs.

The gene construct advantageously comprises for the expression of the other present genes further 3'- and / or 5'-terminals 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 vary depending on the host organism for example, mean that the gene only expresses after induction or is overexpressed or that it expresses immediately and / or is overexpressed.

The regulatory sequences or factors can also preferably an advantageous effect on the expression of the have introduced genes and thus increase them. In this way it is possible to use the regulatory elements strong transcription signals, such as promoters and / or enhancers, advantageously be enhanced at the transcription level. It however, translation is still possible, for example by enhancing mRNA stability.

C. Recombinant Expression Vectors and Host Cells

Another aspect of the invention relates to vectors preferably expression vectors which contain a nucleic acid, encoding a PSE (or part of it). Like here used, the term "vector" refers to a nucleic acid molecule, that can transport another nucleic acid to which it is bound. A vector type is a "plasmid", what there is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another Vector type is a viral vector, with additional DNA segments can be ligated into the viral genome. Certain vectors can in a host cell into which they have been placed replicate autonomously (e.g. bacterial vectors with bacterial Origin of replication and episomal mammalian vectors). Other Vectors (e.g. non-episomal mammalian vectors) are used in the Introduction into the host cell in the genome of a host cell integrated and thereby replicated together with the host genome.

In addition, certain vectors can express genes with to which they are functionally connected. These vectors are referred to here as "expression vectors". Usually have expression vectors used for recombinant DNA techniques are suitable, the form of plasmids. In the present Description can "plasmid" and "vector" interchangeably be used because the plasmid is the most commonly used Is vector shape. However, the invention is intended to address these others Expression vector forms, such as viral vectors (e.g. replication-deficient retroviruses, adenoviruses and adeno-related viruses), perform similar functions. Furthermore, 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 circular DNA.

The recombinant expression vectors according to the invention comprise a nucleic acid according to the invention or one according to the invention Gene construct in a form that is suitable for expression of the Nucleic acid in a host cell, which means that the recombinant expression vectors one or more Regulatory sequences selected based on the expression using host cells with the one to be expressed Nucleic acid sequence is operably linked. In one recombinant expression vector means "functional linked "that the nucleotide sequence of interest is such the regulatory sequence (s) is linked to the expression the nucleotide sequence is possible and bound to each other are so that both sequences are the predicted, the sequence perform the assigned function (e.g. in an in vitro Transcription / translation system or in a host cell, when the vector is introduced into the host cell). The The term "regulatory sequence" is intended to be promoters, enhancers and others Expression control elements (e.g. polyadenylation signals) include. These regulatory 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, including the references therein. Regulatory sequences include those that are constitutive Expression of a nucleotide sequence in many host cell types control, and those that direct expression of Nucleotide sequence only in certain host cells under certain Control conditions. The expert knows that the design the expression vector of factors such as the selection of the transforming host cell, the level of expression of the desired protein, etc., may depend. The invention Expression vectors can be introduced into host cells in order to thereby proteins or peptides, including fusion proteins or peptides to be produced by the nucleic acids as here described, encoded (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. 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: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella and Stylonychia, especially the genus Stylonychia lemnae, with vectors after a transformation process, such as described in WO 98/01572, and cells of multicellular plants (see Schmidt, R. and Willmitzer, L. (1988) "High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants "Plant Cell Rep .: 583-586; Plant Molecular Biology and Biotechnology, 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 cited therein References)) or non-human mammalian cells are expressed. 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 can alternatively, for example using T7 promoter Regulatory sequences and T7 polymerase, transcribed in vitro and be translated.

Proteins are mostly expressed in prokaryotes with vectors that are constitutive or inducible promoters contain the expression of fusion or control non-fusion proteins. Fusion vectors add a series of Amino acids to a protein encoded therein, usually on Amino terminus of the recombinant protein, but also at the C-terminus or fuses 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. In the case of fusion Expression vectors often become a proteolytic cleavage site the junction of the fusion unit and the recombinant Protein introduced so that the separation of the recombinant Protein from the fusion unit after cleaning the Fusion protein is possible. These enzymes and their corresponding ones 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. At a The PSE coding sequence is in one embodiment pGEX expression vector cloned, so that a vector is generated that a Fusion protein encodes that from the N-terminus to the C-terminus GST thrombin cleavage site X protein. The fusion protein can be by affinity chromatography using Glutathione-agarose resin can be cleaned. Recombinant PSE, which is not merged with GST can be split by 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 is based on the transcription by host RNA polymerase from one Hybrid trp-lac fusion promoter. The target gene expression from the pET 11d vector is based on the transcription of a T7-φ10-lac- Fusion promoter generated by a coexpressed viral RNA polymerase (T7 gn1) is mediated. This viral polymerase will from the host strains BL21 (DE3) or HMS174 (DE3) from one resident λ prophage provided a T7 φ1 gene under the Transcription control of the lacUV 5 promoter harbors.

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,? gt11 or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or p774bJ6, or CBDn2A6, in CBDn2A6, in PBD2146 or PBD2146, or CBDn2A6, in PBD2146, or CBDn2A6, in CBD2146, or PBD2146, in CBD2146 or PBD2146, pC194 or pBDn2146

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 Nucleic acid sequence in an expression vector insert nucleic acid so that the individual codons for each Amino acids are those that are preferably used in expression 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 for example 2αM, 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 for expression of 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, one Nucleic acid according to the invention in mammalian cells using a mammalian Expression vector expressed. examples for Mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). In the The control functions of the Expression vector often from viral regulatory elements provided. Commonly used promoters come from e.g. B. from Polyoma, Adenovirus2, Cytomegalovirus 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 certain 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. neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477), pancreatic promoters (Edlund et al., (1985) Science 230: 912-916) and mammary-specific promoters (e.g. Whey promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166). Also development-regulated promoters are included, e.g. B. the hox promoters the mouse (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 those that 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.

A plant expression cassette preferably contains Regulatory sequences, which the gene expression in Can control plant cells and are functionally connected so that each sequence its function, such as termination of transcription, can fulfill, for example polyadenylation signals. Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens-t-DNA are derived, such as 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 others are functionally active terminators in plants suitable.

Because plant gene expression very often does not show up Transcriptional levels is restricted, contains a plant expression cassette preferably other operably linked sequences, such as Translation enhancers, for example the overdrive sequence, which represents the 5'-untranslated leader sequence Tobacco mosaic virus, which 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 (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those derived from plant viruses, such as 35S CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also US 5352605 and WO 84/02913) or plant promoters such as that in US 4,962,028 described the small subunit of the Rubisco.

Other preferred sequences for use in are functional connection in plant gene expression cassettes Targeting sequences that are used to control the gene product appropriate cell compartments are necessary (see one Overview 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, Chloroplasts, chromoplasts, the extracellular space, the Mitochondria, 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 the gene expression is time-specific Way is done. Examples of such promoters are a Salicylic acid-inducible promoter (WO 95/19443) Tetracycline inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol inducible promoter.

Also promoters based on biotic or ablotic Responding to stress 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 the wound-inducible pinII promoter (EP-A-0 375 091).

In particular, those 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 promoter from Arabidopsis (WO 98/45461), the phaseolin promoter Phaseolus vulgaris (US 5,504,200), the Bce4 promoter from Brassica (WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9) and promoters, which seed-specific expression in monocot plants, such as Bring corn, barley, wheat, rye, rice, etc. suitable Notable promoters are the lpt2 or lpt1 gene promoter from barley (WO 95/15389 and WO 95/23230) or those in WO 99/16890 (promoters from the barley hordein gene, the rice Glutelin gene, the rice oryzin gene, the rice prolamin gene, the Wheat gliadin gene, wheat glutelin gene, the maize zein gene, the Oat-glutelin gene, the sorghum-kasirin gene, the rye-secalin gene 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, are described in WO 95/16783 and WO 97/06250, and the clpP- Arabidopsis promoter, described in WO 99/46394.

The invention also provides a recombinant Expression vector ready, comprising a DNA molecule according to the invention, that clones in the antisense direction into the expression vector 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. It can Regulatory sequences are selected that work with an in Antisense direction cloned nucleic acid are linked 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 Control cell type-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 one regulatory area, the activity of which is produced by the Cell type can be determined in which the vector is introduced has been. An explanation of the regulation of gene expression using antisense genes see in Weintraub, H., et al., Antisense 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", "recombinant host cell" and "transgenic host cell" are interchangeable here used. Of course, these terms do not apply only the specific target cell, but also the offspring or potential descendants of this cell. Because in successive Generations due to mutation or environmental influences certain modifications may occur, these are offspring are not necessarily identical to the parental cell, but are still encompassed by the scope of the term as used here.

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 cells (CHO) or COS cells), algae, Ciliates, plant cells, fungi or other microorganisms, such as C. glutamicum. Other suitable Host 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 Introducing foreign nucleic acid (e.g. DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, natural Competence, chemically mediated transfer, electroporation or particle bombardment. Suitable procedures for Transformation or transfection of host cells, including Plant cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual., 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals such as Methods in Molecular Biology, 1995, vol. 44, Agrobacterium protocols, eds .: Gartland and Davey, Humana Press, Totowa, New Jersey.

It is known about the stable transfection of mammalian cells that depending on the expression vector used and Transfection technology only a small part of the cells' foreign DNA integrated into genome. To identify and select them 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 Biosynthetic pathways involved, for example, of 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 use in which these genes are not functional since they 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 is PSE encoded, or can be introduced on a separate vector become. Cells stable with the introduced nucleic acid have been transfected, for example by Drug selection can be identified (e.g. cells that survive the have integrated selectable markers, whereas the others Cells die).

To generate 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 thereby change the PSE gene, e.g. B. functionally disrupt. This PSE gene is preferred a Physcomitrella patens or Phytophthora infestans PSE gene, however, it can be a homologue or analogue from a related one Plant or even from a mammalian, yeast, or insect source be used. In a preferred embodiment the vector is designed to contribute to the endogenous PSE gene homologous recombination is functionally disrupted (i.e. not longer encodes a functional protein, also as a knock-out Vector)). Alternatively, the vector can be designed in this way be the endogenous PSE gene in homologous recombination mutated or otherwise changed, but still a encodes functional protein (e.g., the 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 a 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 one Section of the PSE gene at its 5 'and 3' end from additional Flanked nucleic acid of the PSE gene, making it homologous Recombination between the exogenous PSE gene that is on the vector is present, 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 (both at the 5 'and 3' ends) included (a description for vectors for homologous recombination see e.g. B. in Thomas, K.R., and Capecchi, M.R. (1987) Cell 51: 503 or der Recombination in Physcomitrella patens based on cDNA in Strepp et al., 1998, Proc. Natl. Acad. Sci. USA 95 (8): 4368-4373). The vector is placed in a microorganism or a plant cell (e.g. by means of polyethylene glycol-mediated DNA), and Cells in which the introduced PSE gene with the endogenous PSE Gene is homologously recombined using the Selected field of known techniques.

In another embodiment, recombinant organisms, how microorganisms are made, the selected systems contain, which a regulated expression of the introduced Enable gene. The inclusion of a PSE gene in a vector being brought under the control of the lac operon, enables z. B. expression of the PSE gene only in the presence by IPTG. These regulation systems are known in the art.

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, this includes Process the cultivation of the host cell according to the invention (in the a recombinant expression vector encoding a PSE, has been introduced, or a gene has been introduced into the genome thereof encoding a wild-type or modified PSE) in a suitable medium until the PSE has been produced. In a further embodiment, the method comprises Isolate 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 all suitable prokaryotic or eukaryotic organisms. The host organisms advantageously used are organisms, such as bacteria, Mushrooms, yeasts, non-human animal or plant cells. Further advantageous organisms are not human animals or preferably Plants or parts thereof. Mushrooms, yeasts or plants preferably used, particularly preferably mushrooms or plants, very particularly preferably plants, such as oil fruit plants, the contain large amounts of lipid compounds, such as rapeseed, Evening primrose, canola, peanut, flax, soy, diesel, sunflower, Borage, or plants such as corn, wheat, rye, oats, Triticale, rice, barley, cotton, cassava, pepper, tagetes, Solanaceae plants such as potato, tobacco, eggplant and tomato, Vicia species, peas, alfalfa, bush plants (coffee, cocoa, tea), Salix species, trees (oil plant, coconut) and perennial grasses and fodder crops. Particularly preferred according to the invention Plants are oil fruit plants such as soybean, peanut, rapeseed, canola, Sunflower, safflower, trees (oil palm, coconut).

• a) die erfindungsgemäße Nukleinsäuresequenz, oder
• b) eine mit der erfindungsgemäßen Nukleinsäuresequenz funktionell verknüpfte genetische Kontrollsequenz, zum Beispiel ein Promotor, oder
• c) (a) und (b)

With regard to, for example, a nucleic acid sequence, an expression cassette (= gene construct) or a vector containing said nucleic acid sequence or an organism transformed by means of the nucleic acid sequences, expression cassette or vector according to the invention, “transgene” means all such constructions which have been obtained by genetic engineering methods and in which either

• a) the nucleic acid sequence according to the invention, or
• b) a genetic control sequence functionally linked to the nucleic acid sequence according to the invention, for example a promoter, or
• c) (a) and (b)

are not in their natural, genetic environment or have been modified by genetic engineering methods, whereby the modification exemplarily a substitution, addition, Deletions, inversions or insertions of one or more May be nucleotide residues. Natural genetic environment means the natural chromosomal locus in the organism of origin or being in a genomic library. In the case of one genomic library is the natural, genetic environment the nucleic acid sequence preferably at least partially receive. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferred at least 1000 bp, very particularly preferably at least 5000 bp. A naturally occurring expression cassette - for example the naturally occurring combination of the natural promoter the nucleic acid sequence according to the invention with the corresponding PSE gene - becomes a transgenic expression cassette when this through non-natural, synthetic ("artificial") Procedures such as mutagenization is changed. Corresponding methods are described for example in US 5,565,350 or WO 00/15815.

Another object of the invention relates to transgenic Organisms transformed with at least one according to the invention Nucleic acid sequence, expression cassette or one vector according to the invention, as well as cells, cell cultures, tissues, parts - such for example with plant organisms leaves, roots etc. - or propagation material derived from such organisms.

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 is essentially free of cellular material when it passes through Recombinant DNA technology is produced, or by chemical Precursors or other chemicals if it is chemically synthesized 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 powerful preferably 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. Point in preferred embodiments isolated proteins or biologically active parts thereof none contaminating proteins from the same organism where the PSE comes from. These proteins are usually made by recombinant expression for example of a Phytophthora infestans PSE in other fungi, plants or microorganisms, for example bacteria such as C. glutamicum, fungi such as Mortierella, Yeast, such as Saccharomyces, or ciliates, algae or plants such as Phycomitrella patens or oil crops.

An isolated PSE according to the invention or a part thereof can also on the metabolism of building cell membranes in Phytophthora infestans necessary connections or on transportation of molecules participate through these membranes or on Fatty acid metabolism may be involved or has one or more of those in it Activities indicated in Table I. With preferred In embodiments, the protein or part thereof comprises one Amino acid sequence that is sufficiently homologous to an amino acid sequence SEQ ID NO: 2 is that the protein or part thereof is the Ability to build on cell membranes in metabolism Phytophthora infestans necessary connections or on transportation of molecules across these membranes or on fatty acid metabolism participate, maintains. The part of the protein is preferred a biologically active part as described here. At a another preferred embodiment has an inventive PSE is one of the amino acid sequences shown in SEQ ID NO: 2. at In a further preferred embodiment, the PSE has one Amino acid sequence encoded by a nucleotide sequence who, for example under stringent conditions, to a Nucleotide sequence of SEQ ID NO: 1 hybridized. Another one preferred embodiment, the PSE has an amino acid sequence, which is encoded by a nucleotide sequence which is at least approximately 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 the SEQ ID NO: 2. The preferred PSE according to the invention has preferably also at least one of the PSE described here Activities. For example, one includes preferred PSE an amino acid sequence derived from a nucleotide sequence is encoded which, for example under stringent conditions, hybridized to a nucleotide sequence of SEQ ID NO: 1 and on Metabolism to build cell membranes in Phytophthora infestans necessary connections or at the transport of Molecules can participate in these membranes or on Fatty acid metabolism is involved or one or more of the ones in table activities indicated.

In other embodiments, the PSE is essentially homologous to an amino acid sequence of SEQ ID NO: 2 and retains the functional activity of the protein of one of the sequences of the SEQ ID NO: 2, but their amino acid sequence differs due to natural variation or mutagenesis as detailed described in subsection I above. Another Thus, in one embodiment, the PSE is a protein that is a Amino acid sequence comprising 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 preferred at least about 96%, 97%, 98%, 99% or even more homologous a complete amino acid sequence of SEQ ID NO: 2 and has at least one of the PSE activities described here. In another embodiment, the invention relates to a whole Phytophthora infestans protein, which is essentially homologous to a complete amino acid sequence of SEQ ID NO: 2 is.

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 shown in SEQ ID NO: 2 Amino acid sequence or the amino acid sequence of a protein that leads to a PSE is homologous, which is less amino acids than the full length PSE or have the full length protein homologous to a PSE and have at least one activity of a PSE. Usually include biologically active parts (peptides, e.g., peptides that for example 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, Are 100 or more amino acids long) a domain or a motif with at least one activity of a PSE. Moreover, others can biologically active parts in which other areas of the protein are deleted, produced by recombinant techniques and related to one or more of the activities described here to be examined. The biologically active parts of a PSE preferably comprise one or more selected ones Domains / motifs or parts thereof with biological activity.

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 be replaced by a suitable cleaning scheme using Standard protein purification techniques can be isolated from the cells. As an alternative to recombinant expression can be a PSE, a polypeptide, or a peptide chemically synthesized using standard peptide synthesis techniques become. In addition, native PSE can be derived from cells (e.g. endothelial cells) z. B. isolated using an anti-PSE antibody, which can be produced by standard techniques, one PSE according to the invention or a fragment thereof is used.

The invention also provides chimeric or PSE proteins PSE fusion proteins ready. As used herein, a "chimeric PSE protein "or" PSE fusion protein "is a PSE polypeptide that is operably linked to a non-PSE polypeptide. On "PSE polypeptide" refers to a polypeptide with one Amino acid sequence corresponding to a PSE whereas a "non-PSE- Polypeptide "refers to a polypeptide with an amino acid sequence, which corresponds to a protein that is essentially not is homologous to the PSE, e.g. B. a protein that is different from the PSE differs and from the same or another Organism comes from. The term is intended within the fusion protein "Functionally linked" means that the PSE polypeptide and the non-PSE polypeptide is fused together that both sequences are the predicted, the sequence used perform the assigned function. The non-PSE polypeptide can be at the N-terminus or the C-terminus of the PSE polypeptide be merged. In one embodiment, it is Fusion protein, for example, a GST-PSE fusion protein in which the PSE sequences fused to the C-terminus of the GST sequences are. These fusion proteins can cleanse the facilitate recombinant PSEs. In another embodiment the fusion protein is a PSE that has a heterologous signal sequence has at its N-terminus. In certain host cells (e.g. Mammalian host cells) can express and / or secrete one PSE increased by using a heterologous signal sequence become.

A chimeric PSE protein according to the invention or PSE fusion protein is made using standard recombinant DNA techniques manufactured. For example, DNA fragments are different Encode polypeptide sequences according to conventional techniques in Reading frames ligated together, for example by smooth or overhanging ends for ligation, restriction enzyme cleavage for Provide suitable ends, fill 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 Anchor 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 Encode 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 through, for example, competitive binding to a downstream or - upward element of the metabolic cascade for Cell membrane components comprising the PSE or by binding to a PSE, which is used to transport connections via Cell membranes mediate, inhibit, which inhibits translocation becomes.

In an alternative embodiment, homologs of the PSE by screening combinatorial banks of mutants, e.g. B. Shortening mutants, the PSE with regard to PSE agonists or Antagonist activity can be identified. At a Embodiment is a varied bank of PSE variants through 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 Mixture of synthetic oligonucleotides in gene sequences be made so that a degenerate set 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. It are a variety of processes used to manufacture banks potential PSE homologs from a degenerate Oligonucleotide sequence can be used. The chemical synthesis of a degenerate gene sequence can be in a DNA synthesizer carried out and then the synthetic gene into an appropriate one Expression vector can be ligated. The use of a degenerate set of genes enables the delivery of all Sequences that contain the desired set of potential PSE sequences encode in a mixture. Process for the synthesis of degenerate Oligonucleotides are known in the art (see e.g. 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 are only about done once 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 screening in the art Gene products in combinatorial banks caused by point mutations or shortening have been made, and for screening from cDNA banks for gene products with a selected one Property known. These techniques can be applied to the fast Customize screening of gene banks by combinatorial Mutagenesis of PSE homologs have been generated. The most commonly used techniques for screening large gene banks that can be subjected to a high throughput analysis, usually involve cloning the library into replicable ones Expression vectors, transforming suitable cells with the resulting vector library and expressing the combinatorial Genes under 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 measures frequency functional mutants increased in banks, can be in combination with the screening tests to identify PSE homologs can be used (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

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

E. Uses and methods according to the invention

The nucleic acid molecules, proteins, Protein homologs, fusion proteins, primers, vectors and host cells can be used in one or more of the following procedures are used: identification of Phytophthora infestans and related organisms, mapping the genomes of organisms, related to Phytophthora infestans, identification and Localization of Phytophthora infestans sequences of interest, Evolutionary studies, determination of PSE protein areas that are necessary for the function is necessary, modulation of a PSE activity; Modulation of the metabolism of one or more Cell membrane components; Modulation of the transmembrane transport of one or multiple compounds as well as modulation of cellular production a desired compound, such as a fine chemical. The PSE nucleic acid molecules according to the invention have a large number of uses. You can first use to identify a Organism as Phytophthora infestans or as a close relative of which are used. You can also use it to identify the Presence of Phytophthora infestans or a relative of which used in a mixed population of microorganisms become. The invention provides the nucleic acid sequences one Series of Phytophthora infestans genes ready; by probing the extracted genomic DNA of a culture of one uniform or mixed population of microorganisms among stringent conditions with a probe covering an area a Phytophthora infestans gene or parts thereof overstretched, which is unique for this organism, one can determine whether this organism is present. Phytophthora infestans itself is not duplicated for commercial production unsaturated acids are used, but oomycetes are principled suitable for the production of PUFAs. Therefore are suitable PSE-related DNA sequences especially for use in PUFA production in other organisms.

Furthermore, the nucleic acid and Protein molecules serve as markers for specific areas of the genome. This is not only for mapping the genome, but also for functional studies of Phytophthora infestans proteins suitable. To identify the genome area to which a certain DNA-binding protein from Phytophthora infestans_binds, for example, the Phytophthora infestans genome could be cleaved and the fragments are incubated with the DNA-binding protein become. Those who bind the protein can additionally with the nucleic acid molecules according to the invention, preferably with easily detectable markings. the Binding of such a nucleic acid molecule to the genome fragment enables the fragment to be located on the genome map of Phytophthora infestans and relieved if this happens several times 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 be sufficiently homologous to the sequences of related species that these nucleic acid molecules as markers for construction can serve a genomic map in related fungi.

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 Molecules according to the invention 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 function.

The manipulation of the PSE nucleic acid molecules according to the invention can be used to produce PSEs with functional differences lead to 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 level 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, plants or mushrooms on a large scale is significantly improved when the cell has the desired Connections secreted because these connections from the Culture medium (in contrast to extraction from the mass of the grown Cells) can be easily cleaned. Otherwise, the Improve purification when the cell connects in vivo a specialized compartment with one species Concentration mechanism saves. Plants that express PSEs can an increased transport for better distribution within the Plant tissue and organs. By enlarging the Number or activity of transporter molecules which Exporting fine chemicals from the cell, it may be possible Amount of fine chemical produced in the extracellular Medium is present to increase, thereby harvesting and cleaning or for plants a more efficient distribution can be facilitated. to efficient overproduction of one or more fine chemicals are, on the other hand, increased amounts of cofactors, precursor molecules and intermediates for the appropriate biosynthetic pathways required. By increasing the number and / or the activity of transporter proteins involved in the import of nutrients such as Carbon sources (i.e. sugars), nitrogen sources (i.e. Amino acids, ammonium salts), phosphate and sulfur are the production of a fine chemical due to the Removal of all restrictions on the nutrient supply at Improve the biosynthesis process. Fatty acids, such as PUFAs, and lipids, the PUFAs themselves are 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 are involved, the yield, production and / or Efficiency of the production of fatty acid and lipid molecules in Ciliates, algae, plants, mushrooms, yeasts or others Increase microorganisms.

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 this Can actively disrupt metabolic processes (e.g. nitrided peroxynitrite famously known tyrosine side chains, whereby some enzymes with Tyrosine inactivated in the active center (Groves, J. T. (1999) Curr. Opin. Chem. Biol. 3 (2); 226-235)). This Waste products are usually excreted, but those for cells used for fermentative production on a large scale 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 efficient metabolic activity maintained. 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 that the relative amounts of different lipid and Fatty acid molecules are changed. This can be a crucial one Affect the lipid composition of the cell membrane. There each type of lipid has different physical properties, can change the lipid composition of a membrane Change membrane fluidity significantly. Changes in Membrane fluidity can transport molecules across the membrane affect what, as explained above, the export of Waste products or the fine chemicals produced or the Can modify import of necessary nutrients. These changes The membrane fluidity can also affect the integrity of the cell decisively influence; Cells with comparatively weaker ones Membranes are more susceptible to ablotic and biotic Stress conditions that damage or kill the cell can. By manipulating PSEs involved in the production of Fatty acids and lipids are involved in membrane construction, so the resulting membrane is a membrane composition has those for those in cultures who are producing Fine chemicals are used, prevailing environmental conditions A more proportion of cells should be more receptive survive and reproduce. Larger amounts of producing Cells should be in greater yields, higher production or efficiency of the production of fine chemicals from culture manifest.

The aforementioned mutagenesis strategies for PSEs that lead 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 recombinant or transgenic algae, ciliates, plants, non-human animals, fungi or other microorganisms such as C. glutamicum can be used, the mutant PSE nucleic acid and express protein molecules so that the yield, production and / or efficiency of producing a desired compound is improved. This desired connection can be any natural product of algae, ciliates, plants, animals, mushrooms or C. glutamicum, which is the end product of Biosynthetic pathways and intermediates more naturally occurring Metabolic pathways includes, as well as molecules involved in the metabolism of these cells do not occur naturally, but that of the invention Cells are produced.

A further embodiment according to the invention is a method for producing PUFAs, the method comprising growing an organism which comprises a nucleic acid according to the invention, a gene construct according to the invention or a vector according to the invention which encode a polypeptide which contains C ₁₆ and / or C ₁₈ fatty acids with at least two double bonds in the fatty acid molecule extended by at least two carbon atoms under conditions under which PUFAs are produced in the organism. PUFAs produced by this method can be isolated by harvesting the organisms either from the culture in which they grow or from the field, breaking up and / or extracting the harvested material with an organic solvent. The oil 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 content of PUFAs means at least 5%, preferably 10%, particularly preferably 20%, very particularly preferably 40% more PUFAs than the original organism, for example an oomycete such as Phytophthora or a plant such as an oil fruit plant which does not contain any additional nucleic acid encodes the elongase according to the invention. Furthermore, the aforementioned oils, lipids, phospholipids, sphingolipids, glycolipids, triacylglycerols and / or free fatty acids with a higher PUFA content have a different composition than the composition of the starting organisms. This applies in particular to plants which naturally do not contain longer-chain polyunsaturated C ₂₀ or C ₂₂ fatty acids such as DHA, EPA or ARA.

The PUFAs produced by this process are preferably C ₂₀ or C ₂₂ fatty acid molecules with at least two double bonds in the fatty acid molecule, preferably three or four double bonds, particularly preferably three double bonds. These 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 transgenic plants.

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

Another embodiment of the invention is the use of the oil, lipid or fatty acid composition in Animal feed, food, cosmetics or pharmaceuticals.

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 applications is here 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, Linking DNA fragments, transforming Escherichia coli and yeast cells, bacterial growth and sequence analysis recombinant DNA, were carried out 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).

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 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.

Example 2 Construction of the cDNA bank

The first strand synthesis was used to construct the cDNA library 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 adapter (Pharmacia, Freiburg, Germany) using T4 DNA ligase (Roche, 12 ° C, overnight) ligated to the cDNA ends and by incubation with Polynucleotide kinase (Roche, 37 ° C, 30 min) phosphorylated. This The mixture was mixed on a low-melting agarose gel subjected. DNA molecules over 300 base pairs were extracted from the Gel eluted, phenol extracted, on Elutip-D columns (Schleicher and Schüll, Daßel, Germany) concentrated and on vector arms ligated and in lambda-ZAPII phage or lambda-ZAP-express phage using the Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands) packed, using manufacturer's material and his instructions were followed.

Example 3 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 random sequencing was carried out after the preparative plasmid extraction from cDNA banks via in vivo mass section and retransformation of DH10B on agar plates (details on material and protocol from Stratagene, Amsterdam, Netherlands). Plasmid DNA was obtained from overnight grown E. coli cultures grown in Luria broth with ampicillin (see Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6)) , prepared on a Quiagen DNA preparation robot (Quiagen, Hilden) according to the manufacturer's protocols. Sequencing primers with the following nucleotide sequences were used:
5'-CAGGAAACAGCTATGACC-3 '
5'-CTAAAGGGAACAAAAGCTG-3 '
5'-TGTAAAACGACGGCCAGT-3 '

The sequences were generated using the Standard software package EST-MAX, commercially available from Bio-Max (Munich, Germany) is delivered, processed and commented. A clone with weak homologies to known elongases became more detailed characterized.

Example 4 Identification of the PSE1 gene from P. infestans and Analysis of the cDNA clone PiPSE1

An EST sequence (database entry: PI001002014r) was created due to weak homology with known elongases among others Candidate genes considered as target genes.

The BESTFIT program was used for the sequence comparison, d. H. the BLOSUM amino acid substitution matrices, with Henikoff, S., and Henikoff. J.G. (1992) Amino acid substitution matrices from protein blocks, Proc. Natl. Acad. Sci. United States 89: 10915-10919.

The sequence of the clone with the database no. PI001002014r was used for comparison with the yeast elo1 peptide sequence. Since the new P. infestans clone was incomplete, the corresponding full length clone was isolated from the P. infestans cDNA bank (PiPSEI). For this purpose, a digoxigenin-labeled probe was produced by PCR using the PCR DIG synthesis kit (Roche), PI001002014 serving as the template. The following primers were used for the PCR:
PI-DIGf: cacaccatcatgtacacttactac
PI-DIGr: caacttcttcttcgattcctccac

The isolated labeled probe was used for screening the P. infestans cDNA Bank used (according to the information of the Manufacturer, Stratagene). A fragment of 1046 bp in length was generated isolated and referred to as PiPSE1. The open reading frame is 837 bp long and encodes a protein of 278 amino acids with a calculated molecular mass of 32.1 kDa. Sequence comparisons resulted in the following sequence identities or similarities: 26% / 43% with the Physcomitrella patens PSE1p, 23% / 37% with the human HELOp, 21% / 41% with the GLELOp from Mortierella alpina and 17% / 36% with the elongase from C. elegans.

Example 5 Identification of genes using hybridization

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: Depending on the frequency of the gene of interest, 100,000 to 1,000,000 recombinant bacteriophages were plated and onto a nylon membrane transferred. After denaturing with alkali, the DNA was z. B. immobilized by UV crosslinking. The hybridization took place under highly stringent conditions. In aqueous solution, the hybridization and the washing steps were carried out at an ionic strength of 1 M NaCl and a temperature of 68 ° C. Hybridization probes were e.g. B. by labeling using radioactive ( ³² P-) nick transcription (High Prime, Roche, Mannheim, Germany). The signals were detected by means of autoradiography.

Partially homologous or heterologous genes that are related but not are identical, can be analogous to that described above Methods 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.

The isolation of gene sequences which have homologies only to a single domain of, for example, 10 to 20 amino acids can be carried out using synthetic, radioactively labeled oligonucleotide probes. Radioactively labeled oligonucleotides were prepared by phosphorylating the 5 'end of two complementary oligonucleotides with T4 polynucleotide kinase. The complementary oligonucleotides were hybridized to one another and ligated so that concatemers were formed. The double-stranded concatems were radioactively labeled, for example, by nick transcription. Hybridization was usually carried out under low-stringent conditions using high oligonucleotide concentrations. Oligonucleotide hybridization solution 6 × SSC
0.01 M sodium phosphate
1 mM EDTA (pH 8)
0.5% SDS
100 αg / ml denatured salmon sperm DNA
0.1% low-fat dry milk

During the hybridization, the temperature became gradual to 5 to 10 ° C below the calculated oligonucleotide Tm or to to room temperature (means RT = ~ 23 ° C in all experiments, unless otherwise stated) followed by washing steps and autoradiography. The washing was extremely low Stringency performed, for example 3 washing steps below Using 4 × SSC. More details are like from 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.

Production of specific antibodies, for example under

Example 6 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) separated, transferred 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 / w Vol., 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). 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.

Example 7 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 used using of a tissue-specific promoter. For example seed-specific expression can be achieved by or the LeB4 or USP promoter 5 'of the cDNA is cloned 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 obtained using a Signal peptide, 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 8 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 9 plant transformation

The Agrobacterium-mediated plant transformation can be found at Use of standard transformation and Regeneration techniques (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 Hypocotyl 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 strain. Rapeseed selection is usually carried out using kanamycin performed as a selectable plant marker.

Agrobacterium-mediated gene transfer in flax can be using, for example, one of Mlynarova et al. (1994) Plant Cell Report 13: 282-285 carry out.

The transformation of soy can be done using for example 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 Particle 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 10 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, such as Saccharomyces cerevisiae), in which 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 11 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 required for the Translation of the gene product is available) Carrying out a Northern blot (for reference point see Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York, or the example section mentioned above), where a Primer designed to match the gene of interest binds, with a detectable label (usually radioactive or chemiluminescent) is marked so that if the total RNA of a culture of the organism extracted on a gel separated, transferred to a stable matrix and with it The probe is incubated, binding and the extent of binding Probe the presence and also the amount of mRNA for this gene displays. This information shows the degree of transcription of the transformed gene. Total cellular RNA can be derived from cells Tissues or organs with multiple procedures, all in the Are known in the art, such as that of Bormann, E.R., et al. (1992) Mol. Microbiol. 6: 317-326, prepared become.

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 for example 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, that is easy to prove. The presence and the amount the observed mark shows the presence and the amount of the desired mutant protein present in the cell.

Example 12 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 one Compound (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 Components to the increased production of the desired product (i.e. of lipids or a fatty acid) is examined. This Analysis techniques are known to those 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 Chemistry, 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 Materials, 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 that to produce the desired connection used as intermediates and by-products to the To determine overall efficiency of production of the compound. The Analysis methods include measurements of nutrient levels in the Medium (e.g. sugar, hydrocarbons, Nitrogen sources, phosphate and other ions), measurements of the Biomass composition and growth, analysis of production more common Metabolites of biosynthetic pathways and measurements of gases during fermentation. Standard procedures for this Measurements are in Applied Microbial Physiology; A practical Approach, P.M. Rhodes and P.F. Stanbury, eds., IRL Press, S. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and therein described literature references.

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

The unambiguous proof of the existence 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 References therein (1997, in: Advances on Lipid Methodology, Fourth Ed .: Christie, Oily Press, Dundee, 119-169; 1998 Gas Chromatography Mass Spectrometry, 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% Dimethoxypropane for 1 h at 90 ° C, resulting in hydrolyzed oil and Leads to lipid 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 m, 0.32 mm) a temperature gradient between 170 ° C and 240 ° C for 20 min and Subjected at 240 ° C for 5 min. 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 Fatty acids with triple bond via GC-MS after derivatization with 4,4-Dimethoxyoxazoline derivatives (Christie, 1998, see above) to be shown.

Example 13 Expression constructs in heterologous microbial systems Strains, growth conditions and plasmids

The Escherichia coli strain XL1 Blue MRF 'kan (Stratagene) was for subcloning the new elongase piPSE1 from Phytophthora infestans used. For the functional expression of this We used the Saccharomyces cerevisiae strain INVSc 1 gene (Invitrogen Co.). E. coli was grown in Luria Bertini broth (LB, Duchefa, Haarlem, The Netherlands) at 37 ° C. If needed, ampicillin (100 mg / liter) was added and 1.5% agar (W / V) was added for solid LB media. S. cerevisiae was 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) either 2% (w / v) raffinose or glucose grown. For fixed Media 2% (w / v) Bacto ™ agar (Difco) was added. The plasmids used for cloning and expression were pUC18 (Pharmacia) and pYES2 (Invitrogen Co.).

Cloning and expression of a PUFA-specific elongase Phytophthora infestans

For expression in yeast, the Phytophthora infestans cDNA clone piPSE1, which encodes the PUFA-specific elongase (PSE1) gene, was first modified in such a way that a KpnI 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) in addition to the start codon and an XbaI restriction site flanking the stop codon. A pair of primers complementary to its 5 'and 3' ends were synthesized to amplify the open reading frame.
ppex1f: cgg ggtacc acataatgtcgactgagctactgcag
ppex1r: cactag tctaga ttccaacttcttcttcgattcc

The PCR reaction was 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 30 cycles of 30 s at 96 ° C, 30 s at 55 ° C and 2 min at 72 ° C, 1 cycle with 10 min at 72 ° C and stop at 4 ° C.

The correct size of the amplified DNA fragment of 883 bp was confirmed by agarose TBE gel electrophoresis. The amplified DNA was extracted from the gel with the QIAquick gel extraction kit (QIAGEN) extracted and into the SmaI restriction site of the dephosphorylated vector pUC18 using the Sure Clone Ligation Kit (Pharmacia) ligated to give pUCPSE1. After the transformation of E. coli XL1 Blue MRF 'kan a DNA mini-preparation (Riggs, M.G., & McLachlan, A. (1986) A simplified screening procedure for large numbers of plasmid mini-preparation. BioTechniques 4, 310-313) to 24 ampicillin resistant transformants performed, and positive clones were identified using BamHI restriction analysis. The Sequence of the cloned PCR product was determined using Resequencing using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt) approved.

The plasmid DNA from pUC-PSE1 was also digested with KpnI / XbaI and the obtained ~ 900 bp fragment into the KpnI / XbaI restriction site of dephosphorylated yeast E. coli shuttle vector pYES2 ligated to give pY2PSE1. After the transformation of E. coli and DNA minipreparation from the transformates the orientation of the DNA fragment in the vector by HindIII Cleavage checked. A clone was used for DNA maxi preparation with the Nucleobond® AX 500 plasmid DNA extraction kit (Macherey- Nagel, Düringen).

Saccharomyces INVSc1 was analyzed with pY2PSE1 and pYES2 using a modified PEG / lithium acetate protocol transformed (Ausubel et al., 1995). After selection on CMdum agar plates with 2% Four pY2PSE11 transformants (pY2PSE1a-d) and glucose were a pYES2 transformant for further breeding and functional Expression selected.

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 (pY2PSE1a-d, pYES2) and grown for 3 T at 30 ° C, 200 rpm until an optical density at 600 nm (OD ₆₀₀ ) was reached by 1.5-2.

main Culture

For expression, 20 ml of CMdum liquid medium with 2% raffinose and 1% (v / v) Tergitol NP-40 with γ-linoleic acid (γ-18: 3) were mixed to a final concentration of 0.003% (w / v. ) enriched. The media were 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 h after which the cultures had reached 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. Cells from 5 ml of culture were harvested by centrifugation (1000 × g, 10 min. 4 ° C.) and washed once with 100 mM NaHCO ₃ , pH 8.0, 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 µl petroleum ether. The samples were separated on a ZEBRON-ZB-Wax capillary column (30 m, 0.32 mm, 0.25 αm; Phenomenex) in a Hewlett Packard 6850 gas chromatograph with a flame ionization detector. The oven temperature was from 70 ° C (hold 1 min) to 200 ° C at a rate of 20 ° C / min. then programmed to 250 ° C (hold 5 min) at a rate of 5 ° C / min and finally to 260 ° C 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).

expression analysis

The fatty acid patterns of five transgenic yeast strains in mol% are shown in Table I.

The ratios of the γ-linolenic acid added and taken up are highlighted by numbers in bold, those of the elongated products by red numbers and that of the elongated γ-linolenic acid by numbers in bold (last line). The GC analysis of FAMES, which consists of total lipids of the yeasts transformed with pYES2 (i / control) and pY2PSE1, is shown in FIG. 1. For the analysis, the transgenic yeasts were grown in the presence of γ-18: 3.

The results show that γ-18: 3 has been incorporated in large amounts in all transgenic yeasts. All four transgenic yeast clones that were transformed with pY2PSE1 show an additional peak in the gas chromatogram, which was identified by comparison of the retention times as 20: 3 ^{Δ 8, 11, 14} . Gas chromatography / mass spectroscopy can provide additional support to confirm this identity.

The identified products showed that the nucleotide sequence of PiPSE1 encodes a Δ ⁶ -selective fatty acid elongase from the moss Physcomitrella patens, which leads to the formation of new fatty acids in transgenic yeasts.

Further feeding experiments with various other fatty acids (e.g. linoleic acid (18: 2 ^{Δ 9.12?} ), Stearidonic acid (18: 4 ^{Δ 6,9,12,15} )) can be carried out to confirm the substrate ^{selectivity of} these elongases in more detail.

Example 14 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 The supernatant fraction is kept for further purification.

The supernatant fraction from each cleaning procedure becomes one Chromatography with a suitable resin, which desired molecule either on the chromatography resin is retained, 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 be repeated, the same or different Chromatography resins are used. The expert is in the Selection of suitable chromatography resins and their most effective Application for a specific molecule to be purified. The purified product can be 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 for Example 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. To include high performance liquid chromatography (HPLC), spectroscopic methods, staining methods, Thin layer chromatography, 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 be able to determine many equivalents of the specific embodiments of the invention described herein by using only routine experiments. These equivalents are intended to be encompassed by the claims. SEQUENCE LISTING

Claims (21)

1. Isolated nucleic acid which codes for a polypeptide which extends C ₁₆ or C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms, the fatty acids C _{18: 3} ^{Δ 5t, 9.12} , C _{20: 3} ^{Δ 8,11,14} , C _{20: 4} ^{Δ 5,8,11,14} and C _{20: 5} ^{Δ 8,11,14,17} cannot be extended. 2. Isolated nucleic acid, comprising a nucleotide sequence encoding a polypeptide which extends C ₁₆ or C ₁₈ 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, b) a nucleic acid sequence which is derived from the sequence shown in SEQ ID NO: 2 in accordance with the degenerate genetic code, c) Derivatives of the sequence shown in SEQ ID NO: 1, which encodes polypeptides with at least 50% homology to the sequence which encodes the amino acid sequences in SEQ ID NO: 2, the sequence acting as a C ₁₆ or C ₁₈ elongase. 3. The isolated nucleic acid sequence according to claim 2, wherein the Sequence comes from an oomycete. 4. Isolated nucleic acid sequence according to claim 2 or Claim 3, wherein the sequence is of the genus Phytophhthora comes. 5. Amino acid sequence isolated from an Nucleic acid sequence according to one of claims 2 to 4. 6. 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. 7. gene construct according to claim 6, the gene expression by the regulatory signals are amplified. 8. Vector comprising a nucleic acid according to claim 2 or a gene construct according to claim 6. 9. organism comprising at least one nucleic acid after Claim 2, a gene construct according to claim 6 or one The vector of claim 8. 10. The organism of claim 9, wherein the organism Microorganism, a non-human animal or a plant. 11. The organism of claim 9 or 10, wherein the organism is a transgenic plant. 12. A method for producing PUFAs, the method comprising growing an organism comprising a nucleic acid according to claim 2, a gene construct according to claim 6 or a vector according to claim 8, encoding a polypeptide containing C ₁₈ fatty acids with at least two double bonds extended in the fatty acid molecule by at least two carbon atoms under conditions under which PUFAs are formed in the organism. 13. The method according to claim 12, wherein the PUFAs produced by the method are C ₂₀ or C ₂₂ fatty acid molecules with at least two double bonds in the fatty acid molecule. 14. The method according to claim 13, wherein the 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. 15. The method according to any one of claims 12 to 14, wherein the Organism a microorganism, an animal or a plant is. 16. The method according to any one of claims 12 to 15, wherein the Organism is a transgenic plant. 17. The method according to any one of claims 12 to 16, wherein the C ₁₈ fatty acid is a fatty acid with three double bonds in the molecule. 18. oil, lipids or fatty acids or a fraction thereof, produced by the method according to one of claims 12 until 17th 19. Oil, lipid or fatty acid composition comprising PUFAs and comes from transgenic plants. 20. oil, lipid or fatty acid composition according to claim 19, the PUFAs originating from transgenic plants which have a The nucleotide sequence of claim 2. 21. Use of the oil, lipid or fatty acid composition in feed, food, cosmetics or pharmaceuticals.