WO2003074716A2 - Methods for the production of unsaturated fatty acids - Google Patents

Abstract

The invention relates to methods for the production of unsaturated fatty acids, preferably conjugated multiple-unsaturated fatty acids such as conjugated linoleic acid (CLA) by transgenic expression of desaturases from insects of the Lepidoptera order. The invention also relates to transgenic expression cassettes for transgenic expression of desaturases from insects of the Lepidoptera order, and to transgenic organisms transformed therewith.

Description

 Process for the production of unsaturated fatty acids

The invention relates to processes for the production of unsaturated fatty acids, preferably conjugated polyunsaturated fatty acids such as conjugated linoleic acid (CLA) by transgenic expression of desaturases from insects of the order Lepidoptera. According to the invention are also transgenic expression cassettes for the transgenic expression of desaturases from insects of the order Lepidoptera, and the transgenic organisms transformed with them.

Fatty acids and triglycerides have a large number of applications in the food industry, animal nutrition, cosmetics and pharmaceuticals. Special ^■ valuable and sought-after unsaturated fatty acids are the so-called conjugated unsaturated fatty acids. Conjugated polyunsaturated fatty acids are rather rare compared to other polyunsaturated fatty acids. Examples of conjugated fatty acids are conjugated linoleic acids (CLA; conjugated linoleic acid), α-parinaric acid (18: 4 octa-decatetraenoic acid), eleostearic acid (18: 3 octadecatrienoic acid), conjugated linolenic acids, di orphecolic acid and calendula acid (see Scheme 1).

Scheme 1: Conjugated polyunsaturated fatty acids

calendic

α-parinaric

α-Eleostearinsäure

α-eleostearic

dimorphecolic

CLA is a collective term for positional and structural isomers of linoleic acid, which are characterized by a conjugated double bond system starting at carbon atom 8, 9, 10 or 11. Some examples are given in Scheme 2. Geometric isomers exist for each of these positional isomers, i.e. cis-cis, trans-cis, cis-trans, trans-trans.

Scheme 2: Four isomers of conjugated linoleic acids

The CLA isomers (9Z, IIE) -CLA and (10E, 12Z) -CLA are known as the biologically active isomers. CLA is mainly found in foods of animal origin. V. A. High levels of CLA are contained in meat and dairy products from ruminants: approx. 3 to 4 mg CLA / g fat in beef and lamb (Chin et al. (1992) J Food Comp Anal 5: 185-197) and approx. 3 to 7 mg CLA / g fat in milk products (Dhi an et al. (1999) J Dairy Sei 82: 2146-56), whereby (9Z, 11E) -CLA with approx. 80% is the main isomer. Higher plants only contain traces of CLA, whereby both biologically active CLA isomers have not yet been found in plants.

A number of positive effects have been demonstrated for CLA, for example the administration of CLA reduces body fat in humans and animals or increases feed turnover in body weight in animals (Park et al. (1997) Lipids 32: 853-858; Park et al. (1999) Lipids 34: 235-241; WO 94/16690; WO 96/06605; WO 97/46230; WO 97/46118). By giving CLA, allergies (WO 97/32008) or cancer (Banni et al. (1999) Carcinogenesis 20: 1019-1024, Thompson et al. (1997) Cancer Res 57: 5067-5072) can also be positive influence. Also an antiartheriosclerotic. Effect of CLA has been demonstrated (Wilson et al. (2000) Nutr Res 20: 1795-1805). Investigations were carried out both with isomers and with mixtures of isomers.

CLA can be produced synthetically via alkaline isomerization of linoleic acid. On an industrial scale, vegetable oils with a high content of linoleic acid are used, eg sunflower oil or safflower oil. Heating above 180 ° C under alkaline conditions catalyzes two reactions:

(1) the fatty acid ester linkages of the triglyceride skeleton are hydrolyzed and the free fatty acids are released.

(2) Unconjugated unsaturated fatty acids with two or more double bonds are conjugated.

Commercially available CLA oils contain a mixture of different CLA isomers as well as other saturated and unsaturated fatty acids. Due to the presence of these biologically inactive and unnatural isomers, complex cleaning of the biologically active isomers (9Z, 11E) CLA and (10E, 12Z) CLA is necessary or it must be shown that the mixture of isomers poses no risk to human or animal health out. So far it has not been possible to produce individual CLA isomers using alkaline isomerization in an economically relevant process. Fractional crystallization enables the isomers (9Z, 11E) -CLA and (10E, 12Z) -CLA to be enriched. However, it is not possible to produce high quality individual isomers in all of the processes mentioned. The reaction products are usually converted into methyl or ethyl esters in the processes mentioned, so that the natural form of the CLA, namely the free fatty acids or the triacylglyceride, is not present.

Biocatalysis of linoleic acid to CLA can overcome these disadvantages of chemical conversion. Various rumen rumen microorganisms, for example, are able to convert linoleic acid to CLA in the process of biohydrogenation. This is done, among other things, by the enzymatic activity of a CLA isomerase. This enzyme activity was described in Butyrivibrio fibrisolvens (Kepler and Tovee (1966) J Biol Chem 241: 1350), Propionibacterium acnes (Deng et al., Ist International Conference on CLA, 2001, Alesund, Norway), Clostridium sporogenes and Lactobacillus reuteri (WO 99/32604; WO 01/00846). The CLA isomerases described so far use free fatty acids as a substrate. The genes coding for CLA isomerase from Lactobacillus reuteri and Propionibacterium acnes were cloned, and the isomerase from Propionibacterium could be functionally expressed in heterologous microorganisms. The biological conversion of linoleic acid to CLA by microorganisms has qualitative advantages over alkaline isomerization, but it is economically disadvantageous due to the fermentation costs and only provides free fatty acids but no triglycerides.

However, free fatty acids have disadvantageous sensory properties and are for use in the food or animal feed sector essentially unsuitable. Subsequent conversion of the free fatty acids - for example glycerol or glycerides with lipase catalysis - is possible, but expensive.

It is not absolutely possible to transfer the processes based on bacterial CLA isomerases to other organisms. So far, it has been demonstrated that CLA isomerases convert free linoleic acid to CLA (Cepler and Tove (1967) J Biochem Chem 242: 5686-5692). In higher organisms, such as ^plant-'zen, linoleic acid predominantly exists in esterified form. Lipids like triacylglycerides represent the storage form, thioesters like acyl-CoA the activated form of the fatty acid.

Fatty acids with tr ns double bonds are extremely rare. Fatty acids with double bonds are present in the trans position in the seed oil of some plants. For example, an E5 fatty acid has been detected in the seed oil of various Thalictrum species (Rankoff et al. (1971) J Amer Oil Chem Soc 48: 700-701). In addition, E5 desaturase activity has been described in Aquilegia vulgaris (Longman et al. (2000) Biochem Soc Trans 28: 641-643). However, no plants have been described which contain either trans-vaccenic acid (ell-octadecenoic acid) or ElO-octadecenoic acid.

It is known that the desaturation of fatty acids in plants can mainly be done by two mechanisms:

(1) In plastids, fatty acid ACP esters are desaturated and preferably at position 9 by a soluble desaturase

(2) At the endoplasmic reticulum, membrane lipids, especially phosphatidylcholines, are further desaturated at positions 6, 12 and 15 by membrane-bound desaturases.

In some plants, conjugated fatty acids are produced by the activity of a conjugase (Crombie et al. (1984) J Chem Soc Chem Corr-mun 15: 953-955; Crombie et al. (1985) J Chem Soc Perkin Trans 1 : 2425-2434; Fritsche et al. (1999) FEBS Letters 462: 249-253; Cahoon et al. (2001) J Biol Chem 276: 2637-2643; Qiu et al. (2001) Plant Physiol 125: 847-855 ). The biosynthesis of conjugated fatty acids such as calendulic acid, eleostearic acid or punicinic acid takes place via the desaturation of oleic acid to linoleic acid by a Δ12 desaturase and a further desaturation, combined with a rearrangement of the Z9 or Z12 double bond to the conjugate fatty acid a specific conjugate-forming desaturase (conjugase). In addition to the production of calendulic acid, Qui et al. (2001) Plant Physiol 125: 847-855) also the production of conjugated linoleic acid by the enzymatic ac- Activity of conjugase described. A disadvantage of this side activity, however, is that the undesired 8,10-isomer of the conjugated linoleic acid is produced by the enzymatic action.

Lepidoptera pheromone desaturases show a wide variety of substrate specificities and desaturation mechanisms (Roelofs and Wolf (1988) J Chem Ecol 14: 2019-2031; Roelofs (1995) Proc Nat Acad Sei USA 92: 44-49; Tillman et al. (1999 ) Insect Biochem 29: 481-514). These enzyme activities result in the production of unusual unsaturated fatty acid CoA derivatives of various chain lengths and with double bonds at different positions and with different configurations. These act as a starting material for the biosynthesis of pheromones. Liu et. al describe an ell desaturase from the pheromone gland of a moth species ("light brown apple moth"), which presumably plays a function in pheromone biosynthesis (Liu WT et al. (2002) Proc Natl Acad Sei USA 99 (2): 620 -624.

The task was therefore to provide new processes which lead to trigylcerides with a high content of unsaturated fatty acids, preferably conjugated polyunsaturated fatty acids such as CLA. This object is achieved by the present invention.

A first subject of the invention relates to processes for the production of trigylcerides comprising unsaturated fatty acids by transgenic expression of at least one fatty acid desaturase from insects of the order Lepidoptera.

In many organisms, including in plants, the fatty acids in the cytosol are mainly present as CoA esters. In yeasts and animals, desaturation of acyl-CoA fatty acids is the main synthetic route for unsaturated fatty acids, whereas this mechanism is rather rare in plants (Cahoon EB et al. (2000) Plant Physiol. 124: 243-251). Surprisingly, it could be shown that the transgenic expression of desaturases from Lepidoptera leads to a desaturation of the saturated and. unsaturated fatty acid CoA ester leads. As a result, both active CLA isomers are ultimately produced and incorporated into the storage lipids.

The process according to the invention has the particular advantage that CLA-containing triglycerides can be produced directly from organisms, preferably plants, with a high oil content, such as rape or sunflower. The use of eukaryotic enzymes from insects of the order Lepidoptera enables one good expression without the toxic effects often associated with prokaryotic proteins.

"Fatty acid desaturases" means enzymes which are capable of introducing a double bond into fatty acids or their derivatives, such as, for example, preferably fatty acid CoA esters. Cofactors such as NADPH, NADH or even oxygen may also be required. Desaturases are preferred which can use acyl-CoA fatty acids as substrates, the fatty acids of which have a chain length of 14, 16, 18 or 20 carbon atoms, preferably 18 carbon atoms.

The term fatty acid desaturases preferably includes those enzymes which are capable of generating a double bond at position C8, C9, CIO, ClI or C12 in fatty acids or their derivatives, such as, for example, preferably fatty acid CoA esters. Desaturases which lead to targeted structural isomers, ie specifically to ice or trans double bonds, are also preferred. Specifically, this means that at least 60%, preferably at least 80%, very particularly preferably at least 90%, most preferably at least 95% of the respective structural isomer is formed. Desaturases which generate a double bond in fatty acids or their derivatives, such as preferably fatty acid CoA esters, as occurs in a CLA isomer, are very particularly preferred.

"Preferred essential property" of a fatty acid desaturase from Lepidoptera means enzymes with at least one of the following properties:

i) Specific generation of a cis double bond in position C8, C9, C10, Cll or C12 or a trans double bond in position C8, C9, C10, Cll or C12.

ii) Substrate specificity for fatty acids, fatty acid CoA esters or other fatty acid derivatives with a chain length of the fatty acid of 16 and / or 18 carbon atoms. Substrate specificity means the property of a fatty acid desaturase to convert substrates with the specified chain length faster than substrates with a different chain length. The speed of rotation of the preferred substrate compared to the non-preferred substrates is increased by at least 50%, preferably at least 100%, very particularly preferably at least 200%, most preferably at least 500%. At least one property from i) or ii) is preferably given in each case.

Fatty acid desaturases include enzymes that can introduce an isolated double bond, but also conjugases that can generate a conjugated double bond system based on a double bond in a substrate. The first double bond is preferably shifted.

For example, conjugases which are preferred

a) convert a ZII double bond into two ElO and Z12 double bonds ("ZU- (ElO, Z12) conjugase"),

b) converting an Ell double bond into two ElO and Z12 double bonds ("express (ElO, Z12) conjugase"),

c) convert a ZlO double bond into two ElO and Z12 double bonds ("Z10 (ElO, Z12) conjugase").

In summary, these enzymes can be referred to as (ElO, Z12) conjugases. These enzymes can also be considered as ElO desaturases. The essential property of the very particularly preferred ElO desaturases is the introduction of a trans double bond in position C-10 of a fatty acid or a fatty acid CoA ester, the fatty acid having a chain length of 18 carbon atoms.

Also preferred are conjugases that

d) convert a ZlO double bond into two Z9 and Eil double bonds ("Z10 (Z9, Eil) conjugase"),

e) converts an ElO double bond into two Z9 and Eil double bonds ("ElO (Z9, express) conjugase")

f) converts a Zll double bond into two Z9 and express double bonds ("ZU- (Z9, express) conjugase").

In summary, these enzymes can be referred to as (Z9, Eil) conjugases. These enzymes can also be considered as ell-desaturases. The essential property of the very particularly preferred Ell desaturases is the introduction of a trans double bond in position C1 of a fatty acid or a fatty acid CoA ester, the fatty acid having a chain length of 18 carbon atoms. Most preferred are ElO-, Eil-, Z10- and Zll-desaturases, as well as ZU- (ElO, Z12) -conjugase, Eil- (ElO, Z12) -conjugase, Z10- (Z9, Eil) -conjugase and ElO- (Z9, express) conjugase with a substrate specificity for fatty acid or fatty acid derivatives such as fatty acid CoA esters with a fatty acid chain length of 18 carbon atoms.

The fatty acid desaturases preferably originate from a Lepidoptera family selected from the group consisting of Acrolepiidae, Agaristidae, Arctiidae, Bombycidae, Carposinidae, Cochylidae, Cossidae, Eriocraniidae, Gelechiidae, Geometridae, Gracillarii-, Lasocomidae, Lyialiidaidae, Lasialididaidae, Lasialampidaidae, Hepialidaidae, Hepialidaidae, Lasialidaidae, Lai triidae, Lyonetiidae, Nepticulidae, Noctuidae, Notodontidae, Nymphalidae, Oecophoridae, Papilionidae, Pieridae, Psychidae, Pterophoridae, Pyralidae, Saturniidae, Sesiidae, Sphingidae, Tortrici- dae and Zyponomidae, Yp. Particularly preferred are fatty acid desaturases from a Lepidoptera family selected from the group consisting of Tortricidae, Pyralidae, Papilionidae, Noctuidae and Geometridae.

Ell desaturases or the nucleic acid sequences coding for them are preferably obtained from organisms of the species Diaphania hyelineata, Diaphania nitidalis, Leucinodes orbonalis, Ostrinia nubilalis, Sesamia grisescens, Brachmia macroscopa, Paraargyresthia japonica, Mhesictena edema flavidalis moza, Argyresthia chamaecypariae, Bradina sp., Dichrocrocis punctiferalis, Cryptoblabes gnidiella, Palpita unionalis, Sceliodes cordalis, Anisodes sp. , Caloptilia theivora, Phyllonorycter ringoniella, Deilephila elpenor, Manduca sexta, Scirpophaga excerpalis, Scirpophaga nivella, Andraca bipunctata or Diatraea saccharalis. The ell desaturases or the nucleic acid sequences coding therefor are particularly preferably isolated from the pheromone glands of the aforementioned insects. Examples include the ell-desaturase from the pheromone glands of the "light brown apple moth" (Epiphyas postvittana) (SEQ ID NO: 2), from Ostrinia nubilalis (SEQ ID NO: 4) and from Ostrinia furnicalis (SEQ ID NO: 6 ) to call. The ell-desaturases from the organisms mentioned adopt the vegetable acyl-CoA fatty acid derivatives as a substrate.

ElO desaturases or the nucleic acid sequences coding for these are preferably obtained from organisms of the species Dichrocrocis chlorophanta, Dichrocrocis punctiferalis, Bombyx mandarina, Bombyx mori, Coloradia velda, Hemileuca eglanterina, Hemileuca electra electra, Hemileuca electra mojavens or Notarcha derogata isolated. The ElO desaturases or the nucleic acid sequences coding therefor are particularly preferably isolated from the pheromone glands of the aforementioned insects. E9-desaturases or the nucleic acid sequences coding for them are preferably obtained from organisms of the species Epiplema plaqifera, Phyllonorycter coryli, Phyllonorycter harrisella, Phyllonorycter sylvella, Gelechiinae, Bryotropha sp., Bryotropha terrella, Gelechia appherice, Oroxairasaporana, Exoxadema sula, Exoxadema sula, Exoxadema sula, Exoxadema sula, Exoxadema sula, Exoxadema s , Loxostege neobliteralis, Ostrinia nubilalis, Dioryctria clarioralis, Dioryctria merkeli, Dioryctria resinosella, Spodoptera exigua, Spodoptera triturata or Polia grandis isolated. The E9 desaturases or the nucleic acid sequences coding therefor are particularly preferably isolated from the pheromone glands of the aforementioned insects.

E8 desaturases or the nucleic acid sequences coding for them are preferably obtained from organisms of the types Phyllonorycter saportella, Phyllonorycter sp. or Dichrocrocis punctiferalis isolated. The E8 desaturases or the nucleic acid sequences coding therefor are particularly preferably isolated from the pheromone glands of the aforementioned insects.

Zll desaturases, such as can be used advantageously for the production of Zll octadecenoic acid, have been described for Bombyx mori (Ando et al. (1988) Agric Biol Chem. 52: 473-478), Trichoplu- sia ni and Helicoverpa zea (Knipple et al. (1998) Proc Nat Acad Sei USA 95: 15287-15292). Further Zll desaturases or the nucleic acid sequences coding for them can preferably be isolated from Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutella xylostella, Bombyx mori or Diaphania nitidalis. The desaturases from Helicoverpa zea (SEQ ID NO: 8), Trichoplusia ni (SEQ ID NO: 10) and Argyrotenia velutinana (SEQ ID NO: 12) may be mentioned as examples. Zll desaturases or the nucleic acid sequences coding therefor are particularly preferably isolated from pheromone glands of the aforementioned insects.

Z10 desaturases or the nucleic acid sequences coding for them are preferred. Organisms of the species Ctenopseustis filicis, Pseudexentera spoliana, Hemileuca eglanterina, Hemileuca electra electra, Hemileuca electra mojavensis, Hemileuca nutleti, Eurhodope advenella, Mamestra configurata or Dichrocrocis punctiferalisgen insulated, particularly preferably from the perniferous genes, especially preferred from the perniferous genes. The desaturase from Planototrix octo (SEQ ID NO: 14) may be mentioned as an example.

(ElO, Z12) conjugases or the nucleic acid sequences coding for them can advantageously be obtained from Bombyx mori, Phyllonorycter crataegella, Amorbia euneana, Notocelia incarnatana, Notocelia udmanniana, Bombyx mandarina, Coloradia velda, Hemileuca eglanterinaile, Hemileuca eglanterina, Hemile electra mojavensis, Hemileuca nuttalli, Notarcha derogata, Rondotia menciana, Amphion floridensis, Hyles gallii, Hyloicus pinastri, Manduca sexta, Sphinx drupiferarum, Earias insulana, Nola confusalis or Notarcha basipunctalis can be isolated. The (ElO, Z12) conjugases or the nucleic acid sequences coding therefor are particularly preferably isolated from the pheromone glands of the aforementioned insects.

(Z9, Eil) conjugases or the nucleic acid sequences coding for them can advantageously be obtained from Diatraea saccharalis, Xyrosaris lichneuta, Dioryctria abietella, Steno a cecropia, Phalonidia manniana, Pelenophorus vilis, Dioryctria abietella; Dioryctria rubella, Myelopsis tetricella, Jodis lactearia, Scopula personata, Spodoptera descoinsi, Spodoptera eridania, Spodoptera latifascia, Spodoptera littoralis or Spodoptera litur can be isolated. The (Z9, Eil) conjugases or the nucleic acid sequences coding therefor are particularly preferably isolated from the pheromone glands of the aforementioned insects.

Both Zll desaturases as well. Zll conjugases take the plant-based aeyl-CoA fatty acid derivatives as a substrate, and the combined expression of these two classes of desaturases from Lepidoptera in plants leads to the production of (10E, 12Z) -CLA, which is incorporated into the storage lipids.

There are various ways of combining the conjugases or desaturases with one another or with plant enzymes in order to produce the desired CLA isomers, in particular (9Z, 11E) -CLA and (10E, 12Z) -CLA. The following methods are to be understood as examples, but not by way of limitation:

1) Starting from stearic acid via trans-desaturation at position C-II - for example by an ell-desaturase - trans-vaccenoic acid can be prepared (Scheme 3).

Δ

Scheme 3: Biosynthetic pathways for the production of (9Z, 11E) -CLA and (10E, 12Z) -CLA from stearic acid

Starting from palmitic acid, .pal-mitelaidic acid (9E-hexadecenoic acid) is produced via trans-desaturation at position C-9 - for example by an E9 desaturase. This can then be carried out via the enzymatic. Elongase activity can be extended to trans-vaccenoic acid (Scheme 4).

fransvaccenicacid

fransvaccenicacid

(E10.Z12) CLA. (29, £ I1) CLA

Scheme 4: Biosynthetic pathways for the production of (9Z, 11E) -CLA and (10E, 12Z) -CLA starting from palmitic acid Trans-vaccenic acid can then be converted from a Δ9-desaturase to the (9Z, 11E) -CLA isomer. For mammals and humans it could be shown that trans-vaccenic acid is converted to CLA by the enzymatic activity of an endogenous Δ9-desaturase (WO 99/20123; Santora JE et al. (2000) J

Nutr 130: 208-215; Adlof RO et al. (2000) Lipids 35: 131-135). It was also shown that insect cells expressing an ell desaturase can convert the resulting ell fatty acid educt into a Z9, EU fatty acid (Liu WT et al. (2002) Proc Natl Acad Sei USA 99 (2) .-. 620-624

Δ9-desaturases can also catalyze the conversion of trans-vaccenic acid to the (Z9, Eil) -CLA isomer. In a particularly preferred embodiment, this conversion can be further increased by additional expression of a Z9 desaturase. Cytosolic-active Z9 desaturases, such as, for example, yeast Z9 desaturase, are preferred. The production of the (Z9, Eil) -CLA isomer in plants is therefore possible through the expression of a trans-desaturase. The particularly advantageous (9Z, 11E) -CLA isomer is formed under the influence of plant Z9 desaturases.

2) Starting from stearic acid, the corresponding ElO octadecenoic acid is first formed via trans-desaturation at position C10 (Scheme 3). Alternatively, starting from palitic acid, trans-desaturation at position C8 can be used to produce E8-hexadecenoic acid. This can then be extended to ElO-octadecenoic acid via the enzymatic activity of an elongase (Scheme 4). This fatty acid is converted to the (ElO, Z12) -CLA isomer u by the activity of a Δ12-desaturase. This reaction is also caused by herbal

Δl2 desaturases catalyzed. In a particularly preferred embodiment, this conversion can be further increased by additional expression of a Z12 desaturase.

3) Corresponding (ElO, 12) conjugases from Lepidoptera can also be used. These convert, for example, Zll-octadecenoic acid (cis-vaccenoic acid), Ell-octadecenoic acid (trans-vaccenoic acid) or ZlO-octadecenoic acid into (ElO, Z12) -CLA. For this purpose, stearic acid is first converted to ZU-octadecenoic acid under the action of a Zll-desaturase, as described above, to trans-vaccenoic acid or by a Z10-desaturase to ZlO-octadecenoic acid. ^■

4) Furthermore, (Z9, EU) conjugases from Lepidoptera can also be advantageously combined with (Zll), (Z10) or (ElO) desaturases. Starting from stearic acid, these desaturases produce ZU-octadecenoic acid (cis-vaccenoic acid), ZlO-octadeceno acid or ElO-octadecenoic acid, which are then converted to (Z9, Eil) -CLA by a (Z9 / E11) conjugase.

The CLA fatty acids or their derivatives such as CoA fatty acid esters thus produced are stored in the membrane lipids and triacylglycerides.

The desirable enzyme activities could be localized in insects of the order Lepidoptera, especially in the insect species mentioned above. The test systems provided in the context of this invention were used for this purpose (Examples 2, 3 and 4).

The genes coding for these activities enzymes or the genes encoding these nucleic acid sequences can be used in the manner familiar to those skilled ^■ isolated from the organisms, or derived by mutagenesis from corresponding known sequences.

Finding a protein sequence or a corresponding cDNA sequence for an enzymatic activity, functionality or phenotype is a classic task in biochemistry and molecular biology. Various methods are known to the person skilled in the art how he can achieve this object. These methods are based, for example, on the test for determining the desaturase or conjugase activity provided in the context of this invention (see, inter alia, Example 3). The specific activity of a desaturase / conjugase is determined by analyzing the fatty acid pattern, for example by gas chromatography as described in Examples 2, 3 or 4. Using this test, it is possible, starting from a biological material in which a desaturase or conjugase activity has been detected, to isolate the corresponding protein or the nucleic acid which codes for a protein with desaturase or conjugase activity and analyze.

The expression cloning method can be used as an example. This method has been used in many cases to isolate the gene responsible for this or the corresponding cDNA on the basis of a certain enzymatic activity, a certain functionality or a certain phenotype (Dalboge H (1997) FEMS Microbiology Reviews 21 (1): 29 -42, Simonsen H and Lodish HF (1994) Trends Pharma- col Sei 15 (12) .437-441). The classic method of expression cloning has been widely described, for example for membrane proteins, secreted factors and transmembrane channels (Masu Y et al. (1987) Nature 329 (6142): 836-838; Wong GG et al. (1985) Science 228 (4701): 810-815; Funny KD et al. (1996) Development 122 (12): 4001-4012; Smith WC and Harland RM (1992) Cell 7.0 (5): 829-840; Lemaire P et al. (1995) Cell 81 (l): 85-94; Gillissen B et al. (2000) Plant Cell 12 (2): 291-300; Lotan T and Hirschberg J (1995) FEBS Letters 364: 125-128).

In the generalized method of expression cloning described as a result, common recombination and cloning techniques are used, as described, for example, in Maniatis T, Fritsch EF and Sambrook J, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989 ) as well as in Silhavy TJ, Berman ML and Enquist LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience.

In general, an expression library can be prepared starting from an organism, cells or tissue which are capable of producing a desaturase or conjugase activity. For example, in the manner familiar to the person skilled in the art, mRNA or preferably poly (A) mRNA is first isolated from said organism, cells or tissue and cDNA is prepared on the basis thereof (Gubler U, Hoffman BJ (1983) Gene 25: 263-269). Various systems for mRNA or poly (A) mRNA isolation are known to the person skilled in the art and are commercially available. For example, the synthesis can be carried out using the "Quick Prep Micro mRNA Purification Kit" (Amersham Pharmacia Biotech). The first strand cDNA synthesis is preferably carried out with an oligo (dT) primer using a reverse transcriptase (Borson ND et al. (1992) PCR Methods Appl 2: 144-148; Chenchik A et al. (1994) CLONTECHniques 9 ( 1): 9-12). Various systems are known and commercially available which enable the generation of full-length cDNAs. ^One example is the "SMART ¹ " cDNA Library Construction Kit "(Clontech, Cat. # K1051-1) which uses SMART ™ technology (SMART = Switch Mechanism At the 5 'end of RNA Templates) to generate cDNA Libraries (Herrler M (2000) J Mol Med 78 (7): B23). Certain oligonucleotides and the method of long-distance PCR ("long-distance PCR") are used here. The method is described in Example 4 ,

Adapters with single-strand overhangs can be ligated to the double-stranded cDNAs and cloned, for example, into an expression vector which has been cut with restriction enzymes and has compatible cohesive ends. The cloning is preferably directed so that the reading direction is fixed. is placed and an optionally present promoter sense-RNA is generated starting from the cDNA insertion. In the SMART ™ system used as an example, compatible Sfil (A) and Sfil (B) overhangs are used for directional cloning into a vector cut with Sfil (recognition sequence GGCCNNNN! GGCC). In each case one cDNA molecule is cloned into a vector molecule, so that ultimately a multiplicity of vectors differing in the integrated cDNA, based on the same basic vector, are present, which form the expression library. This expression library also contains vectors which can express a desaturase or conjugase transgene. In principle, all expression vectors are suitable which are capable of transgenically expressing desaturases or conjugases in an active form in a transformed organism. Examples include the lambda TriplEX2 vector (after Excision pTriplEX2 vector; manufacturer Clontech) for transgenic expression in E. coli, or the vector pYES2 (manufacturer: Invitrogen) for transgenic expression in the yeast S.cerevisiae (see Example 4).

If necessary, normalization can be carried out in order to compensate for differences in the expression level of individual genes, so that the cDNA for each expressed gene is represented in the expression library with a similar number of copies, regardless of the actual expression level. Various systems have been described for the production of poly (A) mRNA, cDNA and normalized cDNA (US 5,482,845) (Soares MB et al. (1994) Proc Natl Acad Sei USA 91: 9228-9232; Carninci P et al. (2000 ) Genome Res 10: 1617-1630; Bonaldo MF et al. (1996) Genome Res 6: 791-806).

Alternatively, you can also create a subtractive expression library. Here one compares the cDNAs of an organism, cells or tissue which has a desaturase or conjugase with the cDNAs of an organism, cells or tissue which does not have this activity and is of the same genus and type if possible. Methods are known to the person skilled in the art how to specifically isolate the cDNAs which are only expressed in the biological material with the desaturase or conjugase activity. Appropriate methods and systems have been described and are commercially available. Examples include the "PCR-Select ™ cDNA Subtraction Kit" (manufacturer: Clontech) or "Subtractor ™ Kit" (manufacturer: Invitrogen), which enables up to 1000-fold enrichment of rare and / or selectively expressed cDNAs. Using subtractive hybridization, all of the cDNAs present in both biological materials are removed (Chu ZL et al. (1997) Proc Nat Acad Sei USA 94: 10057-10062; Hudson C et al. (1997) Cell 91: 397 -405; Mueller CGF et al. (1997) J Exp Med 186: 655-663; von Stein OD et al. (1997) Nucleic Acids Res 25: 2598-2602; Wong BR et al. (1997) J ^' Biol Chem 272: 25190-25194; Yokomizo T et al. (1997) Nature 387: 620-624; Z icheng S and Jacobs-Lorena M (1997) J Biol Chem 272: 28895-28900).

The transgenic expression of the cDNAs contained in the expression library can be realized, for example, in prokaryotic or eukaryotic cells, which are then subjected to a test for the desaturase or conjugase protein, for example a test for a desaturase or conjugase activity.

To isolate the desaturase or conjugase transgenically expressing vectors from the expression library, the expression library is preferably transformed into an organism. Individual cells of the said organism are preferably transformed in such a way that each cell or each organism regenerated from this cell is only transformed with one type of vector molecule. In principle, all organisms or cells derived from them are suitable for the transformation which are capable of transgenically expressing an active desaturase or conjugase. These can be prokaryotic and eukaryotic organisms. Preferred are all plants, cells derived from them, but also other photosynthetic organisms, such as algae. Systems which are based on transgenic expression in bacteria such as E.coli, yeasts such as Saccharomyces or Pichia, mammalian cells such as COS or CHO, or cells of photosynthetically active organs such as Synechocystis may be mentioned here by way of example but not by way of limitation. Preferred are eukaryotic organisms, very particularly preferred are eukaryotic organisms which are able to synthesize fatty acids or aeyl-CoA fatty acids. The expression vectors used for transformation preferably carry a selection marker, for example an antibiotic resistance or an amino acid synthesis gene for selection for amino acid deficiency, one select successfully transformier- ^'allows ter cells. Further methods of selection are described further below.

In a preferred embodiment, the expression cloning can be carried out in yeast. For example, the yeast expression system from Invitrogen based on the vector pYES2 can be used for this purpose. pYES2 is a "high-copy" episomal vector for the inducible expression of recombinant proteins in S. cerevisiae. Transformed yeast strains are selected on the basis of uracil deficiency (pYES2 carries the ura3 gene). Further preferred expression vectors and transfection methods for the production of the transformed organisms correspond to the methods generally used for the production of transgenic organisms.

Based on the total number of transformed organisms or cells derived from them, those are enriched and isolated which are capable of transgenically expressing a desaturase or conjugase and / or have a corresponding desaturase or conjugase activity.

In general, the desaturase activity can be demonstrated by cultivating a transformed organism or cells derived from it, digesting in a suitable buffer or solvent, digesting with fatty acids or aeyl-CoA fatty acids and optionally with a co-factor such as NADH or Brings NADPH or oxygen into contact and detects the resulting dehydrated fatty acid or Aeyl-CoA fatty acids. The fatty acids or aeyl-CoA fatty acids can preferably originate from the transformed organism itself if the organism or the cell derived from it are able to synthesize fatty acids or aeyl-CoA fatty acids themselves. Otherwise, fatty acids or Aeyl-CoA fatty acids can also be added.

The detection of the fatty acid or aeyl-CoA fatty acid modified by the deaturase or conjugase can, if appropriate after extraction from the incubation mixture, for example with a solvent such as ethyl acetate, be carried out using conventional methods known to the person skilled in the art. Separation methods such as high pressure liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (DC) and detection methods such as mass spectroscopy (MS or MALDI), UV spectroscopy or auto-radiography can be used. Detection is preferably carried out by gas chromatography as described in Examples 3, 4 or 5.

The isolation of the transformed organisms or cells derived from them, which are able to transgenically express a desaturase or conjugase, can be done, for example, by dividing the total number of cells into subgroups, cultivating these subgroups separately, if necessary, and isolating the desaturase. or conjugase activity of the individual subgroups. The subgroup in which a cell type is present which transgenically expresses a desaturase or conjugase protein in a functionally active form has an increased desaturase or conjugase activity. This subgroup is further divided and the process is repeated until a monoclonal culture is obtained, ie a culture which contains only a vector with a specific cDNA coding for a desaturase or conjugase.

The vector which contains the nucleic acid coding for the desaturase or conjugase can be recovered from the transformed organism or cell. This can be seen, for example, by means of polymerase chain reaction (PCR). OHgonucleotide primers which are complementary to vector sequences flanking the desaturase or conjugase cDNA insert are used for this purpose. Knowledge of the desaturase or conjugase nucleic acid sequence is not necessary for this. Alternatively, if the vector is not integrated into the host's genome, it can be recovered from the cells, transformed into E. coli, propagated and sequenced to. to obtain the DNA encoding the desaturase ^'.or conjugase nucleic acid sequence. Also be isolated from the vector, the desaturase or conjugase nucleic acid sequence according to above-described procedure without knowledge of its sequence using PCR result in isolation ^'. After the PCR reactions, additional linkers can be added to the amplicon, which enable directional cloning. However, the cloning can also be carried out by means of a "blunt-end" ligation or T-overhang ligation in a manner familiar to the person skilled in the art. The cDNA of desaturase or conjugase selectively amplified by means of PCR can thus be cloned directly into a vector which is suitable for producing a transgenic organism expressing a desaturase or conjugase, even without prior sequence elucidation. This cloning can be carried out, for example, as a "blunt end" cloning using techniques familiar to the person skilled in the art. Common recombination and cloning techniques are used, as cited above.

The expression cloning described above can particularly preferably be carried out in the yeast Saccharomyces cerevisiae. This can include systems are used, in which the transgene be integrated to ^"expressing cDNAs by homologous recombination in a generated integration platform (Lagarde D et al (2000) Applied and Environmental Microbiology 66 (1):. 64-72).

Alternatively, the expression cloning can also be carried out in vitro using a non-amplified expression library as described above. Corresponding systems have been described and are commercially available. The “TNT ™ Coupled Reticulocyte Lysate System” (Promega; Promega Notes Number 67, 1998, p. 02) may be mentioned as an example. Kirschner et al. have developed an in vitro approach (IVEC = "in vitro expression cloning") that works without living cells (US 5,654,150; King RW et al. (1997) Science 277, 973). The system has been successfully applied primarily to enzymes and kinases. Kirschner et al. have identified kinase substrates and proteases (Lustig KD et al. (1997) Meth Enzymol 283: 83; Stukenberg PT et al. (1997) Curr Biol 7: 338; Kothakota S et al. (1997) Science 278: 294). "In vitro expression cloning" (IVEC) uses small fractions from the expression library. These fractions of approx. 50 to 100 clones of cDNAs each are contained in plasmids and are translated into their corresponding proteins by means of a coupled in vitro transcription / translation system based on reticolocyte lysate. For this, an oligo (dT) -primed cDNA library is constructed as described above and cloned into a high copy expression plasmid with a T3, T7 or SP6 promoter. This plasmid library is then transformed into E. coli. Approx. 50 to 100 independent transformants are cultivated on an agar plate with the appropriate selection antibiotic up to a colonial size of approx. 1 mm, collected and combined. Plasmid DNA is isolated from some of these bacteria. This plamid DNA is transcribed and translated as a template directly in a reticolocyte system. Details of the procedure can be found, for example, in the manufacturer's manual (TNT ™ Coupled Reticulocyte Lysate Systems Technical Bulletin # TB126, Promega Corporation) ". Depending on the number of" full-length "cDNA clones in the library, approximately 30 to 50 Pro The proteins produced are checked for their desaturase or conjugase activity. Individual batches which have an increased desaturase or conjugase activity are further subdivided. Subdivision of the individual batches leads to the cDNA coding for a desaturase or conjugase. The nucleic acid coding for the desaturase or conjugase can be amplified from the vectors contained in this preparation by means of PCR and - if necessary without knowledge and analysis of the sequence - used as described above for the production of an organism expressing a desaturase or conjugase transgenically.

As an alternative to the rabbit reticolocyte extract, wheat germ extract can also be used for the combined transcription / translation (for example using the TNT ™ Wheat Germ Extract System from Clontech).

In a further advantageous application, the cDNAs can be cloned into a retroviral vector. This allows a highly efficient transformation of the cells. Retroviral expression vectors and expression systems are described (Kitamura T, International Journal of Hematology 1998, 67: 351-359) and are commercially available (for example from Clontech; New Retroviral & ClonCapture Expression Libraries; CLONTECHniques October 1998, XIII (4): 22-23). The transfection is first carried out in a packaging cell line (for example EcoPack ™ -293 or RetroPack ™ PT67 Cell Line from Clontech). With the viruses generated in this way, the corresponding target cell line can be transfected and selected for the desired activity. The inserts can then be obtained, for example, by PCR and analyzed. Furthermore, they can be cloned directly into a suitable expression vector, even without prior sequence analysis, as described above, which can then be used to produce a desaturase or conjugase transgenically expressing organism.

ElO or Ell desaturases can be obtained by changing known cis-desaturases via mutagenesis in such a way that they are specific for the desaturation in the trans position. For example, the following nucleic acid sequences coding for cis-desaturases can be subjected to mutagenesis as starting sequences:

i) a nucleic acid sequence coding for a Zll desaturase .. from Helicoverpa zea with SEQ ID NO: 7,

ii) a nucleic acid sequence coding for a Zll desaturase from Trichoplusia ni with the SEQ ID NO: 9,

iii) a nucleic acid sequence coding for a Zll desaturase from Argyrotaenia velutinana with the SEQ ID NO: 11 or

iv) a nucleic acid sequence coding for a ZlO desaturase from Planotortrix octo with SEQ ID NO: 13.

Ell desaturases can also be modified by mutagenesis in such a way that longer-chain fatty acids can be converted better or at all. For example, this can be used as the starting sequence

a) a nucleic acid sequence coding for an ell desaturase ^• from Epiphyas postvittana with SEQ ID NO: 1,

b) a nucleic acid sequence coding for an ell desaturase from Ostrinia nubilalis with the SEQ ID NO: 3, or

c) a nucleic acid sequence coding for an ell desaturase from Ostrinia furnacalis with SEQ ID NO: 5. It is also conceivable to change the specificity for the position at which the double bond is introduced by the above-mentioned desaturases by mutagenesis.

A method for changing the properties such as substrate specificity of desaturases by mutagenesis is known (Cahoon et al. (1997) Proc Nat Acad Sei USA 94: 4872-4877; WO 98/06735). Other suitable methods are described and transferable for other enzymes of lipid metabolism such as lipoxygenases (Hornung et al. (2000) Biochem Soc Trans 28: 825-826; Schwarz et al. (2001) J Biol Chem 276: 773-779).

Nucleic acid sequences coding for desaturases / conjugases for use in the method according to the invention can also be isolated from cDNA preparations - or banks of the corresponding above-mentioned Lepidoptera species - by means of a polymerase chain reaction using appropriate degenerate oligonucleotide primers. The oligonucleotide-primer pair described by SEQ ID NO: 15 and 16 or that of the oligonucleotide-primer pair described by SEQ ID NO: 17 and 18 are preferably used.

The nucleic acid sequence of the fatty acid desaturase from Lepidptera used in the method according to the invention is preferably characterized in that it

a) contains in its sense strand a sequence motif described by SEQ ID NO: 15 or 17, or

b) contains a sequence motif described by SEQ ID NO: 16 or 18 in its antisense strand.

In a further preferred embodiment, the protein sequence of the fatty acid desaturase from Lepidptera used in the process according to the invention is preferably characterized in that it has a homology of at least 65%, preferably at least 70%, particularly preferably at least 80%, very particularly preferably at least 90 % of one of the fatty acid desaturases described by SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14 and - optionally and preferably - has at least one of the preferred essential properties of a desaturase or conjugase.

The proteins identified in this way can also include natural or artificial mutations of one of the abovementioned desaturase nucleic acid sequences and their homologues from other animal or plant genera and species. Mutations include substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues. Where insertions, deletions or substitutions, such as transitions and transversions, can be used, techniques known per se, such as in vitro mutagenesis, "primer repair", restriction or ligation can be used. Manipulations such as restriction, "chewing back" or filling in overhangs for "blunt ends" can provide complementary ends of the fragments for the ligation. Analogous results can also be obtained using the polymerase chain reaction (PCR) using specific oligonucleotide primers.

In a further preferred embodiment, the nucleic acid sequence coding for the fatty acid desaturase from Lepidptera used in the method according to the invention is preferably characterized in that it has a homology of at least 65%, preferably at least 70%, particularly, preferably at least 80, very particularly preferably has at least 90% of one of the nucleic acid sequences described by SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and - optionally and preferably - codes for a protein which has at least one of the preferred essential properties of a desaturase or conjugase.

Homology between two nucleic acids or polypeptides is understood to mean the identity of the nucleic acid sequence over the entire entire length of the sequence, which can be determined by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA). is calculated by setting the following parameters:

Gap Weight: 12 Length Weight: 4

Average Match: 2,912 Average Mismatch: -2, 003

In a further preferred embodiment, the nucleic acid sequence coding for the fatty acid desaturase from Lepidptera used in the method according to the invention is preferably characterized in that, under standard conditions, it contains one of the above-mentioned nucleic acid sequences coding for desaturases, preferably with the sequence according to SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 hybridized.

Standard hybridization conditions are to be understood broadly and mean stringent as well as less stringent hybridization conditions. Such hybridization conditions are described, inter alia, in Sambrook J, Fritsch EF, Maniatis T et al., In Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. described. By way of example, the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with approximately 2X SSC at 50 ° C.) and those with high stringency (with approximately 0.2X SSC at 50 ° C., preferably at 65 ° C. ) (20X SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). Furthermore, the temperature during the washing step can be raised from low stringent conditions at room temperature, about 22 ° C, to more stringent conditions at about 65 ° C. Both parameters, salt concentration and temperature, can be varied simultaneously, one of the two parameters can also be kept constant and only the other can be varied. Denaturing agents such as formamide or SDS can also be used during hybridization. In the presence of 50% formamide, the hybridization is preferably carried out at 42 ° C. Some exemplary conditions for hybridization and washing step are given as a result:

(1) Hybridization conditions with, for example

a) 4X SSC at 65 ° C, b) 6X SSC at 45 ° C, c) 6X SSC at 68 ° C, 100 μg / ml denatured fish sperm DNA, d) 4X SSC, 50% formamide, at 42 ° C, e) 2X or 4X SSC at 50 ° C (weakly stringent condition), or f) 2X or 4X SSC, 30 to 40% formamide, at 42 ° C (weakly stringent condition).

(2) washing steps with for example

a) 0.1X SSC at 65 ° C, or b) 0.1X SSC, 0.5% SDS at 68 ° C, or c) 0.1X SSC, 0.5% SDS, 50% formamide at 42 ° C , or d) 0.2X SSC, 0.1% SDS at 42 ° C, or e) 2X SSC at 65 ° C (weakly stringent condition).

In the context of this invention, “nucleic acid sequence” means, for example, a genomic or a complementary DNA (cDNA) sequence or an RNA sequence and semisynthetic or fully synthetic analogues thereof. These sequences can be in linear or circular form, extrachromosomal or integrated into the genome. The nucleotide sequences of the expression cassettes or nucleic acids according to the invention can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA constituents, and consist of different heterogeneous gene segments from different organisms. In addition, artificial nucleic acid sequences are suitable as long as they have the have the desired essential property. For example, synthetic nucleotide sequences can be generated with codons which are preferred by plants to be transformed. These codons preferred by plants can be determined in the usual way on the basis of the codon usage from codons with the highest protein frequency. Coding nucleotide sequences which have been obtained by back-translating a polypeptide sequence according to the codon usage specific for the host plant are particularly suitable. In order to avoid undesired plant regulatory mechanisms, one can, for example, start from the amino acid sequence of a desaturase from Lepidoptera insects, taking back into account the plant codon usage, retranslate DNA fragments and use them to produce the complete exogenous desaturase sequence optimized for use in the plant.

All of the above-mentioned nucleotide sequences can be produced in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix. The chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). When preparing a nucleic acid construct, various DNA fragments can be manipulated so that a nucleotide sequence with the correct reading direction and reading frame is obtained. To connect the nucleic acid fragments to one another, adapters or linkers can be attached to the fragments. The addition of synthetic oligonucleotides and the filling of gaps with the aid of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning methods are described in Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.

With regard to, for example, a nucleic acid sequence, an expression cassette, a vector or an organism, “transgene” means all such constructions which have been obtained by genetic engineering methods or their use, in which either

a) the nucleic acid sequence coding for a desaturase,. or

b) a genetic control sequence functionally linked to the nucleic acid sequence coding for a desaturase, 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, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment means the natural chromosomal locus in the organism of origin or the 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 preserved. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1000 bp, very particularly preferably at least 5000 bp. A naturally occurring expression cassette - for example the naturally occurring combination of a gene sequence coding for a desaturase with its natural promoter - becomes a transgenic expression cassette if it is changed by non-natural, synthetic ("artificial") methods such as, for example, mutagenization becomes. Corresponding methods are described (US 5,565,350; WO 00/15815; see also above).

Transgenic expression means the use of a transgenic expression cassette for the expression of a nucleic acid sequence.

The invention further relates to transgenic expression cassettes which contain a nucleic acid sequence coding for a desaturase, and to vectors comprising these expression cassettes.

In said transgenic expression cassettes, a nucleic acid molecule coding for a desaturase is preferably functionally linked to at least one genetic control element (for example a promoter) which transgenic expression (transcription and / or translation) of said desaturase in an organism, preferably in plants , guaranteed. If the transgenic expression cassette is to be introduced directly into the plant and the desaturase is to be expressed in plantae, plant-specific genetic control elements (for example promoters) are preferred. However, desaturase can also be expressed in other organisms or in vitro. In this, all prokaryotic or eukaryotic genetic control elements (for example promoters) are preferred which allow expression in the organism chosen in each case for the production. A functional link is understood to mean, for example, the sequential arrangement of a promoter with the desaturase nucleic acid sequence to be expressed and, if appropriate, further regulatory elements such as a terminator such that each of the regulatory elements can perform its function in the transgenic expression of the nucleic acid sequence. This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as, for example, enhancer sequences, can also exert their function on the target sequence from positions which are further away or even from other DNA molecules. Arrangements are preferred in which the nucleic acid sequence to be expressed transgenically is positioned behind the sequence which acts as a promoter, so that the two sequences are covalently linked to one another. The distance between the promoter sequence and the nucleic acid sequence to be expressed transgenically is preferably less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.

The production of a functional link as well as the production of a transgenic expression cassette can be realized by means of common recombination and cloning techniques, as described for example in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Ausubel FM et al. (1987) Current Protocols in Molecular ^' Biology, Greene Publishing Assoc. and Wiley Interscience and Gelvin et al. (1990) In: Plant Molecular Biology Manual. However, further sequences can also be positioned between the two sequences, which for example have the function of a linker with certain restriction enzyme interfaces or a signal peptide. The insertion of sequences can also lead to the expression of fusion proteins. The transgenic expression cassette, consisting of a linkage of promoter and nucleic acid sequence to be expressed, can preferably be integrated in a vector and inserted into a plant genome by, for example, transformation.

However, a transgenic expression cassette should also be understood to mean constructions in which the nucleic acid sequence coding for a desaturase type is placed behind an endogenous promoter such that the expression of the desaturase takes place under the control of the endogenous promoter. The fusion of endogenous promoter and desaturase nucleotide resulting from the insertion Acid sequence is a transgenic expression cassette in the sense of the invention.

Plant-specific promoters basically means any promoter that can control the expression of genes, in particular foreign genes, in plants or plant parts, cells, tissues or cultures. The expression can, for example, be constitutive, inducible or development-dependent. Preferred are:

a) Constitutive promoters

Vectors that allow constitutive expression in plants are preferred (Benfey et al. (1989) EMBO J 8: 2195-2202). “Constitutive” promoter means those promoters which ensure expression in numerous, preferably all, tissues over a relatively long period of plant development, preferably at all times during plant development. A plant promoter or a promoter derived from a plant virus is preferably used in particular. Particularly preferred is the promoter of the 35S transcript of the CaMV cauliflower mosaic virus (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewaker et al. (1985) Virology 140 : 281-288; Gardner et al. (1986) Plant Mol Biol 6: 221-228) or the 19S CaMV promoter (US 5,352,605; WO 84/02913; Benfey et al.

(1989) EMBO J 8: 2195-2202). Another suitable constitutive promoter is the "Rubisco small subunit (SSU)" promoter (US 4,962,028), the LeguminB promoter (GenBank Acc. No. X03677), the promoter of nopaline synthase from Agrobacterium, the TR double promoter, the OCS (Octopin Synthase) promoter from Agrobacterium, the Ubi uitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649), the Ubiquitin 1 promoter (Christensen et al. (1992) Plant Mol Biol 18: 675-689; Bruce et al. (1989) Proc Natl Acad Sei USA 86: 9692-9696), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter

(US 5,683,439), the promoters of the vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat (WO 91/13991), as well as further promoters of genes whose constitutive expression in plants is known to the person skilled in the art.

b) Tissue-specific promoters

Also preferred are promoters with specificities for the anthers, ovaries, flowers, leaves, stems, roots and seeds. All specific promoters such as the promoter of the phaseoline (US 5,504,200; Bustos MM et al. (1989) Plant Cell 1 (9): 839-53), of the 2S albuming gene (Joseffson LG et al. (1987) J Biol Chem 262: 12196-12201), des Legu ins (Shirsat A et al. (1989) Mol Gen Genet 215 (2): 326-331), the USP (unknown seed protein; Bäumlein H et al. (1991) Mol Gen Genet 225 ( 3): 459-67), the Napin gene (US 5,608,152; Stalberg K ^" et al. (1996) L Planta 199: 515-519), the sucrose binding protein (WO 00/26388) or the legumin B4 promoter (LeB4; Baumlein H et al. (1991) Mol

Gen Genet 225: 121-128; Baeumlein et al. (1992) Plant Journal 2 (2): 233-9; Fiedler U et al. (1995) Biotechnology (NY) 13 (10): 1090f), the oleosin promoter from Arabidopsis (WO 98/45461), the Bce4 promoter from Brassica (WO 91/13980). Further suitable seed-specific promoters are those of the genes coding for "high molecular weight glutenin" (HMWG), gliadin, branching enzyme, ADP glucose pyrophosphatase (AG-Pase) or starch synthase. Also preferred are promoters that allow seed-specific expression in monocots such as corn, barley, wheat, rye, rice, etc. The promoter of the lpt2 or lptl gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, the glutelin gene, the oryzine gene, etc.) can be used advantageously Prolamin gene, glidine gene, glutelin gene, zein gene, kasirin gene or secalin gene).

Tuber-, storage root- or root-specific promoters such as the patatin promoter class I (B33), the promoter of the cathepsin D inhibitor from potato.

Leaf-specific promoters such as a promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (s all subunit) from Rubisco (ribulose-1,5-bisphosphate carboxylase) or the ST-LSI promoter from potato (Stockhaus et al. (1989) EMBO J 8: 2445-2451).

Flower-specific promoters such as the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593).

- Anther-specific promoters such as the 5126 promoter (US 5,689,049, US 5,689,051), the glob-1 promoter and the γ-zein promoter. c) Chemically inducible promoters

The transgenic expression cassettes can also contain a chemically inducible promoter (review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol

48: 89-108), by which the expression of the exogenous gene in the plant can be controlled at a particular point in time. Such promoters, e.g. the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), promoter induced by salicylic acid (WO 95/19443), a promoter induced by benzenesulfonamide (EP 0 388 186), one by Tetra - Cyclin-inducible promoter (Gatz et al. (1992) Plant J 2: 397-404), an abscisic acid-inducible promoter (EP 0 335 528) or an ethanol- or cyclohexanone-inducible promoter (WO 93 / 21334) can also be used.

d) stress or pathogen inducible promoters

Also preferred are promoters that are induced by biotic or abiotic stress, such as the pathogen-inducible promoter of the PRPL gene (Ward et al. (1993) Plant Mol Biol 22: 361-366), the heat-inducible hsp70 or hsp80 promoter from tomato (US Pat. No. 5,187,267), the cold-inducible alpha-amylase promoter from the potato (WO 96/12814), the light-inducible PPDK promoter or the wound-induced pinII promoter (EP375091).

Pathogen-inducible promoters include those of genes induced by pathogen attack such as genes from PR proteins, SAR proteins, β-1, 3-glucanase, chitinase, etc. (e.g. Redolfi et al. (198.3) Neth J Plant Pathol 89: 245-254; Uknes, et al. (1992) The Plant Cell 4: 645-656; Van Loon (1985) Plant Mol Viral 4: 111-116; Marineau et al. (1987) Plant Mol Biol 9: 335-342; Matton et al.

(1987) Molecular Plant-Microbe Interactions 2: 325-342; Soms- sich et al. (1986) Proc Natl Acad Sei USA 83: 2427-2430; Soms- sich et al. (1988) Mol Gen Genetics 2: 93-98; Chen et al. (1996) Plant J 10: 955-966; Zhang and Sing (1994) Proc Natl Acad Sei USA 91: 2507-2511; Warner, et al. (1993) Plant J

3: 191-201; Siebertz et al. (1989) Plant Cell 1: 961-968 (1989).

Also included are wound-inducible promoters such as that of the pinll gene (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498), the wunl and wun2 genes (US 5,428,148), the winl and win2 genes (Stanford et al. (1989) Mol Gen Genet 215: 200-208), the systemin (McGurl et al. (1992) Science 225: 1570-1573), the WIPl gene (Rohmeier et al. (1993) Plant Mol Biol 22: 783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73- 76), the MPI gene (Corderok et al. (1994) The Plant J 6 (2): 141-150) and the like.

e) Development-dependent promoters

Further suitable promoters are, for example, fruit-ripening-specific promoters, such as, for example, the fruit-ripening-specific promoter made from tomato (WO 94/21794, EP 409 625). Development-dependent promoters partly include the tissue-specific promoters, since the formation of individual tissues naturally development dependent.

Further promoters suitable for expression in plants are described ^' (Rogers et al. (1987) Meth in Enzymol 153: 253-277; Schardl et al. (1987) Gene 61: 1-11; Berger et al. (1989) Proc Natl Acad Sei USA 86: 8402-8406). Constitutive and seed-specific promoters are particularly preferred.

Furthermore, other promoters can be functionally linked to the nucleic acid sequence to be expressed, which enable expression in other plant tissues or in other organisms, such as E. coli bacteria. In principle, all promoters described above can be used as plant promoters.

The nucleic acid sequences contained in the transgenic expression cassettes or vectors according to the invention can be functionally linked to further genetic control sequences in addition to a promoter. The concept of genetic control sequences. Zen is to be understood broadly and means all those sequences which have an influence on the formation or the function of the transgenic expression cassette according to the invention. Genetic control sequences modify, for example, transcription and translation in prokaryotic or eukaryotic organisms. Preferably, the inventive transgene expression cassette 5 'include-upstream of the respective transgenically ^■ expressing the nucleic acid sequence the promoter with specificity for the embryonal epidermis and / or the flower and 3' downstream, sequence, a terminator sequence as additional genetic control, and, where appropriate, further customary regulatory Elements, in each case functionally linked to the nucleic acid sequence to be expressed transgenically.

Genetic control sequences also include further promoters, promoter elements or minimal promoters that can modify the expression-controlling properties. Genetic control sequences, for example, allow tissue-specific express sion depending on certain stress factors. Corresponding elements are, for example, for water stress, abscisic acid (Lam E and Chua NH, J Biol Chem 1991; 266 (26): 17131 -17135) and heat stress (Schoffl F et al., Molecular & General Genetics 217 (2-3 ): 246-53, 1989).

Further advantageous control sequences are, for example, in the gram-positive promoters amy and SP02, in the yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.

In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the method according to the invention. In addition, synthetic promoters can also be used advantageously.

Genetic control sequences also include the 5 'untranslated regions, introns or non-coding 3' regions of genes such as the actin-1 intron, or the Adhl-S introns 1, 2 and 6 (general: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). It has been shown that these can play a significant role in regulating gene expression. It has been shown that 5 'untranslated sequences can increase the transient expression of heterologous genes. The 5 'leader sequence from the tobacco mosaic virus may be mentioned as an example of translation amplifiers (Gallie et al. (1987) Nucl Acids Res 15: 8693-8711) and the like. They can also promote tissue specificity (Rouster J et al. (1998) Plant J 15: 435-440).

The transgenic expression cassette can advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable increased transgenic expression of the nucleic acid sequence. Also at the 3 'end of the nucleic acid sequences to be expressed transgenically before additional advantageous sequences may also be inserted, such as further regulatory ^elements' or terminators. The nucleic acid sequences to be expressed transgenically can be contained in one or more copies in the gene construct.

Polyadenylation signals suitable as control sequences are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium turne faciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al. (1984) EMBO J 3: 835 ff) or functional equivalents thereof. Examples of loading particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopalin synthase) terminator.

Control sequences are also to be understood as those which enable homologous recombination or insertion into the genome of a host organism or which allow removal from the genome. Methods such as cre / lox technology allow tissue-specific, possibly inducible removal of the transgenic expression cassette from the genome of the host organism (Sauer B (1998) Methods. 14 (4): 381-92). Here certain flanking sequences are added to the target gene (lox sequences), which later enable removal using the cre recombinase.

A transgenic expression cassette and the vectors derived from it can contain further functional elements. The term functional element is to be understood broadly and means all those elements which have an influence on the production, reproduction or function of the transgenic expression cassettes, vectors or organisms according to the invention. Examples include, but are not limited to:

a) Selection markers which are resistant to a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456), antibiotics or biocides, preferably herbicides, such as, for example, kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc. lend. Particularly preferred selection markers are those which confer resistance to herbicides. Examples include: DNA sequences that code for phosphinothricin acetyltransferases (PAT) and inactivate glutamine synthesis inhibitors (bar and pat gene), 5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes) that are resistant to Glyphosat® (N - (phosphono ethyl) glycine), the gox gene (glyphosate oxidoreductase) coding for the glyphosate® degrading enzymes, the deh gene (coding for a dehalogenase which inactivates dalapon), sulfonylurea and imidazolinone inactivating acetolactate synthases and bxn genes encoding bromoxynil-degrading nitrilase enzymes, the aasa gene conferring resistance to the antibiotic apectinomycin, the streptomycin phosphotransferase (SPT) gene conferring resistance to streptomycin, the neomycin phosphotransferas (NPTII) gene, the one Resistance to kanamycin or geneticidin gives the hygromycin phosphotransferase (HPT) gene, which avoids resistance to hygromycin ttelt, the acetolactate synthas gene (ALS), which confers resistance to sulfonylurea herbicides (eg mutated ALS variants with eg the S4 and / or Hra mutation).

b) Reporter genes which code for easily quantifiable proteins and which, by means of their own color or enzyme activity, ensure an assessment of the transformation efficiency or the expression site or time. Reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1).-29-44) such as "green fluorescence protein" (GFP) (Sheen et al. ( 1995) Plant Journal 8 (5): 777-784;

Haseloff et al. (1997) Proc Natl Acad Sei USA 94 (6). -2122-2127; Reichel et al. (1996) Proc Natl Acad Sei USA 93 (12).-5888-5893; Tian et al. (1997) Plant Cell Rep 16: 267-271; WO 97/41228; Chui WL et al. (1996) Curr Biol 6: 325-330; Leffel SM et al. (1997) Biotechniques. 23 (5): 912-8), chloramphenicol transferase, a luciferase (Ow et al. (1986) Science 234: 856-859; Millar et al. (1992) Plant Mol Biol Rep 10: 324-414), the aequoringen (Prasher et al. (1985) Biochem Biophys Res Commun 126 (3): 1259-1268), the ß-galactosidase, R-locus gene (encode a protein that inhibits the production of antihoanine pigments (red color) regulated in plant tissue and thus enables a direct analysis of the promoter activity without the addition of additional auxiliaries or chromogenic substrates; Dellaporta et al., In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium, 11: 263-282, 1988) ß-glucuronidase (Jefferson et al. (1987) EMBO J 6: 3901-3907) is very particularly preferred.

c) Origins of replication which MAESSEN an increase of erfindungsge- ^'transgenic expression cassettes or vectors in the

Ensure E.coli example. Examples include ORI (origin of DNA replication), pBR322 ori or P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

d) Elements which are required for an agrobacterium-mediated plant transformation, such as, for example, the right or left border of the T-DNA or the vir region.

- For the selection of successfully homologously recombined or also transformed cells, it is generally necessary to additionally introduce a selectable marker which gives the successfully recombined cells resistance to a biocide (for example a herbicide), a metabolism inhibitor such as 2-deoxyglu - Cose-6-phosphate (WO 98/45456) or an antibiotic. The selection marker allows the selection of the transformed cells by untransforced (McCorick et al. (1986) Plant Cell Reports 5: 81-84).

As an example, but not by way of limitation, the transgenic expression cassette according to the invention can have the following structure:

a) 5 'plant-specific promoter / desaturase / terminator-3'

The transgenic expression cassette according to the invention preferably has the following structure:

a) 5'-35S promoter / desaturase / OCS terminator-3 ', or

b) 5 '-LeguminB-Pro otor / Desaturase / NOS-Terminator-3'

For the advantageous processes for CLA biosynthesis according to the invention, a co-transformation with more than one of the above-mentioned examples a) or b) can be advantageous. Furthermore, the transformation with one or more vectors, each of which contains a combination of the above-mentioned transgenic expression cassettes, can be advantageous.

In addition, said transgenic expression cassette can contain a nucleic acid sequence whose transgenic expression leads to an increase in fatty acid biosynthesis (as a result of proOIL). This additionally transgenically expressed proOIL nucleic acid sequence can be selected, for example but not restrictively, from nucleic acids coding for acetyl-CoA carboxylase (ACCase), glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidate acyltransferase (LPAT), diacyltransferase acyl (DAGAT) and phospholipid: diacylglycerol acyltransferase (PDAT).

The proOIL nucleic acid sequences also include those nucleic acids whose transgenic expression produces an antisense-RNA or double-stranded RNA, which likewise increases the fatty acid production.

Preferred examples include vectors that contain the following transgenic expression cassettes:

a) 5'-35S promoter / desaturase / OCS terminator / LeguminB promoter / proOIL / NOS terminator-3 ';

b) 5 '-35S promoter / pro desaturase or conjugase / OCS terminator / LeguminB promoter / proOIL / NOS terminator-3'; Constructs a) and b) allow the simultaneous transformation of the plant with a desaturase and a sequence proOIL which increases the fatty acid biosynthesis.

Can be prepared using ^'mitigation techniques of the above-cited recombination and cloning the novel desaturase or proOIL nucleic acids or transgenic expression cassettes are cloned into suitable vectors, for example, in E. coli that allow their proliferation. Suitable cloning vectors include pBR332, pUC series, Ml3mp series and pACYC184. Binary vectors which can replicate both in E. coli and in agrobacteria are particularly suitable.

The desaturase according to the invention or proOIL nucleic acids or transgenic expression cassettes are preferably used in suitable

Transformation vectors inserted. Suitable vectors are inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, pp. 71-119 (1993). Examples and procedures for transformation are set out below.

Another object of the invention relates to transgenic organisms, transformed with at least one transgenic expression cassette according to the invention or a vector according to the invention, and cells, cell cultures, tissues, parts - such as leaves, roots, etc. in plant organisms - or propagation material derived from such organisms.

Organism, starting or host organisms are understood to mean procaryotic or eukaryotic organisms, such as, for example, microorganisms or plant organisms.

Preferred microorganisms are bacteria, yeast, algae or fungi.

Preferred bacteria are bacteria of the genus Escherichia, Corynebacterium, Bacillus, Clostrridium, Proionibacterium, Butyribibrio, Eubacterium, Lactobacillus, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Phaeodactylum, - Colpidium, Mortierella or Entomophinium, Entomophinium for example the genus Synechocystis.

Especially preferred are microorganisms which are capable of infecting plants and thus of transmitting the constructs according to the invention. Preferred microorganisms are those from the genus Agrobacterium and in particular from the species Agrobacterium tumefaciens. Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.

Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria, Phytophthora infestans or others in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995) on page 15, Table 6 described mushrooms.

Vegetable organisms are particularly preferred as transgenic organisms. "Plant organism" includes any organism that is capable of photosynthesis, as well as the cells, tissues, parts or reproductive material derived from it (such as seeds or fruits). Included in the scope of the invention are all genera and species of higher and lower plants in the plant kingdom. Annual, perennial, monocotyledonous and dieotyledonous plants and gymnosperms are preferred. Included are mature plants, seeds, shoots and seedlings, as well as parts derived therefrom, propagation material (for example bulbs, seeds or fruits), plant organs, tissues, protoplasts, callus and other cultures, for example cell cultures. Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.

"Plant" includes all annual and perennial, monocotyledonous and dicotyledonous plants and includes, by way of example but not by way of limitation, those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linu, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,

Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Calia, Glycumis, Cucumisine Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Seeale, Triticum, Sorghum, Picea and Populus.

Preferred are plants the following plant families: Amaranthaceae, ceae Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Crueiferae, Cueurbitaceae, Labiatae, Legumino- SAE, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanacea , Sterculia- ceae, Tetragoniacea, Theaceae, Umbelliferae.

Preferred monocotyledonous plants are selected in particular from the monocotyledonous crop plants, such as, for example, the Gramineae family, such as alfalfa, rice, maize, wheat or other cereal such as barley, millet, rye, triticale or oats, as well as sugar cane and all types of grass.

The invention is very particularly preferably applied from dicotyledonous plant organisms. Preferred dicotyledonous plants are in particular selected from the dicotyledonous crop plants, such as, for example

Asteraceae such as sunflower, tagetes or calendula and others,

Compositae, especially the genus Lactuca, especially the species sativa (salad) and others,

- Crueiferae, especially the genus Brassica, especially the species napus (rape), campestris (turnip), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other types of cabbage; and the genus Arabidopsis, especially the species thaliana as well as cress or canola and others,

- cueurbitaceae such as melon, pumpkin or zucchini and others,

- Leguminosae especially the genus Glycine, especially the type max (soybean) soy, as well as alfalfa, peas, beans or peanuts and others

Rubiaceae, preferably of the subclass Lamiidae such as, for example, Coffea arabica or Coffea liberica (coffee shrub) and others,

Solanaceae, in particular the genus Lycopersicon, especially the species esculentum (tomato) and the genus Solanum, particularly the species tuberosum (potato) and melongena (eggplant), as well as tobacco or peppers and others,

- Sterculiaceae, preferably the subclass Dilleniidae such as. for example Theobroma cacao (cocoa powder) and others,

Theaceae, preferably of the subclass Dilleniidae, such as, for example, Camellia sinensis or Thea sinensis (tea bush) and others,

- Umbelliferae, especially the genus Daucus (especially the species carota (carrot)) and Apium (especially the species graveolens dulce (Seiarie)) and others; and the genus Capsicum, especially the kind annu (pepper) and others,

as well as flax, soy, cotton, hemp, flax, cucumber, spinach, carrot, sugar beet and the various types of trees, nuts and wines, especially banana and kiwi.

Also included are ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawn. Examples include, but are not limited to, angiosperms, bryophytes such as, for example, hepaticae (liverwort) and musci (mosses); Pteridophytes such as ferns, horsetail and lycopods; Gymnosperms such as conifers, cycads, ginkgo and gnetals, the families of rosaceae such as rose, ericaceae such as rhododendrons and azaleas, euphorbiaceae such as poinsettias and croton, caryophyllaceae such as cloves, solanaceae such as petunias, Gesneriaceae such as the Usamalsamineaeaid as the Usambaramineae , Iridaceae such as gladiolus, iris, freesia and crocus, compositae such as marigold, geraniaceae such as geraniums, liliaceae such as the dragon tree, moraceae such as ficus, araceae such as philodendron and others.

Plant organisms in the sense of the invention are further photosynthetically active capable organisms, such as algae, cyanobacteria and mosses. Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Synechocystis is particularly preferred.

Most preferred are plants which are suitable for oil production, such as, for example, rapeseed, sunflower, sesame, safflower (Carthamus tinctorius), olive tree, soybean, peanut, castor oil, oil palm, corn, wheat, cocoa or various types of nuts such as walnut, coconut or Almond. Arabidopsis, cotton, flax, flax are also most preferred.

Plant organisms in the sense of the invention are further photosynthetically active capable organisms, such as algae or cyanobacteria, and mosses. Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Protozoa such as dinoflagellates are also preferred.

From the above-mentioned organisms, particular preference is given to those which can naturally synthesize oils in large amounts, such as fungi such as Mortierella alpina, Pythium insidiosum or plants such as soybean, rapeseed, coconut, oil palm, safflower, castor bean, peanut, Cocoa bean or sunflower or yeasts such as Saccharomyces cerevisiae, soya, rape, sunflower or Saccharomyces cerevisiae are particularly preferred.

Depending on the host organism, the organisms used in the processes are grown or cultivated in a manner known to those skilled in the art. Microorganisms are usually in a liquid medium, which contains a carbon source mostly in the form of sugars, a nitrogen source mostly 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 possibly vitamins, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C to 60 ° C attracted with oxygen. The pH of the nutrient liquid can be kept at a fixed value, that is to say it can be regulated during cultivation or not. The cultivation can be batch-wise, semi-batch wise or continuous. Nutrients may be ^'the fermentation start submitted or semi-continuous or continuous fed ad.

The introduction of a transgenic expression cassette according to the invention into an organism or cells, tissues, organs, parts or seeds of the same (preferably in plants or plant cells, tissues, organs, parts or seeds) can advantageously be implemented using vectors, in which contain the transgenic expression cassettes. The transgenic expression cassette can be introduced into the vector (for example a plasmid) via a suitable restriction site. The resulting plasmid is first introduced into E. coli. Correctly transformed E. coli are selected, grown and the recombinant plasmid is obtained using methods familiar to the person skilled in the art. Restriction analysis and sequencing can be used to check the cloning step.

The introduction of an expression cassette according to the invention into cells, preferably into plant cells, can advantageously be implemented using vectors. Vectors can be, for example, plasmids, cosmids, phages, viruses or even agrobacteria. In an advantageous embodiment, the expression cassette is introduced by means of plasmid vectors. Preferred vectors are those which enable stable integration of the expression cassette into the host genome. The production of a transformed organism (or a transformed cell or tissue) requires that the corresponding DNA, RNA or protein be introduced into the corresponding host cell. 5

A large number of methods are available for this process, which is referred to as transformation (or transduction or transfection) (Keown et al. (1990) Methods in Enzymology 185: 527-537). For example, the DNA or RNA can go straight through

10 microinjection or by bombardment with DNA-coated microparticles. The cell can also be chemically permeabilized, for example with polyethylene glycol, so that the DNA can get into the cell by diffusion. The DNA can also be obtained by protoplast fusion with other DNA-containing ones

15 units such as mini cells, cells, lysosomes or liposomes are made. Electroporation is another suitable method for introducing DNA in which the cells are reversibly permeabilized by an electrical pulse. Appropriate methods have been described (for example in Bilang et al. (1991) Gene

20 100: 247-250; Scheid et al. (1991) Mol Gen Genet 228: 104-112; Guerche et al. (1987) Plant Science 52: 111-116; Neuhause et al. (1987) Theor Appl Genet 75: 30-36; Klein et al. (1987) Nature 327: 70-73 Howell et al. (1980) Science 208: 1265; Horsch et al. (1985) Science 227: 1229-1231; DeBlock et al. (1989) Plant

25 Physiology 91: 694-701; Methods for Plant Molecular Biology

( ^' Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press Inc. (1989)).

30 The desaturases or conjugases can be used for the genetic engineering modification of a wide spectrum of organisms, preferably of plants, so that they become a de novo producer of one or more products derived from lipids, such as both CLA isomers. Plants are initially transformed

35 regenerated and then grown or grown as usual.

Cloning vectors and techniques for the genetic manipulation of ciliates and algae are known to the person skilled in the art (WO 98/01572; 40 Falciatore et al. (1999) Marine Biotechnology 1 (3) .239-251; Dunahay et al. (1995) J Phycol 31: 10004-1012).

Various methods and vectors for introducing genes into the genome of plants and for regenerating plants from 45 plant tissues or plant cells are known (Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, p. 71- 119 (1993); White FF (1993) Vectors for Gene Transfer to higher plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, ed. : Kung and R. Wu, Academic Press, 15-38; That B et al. (1993) 'Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, ed. : Kung and R. Wu, Academic Press, pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42: 205-225; Haiford NG, Shewry PR (2000) Br Med Bull 56 (1): 62-73). Examples include those mentioned above. In plants, the methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are above all the protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun, the so-called "particle bombardment" method, the electroporation, the incubation of dry embryos in DNA-containing solution and the microinjection. In the case of these "direct" transformation methods, there are no special requirements for the plasmid used. Simple plasmids such as those of the pUC series, pBR322, M13mp series, pACYC184 etc. can be used. If complete plants are to be regenerated from the transformed cells, it is necessary that there is an additional selectable marker gene on the plasmid.

In addition to these "direct" transformation techniques, a transformation can also be carried out by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes. These strains contain a plasmid (Ti or Ri plasmid) which is transferred to the plant after agrobaterium infection. Part of this plasmid, called T-DNA (transferred DNA), is integrated into the genome of the plant cell. Alternatively, binary vectors (mini-Ti plasmids) can also be transferred to plants by Agrobacterium and integrated into their genome. The Agrobacterium-mediated transformation is best suited for dieotyledone, diploid plant cells, whereas the direct transformation techniques are suitable for every cell type. Methods for Agrobacterium-mediated transformation are described, for example, by Horsch RB et al. (1985) Science 225: 1229f. If Agrobacteria are used, the expression cassette is to be integrated into special plasmids, either into an intermediate vector (English: shuttle or intermediate vector) or a binary vector. If a Ti or Ri plasmid is to be used for transformation, at least the right boundary, but mostly the right and the left boundary of the Ti or Ri plasmid T-DNA as the flanking region, is connected to the expression cassette to be introduced. Binary vectors are preferably used for the Agrobacterium transformation. Binary vectors can replicate in both E.coli and Agrobacterium. They usually contain a selection marker gene and a linker or polylinker flanked by the right and left T-DNA restriction sequences. They can be transformed directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163: 181-187). The selection marker gene allows selection of transformed agrobacteria and is, for example, the nptll gene which confers resistance to kanamycin. Which in this case acting as a ^'host organism Agrobacterium should already contain a plasmid with the vir region. This is necessary for the transfer of T-DNA to the plant cell. An Agrobacterium transformed in this way can be used to transform plant cells. The use of T-DNA for the transformation of plant cells has been intensively investigated and described (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters BV, Alblasserda, Chapter V; An et al. ( 1985) EMBO J 4: 277-287). Various binary vectors are known and some are commercially available, for example pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA; Bevan et al. (1984) Nucl Acids Res 12: 8711), pBinAR, pPZP200 or pPTV.

The agrobacteria transformed with such a vector can then be used in a known manner to transform plants, in particular crop plants, such as e.g. of rapeseed can be used, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media. The transformation of plants by agrobacteria is described (White FF, Vectors for Gene

Transfer to higher plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38; Jenes B et al. (1993) Techniques for Gene Transfer, in.- Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42: 205-225). From the transformed cells of the wounded leaves or leaf pieces, transgenic plants can be regenerated in a known manner, which contain the above-described desaturase or conjugase according to the invention, pro-desaturase or conjugase or proOIL nucleic acids or transgenic expression cassettes or vectors.

Stably transformed cells, ie those which contain the introduced DNA integrated into the DNA of the host cell, can be selected from untransformed ones if a selectable marker is part of the introduced DNA. As a marker, Each gene acts playfully that can confer resistance to antibiotics or herbicides (such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc.) (see above). Transformed cells that express such a marker gene are able to survive in the presence of concentrations of an appropriate antibiotic or herbicide that kill an untransformed wild type. Examples are mentioned above and preferably comprise the bar gene which confers resistance to the herbicide phosphinotricin (Rathore KS et al. (1993) Plant Mol Biol

10 21 (5): 871-884), the nptll gene, which confers resistance to kanamycin, the hpt gene, which confers resistance to hygromycin, or the EPSP gene, which confers resistance to the herbicide glyphosate. The selection marker allows the selection of transformed cells from untransformed (McCormick et al. (1986)

15 Plant Cell Reports 5: 81-84). The plants obtained can be grown and crossed in a conventional manner. Two or more generations should preferably be cultivated to ensure that genomic integration is stable and inheritable.

20

Once a transformed plant cell has been made, a whole plant can be obtained using methods known to those skilled in the art. This is based on the example of callus cultures. From these still undifferentiated cell

The mass of shoots and roots can be induced in a known manner. The sprouts obtained can be planted out and grown. Appropriate methods are described (Fennell et al. (1992) Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep. 14: 273-278; Jahne et al. (1994) Theor Appl

30 Genet 89: 525-533).

The effectiveness of the expression of the transgenically expressed nucleic acids can be determined, for example, in vitro by sprout meristem propagation using one of the selection described above.

35 methods can be determined. In addition, a change in the type and level of expression of a desaturase or a proOIL nucleic acid sequence and its effect on the CLA and / or fatty acid biosynthesis performance on test plants can be tested in greenhouse experiments.

40

From the transgenic organisms, those are preferred which have an improved CLA production compared to the untransformed wild type. In the context of the present invention, improved CLA production means, for example, the artificial

The acquired ability of an increased biosynthetic performance of at least one compound from the group of CLA's esters, such as, for example, CoA esters or glyceride esters, in the transgenic Organisms compared to the non-genetically modified parent organism. The CLA production in the transgenic organism is preferably increased by 10%, particularly preferably by 50%, very particularly preferably by 100%, compared to the non-genetically modified starting organism. Improved can also mean an advantageously changed qualitative composition of the CLA mixture, ie an increased relative proportion of (9Z, 11E) -CLA and / or (10E, 12Z) -CLA compared to the starting organism.

According to the invention are also derived from the transgenic organisms described above, cell cultures, parts - such as roots, leaves, etc. in transgenic plant organisms - and transgenic propagation material such as seeds or fruits.

Another object of the invention relates to the use of the transgenic organisms according to the invention described above and the cells, cell cultures, parts derived therefrom - such as roots, leaves etc. in transgenic plant organisms - and transgenic propagation material such as seeds or fruits for the production of food or feed, cosmetics or fine chemicals, such as free fatty acids, especially CLA. Particularly preferred is the use for the production of CLA-containing lipids, preferably triglycerides.

Genetically modified plants according to the invention which can be consumed by humans and animals can also be used as food or feed, for example directly or after preparation known per se.

After culturing, the lipids are usually obtained from the organisms. For this purpose, the organisms can first be digested after harvesting or used directly. The lipids are advantageously mixed with suitable solvents such as apolar solvents such as hexane or ethanol, isopropanol or mixtures such as hexane / isopropanol, phenol / chloroform / isoamyl alcohol at temperatures between 0 ° C to 80 ° C, preferably between 20 ° C ^* and 50 ° C extracted. The biomass is usually extracted with an excess of solvent, for example an excess of solvent to biomass of 1: 4. The solvent is then removed, for example by distillation. The extraction can also be done with supercritical CO ₂ . After extraction, the remaining biomass can be removed, for example, by filtration.

The crude oil obtained in this way can then be further purified, for example in the turbidity by adding polar Ren solvents such as acetone or chloroform and subsequent filtration or centrifugation are removed. Further cleaning via columns is also possible.

To obtain the free fatty acids from the triglycerides, they are usually saponified.

Another object of the invention are vegetable oils, fatty acid mixtures and / or triglyceride mixtures with an increased content of unsaturated fatty acids, preferably CLA, which were produced by the above-mentioned methods according to the invention, and their use for the production of foods, animal feed, cosmetics or pharmaceuticals. For this purpose, they are added to the food, animal feed, cosmetics or pharmaceuticals in the usual amounts.

sequences

1. SEQ ID NO: 1: Nucleic acid sequence coding for an acyl-CoA ell-desaturase from Epiphyas postvittana.

5

2. SEQ ID NO: 2: Protein sequence coding for an acyl-CoA ell-desaturase from Epiphyas postvittana.

3. SEQ ID NO: 3: Nucleic acid sequence coding for an acyl-CoA 10 Z / EU desaturase from Ostrinia nubilalis.

4. SEQ ID NO: 4: Protein sequence coding for an acyl-CoA Z / Ell desaturase from Ostrinia nubilalis.

15 5. SEQ ID NO: 5: Nucleic acid sequence coding for an acyl-CoA Z / Ell desaturase from Ostrinia furnacalis.

6. SEQ ID NO: 6:. Protein sequence coding for an acyl-CoA Z / Ell desaturase from Ostrinia furnacalis.

20

7. SEQ ID NO: 7: Nucleic acid sequence coding for an acyl-CoA Δll desaturase from Helicoverpa zea.

8. SEQ ID NO: 8: Protein sequence coding for an acyl-CoA Δll 25 desaturase from Helicoverpa zea.

9. SEQ ID NO: 9: Nucleic acid sequence coding for an acyl-CoA Δll desaturase from Trichoplusia ni.

30 10. SEQ ID NO: 10: Protein sequence coding for an acyl-CoA Δll desaturase from Trichoplusia ni.

11. SEQ ID NO: 11: Nucleic acid sequence coding for an acyl-CoA Δll desaturase from Argyrbtaenia velutinana.

35

12. SEQ ID NO: 12: Protein sequence coding for an acyl-CoA Δll desaturase from Argyrotaenia velutinana.

13. SEQ ID NO: 13: Nucleic acid sequence coding for an acyl-40 CoA Z10 desaturase from Planotortrix octo.

14. SEQ ID NO: 14: Protein sequence coding for an acyl-CoA Z10 desaturase from Planotortrix octo.

45 15. SEQ ID NO: 15: 5 ^λ -ATYACHGCCGGKKMYCAYMG-3 ^λ 16. SEQ ID NO: 16: 5 -GGRAABDYGTGRTGGWAGTT-3 ^λ

17, SEQ ID NO: ^'17: 5 * -CCCCAYCRNCTSTGGWCNCA-3'

18. SEQ ID NO: 18: 5 ^x -CCCTCTAGARTGRRWARTTRTGRWA-3 '

19. SEQ ID NO: 19: 5 '-TAATACGACTCACTATAG-3 ^λ

20. SEQ ID NO: 20: 5 ^v -ACATAACTAATTACATGAT-3 ^v

Examples

The invention is explained in more detail in the following exemplary embodiments with reference to the accompanying figures. Abbreviations with the following meanings are used: - "

General methods

The cloning steps carried out in the context of the present invention, such as e.g. Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of the bacteria and sequence analysis of recombinant DNA are carried out as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. The chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pp. 896-897). The cloning steps carried out in the context of the present invention, such as e.g. Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, multiplication of phages and sequence analysis of recombinant DNA were carried out as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. The sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from Licor (sales by MWG Biotech, Ebersbach) according to the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sei USA 74: 5463-5467).

Example 1: Cultivation of Lepidoptera insects

The insects are kept in terrariums on suitable host plants. The growth conditions are: 27 ° C, day-night rhythm: 14 h light, 10 hours dark. Insects or larvae are transferred to fresh plants twice a week. Dolls are collected and males and flocks are kept separately in terrariums until the finished insects hatch. The pheromone glands can be isolated about 1 5 to 2 days after hatching.

Example 2: Isolation of desaturase or conjugase cDNAs using degenerate primer 10

The pheromone glands are isolated from the abdomen of adult moths and in N2 liq. frozen until total RNA is isolated using, for example, TRIzol (GIBCO / BRL) according to the manufacturer's instructions. Experience has shown that approximately 60 to 15 80 μg of total RNA can be isolated from approximately 30 mg of fresh tissue. About 5 μg of total RNA is used to produce a first strand cDNA with an oligo (dT) primer. This can be done, for example _. the SMART RACE cDNA Amplification Kit (CLONTECH) can be used according to the manufacturer's instructions. This single-strand cDNA serves as a template for a PCR in which the central region of the desaturase / conjugase cDNA is amplified. Two degenerate primers are designed so that they can amplify the conserved central region of the desaturases / conjugases from Lepidoptera and are used in the following PCR protocol: 25

5 µl diluted single strand cDNA 0.2 mM dATP, dTTP, dGTP, dCTP 0.5 µM 5 'primer (SEQ ID NO 8) 0.5 µM 3' primer (SEQ ID NO 9) 30 10 µl 5X Advantage 2 reaction buffer (CLONTECH) -

1 μl 50X Advantage 2 DNA Polymerase ^J mix (CLONTECH) H20 add. 50 ul

Alternatively, the degenerate primers with the 35 SEQ ID NO: 10 and SEQ ID NO: 11 are used as the primer pair for the amplification of the central desaturase region.

The PCR was carried out under the following cycle conditions:

⁴⁰ 1 step with: 94 ° C (5 min)

35 cycles with: 94 ° C (30 sec), 56 ° C (30 sec), 72 ° C (3 min)

1 step with: 72 ° C (10 min)

Waiting position: 4 ° C

⁴⁵ The PCR product is ligated directly, for example, into the linearized TOPO TA PCR 2.1 Vector (INVITROGEN) and then transformed into E.coli TOP 10 competent cells (INVITROGEN). positive Active colonies are amplified again, the plasmid DNA is purified (QIAGEN Plasmid Mini Kit) and then sequenced.

Based on the sequence information obtained, gene-specific primers can be derived and for the aplification of 5'- and

3 'areas are used (SMART RACE cDNA Amplification Kit (CLONTECH)). These PCR products are also ligated into the TOPO TA PCR 2.1 Vector (INVITROGEN) and then transformed into TOP 10 competent cells (INVITROGEN). Positive colonies are amplified again and the plasmid DNA is purified (QIAGEN Plasmid Mini Kit) and then sequenced.

From the fragments generated in this way, the complete sequence of a desaturase / conjugase can be assembled using standard cloning techniques and, for characterization, converted, for example, into the pYES2 vector (INVITROGEN) for yeast expression. Saccharomyces INVScl (INVITROGEN) are transformed with the corresponding pYES2 expression vectors using a modified PEG / lithium acetate protocol (Ausubel et al. (1996) Current Protocols in Molecular Biology. John Wiley and Sons, New York). After selection on CMdum agar plates with 2% glucose, four pYES2DESAT transformants (pYES2DESATa-d) and one pYES2 transformant are selected for further cultivation and functional expression.

Example 3: Functional expression of a desaturase / conjugase in yeast

Preculture: 20 ml of CMdum liquid medium with 2% (w / v) raffinose were inoculated with the transgenic yeast clones (pYES2DESATa-d, pYES2) and grown for 3 T at 30 ° C., 200 rpm until an optical density of 600 nm (OD600) of 1.5-2 was reached.

Main culture: For the expression, 20 ml of CMdum liquid medium with 2% raffinose and 1% (vol. / Vol.) Tergitol NP-40 with the corresponding substrates, such as stearic acid, palmitic acid or myristic acid, were mixed to a final concentration of 0.003% ( Gew./Vol.) Enriched. The media were inoculated with the precultures to an OD600 of 0.05. Expression was induced at an OD600 of 0.2 with 2% (w / v) galactose for 16 hours after which the cultures had reached an OD600 of 0.8 to 1.2.

Fatty acid analysis: The total fatty acids were extracted from yeast cultures and analyzed by gas chromatography. Cells from 5 ml culture were centrifuged (1000 x g,

10 min, 4 ° C) harvested and washed once with 100 mM NaHC03, pH 8.0 to remove residual medium and fatty acids. For manufacturing Position of the fatty acid methyl esters (FAMES), the cell sediments are snap frozen in N2 liq. and lyophilized under N2 gas at 30 ^a C. The pellet is homogenized in 1% sodium methoxide in methanol and incubated for 20 minutes at room temperature. 5 Then equal volumes of 1 M NaCl and n-heptane are added, mixed and the supernatant transferred to a GC tube. The samples are separated on a DB Wax capillary column (30 m, 0.25 mm, 0.25 μm; Agilent J & W) in a Hewlett Packard 6890 gas chromatograph with a flame ionization detector. The

The oven temperature was raised from 60 ° C (hold 5 min) to 200 ° C at a rate of 20 ° C / min (hold 20 min) and finally to 250 ° C (hold 30 min) at a rate of 20 ° C / min programmed. Nitrogen was used as the carrier gas (1.6 ml / min). The fatty acids were compared to the retention times of FAME standards (SIGMA)

15 identified. The following fatty acids are preferably used as standard: c9-16: l, t9-16: l, 18: 0, c9-18: l, t9-18: l, cll-18: l, tll-18: l, c9 , cl2-18: 2, t9, tl2-18: 2, c9, tll-18: 2, tlO, cl2-18: 2. (The nomenclature of the standards corresponds to that customary for fatty acids and initially gives configuration (c = cis or

20 t = trans) and position of the double bond or chain length of the fatty acid. )

Further feeding experiments with various other fatty acids (e.g. lauric acid, trans-vaccenic acid, cis-vaccenic acid, 25 ElO-octadecenoic acid or ZlO-octadecenoic acid) can be carried out to confirm the substrate selectivity of this desaturase / conjugase in more detail.

Example 4: Expression cloning of desaturase / conjugase from 30 Lepidoptera in Saccharomyces cerevisiae

a) Preparation of the cDNA library

The pheromone glands are isolated from the abdomen of adult moths 35 and in N2 liq. frozen until total RNA is isolated using, for example, TRIzol (GIBCO / BRL) according to the manufacturer's instructions. Experience has shown that approximately 60 to 80 μg of total RNA can be isolated from approximately 30 mg of fresh tissue. About 5 μg of total RNA is used to create a cDNA library. For example, the SMART cDNA Library Construction Kit (CLONTECH) can do this

Manufacturer information can be used. The isolated double-stranded cDNA is finally ligated into the linearized pYES2 (INVITROGEN) yeast expression vector. For this purpose, the multiple cloning site of the pTriplEx2 vector (CLONTECH) is first inserted in pYES2. The double-stranded cDNA is then cloned into the vector modified in this way after digestion with SfilA and SfilB. b) yeast transformation

About 1.5 μg of plasmid DNA is transformed with the Saccharomyces cerevisiae EASY COMP transformation kit (INVITROGEN) into INVScl (INVITROGEN) according to the manufacturer's instructions. Two 50 μl batches are plated on selection medium in a large square petri dish (245 × 245 mm) and incubated for 3 days at 30 ° C.

c) Cultivation of the yeast in microtiter plates

Individual colonies are transferred to microtiter plates (MTP) using a pick robot. The preculture takes place in 200 μl medium [1 x CSM-Ura; 1 x YNB without amino acids and sugar; 0.5% raffinose; 5% glycerin; 40 mg / 1 adenine sulfate; 0.5% ammonium sulfate] for 72 hours at 30 ° C and 250 revolutions / minute. By adding an average volume from the preculture, a. OD600 set from 0.2 in the main culture. The main culture takes place in 1 ml medium [1 x CSM-Ura; 1 x YNB without amino acids and sugar; 0.5% raffinose; 2% galactose; 0.2% Tergitol NP-40; 40 mg / 1 adenine sulfate; 0.5% ammonium sulfate; 0.3 mM fatty acid substrate] for 2 to 3 weeks at 16 ° C and 250 revolutions / minute until an OD600 of 3 to 4 is reached.

d) fatty acid analysis

The yeast cells are sedimented (1000 xg, 10 min, 4 ° C) and stored at -80 ° C until further processing. To produce the fatty acid methyl esters (FAMES), the cell sediments are snap frozen in N2 liq. and lyophyllized under N2 gas at 30 ^e C. The pellet is homogenized in 1% sodium methoxide in methanol and incubated for 20 minutes at room temperature. Then equal volumes of 1 M NaCl and n-heptane are added, mixed and the supernatant transferred to a GC tube. The samples are separated on a DB Wax capillary column (30 m, 0.25 mm, 0.25 μm; Agilent J & W) in a Hewlett Packard 6890 gas chromatograph with a flame ionization detector. The furnace temperature was (min- hold 5) to 200 ° C at a rate of 20 ° C / min (hold min 20) of 60 ° C and finally to 250 ° C (30 hold min) at a rate of ^'20 ° C / min programmed. Nitrogen was used as the carrier gas (1.6 ml / min). The fatty acids were identified by comparison with retention times of FAME standards (SIGMA). The same standards - as listed in Example 3 - are used. Further feeding experiments with various other fatty acids (e.g. lauric acid, trans-vaccenic acid, cis-vaccenic acid, ElO octadecenoic acid or ZlO octadecenoic acid) can be used waisted confirmation of the substrate selectivity of this desaturase or conjugase can be carried out.

e) Plasmid preparation of the positive yeast:

Individual clones which have tested positive are again over 24 hours at 28 ° C in 10 ml minimal medium [1 x CSM-Ura; 1 x YNB w / o AA, sugars, - 2% glucose; 40 mg / 1 adenine sulfate] and sedimented by centrifugation at an OD600 greater than 3. The yeast pellet is soaked for 20 minutes at 37 ° C in 400 ul SCE buffer [1.2 M sorbitol; 0.1 M Na citrate, pH 7.0; 10 mM EDTA; 1 mg / ml lyticase]. Then 400 ul STE buffer [2% SDS; 50 mM Tris / HCl, pH 8.0; 10 mM EDTA] was added to the cell lysis and incubated for 10 minutes at room temperature. For the precipitation of the cell protein, 200 μl of 5 M sodium acetate is added and placed on ice for 30 minutes. After centrifugation, the supernatant is transferred and precipitated with 2.5 times the volume of ethanol. The sedimented pellet is washed with 70% ethanol and finally taken up in sterile water. The sequence of the DESATURASE / CONJUGASE can optionally be determined by sequencing using vector-specific primers (SEQ ID NO: 12 and 13).

Example 5: Manipulation of vegetable fatty acid biosynthesis

a) Generation of DNA constructs for the expression of desaturases / conjugases from Lepidoptera in transgenic plants.

The vector pBinAR is used to produce chimeric DNA constructs for the production of transgenic A.thaliana or B.napus plants which express the desaturases / conjugases from Lepidoptera (Höfgen and Willmitzer (1990) Plant Sei 66: 221- 230). This contains the 35S promoter of the CaMV (cauliflower mosaic virus) (Franck et al. (1980) Cell 21 (1): 285-294) and the termination signal of the octopine synthase gene (Gielen et al. (1984) EMBO J 3: 835-846). After cloning the DESAT / KONJ into this vector, transgenic A.thaliana or Brassica napus plants are generated.

b) Production of transgenic Arabidopis thaliana plants

Wild-type Arabidopsis thaliana plants (Columbia) are transformed with the Agrabacterium tumefaciens strain (GV3101 [pMP90]) on the basis of a modified vacuum filtration method (Clough S and Bent A (1998) Plant J 16 (6): 735-43; Bechtold N et al . (1993) in: Planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CRAcad Sei Pa- ris 1144 (2): 204-212). The Agrobacterium tumefaciens cells used had previously been transformed with the plasmids.

Seeds of the primary transformants are selected on the basis of antibiotic resistance. Antibiotic-resistant seedlings are planted in soil and used as fully developed plants for biochemical analysis.

b) Production of transgenic Brassica napus plants

10

The production of transgenic rapeseed plants is based on a protocol by Bade JB and Damm B (in Gene Transfer to Plants, Polypus I and Spangenberg G (eds.) Springer Lab Manual, Springer Verlag, 1995, 30-38), in which also the composition of the 15 media and buffers used is indicated.

The transformations are carried out with the Agrobacterium tumefaciens strain GV3101 [pMP90]. The plasmids pBinAR-TkTP / Vit E-AT are used for transformation. Brassica napus var. We-

20 stars are made surface-sterile with 70% ethanol (v / v), washed in water for 10 minutes at 55 ° C, in 1% hypochlorite solution (25% v / v Teepol, 0.1% v / v Tween 20) incubated for 20 minutes and washed six times with sterile water for 20 minutes each. The seeds are dried on filter paper for three days and 10 to

25 15 seeds germinated in a glass flask with 15 ml germination medium. The roots and apices are removed from several seedlings (approx. 10 cm in size) and the remaining hypocotyls are cut into pieces approx. 6 mm long. The approx. 600 explants obtained in this way are washed for 30 minutes with 50 ml of basal medium and in

30 transferred a 300 ml flask. After adding 100 ml of callus induction medium, the cultures are incubated for 24 hours at 100 rpm.

An overnight culture of the Agrobacterium strain is set up at 29 ° C. in luria broth medium with kanamycin (20 mg / l), of which 2 ml in 50 ml of luria broth medium without kanamycin for 4 hours at 29 ° C. until an ODεoo ^of Incubated 0.4 to 0.5. After pelleting the culture at 2000 rpm for 25 min, the cell pellet is resuspended in 25 ml of basal medium. The concentration of the bacteria in the solution is adjusted to an ODβoo of 0.3 by adding further basal medium.

The callus induction medium is removed from the rape explants using sterile pipettes, 50 ml of Agrobacterium solution are added, 45 carefully mixed and incubated for 20 min. The agrobacteria suspension is removed, the oilseed rape explants are washed for 1 min with 50 ml of callus induction medium and then 100 ml callus induction medium added. The co-cultivation is carried out for 24 h on a rotary shaker at 100 rpm. The co-cultivation is stopped by removing the callus induction medium and the explants are washed twice for 1 min with 25 ml and twice for 60 min with 100 ml washing medium at 100 rpm. The washing medium with the explants is transferred to 15 cm petri dishes and the medium is removed with sterile pipettes.

For regeneration, 20 to 30 explants are each transferred to 90 mm Petri dishes which contain 25 ml shoot induction medium with kanamycin. The Petri dishes are closed with 2 layers of Leukopor and incubated at 25 ° C and 2000 lux with photoperiods of 16 hours light / 8 hours dark. The developing calli are transferred to fresh petri dishes with shoot induction medium every 12 days. All further steps for the regeneration of whole plants are described by Bade, JB and Damm, B. (in: Gene Transfer to Plants, Potrykus I and Spangenberg G (eds.) Springer Lab Manual, Springer Verlag, 1995, 30-38) -written.

c) Analysis of the fatty acid pattern in the transgenic plants

The seeds of transgenic plants are homogenized directly in 1% sodium methoxide in methanol and incubated for 20 minutes at room temperature. Then equal volumes of 1 M NaCl and n-heptane are added, mixed and the supernatant is transferred to a GC tube. The samples are separated on a DB-Wax capillary column (30 m, 0.25 mm, 0.25 μm; Agilent J & W) in a Hewlett Pakard-6890 gas chromatograph with a flame ionization detector. The oven temperature was raised from 60 ° C (hold 5 min) to 200 ° C at a rate of 20 ° C / min (hold 20 min) and finally to 250 ° C (hold 30 min) at a rate of 20 ° C / min programmed. Nitrogen was used as the carrier gas (1.6 ml / min). The fatty acids were identified by comparison with retention times of FAME standards (SIGMA). The same standards - as listed in Example 3 - are used.

Claims

claims 1. Process for the production of unsaturated fatty acids and comprehensive trigylcerides, characterized in that at least one fatty acid desaturase from insects of the order Lepidoptera is transgenically expressed in a plant organism. 2. The method according to claim 1, characterized in that the fatty acid desaturase in fatty acids, fatty acid CoA esters or other fatty acid derivatives can generate a double bond at the position C8, C9, CIO, Cll or C12. 3. The method according to any one of claims 1 or 2, characterized in that the fatty acid desaturase specifically produces an ice or trans double bond in fatty acids, fatty acid CoA esters or other fatty acid derivatives with a chain length of the fatty acid of 16 or 18 carbon atoms. 4. The method according to any one of claims 1 to 3, characterized in that the fatty acid desaturase has a homology of 'at least 65% to one of the fatty acid desaturases described by SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14. 5. The method according to any one of claims 1 to 4, characterized in that the nucleic acid sequence of the fatty acid desaturase a) contains in its sense strand a sequence motif described by SEQ ID NO: 15 or 17, or b) contains a sequence motif described by SEQ ID NO: 16 or 18 in its antisense strand. 6. The method according to any one of claims 1 to 5, characterized in that the unsaturated fatty acids comprise conjugated linoleic acid. 7. triglycerides produced by a method according to any one of claims 1 to 6. 8. Use of triglycerides according to claim 7 for the production of food, feed, cosmetics or fine chemicals.