WO2008104559A1 - Method for the production of polyunsaturated fatty acids in transgenic organisms - Google Patents

Abstract

The invention relates to a method for producing polyunsaturated fatty acids, particularly long-chain polyunsaturated fatty acids such as arachidonic acid and/or eicosapentaenoic acid, in a transgenic organism by introducing nucleic acid that code for polypeptides having ?-6 desaturase, ?-6 elongase, and/or ?-5 desaturase activity into the organism. Advantageously, the ?-6 desaturase and the ?-5 desaturase are obtained from Mantoniella squamata while the ?-6 elongase is obtained from Physcomitrella patens. Advantageously, a gene coding for a ?-3 desaturase is also expressed in the organism. In another advantageous embodiment of said method, other nucleic acid sequences coding for polypeptides of fatty acid and lipid metabolism biosynthesis can be expressed in the organism. The nucleic acid sequences coding for a ?-8 desaturase, ?-12 desaturase, ?-15 desaturase, ?-4 desaturase, ?-9 elongase, and/or ?-5 elongase activity are particularly advantageous therefor.

Description

 Process for the production of polyunsaturated fatty acids in transgenic organisms

The present invention relates to a process for the preparation of polyunsaturated fatty acids, in particular long-chain polyunsaturated fatty acids such as arachidonic acid and / or eicosapentaenoic acid, in a transgenic organism by introducing into the organism nucleic acids which are suitable for polypeptides having Δ6-desaturase, Δ6-elongase and / or Δ5-desaturase activity. Advantageously, the Δ6-desaturase and Δ5-desaturase are from Mantoniella squamata and the Δ6 elongase is from Physcomitrella patens. Advantageously, a gene which codes for a ω3-desaturase is also expressed in the organism. In a further advantageous embodiment of the method, further nucleic acid sequences coding for polypeptides of the biosynthesis of the fatty acid and lipid metabolism can be expressed in the organism. Particularly advantageous for this purpose are the nucleic acid sequences which code for a Δ8-desaturase, Δ12-desaturase, Δ15-desaturase, Δ4-desaturase, Δ9-elongase and / or Δ5-elongase activity. The

The invention further relates to the nucleic acid sequences according to the invention, vectors comprising the nucleic acid sequences and / or the nucleic acid constructs and transgenic organisms containing the abovementioned nucleic acid sequences, nucleic acid constructs and / or vectors. Furthermore, the invention relates to a process for the preparation of oils, lipids and / or triacylglycerides having an increased content of long-chain polyunsaturated fatty acids. Another part of the invention relates to oils, lipids and / or fatty acids prepared by the process according to the invention and their use, in particular the use in feed or food, cosmetics or pharmaceuticals.

Fatty acids and triacylglycerides have a variety of uses in the food, animal nutrition, cosmetics and pharmaceutical industries. Depending on whether they are free saturated and unsaturated fatty acids or triacylglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a wide variety of applications. Polyunsaturated fatty acids such as linoleic and linolenic acid are essential for - J -

animals are essential because they can not be produced by them. Therefore, polyunsaturated ω3 fatty acids and 006 fatty acids are an important component of animal and human food.

Polyunsaturated long-chain ω3-fatty acids such as eicosapentaenoic acid (= EPA,

C20: 5 ^Δ5 ' ⁸ ' ⁿ ' ¹⁴ ' ¹⁷ ) or docosahexaenoic acid (= DHA, _C2 2: 6 ^Δ4 ' ⁷ ' ¹⁰ ' ¹³ ' ^{16 '19} ) are important components of the human diet because of their different roles in health Aspects such as the development of the child's brain, the functionality of the eye, the synthesis of hormones and other signaling substances, as well as the prevention of cardiovascular complaints, cancer and diabetes include. There is therefore a need for the production of polyunsaturated long-chain fatty acids.

Due to the customary composition of human food today, addition of ω3 polyunsaturated fatty acids, which are preferred in fish oils, is particularly important for food. Thus, for example, polyunsaturated fatty acids DHA or EPA baby food are added to increase the nutritional value. The unsaturated fatty acid DHA is thereby attributed a positive effect on the development and maintenance of brain functions.

The following are polyunsaturated fatty acids as PUFA, PUFAs, LCPUFA or

LCPUFAs (poly unsaturated fatty acids, PUFA, polyunsaturated fatty acids, LCPUFA, long-chain polyunsaturated fatty acids).

Mainly the different fatty acids and triglycerides are from microorganisms like Mortierella or Schizochytrium or from oil-producing plants like soy or rapeseed, algae such as Crypthecodinium or Phaeodactylum and others obtained, they usually incurred in the form of their triacylglycerols (= triglycerides = triglycerols). But they can also be obtained from animals such as fish. The free fatty acids are advantageously prepared by saponification. Very long-chain polyunsaturated fatty acids such as DHA, EPA, arachidonic acid (= ARA, C20: 4 ^Δ5 ' ⁸ ' ^{π '14} ), dihomo-γ-linolenic acid (C20: 3 ^Δ8 ' ^{n '14} ) or

Docosapentaenoic acid (DPA, Q22: 5 ^Δ7 ' ¹⁰ ' ¹³ ' ¹⁶ ' ¹⁹ ) is not synthesized in oilseed crops such as rapeseed, soybean, sunflower, safflower. Common natural sources of these fatty acids are fish such as herring, salmon, sardine, perch, eel, carp, trout, halibut, mackerel, zander or tuna or algae.

Depending on the application, oils with saturated or unsaturated fatty acids are preferred. Thus, e.g. in the human diet lipids with unsaturated fatty acids, in particular polyunsaturated fatty acids are preferred. The polyunsaturated ω3-fatty acids thereby a positive effect on the cholesterol level in the blood and thus the possibility of preventing heart disease is attributed. By adding these ω3-fatty acids to

Food can significantly reduce the risk of heart disease, stroke or high blood pressure. Also, inflammatory, especially chronic inflammatory processes in the context of immunological diseases such as rheumatoid arthritis can be positively influenced by ω3 fatty acids. They are therefore added to foods, especially dietary foods, or are used in medicines.

ω3 and oo6 fatty acids are precursors of tissue hormones, the so-called eicosanoids such as the prostaglandins derived from dihomo-γ-linolenic acid, arachidonic acid and eicosapentaenoic acid, and thromboxanes and leukotrienes derived from arachidonic acid and eicosapentaenoic acid , Eicosanoids (so-called PG ₂ -SeHe), which are made of ω6- Fatty acids are formed, usually promote inflammatory reactions, while eicosanoids (so-called PG3 series) of ω3 fatty acids have little or no pro-inflammatory effect.

Due to their positive properties, there have been no shortage of attempts in the past to make genes involved in the synthesis of fatty acids or triglycerides available for the production of oils in various organisms with modified levels of unsaturated fatty acids. Thus, WO 91/13972 describes a Δ9-desaturase, in WO 93/11245 a Δ15-desaturase and in WO 94/11516 a Δ12-desaturase. Further desaturases are described, for example, in EP-AO 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. As a rule, the characterization of membrane-bound desaturases takes place by introduction into a suitable organism, which is subsequently examined for enzyme activity by means of reactant and product analysis. Δ6-desaturases are described in WO 93/06712, US 5,614,393, US 5,614,393, WO 96/21022, WO 00/21557 and WO 99/27111 and also the application for production in transgenic organisms is described as in WO 98/46763 WO 98 / 46764, WO 98/46765. It also describes the expression of various desaturases as in WO 99/64616 or WO 98/46776 and the formation of polyunsaturated fatty acids.

The polyunsaturated fatty acids can be classified according to their desaturation pattern into two broad classes, the ω6 or ω3 fatty acids, which have metabolically and functionally different activities (Figure 1). The fatty acid linoleic acid (18: 2 ') acts as the starting material for the co-pathway, while the ω3 pathway proceeds via linolenic acid (18: 3 ^Δ9 ' ^{12 '15} ). Linolenic acid is formed by the activity of Δ15- or ω3-desaturase (Tocher et al., 1998, Prog. Lipid Res., 37, 73-117, Domergue et al., 2002, Eur. J. Biochem., 269, 4105-4113 ).

Mammals and therefore humans do not have the corresponding desaturase activity (Δ12- and ω3-desaturase) and must ingest these fatty acids (essential fatty acids) through the diet. Through the succession of desaturase and elongase reactions, the physiologically important polyunsaturated fatty acids ARA, an oo6 fatty acid, and the two ω3-fatty acids EPA and DHA are then synthesized from these precursors. The application of ω3-fatty acids shows the above-described therapeutic effect in the treatment of cardiovascular diseases.

The elongation of fatty acids by elongases of 2 and 4 C atoms, respectively, is of crucial importance for the production of C20 and C22 PUFAs, respectively. This process is over

4 steps. The first step is the condensation of malonyl-CoA on the fatty acid acyl-CoA by ketoacyl-CoA synthase (KCS, hereinafter referred to as elongase). This is followed by a reduction step (ketoacyl-CoA reductase, KCR), a dehydration step (dehydratase) and a final reduction step (enoyl-CoA reductase). It has been postulated that the activity of elongase affects the specificity and speed of the whole process.

The previously known processes for the preparation of ARA and / or EPA have some disadvantages. In general, both fatty acids are obtained as a mixture in the process. The only known process for producing arachidonic acid with low levels of EPA is a fungal, fermentative process. This is an oil source that is sub-optimal from a nutritional point of view because it contains fatty acids that are otherwise absent in human food. As further arachidonic acid source Eilipide come into question. However, these contain high levels of phospholipids, such as cholesterol, which tend to be detrimental to widespread use in foods.

Higher plants contain polyunsaturated fatty acids such as linoleic acid (Cl 8: 2) and linolenic acid (C18: 3). ARA, EPA and / or DHA are not present in the seed oil of higher plants or only in traces. However, it would be advantageous to produce LCPUFAs in higher plants, preferably in oilseeds such as oilseed rape, linseed, sunflower and soybeans, as this will enable large quantities of high quality LCPUFAs to be obtained inexpensively for the food, animal and pharmaceutical industries. For this purpose, gene coding for enzymes of the biosynthesis of LCPUFAs in oilseeds must be advantageously introduced and expressed via genetic engineering methods. These are genes which encode, for example, Δ6-desaturases, Δ6-elongases, Δ5-desaturases or Δ4-desaturases. These genes can be advantageously isolated from microorganisms and lower plants that produce LCPUFAs and incorporate them into membranes or triacylglycerides. For example, Δ6-desaturase genes from the moss Physcomitrella patens and Δ6 elongase genes from P. patens and the nematode C. elegans have already been isolated.

First transgenic plants which contain and express genes encoding enzymes of the LCPUF A biosynthesis and produce LCPUFAs have for the first time been described in DE 102 19 203, for example. However, these plants produce LCPUFAs in quantities that need further optimization for processing. Therefore, in order to facilitate fortification of food and feed with these polyunsaturated fatty acids, there is still a great need for a simple, inexpensive process for preparing these polyunsaturated fatty acids, especially ARA and EPA, and especially in eukaryotic systems.

It was therefore an object to develop a simple, inexpensive, economical process for the production of ARA and / or EPA, which does not have the aforementioned disadvantages. In addition, such a method should allow the synthesis of fatty acids inexpensively in almost any amount. In addition to the value products ARA and / or EPA as few other PUFAs, advantageously only either ω3 or ω6 fatty acids, advantageously only ω3 fatty acids should be included.

A further object was to provide further genes or enzymes which are suitable for the synthesis of LCPUFAs, in particular genes which encode a Δ5-desaturase or Δ6-desaturase activity, for the production of polyunsaturated fatty acids.

Another object of this invention has been to provide genes and enzymes, respectively, which permit a shift from the ω6 fatty acids to the ω3 fatty acids.

The objects of the invention were achieved, inter alia, by the process according to the invention for the production of arachidonic acid or eicosapentaenoic acid in transgenic organisms, characterized in that the content of arachidonic acid and / or eicosapentaenoic acid in the transgenic organism is at least 1% by weight, based on the total lipid content of the transgenic organism and the method comprises the following method step (s): a) introduction of a nucleic acid sequence into the organism which codes for a polypeptide having the activity of a Δ6-desaturase, selected from the group consisting of a nucleic acid sequence having the sequence shown in SEQ ID NO: 1, nucleic acid sequences resulting as a result of degenerate genetic codes from the amino acid sequence shown in SEQ ID NO: 2, and derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, which code for polypeptides having at least 40% identity with SEQ ID NO: 2 at amino acid level and a Δ6 Desaturase activity, and / or b) introducing into the organism a nucleic acid sequence which codes for a polypeptide having the activity of a Δ5-desaturase, selected from the group consisting of a nucleic acid sequence having the sequence shown in SEQ ID NO: 3, Nucleic acid sequences resulting from the degenerate genetic code of the amino acid sequence shown in SEQ ID NO: 4 derive nucleic acid sequence, and derivatives of the nucleic acid sequence shown in SEQ ID NO: 3, which encode polypeptides having at least 40% identity with SEQ ID NO: 4 at the amino acid level and have a Δ-5-desaturase activity.

The present invention also relates to a process for producing ω3-fatty acids in transgenic organisms, characterized in that it comprises the following process step (s): a) introduction of a nucleic acid sequence into the organism which represents a polypeptide with the activity of a Δ-6 Desaturase encoded selected from the group consisting of a nucleic acid sequence having the sequence shown in SEQ ID NO: 1, nucleic acid sequences which can be derived as the result of the degenerate genetic code of the amino acid sequence shown in SEQ ID NO: 2, and derivatives of in SEQ ID NO: 1 shown Nucleic acid sequence encoding polypeptides having at least 40% identity with SEQ ID NO: 2 at amino acid level and having a Δ6-desaturase activity, and / or b) introducing into the organism a nucleic acid sequence encoding a polypeptide having the activity of a Δ 5-desaturase selected from the group consisting of a

Nucleic acid sequence having the sequence shown in SEQ ID NO: 3, nucleic acid sequences which can be derived as a result of the degenerate genetic code of the amino acid sequence shown in SEQ ID NO: 4, and derivatives of the nucleic acid sequence shown in SEQ ID NO: 3, which polypeptides which have at least 40% identity with SEQ ID NO: 4 at the amino acid level and have Δ5-desaturase activity.

The steps a) and b) are advantageously carried out in each case. In a further preferred embodiment, in addition to the Δ 6-desaturase activity according to the invention and the Δ 5-desaturase activity according to the invention, a Δ 6-elongase activity is introduced into the organism. The organism is preferably a microorganism, a yeast or a plant, with useful plants being particularly preferred.

Nucleic acid sequences which code for polypeptides having the activity of a Δ6-elongase and which can be used in the context of the invention are described, for example, in WO 2007/017419, WO 2006/069710, WO 2006/100241, WO 2005/083053, WO2006 / 069936, WO2005 / 12316, Domergue et al. (2002) Eur J Biochem 269: 4105-4113; Girke et al. (1998) Plant J 15: 39-48. Preferably, the Δ6-elongase is a Δ6-elongase from the moss Physcomitrella patens, as described, for example, in Zank et al. (2002) Plant J. 31: 255-268. Particularly preferably, the Δ-6 elongase has the sequence shown in SEQ ID NO. 14 indicated amino acid sequence. In a preferred embodiment of the method, a nucleic acid sequence which codes for an ω-3-desaturase is additionally introduced into the organisms, especially the plants. Suitable nucleic acid sequences coding for an ω-3-desaturase are, for example. described in WO 2007/017419, WO 2006/069710, WO 2006/100241, WO 2005/083053, Michaelson et al. (1998) J Biol Chem 273: 19055-19059; Kaewsuwan et al. (2006) J Biol Chem. 281: 21988-97.

Moreover, the person skilled in the art can take suitable sequences of the current scientific literature and in particular the gene databases and use them for the purposes of this invention.

Advantageously, the polyunsaturated fatty acids ARA and / or EPA produced in the process according to the invention and further LCPUFAs of the ω-3 or ω-6 fatty acid series contain at least two, advantageously three, four, five or six double bonds. Particularly advantageously, the fatty acids contain four, five or six double bonds. The fatty acids produced in the process advantageously have 18, 20 or 22 carbon atoms in the fatty acid chain, preferably the fatty acids contain 20 or 22 carbon atoms in the fatty acid chain. Advantageously, it is ARA and / or EPA.

Advantageously, saturated fatty acids with the nucleic acids used in the process little or not at all, advantageously not reacted. Little is to be understood that, compared to polyunsaturated fatty acids, the saturated fatty acids having less than 5% of the activity, advantageously less than 3%, particularly advantageously less than 2%, very particularly preferably less than 1, 0.5, 0.25 or 0.125% of the activity are reacted. These fatty acids prepared in addition to the fatty acids ARA and / or EPA produced in the process can additionally be prepared as individual fatty acids in the process or be present in a fatty acid mixture. In the context of the invention, "ω3-fatty acids" are understood as meaning those unsaturated fatty acids in which the last double bond in the carbon chain is present at the third-last carbon bond from the carboxyl end. Examples of ω3 fatty acids are α-linolenic acid (18: 3 ^Δ9 ' ¹² ' ¹⁵ ), eicosapentaenoic acid (EPA; 20: 5 ^Δ5 ' ⁸ ' ⁿ ' ¹⁴ ' ¹⁷ ) and docosahexaenoic acid (DHA; 22: 6 ^Λ4 ' ⁷ ' ¹⁰ ' ¹³ ' ^{16 '19} ). Further ω3 -fatty acids are known to the person skilled in the art.

In the process according to the invention, predominantly ω3-fatty acids are produced, i. the ratio of ω3 fatty acids produced to ω6 fatty acids produced is at least 5: 1, 6: 1 or 7: 1, preferably at least 8: 1, 9: 1 or 10: 1, more preferably at least 12: 1, 15: 1 , 18: 1 or 20: 1. Most preferably, no ω6 fatty acids are produced by the process of the present invention. Thus, the Δ-6-desaturases and Δ5-desaturases of Mantoniella squamata according to the invention have the advantage of ω3 specificity over the desaturases of the prior art, such as, for example, the desaturases from Ostreococcus tauri and Phaeodactylum tricornutum (see, for example, Figures 15 and 16).

Advantageously, the abovementioned nucleic acid sequences which code for Δ-6-desaturases, Δ-5-desaturases and / or Δ-6-elongases and / or ω-desaturases are expressed in combination with further genes of the fatty acid and / or lipid metabolism, such as eg Nucleic acid sequences corresponding to polypeptides having Δ-8-desaturase, Δ-12-desaturase, Δ-15-desaturase, Δ-4-desaturase, Δ-9 elongase and / or Δ-5 elongase activity encode.

Suitable nucleic acid sequences which code for polypeptides having Δ-5-elongase, Δ-12-desaturase or Δ-4-desaturase activity are described in WO 2006/069710, WO 2006/100241, WO 2005/083053, WO2006 / 069936, WO2005 / 012316, Meyer et al. (2004) J. Lipid Res. 45: 1899-1909, Meyer et al. (2003) Biochemistry 42: 9779-88. The amino acid sequence of a suitable Δ-5 elongase is also shown in SEQ ID no. 16 indicated.

Moreover, the person skilled in the art can take suitable sequences of the current scientific literature and in particular the gene databases and use them for the purposes of this invention. This also applies to the other genes useful in this invention for enzymes involved in fatty acid and lipid biosynthesis.

Advantageously, the nucleic acids used in the method according to the invention are expressed in transgenic plants in the vegetative tissue (= somatic tissue). For the purposes of this invention, vegetative tissue is to be understood as meaning that the tissue is characterized by multiplying by mitotic divisions. Such tissue also arises from asexual reproduction (= apomixis) and multiplication. Propagation is when the number of individuals increases in successive generations. These individuals, born of asexual reproduction, are largely identical to their parents.

Examples of such tissues are leaf, flower, root, stem, aboveground or subterranean shoots (lateral shoots, stolons), rhizomes, buds, tubers such as tubers or tubers, onion, broodstock, brood buds, bulbils, or turions. Such tissues can also be caused by spurious, real or man-made viviparous. But also seeds, which are caused by agamospermia, as they are typical for Asteraceae, Poaceae or Rosaceae, belong to the vegetative tissues, in which the expression takes place favorably. To a lesser extent or not at all, the nucleic acids used in the method according to the invention are expressed in the generative tissue (germline tissue). For the purposes of this invention, generative tissue is to be understood as meaning that the tissue is formed by meiotic division. Examples of such tissues are tissues which are damaged by sex. Reproduction, that is, meiotic cell divisions arise, such as seeds, which have arisen through sexual processes. To a lesser extent, it is to be understood that, as compared to the vegetative tissue, the expression measured at the RNA and / or protein level is less than 5%, advantageously less than 3%, particularly advantageously less than 2%, very particularly preferably less than 1, 0.5, 0.25 or 0.125%.

The polyunsaturated fatty acids produced in the process are advantageously bound in membrane lipids and / or triacylglycerides, but may also be present as free fatty acids or bound in the form of other fatty acid esters in the organisms. They may be present as "pure products" or advantageously in the form of mixtures of different fatty acids or mixtures of different phospholipids such as phosphatidylglycol, phosphatidylcholine, phosphatidylethanolamine and / or phosphatidylserine and / or triacylglycerides, monoacylglycerides and / or diacylglycerides. The LCPUFAs ARA and / or EPA produced in the process are advantageously present in phosphatidylcholine and / or phosphatidylethanolamine and / or in the triacylglycerides. The triacylglycerides may also contain other fatty acids such as short-chain fatty acids having 4 to 6 carbon atoms, medium-chain fatty acids having 8 to 12 carbon atoms or long-chain fatty acids having 14 to 24 carbon atoms, preferably containing long-chain fatty acids, particularly preferably the long-chain Fatty acids LCPUFAs of C 18, C 20 or C 22 fatty acids.

Advantageously in the process according to the invention are fatty acid esters with polyunsaturated C 18, C 20 and / or C 22 fatty acid molecules having at least two double bonds in the fatty acid ester, advantageously having at least three, four, five or six double bonds in the fatty acid ester, more preferably at least five or six double bonds produced in the fatty acid ester and lead advantageously to the synthesis of linoleic acid (= LA, C18: 2Δ9,12), γ-linolenic acid acid (= GLA, C18: 3Δ6, 9, 12), stearidonic acid (= SDA, C18: 4Δ6.9, 12.15), dihomo-γ-linolenic acid (= DGLA, 20: 3Δ8, 1.14) , ω-3-eicosatetraenoic acid (= ETA, C20: 4Δ5.8, 1.14), arachidonic acid (ARA, C20: 4Δ5.8, 11.14), eicosapentaenoic acid (EPA, C20: 5Δ5.8, l 1,14,17), ω-6-docosapentaenoic acid (C22: 5Δ4,7, 10,13,16), ω-6-docosatetraenoic acid (C22: 4Δ7, 10,13,16), ω-3-docosapentaenoic acid (= DPA, C22: 5Δ7, 10, 13, 16, 19), docosahexaenoic acid (= DHA,

C22: 6Δ4, 7, 10, 13, 16, 19) or mixtures thereof, preferably ARA and / or EPA. Most preferably, the ω-3 fatty acid EPA is prepared.

The fatty acid esters with polyunsaturated C 18, C 20 and / or C 22 fatty acid molecules can be prepared from the organisms used for the preparation of the fatty acid esters in the form of an oil or lipid, for example in the form of compounds such as sphingolipids, phosphoglycerides, lipids, glycolipids such as glycosphingolipids, phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters such as the acetyl-coenzymeA esters containing the polyunsaturated fatty acids having at least two, three, four, five or six preferably five or more six double bonds are isolated, advantageously they are isolated in the form of their diacylglycerides, triacylglycerols and / or in the form of phosphatidylcholine, particularly preferably in the form of the triacylglycerides. In addition to these esters, the polyunsaturated fatty acids are also included as free fatty acids or bound in other compounds in the organisms beneficial to the plants. In general, the various aforementioned compounds (fatty acid esters and free fatty acids) in the organisms in an approximate distribution of 80 to 90 wt .-% triglycerides, 2 to 5 wt .-% diglycerides, 5 to 10 wt .-% monoglycerides, 1 to 5 wt .-% of free fatty acids, 2 to 8 wt .-% phospholipids ago, wherein the sum of the various compounds to 100 wt .-% complements. In the process according to the invention, the LCPUFAs produced have a content of at least 1, 2, 3 or 4% by weight, advantageously of at least 5, 6, 7, 8, 9 or 10% by weight, preferably of at least 11.12, 13, 14 or 15 wt .-%, particularly preferably of at least 16, 17, 18, 19 or 20 wt .-%, most preferably of at least 25, 30, 35 or 40 wt .-% based on the total fatty acids in the transgenic organisms, advantageously produced in a transgenic plant.

The fatty acids produced in the process according to the invention are ARA and / or EPA with a content of at least 10% by weight. preferably at least 11, 12, 13, 14 or 15% by weight, more preferably at least 16, 17, 18, 19 or 20% by weight, most preferably at least 25, 26, 27, 28, 29 , 30 or 31 wt .-%, most preferably of at least 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 wt .-% based on the total fatty acids in the triacylglycerols and / or phosphatidylglycerides advantageously contained in the phosphatidylcholine. Advantageously, the fatty acids are prepared in bound form. With the aid of the nucleic acids used in the method according to the invention, these unsaturated fatty acids can be brought to the snl, sn2 and / or sn3 position of the advantageously prepared triglycerides. Advantageously, at least 11% of the triacylglycerols are doubly substituted, that is substituted at snl and sn2 or sn2 and sn3 positions. Trisubstituted triacylglycerides are also detectable. Since the starting compounds linoleic acid (C 18: 2) or linolenic acid (C 18: 3) undergo several reaction steps in the process according to the invention, the end products of the process, such as, for example, arachidonic acid (ARA) or eicosapentaenoic acid (EPA), are not produced as absolute pure products. There are always traces or larger amounts of precursors in the final product. If, for example, both linoleic acid and linolenic acid are present in the starting plant, the end products such as ARA or EPA are present as mixtures. The precursors should not be beneficial more than 20 wt .-%, preferably not more than 15 wt .-%, more preferably not more than 10 wt .-%, most preferably not more than 5 wt .-%, based on the amount of the respective end product. Advantageously, only ARA or EPA are bound in the process of the invention in a transgenic plant as end products or prepared as free acids.

Fatty acid esters or fatty acid mixtures which have been prepared by the process according to the invention advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid; 7 - 85% oleic acid; 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachidic acid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acids and 60 to 85% of polyunsaturated fatty acids, based in each case on 100% and on the total fatty acid content of the organisms , As an advantageous polyunsaturated fatty acid in the fatty acid esters or fatty acid mixtures are preferably at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1% based on the total fatty acid content of arachidonic acid. Furthermore, the fatty acid esters or fatty acid mixtures which have been prepared by the process according to the invention advantageously contain fatty acids selected from the group of the fatty acids erucic acid (13-docosaic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8 , 9-methylene heptadec-8-enoic acid), chaulmoogric acid (cyclopentendodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,1-dienanoic acid), vernoic acid (9,10-epoxyoctadec-12-enoic acid), taranic acid (6-octadecynonic acid), 6-nonadecynoic acid, santalbinic acid (tl 1-octadecen-9-ynoic acid), 6,9-octadecenynonic acid, pyrulic acid (tl0-heptadecen-8-ynonic acid), crepenynic acid (9-octadecene acid). 12-ynonic acid), 13,14-dihydrooropheic acid, octadecene-13-ene-9,1-l-diynonic acid, petroselenoic acid (cis-6-octadecenoic acid), 9c, 12t-octadecadienoic acid, calendulic acid (8-octyl-2-octadecatrienoic acid), catalpinic acid ( 9% of octadecatrienoic acid), ele- losteric acid (9% of octadecatrienc ureine), jacric acid (8cl-oct2c-octadecatrienoic acid), punicic acid (9cl Itl3c Octadecatrienoic acid), parinaric acid (9 cc octyl-octadecatetraenoic acid), pinolenic acid (all-cis-5,9,12-octadecatrienoic acid), labialic acid (5,6-octadecadienenoic acid), ricinoleic acid (12-hydroxyoleic acid) and / or coriolinic acid (13-hydroxybenzoic acid). 9c, 1 lt octadecadienoic acid). The abovementioned fatty acids are generally advantageously present only in traces in the fatty acid esters or fatty acid mixtures prepared by the process according to the invention, that is to say they come to less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids. , 22% or 21%, more preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, most preferably less than 4%, 3%, 2% or 1% ago. In a further preferred embodiment of the invention, these abovementioned fatty acids come to less than 0.9%, 0.8%, 0.7%, 0.6% or 0.5%, more preferably less than 0, relative to the total fatty acids. 4%, 0.3%, 0.2%, 0.1% ago. Advantageously, the fatty acid esters or mixtures of fatty acids prepared by the process according to the invention contain less than 0.1% based on the total fatty acids or no butyric acid, no cholesterol, no clupanodonic acid (= docosapentaenoic acid, C22: 5Δ4, 8, 12, 15, 21) and no nisic acid (Tetracosahexaenoic acid, C23: 6Δ3, 8, 12, 15, 18, 21).

By the nucleic acid sequences according to the invention or in the method according to the invention used nucleic acid sequences can increase the yield of polyunsaturated fatty acids of at least 50%, preferably at least 80%, more preferably at least 100%, most preferably at least 150% compared to the non-transgenic starting organism For example, a yeast, an alga, a fungus, or a plant such as Arabidopsis or flax can be obtained by comparison in GC analysis.

Also, chemically pure polyunsaturated fatty acids or fatty acid compositions can be prepared by the methods described above. For this purpose, the fatty acids or the fatty acid compositions from the organism such as the microorganisms or the plants or the culture medium in which or on which the organisms were grown, or isolated from the organism and the culture medium in a known manner, for example via extraction, distillation, crystallization, chromatography or combinations of these methods. These chemically pure fatty acids or fatty acid compositions are advantageous for applications in the food industry, the cosmetics industry and especially the pharmaceutical industry.

In principle, all organisms such as microorganisms, non-human animals or plants come into question as organism for the preparation in the process according to the invention. As plants, in principle, all plants are in question, which are able to synthesize fatty acids as all dicotyledonous or monocotyledonous plants, algae or mosses.

The plants are advantageously crops. Agricultural crops are plants that are used for food production for humans and animals, the production of luxury foods, fibers and pharmaceuticals such as cereals such as corn, rice, wheat, barley, millet, oats, rye, buckwheat; such as tubers such as potatoes, cassava, potatoes, yams, etc .; such as sugar beets such as sugar cane or sugar beet; such as legumes such as beans, peas, broad bean, etc .; such as oil and fatty fruits such as soybean, rapeseed, sunflower, safflower, flax, camelina, etc., to name but a few. Advantageous plants are selected from the group of plant families consisting of the families of Aceraceae, Actinidiaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Arecaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Cannaceae, Caprifoliaceae, Chenopodiaceae, Convolvulaceae, Cucurbitaceae, Dioscoreaceae, Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Fagaceae, Geraniaceae, Gramineae, Grossulariaceae, Juglandaceae, Lauraceae, Leguminosae, Liliaceae, Linaceae, Malvaceae, Moraceae, Musaceae, Oleaceae, Oxalidaceae, Papaveraceae, Poaceae, Polygonaceae, Prasinophyceae, Punicaceae, Rosaceae, Rubiaceae, Rutaceae, Scrophulariaceae, Solanaceae, Sterculiaceae and Valerianaceae.

By way of example, the following plants may be selected from the group Adelotheciaceae, such as the genera Physcomitrella, e.g. the genus and species Physcomitrella patens, Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium e.g. the genus and species Pistacia vera [pistachio], Mangifer indica [Mango] or Anacardium occidentale [cashew], Asteraceae such as the genera Calendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta, Tagetes, Valeriana e.g. the genus and species Calendula officinalis [garden calendula], Carthamus tinctorius [safflower], Centaurea cyanus [cornflower], Cichorium intybus [chicory], Cynara scolymus [Artichoke], Helianthus annus [sunflower], Lactuca sativa, Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp. sativa, Lactuca scariola L. var. integrata, Lactuca scariola L. var. integrifolia, Lactuca sativa subsp. romana, Locusta communis, Valeriana locusta [lettuce], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [marigold], Apiaceae such as the genus Daucus e.g. the genus and species Daucus carota [carrot], Betulaceae such as the genus Corylus e.g. the genera and species Corylus avellana or Corylus colurna [hazelnut], Boraginaceae such as the genus Borago e.g. the genus and species Borago officinalis [borage], Brassicaceae such as the genera Brassica, Camelina, Melanosinapis, Sinapis, Arabidopsis e.g. the genera and species Brassica napus,

Brassica rapa ssp. [Rapeseed], Sinapis arvensis, Brassica juncea, Brassica juncea var. Juncea, Brassica juncea var. Crispifolia, Brassica juncea var. Foliosa, Brassica nigra, Brassica sinapioides, Camelina sativa, Melanosinapis communis [mustard], Brassica oleracea [feeder] or Arabidopsis thaliana , Bromeliaceae such as the genera Anana, Bromelia (pineapple) eg the genera and species Anana comosus, pineapple pineapple or Bromelia comosa [pineapple], Caricaceae as the genus Carica as the genus and species Carica papaya [Papaya], Cannabaceae as the genus Cannabis as the genus and species Cannabis sative [hemp], Convolvulaceae as the genera Ipomea, Convolvulus eg the genera and species Ipomoea batatus, Ipomoea pandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus [Sweet potato, Batata],

Chenopodiaceae such as the genus Beta such as the genera and species Beta Vulgaris, Beta vulgaris var. Altissima, Beta vulgaris var. Vulgaris, Beta maritima, Beta vulgaris var. Perennis, Beta vulgaris var. Conditiva or Beta vulgaris var. Esculenta [sugar beet], Crypthecodiniaceae such as the genus Crypthecodinium eg the genus and species Cryptecodinium cohnii, Cucurbitaceae such as the genus Cucubita eg the genera and species Cucurbita maxima, Cucurbita mixta, Cucurbita pepo or Cucurbita moschata [squash], Cymbellaceae such as the genera Amphora, Cymbella, Okedenia, Phaeodactylum, Reimeria eg the genus and species Phaeodactylum tricornutum, Ditrichaceae such as the genera Ditrichaceae, Astomiopsis, Ceratodon, Chrysoblastella, Ditrichum, Distichium, Eccremidium, Lophidion, Philibertiella, Pleuridium, Saelania, Trichodon, Skottsbergia eg the genera and species Ceratodon antarcticus, Ceratodon columbiae, Ceratodon heterophyllus , Ceratodon pu [phi] urascens, Ceratodon purpureus, Ceratodon purpureus ssp. convolutus, Ceratodon purpureus ssp. stenocarpus, Ceratodon purpureus var. rotundifolius, Ceratodon ratodon, Ceratodon stenocarpus, Chrysoblastella chilensis, Ditrichum ambiguum, Ditrichum brevisetum, Ditrichum crispatissimum, Ditrichum difficile, Ditrichum falcifolium, Ditrichum fiexicaule, Ditrichum giganteum, Ditrichum heteromallum, Ditrichum linear, Ditrichum linear, Ditrichum montanum, Ditrichum montanum, Ditrichum pallidum, Ditrichum punctulatum, Ditrichum pusillum, Ditrichum pusillum var. Tortile, Ditrichum rhynchostegium, Ditrichum schimperi, Ditrichum tortile, Distichium capillaceum, Distichium hagenii, Distichium inclinatum, Distichium macounii, Eccremidium fioridanum, Eccremidium whiteleggei, Lophidion strictus, Pleuridium acuminatum, Pleuridium alternifolium, Pleuridium holdridgei, Pleuridium mexicanum, Pleuridium ravenelii, Pleuridium subulatum, Saelania glaucescens, Trichodon borealis, Trichodon cylindricus or Trichodon cylindricus var. Oblongus, Elaeagnaceae such as the genus Elaeagnus eg the genus and species Olea europaea [Olive], Ericaceae like the genus Kalmia eg the genera and species Kalmia latifolia, Kalmia angustifolia, Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistus chamaerhodendros or Kalmia lucida [Berglorbeer], Euphorbiaceae such as the genera Manihot, Janipha, Jatropha, Ricinus eg the genera and species Manihot utilissima, Janipha manihot, Manathot manihot, Manihot aipil, Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot esculenta [Manihot] or Ricinus communis [Castor], Fabaceae such as the genera Pisum, Albizia, Cathormion, Feuillea, Inga, Pithecolobium, Acacia , Mimosa, Medicajo, Glycine, Dolichos, Phaseolus, Soy eg the genera and species Pisum sativum, Pisum arvense, Pisum humile [pea], Albizia berteriana, Albizia julibrissin, Albizia lebbeck, Acacia berteriana, Acacia littoralis, Albizia berteriana, Albizzia berteriana, Cathormion berteriana, Feuillea berteriana, Inga fragrans, Pithecellobium berterianum, Pithecellobium fragrans, Pithecolobium berterianum , Pseudalbizzia berteriana,

Acacia julibrissin, Acacia nemu, Albizia nemu, Feuilleea julibrissin, Mimosa julibrissin, Mimosa speciosa, Sericanrda julibrissin, Acacia lebbeck, Acacia macrophylla, Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa [Seidenbaum], Medicago sativa, Medicago falcata, Medicago varia [Alfalfa] Glycine max Dolichos soy, Glycine gracilis, Glycine hispida, Phaseolus max, Soy hispida or Soy max [Soybean], Funariaceae as the genera

Aphanorrhegma, Entosthodon, Funaria, Physcomitrella, Physcomitrium eg bolanderi the genera and species Aphanorrhegma serratum, Entosthodon attenuatus, Entosthodon, Entosthodon bonplandii, Entosthodon californicus, Entosthodon drummondii, jamesonii Entosthodon, Entosthodon leibergii, tucsoni Entosthodon neoscoticus, Entosthodon rubrisetus, Entosthodon spathulifolius, Entosthodon, Funaria americana, Funaria bolanderi, Funaria calcarea, - YY -

Funaria californica, Funaria calvescens, Funaria convoluta, Funaria flavicans, Funaria groutiana, Funaria hygrometrica, Funaria hygrometrica var. Arctica, Funaria hygrometrica var. Calvescens, Funaria hygrometrica var. Convoluta, Funaria hygrometrica var. Muralis, Funaria hygrometrica var. Utahensis, Funaria microstoma , Funaria microstoma var. Obtusifolia, Funaria muhlenbergii, Funaria orcuttii, Funaria piano convexa, Funaria polaris, Funaria ravenelii, Funaria rubriseta, Funaria serrata, F [upsilon] naria sonorae, Funaria sublimbatus, Funaria tucsoni, Physcomitrella californica, Physcomitrella patens, Physcomitrella readeri, Physcomitrium spp., Physcomitrium californicum, Physcomitrium collenchymatum, Physcomitrium coloradense, Physcomitrium cupuliferum, Physcomitrium drummondii, Physcomitrium eurystomum, Physcomitrium flexifolium, Physcomitrium hookeri, Physcomitrium hookeri var. serratum, Physcomitrium immersum, Physcomitrium kellermanii, Physcomitrium megalocarpum, Physcomitrium pyriforme, Physcomitrium pyriforme var. serratum, Physcomitrium rufipes, Physcomitrium sandbergii, Physcomitrium subsphaericum, Physcomitrium washingtoniense, Geraniaceae such as the genera Pelargonium, Cocos, Oleum e.g. the genera and species Cocos nucifera, Pelargonium grossularioides or Oleum cocois [coconut], Gramineae such as the genus Saccharum e.g. the genus and species Saccharum officinarum, Juglandaceae such as the genera Juglans, Wallia e.g. the genera and species of Juglans regia, Juglans ailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea, Juglans bixbyi, Juglans californica, Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa, Juglans nigra or Wallia nigra [Walnut], Lauraceae Like the genera Persea, Laura eg the

Genera and species Laurus nobilis [Laurel], Persea ame [eta] cana, Persea gratissima or Persea persea [avocado], Leguminosae such as the genus Arachis eg the genus and species Arachis hypogaea [peanut], Linaceae such as the genera Linum, Adenolinum eg the Genera and species Linum usitatissimum, Linum humile, Linum aust [eta] acum, Linum bienne, Linum angustifolium, Linum catharticum, Linum fiavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var. lewisii, Linum pratense or Linum trigynum [flax], Lythrarieae as the genus Punica eg the genus and species Punica granatum [pomegranate], Malvaceae as the genus Gossypium eg the genera and species Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum or Gossypium thurberi [cotton], Marchantiaceae such as the genus Marchantia eg the genera and species Marchantia berteroana, Marchantia foliacea, Marchantia macropora, Musaceae as the genus Musa eg the genera and species Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [Banana], Onagraceae such as the genera Camissonia, Oenothera eg the genera and species Oenothera biennis or Camissonia brevipes [evening primrose], Palmae as the genus Elacis eg the genus and species Elaeis guineensis [oil palm], Papaveraceae as the genus Papaver eg the genera and Species Papaver Oriental, Papaver rhoeas, Papaver dubium [poppy], Pedaliaceae as the genus Sesamum eg the genus and species Sesamum indicum [sesame], Piperaceae as the genera Piper, Artanthe, Peperomia, Steffensia eg the genera and species Piper ad [upsilon] Piper amalago, Piper angustifolium, Piper auritum, Piper betel, Piper cubeba, Piper longum, Piper nigrum, Piper retrofractum, Artanthe adunca, Artanthe elongata, Peperomia elongata, Piper elongatum, Steffensia elongata [Cayenne pepper], Poaceae such as the genera Hordeum, Sea ale, Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea (corn), Triticum eg the genera and species Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeum secalinu m, Hordeum distichon Hordeum aegiceras, Hordeum hexastichon, Hordeum hexastichum, Hordeum irregular, Hordeum sativum, Hordeum secalinum [barley], Seeale cereale [rye], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida [oats Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghum vulgaris, Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghum aethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum, Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense, Sorghum lanceolatum, Sorghum nervosa, Sorghum saccharatum, Sorghum subglabrescens, Sorghum verticilliflorum, Sorghum vulgaris, Holcus halepensis, Sorghum miliaceum, Panicum militaceum [millet], Oryza sativa, Oryza latifolia [rice], Zea mays [maize] Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare [wheat], Porphyridiaceae as the genera

Chroothece, Flintiella, Petrovanella, Porphyridium, Rhodella, Rhodosorus, Vanhoeffenia e.g. the genus and species Porphyridium cr [upsilon] entum, Proteaceae such as the genus Macadamia e.g. the genus and species Macadamia intergrifolia [Macadamia], Prasinophyceae such as the genera Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus e.g. the genera and species Nephroselmis olivacea, Prasinococcus capsulatus, Scherffelia dubia,

Tetraselmis chui, Tetraselmis suecica, Mantoniella squamata, Ostreococcus tauri, Rubiaceae such as the genus Coffea eg the genera and species Cofea spp., Coffea arabica, Coffea canephora or Coffea liberica [coffee], Scrophulariaceae such as the genus Verbascum eg the genera and species Verbascum blattaria Verbascum chaixii, Verbascum densifiorum, Verbascum lagurus, Verbascum longifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum, Verbascum phlomoides, Verbascum phenicum, Verbascum pulverulentum or Verbascum thapsus, Solanaceae such as the genera Capsicum, Nicotiana, Solanum, Lycopersicon Genera and species Capsicum annuum, Capsicum annuum var. Glabriusculum, Capsicum frutescens [pepper], Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata, Nicotiana attenuata, Nicotiana glauca, Nicotiana slowdorifii, Nicotiana obtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotiana rustica , Nicotiana sylvestris [tobacco], Solanum tuberosum [Potato], Solanum melongena [eggplant], Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon py [eta] forme, Solanum integrifolium or Solanum lycopersicum [tomato], Sterculiaceae such as the genus Theobroma eg the genus and species Theobroma cacao [cocoa] or Theaceae like the genus Camellia eg the genus and species Camellia sinensis [tea].

Examples of advantageous microorganisms are fungi selected from the families of the families Chaetomiaceae, Choanephoraceae, Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae, Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae or Tuberculariaceae. By way of example, the following microorganisms selected from the group: Choanephoraceae such as the genera Blakeslea, Choanephora e.g. the genera and species Blakeslea trispora, Choanephora cueurbitarum, Choanephora infundibulifera var. cueurbitarum, Mortierellaceae such as the genus Mortierella e.g. the genera and species Mortierella isabellina, Mortierella polycephala,

Mortierella ramanniana, Mortierella vinacea, Mortierella zonata, Pythiaceae such as the genera Phytium, Phytophthora e.g. the genera and species Pythium debaryanum, Pythium intermedium, Pythium irregular, Pythium megalacanthum, Pythium paroecandrum, Pythium sylvaticum, Pythium ultimum, Phytophthora cactorum, Phytophthora cinnamomi, Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri,

Phytophthora erythroseptica, Phytophthora lateralis, Phytophthora megasperma, Phytophthora nicotianae, Phytophthora nicotianae var. Parasitica, Phytophthora palmivora, Phytophthora parasitica, Phytophthora syringae, Saccharomycetaceae such as the genera Hansenula, Pichia, Saccharomyces, Saccharomyces, Yarrowia eg the genera and species Hansenula anomala, Hansenula californica Hansenula canadensis, Hansenula capsulata, Hansenula ciferrii, Hansenula glucozyma, Hansenula henricii, Hansenula holstii, Hansenula minuta, Hansenula nonfermentans, Hansenula philodendri, Hansenula polymorph, Hansenula saturnus, Hansenula subpelliculosa, Hansenula wickerhamii, Hansenula wingei, Pichia alcoholophila, Pichia angusta, Pichia anomala, Pichia bispora, Pichia burtonii, Pichia canadensis, Pichia capsulata, Pichia carsonii, Pichia cellobiosa, Pichia ciferrii, Pichia farinosa, Pichia fermentans, Pichia finlandica, Pichia glucozyma, Pichia guilliermondii, Pichia haplophila, Pichia henricii, Pichia holstii, Pichia jadinii, Pichia lindnerii, Pichia membranaefaciens, Pichia methanolica, Pichia minuta var. minuta, Pichia minuta var. nonfermentans, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia philodendri, Pichia pini, Pichia polymorphe, Pichia quercuum, Pichia rhodanensis, Pichia sargentensis, Pichia stipitis, Pichia strasburgensis, Pichia subpelliculosa, Pichia toletana, Pichia trehalophila, Pichia vini, Pichia xylosa, Saccharomyces aceti, Saccharomyces bailii, Saccharomyces bayanus, Saccharomyces bisporus, Saccharomyces capensis , Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces cerevisiae var. Ellipsoideus, Saccharomyces chevalieri, Saccharomyces delbrueckii, Saccharomyces diastaticus, Saccharomyces drosophilarum, Saccharomyces elegans, Saccharomyces ellipsoideus,

Saccharomyces fermentati, Saccharomyces florentine, Saccharomyces fragilis, Saccharomyces heterogenicus, Saccharomyces hienipiensis, Saccharomyces inusitatus, Saccharomyces italicus, Saccharomyces kluyveri, Saccharomyces krusei, Saccharomyces lactis, Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomyces montanus, Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomyces paradoxus, Saccharomyces pastorianus , Saccharomyces pretoriensis, Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces uvarum, Saccharomyces ludwigii, Yarrowia lipolytica, Schizosacharomycetaceae such as the generic Schizosaccharomyces eg the species Schizosaccharomyces japonicus var. japonicus, Schizosaccharomyces japonicus var. versatilis, Schizosaccharomyces malidevorans, Schizosaccharomyces octosporus, Schizosaccharomyces pombe var. malidevorans,

Schizosaccharomyces pombe var. Pombe, Thraustochytriaceae: such as the genera Althomia, limacinum Aplanochytrium, Japonochytrium, Schizochytrium, Thraustochytrium eg the species Schizochytrium aggregatum, Schizochytrium, Schizochytrium minutum mangrovei, Schizochytrium, Schizochytrium octosporum, aggregatum Thraustochytrium, amoeboideum Thraustochytrium, Thraustochytrium antacticum, arudimentale Thraustochytrium, Thraustochytrium aureum, Thraustochytrium benthicola, Thraustochytrium globosum, Thraustochytrium indicum, Thraustochytrium kerguelense, Thraustochytrium kinnei, Thraustochytrium motivum, Thraustochytrium multi Rudi mental, Thraustochytrium pachydermum, Thraustochytrium proliferum, Thraustochytrium roseum, Thraustochytrium rossii, visurgense Thraustochytrium Striaton or Thraustochytrium. Further advantageous microorganisms are, for example, bacteria selected from the group of the families Bacillaceae, Enterobacteriacae or Rhizobiaceae.

Examples include the following microorganisms selected from the group: Bacillaceae such as the genus Bacillus z.Beg the genera and species Bacillus acidocaldarius,

Bacillus acidoterrestris, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus sphaericus subsp. fusiformis, Bacillus galactophilus, Bacillus globisporus, Bacillus globisporus subsp. marinus, Bacillus halophilus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis subsp. spizizenii, Bacillus subtilis subsp. subtilis or Bacillus thuringiensis; Enterobacteriacae such as the genera Citrobacter, Edwardsieila, Enterobacter, Erwinia, Escherichia, Klebsiella, Salmonella or Serratia eg the genera and species Citrobacter amalonaticus, Citrobacter diversus, Citrobacter freundii, Citrobacter genomospecies, Citrobacter gillenii, Citrobacter intermedium, Citrobacter koseri, Citrobacter murliniae, Citrobacter sp , Edwardsiella hoshinae, Edwardsieila ictaluri, Edwardsiella tarda, Erwinia alni, Erwinia amylovora, Erwiniaananatis, Erwinia aphidicola, Erwinia billingiae, Erwinia cacticida, Erwinia carcinogena, Erwinia carnegieana, Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. betavasculorum, Erwinia carotovora subsp. odorifera, Erwinia carotovora subsp. wasabiae, Erwinia chrysanthemi, Erwinia cypripedii, Erwinia dissolvens, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia nimipressuralis, Erwinia persicina, Erwinia psidii, Erwinia pyrifoliae, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia tracheiphila, Erwinia uredovora, Escherichia adecarboxylata, Escherichia anindolica, Escherichia aurescens, Escherichia blattae, Escherichia coli, Escherichia coli var. Communior, Escherichia coli mutabile, Escherichia fergusonii, Escherichia hermannii, Escherichia sp., Escherichia vulneris, Klebsiella aerogenes, Klebsiella edwardsii subsp. atlantae, Klebsiella ornithinolytica, Klebsiella oxytoca, Klebsiella planticola, Klebsiella pneumoniae, Klebsiella pneumoniae subsp. pneumoniae, Klebsiella spp., Klebsiella terrigena, Klebsiella trevisanii, Salmonella abony, Salmonella arizonae, Salmonella bongori, Salmonella choleraesuis subsp. arizonae, Salmonella choleraesuis subsp. bongori, Salmonella choleraesuis subsp. cholereasuis, Salmonella choleraesuis subsp. diarizonae, Salmonella choleraesuis subsp. houtenae, Salmonella choleraesuis subsp. indica, Salmonella choleraesuis subsp. Salamae, Salmonella daressalaam, Salmonella enterica subsp. houtenae, Salmonella enterica subsp. salamae, Salmonella enteritidis, Salmonella gallinarum, Salmonella heidelberg, Salmonella panama, Salmonella senftenberg, Salmonella typhimurium, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia liquefaciens, Serratia marcescens, Serratia marcescens subsp. marcescens, Serratia marinorubra, Serratia odorifera, Serratia plymouthensis, Serratia plymuthica, Serratia proteamaculans, Serratia proteamaculans subsp. quinovora, Serratia quinivorans or Serratia rubidaea; Rhizobiaceae such as the genera Agrobacterium, Carbophilus, Chelatobacter, Ensifer, Rhizobium, Sinorhizobium eg the

Species and Species: Agrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacterium gelatinovorum, Agrobacterium larrymoorei, Agrobacterium meteori, Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium rubi, Agrobacterium stellulatum, Agrobacterium tumefaciens, Agrobacterium vitis, Carbophilus carboxidus, Chelatobacter heintzii, Ensifer adhaerens, Ensifer arboris, Ensifer fredii , Ensifer kostiensis, Ensifer kummerowiae, Ensifer Rhizobium eti, Rhizobium fredii, Rhizobium galegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobium hainanense, Rhizobium huakuii, Rhizobium huautlense, Rhizobium indigoferae, Rhizobium japoni cum, Rhizobium leguminosarum, Rhizobium loessense, Rhizobium loti, Rhizobium lupini, Rhizobium mediterraneum, Rhizobium meliloti, Rhizobium mongolense, Rhizobium phaseoli, Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium rubi, Rhizobium sullae, Rhizobium tianshanense, Rhizobium trifolii, Rhizobium tropici, Rhizobium undicola, Rhizobium vitis, Sinorhizobium adhaerens , Sinorhizobium arboris, Sinorhizobium fredii, Sinorhizobium kostiense, Sinorhizobium kummerowiae, Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium morelense, Sinorhizobium saheli or Sinorhizobium xinjiangense.

Further advantageous microorganisms for the method according to the invention are, for example, protists or diatoms selected from the group of the families Dinophyceae, Turaniellidae or Oxytrichidae such as the genera and species: Crypthecodinium cohnii, Phaeodactylum tricornutum, Stylonychia mytilus, Stylonychia pustulata, Stylonychia putrina, Stylonychia notophora, Stylonychia sp. , Colpidium campylum or Colpidium sp.

Advantageous in the process according to the invention are transgenic organisms such as fungi such as Mortierella or Traustochytrium, yeasts such as Saccharomyces or Schizosaccharomyces, mosses such as Physcomitrella or Ceratodon, non-human animals such as Caenorhabditis, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium or Phaeodactylum or plants such as dicotyledonous or monocotyledonous plants. It is particularly advantageous to use organisms in the process according to the invention which belong to the oil-producing organisms, ie be used for the production of oils, such as fungi such as Mortierella or Thraustochytrium, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium, Phaeodactylum or plants, especially plants, preferably oil crops containing large amounts of lipid compounds contain, such as peanut, rapeseed, canola, sunflower, safflower (Carthamus tinctoria), poppy, mustard, hemp, castor, olive, sesame, calendula, punica, evening primrose, mullein, thistle, wild roses, hazelnut, almond, macadamia, avocado, laurel , Pumpkin, flax, soy, pistachio, borage, trees (oil palm, coconut or walnut) or crops such as corn, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, tagetes, solanaceae plants, such as Potato, tobacco, aubergine and tomato, Vicia species, pea, alfalfa or bush plants

(Coffee, cocoa, tea), Salix species and perennial grasses and forage crops. Preferred plants according to the invention are oil crop plants, such as peanut, rapeseed, canola, sunflower, safflower, poppy, mustard, hemp, castor, olive, calendula, punica, evening primrose, pumpkin, flax, soy, borage, trees (oil palm, coconut). Particularly preferred are C 18: 2 and / or C 18: 3 fatty acid-rich plants such as sunflower, safflower, tobacco, mullein, sesame seed,

Cotton, pumpkin, poppy, evening primrose, walnut, flax, hemp, thistle or safflower. Very particularly preferred are plants such as safflower, sunflower, poppy, evening primrose, walnut, flax or hemp.

For the method according to the invention described, it is advantageous in the plants in addition to the introduced under step (a) and (b) nucleic acids and optionally introduced nucleic acid sequences encoding the Δ-6 elongase and / or the ω-3-desaturases to additionally introduce further nucleic acids which code for enzymes of the fatty acid or lipid metabolism. In principle, all genes of the fatty acid or lipid metabolism can advantageously be used in combination with the nucleic acid sequences used in the inventive method, which are suitable for Δ6-desaturase (s), Δ5-desaturase (s), Δ6-elongase (s) and / or ω-3-desaturase (s) [in the context of this application, the plural shall encode the singular and vice versa]; Advantageously, genes of the fatty acid or lipid metabolism are selected from the group of acyl-CoA dehydrogenase (s), acyl-ACP [= acyl carrier protein] desaturase (s), acyl-ACP thioesterase (s), fatty acid acyl transferase ( n), acyl-CoAlysophospholipid acyltransferases, fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A-carboxylase (s), acyl coenzyme A oxidase (s), fatty acid desaturase (n), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allene oxide synthases, hydroperoxide lyases or fatty acid elongase (s) in combination with Δ6-elongase, Δ6-desaturase, Δ5-desaturase and or ω-3-desaturase used. Particular preference is given to genes selected from the group of Δ-4-desaturases, Δ-8-desatuases, Δ-9-desaturases, Δ-12-desaturases, Δ-5-elongases or Δ-9-elongases in combination with the abovementioned genes for the Δ-6 elongase, Δ-6-desaturase, Δ-5-desaturase and / or ω-3-desaturase, wherein single genes or multiple genes can be used in combination.

The ω-3-desaturase used in the method according to the invention should advantageously allow a shift from the ω-6 biosynthetic pathway to the ω-3 biosynthetic pathway, which advantageously leads to a shift of Cl 8: 2 to C18: 3 fatty acids. By virtue of these properties of omega-3-desaturase, it is advantageously possible to shift the fatty acid spectrum within an organism, advantageously within a plant or a fungus, from the omega-6 fatty acids to the omega-3 fatty acids. Furthermore, it is advantageous that the ω-3-desaturase converts a wide range of phospholipids such as phosphatidylcholine (= PC), phosphatidylinositol (= PIS) or phosphatidylethanolamine (= PE). Finally, desaturation products can also be used in the Neutral lipids (= NL), that is found in the triglycerides.

The Δ5-desaturases and Δ6-desaturases according to the invention from Mantoniella squamata have the advantage over the known Δ5-desaturases and Δ6-desaturases, for example from Phaeodactylum tricornutum, that they bind fatty acids to phospholipids or CoA-

Fatty acid esters, advantageously CoA-F ettsäureester implement. Advantageously, the desaturases used in the process according to the invention convert their respective substrates in the form of the Co A-fatty acid esters (see Examples 10 and 11). This leads, if previously a Elongationsschritt has taken place, advantageously to an increased product yield. The respective desaturation products are thereby synthesized in higher amounts, since the elongation step usually takes place on the CoA fatty acid esters, while the desaturation step takes place predominantly on the phospholipids or on the triglycerides. An exchange reaction between the CoA fatty acid esters and the phospholipids or triglycerides, which would require a further possibly limiting enzyme reaction, is thus not necessary.

By the enzymatic activity of the nucleic acids used in the method according to the invention which code for polypeptides with Δ-6-elongase, Δ-6-desaturase, Δ-5-desaturase and / or ω-3-desaturase activity, advantageously in combination with nucleic acid sequences which are polypeptides of fatty acid or lipid metabolism such as other polypeptides with Δ-4, Δ-5, Δ-6, Δ-8, Δ-12-desaturase or Δ-5, Δ-6 or Δ -9-Elongaseaktivität encode, a variety of polyunsaturated fatty acids can be prepared in the process according to the invention. Depending on the selection of the crop plants used for the process according to the invention, mixtures of the various polyunsaturated fatty acids or individual polyunsaturated fatty acids such as EPA or ARA can be prepared in free or bound form. Depending on which fatty acid composition prevails in the starting plant (Cl 8: 2 or C18: 3 fatty acids), fatty acids derived from C18: 2 fatty acids, such as GLA, DGLA or ARA or those derived from C18: 3 Derived fatty acids, such as SDA, ETA or EPA. If only linoleic acid (= LA, C18: 2 ^{Δ9 '12} ) is present as unsaturated fatty acid in the plant used for the process, only GLA, DGLA and ARA can arise as products of the process which may be present as free fatty acids or bound. If only α-linolenic acid (= ALA, C18: 3Δ9,12,15) is present as unsaturated fatty acid in the plant used in the process, for example as in flax, the products of the process can only be SDA, ETA and / or EPA, which as described above may be present as free fatty acids or bound. By modifying the activity of the enzymes used in the process and involved in the synthesis Δ6-elongase, Δ6-desaturase, Δ5-desaturase and / or ω-3-desaturase advantageous in combination with other genes of lipid or fatty acid metabolism can be specifically produced in the plants only individual products. Advantageously, only ARA or EPA or their mixtures are synthesized, depending on the fatty acid present in the organism or in the plant, which serves as the starting substance for the synthesis. Since it is about

Biosynthesis chains, the respective end products are not present as pure substances in the organisms. There are always small amounts of precursor compounds in the final product. These small amounts are less than 20 wt .-%, advantageously less than 15 wt .-%, more preferably less than 10 wt .-%, most preferably less than 5, 4, 3, 2 or 1 wt .-% based on the end product EPA or ARA or mixtures thereof.

In addition to the production of the starting fatty acids for the process according to the invention directly in the organism, especially the plant, the fatty acids can in principle also be fed from the outside. For cost reasons, production in the organism, especially in the plant, is preferred. Preferred substrates are linoleic acid (C18: 2Δ9,12), γ-linolenic acid (C18: 3Δ6,9,12), Eicosadienoic acid (C20: 2Δ11,14), dihomo-γ-linolenic acid (C20: 3Δ8, 11,14), arachidonic acid (C20: 4Δ5,8, 11,14), docosatetraenoic acid (C22: 4Δ7, 10,13, 16) and docosapentaenoic acid (C22: 5Δ4,7,10,13,15).

To increase the yield in the described process for the preparation of oils and / or triglycerides having an advantageously increased content of polyunsaturated fatty acids, it is advantageous to increase the amount of starting material for the fatty acid synthesis, this can be achieved, for example, by introducing a nucleic acid into the organism for a polypeptide encoded with Δ-12-desaturase activity. This is particularly advantageous in oil-producing organisms such as the Brassicaceae family such as the genus Brassica e.g. rape; the family of Elaeagnaceae such as the genus Elaeagnus e.g. the genus and species Olea europaea or the family Fabaceae such as the genus Glycine e.g. the genus and species Glycine max, which have a high oleic acid content. Since these organisms have only a low content of linoleic acid, the use of said Δ-12-desaturases for the preparation of the starting product linoleic acid is advantageous.

Nucleic acids used in the method according to the invention are advantageously derived from plants such as algae, for example algae of the family Prasinophyceae as from the genera Heteromastix, Mammella, Mantoniella, Micromonas, Nephroselmis, Ostreococcus, Prasinocladus, Prasinococcus, Pseudoscourfielda, Pycnococcus, Pyramimonas, Scherffelia or Tetraselmis such as the genera and Heteromastix longifillis, Mamiella gilva, Mantoniella squamata, Micromonas pusilla, Nephroselmis olivacea, Nephroselmis pyriformis, Nephroselmis rotunda, Ostreococcus tauri, Ostreococcus sp. Prasinocladus ascus, Prasinocladus lubricus, Pycnococcus provasolii, Pyramimonas amylifera, Pyramimonas disomata, Pyramimonas obovata, Pyramimonas orientalis, Pyramimonas parkeae, Pyramimonas spinifera, Pyramimonas sp., Tetraselmis apiculata, Tetraselmis carteriaformis, Tetraselmis Chui, convolutae Tetraselmis, Tetraselmis desikacharyi, Tetraselmis gracilis, Tetraselmis hazeni, Tetraselmis impellucida, Tetraselmis inconspicua, Tetraselmis levis, Tetraselmis maculata, Tetraselmis marina, Tetraselmis striata, Tetraselmis subcordiformis, Tetraselmis suecica, Tetraselmis tetrabrachia, Tetraselmis tetrathele , Tetraselmis verrucosa, Tetraselmis verrucosa fo. rubens or tetraselmis sp. or algae of the Euglenaceae family such as the genera Ascoglena, Astasia, Colacium, Cyclidiopsis, Euglena, Euglenopsis, Hyalophacus, Khawkinea, Lepocinclis, Phacus, Strombomonas or Trachelomonas such as the genera and species Euglena acus, Euglena geniculata, Euglena gracilis, Euglena mixocylindracea, Euglena rostrifera, Euglena viridis, Colacium stentorium, Trachelomonas cylindrica or Trachelomonas volvocina. Advantageously, the nucleic acids used are derived from algae of the genera Euglena, Mantoniella or Ostreococcus.

Further advantageous plants are algae such as Isochrysis or Crypthecodinium, algae / diatoms such as Thalassiosira or Phaeodactylum, mosses such as Physcomitrella or Ceratodon or higher plants such as Primulaceae such as Aleuritia, Calendula stellata, Osteospermum spinescens or Osteospermum hyoseroides, microorganisms such as fungi such as Aspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor or Mortierella, bacteria such as Shewanella, yeast or animals such as nematodes such as Caenorhabditis, insects, frogs, sea cucumbers or fish. Preferably, the nucleic acid sequences are of the vertebrate class; Euteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei, Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus or Vertebrata, Amphibia, Anura, Pipidae, Xenopus or Evertebrata such as Protochordata, Tunicata, Holothuroidea, Cionidae such as Amaroucium constellatum, Botryllus schlössen, Ciona intestinalis, Molgula citrina, Molgula manhattensis, Perophora viridis or Styela partita. Particularly advantageous are the Nucleic acids from fungi, animals or from plants such as algae or mosses, preferably from the order of Salmoniformes such as the family Salmonidae such as the genus Salmo, for example, from the genera and species Oncorhynchus mykiss, Trutta trutta or Salmo trutta fario, from algae such as the genera Mantoniella or Ostreococcus or from the diatoms such as the genera Thalassiosira or Phaeodactylum or from algae such as Crypthecodinium.

Advantageously, in the method according to the invention, the abovementioned nucleic acid sequences or their derivatives or homologs which code for polypeptides which still have the enzymatic activity of the proteins encoded by the nucleic acid sequences are expressed in the organism. These sequences are used alone or in combination with those for Δ12-desaturase, Δ4-desaturase, Δ5-desaturase, Δ6-desaturase, Δ5-elongase, Δ6-elongase and / or ω 3-desaturase-encoding nucleic acid sequences are cloned into expression constructs and used for introduction and expression in organisms. By their construction, these expression constructs enable a favorable optimal synthesis of the polyunsaturated fatty acids produced in the process according to the invention.

In a preferred embodiment, the method further comprises the step of obtaining a cell or a whole organism, especially a whole plant, which comprises the nucleic acid sequences used in the method which are responsible for a Δ6-desaturase, Δ6-elongase, Δ-5 Desaturase and / or ω-3-desaturase encoded, wherein the cell and / or the organism may contain other nucleic acid sequences of the lipid or fatty acid metabolism. These nucleic acid sequences preferably used in the method are advantageously expressed for expression in at least one gene construct and / or a vector as described below, alone or in combination with other nucleic acid sequences which are suitable for proteins of the fatty acid or lipid metabolism, incorporated and finally transformed into the cell or organism. In a further preferred embodiment, this method further comprises the step of recovering the oils, lipids or free fatty acids from the organism or from the culture. The culture may be, for example, a fermentation culture, for example, in the case of culturing microorganisms such as Mortierella, Thalassiosira, Mantoniella, Ostreococcus, Saccharomyces or Thraustochytrium, or a greenhouse or field crop of a plant. The cell or organism thus produced is advantageously a cell of an oil-producing organism such as an oil crop such as peanut, canola, canola, flax, hemp, peanut, soybean, safflower, hemp, sunflower or borage.

Cultivation is, for example, culturing in the case of plant cells, tissue or organs on or in a nutrient medium or the whole plant on or in a substrate, for example in hydroponics, potting soil or on arable land.

"Transgene" or "recombinant" in the sense of the invention means, for example, a nucleic acid sequence, an expression cassette (= gene construct) or a vector containing the nucleic acid sequence according to the invention or an organism transformed with the nucleic acid sequences, expression cassette or vector according to the invention all such by genetic engineering methods Constructions 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, the modification being exemplified by substitution, addition, deletion, inversion or insertion of one or more nucleotide residues can. Natural genetic environment means the natural genomic or chromosomal locus in the source organism or presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably at least partially conserved. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, more preferably at least 1000 bp, most preferably at least 5000 bp.

A naturally occurring expression cassette - for example, the naturally occurring combination of the natural promoter of the nucleic acid sequence used in the method according to the invention, which is suitable for proteins with appropriate Δ6-desaturase, Δ6-elongase, Δ5-desaturase and / or ω-3 Desaturase activity, advantageously in combination with nucleic acid sequences which are suitable for proteins having Δ-12-desaturase, Δ-4-desaturase, Δ-8-desaturase, Δ-9 elongase, and / or Δ-5. Elongase activity becomes a transgenic expression cassette when it is changed by non-natural, synthetic ("artificial") methods such as a mutagenization. Corresponding methods are described, for example, in US Pat. No. 5,565,350 or WO 00/15815.

As mentioned above, the term "transgenic organism" or "transgenic plant" within the meaning of the invention means that the nucleic acids used in the method are not in their natural position in the genome of an organism, in which case the nucleic acids can be expressed homologously or heterologously. However, as mentioned above, transgene also means that the nucleic acids according to the invention are in their natural place in the genome of an organism, but that the sequence has been changed compared to the natural sequence and / or that the regulatory sequences of the natural sequences have been altered. The transgenic expression of the nucleic acids according to the invention at non-natural sites in the genome is preferred understand, that is, a homologous or preferably heterologous expression of the nucleic acids is present. Preferred transgenic organisms are fungi such as Mortierella or Phytophthora, mosses such as Physcomitrella, algae such as Mantoniella or Ostreococcus, diatoms such as Thalassiosira or Crypthecodinium or plants such as the oil crops and oil-producing plants, vegetable, salad or ornamental plants, which are advantageously selected from the A group of plant families consisting of the families of Aceraceae, Actinidiaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Arecaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Cannaceae, Caprifoliaceae, Chenopodiaceae, Convolvulaceae, Cucurbitaceae, Dioscoreaceae, Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Fagaceae, Grossulahaceae, Juglandaceae, Lauraceae, Liliaceae, Linaceae, Malvaceae, Moraceae, Musaceae, Oleaceae,

Oxalidaceae, Papaveraceae, Poaceae, Polygonaceae, Punicaceae, Rosaceae, Rubiaceae, Rutaceae, Scrophulariaceae, Solanaceae, Sterculiaceae and Valerianaceae.

In principle, all organisms which are able to synthesize fatty acids, especially unsaturated fatty acids, or which are suitable for the expression of recombinant genes, are suitable in principle as organisms or host organisms for the nucleic acids, the expression cassette or the vector used in the process according to the invention. Examples include plants such as Arabidopsis, Asteraceae such as calendula or crops such as soybean, peanut, castor, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, dyer safflower (Carthamus tinctorius) or cocoa bean, microorganisms such as fungi, for example, the genus Mortierella, Thraustochytrium, Saprolegnia, Phytophthora or Pythium, bacteria such as the genus Escherichia or Shewanella, yeasts such as the genus Saccharomyces, cyanobacteria, ciliates, algae such as Mantoniella or Ostreococcus or protozoans such as dinoflagellates such as Thalassiosira or Crypthecodinium called. Further advantageous organisms, especially plants, are mentioned elsewhere in this application. Preference is given to organisms that naturally Such as Mushrooms such as Mortierella alpina, Pythium insidiosum, Phytophthora infestans or plants such as soybean, rapeseed, coconut, oil palm, dyeing safflower, flax, hemp, castor, calendula, peanut, cocoa bean or sunflower or yeasts such as Saccharomyces cerevisiae, especially Soy, flax, rapeseed, safflower, sunflower, calendula, Mortierella or Saccharomyces cerevisiae are preferred. In principle, transgenic animals, advantageously non-human animals, such as, for example, C. elegans, are suitable as host organisms in addition to the abovementioned transgenic organisms.

Useful host cells are also mentioned in: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). usable

Expression strains e.g. those which have lower protease activity are described in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128.

These include plant cells and certain tissues, organs and parts of plants in all their forms, such as anthers, fibers, root hairs, stems, embryos, calli, cotyledons, petioles, crops, plant tissue, reproductive tissue and cell cultures, that of the actual transgenic plant is derived and / or can be used to produce the transgenic plant.

Transgenic plants which contain the polyunsaturated fatty acids synthesized in the process according to the invention can advantageously be marketed directly, without the synthesized oils, lipids or fatty acids having to be isolated. This form of marketing is particularly beneficial. Plants in the process according to the invention include whole plants and all plant parts, plant organs or plant parts such as leaves, stems, seeds, roots, tubers, anthers, fibers, root hairs, stems, embryos, calli, cotyledons, petioles, crop material, plant tissue, reproductive tissue, Cell cultures derived from the transgenic plant and / or used to produce the transgenic plant. The seed includes all seed parts such as the seed shells, epidermis and sperm cells, endosperm or embryonic tissue. However, the compounds prepared in the process according to the invention can also be isolated from the organisms advantageously plants in the form of their oils, fat, lipids and / or free fatty acids. Polyunsaturated fatty acids produced by this process can be harvested by harvesting the organisms either from the culture in which they grow or from the field. This can be done by pressing or extraction of the plant parts, preferably the plant seeds. In this case, the oils, fats, lipids and / or free fatty acids by so-called cold beat or cold pressing can be obtained without supplying heat by pressing. To make it easier to dig up the plant parts, especially the seeds, they are first crushed, steamed or roasted. The pretreated seeds can then be pressed or extracted with solvents such as warm hexane. Subsequently, the solvent is removed again. In the case of microorganisms, these are harvested after harvesting, for example, directly without further working steps, or else extracted after digestion by various methods known to the person skilled in the art. In this way, more than 96% of the compounds prepared in the process can be isolated.

Subsequently, the products thus obtained are further processed, that is refined. First, for example, the Pfieszenschleime and turbidity are removed. The so-called degumming can be carried out enzymatically or, for example, chemically / physically by adding acid, such as phosphoric acid. Subsequently, the free fatty acids are removed by treatment with a base, for example sodium hydroxide solution. The product obtained is for removal The lye remaining in the product is thoroughly washed with water and dried. In order to remove the dyes still contained in the product, the products are subjected to bleaching with, for example, bleaching earth or activated carbon. Finally, the product is deodorized, for example, with steam.

The PUFAs or LCPUFAs produced by this process are preferably C 18, C 20 and / or C 22 fatty acid molecules, advantageously C 20 -fatty acid molecules having at least two double bonds in the fatty acid molecule, preferably three, four, five or six double bonds. These C18, C20 or C22 fatty acid molecules can be isolated from the organism in the form of an oil, lipid or free fatty acid. Suitable organisms are, for example, those mentioned above. Preferred organisms are transgenic plants.

An embodiment of the invention are therefore oils, lipids or fatty acids or fractions thereof, which have been prepared by the method described above, more preferably oil, lipid or a fatty acid composition comprising PUFAs and transgenic

Plants come from. Another embodiment of the invention is the use of these oils, lipids or fatty acids or fractions thereof, which have been prepared by the process described above, for the production of feed, food, cosmetics, dietary supplements or pharmaceuticals.

These oils, lipids or fatty acids advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid as described above; 7 to 85% oleic acid; 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachidic acid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acids and 60 to 85% of polyunsaturated fatty acids, based in each case on 100% and on the total fat acid content of the organism. As an advantageous polyunsaturated fatty acid are in the fatty acid esters or fatty acid mixtures such as phosphatidyl fatty acid esters or triacylglyceride esters preferably at least 1, 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 wt .-% based on the total fatty acid content of arachidonic acid and / or at least 20, 22, 24 or 25, advantageously at least 26, 28 or 30, more preferably at least 32, 34, 36, 38 or 40, most preferably at least 42, 44, 45 wt .-% or more based on the total fatty acid content of eicosapentaenoic acid.

Furthermore, the fatty acid esters or fatty acid mixtures prepared by the process according to the invention advantageously contain fatty acids selected from the group of the fatty acids erucic acid (13-docosaic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8,9 -Methylene heptadec-8-enoic acid), chaulmoogric acid (cyclopentenodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,1-l-dienoic acid), vernonic acid (9,10-epoxyoctadec-12-enoic acid), tartric acid (6-octadecynoic acid), 6-nonadecynoic acid, santalbic acid (tl 1-octadecen-9-ynoic acid), 6,9-octadecenynoic acid, pyrulic acid (tlO-heptadecen-8-ynonic acid), crepenyninic acid (9-octadecen-12-ynonic acid) , 13,14-dihydro-oropheic acid, octadecene-13-ene-9,1-l-diynoic acid, petroselenoic acid (cis-6-octadecenoic acid), 9c, 12t-octadecadienoic acid, calendulic acid (8-octylcadecatrienoic acid), catalpinic acid (9 ml of octadecatrienoic acid ), Eleosteric acid (9cl Itl3t octadecatrienoic acid), Yes carcinic acid (8C1 oct2c octadecatrienoic acid), punicic acid (9cl Itl3c octadecatrienoic acid), parinaric acid (9cl Itl3tl5c octadecatetraenoic acid), pinolenic acid (all-cis-5,9,12-octadecatrienoic acid),

Labaliensäure (5,6-Octadecadienallensäure), Ricinolsäure (12-Hydroxyölsäure) and / or Coriolinsäure (13-Hydroxy-9c, l ltOctadecadienonsäure). The abovementioned fatty acids are generally advantageously present only in traces in the fatty acid esters or fatty acid mixtures prepared by the process according to the invention, that is to say they are less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids. , 22% or 21%, more preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, most preferably less than 4%, 3%, 2% or 1% , In a further preferred form of the invention, these abovementioned fatty acids come to less than 0.9%, 0.8%, 0.7%, 0.6% or 0.5%, more preferably less than 0, relative to the total fatty acids, 4%, 0.3%, 0.2%, 0.1% ago. Advantageously, the fatty acid esters or mixtures of fatty acids prepared by the process according to the invention contain less than 0.1% based on the total fatty acids and / or no butter butyric acid, no cholesterol, no clupanodonic acid (= docosapentaenoic acid, C22: 5Δ4, 8, 12, 15, 21) ) and no nisic acid (tetracosahexaenoic acid, C23: 6Δ3,8,12,15,18,21).

The oils, lipids or fatty acids according to the invention advantageously contain at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least 6%, 7%, 8%, 9% or 10%, particularly advantageously at least 11%, 12%, 13%, 14% or 15% ARA or at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least 6%, or 7%, more preferably at least 8 %, 9% or 10% EPA and / or DHA based on the total fatty acid content of the production organism advantageously a plant, particularly advantageous an oil crop such as soybean oilseed rape, coconut, oil palm, Färbersafflor, flax, hemp, castor, calendula, peanut, cocoa bean, sunflower or the above other monocotyledonous or dicotyledonous oil crops.

Another embodiment of the invention is the use of the oils, lipids, the

Fatty acids and / or the fatty acid composition prepared by the process according to the invention in feed, food, cosmetics or pharmaceuticals. The oils, lipids, fatty acids or fatty acid mixtures obtained in the process according to the invention can be used in the manner known to those skilled in the art for blending with other oils, lipids, fatty acids or fatty acid mixtures of animal origin, such as fish oils. These too produced oils, lipids, fatty acids or fatty acid mixtures, which consist of vegetable and animal components, can be used for the production of feed, food, cosmetics or pharmaceuticals.

The term "oil", "lipid" or "fat" is understood as meaning a fatty acid mixture which contains unsaturated, saturated, preferably esterified fatty acid (s). It is preferred that the oil, lipid or fat contains a high proportion of polyunsaturated free or advantageously esterified fatty acid (s), in particular linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, α-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, Docosapentaenoic acid or docosahexaenoic acid has. Preferably, the proportion of unsaturated esterified fatty acids is about 30%, more preferred is a proportion of 50%, even more preferred is a proportion of 60%, 70%, 80% or more. For determination, e.g. the proportion of fatty acid after conversion of the fatty acids into the methyl esters can be determined by gas chromatography by transesterification. The oil, lipid or fat may contain various other saturated or unsaturated fatty acids, e.g. Calendulic acid, palmitic, palmitoleic, stearic, oleic acid, etc. included. In particular, depending on the starting organism, the proportion of the various fatty acids in the oil or fat may vary.

The polyunsaturated fatty acids prepared in the process and advantageously having at least two, three, four or five, particularly preferably four or five double bonds are, as described above, advantageously fatty acid esters, for example sphingolipid esters, phosphoglyceride esters, lipid esters, glycolipid esters, phospholipid esters, monoacylglycerol esters, diacylglycerol esters , Triacylglycerinester or other fatty acid esters, it is preferably phospholipid esters and / or Triacylglycerinester. From the fatty acid esters thus prepared in the process according to the invention, the polyunsaturated fatty acids present can be advantageously with at least five or six double bonds, for example via an alkali treatment, for example, aqueous KOH or NaOH or acid hydrolysis in the presence of an alcohol such as methanol or ethanol or release via an enzymatic cleavage and isolate via, for example, phase separation and subsequent acidification over, for example, H ₂ SO ₄ . The release of the fatty acids can also be carried out directly without the workup described above.

After incorporation into an organism, advantageously a plant cell or plant, the nucleic acids used in the method can either lie on a separate plasmid or can advantageously be integrated into the genome of the host cell. When integrated into the genome, integration may be at random or by such recombination as to replace the native gene with the incorporated copy, thereby modulating the production of the desired compound by the cell, or by using a gene in trans such that Gene having a functional expression unit, which contains at least one expression of a gene ensuring sequence and at least one polyadenylation of a functionally transcribed gene ensuring sequence is operably linked. Advantageously, the nucleic acids are brought into the plants via multi-expression cassettes or constructs for multiparallel expression in the organisms, advantageously for multiparallel seed-specific expression of genes.

Moose and algae are the only known pesticide systems that produce significant amounts of polyunsaturated fatty acids, such as arachidonic acid (ARA) and / or eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA). Moose contain PUFAs in membrane lipids, while algae, algae-related organisms and some fungi also contain significant amounts Accumulate levels of PUFAs in the triacylglycerol fraction. Therefore, nucleic acid molecules isolated from strains that also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for the method of the invention and thus for modification of the lipid and PUFA production system in a host, especially plants such as oil crop plants, for example Rapeseed, canola, flax, hemp, soy, sunflower, borage. They are therefore advantageous for use in the process according to the invention.

As substrates of the nucleic acids used in the method according to the invention which code for polypeptides with Δ-6-desaturase, Δ-5-desaturase, Δ-6-elongase and / or ω-3-desaturase activity, and / or the other used nucleic acids such as the nucleic acids selected for polypeptides of fatty acid or lipid metabolism from the group acyl CoADehydrogenase (s), acyl-ACP acyl carrier protein desaturase (s), acyl-ACP thioesterase (s), fatty acid Acyl-transferase (s), acyl-CoA: lysophospho lipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (n), fatty acid desaturase (s), fatty acid acetylenase (s), lipoxygenase (s), triacylglycerol lipase (s), allene oxide synthase (s), hydroperoxide lyase (s) or fatty acid elongase (s) C 16, C 18 or C 20 fatty acids are advantageously suitable. The fatty acids reacted as substrates in the process are preferably reacted in the form of their acyl-CoA esters and / or their phospholipid esters.

For the preparation of the long-chain PUFAs according to the invention, the saturated, monounsaturated C 16 -fatty acids and / or polyunsaturated C 18 -fatty acids must first be desaturated and / or elongated or only desaturated, depending on the substrate, by the enzymatic activity of a desaturase and / or elongase, and then be further desaturated Elongase be extended by at least two carbon atoms. After a round of elongation this leads Enzyme activity either from C16 fatty acids to C18 fatty acids or from C18 fatty acids to C20 fatty acids, and after two elongation cycles from C16 fatty acids to C20 fatty acids. The activity of the desaturases and elongases used in the process according to the invention preferably leads to C 18- and / or C 20 -fatty acids advantageously having at least two double bonds in the fatty acid molecule, preferably having three, four or five double bonds, more preferably C 20 -fatty acids having at least four double bonds in the fatty acid molecule. Particularly preferred as products of the method according to the invention are dihomo-γ-linolenic acid, arachidonic acid and / or eicosapentaenoic acid. The C18 fatty acids having at least two double bonds in the fatty acid can be extended by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.

The preferred biosynthesis site of fatty acids, oils, lipids or fats in the advantageously used plants is, for example, generally the seed or cell layers of the

Samens, so that a seed-specific expression of the nucleic acids used in the method makes sense. However, it is obvious that the biosynthesis of fatty acids, oils or lipids need not be limited to the seed tissue, but may also be tissue-specific in all other parts of the plant - for example in epidermal cells or in the tubers. Advantageously, the synthesis according to the inventive method in the vegetative (somatic) tissue instead.

In the method according to the invention as organisms microorganism such as yeasts such as Saccharomyces or Schizosaccharomyces, fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Thraustochytrium or algae such as Isochrysis, Mantonielia, Ostreococcus, Phaeodactylum or Crypthecodinium used, these organisms are advantageously attracted to fermentation.

By using the nucleic acids according to the invention which code for a Δ5-desaturase or a Δ6-desaturase, the polyunsaturated fatty acids produced in the process can be at least 5%, preferably at least 10%, particularly preferably at least 20%. , most preferably by at least 50% over the wild-type of organisms which do not recombinantly contain the nucleic acids.

By the method according to the invention, the polyunsaturated fatty acids produced in the organisms used in the process can in principle be increased in two ways. Advantageously, the pool of free polyunsaturated fatty acids and / or the proportion of esterified polyunsaturated fatty acids produced by the process can be increased. Advantageously, the inventive method increases the pool of esterified polyunsaturated fatty acids in the transgenic organisms, advantageously in the form of the phosphatidyl esters and / or triacyl esters.

If microorganisms are used as organisms in the process according to the invention, they are grown or grown, depending on the host organism, in a manner known to the person skilled in the art. Microorganisms are usually in a liquid medium containing a carbon source usually in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally vitamins, at temperatures between 0 ⁰ C and 100 ⁰ C, preferably between 10 ⁰ C and 60 ⁰ C attracted under oxygen fumigation. The pH of the nutrient fluid can be kept at a fixed value be regulated during breeding or not. The cultivation can be batchwise, semi-batch wise or continuous. Nutrients can be presented at the beginning of the fermentation or fed in semi-continuously or continuously. The polyunsaturated fatty acids prepared can be isolated from the organisms by methods known to those skilled in the art as described above. For example, extraction, distillation, crystallization, optionally salt precipitation and / or chromatography. The organisms can be opened up for this purpose yet advantageous.

The erfmdungs proper method, when it is in the host organisms are microorganisms, advantageously carried out at a temperature between 0 ⁰ C and 95 ° C, preferably between 10 ⁰ C and 85 ° C, more preferably between 15 ° C and 75 ° C, most preferably carried out between 15 ° C and 45 ° C.

The pH is advantageously maintained between pH 4 and 12, preferably between pH 6 and 9, more preferably between pH 7 and 8.

The process according to the invention can be operated batchwise, semi-batchwise or continuously. A summary of known cultivation methods is in the textbook by Chmiel (bioprocess 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994) ) to find.

The culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are given in the Manual of Methods for General Bacteriology of the Merican Society for Bacteriology (Washington D.C, USA, 1981).

As described above, these media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of very good carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It may also be advantageous to add mixtures of different carbon sources. Other possible sources of carbon are oils and fats, e.g. Soybean oil, sunflower oil, peanut oil and / or coconut fat, fatty acids such as e.g. Palmitic acid, stearic acid and / or linoleic acid, alcohols and / or polyhydric alcohols such. Glycerol, methanol and / or ethanol and / or organic acids such as e.g. Acetic acid and / or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds. Exemplary nitrogen sources include ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soy protein, yeast extract, meat extract and others. The nitrogen sources can be used singly or as a mixture.

Inorganic salt compounds that may be included in the media include the Chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

As a sulfur source for the production of sulfur-containing fine chemicals, in particular methionine, inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols can be used.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source. Chelating agents can be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

The fermentation media used according to the invention for the cultivation of microorganisms usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthotenate and pyridoxine. Growth factors and salts are often derived from complex media components, such as yeast extract, molasses, corn steep liquor, and the like. In addition, suitable precursors can be added to the culture medium. The exact composition of

Media connections strongly depend on the particular experiment and are decided individually for each specific case. Information about the media optimization is available from the textbook "Applied Microbiol Physiology, A Practical Approach" (Ed PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.

All media components are sterilized either by heat (20 min at 1.5 bar and 121 ⁰ C) or by sterile filtration. The components can either be sterilized together or, if necessary, sterilized separately. All media components may be present at the beginning of the culture or added randomly or batchwise, as desired.

The temperature of the culture is usually between 15 ° C and 45 ° C, preferably at 25 ° C to 40 ⁰ C and can be kept constant or changed during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably 7.0. The pH for cultivation can be controlled during cultivation by addition of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid. To control the foam development antifoams such. B. fatty acid polyglycol esters are used. To maintain the stability of plasmids, the medium can be selected selectively acting substances such. As antibiotics, are added. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as ambient air, are introduced into the culture. The temperature of the culture is normally from 20 ⁰ C to 45 ° C and preferably at 25 ° C to 40 ⁰ C. The culture is continued until a maximum of the desired product has formed. This goal is usually reached within 10 hours to 160 hours.

The fermentation broths thus obtained, in particular containing polyunsaturated fatty acids, usually have a dry matter content of 7.5 to 25% by weight. The fermentation broth can then be further processed. Depending on the requirement, the biomass can be wholly or partly by separation methods, such. As centrifugation, filtration, decantation or a combination of these methods are removed from the fermentation broth or completely left in it. Advantageously, the biomass is worked up after separation.

The fermentation broth can also without cell separation with known methods such. B. with the aid of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, or by nanofiltration, thickened or concentrated. This concentrated fermentation broth may eventually be worked up to recover the fatty acids contained therein.

The fatty acids obtained in the process are also suitable as starting material for the chemical synthesis of further products of value. They may be used, for example, in combination with each other or solely for the manufacture of pharmaceuticals, foods, animal feed or cosmetics.

A further subject of the invention are isolated nucleic acid molecules comprising a nucleic acid sequence which codes for polypeptides having Δ-6-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 1, b) nucleic acid sequences which are known as Deriving the result of the degenerate genetic code from the amino acid sequence shown in SEQ ID NO: 2, and c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1 which encode polypeptides having at least 68% identity at the amino acid level with SEQ ID NO: 2 and a Δ-6 Have desaturase activity.

The nucleic acid sequences found in the context of this invention, which code for a polypeptide with Δ-6-desaturase activity, encode a polypeptide which is distinguished from known lipid-dependent desaturases by a very high substrate turnover (35% desaturation). In contrast to, for example, the Δ-6-desaturase from Ostreococccus tauri, the Δ-6-desaturase according to the invention from M. squamata shows a significantly higher substrate specificity. The Δ-6-desaturase according to the invention converts only 18: 3 '' to 18: 4 ''', whereas the Δ6-desaturase from Ostreococccus tauri also accepts 18: 2 ^Δ9 ' ¹² as substrate. The advantage is that one can achieve a more targeted production of .omega.3-fatty acids by the Δ-6-desaturase according to the invention from M. squamata (see FIG. 1).

High substrate specificity of the Δ 6-desaturase of the present invention means in this invention that the substrate 18: 3 ^{Δ9 '12 '15} is reacted significantly more than 18: 2 ^{Δ9' 12th} Preference is given to 18: 3 ^Δ9 ' ¹² ' ¹⁵ 1, 5-fold, 2-fold, 4-fold, 6-fold, 8-fold or 10-fold, more preferably 12-fold, 15-fold or 20-fold stronger accepted and implemented as 18: 2 ^{Δ9 '12} .

A further subject of the invention are isolated nucleic acid molecules comprising a nucleic acid sequence which codes for polypeptides with Δ-5-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 3, b) nucleic acid sequences which are known as Derive the result of the degenerate genetic code from the amino acid sequence shown in SEQ ID NO: 4, and c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 3, which code for polypeptides having at least 67% identity at the amino acid level with SEQ ID NO: 4 and have a Δ-5 desaturase activity.

The Δ 5-desaturase activity found in the context of this invention, like the Δ 6-desaturase according to the invention, is acyl-CoA-dependent. It is the first Δ5-desaturase activity found in a microalgae.

By "derivatives" of the nucleic acid sequences according to the invention are meant in particular nucleic acid sequences which under stringent conditions with a

Nucleic acid sequence according to SEQ ID no. 1 or SEQ ID NO. 3 hybridize, as well as fragments of the nucleic acid sequences according to SEQ ID NO. 1 or SEQ ID NO. Third

A further subject of the invention are gene constructs which contain the nucleic acid sequences according to the invention SEQ ID NO: 1 and / or SEQ ID NO: 3, wherein the nucleic acid is in each case operably linked to one or more regulatory signals. In addition, further biosynthesis genes of the fatty acid or lipid metabolism can be selected from the group consisting of acyl-CoA dehydrogenase (s), acyl-ACP [= acyl carrier protein] -desaturase (s), acyl-ACP-thioesteraseCn, fatty acid-acyl- Transferase (s), acyl CoA: lysophospholipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (s), Fatty acid desaturase (s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allene oxide synthases, hydroperoxide lyases or fatty acid elongase (s) may be included in the gene construct. Also advantageous are biosynthesis genes of the fatty acid or lipid metabolism selected from the group of Δ-4-desaturase, Δ 8-desaturase, Δ-9-desaturase, Δ-12-desaturase, Δ-5-elongase, Δ- 6-elongase, Δ-9 elongase, ω3-desaturase and / or Δ15-desaturase. Preferably, at least one Δ6-elongase is contained in the gene construct.

Advantageously, all of the nucleic acid sequences used in the method of the invention are derived from a eukaryotic organism such as a plant, a microorganism or an animal. The nucleic acid sequences are preferably derived from the order Salmoniformes, algae such as Mantoniella or Ostreococcus, fungi such as the genus Phytophtora or diatoms such as the genera Thalassiosira or Crypthecodinium.

The nucleic acid sequences used in the method which code for proteins with Δ5-desaturase, Δ6-desaturase, Δ6-elongase activity or another enzyme activity of the fatty acid and lipid metabolism are advantageously used alone or preferably in combination in an expression cassette (= Nucleic acid construct), which allows the expression of the nucleic acids in an organism advantageously a plant or a microorganism introduced. It may contain more than one nucleic acid sequence of an enzymatic nucleic acid construct

Activity such as a Δ5 -desaturase, Δ6-desaturase and / or Δ6-elongase may be included.

For introduction, the nucleic acids used in the method are advantageously subjected to amplification and ligation in a known manner. Preferably, the procedure is based on the protocol of the Pfu DNA polymerase or of a Pfu / Taq DNA polymerase mixture. The primers are selected on the basis of the sequence to be amplified. Conveniently, the primers should be chosen so that the amplificate comprises the entire codogenic sequence from the start to the stop codon. Following amplification, the amplificate is conveniently analyzed. For example, the analysis can be carried out after gel electrophoretic separation in terms of quality and quantity. In connection the amplificate can be purified according to a standard protocol (eg Qiagen). An aliquot of the purified amplificate is then available for subsequent cloning. Suitable cloning vectors are well known to those skilled in the art. These include, in particular, vectors which can be replicated in microbial systems, ie in particular vectors which ensure efficient cloning in yeasts or fungi, and which enable stable transformation of plants. In particular, various suitable for T-DNA-mediated transformation, binary and co-integrated vector systems. Such vector systems are generally characterized in that they contain at least the vir genes required for the Agrobacterium-mediated transformation as well as the T-DNA limiting sequences (T-DNA border). Preferably, these vector systems also include other cis-regulatory regions such as promoters and terminators and / or selection markers, with which correspondingly transformed organisms can be identified. Whereas in co-integrated vector systems vir genes and T-DNA sequences are arranged on the same vector, binary systems are based on at least two vectors, one of them vir genes, but no T-DNA and a second T-DNA, but none carries vir gene. As a result, the latter vectors are relatively small, easy to manipulate and replicate in both E.coli and Agrobacterium. These binary vectors include vectors of the series pBIB-HYG, pPZP, pBecks, pGreen. Binl9, pBHO1, pBinAR, pGPTV and pCAMBIA are preferably used according to the invention. For a review of binary vectors and their use, see Hellens et al., Trends in Plant Science (2000) 5: 446-451. For the vector preparation, the vectors can first be linearized with restriction endonuclease (s) and then enzymatically modified in a suitable manner. The vector is then purified and an aliquot used for cloning. In cloning, the enzymatically cut and, if necessary, purified amplicon is cloned with similarly prepared vector fragments using ligase. In this case, a particular nucleic acid construct or vector or plasmid construct a or also have several codogenic gene segments.

Preferably, the codogenic gene segments in these constructs are functionally linked to regulatory sequences. The regulatory sequences include, in particular, plant sequences such as the promoters and terminators described above. The

Constructs can advantageously be stably propagated in microorganisms, in particular Escherichia coli and Agrobacterium tumefaciens, under selective conditions and enable a transfer of heterologous DNA into plants or microorganisms.

With the advantageous use of cloning vectors, the nucleic acids used in the method, the inventive nucleic acids and nucleic acid constructs can be introduced into organisms such as microorganisms or advantageously plants and thus used in pitch transformation, such as those published in and cited herein: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-119 (1993); FF White, Vectors for Gene Transfer to Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, 15-38; Genes Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225)). The nucleic acids used in the method, the inventive nucleic acids and nucleic acid constructs and / or vectors can thus be used advantageously for genetically modifying a broad spectrum of organisms to plants so that they become better and / or more efficient producers of PUFAs. By introducing an ω3-desaturase, Δ6-desaturase, Δ6-elongase and / or Δ5-desaturase gene into an organism alone or in combination with other genes, not only the biosynthesis flux to the final product can be increased, but also the corresponding triacyl- glycerol and / or phosphatidyl ester composition increased or created de novo. Likewise, the number or activity of other genes necessary for the import of nutrients necessary for the biosynthesis of one or more fatty acids, oils, polar and / or neutral lipids may be increased, such that the concentration of these precursors, cofactors or intermediates within the cells or within the storage compartment, thereby further increasing the ability of the cells to produce PUFAs, as described below. By optimizing the activity or increasing the number of one or more ω3-desaturase, Δ6-desaturase, Δ6-elongase and / or Δ5-desaturase genes involved in the biosynthesis of these compounds, or by destroying the activity of one or more Genes involved in the degradation of these compounds may be able to increase the yield, production and / or efficiency of the production of fatty acid and lipid molecules from organisms, and advantageously from plants.

The nucleic acid molecules used in the method according to the invention encode proteins or parts thereof, the proteins or the individual protein or parts thereof containing an amino acid sequence which is sufficiently homologous to an amino acid sequence which is present in the sequences SEQ ID NO: 2 or SEQ ID NO: 4, so that the proteins or parts thereof still have Δ6-desaturase and / or Δ5-desaturase activity. Preferably, the proteins or portions thereof encoded by the nucleic acid molecule (s) still retain their essential enzymatic activity and the ability to catabolize cell membranes or lipid bodies in organisms advantageously participate in plants necessary compounds or in the transport of molecules through these membranes.

The enzymatic activity of the Δ-6-desaturase according to the invention and Δ-5-desaturase or derivatives thereof can be determined, for example, by expressing the enzyme to be tested in a suitable host organism such as yeast, exogenous substrates (18: 3 ''). in the case of Δ-6-desaturase and 20: 3 "in the case of Δ-5-desaturase) and, after a certain incubation time, analyzes the fatty acid pattern. The appearance of the products 18: 4 ^Λ6 ' ⁹ ' ^{12 '15} or 20: 4 ^Λ5 ' ⁸ ' ^π ' ¹⁴ indicates a Δ-6-desaturase or Δ-5-desaturase activity in the yeast cultures. Details on the experimental procedure can be found in the exemplary embodiments of the present application.

A Δ6-desaturase or Δ5-desaturase in the sense of the present invention has an enzymatic activity of at least 10%, 15%, 20% or 25%, preferably 30%, 35%, 40% or 45%, especially preferably 50%, 55%, 60%, 65%, 70% or 75%, most preferably 80%, 82%, 84%, 86% or 88%, and most preferably 90%, 92%, 94%, 96% , 98%, 100%, 110%, 120%, 130%, 150%, 180% or 200% of the enzymatic activity of the enzymes encoded by SEQ ID NO: 1 and 3, respectively.

Advantageously, the proteins encoded by the nucleic acid molecules are at least 67%, 68% or 69% and preferably at least about 70%, 74%, 78%, 80% or 84%, and more preferably at least about 85%, 86%, 87%, 88%, 89% or 90%, and most preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to those shown in SEQ ID NO: 2 or SEQ ID NO: 4 amino acid sequences shown. For the purposes of the invention is meant homology or homologous, identical or identical. Homology was calculated over the entire amino acid or nucleic acid sequence range. For the comparison of different sequences, a number of programs that are based on different algorithms are available to the person skilled in the art. The algorithms of Needleman and Wunsch or Smith and Waterman provide particularly reliable results. For the sequence comparisons, the program PiIeUp was used (J. Mol.

Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5, 1989: 151-153) or the Gap and BestFit programs [Needleman and Wunsch (J. Mol. Biol. 48: 443-453 (1970) and Smith and Waterman (Adv. Appl.Math.2482-489 (1981)], which are included in the GCG Software package [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)] Percent sequence homology scores were determined using the GAP program over the entire sequence range, with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10,000, and Average Mismatch: 0.000 These settings are always defaults unless otherwise noted used for sequence comparisons.

Significant enzymatic activity of the Δ-6-desaturase or Δ-5-desaturase used in the process according to the invention is to be understood as meaning that it has opposite the proteins / enzymes coded by the sequence with SEQ ID NO: 1 or SEQ ID NO: 3 and their derivatives in comparison, at least one enzymatic activity of at least 10%, preferably 20%, more preferably 30% and very particularly 40% and thus the metabolism of fatty acids for the construction of fatty acid esters such as diacylglycerols and / or triacylglycerides in an organism advantageously a plant or Plant cell necessary compounds or participate in the transport of molecules via membranes, wherein Cl 8, C20 or C22 carbon chains in the fatty acid molecule with double bonds at least two, preferably three, four, five or six digits are meant. Nucleic acids useful in the method are derived from bacteria, fungi, diatoms, animals such as Caenorhabditis or Oncorhynchus or plants such as algae or mosses such as the genera Shewanella, Physcomitrella, Thraustochytrium, Fusarium, Phytophthora, Ceratodon, Mantoniella, Ostreococcus, Isochrysis, Aleurita, Muscarioides, Mortierella , Borago, Phaeodactylum, Crypthecodinium, especially of the genera and species Oncorhynchus mykiss, Thalassiosira pseudonona, Mantoniella squamata, Ostreococcus sp., Ostreococcus tauri, Euglena gracilis, Physcomitrella patens, Phytophthora infestans, Fusarium graminaeum, Cryptocodinium cohnii, Ceratodon purpureus, Isochrysis galbana, Aleurita farinosa, Thraustochytrium sp., Muscarioides viallii, Mortierella alpina, Borago officinalis, Phaeodactylum tricornutum, Caenorhabditis elegans, or more particularly from Oncorhynchus mykiss, Thalassiosira pseudonona or Crypthecodinium cohnii.

Alternatively, nucleotide sequences encoding a Δ6-desaturase or Δ5-desaturase and which hybridize to a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3, advantageously under stringent conditions, may be used in the method of the invention ,

Nucleic acid molecules advantageous for the method according to the invention can be isolated on the basis of their homology to the desaturase nucleic acids disclosed herein using the sequences or a part thereof as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. For example, isolated nucleic acid molecules can be used that are at least 15 nucleotides long and hybridize under stringent conditions with the nucleic acid molecules comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Nucleic acids of at least 25, 50, 100, 250 or more nucleotides may also be used. The term "hybridized under stringent conditions" as used herein is intended to describe hybridization and washing conditions under which nucleotide sequences that are at least 60% homologous to one another usually remain hybridized to one another. The conditions are preferably such that sequences that are at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous, are usually hybridized to each other. These stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridizations in 6 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 0.2 x SSC, 0.1%. SDS at 50 to 65 ° C. It is known to those skilled in the art that these hybridization conditions differ with respect to the type of nucleic acid and, for example, when organic solvents are present, with respect to the temperature and the concentration of the buffer. The temperature differs, for example under "standard hybridization conditions" depending on the type of nucleic acid between 42 ° C and 58 ° C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2). If organic solvent is present in the above buffer, for example 50% formamide, the temperature is about 42 ° C under standard conditions. Preferably, the hybridization conditions for DNA: DNA hybrids are, for example, 0.1 x SSC and 20 ⁰ C to 45 ° C, preferably between 30 ⁰ C and 45 ° C. Preferably, the hybridization conditions for DNA: RNA hybrids are, for example, 0.1 x SSC and 30 ⁰ C to 55 ° C, preferably between 45 ° C and 55 ° C. The above-mentioned hybridization temperatures are determined, for example, for a nucleic acid of about 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. The person skilled in the art knows how the required hybridization conditions based on textbooks, such as the above-mentioned or from the following textbooks Sambrook et al., "Molecular Cloning", Colard Spring Harbor Laboratory, 1989; Harnes and Higgins (Eds.) 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed.) 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

Alternatively, nucleotide sequences coding for an ω-3-desaturase, Δ12-desaturase, Δ9-elongase, Δ8-desaturase or Δ-4-desaturase can additionally be used in the method according to the invention. The nucleic acid sequences used in the method are advantageously introduced into an expression cassette which enables expression of the nucleic acids in organisms such as microorganisms or plants.

The nucleic acid sequences which are responsible for one of the abovementioned enzyme activities, advantageously an ω-3-desaturase, Δ6-desaturase, Δ5-desaturase, Δ6-elongase, Δ12-desaturase, Δ5-elongase, Δ-9 elongase, Δ-8-desaturase or Δ-4-desaturase, with one or more regulatory signals advantageously functionally linked to increase gene expression. These regulatory sequences are intended to allow the targeted expression of genes and protein expression. Depending on the host organism, this may mean, for example, that the gene is expressed and / or overexpressed only after induction, or that it is immediately expressed and / or overexpressed. For example, these regulatory sequences are sequences to which inducers or repressors bind, and so

Regulate expression of the nucleic acid. In addition to these new regulatory sequences, or instead of these sequences, the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically altered so that natural regulation is eliminated and gene expression increased. The expression cassette (= expression construct = gene construct) can also be simpler be constructed, that is, no additional regulatory signals before the nucleic acid sequence or its derivatives were inserted and the natural promoter with its regulation was not removed. Instead, the natural regulatory sequence has been mutated to no longer regulate and / or increase gene expression. These modified promoters can be brought in the form of partial sequences (= promoter with parts of the inventive nucleic acid sequences) alone before the natural gene to increase the activity. In addition, the gene construct may advantageously also contain one or more so-called enhancer sequences functionally linked to the promoter, which allow increased expression of the nucleic acid sequence. Additional advantageous sequences can also be inserted at the 3 'end of the DNA sequences, such as further regulatory elements or terminators. The ω-3-desaturase, Δ-6-desaturase, Δ-5-desaturase, Δ-6 elongase, Δ-12-desaturase, Δ-5 elongase, Δ-9 elongase, Δ-8-desaturase or Δ-4-desaturase genes can be present in one or more copies in the expression cassette (= gene construct). Advantageously, only one copy of the genes is present in the expression cassette. This gene construct or gene constructs can be expressed together in the host organism. In this case, the gene construct or the gene constructs can be inserted in one or more vectors and be present freely in the cell or else be inserted in the genome. It is advantageous for the insertion of additional genes in the host genome when the genes to be expressed are present together in a gene construct.

The regulatory sequences or factors can, as described above, preferably positively influence the gene expression of the introduced genes and thereby increase them. Thus, enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers. In addition, however, an enhancement of the translation is possible by, for example the stability of the mRNA is improved.

A further embodiment of the invention are one or more gene constructs which contain one or more sequences which are defined by SEQ ID NO: 1 or SEQ ID NO: 3 or its derivatives and for polypeptides according to SEQ ID NO: 2 or SEQ ID NO: 4 code. The abovementioned Δ6-desaturase and Δ5-desaturase proteins advantageously result in the desaturation or elongation of fatty acids in interaction with other enzymes of fatty acid and lipid biosynthesis, the substrate advantageously having one, two, three, four, five or six Double bonds and advantageously has 18, 20 or 22 carbon atoms in the fatty acid molecule. The same applies to their homologs, derivatives or analogs which are operably linked to one or more regulatory signals, advantageously to increase gene expression.

Advantageous regulatory sequences for the novel process are, for example, in promoters, such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5 , T3, gal, trc, ara, SP6, γ-PR or γ-PL promoter and are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are, for example, in the gram-positive promoters amy and SP02, in the yeast or fungal promoters ADC1, MFa, AC, P-60, CYCl, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV / 35S [Franck et al, Cell 21 (1980) 285-294], PRPl [Ward et al., Plant. Biol. 22 (1993)], SSU, OCS, Iib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter. Also advantageous in this connection are inducible promoters, such as those described in EP-AO 388 186 (benzylsulfonamide-inducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracycline Inducible), EP-AO 335 528 ( Abzisinic inducible) or WO 93/21334 (ethanol or cyclohexenol inducible) promoters. Other suitable plant promoters are Promoter of cytosolic FBPase or potato ST-LSIPromotor (Stockhaus et al., EMBO J. 8, 1989, 2445), the glycine max phosphoribosyl-pyrophosphate amidotransferase promoter (Genbank Accession No. U87999) or described in EP-A-0 249 676 nodea specific promoter. Particularly advantageous promoters are promoters which allow expression in tissues involved in fatty acid biosynthesis. Especially advantageous are seed-specific promoters, such as the USP promoter according to the embodiment, but also other promoters such as the LeB4, DC3, phaseolin or napin promoter. Further particularly advantageous promoters are seed-specific promoters which can be used for monocotyledonous or dicotyledonous plants and in US Pat. No. 5,608,152 (rapeseed napin promoter), WO 98/45461 (oleosin promoter from Arobidopsis), US Pat. No. 5,504,200

(Phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoter from Brassica), von Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (LeB4 promoter from a legume) are described , where these promoters are suitable for dicotyledons. The following promoters are suitable, for example, for monocots: barley lpt-2 or lpt-1 promoter (WO 95/15389 and WO 95/23230), barley hordein promoter and other suitable promoters described in WO 99/16890.

It is possible in principle to use all natural promoters with their regulatory sequences, such as those mentioned above, for the new method. It is also possible and advantageous to use, in addition or alone, synthetic promoters, especially if they mediate seed-specific expression, e.g. described in WO 99/16890.

In order to achieve a particularly high content of PUFAs, especially in transgenic plants, the PUFA biosynthesis genes should advantageously be seed-specifically expressed in oilseeds. For this purpose, seed-specific promoters can be used, or such promoters, the are active in the embryo and / or in the endosperm. In principle, seed-specific promoters can be isolated from both dicotolydone and monocotolydonous plants. Advantageous preferred promoters are listed below: USP (= unknown seed protein) and Vicilin (Vicia faba) [Baumlein et al, Mol. Gen Genet, 1991, 225 (3)], napin (rapeseed) [US Pat. No. 5,608,152], acyl- Carrier protein (rape) [US 5,315,001 and WO 92/18634], oleosin (Arabidopsis thaliana) [WO 98/45461 and WO 93/20216], phaseolin (Phaseolus vulgaris) [US 5,504,200], Bce4 [WO 91/13980], Legumes B4 (LegB4 promoter) [Bäumlein et al., Plant J., 2,2, 1992], Lpt2 and lpt1 (barley) [WO 95/15389 and others]. WO95 / 23230], seed-specific promoters from rice, maize and the like. Wheat [WO 99/16890], Amy32b, Amy 6-6 and Aleurain [US 5,677,474], Bce4 (rape) [US 5,530,149], glycinin (soybean) [EP 571 741], phosphoenol pyruvate carboxylase (soybean)

[JP 06/62870], ADR12-2 (soybean) [WO 98/08962], isocitrate lyase (rapeseed) [US 5,689,040] or [alpha] -amylase (barley) [EP 0 781 849]. Plant gene expression can also be facilitated by a chemically inducible promoter (see review in Gatz 1997, Annu Rev. Plant Physiol Plant Mol. Biol., 48: 89-108). Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.

In another embodiment, sequences are advantageously used for the expression that allow constitutive expression in as many tissues of the plant as the CaMV35S, CaMV36S, CaMV35Smas, nos, mas, ubi, stpt, lea or super promoter. Preferably, the expression is carried out in the vegetative tissue as described above. For example, the 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 or instead of these sequences, the natural regulation of these sequences before the actual structural genes may still be present and possibly genetically altered, so that the natural regulation was switched off and the expression of genes was increased. In addition, the gene construct may advantageously also contain one or more so-called enhancer sequences functionally linked to the promoter, which allow increased expression of the nucleic acid sequence. Additional advantageous sequences can also be inserted at the 3 'end of the DNA sequences, such as further regulatory elements or terminators. Advantageous terminators are, for example, viral terminators such as the 35S terminator or others. The enzymes or nucleic acids which code for these enzymes in the method according to the invention can be contained in one or more copies in the expression cassette (= gene construct). Advantageously, only one copy of the genes is present in the expression cassette. This gene construct or gene constructs can be introduced simultaneously or sequentially into the plant and expressed together in the host organism. In this case, the gene construct or the gene constructs can be inserted in one or more vectors and be present freely in the cell or else be inserted in the genome. It is advantageous for the insertion of further genes into the plant if the genes to be expressed are present together in a gene construct.

It is understood that the nucleic acid sequences which code for the Δ6-desaturase or Δ5-desaturase according to the invention and optionally further enzymes of the lipid metabolism can also be present individually in a gene construct and introduced into a suitable host plant. These transformed host plants can then be crossed with each other to obtain the desired combination of enzymes in the offspring. In order to ensure stable integration of the biosynthesis genes into the transgenic plant over several generations, each of the nucleic acids used in the process should be expressed under the control of its own, preferably a different promoter, since repeating sequence motifs lead to instability of the T-DNA or to recombination events can. The expression cassette is advantageously constructed so that a promoter follows a suitable interface for insertion of the nucleic acid to be expressed, advantageously in a polylinker. Optionally, a terminator may be behind the polylinker. This sequence is repeated several times, preferably three, four or five times, so that up to five genes are combined in one construct and thus can be introduced into the transgenic plant for expression. Advantageously, the sequence is repeated up to three times. The nucleic acid sequences are inserted for expression via the appropriate interface, for example in the polylinker downstream of the promoter. Advantageously, each nucleic acid sequence has its own promoter and optionally its own terminator. Such advantageous constructs are disclosed for example in DE 10102337, DE 10102338 or WO 2007/017419. However, it is also possible to insert several nucleic acid sequences behind a promoter and possibly in front of a terminator. In this case, the insertion site or the sequence of the inserted nucleic acids in the expression cassette is not of decisive importance, that is, a nucleic acid sequence may be inserted at the first or last position in the cassette, without this significantly affecting the expression. Advantageously, different promoters can be used in the expression cassette, for example the USP, LegB4 or DC3 promoter and different ones

Terminators are used. But it is also possible to use only one type of promoter in the cassette. For constitutive expression, the CaMV35S promoter can also be used several times. As described above, transcription of the introduced genes should be advantageously aborted by suitable terminators at the 3 'end of the introduced biosynthetic genes (beyond the stop codon). For example, the OCSl terminator can be used here. As for the promoters, different terminator sequences should be used for each gene.

The gene construct may, as described above, also include other genes to be introduced into the organisms. It is possible and advantageous to introduce into the host organisms regulatory genes, such as genes for inducers, repressors or enzymes, which intervene by their enzyme activity in the regulation of one or more genes of a biosynthetic pathway, and to express therein. These genes may be heterologous or of homologous origin. Furthermore, further biosynthesis genes of the fatty acid or lipid metabolism can advantageously be contained in the nucleic acid construct or gene construct, or else these genes can be located on a further or several further nucleic acid constructs. Advantageously, as biosynthesis genes of the fatty acid or lipid metabolism, a gene selected from the group acyl-CoA-dehydrogenase (s), acyl-ACP [= acyl carrier protein] - desaturase (s), acyl-ACP-thioesterase (s), fatty acid Acyltransferase (s), acyl-CoA: lysophospholipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (n), fatty acid desaturase (s), fatty acid acetylenase (s), lipoxygenase (s), triacylglycerol lipase (s), allene oxide synthase (s),

Hydroperoxide lyase (s) or fatty acid elongase (s) or combinations thereof. Particularly advantageous nucleic acid sequences are biosynthesis genes of the fatty acid or lipid metabolism selected from the group of acyl-CoA: lysophospholipid acyltransferase, Δ-4-desaturase, Δ-5-desaturase, Δ-6-desaturase, Δ-9-desaturase, Δ-12 -Desaturase, Δ-5- Elongase and / or Δ-6 elongase.

In this case, the abovementioned nucleic acids or genes can be cloned in combination with other elongases and desaturases in expression cassettes, such as those mentioned above, and used for the transformation of plants with the aid of Agrobacterium.

The expression cassettes can be used in principle directly for introduction into the plant or else be introduced into a vector. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is attached. One type of vector is a "plasmid," which is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors may autonomously replicate in a host cell into which they have been introduced (eg bacterial vectors of bacterial origin of replication). Other vectors are advantageously integrated into the genome of a host cell upon introduction into the host cell and thereby replicated together with the host genome. In addition, certain vectors may direct the expression of genes to which they are operably linked. These vectors are referred to herein as "expression vectors". Usually, expression vectors suitable for recombinant DNA techniques are in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably because the plasmid is the most commonly used vector form. However, the invention is intended to encompass other forms of expression vectors, such as viral vectors that perform similar functions. Furthermore, the term vector is also intended to mean other vectors known to the person skilled in the art, such as phages, viruses, such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, Cosmids, linear or circular DNA.

The recombinant expression vectors advantageously used in the method comprise the nucleic acid sequences or the described gene construct used in the method in a form suitable for expression of the nucleic acids used in a host cell, which means that the recombinant expression vectors comprise one or more regulatory sequences selected on the basis of For expression to be used host cells, which is operably linked to the nucleic acid sequence to be expressed include. In a recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is bound to the regulatory sequence (s) such that expression of the nucleotide sequence is possible and they are linked to each other such that both sequences correspond to the predicted sequences attributed to the sequence Function (eg in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Florida, Ed .: Glick and Thompson, chapter 7, 89-108, including references therein. Regulation sequences include those that direct the constitutive expression of a nucleotide sequence in many types of host cells and those that direct the direct expression of the nucleotide sequence only in certain host cells under certain conditions. One skilled in the art will appreciate that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed, the level of expression of the desired protein, etc. The recombinant expression vectors used may be designed to express the nucleic acid sequences used in the method in prokaryotic or eukaryotic cells. This is advantageous since intermediate steps of the vector construction are often carried out in microorganisms for the sake of simplicity. For example, the genes of the invention and other genes of fatty acid and lipid metabolism can be expressed in bacterial cells, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, MA, et al., 1992, Foreign gene expression in yeast : a review ", Yeast 8: 423-488; van den Hondel, CAMJJ, et al. (1991)" Heterologous gene expression in filamentous fungi ", in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure, eds. Pp. 396-428: Academic Press: San Diego, and van den Hondel, CAMJJ, & Punt, PJ. (1991) Gene transfer systems and vector development for filamentous fungi, in Applied Molecular Genetics of Fungi, Peberdy, JF. et al., eds. 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, Desaturaseudocohnilembus, Eupl otes, Engelmanieila and Stylonychia, in particular of the genus Stylonychia lemnae, with vectors according to a transformation method as described in WO 98/01572, and preferably in 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); FF White, B. Jenes et al., Techniques for Gene Transfer, Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225 (and references cited therein). Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Of the Alternatively, recombinant expression vector may be transcribed and translated in vitro using, for example, T7 promoter regulatory sequences and T7 polymerase.

The expression of proteins in prokaryotes is usually done with vectors containing constitutive or inducible promoters which direct the expression of fusion or non-fusion proteins. Typical fusion expression vectors include i.a. pGEX (Pharmacia Biotech Inc., Smith, DB, and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), in which glutathione-S Transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein.

Examples of suitable inducible non-fusion E. coli expression vectors are i.a. pTrc (Amann et al., (1988) Gene 69: 301-315) and pET Id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Target gene expression from the pTrc vector is based on transcription by host RNA polymerase from a hybrid trp-lac fusion promoter. Target gene expression from the pET 1 Id vector is based on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is produced in the host strains BL21 (DE3) or HMS 174 (DE3) by a resident [lambda]

Prophagen provided a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. Other suitable vectors in prokaryotic organisms are known to those 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 Ml 13mp series, pKC30, pRep4, pHSl , pHS2, _P PLc236, pMBL24, pLG200, pUR290, pN-111113-Bl, γgtl 1 or pBdCI, in Streptomyces pIJ101, pI364, pIJ702 or pIJ361, in Bacillus pUB10, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667. In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in the yeast S. cerevisiae include pYeDesaturased (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 methods for constructing vectors suitable for use in other fungi, such as filamentous fungi, include those described in detail in: van den Hondel, CAMJJ, & Punt, PJ. (1991) Gene transfer systems and vector development for filamentous fungi, Applied Molecular Genetics of Fungi, JF Peberdy et al., Eds., Pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi [JW Bennet & LL Lasure, Eds., pp. 396-428: Academic Press: San Diego] Other suitable yeast vectors are, for example, pAG-1, YEp6, YEpI 3 or _P EMBLYe23.

Alternatively, the nucleic acid sequences used in the method of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (eg, 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 provide only a brief overview of possible suitable vectors. Other plasmids are known to the person skilled in the art and are described, for example, in: Cloning Vectors (Eds. Pouwels, PH, et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). For other suitable expression systems for prokaryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Ed., CoId Spring Harbor Laboratory, Col. Spring Harbor Laboratory Press, Col. Spring Harbor, NY, 1989.

Also, the genes used in the method can be found in unicellular plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251 and references cited therein, and higher plant pesticides (eg, spermatophytes, such as crops). are expressed. Examples of plant expression vectors include those described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left Border ", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 8711-8721; Vectors for Gene Transfer to Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, pp. 15-38.

A plant expression cassette preferably contains regulatory sequences that can control gene expression in clones and are operably linked so that each sequence can fulfill its function, such as termination of transcription, for example, polyadenylation signals. Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens T-DNA, such as the gene 3 of the Ti plasmid pTiACH [delta] known as octopine synthase (Gielen et al. (1984) EMBO J. 3: 835ff.) Or functional equivalents thereof , but also all other terminators functionally active in plants are suitable.

Since plant gene expression is very often not limited to the level of transcription, a plant expression cassette preferably contains other operably linked sequences, such as Translational enhancers, such as the overdrive sequence, which contains the 5'-untranslated tobacco mosaic virus leader sequence which increases the protein / RNA ratio (Gallie et al., (1987) Nucl. Acids Research 15: 8693-8711).

The inserted gene, as described above, must be operably linked to a suitable promoter that performs gene expression in an upright, cell or tissue-specific manner. Useful promoters are constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those derived from plant viruses, such as 35S CaMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also US 5,352,605 and WO 84/02913) or plant promoters, such as the Rubisco small subunit described in US 4,962,028.

Other preferred sequences for use in the functional compound in plant gene expression cassettes are targeting sequences necessary to direct the gene product into its corresponding cell compartment (see review in Kermode, Crit., Plant, 15, 4 (1996) 285) -423 and references cited therein), for example to the vacuole, the nucleus, all types of plastids such as amyloplasts, chloroplasts, chromoplasts, extracellular space, mitochondria, endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.

The plant gene expression can also be facilitated as described above via a chemically inducible promoter (see a review in Gatz 1997, Annu Rev. Plant Physiol Plant Mol. Biol., 48: 89-108). Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline Inducible promoter (Gatz et al. (1992) Plant J. 2: 397-404) and an ethanol-inducible promoter. Promoters which respond to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-induced PRPl gene promoter (Ward et al. (1993) Plant Mol. Biol. 22: 361-366), the heat-inducible hsp80 promoter from tomato (US Pat. No. 5,187,267), the potato-alpha-amylase-inducible promoter (WO 96/12814) or the wound-inducible pinII promoter (EP-A-0 375 091).

In particular, those promoters which induce gene expression in tissues and organs in which fatty acid, lipid and oil biosynthesis take place are preferred in seed cells such as the cells of the endosperm and the developing embryo. suitable

Promoters are the rapeseed napkin promoter (US 5,608,152), the Vicia faba USP promoter (Baeumlein et al (1991) Mol Gen Genet, 225 (3): 459-67), the Arabidopsis oleosin promoter (WO 98/45461), the phaseolin promoter from Phaseolus vulgaris (US 5,504,200), the Brassica Bce4 promoter (WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al. (1992) Plant Journal 2 (1992); 2): 233-9) as well as promoters which induce seed-specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice and the like. Suitable noteworthy promoters are the lpt2 or lpt1 gene promoter from barley (WO 95/15389 and WO 95/23230) or the promoters described in WO 99/16890 from the barley hordein gene, the rice glutelinase. Gene, rice oryzine gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, corn zein gene, oat glutelin gene, sorghum kasirin gene, rye secalin -Gene).

In particular, the multiparallel expression of the nucleic acid sequences used in the method may be desired. The introduction of such expression cassettes can be carried out or preferred via a simultaneous transformation of a plurality of individual expression constructs by combining several expression cassettes on a construct. It is also possible to transform a plurality of vectors each having a plurality of expression cassettes and to transfer them to the host cell.

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

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection", conjugation and transduction are intended to encompass a variety of methods known in the art for introducing foreign nucleic acid (eg 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 methods for transforming or transfecting host cells, including plant cells, can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., CoId 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.

The term "nucleic acid (molecule)" as used herein also includes, in an advantageous embodiment, those located at the 3 'and 5' ends of the coding gene region untranslated sequence: at least 500, preferably 200, more preferably 100 nucleotides of the sequence upstream of the 5 'end of the coding region and at least 100, preferably 50, more preferably 20 nucleotides of the sequence downstream of the 3' end of the coding gene region. An "isolated" nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid. An "isolated"

Nucleic acid preferably does not have sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (e.g., sequences located at the 5 'and 3' ends of the nucleic acid). For example, in various embodiments, the isolated nucleic acid molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences that naturally comprise the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived flank.

The nucleic acid molecules used in the method, eg, a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. It is also possible with the aid of comparative algorithms to identify, for example, a homologous sequence or homologous, conserved sequence regions at the DNA or amino acid level. These can be used as hybridization probes in standard hybridization techniques (such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CoId Spring Harbor Laboratory, Col. Spring Harbor Laboratory Press, ColD Spring Harbor, NY, 1989 ) can be used to isolate further nucleic acid sequences useful in the method. Moreover, a nucleic acid molecule comprising a complete sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof can be isolated by polymerase chain reaction, wherein oligonucleotide primers based on this sequence or parts thereof (eg, a nucleic acid molecule comprising the complete sequence or a portion thereof can be isolated by polymerase chain reaction using oligonucleotide primers prepared on the basis of this same sequence). For example, mRNA can be isolated from cells (eg, by the guanidinium thiocyanate extraction method of Chirgwin et al., (1979) Biochemistry 18: 5294-5299) and cDNA by reverse transcriptase (eg, Moloney MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV Reverse Transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be prepared on the basis of one of the sequences shown in SEQ ID NO: 1 or SEQ ID NO: 3 or with the aid of the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4. A nucleic acid of the invention may be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques. The thus amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis. Oligonucleotides corresponding to a desaturase nucleotide sequence may be prepared by standard synthetic methods, for example, with an automated DNA synthesizer.

Homologs of the desaturase nucleic acid sequences used having the sequence SEQ ID NO: 1 or SEQ ID NO: 3 means for example allelic variants with at least 67%, 68% or 69%, preferably at least about 70%, 74%, 78%, 80% or 84%, more preferably at least about 85%, 86%, 87%, 88%, 89% or 90%, and most preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity or homology to a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or their homologs, derivatives or analogs or parts thereof. Furthermore, isolated nucleic acid molecules of a nucleotide sequence which hybridize to one of the nucleotide sequences shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof, eg hybridized under stringent conditions. A part according to the invention is understood to mean that at least 25 base pairs (= bp), 50 bp, 75 bp, 100 bp, 125 bp or 150 bp, preferably at least 175 bp, 200 bp, 225 bp, 250 bp, 275 bp or 300 bp, more preferably 350 bp, 400 bp, 450 bp, 500 bp or more base pairs are used for the hybridization. It may also be advantageous to use the overall sequence. In particular, allelic variants include functional variants which can be obtained by deletion, insertion or substitution of nucleotides from / in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, but the intention is that the enzyme activity should be that of the one resulting therefrom synthesized proteins for the insertion of one or more genes is advantageously retained. Proteins which still have the enzymatic activity of Δ-6-desaturase or Δ-5-desaturase, ie whose activity is essentially not reduced, means proteins with at least 10%, preferably 20%, particularly preferably 30%, very particularly preferably 40%, 50%, 60%, 70%, 80% of the original enzyme activity compared to the protein encoded by SEQ ID NO: 1 or SEQ ID NO: 3.

For example, homologues of SEQ ID NO: 1 or SEQ ID NO: 3 also mean bacterial, fungal and plant homologs, truncated sequences, single-stranded DNA or RNA of the coding and non-coding DNA sequence.

Homologs of SEQ ID NO: 1 or SEQ ID NO: 3 also mean derivatives, such as promoter variants. The promoters upstream of the indicated nucleotide sequences may be modified by one or more nucleotide substitutions, insertion (s) and / or deletion (s) without, however, interfering with the functionality or activity of the promoters becomes. It is also possible that the activity of the promoters is increased by modification of their sequence or that they are completely replaced by more active promoters, even from heterologous organisms.

The abovementioned nucleic acids and protein molecules with Δ-6-desaturase or Δ-5-desaturase activity, which are involved in the metabolism of lipids and fatty acids, PUFA cofactors and enzymes or in the transport of lipophilic compounds via membranes, are used in the method according to the invention for modulation the production of PUFAs in transgenic organisms advantageous in plants such as maize, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, Linum species such as oil or fiber kidney, Brassica species such as rapeseed, canola and Turnip rape, pepper, sunflower, borage, evening primrose and tagetes, solanacea plants such as potato, tobacco, aubergine and tomato, vicia species, pea, manioc, alfalfa, shrimp (coffee, cocoa, tea), salix species, trees ( Oil palm, coconut) and perennial grasses and forage crops, either directly (eg, if overexpression or optimization of a fatty acid biosynthesis protein has a direct effect on the yield,

Production and / or production efficiency of the fatty acid from modified organisms) and / or may have an indirect effect which nevertheless results in an increase in the yield, production and / or efficiency of the production of the PUFAs or a decrease in undesired compounds (eg modulation of the metabolism of lipids and fatty acids, cofactors and enzymes results in changes in the yield, production and / or efficiency of production or composition of the desired compounds within the cells, which in turn may affect the production of one or more fatty acids). The combination of different precursor molecules and biosynthetic enzymes leads to the production of various fatty acid molecules, which has a decisive effect on the composition of the lipids.

Particularly suitable for the production of PUFAs, for example stearidonic acid, eicosapentaenoic acid, arachidonic acid and docosahexaenoic acid, are Brassicaceae, Boraginaceae, Primulaceae, or Linaceae. Lein (Linum usitatissimum) is particularly advantageously suitable for the production of PUFAS with the nucleic acid sequences according to the invention, preferably as described, in combination with other desaturases and elongases.

The lipid synthesis can be divided into two sections: the synthesis of fatty acids and their binding to sn-glycerol-3-phosphate as well as 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-Co A into malonyl-CoA by the acetyl-Co A carboxylase or in

Acetyl-ACP by acetyl transacylase. After a condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydration reactions to give a saturated fatty acid molecule of the desired chain length. The production of unsaturated fatty acids from these molecules is catalyzed by specific desaturases, either aerobically by molecular oxygen or anaerobically (for fatty acid synthesis in microorganisms see FC Neidhardt et al., (1996) E. coli and Salmonella ASM Press: Washington, D See pages 612-636 and references therein, Lengeier et al., (Eds.) (1999) Biology of Procaryotes, Thieme: Stuttgart, New York, and the references therein, and Magnuson, K., et al ) Microbiological Reviews 57: 522-542 and the references included). The fatty acids thus bound to phospholipids must then be converted again for the further elongations from the phospholipids into the fatty acid CoA ester pool. This is facilitated by acyl-CoA: lysophospholipid acyltransferases. Furthermore, these enzymes can transfer the elongated fatty acids again from the CoA esters to the phospholipids. This reaction sequence can optionally be run through several times.

Precursors for the PUFA biosynthesis are, for example, oleic acid, linoleic acid and linolenic acid. These C18 carbon fatty acids must be extended to C20 and C22 to obtain Eicosa and Docosa chain type fatty acids, especially ARA and EPA. Arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid, advantageously eicosapentaenoic acid, arachidonic acid and / or docosahexaenoic acid, can be prepared with the aid of the desaturases and / or elongases used in the process and subsequently used for various purposes in food, feed, cosmetic or pharmaceutical applications become. C20 and / or C22 fatty acids having at least two, advantageously at least three, four, five or six double bonds in the fatty acid molecule, preferably C20 or C22 fatty acids with advantageously four, five or six double bonds in the fatty acid molecule, can be produced with the abovementioned enzymes. The desaturation can be carried out before or after elongation of the corresponding fatty acid. Thus, the products of desaturase activities and possible further desaturation and elongation result in preferred PUFAs having a higher desaturation level, including a further elongation of C20 to C22 fatty acids, to fatty acids such as γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, Stearidonic acid, eicosatetraenoic acid or eicosapentaenoic acid. Substrates of the desaturases and elongases used in the process according to the invention are C16, C18 or C20 fatty acids such as, for example, linoleic acid, γ-linolenic acid, α-linolenic acid, dihomo-γ-linolenic acid, eicosatetrape acid or stearidonic acid. Preferred substrates are linoleic acid, γ-linolenic acid and / or α-linolenic acid, dihomo-γ-linolenic acid or arachidonic acid, eicosatetraenoic acid or eicosapentaenoic acid. The synthesized C20 or C22 fatty acids having at least two, three, four, five or six double bonds in the fatty acid are obtained in the novel process in the form of the free fatty acid or in the form of their esters, for example in the form of their glycerides.

The term "glyceride" is understood to mean a glycerol esterified with one, two or three carboxylic acid residues (mono-, di- or triglyceride). By "glyceride" is also meant a mixture of different glycerides. The glyceride or glyceride mixture may contain other additives, e.g. contain free fatty acids, antioxidants, proteins, carbohydrates, vitamins and / or other substances.

A "glyceride" in the sense of the method according to the invention is also understood to mean derivatives derived from glycerol. In addition to the fatty acid glycerides described above, these also include glycerophospholipids and glyceroglycolipids. The glycerophospholipids, such as lecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol, phosphatidylserine and alkylacylglycerophospholipids, may be mentioned by way of example here.

Furthermore, fatty acids must then be transported to various modification sites and incorporated into the triacylglycerol storage lipid. 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).

Publications on plant fatty acid biosynthesis, desaturation, lipid metabolism and membrane transport of fatty compounds, beta-oxidation, Fatty acid modification and cofactors, triacylglycerol storage and assembly, including references therein, see the following articles: Kinney, 1997, Genetic Engineering, eds .: 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, eds .: 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, eds .: Murata and Somerville, Rockville, American Society of Plant Physiologists, 150-158, Murphy & Ross 1998, Plant Journal. 13 (1): 1-16.

The PUFAs produced in the process comprise a group of molecules that are no longer able to synthesize, and therefore need to take up, higher animals, or that can no longer sufficiently produce higher animals themselves, and thus have to additionally take up, even though they are readily synthesized by other organisms, such as bacteria For example, cats can no longer synthesize arachidonic acid.

For the purposes of the invention, phospholipids are to be understood as meaning phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and / or phosphatidylinositol, advantageously phosphatidylcholine. The terms production or productivity are in the

In the art and include the concentration of the fermentation product formed in a given period of time and fermentation volume (eg, kg of product per hour per liter). It also includes the productivity within a plant cell or plant, that is the content of the desired fatty acids produced in the process based on the content of all fatty acids in that cell or plant. The term Efficiency of production includes the time it takes to produce a certain amount of production (eg how long the cell needs to set up a specific throughput rate of a fine chemical). The term yield or product / carbon yield is known in the art and includes the efficiency of converting the carbon source into the product (ie, the fine chemical). This is usually expressed, for example, as kg

Product per kg of carbon source. By increasing the yield or production of the compound, the amount of the recovered molecules or molecules of this compound obtained in a given amount of culture is increased over a fixed period of time. The terms biosynthesis or biosynthetic pathway are known in the art and involve the synthesis of a compound, preferably an organic compound, by a cell from intermediates, for example in a multi-step and highly regulated process. The terms degradation or degradation pathway are well known in the art and involve the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), for example in a multi-step and highly regulated process. The term metabolism is known in the art and includes the entirety of the biochemical reactions that take place in an organism. The metabolism of a particular compound (e.g., the metabolism of a fatty acid) then comprises all of the biosynthetic, modification, and degradation pathways of that compound in the cell that affect that compound.

The desaturation efficiency in the context of the invention can be calculated by the formula (product x 100) / (educt + product).

To determine the percent homology (= identity) of two amino acid sequences (eg one of the sequences of SEQ ID NO: 2 or SEQ ID NO: 4) or of two nucleic acids (eg SEQ ID NO: 1 or SEQ ID NO: 3), the sequences are written one below the other for the purpose of optimal comparison (eg, gaps may be inserted into the sequence of one protein or one nucleic acid for optimal alignment with the other protein or nucleic acid produce). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding site in the other sequence, then the molecules are homologous at that position (ie, amino acid or nucleic acid 'homology' as used herein corresponds to amino acid or nucleic acid "identity".) The percent homology between the two sequences is a function of the number of identical positions common to the sequences (ie% homology = number of identical positions / total number of positions x 100) Identities are thus to be regarded as synonymous The programs or algorithms used are described above.

An isolated nucleic acid molecule encoding a Δ6-desaturase or Δ5-desaturase homologous to a protein sequence of SEQ ID NO: 2 or SEQ ID NO: 4 may be prepared by introducing one or more nucleotide substitutions, additions, or deletions are generated in a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into any of the sequences of SEQ ID NO: 1 or SEQ ID NO: 3 by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made on one or more of the predicted nonessential amino acid residues. In a "conservative amino acid substitution", the amino acid residue is replaced with an amino acid residue having a similar side chain. In the field are families of Amino acid residues have been defined with similar side chains. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg Alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). A predicted nonessential amino acid residue in a Δ6-desaturase or Δ5-desaturase is thus preferably exchanged for another amino acid residue from the same side-chain family. Alternatively, in another embodiment, the mutations may be introduced randomly over all or part of the Δ6-desaturase or Δ5-desaturase coding sequence, for example, by saturation mutagenesis, and the resulting mutants may be prepared according to the Δ-6 described herein - Desaturase or Δ-5-desaturase activity are screened to identify mutants that have retained the Δ-6-desaturase or Δ-5-desaturase activity. After mutagenesis of any of SEQ ID NO: 1 or SEQ ID NO: 3, the encoded protein can be recombinantly expressed, and the activity of the protein can be determined using, for example, the assays described herein. Thereafter, as a rule, the activity is detected by expression of the corresponding cDNA in an organism such as yeast and subsequent fatty acid analysis.

Further subjects of the invention are transgenic non-human organisms which contain the nucleic acids according to the invention according to SEQ ID NO: 1 or SEQ ID NO: 3 or a gene construct or a vector which contain these nucleic acid sequences according to the invention. Advantageously, the non-human organism is a microorganism, a non-human animal or a plant, more preferably a plant. examples for suitable plants have already been mentioned.

This invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references cited in this patent application, patent applications, patents, and published patent applications are incorporated herein by reference.

Examples

Example 1: General Cloning Methods

The cloning methods, e.g. Restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, attachment of DNA fragments, transformation of Escherichia coli cells, culture of bacteria and sequence analysis of recombinant DNA were performed as described in Sambrook et al , (1989) (CoId Spring Harbor Laboratory Press: ISBN 0-87969-309-6).

Example 2 Sequence Analysis of Recombinant DNA

The sequencing of recombinant DNA molecules was carried out using a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al. (1977) Proc. Natl. Acad., See, USA74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced to avoid polymerase defects in constructs to be expressed and checked.

Example 3 Lipid Extraction from Yeasts and Seeds

The effect of genetic modification in plants, fungi, algae or ciliates on the production of a desired compound (such as a fatty acid) may be determined by culturing the modified microorganism or modified plant under suitable conditions (such as those described above) and the medium and / or the cellular components are assayed for increased production of the desired product (ie, lipids or a fatty acid). These analytical techniques are known to the person skilled in the art and include spectroscopy, thin-layer chromatography, staining methods of various types, enzymatic and microbiological methods and analytical chromatography, such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial 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, PA, et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, JF, and Cabral, JMS (1992) Recovery processes for biological materials, John Wiley and Sons, Shaeiwitz, JA, and Henry, JD (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11 , Pp. 1-27, VCH : Weinheim; and Dechow, FJ. (1989) Separation and purification techniques in biotechnology, Noyes Publications). In addition to the above-mentioned methods, plant lipids derived from plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Be. 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 in Christie, William W., Advances in Lipid Method, Logy, Ayr / Scotland: OiIy Press (OiIy Press Lipid Library, 2); Christie, William W., Gas Chromatography and Lipids. A Practical Guide - Ayr, Scotland: OiIy Press, 1989, Repr. 1992, DC, 307 p. (OiIy Press Lipid Library; 1); "Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952) - 16 (1977) udT: Progress in the Chemistry of Fats and Other Lipids CODES.

In addition to measuring the end product of the fermentation, it is also possible to analyze other components of the metabolic pathways used to produce the desired compound, such as by-products and by-products, to determine the overall efficiency of production of the compound. The analysis methods include measurements of

Nutrient levels in the medium (e.g., sugars, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of biomass composition and growth, analysis of the production of common metabolites of biosynthetic pathways, and measurements of gases produced during fermentation. Standard methods for these measurements are in Applied Microbial Physiology; A Practical Approach, P.M. Rhodes and P.F. Stanbury, eds., IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and references cited therein.

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

The unequivocal evidence of the presence of fatty acid products can be obtained by analysis of recombinant organisms by standard analytical methods: GC, GC-MS or TLC as variously described by Christie and the references therein (1997, in: Advances on Lipid Methodology, Fourth Edition: Christie, OiIy Press, Dundee, 119-169; 1998, gas chromatography-mass spectrometry method, Lipids 33: 343-353). The material to be analyzed may be broken up by sonication, milling in the glass mill, liquid nitrogen and milling or other applicable methods. The material must be centrifuged after rupture. The sediment is distilled in aqua. re-suspended, heated at 100 ^° C. for 10 minutes, cooled on ice and recentrifuged, followed by extraction into 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 hour at 90 ^° C., resulting in hydrolyzed oil and lipid compounds, which give transmethylated lipids. These fatty acid methyl ester are extracted in petroleum ether and finally subjected to GC analysis (mikrom Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25, 0.32 mm) using a capillary column with a temperature gradient of between 170 ⁰ C and 240 ⁰ C for 20 min and 5 min at 240 ⁰ C subjected. The identity of the resulting fatty acid methyl esters must be defined using standards available from commercial sources (ie Sigma).

Plant material is first mechanically homogenized by mortars to make it more accessible to extraction. Then it is heated for 10 min at 100 ⁰ C and sedimented again after cooling on ice. The cell sediment is hydrolyzed for 1 h at 90 ^° C. with 1 M methanolic sulfuric acid and 2% dimethoxypropane, and the lipids are trans methylated. The resulting fatty acid methyl esters (FAME) are extracted into petroleum ether. The extracted FAME are purified by gas chromatography using a capillary column (Chrompack, WCOT fused silica, CP-Wax-52 CB, 25 m, 0.32 mm) and a temperature gradient of 170 ^° C. to 240 ^° C. in 20 minutes and 5 minutes at 240 ° C. ⁰ C analyzed. The identity of the fatty acid methyl esters is confirmed by comparison with corresponding FAME standards (Sigma). The identity and position of the double bond can be determined by appropriate chemical Derivatization of the FAME mixtures, for example to 4,4-dimethoxyoxazoline derivatives (Christie (1998) Chem. Phys. Lipids 94, 35-41) can be further analyzed by GC-MS.

Example 4: Cloning of desaturase genes from Mantoniella squamata

Mantoniella squamata (Manton et Parke), Desikachary, was purchased from the algae culture collection Göttingen (SAG, Germany). Non-axenic cultures in batch cultures under Langtaglicht (14 h) conditions with 45 micromol photons m ^"s" in 50-200 ml Brackish Water medium (1/2 SWLS) supplemented with soil extract, grown at 20 ⁰ C , The biomass was harvested by centrifugation and used for total RNA isolation.

Total RNA was extracted from a 7 day old M. squamata culture using the RNAeasy Kit (Qiagen, Valencia, CA, USA). Poly A + RNA was isolated from total RNA using oligo dT cellulose (Sambrook et al., 1989, vide supra). Reverse transcription was performed using Promega's Reverse Transcription Kit and the resulting cDNA was inserted into lambda ZAP-Vector (lambda ZAP Gold, Stratagene) according to the manufacturer's protocol. Following in vivo mass excision of the cDNA library, plasmid recovery and transformation of Escherichia coli, plasmid DNA was prepared on a Qiagen DNA purification robot (Qiagen, Hilden, Germany) according to the manufacturer's instructions and subjected to random sequencing by means of the chain termination method Use of the ABI

PRISM Big Dye Termination Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany). Evaluation and annotation of the EST sequences between 100 and 500 base pairs yielded an EST database with non-redundant sequences. In this database successfully new desaturase sequences from M. squamata could be identified. 5 μl of total RNA isolated from M. squamata was transcribed and ligated to adapter-ligated double-stranded cDNA (ds-cDNA) using the Marathon cDNA amplification kit (BD Bioscience) according to the manufacturer's instructions. The adapter-ligated ds cDNA was diluted (1: 250) with sterile dd H ₂ O and used as template for 5 'and 3' RACE PCR reactions to eliminate the missing 5 'prime and 3' prime To obtain ends of the coding sequences for different desaturases. For the RACE reactions, a Marathon cDNA adapter primer 1 (API) and a gene-specific primer were designed using the EST sequence information. The primers used in 5 'and 3' RACE reactions were as follows: for MsΔ6 (MsI, Δ-6-desaturase from M. squamata) as 5'-RACE primer 5'-CATCCGGGCGGCAGCGTCATCTTCTAC-S 'and as

3'-RACE primer 5'-GGAGAAGAGGTGGTGGATGACCTGG-S '; for MsΔ5 (MsII, Δ-5-desaturase from M. squamata) as 5'-RACE primer 5'-CCGAGTGAGGGGAGTACGTGGCGGG-S 'and as

3'-RACE primer 5'-CACTCTCCGGCGGGCTCAACTACC-S '.

A 50 μl standard reaction mixture contained 1 × Ex Taq DNA polymerase buffer, 1 × Ex Taq DNA polymerase (TaKaRa Bio), 0.2 mM of each dNTP, 0.5 μM 5 'primer or 3' primer, 0, 5 μM API and 5 μl of the diluted adapter-ligated ds cDNA. RACE PCR amplification was performed as follows: 30 sec. At 94 ° C; 5 cycles of 5 s at 94 ⁰ C, 3 min at 72 ⁰ C; 5 cycles of 5 s at 94 ⁰ C, 3 min at 70 ⁰ C; 20 cycles of 5 s at 94 ⁰ C, 3 min at 68 ⁰ C. The amplifϊzierten products were isolated from agarose gels by means of a kit (GE Healthcare Bioscience) and then purified in the plasmid pGEM-T (Promega). From the 5 'and / or 3' cDNA sequence data obtained by the RACE-PCR, the putative translation start codons and stop codons were identified, and this sequence information was used to obtain full-length cDNA clones of the putative desaturases from M. squamata receive.

The tracked EST sequencing strategy generated more than 3,500 non-redundant sequences. The sequences of the two isolated full length cDNA clones for desaturases from M. squamata are given in SEQ ID NO: 1 and 3. In this case, the MsI cDNA represents a Δ6-desaturase, shown in SEQ ID NO: 1, which was annotated as MsΔ6 (M squamata Δ6-desaturase). The amino acid sequence shown in SEQ ID NO: 2 shows an ORF of 450 amino acids. The MsII cDNA shown in SEQ ID NO: 3 encodes a Δ5-desaturase whose amino acid sequence shown in SEQ ID NO: 4 has an ORF of 483 amino acids. Similarly, the MsII clone was annotated as MsΔ5 (M squamata Δ5-desaturase).

Comparing the sequences with known desaturases reveals similarities, including an HPGG N-terminal motif (cytochrome b5 binding domain), the three histidine boxes that are likely to play a role in the coordination of the active site diiron center Biol. 49, 611-641) and the more variable third histidine box with a typical H- to Q-substitution. These properties are characteristic of front-end desaturases. Example 5: Expression of Desaturases in Yeast

Gene-specific primers were designed to the 5 'and 3' ends of the coding regions of the corresponding nucleotide sequences introducing restriction sites for subsequent cloning into the various yeast expression vectors. For the first characterization of MsΔ6, the ORF (open reading frame) of MsΔ6 was cloned with the primers given in Table 1 in pYES2. For further characterization, the forward primers of MsΔ6 and MsΔ5 were designed such that the nucleotide sequence ACATA was included before the ATG start codon (Table 1) to enhance translation initiation in eukaryotic cells. The ORFs of MsΔ6 and MsΔ5 were amplified by PCR with the described primers (see Table 1) using the Expand High Fidelity ^{1 "1118} PCR system (Roche Diagnostics), cDNA template and a standard PCR protocol (2 min at 94 ⁰ C, 10 cycles of 10 s at 94 ⁰ C, 30 s at 70 ⁰ C, 80 s at 72 ⁰ C, followed by 20 cycles of 10 s at 94 ⁰ C and 3 min at 72 ⁰ C, and a final elongation step of 7 min at 72 ⁰ C) modified. amplified cDNAs were cloned into the pGEM-T vector (Promega) before they cut again and cloned into a yeast expression vector (pYES2, Invitrogen, pESC-LEU or pESC-TRP, Stratagene) which resulted in the vectors pYES2-MsΔ6, pESC-LEU-MsΔ6 and pESC-TRP-MsΔ5.

For co-expression experiments, the Δ6 elongase from Physcomitrella patens, PSE1 (Zank et al. (2002) Plant J. 31: 255-268) was cloned into the yeast expression vector pESC-LEU to add the vector pESC-LEU-PSE1 -MsΔ6 before the ORF of MsΔ6 was inserted as explained above. For the purpose of comparison with the known desaturases from Phaeodactylum tricornutum, PtΔ6 and PtΔ5, the P. tricornutum cDNA clones were cloned in yeast expression vectors as described above for M. squamata desatmases, yielding pESC-LEU-PSEI-PtΔ6 and pESC-TRP -PtΔ5 were obtained. All primers for fill length cDNAs and the yeast expression constructs are listed in Table 1.

S. cerevisiae cells of the INVScI strain from Invitrogen (Karlsruhe, Germany) were prepared by the method of Dohmen et al. (Dohmen et al. (1991) Yeast 7: 691-692). For purposes of inducing expression cultures for 72 h were at 21-23 ⁰ C in the presence of 2% (w / v) galactose, supplemented with 350 uM of a suitable fatty acid substrate and in the presence of 1% Igepal CA 630 ( 'NP 40') of Attracted Sigma-Aldrich. The cells were harvested by centrifugation at 1200 g for 5 min and the pellets washed twice with sterile dd H ₂ O before being used for further analysis. The host strain transformed with the empty vector (s) was used as a negative control in all experiments.

Example 6: Fatty acid analysis

Fatty acid methyl esters (FAMEs) were obtained by transmethylation of the yeast cell pellets with 0.5 M sulfuric acid in methanol containing 2% (v / v) dimethoxypropane at 80 ^° C. for 1 h. FAMEs were extracted in hexane and analyzed by gas chromatography (GC). GC analysis was performed on an Agilent GC 6890 system coupled with a FID detector equipped with a 122-2332 DB-23 capillary column (30 mx 0.32 mm, 0.5 μm coating thickness, Agilent). Helium was ^"used samples were injected at 220 ⁰ C The temperature gradient was as follows: 150 ⁰ C for 1 min, 150 ⁰ C - 200 ⁰ C at 15 ⁰ C min., As the carrier gas (1 mL ^{min)" 1} 200 ⁰ C - 250 ⁰ C at 2 ⁰ C min ^"1 , and 250 ⁰ C for 10 min. The data were evaluated using HP ChemStation Rev. A09.03. FAMEs were identified by comparison with appropriate reference substances of FAMEs.

To confirm the substrate and regiospecificity of the putative desaturases, the filling length cDNAs of MsΔ6 and MsΔ5 had been cloned into pYES2, pESC-LEU and pESC-TRP. In these vectors, the desaturase sequences are under the control of the galactose-inducible promoter GAL1 or GAL10. The plasmids obtained were designated MsΔ6-pYES2, MsΔ6-pESC-LEU and MsΔ5-pESC-TRP. The clones were individually expressed in S. cerevisiae strain INVScI. Both transformants were incubated in the presence of potential fatty acid substrates for Δ-6 and Δ-5-desaturases, namely 18: 2 ^{Δ9 '12} , 18: 3 ^Δ9 ' ^{12 '15} , 20: 3 ^Δ8 ' ^{n '14} and 20: 4 ^Δ8 ' ⁿ ' ^{14 '17} . Upon feeding α-linolenic acid (18: 3 ^Δ9 ' ¹² ' ¹⁵ ), the yeast cells expressing the ORF of MsΔ6 produced a new fatty acid. By GC-MS analysis (Christie (1998) vide supra), this new fatty acid could be identified as stearidonic acid (1 g _: 4 ^Δ6 ' ⁹ ' ^{12 '15} ^), demonstrating that the MsΔ6 cDNA was responsible for a Δ-6 The corresponding feeding experiments are shown in FIG.

Expression of MsΔ5 feeding dihomo-γ-linolenic acid (20: 3 ^Δ8 ' ⁿ ' ^H ) and ω3-arachidonic acid (20: 4 ^Δ8 ' ⁿ ' ^{14 '17} ) also resulted in ^novel fatty acid products containing arachidonic acid (ARA, 20 : 4 ^{Δ5 '8' n '14)} or eicosapentaenoic acid (EPA, ₂ 0: 5 ^{A5' 8 'n' 14 '17)} corresponded. The structure was confirmed by GC-MS analysis to show that MsΔ5 is a Δ5-desaturase. The corresponding feeding experiments are shown in FIG.

The MsΔ6 enzyme showed very high specificity, only the fed α-linolenic acid was used as substrate and in stearidonic acid with 17% desaturation efficiency (pYES2-MsΔ6) and 35% desaturation efficiency (pESC-LEU-MsΔ6, which is the translation initiation sequence ACATA) (Figure 4A). In contrast, MsΔ5 appears to be less specific and converts both 20: 3 ^Δ8 ' ⁿ ' ¹⁴ and 20: 4 ^Δ8 ' ⁿ ' ^{14 '17} as a substrate for desaturation. The desaturation efficiency of MsΔ5 with 20: 4 '''as a substrate was comparable to that with _{20: 3} ^Δ8, π ^{, i4} ^ _{9 o / o and} η o _{/ o} conversion, _respectively , as can be seen from FIG. 4B.

Example 7: Lipid analysis

For lipid analysis, expression was carried out in 120 ml cultures. Harvested cell pellets were homogenized in 5 ml chloroform / methanol (1: 2) and the lipids extracted on a shaker for 4 h and then for 20 h with 5 ml chloroform / methanol (2: 1) at 4 ^° C. The resulting organic phases were combined and evaporated under nitrogen. The remaining lipids were dissolved in 1 ml of chloroform. Separation of the lipid classes (neutral lipids and phospholipids) was achieved using a silica gel column (Bond Elut SI, 100 mg / ml, Varian, Darmstadt). The lipid extracts were loaded onto the silica gel column previously pre-equilibrated with chloroform and then fractionated into the lipid classes by elution as follows: neutral fats with chloroform and phospholipids with methanol / glacial acetic acid (9: 1). The isolation of the individual components from the phospholipid class was achieved by means of thin-layer chromatography (TLC) with methanol / chloroform / glacial acetic acid (65: 25: 8) as the eluent and with suitable standards.

A comparison of the isolated desaturases with known desaturases suggested an acyl-CoA-dependent fatty acid desaturation in M. squamata. The distribution of the produced fatty acids into lipid classes may be an indication of an acyl-CoA-specific desaturation. For this reason, the desaturase-expressing yeast cells were fed with exogenous 18: 3 ^Δ9 ' ¹² ' ¹⁵ or 20: 3 '' and analyzed the fatty acid distribution in different lipid classes. In the fraction of the neutral lipids and phospholipids in all 4 ^{Δ6 '9' 12 '15} be detected: in the case of expression MsΔ6 the newly formed 18 could. This result is shown in FIG. The proportions of phospholipids vary from 14% in phosphatidylethanolamine (PE) and phosphatidylinositol / phosphatidylserine (PI / PS) to 22% of total lipid-associated stearidonic acid in phosphatidylcholine (Figure 5A). A similar distribution of 20: 4 ^Δ5 ' ⁸ ' ^{n '14} was found after expression of MsΔ5 in the components of the individual lipid classes. The desaturation product 20: 4 '''could be detected in all phospholipids and in the neutral lipid fraction. Furthermore, in the fractions PC (27%), PI / PS (36%) and neutral fats (29%) an accumulation of arachidonic acid was observed in equal amounts, as shown in Figure 5C, while the enrichment in the fraction PE with 8% lower.

The separate expression of the two desaturases, MsΔ6 and MsΔ5, as shown in Figure 5, shows that the desaturation products of the two desaturases in the PC fraction were not particularly highly enriched, but rather could be detected in relatively high proportions in all of the various lipid species analyzed , This observation suggests that MsΔ6 and MsΔ5 accept acyl-CoA thioesters as substrates rather than lipid-bonded acyl groups, as observed for the acyl-CoA specific Δ-6 desaturase from O. tauri.

The presumption of acyl-CoA-specific desaturation by the isolated desaturases from M. squamata was further investigated in co-expression experiments. Both enzymes MsΔ6 and MsΔ5 were expressed in yeast together with the acyl-CoA-dependent Δ-6 elongase from P. patens, PSE1 (Zank et al. (2002) vide supra). Feeding 18: 3 ^Δ9 ' ¹² ' ¹⁵ as an exogenous substrate revealed efficient biosynthesis of eicosapentaenoic acid (EPA), see Figure 6B, wherein EPA accumulated to approximately 0.7% of the total fatty acids (Figure 7). The accumulation of EPA was thus approximately 3-fold higher when MsΔ6 and MsΔ5 were expressed than when expression of the corresponding lipid-dependent desaturases from P. tricornutum (FIG. 7) was also reported by Domergue et al., 2002 (Eur. J. Biochem 269: 4105-4113). The higher production rates of EPA upon co-expression of desaturases from M. squamata and P. patens Δ6 elongase resulted from the efficient Δ6 desaturation of the 18: 3 ^Δ9 ' ¹² ' ¹⁵ substrate (16% conversion) by MsΔ6 , the efficient elongation (97% conversion) of the Δ6 desaturation product 18: 4 '''to the corresponding C20 fatty acid as well as the efficient Δ5 desaturation (20% conversion).

Using lipid-dependent desaturases in combination with a CoA-specific elongase, EPA accumulation was not as significant as the fatty acid intermediates are intermediates between the PC pool and the acyl-CoA pool because of the additional transacylation step required to move back and forth, probably less efficiently presented to the affected enzymes. The indicative fatty acid ^intermediate 18: 4 ^Δ6 ' ⁹ ' ^{12 '15} accumulated to a greater extent than could be observed with expression of M. squamata enzyme Q, indicating less efficient conversion for Δ-6 desaturation (6% conversion) Δ6 elongation (61% conversion) and consequently lower Δ5 desaturation (14% conversion).

The presence of 20: 3 ^Δ11 ' ¹⁴ ' ¹⁷ and 20: 4 ^Δ5 ' ⁿ ' ^{14 '17} in both profiles was due to the elongation of 18: 3'' ⁵ by PSEl and subsequent desaturation by MsΔ5. These by-products of the Δ-6 elongation reaction were unexpected and only accumulated for the reason that a large amount of 18: 3 ^Δ9 ' ¹² ' ¹⁵ had been fed. The synthesis of 20: 4 ^Δ5 ' ⁿ ' ^{14 '17} shows the broad substrate spectrum of the MsΔ5 enzyme. Example 8: Cloning into Plant Expression Vectors and Generation of Transgenic Plants and Fatty Acid Analysis of Transgenic Plants

For the production of long-chain, polyunsaturated fatty acids in plants, the nucleic acid sequences used in the method according to the invention which code for a Δ6-desaturase and a Δ5-desaturase activity are expressed in an expression cassette alone or in a preferred manner in combination with other nucleic acid sequences, for a Δ6 elongase, a Δ5 elongase, a Δ4-desaturase, an ω3-desaturase, a Δ12-desaturase, a Δ15-desaturase, a bifunctional Δ12 and Δ15-desaturase, a lysophospholipid acyltransferase, a diacylglycerol acyltransferase or a phospholipid diacylglycerol acyltransferase, are introduced into and expressed in a suitable plant. There may be more than one nucleic acid sequence of an enzymatic activity, e.g. a Δ6-desaturase, Δ5-desaturase, Δ6-elongase, Δ5-elongase, Δ4-desaturase, ω-3-desaturase, Δ12-desaturase, Δ15-desaturase, bifunctional Δ 12 and Δ 15 desaturase, lysophospholipid acyltransferase, diacylglycerol acyltransferase and / or phospholipid

Diacylglycerol acyltransferase be included. For expression of the nucleic acid sequences mentioned in this invention in plants such as Brassica species such as oilseed rape, sunflower, soybean, linum species such as oillein, corn, rye, wheat, oats, crambe, triticale, rice, greens, potatoes, field beans such as Vicia faba, pea, oil palm, coconut, peanut, borage are promoters that are active in plant cells and the expression of the

Ensure nucleic acid sequence (s) in plant cells. These are preferably promoters with a constant and / or seed-specific and / or tissue-specific activity. For introducing the nucleic acids or nucleic acid constructs, the nucleic acids used in the context of this invention are preferably amplification and ligation by means of polymerase chain reaction (for example, following the protocol of the Pfu-DNA reaction). Polymerase or Taq polymerase) or T4 DNA ligase or other DNA-ligating enzymes. The primers should be chosen to allow amplification of the entire coding region of the nucleic acids described in this method. The nucleic acids can then be introduced into suitable for expression in microorganisms or plant cells Verktorsysteme. These include, in particular, vectors which are replicable in microbial systems and, above all, vectors which ensure efficient cloning in yeasts, microorganisms or fungi, and the stable transformation of plants, such as Agrobacterium-mediated transformation. Suitable transformation methods for plants are known in the art and are based on T-DNA mediated transformation suitable binary and co-integrated vector systems. The

Vector systems include additional cis-regulatory elements and selection markers. The transformation of the plants is based on standard protocols and the selection of trans formants is allowed by means of the selection marker.

The fatty acid analysis of the transgenic plants is suitably carried out in the form of a

Fatty acid analysis of transgenic seeds, e.g. transgenic oilseed rape seeds (Brassica napus). The following protocol of a single seed analysis can be carried out:

Single transgenic seeds are disrupted using a needle and the fatty acids are derivatized by the addition of 10 μl of TMSH (Macherey-Nagel). After evaporation of the TMSH, the single seed samples are taken up in 5 μl acetonitrile. The fatty acid determination is carried out by GC analysis (see Example 6 "fatty acid analysis). Example 9: Acyl-CoA species, synthesis, extraction and analysis

Authentic standards for saturated and monounsaturated acyl-CoA esters with acyl chain lengths from C 12 to Cl 8 were obtained from Sigma. Standards for polyunsaturated acyl-CoAs (18: 3 ^Δ9 ' ¹² ' ¹⁵ , 18: 4 ^Δ6 ' ⁹ ' ^{12 '15} , 20: 3 ^Δ8 ' ^{n '14} , 20: 4 ^Δ8 ' ⁿ ' ¹⁴ ' ¹⁷ and ₂ 0 : 5 ^Δ5 ' ⁸ ' ⁿ ' ¹⁴ ' ¹⁷ ) were ^purified enzymatically using a recombinant acyl-CoA synthetase from Pseudomonas sp. (Sigma) synthesized. The reaction mixture (200 μl) containing 100 mM Tris-HCl, pH 8.1, 10 mM MgCl ₂ , 5 mM ATP, 5 mM CoASH, 2 mM DTT, 25 μM free fatty acid, and 2.5 units of the acyl-CoA Synthase was incubated for two hours at 37 ° C. The reaction was stopped with 50 μl of glacial acetic acid / ethanol 1: 1 (v / v), the desired aqueous phases were washed with petrol to remove residual free fatty acids. After purification of the acyl-CoA species on a Seppak column (Strata C18-E, Phenomenex) using acetonitrile as the eluent, the samples were dried under argon and dissolved in 50 mM Mes, pH 5.0.

For acyl-CoA analysis of yeast cells, 20 ml of the liquid _{cultures were} harvested at an _OD0000 of _1.5 to 2.0 and the acyl-CoA species were extracted as described in Domergue et al. (2005) Biochem. J. 389 (2): 483-490. Conversion of the acyl-CoA esters to their etheno derivatives and acyl-CoA analysis were performed as described by Larson and Graham (2001) Plant J. 25 (1): 115-125.

Example 10: Distribution of MsΔ6 and MsΔ5 desaturation products in different lipid classes in yeast

Since the distribution of product fatty acids in different lipid classes can serve as an indicator of desaturation in the acyl-CoA pool, yeast cultures expressing MsΔ6 and MsΔ5 were treated with exogenous 18: 3 ^Δ9 ' ¹² ' ¹⁵ and 20: 3 ^Δ8 ' ^n, respectively ^'14 and the fatty acid distribution in the individual lipid classes was analyzed. In parallel, the same experiments were performed with the acyl-CoA-dependent OtΔ6 and the lipid-dependent PtΔ6 and PtΔ5 desaturase. For all Δ6-desaturases tested, 18: 4 ^Δ6 ' ⁹ ' ^{12 '15} , which was produced by Δ6 desaturation of the 18: 3 ^Δ9 ' ^{12 '15} substrate, was predominantly in the neutral

Lipid fraction and in PC and to a lesser degree in the other phospholipids analyzed (Figure 8A). As expected for a lipid-dependent desaturase, the expression of PtΔ6 in the accumulation of the 18: 4 ^Δ6 ' ⁹ ' ^{12 '15} product ^resulted mainly in phosphatidylcholine (PC), whereas 18: 4 ^Δ6 ' ⁹ ' ¹² ' ¹⁵ in the expression of the CoA-dependent OtΔ6 between neutral lipids and PC was approximately equally distributed. The expression of MsΔ6 even resulted in an increased accumulation of 18: 4 '''in neutral lipids over PC, indicating that MsΔ6 desaturation is not lipid dependent. The 18: 4 "' ⁵ product was approximately evenly distributed in all Δ6-desaturase expression cultures in phosphatidylinositol with phosphatidylserine (PI / PS), phosphatidylethanolamine (PE) and diphosphatidylglycerol (CL).

When the Δ5-desaturases were tested for the distribution of the desaturation product 20: 4 '''in the lipid classes, the product was detected in the neutral lipid fraction and in all the phospholipids analyzed (Figure 8B). If MsΔ5 was expressed, accumulated 20: 4 ^{Δ5 '8' n '14} approximately equally in PC PI / PS, PE, CL and in the neutral Lipid fraction as shown in Fig. 8B. In contrast, 20: 4 ''', which was formed by the expression of PtΔ5, accumulated mainly in PC and was only found in small amounts in PI / PS (10%), PE (5%) or DPG (6%). demonstrated. In contrast to the results for the lipid-specific desaturases (such as, for example, PtΔ5), in which the desaturation products are enriched in particular in PC (Domergue et al. (2003) J. Biol. Chem. 278: 35115-35126), The data show that fatty acids produced by MsΔ6 and MsΔ5 are not primarily associated with PC and can be detected in approximately equal amounts in all lipid species analyzed. These results indicate that MsΔ6 and MsΔ5 use acyl-CoA thioesters instead of lipid-bonded acyl groups as substrates.

The comparison of OtΔ6 with the new MsΔ6 revealed that the latter desaturase is highly specific for the ω-3 fatty acid 18: 3 '', while OtΔ6 also converts the fatty acid 18: 2 ^{Δ9 '12} (Figure 9) ).

Example 11: Detection of Desaturation Products of MsΔ6 and MsΔ5 in the Acyl-CoA Pool

For direct confirmation of the acyl-CoA dependence of MsΔ6 and MsΔ5, the acyl-CoA profiles were determined for the respective yeast expression cultures. Control cultures expressing OtΔ6, PtΔ6 or PtΔ5 were tested in parallel. The data presented were obtained by the method described in Domergue et al. (2005) Biochem. J. 389 (2): 483-490. Exogenous substrates (18: 3 ^Δ9 ' ¹² ' ¹⁵ for Δ6-desaturases or 20: 3 ^Δ8 ' ⁿ ' ¹⁴ for Δ5-desaturases) were added to the induced yeast cultures at an optical density of 1.5 to 2.0. Within five minutes after adding exogenous 18: 3 ^Δ9 ' ¹² ' ¹⁵ substrate to the cultures expressing MsΔ6, OtΔ6 or PtΔ6, 18: 3 ^Δ9 ' ¹² ' ¹⁵ became the new peak in the Total fatty acid pools and in the acyl CoA pool of the respective cultures detected (Fig. 10 A). In addition, a second new peak corresponding to 18: 4 ^A6 ' ⁹ ' ^{12 '15} , the product of Δ6 desaturation, appeared simultaneously in the yeast acyl-CoA pool expressing MsΔ6 and OtΔ6, but not in the acyl-CoA pool of the yeast expressing PtΔ6 (Figure 10 A). The 18: 4 ^Δ6 ' ⁹ ' ^{12 '15} product was not detectable in any of the total fatty acid pool cultures at this early stage and 18: 4 ^Δ6 ' ⁹ ' ¹² ' ¹⁵ appeared in the total fatty acid profiles of the cultures, MsΔ6, OtΔ6 and PtΔ6 expressed, respectively, at times after one hour after addition of 18: 3 ". After one hour, traces of 18: 4 ^Δ6 ' ⁹ ' ^{12 '15} also appeared in the acyl-CoA pool of the yeast expressing PtΔ6 (data not shown). The appearance of the Δ6-desaturase product in the acyl-Co A pool prior to its appearance in the total fatty acid pool suggests that MsΔ6

18: 3 ^Δ9 ' ¹² ' ¹⁵ to 18: 4 ^Δ6 ' ⁹ ' ^{12 '15} in an acyl-CoA dependent manner, which is consistent with the data for OtΔ6 described by Domergue et al. (2005) Biochem. J. 389 (2): 483-490. Conversely, the delayed appearance of the 18: 4 ^Δ6 ' ⁹ ' ^{12 '15} product in the acyl-CoA pool in the expression of PtΔ6 indicates that desaturation by PtΔ6 occurs on fatty acids attached to phospholipids rather than those associated with CoA.

Changes in the total fatty acids and the acyl-CoA pool in the expression of the Δ5-desaturases MsΔ5 and PtΔ5 were analyzed immediately before the addition of exogenous 20: 3 ^Δ8 ' ⁿ ' ¹⁴ substrate and then at 1, 4, 8 and 24 h since these desaturases were not as efficient as the Δ6-desaturases and the Δ5 desaturation products were detectable only at these later times. Within 1 h after addition of exogenous 20: 3 "substrate to cultures expressing MsΔ5 or PtΔ5, 20: 3 ^Δ8 ' ⁿ ' ¹⁴ became the new peak in the total fatty ^acid pools and in the acyl-CoA pools of all Proven cultures. After 4h, a second new peak corresponding to the 20 ^ ⁵ ' ⁸ ' ^{1 lll4} product of Δ5 desaturation appeared, in the total fatty acid pool of the yeast expressing PtΔ5, but not that of the yeast expressing MsΔ5 (Figure 10B). In MsΔ5 cultures, the 20: 4 ^Δ5 ' ⁸ ' ^{π '14} product was undetectable until eight hours after substrate ^addition , when 20: 4''' appeared in the acyl-CoA pool but still was not in the total fatty acid pool detectable (Fig. 10 B). Only at times ^exceeding eight hours after the addition of 20: 3 ^Δ8 ' ⁿ ' ¹⁴ were traces of 20: 4 ^Δ5 ' ⁸ ' ^{n '14} in the total fatty acid pool of yeast expressing MsΔ5 detectable (Figure 10B ).

Example 12: Extension of Desaturation Products of MsΔ6 and MsΔ5

To provide further evidence that MsΔ6 and MsΔ5 are acyl-CoA dependent

Desaturases, the desaturases were individually coexpressed with acyl-CoA elongases. The background of this experiment is that fatty acid extension takes place in the acyl-CoA pool (Domergue et al (2003) J. Biol. Chem. 278: 35115-35126) and that fatty acids desaturated in the acyl-CoA pool be extended more efficiently than those that have been denatured by lipid-dependent enzymes and thereby require additional acyl-transferase activities. The efficiency of the combined desaturations / extensions was compared in the same way to that resulting from the expression of the known desaturases OtΔ6, PtΔ6 or PtΔ5.

The Δ6-desaturases were isolated in yeast with the Δ6-elongase PSE1 from the moss

Physcomitrella patens (Zank et al. (2002) Plant J. 31: 255-268, Fig. 11 A). In the case of MsΔ6 and OtΔ6, the desaturation product 18: 4 ^Δ6 ' ⁹ ' ^{12 '15 was} barely detectable and was nearly 100% extended to the end product of the elongation, 20: 4 ^Δ8 ' ⁿ ' ¹⁴ ' ¹⁷ . The additional elongation ^product , 20: 3 ^Δ11 ' ¹⁴ ' ¹⁷ , corresponds to the fed fatty acid, 18: 3 ^Δ9 ' ¹² ' ¹⁵ , and shows that Δ9-desaturated fatty acids also serve as substrates for PSE1, as before (Zank et al. (2002) Plant J. 31: 255-268). In contrast to the situation with the expression of MsΔ6 PtΔ6 resulted in the accumulation of the Δ6-Desaturierungsprodukts _{18: 4} ^{Δ6,9, i2, i5} _{and a} g _eri ngerer part (about 60%) of 18: 4 ^{Δ6 '9' 12} ' ¹⁵ was extended to 20: 4 ^Δ8 ' ⁿ ' ^{14 '17} . Exogenously added 18: 3 "was better and more efficiently elongated by PSEI than the desaturation product of PtΔ6, which is consistent with the activation of exogenously added fatty acids to acyl-CoA forms.

The Δ5-desaturases MsΔ5 and PtΔ5 were coexpressed in yeast with the OtELO5 Δ5 elongase from O. tauri (Meyer et al. (2004) J Lipid Res. 45 (10): 1899-1909). 20: 4 ^Δ8 ' ⁿ ' ^{14 '17} , the ω3 substrate of the Δ5-desaturases, was added to the expression cultures, since the O. tauri Δ5 elongase omega3 fatty acid substrates is preferred over ω6 substrates (Meyer et al ) J Lipid Res. 45 (10): 1 899-1909). As shown in Fig. 11 B, the desaturation product of MsΔ5, _{20: 5} ^Δ5,8, n ^{, i4, i7} ( _EPA ) _{5 was almost} completely extended to 22: 5 ^Δ7 ' ¹⁰ ' ¹³ ' ¹⁶ ' ¹⁹ (extension ca 90 %), whereas the lipid-dependent desaturase PtΔ5 led to an accumulation of the lipid-bound desaturation intermediates (here 20: 5 ^Δ5 ' ⁸ ' ⁿ ' ¹⁴ ' ¹⁷ (EPA)) (Figure 11 B) and the elongation efficiency was low (about 24%).

Collectively, all data show that both MsΔ6 and MsΔ5 are acyl-CoA-dependent fatty acid desaturases.

Example 13 Establishment of Acyl-CoA-dependent EPA Biosynthesis in Arabidopsis thaliana

The establishment of an acyl-CoA-dependent EPA biosynthesis pathway in yeast has shown that it is possible to circumvent the bottleneck of substrate / product transport between CoA and lipids by exclusively acyl-CoA specific enzyme activities and that in lipid dependent desaturation additionally required enzymatic activities of the acyl-CoA: lysophosphatidylcholineacyltransferases (LPCAT) to avoid. Since yeast is not an 01-accumulating organism and the starting substrate of the reaction sequence is not endogenous nature, but yeast is added in high amounts to the expression system, the experiments described so far serve as a review of the overall concept for the new VLCPUF A biosynthetic pathway.

By establishing the biosynthetic pathway with acyl-CoA-dependent enzyme reactions in Arabidopsis, the described bottleneck of VLCPUF A biosynthesis (Abbadi et al. (2004) Plant Cell 16 (10): 2734-2748) was also to be tested in a plant Organism can be bypassed. For this purpose, vectors for the transgenic expression of the new desaturase enzymes were prepared and transformed by Agrobacterium-mediated gene transfer in Arabidopsis plants.

For plant transformation, plasmids were constructed based on the pUC 19 vector. For the preparation of the constructs, a triple cassette containing three seed-specific USP promoter copies (Bäumlein et al. (1991) Mol. Gen. Gent. 225 (3): 459-67), three OCS terminator copies (MacDonald et al., (1991 ) Nucleic Acids Res. 19 (20): 5575-5581) and three different polylinkers between each promoter and terminator were first introduced into the vector pUC19 (Pharmacia) to give the plasmid USP123OCS. The open reading frames of the various desaturases and elongases were modified by PCR to provide appropriate restriction sites adjacent to the start and stop codons, cloned into the pGEM-T vector (Promega) and sequenced to confirm the correctness. The primers used were (restriction sites shown in bold): MsΔ6, forward 5'-ATGCGCGGCCGCACATAATGTGTCCTCCCAAGGAAT-S 'reverse 5'-GCATTCTAGACTAGTGAGCGTGCGCCTTC-3'; MsΔ5, forward 5'-ATGCCCATGGACATAATGCCCCCGCGCGAGACCACCAC-S 'reverse 5'-GCATACCGGTTCACCCGATGGTTTGAAGGC-S'; PtΔ6, forward 5'-ATGCGCGGCCGCACATAATGGGCAAAGGAGGGGACGC-S 'reverse 5'-GCATTCTAGATTACATGGCGGGTCCATCGCGTA-S'; PtΔ5, forward 5'-ATGCCCATGGACATAATGGCTCCGGATGCGGATAAGC-S 'reverse 5'-GCATGCGATCGCTTACGCCCGTCCGGTCAA-S', PSE1, forward 5'-AGTCGGATCCTATGGAGGTCGTGGAGAGAT-S 'reverse 5'-GACTGCTAGCTCACTCAGTTTT AGCTCCCTT -3'.

The open reading frames were then released using the restriction sites generated by the PCR and then inserted into the same sites of the polylinker of the USP123OCS plasmid. The resulting cassette containing the three genes, each under the control of the USP promoter, was released by digesting the USP123OCS plasmid with Sbfi or SacI and cloned into the appropriate sites of the pCAMBIA3300 binary vector. The binary vector pCAMBIA3300 (CAMBIA, Canberra, Australia) uses the bar gene together with the CaMV 35S promoter as a selection marker in plants. The resulting binary plasmid constructs (Figure 12) were transformed into chemically competent Agrobacterium tumefaciens' cells (strain EH105).

Plants of the Arabidopsis thaliana ecotype Columbia (CoI-O) were transformed by floral dipping (Clough and Bent (1998) Plant J. 16 (6): 735-43.). T2 seeds were collected from single ammonium glufosinate-resistant Tl plants and analyzed individually by GC. For lipid analysis, 10 mg of seed were homogenized in 4 ml of chloroform / methanol / glacial acetic acid (2: 1: 0, v / v / v) and incubated for 24 h at 4 ° C. Seed residues were pelleted (2 min, 3000 xg). The supernatant was collected and the pelleted seed residues were incubated with 2 ml of hexane for 30 min at room temperature. The resulting organic phases were combined and dried under nitrogen. The dried lipids were dissolved in 200 μl of chloroform. The separation of the lipid classes (TAG and various phospholipids) was achieved by thin layer chromatography (TLC) with methanol / chloroform / glacial acetic acid (65: 25: 8) as eluent. The lipids (TAG, PC, PI / PS and PE) were identified according to authentic standards (xy), scraped from the TLC plates and reextracted with the eluent for subsequent fatty acid analysis.

Fatty acid methyl esters (FAMEs) from single or pooled Arabidopsis seeds were prepared by transesterification with trimethylsulfonium hydroxide (TMSH) (Butte et al., 1982, Lett., 15: 841-850). FAMEs of DC-separated individual lipids were prepared by transmethylation with 333 ul of toluene / methanol (1: 2 v / v) and 167 ul 0.5 M NaOCH ₃ in

Room temperature for 20 min. Received. FAMEs were extracted into 500 μl NaCl, 50 μl HCl (37%) and 2 ml hexane, dried under nitrogen and analyzed by GC. GC analysis was performed on an Agilent GC 6890 system coupled to a FID detector equipped with a capillary 122-2332 DB-23 column (30 mx 0.32 mm, 0.5 μm coating thickness ; Agilent). As the carrier gas, helium was used ^"The samples were injected at 220 ⁰ C The temperature gradient was 15O ⁰ C for 1 min, 15O ⁰ C ^(1.... - 200 ⁰ C at 15 ⁰ C Min 1 ml ^{min.)" 1,} 200 ⁰ C -.. 25O ⁰ C at 2 ⁰ C min ^"1 and 25O ⁰ C for 10 min the data were processed using the HP ChemStation Rev. A09.03 FAMEs were identified by appropriate standards.. Many higher plants synthesize 18: 2 'and 18: 3''in their seed oils, but do not incorporate VLCPUFA with a chain length of more than 18 carbon atoms or more than three double bonds in TAG. Therefore, there is considerable interest in enabling the synthesis of omega-3 fatty acids in agronomically interesting oilseed species. To ^produce the omega-3 fatty acid 20: 5 ^Δ5 ' ⁸ ' ⁿ ' ¹⁴ ' ¹⁷ (EPA) in seed oils, it is necessary to incorporate a further elongation and two desaturation steps. To avoid the accumulation of ω6 by-products of desaturation, the enzymes selected should be specific to ω3 substrates. Another limitation to avoid is the limiting acyl chain shuttling between PC and CoA pools described by Abbadi et al. (2004) Plant Cell 16 (10): 2734-2748 was observed.

The acyl-CoA dependent Δ6 and Δ5 desaturases from the M. squamata algae were coexpressed with a Δ6 elongase from the moss P. patens in Arabidopsis plants (referred to as triple Ms). For a corresponding control of the efficiency of the lipid-dependent EPA biosynthesis, the Δ6- and Δ5-desaturases from the diatom P. tricornutum were labeled with the Δ6 elongase from the moss P. patens in Arabidopsis plants (referred to as triple-Pt). according to Abbadi et al. (2004) Plant Cell 16 (10): 2734-2748 coexpressed. The plant transformation constructs encoded each gene under the control of the seed-specific USP promoter (Bäumlein et al (1991) Mol Gen Genet 225 (3): 459-67). The fatty acid analysis of the individual T2 seeds revealed different new fatty acids, which were classified as ω3-18: 4 ^Δ6 ' ⁹ ' ^{12 '15} , ω3-20: 3 ^Δ11 ' ^{14 '17} , ω3- 20: 4 ^Δ8 ' ^{1 U4} ' ¹⁷ and ω3-20: 5 ^Δ5 ' ⁸ ' ⁿ ' ¹⁴ ' ¹⁷ (for triple Ms, see Figure 13A and B) and for ω6-18: 3 ^Δ6 ' ⁹ ' ¹² , ω6- for triple Pt. 20: 3 ^Δ8 ' ⁿ ' ¹⁴ and ω6-20: 4 ^Δ5 ' ⁸ ' ^{n '14} (Fig. 13C). These results show that in omega-3 triple-Ms plants and in triple-Pt plants, both the omega-6 and omega-3 pathways were successfully reconstituted. Among the new fatty acids found in the Triple Ms Arabidopsis seed ω3-20: 4 '''occurred most frequently. In contrast, in the triple-Pt Arabidopsis seeds the most common fatty acids were ω6-18: 3 ^Δ6 ' ⁹ ' ¹² , ω3-20: 3 ^Δ11 ' ¹⁴ ' ¹⁷ and at least the first desaturation product in the omega-3 pathway, 18: 4 '''. The accumulation of both Δ6-desaturated fatty acids in the triple-Pt plants is consistent with the results of Abbadi et al. (2004) Plant Cell 16 (10): 2734-2748 and confirms the presence of a bottleneck in lipid-dependent VLCPUF A biosynthesis.

In contrast, no accumulation of the Δ6 desaturation product 18: 4 ^Δ6 ' ⁹ ' ^{12 '15 was} detected in the triple Ms, since all the 18: 4 ^Δ6 ' ⁹ ' ¹² ' ¹⁵ was produced by the Δ6 elongase with very high efficiency (97% conversion) to the corresponding ω3-20: 4 ^Δ8 ' ⁿ ' ^{14 '17} product extended. These results indicate that the acyl-CoA and PC pool bottleneck has been successfully bypassed by the use of strictly CoA-dependent desaturation. The low levels of ω3-20: 5 (EPA) in the triple Ms plants indicate that the Δ5-desaturase activity of MsΔ5 still limits the accumulation of EPA in this Arabidopsis seed. This result is consistent with results from yeast expression studies where MsΔ5 also showed only low desaturation efficiency.

In another set of experiments, in addition to the above-mentioned triple Ms and triple Pt plants, also Triple Ot Arabidopsis plants producing the Δ6 and Δ5 desaturases from Ostreococcus tαuri and the Δ6 elongase from the Express moss P.pαtens. The cloning was done as described above for the triple-Ms and triple-Pt vectors using the following primers:

OtΔ6, forward 5'-ATGCGCGGCCGCMCMTMATGTGCGTGGAGACGGAAAAT-S 'OtΔ6, reverse 5'-ATGCTCTAGATTACGCCGTCTTTCCGGAGTGT-S' OtΔ5, forward 5'-ATGCCCATGGACATAATGTGGACGCCCGCGCGCGA-S ' OtΔ5, reverse 5'-ATGCACCGGTTCATCCGACGGTTTGGAGGG-S '

The fatty acid profiles of single seeds of the triple-Ms, triple-Ot and triple-Pt plants showed new non-Arabidopsis own fatty acid signals, known as ω3-18: 4 ^Δ6 ' ⁹ ' ^{12 '15} , ω3-20: 4 ^Δ8 ' ⁿ ' ^{14 '17} and EPA could be identified (Figure 14). In addition, ω6-VLCPUFA were identifiable in the Triple-Ot and Triple-Pt plants, namely ω6-18: 3 ^Δ6 ' ⁹ ' ¹² , ω6-20: 3 ^Δ8 ' ⁿ ' ¹⁴ and α> 6- 20: 4 '''. Accordingly, the ω3 biosynthesis pathway could be established in the transgenic triple M plants, since the Δ6-desaturase from M. squamata specifically desaturates the oo3 fatty acid 18: 3 ^Δ9 ' ¹² ' ¹⁵ and thus does not produce ω6 fatty acids. In contrast, both the ω3 and the ω6 biosynthetic pathway could be established in the Triple-Ot and Triple-Pt plants, as the Δ6-desaturases from O. tauri and P. tricornutum both the oo3 fatty acid 18: 3 '' and use the ω6 fatty acid 18: 2 ^{Δ9 '12} as substrate.

The percentage of endogenous fatty acids 18: 2 ^{Δ9 '12} and 18: 3 ^Δ9 ' ^{12 '15} and the newly formed ω3 and 006 VLCPUFA in the total fatty acid content of the transgenic Arabidopsis seeds are shown in Figures 15 and 16. The endogenous fatty acids 18: 2 ^{Δ9 '12} and 18: 3 ^Δ9 ' ^{12 '15} served as starting substrates of the newly introduced VLCPUF A biosynthesis. The percentages of the two fatty acids were the same in all three transgenic approaches (Fig. 15 A and 16 A). The most accumulating newly formed fatty acids in the Triple-Ms and Triple-Ot plants were the elongation products of the Δ6 elongase, ω3-20: 4 ^Δ8 ' ⁿ ' ^{14 '17} and ω3- in the Triple-Ot plants, respectively. 20: 4 ^Δ8 ' ⁿ ' ^{14 '17} and ω6-20: 3 ^Δ8 ' ^{n '14} (Fig. 15 C and Fig. 16 C). In contrast, the Δ6-desaturated fatty acids ω3-l 8: 4 ''' ⁵ and 006-18: 3 ^Δ6 ' ^{9 '12 were} the most ^abundant in the triple-Pt plants (Figure 15B and Figure 16B). The accumulation of the two Δ6-desaturated fatty acids in the triple-Pt plants confirmed the observations already made in linseed plants by Abbadi et al. (2004) and shows that also in Arabidopsis the Transfer of fatty acids between acyl-CoA and lipid pool by LPCAT enzymes represents a bottleneck in lipid-dependent VLCPUF A biosynthesis. By almost complete elongation of ω3-18: 4 ^Δ6 ' ⁹ ' ^{12 '15} or ω6-18: 3 ^Δ6 ' ^{9 '12} to the corresponding C20-VLCPUFAs in the Triple-Ms and Triple-Ot plants, in contrast no or only small amounts of the Δ6-desaturase products are detected for the triple-Pt plants (FIG. 15 B and FIG. 16 B). These results demonstrate that the inefficient transesterification of fatty acids between the site of desaturation (PtdCho) and the site of elongation (CoA) in the lipid-dependent VLCPUF A synthesis by the introduction of an exclusively acyl-CoA dependent VLCPUF A biosynthetic pathway ( Triple-Ms and Triple-Ot) could also be bypassed in the plant fatty acid synthesis.

Table 1. Primers and expression constructs used in the embodiments

Enzyme Yeast Expression Primer used vector restriction site

MsΔ6 pYES2 5'-GGGAATTCATGTGTCCTCCCAAGGAATCCACGAG-S 'ECORI

5'-GGGCGGCCGCCTAGTGAGCGTGCGCCTTCCCGTGC-S 'NotI

MsΔ6 pESC-LEU 5'-ATGCGCGGCCGCACATAATGTGTCCTCCCAAGGAAT-S 'NotI

5'-GCATAGATCTCTAGTGAGCGTGCGCCTTC-S 'BglII

MsΔ5 pESC-TRP 5'-ATGCGGATCCACATAATGCCCCCGCGCGAGACCA-S 'BamHI

5 '-GCATGCTAGCTCACCCGATGGTTTGAAGG-S' Nhel

PtΔ6 pESC-LEU 5'-ATGCGCGGCCGCACATAATGGGCAAAGGAGGGGACGC-S 'NotI

5'-GCATTCTAGATTACATGGCGGGTCCATCGCGTA-S 'BglII

PtΔ5 pESC-TRP 5'-ATGCGCGGCCGCACATAATGGCTCCGGATGCGGATA-S 'NotI

5'-GACTTCTAGATTACGCCCGTCCGGTCAAGGG-S 'BglII

PSE1 pESC-LEU 5'-GGGGATCCATGGAGGTCGTGGAGAGA-S 'BamHI

5'-GCK] CTACJCTCACTCAGTTTTAGCTCCC-S 'Nhel

Explanations of the figures

Figure 1: co6 and ω3 fatty acid biosynthesis

Figure 2: Fatty acid profile of yeast expressing MsΔ6 with the translation initiation sequence ACATA before the ATG start codon.

MsΔ6 activity in the presence of 350 μM 18: 3 ^Δ9 ' ¹² ' ¹⁵ . These analyzes reflect the results of three independent expression experiments.

Figure 3: Fatty acid profile of yeast expressing MsΔ5.

A and C) MsΔ5 activity in the presence of each 350 μM 20: 3 ^Δ8 ' ⁿ ' ¹⁴ and

2Q. ^ ^{Δ8, ii,} 14,17 _These analyzes reflect the results of three independent analyzes

Expression experiments.

B and D) Fatty acid profiles of yeast transformed with the empty vector pESC-TRP, in

Presence of each 350 μM 20: 3 ^Δ8 ' ⁿ ' ¹⁴ and 20: 4 ^Δ8 ' ⁿ ' ^{14 '17} .

Figure 4: Conversion efficiency of MsΔ6 and MsΔ5.

Desaturation of MsΔ6 (A) and MsΔ5 (B) is reported as (product x 100) / (educt + product). Each value is the mean ± standard deviation from three independent experiments.

Figure 5: Distribution of fatty acids produced in yeast cultures expressing MsΔ6 or MsΔ5 on lipid classes.

Yeast cultures expressing the M. squamata DQsaturasm were each supplied with exogenous 18: 3 ^Δ9 ' ¹² ' ¹⁵ or 20: 3 ^Δ8 ' ⁿ ' ¹⁴ and the distribution of the fatty acids to individual lipid classes was analyzed. A) and B) show the results of MsΔ6 expression in yeast. C) and D) show the results of MsΔ5 expression in yeast. The fatty acid produced is expressed in terms of mole percent of total fatty acids in each lipid class (A and C) and in mole percent of total lipid-associated produced fatty acids (B and D). Phosphatidylcholine (PC); Phosphatidylserine (PS); Phosphatidylinositol (PI); Phosphatidylethanolamine (PE); neutral lipids (NL).

FIG. 6: The establishment of an EPA biosynthesis pathway in yeast.

The yeast strain INVScI was either transformed with the empty vector (A) or the various co-expression constructs were supplemented with the fatty acid 18: 3 ^Δ9 ' ¹² ' ¹⁵ (B and C). Cell pellet FAMEs were analyzed by GC.

These analyzes reflect the results of three independent ones

Expression experiments.

FIG. 7: Enrichment of EPA in yeast.

The yeast strain INVScI transformed with the M. sgwαmαta-desaturases together with the P. / ααγε-Δ6-elongase or with the P. tricornutum-DesaturasGri together with the P.pαtens Δ6-elongase, was grown in the presence of 18: 3 ^Δ9 ' ¹² ' ¹⁵ . Cell pellet FAMEs were analyzed by GC. These analyzes reflect the results of three independent expression experiments.

Figure 8: Distribution of stearidonic acid and arachidonic acid in different

Lipid classes.

The distribution of stearidonic acid, the product of Δ6-desaturase, or

Arachidonic acid, the product of Δ5-desaturase, in the various lipid classes was detected in yeast cultures containing MsΔ6 (Δ6-desaturase from M. squαmαtα), OtΔ6 (Δ6-

Desaturase from O. tαurϊ) or PtΔ6 (Δ6-desaturase from P. tricornutum)

(A), or in yeast cultures expressing MsΔ5 or PtΔ5 (B). NL: neutral lipids, PC: phosphatidylcholine, PI / PS: phosphatidylinositol with phosphatidylserine, CL: diphosphatidylglycerol

FIG. 9: Substrate specificity of MsΔ6 and OtΔ6

Yeast cultures were transformed with MsΔ6, OtΔ6 and the corresponding empty vector pYES2 and the FAMEs from the cultures analyzed by GC.

Figure 10: Acyl-Co A dependence of MsΔ6 and MsΔ5

Yeast expression cultures expressing MsΔ6 and control cultures expressing PtΔ6 and OtΔ6 were, respectively, after 5 and 60 minutes after addition of exogenous 18: 3 ^Δ9 ' ¹² ' ¹⁵ substrate with respect to total fatty acids (left) and CoA-bound fatty acids (right) analyzed by GC (A). Yeast expression cultures expressing MsΔ5 (on the left) and control cultures expressing PtΔ5 (on the right, respectively) were observed after 1, 4, 8 and 24 hours, respectively, after addition of exogenous 20: 3 ^Δ8 ' ⁿ ' ¹⁴ substrate with respect to total fatty acids (left) and the CoA-bound fatty acids (right) analyzed by GC (B).

FIG. 11: Coexpression of Δ6- and Δ5-desaturases with acyl-Co A-elongases The desaturases MsΔ6, OtΔ6 and PtΔ6 were in each case coexpressed with the Δ6 elongase PSE1 from the moss P. patens in yeast, the fatty acid 18: 3 ^Δ9 ' ^{12 '15} fed and analyzed the fatty acid composition by GC (A). As a control served a culture that had been transformed with the empty vector pESC-LEU. The desaturases MsΔ5 and PtΔ5 were each coexpressed with the Δ5 elongase OtELO5 from O. tauri in yeast, the fatty acid fed in 20: 4 ^Δ8 ' ^π ' ^{14 '17,} and the fatty acid composition analyzed by GC (B). As a control served a Culture transformed with the empty vectors pESC-TRP and pESC-URA.

FIG. 12: Binary pCAMBIA vectors for the transformation of plants

A) Binary vector with expression cassettes for MsΔ6, PSE1 and MsΔ5 under the control of the USP promoter, the transformation of which led to triple Ms plants.

B) Binary vector with expression cassettes for PtΔ6, PSE1 and PtΔ5 under the control of the USP promoter, the transformation of which led to triple-Pt plants.

LB and RB: left and right T-DNA boundaries; USP: Viciafaba USP promoter; MsΔ6 and PtΔ6: Δ6-desaturases from M. squamata and P. tricornutum, respectively; MsΔ5 and PtΔ5: Δ5-desaturases from M. squamata and P. tricornutum, respectively; PSE1: Δ6 elongase from P. patens; OCS: terminator region of the octopine synthase gene of A. tumefaciens; 35S-Prom: 35S promoter of CaMV; 35S term: CaMV 35S terminator; bar: glufosinate resistance gene; Kan: kanamycin resistance gene

FIG. 13: Fatty acid analysis of Arabidopsis seeds

The amount of fatty acids 18: 2 ^{Δ9 '12} , 18: 3 ^Δ9 ' ^{12 '15} , 20: 3 ^Δ11 ' ^{14 '17} (A), 18: 4 ^Δ6 ' ⁹ ' ¹² ' ¹⁵ ,

₂₀ . ₄ Δ8, II, 14, π ₂₀ . ₅ Δ5,8, ll, 14,17 _(ß) ^^ ^ jg .U ^ ΔS.ll, 14 _and ^. ^ 5,8,11,14 _(Q was in seeds from individual Arabidopsis plants MsΔ6 PSE1 and MsΔ5 (Ms3er-29, Ms3er-2, Ms3er-22) and PtΔ6, PSE1 and PtΔ5 (Pt3er-16, Ot3er-7, Pt3er-24), respectively, were determined by GC, in addition, the mean of the respective fatty acid calculated from the values of the individual seeds (in each case right-most graphic).

FIG. 14: Fatty acid analysis of triple Ms, triple Ot and triple Pt Arabidopsis seeds Fatty Acid Profiles of Single Seed Analyzes of Triple-Ms, Triple-Ot and Triple-Pt Plants. Representative GC fatty acid profiles of single seeds of the transgenic triple Ms (A), triple-Ot (B) and triple-Pt (C) Arabidopsis plants are shown from three to eight independent measurements. The fatty acids were compared and identified by comparison with an authentic standard mixture (D).

FIG. 15: ω3 fatty acid content of transgenic Arαbidopsis plants

Single seed analysis of two or three independent Triple Ms (Triple-Ms-29, Triple-Ms-2, Triple-Ms-22), Triple-Ot (Triple-Ot-2, Triple-Ot-4) and Triple-Ms Pt (Triple-Pt-16, Triple-Pt-7, Triple-Pt-24) Arαbidopsis lines was performed. (A) averages and individual values of seed analyzes of ω3-18: 3 ^Δ9 ' ¹² ' ¹⁵ , (B) the Δ6 desaturation product ω3-18: 4 ^Δ6 ' ⁹ ' ^{12 '15} , (C) the elongation product of the Δ6 elongase, ω3-20: 4 ^Δ8 ' ^π ' ^{14 '17} and (D) the final product of the ω3-VLCPUFA biosynthetic pathway EPA, ω3-20: 5 ^Δ5 ' ⁸ ' ^π ' ^{14 '17} .

Figure 16: ω6-F acid content of transgenic Arαbidopsis plants Single seed analysis of two or three independent Triple-Ot and Triple-Pt Arαbidopsis lines was performed. (A) averages and individual values of seed analyzes of ω6-18: 2 ^{Δ9 '12} , (B) the Δ6 desaturation product ω6-18: 3 ^Δ6 ' ^{9 '12} , (C) the elongation product of the Δ6 elongase, ω6-20: 3 ^Δ8 ' ^π ' ¹⁴ and (D) the final product of the ω6-VLCPUFA biosynthetic pathway AA, ω6-20: 4 ^Δ5 ' ⁸ ' ^{n '14} .

Claims

PATENT APPLICATIONS A process for the preparation of 0) 3 -fatty acids in transgenic organisms, characterized in that it comprises the following process step / s: a) introducing into the organism a nucleic acid sequence which is responsible for a polypeptide having the activity of a Δ6-desaturase selected from the group consisting of a nucleic acid sequence having the sequence shown in SEQ ID NO: 1, nucleic acid sequences which can be derived as the result of the degenerate genetic code of the amino acid sequence shown in SEQ ID NO: 2, and derivatives of SEQ ID NO: 1 which encodes polypeptides having at least 40% identity with SEQ ID NO: 2 at amino acid level and having a Δ6-desaturase activity, and / or b) introducing into the organism a nucleic acid sequence encoding a polypeptide with the activity of a Δ-5-desaturase selected from the group consisting of a nucleic acid sequence having the sequence shown in SEQ ID NO: 3, nucleic acid sequences which can be deduced as a result of the degenerate genetic code from the amino acid sequence shown in SEQ ID NO: 4, and derivatives of the nucleic acid sequence shown in SEQ ID NO: 3, which code for polypeptides which at amino acid level have at least 40% identity with SEQ ID NO: 4 and have Δ5-desaturase activity. 2. A process for the preparation of polyunsaturated fatty acids, in particular of arachidonic acid, eicosapentaenoic acid, stearidonic acid, gamma-linolenic acid, docosapentaenoic acid and / or docosahexaenoic acid in transgenic organisms with a content of at least 1% by weight of these compounds based on the total lipid content of the transgenic organism, characterized in that it comprises the following process step (s): a) introduction of a nucleic acid sequence into the organism which codes for a polypeptide having the activity of a Δ6-desaturase selected from the group from a nucleic acid sequence having the sequence shown in SEQ ID NO: 1, nucleic acid sequences which can be deduced as a result of the degenerate genetic code of the amino acid sequence shown in SEQ ID NO: 2, and derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, the encode polypeptides which have at least 40% identity with SEQ ID NO: 2 at amino acid level and have a Δ6-desaturase activity, and / or b) introducing into the organism a nucleic acid sequence which represents a polypeptide with the activity of Δ5 Desaturase selected from the group consisting of a nucleic acid sequence with the SEQ ID NO: 3 Darge set sequence, nucleic acid sequences which can be deduced as a result of the degenerate genetic code from the amino acid sequence shown in SEQ ID NO: 4, and derivatives of the nucleic acid sequence shown in SEQ ID NO: 3, which code for polypeptides which at least 40% identity at the amino acid level having SEQ ID NO: 4 and have a Δ-5-desaturase activity. The method of claim 1 or 2, further comprising introducing into the organism a nucleic acid sequence encoding a polypeptide having the activity of a Δ6-elongase. The method of claim 3, wherein the polypeptide is derived from P. patens Δ6-elongase activity. The method of any one of the preceding claims, further comprising introducing into the organism a nucleic acid sequence encoding a polypeptide having the activity of a C0-3 desaturase. 6. A method according to any one of the preceding claims, further comprising introducing into the organism a nucleic acid sequence encoding a polypeptide having the activity of a Δ5 elongase, a Δ4-desaturase, a Δ12-desaturase, a Δ- 15-desaturase, a bifunctional Δ-12 and Δ-15 desaturase, a lysophospholipid acyltransferase, a diacylglycerol acyltransferase and / or a phospholipid diacylglycerol acyltransferase. A method according to any preceding claim, wherein the organism is a microorganism, a fungus, a yeast or a plant. 8. The method according to any one of the preceding claims, wherein the organism is an oil-producing plant, vegetable, salad or ornamental plant. 9. The method according to any one of the preceding claims, wherein the organism is a crop selected from the group of plant families consisting of the families of Aceraceae, Actinidiaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Arecaceae, Betulaceae, Boraginaceae, Brassicaceae , Bromeliaceae, Cannabaceae, Cannaceae, Caprifoliaceae, Chenopodiaceae, Convolvulaceae, Cucurbitaceae, Dioscoreaceae, Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Fagaceae, Grossulariaceae, Juglandaceae, Lauraceae, Liliaceae, Linaceae, Malvaceae, Moraceae, Musaceae, Oleaceae, Oxalidaceae, Papaveraceae, Poaceae , Polygonaceae, Punicaceae, Rosaceae, Rubiaceae, Rutaceae, Scrophulariaceae, Solanaceae, Sterculiaceae and Valerianaceae. 10. The method according to any one of the preceding claims, wherein the content of arachidonic acid, eicosapentaenoic acid, stearidonic acid, gamma-linolenic acid, docosapentaenoic acid and / or docosahexaenoic acid in the transgenic organisms at least 4 wt.% Of these compounds based on the total lipid content of the organism. 11. The method according to any one of the preceding claims, characterized in that the arachidonic acid, eicosapentaenoic acid, stearidonic acid, gamma-linolenic acid, docosapentaenoic acid and / or docosahexaenoic acid present in the organism predominantly bound as an ester in phospholipids or triacylglycerides. 12. The method according to any one of the preceding claims, characterized in that the arachidonic acid, eicosapentaenoic acid, stearidonic acid, gamma-linolenic acid, docosapentaenoic acid and / or docosahexaenoic acid from the organism in the form of their oils, lipids or free fatty acids is isolated. 13. The method according to any one of the preceding claims, characterized in that additional additional biosynthesis genes of the fatty acid or lipid metabolism are introduced, selected from the group acyl-CoA dehydrogenase (s), acyl-ACP [= acyl carrier protein] desaturase (n Acyl-ACP thioesterase (s), fatty acid acyltransferase (s), acyl CoA: lysophospholipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase ( n), acyl coenzyme A oxidase (s), fatty acid desaturase (s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, Allene synthases, hydroperoxide lyases or fatty acid elongase (s). 14. Oil, lipids or fatty acids or a fraction thereof, produced by the process according to one of claims 1 to 13. An oil, lipid or fatty acid composition comprising long chain polyunsaturated fatty acids prepared by the process of any one of claims 1 to 13 and derived from transgenic plants. 16. A process for the preparation of oils, lipids or fatty acid compositions by mixing oil, lipids or fatty acids according to claim 14 or oil, lipid or fatty acid composition according to claim 15 with animal oils, lipids or fatty acids. 17. Use of oil, lipids or fatty acids according to claim 14 or oil, lipid or fatty acid compositions according to claim 15 or oils, lipids or fatty acid compositions prepared according to claim 16 in feed, food, cosmetics or pharmaceuticals. 18. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having the activity of a Δ6-desaturase selected from the group consisting of a nucleic acid sequence having the sequence shown in SEQ ID NO: 1, nucleic acid sequences resulting from degeneracy derive genetic codes from the amino acid sequence shown in SEQ ID NO: 2, and derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, which code for polypeptides having at least 68% identity with SEQ ID NO: 2 at the amino acid level and a Δ- Have 6- desaturase activity. 19. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having the activity of a Δ5-desaturase selected from the group consisting of a nucleic acid sequence having the sequence shown in SEQ ID NO: 3, nucleic acid sequences resulting from degeneracy derive genetic code from the amino acid sequence shown in SEQ ID NO: 4, and derivatives of the nucleic acid sequence shown in SEQ ID NO: 3, which code for polypeptides having at least 67% identity with SEQ ID NO: 4 at the amino acid level and a Δ- Have 5- desaturase activity. 20. Nucleic acid molecule according to claim 18 or 19, wherein the sequence is derived from an alga, a fungus, a microorganism, a plant or a non-human animal. 21. An amino acid sequence which is encoded by a nucleic acid sequence according to any one of claims 18 to 20. 22. A gene construct comprising a nucleic acid sequence according to any one of claims 18 to 20, wherein the nucleic acid sequence is operably linked to one or more regulatory signals. 23. A vector containing a nucleic acid sequence according to any one of claims 18 to 20 or a gene construct according to claim 22. 24. Transgenic non-human organism comprising at least one nucleic acid sequence according to any one of claims 18 to 20, a gene construct according to claim 22 or a vector according to claim 23. 25. Transgenic non-human organism according to claim 24, wherein the organism is a plant.