DE10208812A1 - Process for the production of unsaturated fatty acids - Google Patents

Abstract

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.

Description

The invention relates to processes for the production of unsaturated fatty acids, preferably conjugated polyunsaturated Fatty acids such as conjugated linoleic acid (CLA) through transgenic Expression of desaturases from insects of the order Lepidoptera. According to the invention, transgenic expression cassettes are also used transgenic expression of desaturases from insects of the order Lepidoptera, as well as the transgenes transformed with them Organisms.

Fatty acids and triglycerides have a large number of applications in the food industry, animal nutrition, cosmetics and in the pharmaceutical sector. The so-called conjugated unsaturated fatty acids are particularly valuable and sought-after 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), α-parinaric acid (18: 4 octadecatetraenoic acid), eleostearic acid (18: 3 octadecatrienoic acid), conjugated linolenic acids, dimorphhecolic acid and calendulic acid (see scheme 1). Scheme 1 Conjugated polyunsaturated fatty acids

CLA is a collective term for positional and structural Isomers of linoleic acid, which are conjugated Double bond system starting at carbon atom 8, 9, 10 or 11 distinguished. 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, 11E) -CLA and (10E, 12Z) -CLA are as that known biologically active isomers. CLA is predominantly in Contain food of animal origin. V. a. in meat and in Ruminant dairy products are high concentrations of CLA contain: approx. 3 to 4 mg CLA / g fat in beef and lamb (Chin et al. (1992) J Food Comp Anal 5: 185-197) and about 3 to 7 mg CLA / g fat in milk products (Dhiman et al. (1999) J Dairy Sci 82: 2146-56), whereby (9Z, 11E) -CLA with approx. 80% each Main isomer is. Higher plants only contain traces of CLA with both biologically active CLA isomers not yet in Plants have been found.

A number of positive effects have been demonstrated for CLA, so the administration of CLA reduces body fat in humans and Animal or increases the 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). Allergies can also be avoided by giving CLA (WO 97/32008) or Krebs (Banni et al. (1999) Carcinogenesis 20: 1019-1024, Thompson et al. (1997) Cancer Res 57: 5067-5072) influence positively. Also an antiartheriosclerotic effect of CLA is documented (Wilson et al. (2000) Nutr Res 20: 1795-1805). Investigations were carried out with both isomers and Isomer mixtures carried out.

• 1. die Fettsäure-Esterbindungen des Triglyceridgerüsts werden hydrolisiert und die freien Fettsäuren werden freigesetzt.
• 2. unkonjugierte, ungesättigte Fettsäuren mit zwei oder mehr Doppelbindungen werden konjugiert.

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, e.g. B. sunflower oil or safflower oil. Heating above 180 ° C under alkaline conditions catalyzes two reactions:

• 1. the fatty acid ester bonds of the triglyceride structure 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 various CLA isomers as well as other saturated and unsaturated Fatty acids. Due to the presence of these biologically inactive and unnatural isomers is a complex biological cleanse active isomers (9Z, 11E) CLA and (10E, 12Z) CLA necessary or it must be shown that there is no danger from the mixture of isomers for the health of humans and animals. So far it is not possible via alkaline isomerization in an economical way relevant processes to produce individual CLA isomers. By fractional crystallization it is possible to get the isomers Enrich (9Z, 11E) -CLA or (10E, 12Z) -CLA. In all of the above However, it is not possible to use single isomers in high processes To manufacture quality. The reaction products are in the mentioned methods usually to methyl or ethyl esters implemented so that not the natural form of the CLA, namely the free fatty acids or the triacylglyceride are present.

Biocatalysis of linoleic acid to CLA can overcome these disadvantages chemical transformation can be overcome. Various Rumen rumen microorganisms are, for example, in the Able to process linoleic acid to CLA in the process of biohydrogenation implement. This happens among other things through the enzymatic CLA isomerase activity. This enzyme activity has been described in Butyrivibrio fibrisolvens (Kepler and Tovee (1966) J Biol Chem 241: 1350), Propionibacterium acnes (Deng et al., 1st 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 Lactobacillus reuteri and Propionibacterium acnes were cloned, and the isomerase from Propionibacterium could be heterologous Microorganisms are functionally expressed. The biological The conversion of linoleic acid to CLA by microorganisms has indeed qualitative advantages over alkaline isomerization, it is but economically disadvantageous due to the fermentation costs and provides only free fatty acids, but no triglycerides. However, free fatty acids have adverse sensory effects Properties and are for use in the food or animal feed sector essentially unsuitable. A subsequent implementation of the free fatty acids - for example glycerin or glycerides under Lipase catalysis - is possible, but expensive.

The transfer of bacterial CLA isomerases Procedure on other organisms is not absolutely possible. So far, it has been demonstrated that CLA isomerases are free Convert linoleic acid to CLA (Cepler and Tove (1967) J Biochem Chem 242: 5686-5692). In higher organisms, such as Plants, linoleic acid is mainly in esterified form in front. Lipids like triacylglycerides are the storage form Thioesters like Acyl-CoA the activated form of the fatty acid.

Fatty acids with trans double bonds are extremely rare. In some plants' seed oil there are fatty acids with double bonds available in trans position. So there is different in the seed oil Thalictrum species an E5 fatty acid has been detected (Rankoff et al. (1971) J Amer Oil Chem Soc 48: 700-701). In addition, in Aquilegia vulgaris an E5 desaturase activity has been described (Longman et al. (2000) Biochem Soc Trans 28: 641-643). There are but no plants have been described either trans-vaccenic acid (E11 octadecenoic acid) or E10 octadecenoic acid.

• 1. In Plastiden werden Fettsäuren-ACP-Ester durch eine lösliche Desaturase vorzugsweise an Position 9 desaturiert und
• 2. am Endoplasmatischen Reticulum werden Membranlipide, vor allem Phosphatidylcholine, durch Membran-gebundene Desaturasen bevorzugt weiter an Positionen 6, 12 und 15 desaturiert.

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 desatured and preferably at position 9 by a soluble desaturase
• 2. on the endoplasmic reticulum, membrane lipids, especially phosphatidylcholines, are preferably further desaturated at positions 6, 12 and 15 by membrane-bound desaturases.

In some plants, the conjugated fatty acids Activity of a conjugase produced (Crombie et al. (1984) J Chem Soc Chem Commun 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 Punicic acid runs through the desaturation of oleic acid to linoleic acid through a Δ12 desaturase and a further desaturation, combined with a rearrangement of the Z9 or Z12 double bond to Conjugate fatty acid through a specific conjutia-forming Desaturase (conjugase). In addition to the production of calendulic acid is published by Qui et al. (2001) Plant Physiol 125: 847-855) also the Production of conjugated linoleic acid by the enzymatic Activity of conjugase described. The disadvantage of this Side activity, however, that the enzymatic effect unwanted 8,10-isomer of conjugated linoleic acid is formed.

Lepidoptera pheromone desaturases show great diversity of substrate specificities and desaturation mechanisms (Roelofs and Wolf (1988) J Chem Ecol 14: 2019-2031; Roelofs (1995) Proc Nat Acad Sci USA 92: 44-49; Tillman et al. (1999) Insect Biochem 29: 481-514). These enzyme activities cause the production of unusual unsaturated fatty acid CoA derivatives various chain lengths and with double bonds to different Positions and with different configurations. This act as a starting material for the biosynthesis of pheromones. Liu et. al describe an E11 desaturase from the pheromone gland Moth species ("light brown apple moth"), which is believed to have a function in pheromone biosynthesis (Liu WT et al. (2002) Proc Natl Acad Sci USA 99 (2): 620-624.

The task was therefore to prepare new processes make the trigylcerides with a high content of unsaturated Fatty acids, preferably conjugated polyunsaturated Lead fatty acids like CLA. This task is accomplished by the present Invention solved.

A first object of the invention relates to methods for 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 a. in plants, the fatty acids are in Cytosol mainly as CoA ester. In yeast and animals represents the desaturation of acyl-CoA fatty acids Main synthetic route for unsaturated fatty acids while in plants this Mechanism is rather rare (Cahoon EB et al. (2000) Plant Physiol. 124: 243-251). Surprisingly it could be shown that the transgenic expression of desaturases from Lepidoptera too desaturation of saturated and unsaturated fatty acids CoA ester leads. This ultimately leads to both being active CLA isomers produced and incorporated into the storage lipids become.

The method according to the invention has the particular advantage that starting from organisms, preferably plants, with a high oil content, such as rapeseed or sunflower, directly containing CLA Triglycerides can be produced. The usage eukaryotic enzymes from insects of the order Lepidoptera enable one good expression without those with prokaryotic proteins often associated toxic effects.

"Fatty acid desaturases" means enzymes that are capable of Fatty acids or their derivatives, such as preferred Fatty acid CoA esters to introduce a double bond. You can optionally cofactors such as NADPH, NADH or else Oxygen may also be required. Such desaturases are involved preferred, which can use acyl-CoA fatty acids as substrate, whose fatty acid has a chain length of 14, 16, 18 or 20 carbon atoms has, preferably 18 carbon atoms.

The term fatty acid desaturases preferably includes such Enzymes that are able to convert into fatty acids or their derivatives, such as preferably fatty acid CoA esters, a To generate a double bond at position C8, C9, C10, C11 or C12. Desaturases which are too targeted are also preferred Structural isomers, i.e. specific to cis or trans double bonds to lead. Specific means that the respective structural isomer at least 60%, preferably at least 80%, very particularly preferably at least 90%, most preferably at least 95% arises. Desaturases which are particularly preferred are those in Fatty acids or their derivatives, such as preferably fatty acid CoA esters generate a double bond as they do in a CLA Isomer occurs.

• a) Spezifische Erzeugung einer cis-Doppelbindung in Position C8, C9, C10, C11 oder C12 oder einer trans-Doppelbindung in Position C8, C9, C10, C11 oder C12.
• b) Substratspezifität für Fettsäuren, Fettsäure-CoA-Ester oder andere Fettsäurederivate mit einer Kettenlänge der Fettsäure von 16 und/oder 18 C-Atomen. Substratspezifität meint dabei die Eigenschaft einer Fettsäuredesaturase, Substrate mit der angegebenen Kettenlänge schneller umzusetzen als Substrate mit anderer Kettenlänge. Dabei ist die Umdrehungsgeschwindigkeit des bevorzugten Substrates gegenüber den nicht-bevorzugten Substraten um mindestens 50%, bevorzugt mindestens 100%, ganz besonders bevorzugt mindestens 200%, am meisten bevorzugt mindesten 500% erhöht.

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

• a) Specific generation of a cis double bond in position C8, C9, C10, C11 or C12 or a trans double bond in position C8, C9, C10, C11 or C12.
• b) 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 preferred in each case given.

Fatty acid desaturases include enzymes that are isolated Can introduce double bonds, but also conjugases that start out a conjugate of a double bond in a substrate Can generate double bond system. The first one Preferred double bond shifted.

• a) eine Z11-Doppelbindung in zwei E10 und Z12 Doppelbindungen umwandeln ("Z11-(E10,Z12)-Konjugase"),
• b) eine E11-Doppelbindung in zwei E10 und Z12 Doppelbindungen umwandeln ("E11-(E10,Z12)-Konjugase"),
• c) eine Z10-Doppelbindung in zwei E10 und Z12 Doppelbindungen umwandeln ("Z10-(E10,Z12)-Konjugase").

For example, conjugases which are preferred

• a) convert a Z11 double bond into two E10 and Z12 double bonds ("Z11 (E10, Z12) conjugase"),
• b) converting an E11 double bond into two E10 and Z12 double bonds ("E11- (E10, Z12) conjugase"),
• c) convert a Z10 double bond into two E10 and Z12 double bonds ("Z10 (E10, Z12) conjugase").

In summary, these enzymes can be called (E10, Z12) conjugases be designated. These enzymes can also be called E10 desaturases be valid. The essential property of the very special preferred E10 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 has a chain length of 18 carbon atoms.

• a) eine Z10-Doppelbindung in zwei Z9 und E11 Doppelbindungen umwandeln ("Z10-(Z9,E11)-Konjugase"),
• b) eine E10-Doppelbindung in zwei Z9 und E11 Doppelbindungen umwandelt ("E10-(Z9,E11)-Konjugase")
• c) eine Z11-Doppelbindung in zwei Z9 und E11 Doppelbindungen umwandelt ("Z11-(Z9,E11)-Konjugase").

Also preferred are conjugases that

• a) convert a Z10 double bond into two Z9 and E11 double bonds ("Z10 (Z9, E11) conjugase"),
• b) converts an E10 double bond into two Z9 and E11 double bonds ("E10- (Z9, E11) conjugase")
• c) converts a Z11 double bond into two Z9 and E11 double bonds ("Z11 (Z9, E11) conjugase").

In summary, these enzymes can be called (Z9, E11) conjugases be designated. These enzymes can also be called E11 desaturases be valid. The essential property of the very special preferred E11 desaturases is the introduction of a trans double bond in position C11 of a fatty acid or a fatty acid CoA ester, the fatty acid has a chain length of 18 carbon atoms.

Most preferred are E10, E11, Z10 and Z11 desaturases, and Z11 (E10, Z12) conjugase, E11 (E10, Z12) conjugase, Z10 (Z9, E11) conjugase and E10 (Z9, E11) conjugase with one 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, Gracillariidae, Hepialidae, Ithomiidae, Lasiocampidae, Lycaenidae, Lymantriidae, Lyonetiidae, Nepticulidae, Noctuidae, Notodontidae, Nymphalidae, Oecophoridae, Papilionidae, Pieridae, Psychidae, Pterophoridae, Pyralidae, Saturniidae, Sesiidae, Sphingidae, Tortricidae, Yponomeutidae and Zygaenidae. Are particularly preferred Fatty acid desaturases selected from a Lepidoptera family the group consisting of Tortricidae, Pyralidae, Papilionidae, Noctuidae and Geometridae.

E11 desaturases or those coding for them Nucleic acid sequences are preferred from organisms of the Diaphania species hyalinata, Diaphania nitidalis, Leucinodes orbonalis, Ostrinia nubilalis, Sesamia grisescens, Brachmia macroscopa, Paraargyresthia japonica, Mnesictena flavidalis, Telorta edentata, Epiplema 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 isolated. The E11 desaturases are particularly preferred or the nucleic acid sequences coding therefor from the Pheromone glands of the aforementioned insects isolated. The example is E11 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) call. The E11 desaturases from the organisms mentioned take them vegetable acyl-CoA fatty acid derivatives as a substrate.

E10 desaturases or those coding for them Nucleic acid sequences are preferred from organisms of the dichrocrocis species chlorophanta, dichrocrocis punctiferalis, bombyx mandarina, Bombyx mori, Coloradia velda, Hemileuca eglanterina, Hemileuca electra electra, Hemileuca electra mojavensis, Hemileuca nuttalli or Notarcha derogata isolated. The are particularly preferred E10 desaturases or the nucleic acid sequences coding therefor isolated from the pheromone glands of the aforementioned insects.

E9 desaturases or those coding for them Nucleic acid sequences are preferred from organisms of the species Epiplema plaqifera, Phyllonorycter coryli, Phyllonorycter harrisella, Phyllonorycter sylvella, Gelechiinae, Bryotropha sp., Bryotropha terrella, Gelechia betulae, Adoxophyes orana, Exartema appendiceum, Zeiraphera canadensis, Loxostege neobliteralis, Ostrinia nubilalis, Dioryctria clarioralis, Dioryctria merkeli, Dioryctria resinosella, Spodoptera exigua, Spodoptera triturata or Polia grandis isolated. The E9 desaturases and the nucleic acid sequences encoding it from the pheromone glands isolated insects.

E8 desaturases or those coding for them Nucleic acid sequences are preferred from organisms of the phyllonorycter species saportella, Phyllonorycter sp. or Dichrocrocis punctiferalis isolated. The E8 desaturases and the nucleic acid sequences encoding it from the pheromone glands isolated insects.

Z11 desaturases as used for the production of Z11 octadecenoic acid can be used advantageously are described for Bombyx mori (Ando et al. (1988) Agric Biol Chem. 52: 473-478), Trichoplusia ni and Helicoverpa zea (Knipple et al. (1998) Proc Nat Acad Sci USA 95: 15287-15292). Other Z11 desaturases or for them coding nucleic acid sequences can preferably from Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutella xylostella, Bombyx mori or Diaphania nitidalis be isolated. The desaturases from Helicoverpa are exemplary zea (SEQ ID NO: 8), Trichoplusia ni (SEQ ID NO: 10) and Argyrotaenia velutinana (SEQ ID NO: 12). Particularly preferred become Z11 desaturases or the coding for them Nucleic acid sequences isolated from the pheromone glands of the aforementioned insects.

Z10 desaturases or those coding for them Nucleic acid sequences are preferred from organisms of the species Ctenopseustis filicis, Pseudexentera spoliana, Hemileuca eglanterina, Hemileuca electra electra, Hemileuca electra mojavensis, Hemileuca nuttalli, Eurhodope advenella, Mamestra configurata or Dichrocrocis punctiferalis isolated, particularly preferably from the Pheromone glands of the aforementioned insects. The desaturase is an example Planototrix octo (SEQ ID NO: 14).

(E10, Z12) conjugases or those coding for them Nucleic acid sequences can advantageously be from Bombyx mori, Phyllonorycter crataegella, Amorbia cuneana, Notocelia incarnatana, Notocelia uddmanniana, Bombyx mandarina, Coloradia velda, Hemileuca eglanterina, Hemileuca electra electra, Hemileuca 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 to be isolated. The are particularly preferred (E10, Z12) conjugases or the coding for them Nucleic acid sequences isolated from the pheromone glands of the aforementioned insects.

(Z9, E11) conjugases or those coding for them Nucleic acid sequences can advantageously be obtained from Diatraea saccharalis, Xyrosaris lichneuta, Dioryctria abietella, Stenoma cecropia, Phalonidia manniana, Peternophorus vilis, Dioryctria abietella; Dioryctria rubella, Myelopsis tetricella, Jodis lactearia, Scopula personata, Spodoptera descoinsi, Spodoptera eridania, Spodoptera latifascia, Spodoptera littoralis or Spodoptera litura isolated become. The (Z9, E11) conjugases or the nucleic acid sequences coding therefor from the Pheromone glands of the aforementioned insects isolated.

Both Z11 desaturases and Z11 conjugases take the vegetable acyl-CoA fatty acid derivatives as a substrate, and the combined expression of these two classes from desaturases Lepidoptera in plants leads to the production of (10E, 12Z) -CLA, which is built into the storage lipids.

• 1. Ausgehend von Stearinsäure über trans-Desaturierung an Position C-11 - beispielsweise durch eine E11-Desaturase - trans- Vaccensäure hergestellt werden (Schema 3).
  Schema 3 Biosynthesewege zur Herstellung von (9Z,11E)-CLA und (10E,12Z)-CLA ausgehend von Stearinsäure
  
  Ausgehend von Palmitinsäure wird über trans-Desaturierung an Position C-9 - beispielsweise durch eine E9-Desaturase - Palmitelaidinsäure (9E-Hexadecensäure) hergestellt. Diese kann anschließend über die enzymatische Aktivität einer Elongase zu trans-Vaccensäure verlängert werden (Schema 4).
  Schema 4 Biosynthesewege zur Herstellung von (9Z,11E)-CLA und (10E,12Z)-CLA ausgehend von Palmitinsäure
  
  Trans-Vaccensäure kann dann von einer Δ9-Desaturase zum (9Z,11E)-CLA Isomer umgesetzt werden. Für Säugetiere und Mensch konnte gezeigt werden, dass trans-Vaccensäure durch die enzymatische Aktivität einer endogenen Δ9-Desaturase zu CLA umgesetzt wird (WO 99/20123; Santora JE et al. (2000) J Nutr 130: 208-215; Adlof RO et al. (2000) Lipids 35: 131-135). Ebenso konnte gezeigt werden das Insektenzellen die eine E11-Desaturase exprimieren, das entstandene E11-Fettsäure- Edukt in ein Z9, E11-Fettsäure umwandeln können (Liu WT et al. (2002) Proc Natl Acad Sci USA 99(2): 620-624. Pflanzliche Δ9-Desaturasen können ebenso die Umsetzung von trans-Vaccensäure zum (Z9,E11)-CLA Isomer katalysieren. Diese Umsetzung kann in einer besonders bevorzugten Ausführungsform durch zusätzliche Expression einer Z9-Desaturase weiter gesteigert werden. Bevorzugt sind dabei zytosolisch-aktive Z9-Desaturasen, wie beispielsweise die Z9-Desaturase aus Hefe. Die Produktion des (Z9,E11)-CLA Isomers in Pflanzen ist also möglich durch die Expression einer trans-Desaturase. Das besonders vorteilhafte (9Z,11E)-CLA Isomer wird dabei unter Einfluss pflanzlicher Z9-Desaturasen gebildet.
• 2. Ausgehend von Stearinsäure wird zunächst über trans-Desaturierung an Position C10 die entsprechende E10-Octadecensäure gebildet (Schema 3). Alternativ kann ausgehend von Palmitinsäure wird über trans-Desaturierung an Position C8 E8-Hexadecensäure hergestellt. Diese kann anschließend über die enzymatische Aktivität einer Elongase zu E10-Octadecensäure verlängert werden (Schema 4). Diese Fettsäure wird durch die Aktivität einer Δ12-Desaturase zum (E10,Z12)-CLA Isomer umgesetzt werden. Diese Reaktion wird auch durch pflanzliche Δ12-Desaturasen katalysiert. Diese Umsetzung kann in einer besonders bevorzugten Ausführungsform durch zusätzliche Expression einer Z12-Desaturase weiter gesteigert werden.
• 3. Es können auch entsprechende (E10,Z12)-Konjugasen aus Lepidoptera eingesetzt werden. Diese wandeln beispielsweise Z11- Octadecensäure (cis-Vaccensäure), E11-Octadecensäure (trans- Vaccensäure) oder Z10-Octadecensäure in (E10,Z12)-CLA um. Dazu wird zunächst Stearinsäure unter Wirkung einer Z11-Desaturase zu Z11-Octadecensäure, wie oben beschrieben zu trans- Vaccensäure oder durch eine Z10-Desaturase zu Z10-Octadecensäure umgesetzt.
• 4. Ferner können auch (Z9,E11)-Konjugasen aus Lepidoptera vorteilhaft mit (Z11)-, (Z10)- oder (E10)-Desaturasen kombiniert werden. Diese Desaturasen produzieren ausgehend von Stearinsäure Z11-Octadecensäure (cis-Vaccensäure), Z10-Octadecensäure oder E10-Octadecensäure, welche anschließend durch eine (Z9/E11)-Konjugase zu (Z9,E11)-CLA umgesetzt werden.

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-11 - for example by an E11-desaturase - trans-vaccenic acid can be prepared (Scheme 3).
  Scheme 3 Biosynthetic pathways for the production of (9Z, 11E) -CLA and (10E, 12Z) -CLA from stearic acid
  
  Starting from palmitic acid, palmitelaidic acid (9E-hexadecenoic acid) is produced via trans-desaturation at position C-9, for example by means of an E9 desaturase. This can then be extended to trans-vaccenoic acid via the enzymatic activity of an elongase (Scheme 4).
  Scheme 4 Biosynthetic pathways for the production of (9Z, 11E) -CLA and (10E, 12Z) -CLA 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 E11 desaturase can convert the resulting E11 fatty acid starting material into a Z9, E11 fatty acid (Liu WT et al. (2002) Proc Natl Acad Sci USA 99 (2): 620- 624. Plant Δ9-desaturases can also catalyze the conversion of trans-vaccenic acid to the (Z9, E11) -CLA isomer, which in a particularly preferred embodiment can be further increased by additional expression of a Z9-desaturase, cytosolic-active being preferred Z9 desaturases, such as, for example, yeast Z9 desaturase. The production of the (Z9, E11) -CLA isomer in plants is therefore possible by expression of a trans-desaturase. The particularly advantageous (9Z, 11E) -CLA isomer is thereby formed under the influence of vegetable Z9 desaturases.
• 2. Starting from stearic acid, the corresponding E10 octadecenoic acid is first formed via trans-desaturation at position C10 (Scheme 3). Alternatively, starting from palmitic acid, trans-desaturation at position C8 produces E8-hexadecenoic acid. This can then be extended to E10 octadecenoic acid via the enzymatic activity of an elongase (Scheme 4). This fatty acid is converted to the (E10, Z12) -CLA isomer by the activity of a Δ12 desaturase. This reaction is also catalyzed by plant Δ12 desaturases. In a particularly preferred embodiment, this conversion can be further increased by additional expression of a Z12 desaturase.
• 3. Corresponding (E10, Z12) conjugases from Lepidoptera can also be used. These convert, for example, Z11 octadecenoic acid (cis-vaccenoic acid), E11 octadecenoic acid (trans-vaccenoic acid) or Z10 octadecenoic acid into (E10, Z12) -CLA. For this purpose, stearic acid is first converted to Z11 octadecenoic acid under the action of a Z11 desaturase, as described above to trans-vaccenoic acid or by Z10 desaturase to Z10 octadecenoic acid.
• 4. Furthermore, (Z9, E11) conjugases from Lepidoptera can also advantageously be combined with (Z11), (Z10) or (E10) desaturases. These desaturases produce Z11-octadecenoic acid (cis-vaccenoic acid), Z10-octadecenoic acid or E10-octadecenoic acid starting from stearic acid, which are then converted to (Z9, E11) -CLA by a (Z9 / E11) conjugase.

The CLA fatty acids or their derivatives such as CoA Fatty acid esters are found in membrane lipids and triacylglycerides saved.

The desirable enzyme activities could be found in insects Order Lepidoptera, especially in the above Insect species can be localized. For this purpose, the under this Test systems provided invention used (Example 2, 3rd and 4).

The enzymes coding for these activities, or those for them coding nucleic acid sequences can in the expert in a familiar manner isolated from the corresponding organisms be, or by mutagenesis from corresponding known sequences be derived.

Finding a protein sequence or a corresponding one cDNA sequence for an enzymatic activity, functionality or phenotype is a classic task of biochemistry and molecular biology. Various methods are known to the person skilled in the art known how he can solve this task. This procedure are based, for example, on that available within the scope of this invention tests for the determination of desaturase or conjugase Activity (see, inter alia, Example 3). The determination of each specific activity of a desaturase / conjugase takes place via the analysis of the fatty acid pattern for example Gas chromatography as described in Examples 2, 3 or 4. Under Using this test it is possible to start from one biological material in which a desaturase or conjugase activity was detected, the corresponding protein or Nucleic acid required for a protein with desaturase or conjugase activity encodes, isolates and analyzes.

The method of expression cloning can be exemplified ("expression cloning") apply. This method is often one of them have been applied, starting from a certain enzymatic Activity, a certain functionality or a certain Phenotype, the gene responsible for it or the isolate corresponding cDNA (Dalboge H (1997) FEMS Microbiology Reviews 21 (1): 29-42, Simonsen H and Lodish HF (1994) Trends Pharmacol Sci 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 70 (5): 829-840; Lemaire P et al. (1995) Cell 81 (1): 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 procedure of the Expression cloning uses common recombination and Cloning techniques such as those used 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 Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience are described.

Generally one can start from an organism, cells or Tissues used to produce a desaturase or Conjugase activity are capable of producing an expression library. Examples are isolated in the manner familiar to the person skilled in the art initially mRNA or preferably poly (A) mRNA from said organism, Cells or tissue and uses them to produce cDNA (Gubler U, Hoffman BJ (1983) Gene 25: 263-269). Different systems for mRNA or poly (A) mRNA isolation are known to the person skilled in the art and commercially available. The synthesis with the "Quick Prep Micro mRNA Purification Kit" (Amersham Pharmacia Biotech). The first strand cDNA synthesis is preferably with an oligo (dT) primer using a reverse Transcriptase carried out (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 commercial available to generate complete cDNAs ("fulllength cDNAs") enable. The "SMART ™ cDNA Library Construction Kit "(Clontech, Cat. # K1051-1). This uses SMART ™ technology (SMART = Switch Mechanism At the 5 'end of RNA templates) for the generation of cDNA libraries (Herrler M (2000) J Mol Med 78 (7): B23). Here come certain Oligonucleotides and the method of long-range PCR ("long-distance PCR"). The procedure is in Example 4 described.

Adapters can be attached to the double-stranded cDNAs Single strand overhangs are ligated that exemplify cloning in an expression vector cut with restriction enzymes and has compatible cohesive ends. Prefers the cloning is directed so that the reading direction is fixed and a possibly present promoter sense-RNA generated from the cDNA insertion. In the example used SMART ™ system compatible SfiI (A) and SfiI (B) - Overhangs for directional cloning into one with SfiI (Recognition sequence GGCCNNNN! NGGCC) cut vector used. One cDNA molecule is cloned into one vector molecule, see above that ultimately a multitude is in the integrated cDNA discriminating vectors, based on the same basic vector, are present, which form the expression library. In this Expression library also contain vectors that contain a Can express desaturase or conjugase transgenes. Are principally suitable for all expression vectors which are able to Desaturases or conjugases in an active form in a transformed To express the organism transgenically. The lambda is an example TriplEX2 vector (after Excision pTriplEX2 vector; manufacturer Clontech) for transgenic expression in E. coli, or the vector pYES2 (Manufacturer: Invitrogen) for transgenic yeast S. cerevisiae called (see example 4).

If necessary, one can carry out a normalization to To compensate for differences in the level of expression of individual genes, so that the cDNA to every expressed gene - regardless of the actual expression level - with a similar copy number in the expression library is represented. For the production of poly (A) mRNA, cDNA and normalized cDNA are different Systems described (US 5,482,845) (Soares MB et al. (1994) Proc Natl Acad Sci USA 91: 9228-9232; Carninci P et al. (2000) genomes Res 10: 1617-1630; Bonaldo MF et al. (1996) Genome Res 6: 791-806).

Alternatively, you can also use a subtractive expression library make. Here you compare the cDNAs from an organism, Cells or tissue that contains a desaturase or conjugase, with the cDNAs of an organism, cells or tissue that Activity does not have and if possible of the same genus and Is kind. Methods are known to the person skilled in the art as to how the Can isolate cDNAs that are only in the biological material of desaturase or conjugase activity are expressed. Appropriate methods and systems have been described and are commercial available. The "PCR-Select ™ cDNA Subtraction is exemplary Kit "(manufacturer: Clontech) or" Subtractor ™ Kit "(manufacturer: Invitrogen), which is up to more than 1000 times Enrichment of rare and / or selectively expressed cDNAs enabled. Using subtractive hybridization, all of these cDNAs removed that are present in both biological materials (Chu ZL et al. (1997) Proc Nat Acad Sci USA 94: 10057-10062; Hudson C et al. (1997) Cell 91: 397-405; Mueller CGF et al. (1997) J Exp Med 186: 655-663; by Stein OD et al. (1997) Nucleic Acids Res 25: 2598-2602; Wong BR et al. (1997) J Biol Chem 272: 25,190 to 25,194; Yokomizo T et al. (1997) Nature 387: 620-624; Zhicheng S and Jacobs-Lorena M (1997) J Biol Chem 272: 28895-28900).

Transgenic expression in the expression library cDNAs contained can be exemplified in prokaryotic or eukaryotic cells can be realized, which is then tested on the desaturase or conjugase protein, for example a test for desaturase or conjugase activity.

Transgenic to isolate the desaturase or conjugase expressing vectors from the expression library, the Expression library preferably transformed into an organism. Individual cells of said one are preferably transformed Organism in such a way that every cell or everyone emanating from this cell regenerated organism with only one kind of vector molecule is transformed. In principle, all are organisms or of them derived cells suitable for the transformation in the Are able to transgene an active desaturase or conjugase express. These can be prokaryotic and eukaryotic Be organisms. All plants derived from them are preferred Cells, but also other photosynthetic organisms, such as Example algae. Here are exemplary but not restrictive Systems called 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 organisms such as Synechocystis based. Eukaryotic organisms are preferred, eukaryotic organisms which are particularly preferred are those which are able to fatty acids or acyl-CoA fatty acids synthesize. Those used for transformation are preferred Expression vectors a selection marker, for example one Antibiotic resistance or an amino acid synthesis gene for selection Amino acid deficiency that makes a selection successful transformed cells. Other methods of selection are described below.

In a preferred embodiment, the Expression cloning can be done in yeast. This can be exemplified by the the company's pYES2-based yeast expression system Invitrogen can be used. pYES2 is a "high-copy" episomal Vector for the inducible expression of recombinant proteins in S. cerevisiae. Transformed yeast strains are selected based on uracil deficiency (pYES2 carries the ura3 gene).

More preferred expression vectors and transfection methods to produce the transformed organisms commonly used for the production of transgenic organisms Method.

From the total number of organisms transformed or from them derived cells, those are enriched and isolated that are capable of a desaturase or conjugase to express transgene and / or a corresponding desaturase or conjugase activity.

In general, the detection of desaturase activity can thereby done that you have a transformed organism or from it derived cells cultured in a suitable buffer or Disintegrates solvent, the digestion with fatty acids or Acyl-CoA fatty acids and optionally with a co-factor such as Connects NADH or NADPH or oxygen and the resulting desaturated fatty acid or acyl-CoA fatty acids prove. The fatty acids or acyl-CoA fatty acids can be preferred come from the transformed organism itself if the Organism or the cell derived from it are able to to synthesize fatty acids or acyl-CoA fatty acids themselves. Otherwise, fatty acids or acyl-CoA fatty acids can also be added.

Detection of those modified by the deaturase or conjugase Fatty acid or acyl-CoA fatty acid can, if necessary, after Extraction from the incubation approach, for example with a Solvents such as ethyl acetate, over conventional ones known to the person skilled in the art Methods are done. Separation methods such as High pressure liquid chromatography (HPLC), gas chromatography (GC), Thin layer chromatography (DC) and detection methods such as how Mass spectroscopy (MS or MALDI), UV spectroscopy or Autoradiography are used. Detection is preferred under by gas chromatography as in Examples 3, 4 or 5 described.

Isolation of the transformed organisms or from them derived cells that are capable of a desaturase or Transgenic expression of conjugase can be exemplified thereby happen that you have the total number of cells in subgroups divided, these sub-groups, if necessary, cultivated separately and the desaturase or conjugase activity of the individual Measures subgroups. The subset in which a cell type is present, the a desaturase or conjugase protein in functionally active Form expressed transgenically, has an increased desaturase or Conjugase activity. This subgroup is further divided and repeating the process until you get one monoclonal culture d. H. a culture that is only one Vector with a specific one for a desaturase or conjugase encoding cDNA contains.

From the transformed organism or cell, the vector, which encodes the nucleic acid for desaturase or conjugase contains can be recovered. This can be done using, for example Polymerase chain reaction (PCR). For this you use Oligonucleotide primers that lead to vector sequences that contain the desaturase or flank the conjugase cDNA insert are complementary. The Knowledge of the desaturase or conjugase nucleic acid sequence not necessary for this. Alternatively, the vector can if it doesn't is integrated into the genome of the host, from the cells recovered, transformed into E.coli, propagated and sequenced, the coding for the desaturase or conjugase Obtain nucleic acid sequence. Also from the isolated vector Desaturase or conjugase nucleic acid sequence up described methods without knowing their sequence sequence by means of PCR be isolated. Linkers can also be added after the PCR reactions the amplicon will be added which is a directional cloning enable. 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 Way. The cDNA selectively amplified by PCR Desaturase or conjugase can be used without prior Sequence elucidation can be cloned directly into a vector which is used for Production of a transgenic, a desaturase or conjugase expressing organism is suitable. This cloning can for example as a "blunt end" cloning using the Techniques familiar to those skilled in the art. For this you use common recombination and cloning techniques, as cited above.

The one described above can be particularly preferred Expression cloning can be carried out in the yeast Saccharomyces cerevisiae. Systems can also be used for this, in which the transgenic cDNAs to be expressed by homologous recombination in a integration platform generated (Lagarde D et al. (2000) Applied and Environmental Microbiology 66 (1): 64-72).

Alternatively, expression cloning can also be used one not amplified as described above Expression library can be made in vitro. Corresponding systems are described and commercially available. Be exemplary the "TNT ™ Coupled Reticulocyte Lysate System" (Promega; Promega Notes Number 67, 1998, p. 02). Kirschner et al. to have an in vitro approach (IVEC = "in vitro expression cloning") developed, which manages without living cells (US 5,654,150; King RW et al. (1997) Science 277, 973). The system is successful all have been applied 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). When "in vitro expression cloning "(IVEC) are small fractions of the Expression library used. These fractions of approx. 50 to 100 clones of cDNAs are contained in plasmids and are coupled using an in vitro Transcription / translation system based on reticolocyte lysate in your corresponding proteins translated. This is done as described above an oligo (dT) primed cDNA library was constructed and inserted into a high copy expression plasmid with a T3, T7 or SP6 promoter cloned. This plasmid library is then in E. coli transformed. Approx. 50 to 100 independent transformants each on an agar plate with the corresponding one Selection antibiotic cultivated up to a colony size of approx. 1 mm, collected and united. A part of these bacteria becomes plasmid DNA isolated. This plamid DNA is used as a template directly in one Reticolocyte system transcribed and translated. Details of the Procedures can, for example, the manufacturer's manual (TNT ™ Coupled Reticulocyte Lysate Systems Technical Bulletin # TB126, Promega Corporation) ". Depending on the number of "fulllength" cDNA clones in the library are approximately 30 to 50 Proteins synthesized in every single batch. The produced Proteins are labeled for their desaturase or conjugase Activity checked. Individual approaches that have an increased desaturase or Conjugase activity are further subdivided. Subdivision of the individual approaches leads to that for a desaturase or CDNA encoding conjugase. From those in this preparation Contained vectors can by means of PCR for desaturase or Nucleic acid encoding conjugase are amplified and - possibly without knowledge and analysis of the sequence - as above described for the preparation of a desaturase or conjugase transgenic organism can be used.

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

In a further advantageous application, the cDNAs can be used in be cloned a retroviral vector. This allows one highly efficient transformations of the cells. Retroviral Expression vectors and expression systems are described (Kitamura T, International Journal of Hematology 1998, 67: 351-359) and commercially available (for example from Clontech; New Retroviral & ClonCapture Expression Libraries; CLONTECHniques October 1998, XIII (4): 22-23). The transfection takes place first 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 transfected and regarding the desired activity. The inserts can then, for example, obtained via PCR and analyzed. Furthermore, you can - even without prior sequence analysis as above described cloned directly into a suitable expression vector which is then used to produce a desaturase or conjugase transgene-expressing organism can be used.

• a) eine Nukleinsäuresequenz kodierend für eine Z11-Desaturase aus Helicoverpa zea mit der SEQ ID NO: 7,
• b) eine Nukleinsäuresequenz kodierend für eine Z11-Desaturase aus Trichoplusia ni mit der SEQ ID NO: 9,
• c) eine Nukleinsäuresequenz kodierend für eine Z11-Desaturase aus Argyrotaenia velutinana mit der SEQ ID NO: 11 oder
• d) eine Nukleinsäuresequenz kodierend für eine Z10-Desaturase aus Planotortrix octo mit der SEQ ID NO: 13.

E10 or E11 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:

• a) a nucleic acid sequence coding for a Z11 desaturase from Helicoverpa zea with SEQ ID NO: 7,
• b) a nucleic acid sequence coding for a Z11 desaturase from Trichoplusia ni with the SEQ ID NO: 9,
• c) a nucleic acid sequence coding for a Z11 desaturase from Argyrotaenia velutinana with the SEQ ID NO: 11 or
• d) a nucleic acid sequence coding for a Z10 desaturase from Planotortrix octo with SEQ ID NO: 13.

• a) eine Nukleinsäuresequenz kodierend für eine E11-Desaturase aus Epiphyas postvittana mit der SEQ ID NO: 1,
• b) eine Nukleinsäuresequenz kodierend für eine E11-Desaturase aus Ostrinia nubilalis mit der SEQ ID NO: 3, oder
• c) eine Nukleinsäuresequenz kodierend für eine E11-Desaturase aus Ostrinia furnacalis mit der SEQ ID NO: 5.

E11 desaturases can also be changed 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 E11 desaturase from Epiphyas postvittana with the SEQ ID NO: 1,
• b) a nucleic acid sequence coding for an E11 desaturase from Ostrinia nubilalis with the SEQ ID NO: 3, or
• c) a nucleic acid sequence coding for an E11 desaturase from Ostrinia furnacalis with SEQ ID NO: 5.

It is also conceivable the specificity for the position in which the Double bond is introduced by the above desaturases, to change through mutagenesis.

Process for changing properties such as substrate specificity of desaturases by mutagenesis is known (Cahoon et al. (1997) Proc Nat Acad Sci USA 94: 4872-4877; WO 98/06735). Further suitable methods are for other enzymes of lipid metabolism such as described and transferable 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 carried out using Polymerase chain reaction using appropriate degenerate oligonucleotide primer from cDNA preparations - or banks of the corresponding Lepidoptera species mentioned above become. The oligonucleotide-primer pair are preferred described by SEQ ID NO: 15 and 16 or that the oligonucleotide Primer pair described by SEQ ID NO: 17 and 18 used.

• a) in ihren sense-Strang eine Sequenzmotiv beschrieben durch SEQ ID NO: 15 oder 17 enthält, oder
• b) in ihren antisense-Strang eine Sequenzmotiv beschrieben durch SEQ ID NO: 16 oder 18 enthält.

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 used in the method according to the invention preferred fatty acid desaturase from Lepidptera characterized in that it prefers a homology of at least 65% at least 70%, particularly preferably at least 80%, entirely 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 - optional and preferred - via at least one of the preferred essential properties of a desaturase or conjugase.

The proteins labeled in this way can also be natural or artificial mutations of any of the above Desaturase nucleic acid sequences and their homologues from other animal or Plant genera and species include. Include mutations Substitutions, additions, deletions, inversions or insertions one or more nucleotide residues. Where insertions, deletions or substitutions such as. B. transitions and transversions, in Techniques known per se, such as in vitro mutagenesis, "primer repair", restriction or ligation used become. Through manipulations, such as. B. restriction, "chewingback" or filling up overhangs for "blunt ends" complementary ends of the fragments are available for ligation be put. Analog results can also be found under Using the polymerase chain reaction (PCR) using specific oligonucleotide primers come.

In a further preferred embodiment, the Nucleic acid sequence coding for those in the method according to the invention fatty acid desaturase from Lepidptera preferably characterized in that it has a homology of at least 65%, preferably at least 70%, particularly preferred at least 80, very particularly preferably at least 90% of one of the Nucleic acid sequence described by SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and - optional and preferred - for a protein encodes that via at least one of the preferred essentials Features 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 is set by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) the following parameter is calculated:
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 those in the method according to the invention fatty acid desaturase from Lepidptera preferably characterized in that it works under standard conditions with one of the above coding for desaturases Nucleic acid sequence, preferably with the sequence according to SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 hybridizes.

• 1. Hybridisierungbedingungen mit zum Beispiel
  • a) 4 × SSC bei 65°C,
  • b) 6 × SSC bei 45°C,
  • c) 6 × SSC bei 68°C, 100 µg/ml denaturierter Fischsperma-DNA,
  • d) 4 × SSC, 50% Formamid, bei 42°C,
  • e) 2 × oder 4 × SSC bei 50°C (schwach stringente Bedingung), oder
  • f) 2 × oder 4 × SSC, 30 bis 40% Formamid, bei 42°C (schwach stringente Bedingung).
• 2. Waschschritte mit zum Beispiel
  • a) 0,1 × SSC bei 65°C, oder
  • b) 0,1 × SSC, 0,5% SDS bei 68°C, oder
  • c) 0,1 × SSC, 0,5% SDS, 50% Formamid bei 42°C, oder
  • d) 0,2 × SSC, 0,1% SDS bei 42°C, oder
  • e) 2 × SSC bei 65°C (schwach stringente Bedingung).

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 2 × SSC at 50 ° C.) and those with high stringency (with approximately 0.2 × SSC at 50 ° C., preferably at 65 ° C. C) (20 x SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0). In addition, 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 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) 4 × SSC at 65 ° C,
  • b) 6 × SSC at 45 ° C,
  • c) 6 × SSC at 68 ° C, 100 µg / ml denatured fish sperm DNA,
  • d) 4 × SSC, 50% formamide, at 42 ° C,
  • e) 2 × or 4 × SSC at 50 ° C (weakly stringent condition), or
  • f) 2 × or 4 × SSC, 30 to 40% formamide, at 42 ° C (weakly stringent condition).
• 2. Washing steps with for example
  • a) 0.1 × SSC at 65 ° C, or
  • b) 0.1 × SSC, 0.5% SDS at 68 ° C, or
  • c) 0.1 × SSC, 0.5% SDS, 50% formamide at 42 ° C, or
  • d) 0.2 × SSC, 0.1% SDS at 42 ° C, or
  • e) 2 × SSC at 65 ° C (weakly stringent condition).

“Nucleic acid sequence” means in the context of this invention for example a genomic or a complementary DNA (cDNA) sequence or an RNA sequence and semisynthetic or fully synthetic analogues from that. These sequences can be in linear or circular form, extrachromosomal or integrated into the genome. The Nucleotide sequences of the expression cassettes according to the invention or Nucleic acids can be made synthetically or naturally be obtained or a mixture of synthetic and contain natural DNA components, as well as from various heterologous gene segments from different organisms exist. Furthermore artificial nucleic acid sequences are suitable as long as they have the have the desired essential property. For example, you can synthetic nucleotide sequences are generated with codons that of plants to be transformed are preferred. This from Plants' preferred codons can be identified based on codon usage Codons with the highest protein frequency in the usual way be determined. Coding are particularly suitable Nucleotide sequences which are obtained by back-translating a polypeptide sequence the codon usage specific to the host plant were. To avoid unwanted herbal regulatory mechanisms can be avoided, for example, starting from the amino acid sequence of a desaturase from Lepidoptera insects under consideration of vegetable codon usage, translate back DNA fragments and from this the complete one, for use in the plant Create optimized exogenous desaturase sequence.

All of the nucleotide sequences mentioned above are in themselves known manner by chemical synthesis from the Nucleotide building blocks such as by fragment condensation of individual ones overlapping, complementary nucleic acid building blocks of the Double helix can be produced. Chemical synthesis of oligonucleotides can, for example, in a known manner, after the Phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, page 896-897). When preparing a Different DNA fragments can be manipulated in this way be that a nucleotide sequence with correct reading direction and correct reading frame is obtained. For connecting the Nucleic acid fragments among themselves can be attached to the fragments Adapters or linkers can be used. The attachment synthetic oligonucleotides and filling gaps using the Klenow fragment of DNA polymerase and ligation reactions and general cloning procedures are described in Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.

• a) die für eine Desaturase kodierende Nukleinsäuresequenz, oder
• b) eine mit der für eine Desaturase kodierenden Nukleinsäuresequenz funktionell verknüpfte genetische Kontrollsequenz, zum Beispiel ein Promotor, oder
• c) (a) und (b)

sich nicht in ihrer natürlichen, genetischen Umgebung befinden oder durch gentechnische Methoden modifiziert wurden, wobei die Modifikation beispielhaft eine Substitutionen, Additionen, Deletionen, Inversion oder Insertionen eines oder mehrerer Nukleotidreste sein kann. Natürliche genetische Umgebung meint den natürlichen chromosomalen Locus in dem Herkunftsorganismus oder das Vorliegen in einer genomischen Bibliothek. Im Fall einer genomischen Bibliothek ist die natürliche, genetische Umgebung der Nukleinsäuresequenz bevorzugt zumindest noch teilweise erhalten. Die Umgebung flankiert die Nukleinsäuresequenz zumindest an einer Seite und hat eine Sequenzlänge von mindestens 50 bp, bevorzugt mindestens 500 bp, besonders bevorzugt mindestens 1000 bp, ganz besonders bevorzugt mindestens 5000 bp. Eine natürlich vorkommende Expressionskassette - beispielsweise die natürlich vorkommende Kombination einer für eine Desaturase kodierenden Gensequenz mit ihrem natürlichen Promotor - wird zu einer transgenen Expressionskassette, wenn diese durch nicht-natürliche, synthetische ("künstliche") Verfahren wie beispielsweise einer Mutagenisierung geändert wird. Entsprechende Verfahren sind beschrieben (US 5,565,350; WO 00/15815; siehe auch oben). 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 this is changed by non-natural, synthetic ("artificial") methods such as, for example, mutagenization. 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 a nucleic acid sequence coding for a desaturase contain, as well as vectors comprising these expression cassettes.

In said transgenic expression cassettes, one stands for one Desaturase-encoding nucleic acid molecule preferably in functional linkage with at least one genetic Control element (for example a promoter) that the transgenic Expression (transcription and / or translation) of said desaturase in an organism, preferably in plants. Should the transgenic expression cassette inserted directly into the plant and the desaturase are expressed in plantae plant-specific genetic control elements (e.g. Promoters) preferred. However, desaturase can also be found in others Organisms or in vitro. In this are all prokaryotic or eukaryotic genetic controls (for example promoters) preferred, the expression in the Allow organism chosen for production.

A functional link is, for example the sequential arrangement of a promoter with the expressing desaturase nucleic acid sequence and possibly other regulatory elements such as a terminator such that each of the regulatory elements its function in the transgenic Expression of the nucleic acid sequence can meet. This is not necessarily a direct link in the chemical sense required. Genetic control sequences, such as Enhancer sequences can also function from further away Positions or even from other DNA molecules on the target sequence exercise. Arrays are preferred in which the transgene is too expressing nucleic acid sequence behind that as a promoter acting sequence is positioned so that both sequences are covalently linked. The distance is preferred between the promoter sequence and the transgene to be expressed Nucleic acid sequence less than 200 base pairs, especially preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.

Establishing a functional link as well Production of a transgenic expression cassette can be carried out using common recombination and cloning techniques are realized, as 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. Between the two But sequences can also be positioned, for example, the function of a linker with certain Restriction enzyme interfaces or a signal peptide. The insertion of sequences for the expression of Lead fusion proteins. Preferably, the transgenic Expression cassette, consisting of a link between promoter and expressing nucleic acid sequence integrated in a vector and through, for example, transformation into a vegetable Genome can be inserted.

However, there are also those under a transgenic expression cassette Understand constructions in which the nucleic acid sequence coding for a desaturase type behind an endogenous The promoter is placed under the expression of desaturase The endogenous promoter is checked. The through the insertion resulting fusion of endogenous promoter and Desaturase nucleic acid sequence is a transgenic expression cassette in the sense of Invention.

• a) Konstitutive Promotoren
  Bevorzugt sind Vektoren, die eine konstitutive Expression in Pflanzen ermöglichen (Benfey et al. (1989) EMBO J 8: 2195-2202). "Konstitutiver" Promotor meint solche Promotoren, die eine Expression in zahlreichen, bevorzugt allen, Geweben über einen größeren Zeitraum der Pflanzenentwicklung, bevorzugt zu allen Zeitpunkten der Pflanzenentwicklung, gewährleisten. Vorzugsweise verwendet man insbesondere einen pflanzlichen Promotor oder einen Promotor, der einem Pflanzenvirus entstammt. Insbesondere bevorzugt ist der Promotor des 35S-Transkriptes des CaMV Blumenkohlmosaikvirus (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewmaker et al. (1985) Virology 140: 281-288; Gardner et al. (1986) Plant Mol Biol 6: 221-228) oder der 19S CaMV Promotor (US 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J 8: 2195-2202). Ein weiterer geeigneter konstitutiver Promotor ist der "Rubisco small subunit (SSU)"-Promotor (US 4,962,028), der LeguminB-Promotor (GenBank Acc.-Nr. X03677), der Promotor der Nopalinsynthase aus Agrobacterium, der TR-Doppelpromotor, der OCS (Octopin Synthase) Promotor aus Agrobacterium, der Ubiquitin Promotor (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649), den Ubiquitin 1 Promotor (Christensen et al. (1992) Plant Mol Biol 18: 675-689; Bruce et al. (1989) Proc Natl Acad Sci USA 86: 9692-9696), den Smas Promotor, den Cinnamylalkoholdehydrogenase-Promotor (US 5,683,439), die Promotoren der vakuolärer ATPase Untereinheiten oder der Promotor eines prolinreichen Proteins aus Weizen (WO 91/13991), sowie weitere Promotoren von Genen, deren konstitutive Expression in Pflanzen dem Fachmann bekannt ist.
• b) Gewebespezifische Promotoren
  Bevorzugt sind ferner Promotoren mit Spezifitäten für die Antheren, Ovarien, Blüten, Blätter, Stengel, Wurzeln und Samen.
  Samenspezifische Promotoren
  wie zum Beispiel der Promotor des Phaseolins (US 5,504,200; Bustos mm et al. (1989) Plant Cell 1(9): 839-53), des 25 Albumingens (Joseffson LG et al. (1987) J Biol Chem 262: 12196-12201), des Legumins (Shirsat A et al. (1989) Mol Gen Genet 215(2): 326-331), des USP (unknown seed protein; Bäumlein H et al. (1991) Mol Gen Genet 225(3): 459-67), des Napin Gens (US 5,608,152; Stalberg K et al. (1996) L Planta 199: 515-519), des Saccharosebindeproteins (WO 00/26388) oder der Legumin B4-Promotor (LeB4; Bäumlein 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), der Oleosin-Promoter aus Arabidopsis (WO 98/45461), der Bce4-Promoter aus Brassica (WO 91/13980). Weitere geeignete samenspezifische Promotoren sind die der Gene kodierend für das "High Molecular Weight Glutenin" (HMWG), Gliadin, Verzweigungsenzym, ADP Glucose Pyrophosphatase (AG- Pase) oder die Stärkesynthase. Bevorzugt sind ferner Promotoren, die eine samenspezifische Expression in Monokotyledonen wie Mais, Gerste, Weizen, Roggen, Reis etc. erlauben. Vorteilhaft eingesetzt werden können der Promoter des lpt2 oder lpt1-Gen (WO 95/15389, WO 95/23230) oder die Promotoren beschrieben in WO 99/16890 (Promotoren des Hordein-Gens, des Glutelin-Gens, des Oryzin-Gens, des Prolamin-Gens, des Gliadin-Gens, des Glutelin-Gens, des Zein-Gens, des Kasirin-Gens oder des Secalin-Gens).
  Knollen-, Speicherwurzel- oder Wurzel-spezifische Promotoren wie beispielsweise der Patatin Promotor Klasse I (B33), der Promotor des Cathepsin D Inhibitors aus Kartoffel.
  Blattspezifische Promotoren
  wie Promotor der cytosolischen FBPase aus Kartoffel (WO 97/05900), der SSU Promotor (small subunit) der Rubisco (Ribulose-1,5-bisphosphatcarboxylase) oder der ST-LSI Promotor aus Kartoffel (Stockhaus et al. (1989) EMBO J 8: 2445-2451).
  Blütenspezifische Promotoren
  wie beispielsweise der Phytoen Synthase Promotor (WO 92/16635) oder der Promotor des P-rr Gens (WO 98/22593).
  - Antheren-spezifische Promotoren wie den 5126-Promotor (US 5,689,049, US 5,689,051), den globl Promotor und den γ-Zein Promotor.
• c) Chemisch induzierbare Promotoren
  Die transgenen Expressionskassetten können auch einen chemisch induzierbaren Promotor enthalten (Übersichtsartikel: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48: 89-108), durch den die Expression des exogenen Gens in der Pflanze zu einem bestimmten Zeitpunkt gesteuert werden kann. Derartige Promotoren, wie z. B. der PRP1 Promotor (Ward et al. (1993) Plant Mol Biol 22: 361-366), durch Salicylsäure induzierbarer Promotor (WO 95/19443), ein durch Benzolsulfonamid-induzierbarer Promotor (EP 0 388 186), ein durch Tetrazyklin-induzierbarer Promotor (Gatz et al. (1992) Plant J 2: 397-404), ein durch Abscisinsäure induzierbarer Promotor (EP 0 335 528) bzw. ein durch Ethanol- oder Cyclohexanon-induzierbarer Promotor (WO 93/21334) können ebenfalls verwendet werden.
• d) Stress- oder Pathogen-induzierbare Promotoren
  Ferner sind Promotoren bevorzugt, die durch biotischen oder abiotischen Stress induziert werden wie beispielsweise der pathogen-induzierbare Promotor des PRP1-Gens (Ward et al. (1993) Plant Mol Biol 22: 361-366), der hitzeinduzierbare hsp70- oder hsp80-Promoter aus Tomate (US 5,187,267), der kälteinduzierare alpha-Amylase Promoter aus der Kartoffel (WO 96/12814), der licht-induzierbare PPDK Promotor oder der verwundungsinduzierte pinII-Promoter (EP 375091).
  Pathogen-induzierbare Promotoren umfassen die von Genen, die infolge eines Pathogenbefalls induziert werden wie beispielsweise Gene von PR-Proteinen, SAR-Proteinen, β-1,3-Glucanase, Chitinase usw. (beispielsweise Redolfi et al. (1983) 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; Somssich et al. (1986) Proc Natl Acad Sci USA 83: 2427-2430; Somssich 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 Sci USA 91: 2507-2511; Warner, et al. (1993) Plant J 3: 191-201; Siebertz et al. (1989) Plant Cell 1: 961-968(1989).
  Umfasst sind auch verwundungs-induzierbare Promotoren wie der des pinII Gens (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498), des wun1 und wun2-Gens (US 5,428,148), des win1- und win2-Gens (Stanford et al. (1989) Mol Gen Genet 215: 200-208), des Systemin (McGurl et al. (1992) Science 225: 1570-1573), des WIP1-Gens (Rohmeier et al. (1993) Plant Mol Biol 22: 783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76), des MPI-Gens (Corderok et al. (1994) The Plant J 6(2): 141-150) und dergleichen.
• e) Entwicklungsabhängige Promotoren
  Weitere geeignete Promotoren sind beispielsweise fruchtreifung-spezifische Promotoren, wie beispielsweise der fruchtreifung-spezifische Promotor aus Tomate (WO 94/21794, EP 409 625). Entwicklungsabhängige Promotoren schließt zum Teil die Gewebespezifischen Promotoren ein, da die Ausbildung einzelner Gewebe naturgemäß entwicklungsabhängig erfolgt.

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 be constitutive, inducible or development-dependent, for example. Preferred are:

• a) Constitutive promoters
  Vectors which 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. In particular, a plant promoter or a plant virus-derived promoter is preferably used. 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; Shewmaker 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 ubiquitin 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 Sci 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.
  Seed-specific promoters
  such as the promoter of phaseoline (US 5,504,200; Bustos mm et al. (1989) Plant Cell 1 (9): 839-53), of 25 albumingen (Joseffson LG et al. (1987) J Biol Chem 262: 12196- 12201), leguminum (Shirsat A et al. (1989) Mol Gen Genet 215 (2): 326-331), 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; Bäumlein 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 lpt1 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, gliadin 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 (small subunit), the 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 globl 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 means of which the expression of the exogenous gene in the plant is controlled at a specific point in time can. Such promoters, such as. B. the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186), a tetracycline inducible promoter (Gatz et al. (1992) Plant J 2: 397-404), a promoter inducible by abscisic acid (EP 0 335 528) or a promoter inducible by ethanol or cyclohexanone (WO 93/21334) can also be used 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 PRP1 gene (Ward et al. (1993) Plant Mol Biol 22: 361-366), the heat-inducible hsp70 or hsp80 promoter from tomato (US 5,187,267), the cold-inducing alpha-amylase promoter from the potato (WO 96/12814), the light-inducible PPDK promoter or the wound-induced pinII promoter (EP 375091).
  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. (1983) 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; Somssich et al. (1986) Proc Natl Acad Sci USA 83: 2427-2430; Somssich 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 Sci 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 pinII gene (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498), the wun1 and wun2 genes (US 5,428,148), the win1 and win2 genes (Stanford et al. (1989) Mol Gen Genet 215: 200-208), the Systemin (McGurl et al. (1992) Science 225: 1570-1573), the WIP1 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 from tomato (WO 94/21794, EP 409 625). Development-dependent promoters partly include the tissue-specific promoters, since the formation of individual tissues is naturally development-dependent.

Further promoters suitable for expression in plants are (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 Sci USA 86: 8402-8406). Are particularly preferred constitutive and seed-specific promoters.

Further promoters can also functionally function with the expressing nucleic acid sequence linked to expression in other plant tissues or in other organisms, such as Example E.coli enable bacteria. As plant promoters in principle all promoters described above come into question.

The in the transgenic expression cassettes according to the invention or nucleic acid sequences containing vectors can with other genetic control sequences in addition to a promoter be functionally linked. The concept of genetic Control sequences is to be understood broadly and means all those sequences that an influence on the formation or the function of the have transgenic expression cassette according to the invention. genetic Control sequences modify, for example, the transcription and Translation in prokaryotic or eukaryotic organisms. The transgenes according to the invention preferably comprise Expression cassettes 5 'upstream from the respective transgene expressing nucleic acid sequence the promoter with specificity for the embryonic epidermis and / or the flower and 3'-downstream a terminator sequence as an additional genetic Control sequence, as well as any other usual regulatory Elements, each functionally linked to the transgene expressing nucleic acid sequence.

Genetic control sequences also include other promoters Promoter elements or minimal promoters that the can modify expression-controlling properties. So through genetic control sequences, for example the tissue-specific ones Expression also depend 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 SPO2, in the yeast or Fungus promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.

In principle, all natural promoters can with their Regulation sequences such as those mentioned above for the inventive Procedures are used. You can also synthetic promoters can be used advantageously.

Genetic control sequences also include the 5 'untranslated regions, introns or 3' non-coding region of Genes such as the Actin-1 intron or the Adh1-S introns 1, 2 and 6 (general: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). It is shown that these have significant functions in regulation of gene expression can play. So it was shown that 5'-untranslated sequences make the transient expression more heterologous Can amplify genes. Exemplary for translation amplifiers the 5 'leader sequence from the tobacco mosaic virus should be mentioned (Gallie et al. (1987) Nucl Acids Res 15: 8693-8711) and like. They can also promote tissue specificity (Rouster J et al. (1998) Plant J 15: 435-440).

The transgenic expression cassette can advantageously be a or several so-called "enhancer sequences" functionally linked to the promoter containing an increased transgenic Enable expression of the nucleic acid sequence. Also at the 3 'end of the Nucleic acid sequences to be expressed transgenically additional advantageous sequences are inserted, like others regulatory elements or terminators. The transgenic too expressing nucleic acid sequences can be in one or more copies in the Gene construct may be included.

Suitable polyadenylation signals as control sequences vegetable polyadenylation signals, preferably those which are in the essential T-DNA polyadenylation signals from Agrobacterium tumefaciens, especially 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 for the OCS is a particularly suitable terminator sequence (Octopine synthase) terminator and the NOS (nopaline synthase) terminator.

Control sequences are also to be understood as those which a homologous recombination or insertion into the genome of a Allow host organism or removal from the genome allow. Methods like cre / lox technology allow one 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 are certain flanking sequences added to the target gene (lox sequences), the later allow removal using the cre recombinase.

• a) Selektionsmarker, die eine Resistenz gegen einen Metabolismusinhibitor wie 2-Desoxyglucose-6-phosphat (WO 98/45456), Antibiotika oder Biozide, bevorzugt Herbizide, wie zum Beispiel Kanamycin, G 418, Bleomycin, Hygromycin, oder Phosphinotricin etc. verleihen. Besonders bevorzugte Selektionsmarker sind solche die eine Resistenz gegen Herbizide verleihen. Beispielhaft seien genannt: DNA Sequenzen, die für Phosphinothricinacetyltransferasen (PAT) kodieren und Glutaminsynthaseinhibitoren inaktivieren (bar und pat Gen), 5-Enolpyruvylshikimat-3-phosphatsynthasegene (EPSP Synthasegene), die eine Resistenz gegen Glyphosat® (N-(phosphonomethyl)glycin) verleihen, das für das Glyphosat® degradierende Enzyme kodierende gox Gen (Glyphosatoxidoreduktase), das den Gen (kodierend für eine Dehalogenase, die Dalapon inaktiviert), Sulfonylurea- und Imidazolinon inaktivierende Acetolactatsynthasen sowie bxn Gene, die für Bromoxynil degradierende Nitrilaseenzyme kodieren, das aasa-Gen, das eine Resistenz gegen das Antibiotikum Apectinomycin verleih, das Streptomycinphosphotransferase (SPT) Gen, das eine Resistenz gegen Streptomycin gewährt, das Neomycinphosphotransferas (NPTII) Gen, das eine Resistenz gegen Kanamycin oder Geneticidin verleiht, das Hygromycinphosphotransferase (HPT) Gen, das eine Resistenz gegen Hygromycin vermittelt, das Acetolactatsynthas Gen (ALS), das eine Resistenz gegen Sulfonylharnstoff-Herbizide verleiht (z. B. mutierte ALS-Varianten mit z. B. der 54 und/oder Hra Mutation).
• b) Reportergene, die für leicht quantifizierbare Proteine kodieren und über Eigenfarbe oder Enzymaktivität eine Bewertung der Transformationseffizienz oder des Expressionsortes oder -zeitpunktes gewährleisten. Ganz besonders bevorzugt sind dabei Reporter-Proteine (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(1): 29-44) wie das "green fluorescence protein" (GFP) (Sheen et al. (1995) Plant Journal 8(5): 777-784; Haseloff et al. (1997) Proc Natl Acad Sci USA 94(6): 2122-2127; Reichel et al. (1996) Proc Natl Acad Sci 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), die Chloramphenicoltransferase, eine Luziferase (Ow et al. (1986) Science 234: 856-859; Millar et al. (1992) Plant Mol Biol Rep 10: 324-414), das Aequoringen (Prasher et al. (1985) Biochem Biophys Res Commun 126(3): 1259-1268), die β-Galactosidase, R- Locus Gen (kodieren ein Protein, das die Produktion von Anthocyaninpigmenten (rote Färbung) in pflanzlichen Gewebe reguliert und so eine direkte Analyse der Promotoraktivität ohne Zugabe zusätzlicher Hilfstoffe oder chromogener Substrate ermöglicht; Dellaporta et al., In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium, 11: 263-282, 1988), ganz besonders bevorzugt ist die β-Glucuronidase (Jefferson et al. (1987) EMBO J 6: 3901-3907).
• c) Replikationsursprünge, die eine Vermehrung der erfindungsgemässen transgenen Expressionskassetten oder Vektoren in zum Beispiel E.coli gewährleisten. Beispielhaft seien genannt ORI (origin of DNA replication), der pBR322 ori oder der P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
• d) Elemente, die für eine Agrobakterium vermittelte Pflanzentransformation erforderlich sind, wie zum Beispiel die rechte oder linke Begrenzung der T-DNA oder die vir-Region.

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 confer resistance 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. Particularly preferred selection markers are those which confer resistance to herbicides. Examples include: DNA sequences which code for phosphinothricin acetyltransferases (PAT) and inactivate glutamine synthase inhibitors (bar and pat gene), 5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes) which have resistance to Glyphosat® (Ncin) phosphonomethyl lend the gox gene (glyphosate oxidoreductase) coding for the glyphosate®-degrading enzyme, the gene (coding for a dehalogenase which inactivates dalapon), sulfonylurea and imidazolinone-inactivating acetolactate synthases, and bxn genes for nitrodinase-degrading nitrodenzenzene Gene conferring resistance to the antibiotic apectinomycin, the streptomycin phosphotransferase (SPT) gene conferring resistance to streptomycin, the neomycin phosphotransferas (NPTII) gene conferring resistance to kanamycin or geneticidin, the hygromycin phosphotransferase (HPT) gene, the Resistance to hygromycin mediates the Acetolacta tsynthas gene (ALS), which confers resistance to sulfonylurea herbicides (e.g. B. mutated ALS variants with z. B. the 54 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 place or time of expression. Reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1): 29-44) such as the "green fluorescence protein" (GFP) (Sheen et al. (1995) Plant Journal 8 (5): 777-784; Haseloff et al. (1997) Proc Natl Acad Sci USA 94 (6): 2122-2127; Reichel et al. (1996) Proc Natl Acad Sci 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 β-galactosidase, R locus gene (encode a protein which regulates the production of anthocyanin pigments (red color) in plant tissue and thus a direct one Analysis of promoter activity possible without the addition of additional auxiliary substances or chromogenic substrates ht; Dellaporta et al., In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium, 11: 263-282, 1988), β-glucuronidase (Jefferson et al. (1987) EMBO J 6: 3901-3907).
• c) origins of replication, which ensure an increase in the transgenic expression cassettes or vectors according to the invention in, for example, E. coli. 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 transformed cells, it is usually necessary to use one selectable marker to introduce the successful recombined cells resistance to a biocide (for Example a herbicide), a metabolism inhibitor such as 2-Deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic. The Selection marker allows the selection of the transformed cells from untransformed (McCormick et al. (1986) Plant Cell Reports 5: 81-84).

• a) 5'-Pflanzenspezifischer Promotor/Desaturase/Terminator-3'

By way of 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 '

• a) 5'-35S-Promotor/Desaturase/OCS-Terminator-3', oder
• b) 5'-LeguminB-Promotor/Desaturase/NOS-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 promoter / desaturase / NOS terminator-3 '

For the advantageous method according to the invention for CLA Biosynthesis can co-transform with more than one of the Examples a) or b) above may be advantageous. Furthermore, the transformation with one or more vectors, each one Combination of the above-mentioned transgenic expression cassettes included, be advantageous.

In addition, said transgenic expression cassette can be a Contain nucleic acid sequence, their 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 from exemplary but not restrictive Nucleic acids coding for acetyl-CoA carboxylase (ACCase), Glycerol-3-phosphate acyltransferase (GPAT), Lysophosphatidate acyltransferase (LPAT), diacylglycerol acyltransferase (DAGAT) and phospholipid: diacylglycerol acyltransferase (PDAT).

The proOIL nucleic acid sequences also include such Nucleic acids whose transgene expression is an antisense RNA or generates double-stranded RNA, which also increases the fatty acid Production causes.

• a) 5'-35S-Promotor/Desaturase/OCS-Terminator/LeguminB- Promotor/proOIL/NOS-Terminator-3';
• b) 5'-35S-Promotor/proDesaturase oder Konjugase/OCS-Terminator/LeguminB-Promotor/proOIL/NOS-Terminator-3';

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 simultaneous Transformation of the plant with a desaturase and a fatty acid Biosynthesis-enhancing sequence proOIL.

Using the recombination and Cloning techniques can the desaturase according to the invention or OIL nucleic acids or transgenic expression cassettes in suitable vectors are cloned, their propagation, for example in E.coli. Suitable cloning vectors are u. a. pBR332, pUC series, M13mp series and pACYC184. Especially binary vectors which are suitable both in E. coli and in Can replicate agrobacteria.

The desaturase according to the invention or proOIL nucleic acids or Transgenic expression cassettes are preferred in suitable ones Transformation vectors inserted. Suitable vectors are below others in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), chap. 6/7, pp. 71-119 (1993). Examples and transformation methods are detailed below.

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

Under organism, parent or host organisms prokaryotic or eukaryotic organisms, such as Microorganisms or plant organisms understood.

Preferred microorganisms are bacteria, yeast, algae or Mushrooms.

Preferred bacteria are bacteria of the genus Escherichia, Corynebacterium, Bacillus, Clostrridium, Proionibacterium, Butyrivibrio, Eubacterium, Lactobacillus, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Phaeodactylum, Colpidium, Mortierella, Entomophthora, Mucor, Crypthecodinium or cyanobacteria for Example of the genus Synechocystis.

Especially preferred are microorganisms that are used for infection of plants and thus for the transfer of the invention Constructs are capable. Preferred microorganisms are those from the genus Agrobacterium and in particular from Art Agrobacterium tumefaciens.

Preferred yeasts are Candida, Saccharomyces, or Hansenula Pichia.

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

Vegetable in particular are preferred as transgenic organisms Organisms. "Plant organism" includes any organism who is capable of photosynthesis, as well as that of it derived cells, tissues, parts or reproductive material (such as seeds or Fruit). All are included within the scope of the invention Genera and species of higher and lower plants of the Plant Kingdom. Annuals, perennials, monocotyledons and dicotyledons Plants as well as gymnosperms are preferred. Are included ripe plant, seeds, shoots and seedlings, as well as thereof derived parts, propagation material (for example tubers, seeds or Fruits), plant organs, tissues, protoplasts, callus and others Cultures, for example cell cultures. Ripe plants means Plants at any stage of development beyond Seedling. Seedling means a young, immature plant at an early stage Development.

"Plant" includes all annual and perennial, monocot and dicot plants and closes, however, by way of example not restricting those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, 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, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea and Populus.

Plants from the following plant families are preferred: Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanacea, Sterculiaceae, Tetragoniacea, Theaceae, Umbelliferae.

Preferred monocot plants are selected in particular from the monocotyledonous crops, such as the Gramineae such as alfalfa, rice, corn, wheat or others Cereals such as barley, millet, rye, triticale or oats and the Sugar cane and all types of grass.

• - Asteraceae wie Sonnenblume, Tagetes oder Calendula und andere mehr,
• - Compositae, besonders die Gattung Lactuca, ganz besonders die Art sativa (Salat) und andere mehr,
• - Cruciferae, besonders die Gattung Brassica, ganz besonders die Arten napus (Raps), campestris (Rübe), oleracea cv Tastie (Kohl), oleracea cv Snowball Y (Blumenkohl) und oleracea cv Emperor (Broccoli) und weitere Kohlarten; und der Gattung Arabidopsis, ganz besonders die Art thaliana sowie Kresse oder Canola und andere mehr,
• - Cucurbitaceae wie Melone, Kürbis oder Zucchini und andere mehr,
• - Leguminosae besonders die Gattung Glycine, ganz besonders die Art max (Sojabohne) Soja sowie Alfalfa, Erbse, Bohnengewächsen oder Erdnuss und andere mehr
• - Rubiaceae, bevorzugt der Unterklasse Lamiidae wie beispielsweise Coffea arabica oder Coffea liberica (Kaffestrauch) und andere mehr,
• - Solanaceae besonders die Gattung Lycopersicon, ganz besonders die Art esculentum (Tomate) und die Gattung Solanum, ganz besonders die Art tuberosum (Kartoffel) und melongena (Aubergine) sowie Tabak oder Paprika und andere mehr,
• - Sterculiaceae, bevorzugt der Unterklasse Dilleniidae wie beispielsweise Theobroma cacao (Kakaostrauch) und andere mehr,
• - Theaceae, bevorzugt der Unterklasse Dilleniidae wie beispielsweise Camellia sinensis oder Thea sinensis (Teestrauch) und andere mehr,
• - Umbelliferae, besonders die Gattung Daucus (ganz besonders die Art carota (Karrotte)) und Apium (ganz besonders die Art graveolens dulce (Selarie)) und andere mehr; und die Gattung Capsicum, ganz besonders die Art annum (Pfeffer) und andere mehr,

sowie Lein, Soya, Baumwolle, Hanf, Flachs, Gurke, Spinat, Möhre, Zuckerrübe und den verschiedenen Baum-, Nuss- und Weinarten, insbesondere Banane und Kiwi. 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 (lettuce) and others,
• - Cruciferae, 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,
• - Cucurbitaceae 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 especially the genus Lycopersicon, especially the species esculentum (tomato) and the genus Solanum, especially the species tuberosum (potato) and melongena (eggplant) as well as tobacco or paprika and others,
• Sterculiaceae, preferably of the subclass Dilleniidae such as Theobroma cacao (cocoa bush) 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 (selaria)) and others; and the genus Capsicum, especially the species annum (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 includes ornamental plants, useful or ornamental trees, Flowers, cut flowers, shrubs or lawn. Exemplary but not restrictive include angiosperms, bryophytes such as Example Hepaticae (liverwort) and Musci (mosses); Pteridophytes such as ferns, horsetail and lycopods; Gymnosperms like Conifers, cycads, ginkgo and gnetals, the families of the Rosaceae like rose, ericaceae like rhododendrons and azaleas, euphorbiaceae like poinsettias and croton, Caryophyllaceae like cloves, Solanaceae like petunias, Gesneriaceae like the African violet, Balsaminaceae like balsam, Orchidaceae like orchids, Iridaceae like gladiolus, iris, freesia and crocus, compositae like Marigold, Geraniaceae like geraniums, Liliaceae like that Dragon tree, Moraceae like Ficus, Araceae like Philodendron and others more.

Plant organisms in the sense of the invention are still other photosynthetically active competent organisms, such as Example algae, cyanobacteria and mosses. Preferred algae are Green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. In particular Synechocystis is preferred.

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

Plant organisms in the sense of the invention are still other photosynthetically active competent organisms, such as Example algae or cyanobacteria, as well as mosses. Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Further protozoa such as dinoflagellates are preferred.

From the aforementioned organisms, those are particularly preferred can naturally synthesize oils in large quantities such as Mushrooms such as Mortierella alpina, Pythium insidiosum or plants such as soybean, rapeseed, coconut, oil palm, safflower, castor bean, peanut, Cocoa bean or sunflower or yeast such as Saccharomyces cerevisiae, soya, rape, sunflower or Saccharomyces cerevisiae.

The organisms used in the procedures vary depending on Host organism in a manner known to those skilled in the art or bred. Microorganisms are usually in a liquid Medium, which is 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 contains, at temperatures between 0 ° C and 100 ° C, preferred attracted between 10 ° C to 60 ° C with gassing with oxygen. It can the pH of the nutrient liquid is kept at a fixed value, that is, be regulated or not during cultivation. The Cultivation can be batch, semi batch or continuous respectively. Nutrients can be submitted at the start of fermentation or fed semi-continuously or continuously become.

The introduction of a transgenic according to the invention Expression cassette in an organism or cells, tissues, organs, parts or seeds of the same (preferably in plants or vegetable Cells, tissues, organs, parts or seeds), can be beneficial under Using vectors can be realized in which the transgenic Expression cassettes are included. The transgenic Expression cassette can be inserted into the vector (for example a plasmid) via a appropriate restriction interface are introduced. The The resulting plasmid is first introduced into E. coli. Correctly transformed E. coli are selected, grown and the recombinant plasmid obtained by methods familiar to the person skilled in the art. Restriction analysis and sequencing can serve the Check cloning step.

The introduction of an expression cassette according to the invention in Cells, preferably in plant cells, can be beneficial under Using vectors can be realized. Vectors can exemplary plasmids, cosmids, phages, viruses or Agrobacteria. In an advantageous embodiment, the Introduction of the expression cassette using plasmid vectors realized. Preferred vectors are those which are stable Enable integration of the expression cassette into the host genome.

The production of a transformed organism (or a transformed cell or tissue) requires that the appropriate DNA, RNA or protein into the corresponding host cell is introduced.

For this process, which is called transformation (or transduction or transfection), there is a multitude of Methods available (Keown et al. (1990) Methods in Enzymology 185: 527-537). For example, the DNA or RNA can go straight through Microinjection or by bombardment with DNA-coated Microparticles are introduced. The cell can also be chemically for example with polyethylene glycol, can be permeabilized, so that the DNA can get into the cell by diffusion. The DNA can also by protoplast fusion with other DNA-containing Units such as minicells, cells, lysosomes or liposomes respectively. Electroporation is another suitable method for Introduction of DNA in which the cells are reversible through a electrical impulse can be permeabilized. Appropriate procedures are described (for example in Bilang et al. (1991) genes 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 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)).

The desaturases or conjugases can be used for genetic modification of a wide range of organisms, preferably use of plants, so that this is a de novo producer one or more products derived from lipids, such as both CLA isomers. Plants are initially transformed regenerated and then grown as usual or grown.

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

Different methods and vectors for introducing genes into the genome of plants as well as for the regeneration of plants Plant tissues or plant cells are known (Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 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; Halford NG, Shewry PR (2000) Br Med Bull 56 (1): 62-73). These include, for example, the mentioned above. In plants, the described are Methods for transforming and regenerating plants Plant tissues or plant cells for transient or stable Transformation used. Suitable methods are, above all Protoplast transformation induced by polyethylene glycol DNA uptake, the biolistic method with the gene gun, the so-called "particle bombardment" method, electroporation, the incubation of dry embryos in DNA-containing solution and the Microinjection. In the case of this "direct" Transformation methods are no special requirements for the plasmid used posed. Simple plasmids like the pUC series, pBR322, M13mp Series, pACYC184 etc. can be used. Should be complete Plants are regenerated from the transformed cells, so it is necessary that there is an additional one on the plasmid selectable marker gene is located.

In addition to these "direct" transformation techniques, a Transformation also by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes can be performed. These strains contain a plasmid (Ti or Ri plasmid) which is based on the plant is transmitted after agrobaterium infection. A part this plasmid, called T-DNA (transferred DNA), is inserted into the Plant cell genome integrated. Alternatively, through Agrobacterium also binary vectors (Mini-Ti plasmids) on plants transmitted and integrated into their genome. The Agrobacterium -mediated transformation is best for dicotyledons, diploid plant cells are suitable, whereas the direct ones Transformation techniques are suitable for every cell type. Procedure for Agrobacterium mediated transformation are for example described by Horsch RB et al. (1985) Science 225: 1229f. Become Agrobacteria are used, so the expression cassette is in integrate special plasmids, either into an intermediate vector (English: shuttle or intermediate vector) or a binary Vector. If a Ti or Ri plasmid is used for transformation is at least the right limit, but mostly the right and left borders of the Ti or Ri plasmid T-DNA as a flanking region with the one to be introduced Expression cassette connected.

Binary are preferred for the Agrobacterium transformation Vectors used. Binary vectors can be found in both E.coli and replicate in Agrobacterium. They usually contain one Selection marker genes and a linker or polylinker flanked by the right and left T-DNA delimitation sequence. You can can be transformed directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163: 181-187). The selection marker gene allows a selection of transformed agrobacteria and is for Example the nptII gene, which is resistant to kanamycin gives. The one that acts as the host organism in this case Agrobacterium should already be a plasmid with the vir region contain. This is for the transfer of T-DNA to the plant Cell required. An Agrobacterium transformed in this way can be used for Plant cell transformation can be used. The Use of T-DNA to transform plant cells intensively examined and described (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J 4: 277-287). Various binary vectors are known and some are commercial available such as 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 in a known way to transform plants, especially of crops, such as. B. rapeseed used by, for example, wounded leaves or pieces of leaf in bathed in an agrobacterial solution and then in suitable Media are cultivated. The transformation of plants through 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; That 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 pieces of leaf can in a known manner transgenic plants are regenerated, which integrates the above Desaturase or conjugase according to the invention described, pro- Desaturase or conjugase or proOIL nucleic acids or Contain transgenic expression cassettes or vectors.

Stably transformed cells d. H. those that have the introduced DNA can be integrated into the DNA of the host cell untransformed can be selected if a selectable Marker is part of the DNA introduced. Can be used as a marker exemplary each gene act that resistance to Antibiotics or herbicides (such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc.) is able to confer (see above). Transformed cells that express such a marker gene are in the Able to in the presence of concentrations of an appropriate Antibiotic or herbicide to survive that one kill untransformed wild type. Examples are mentioned above and include prefers the bar gene that resistance to the herbicide Phosphinotricin confers (Rathore KS et al. (1993) Plant Mol Biol 21 (5): 871-884), the nptII gene that resistance to kanamycin conferred the hpt gene conferring resistance to hygromycin, or the EPSP gene, which is resistant to the herbicide glyphosate gives. The selection marker allows the selection of transformed cells from untransformed (McCormick et al. (1986) Plant Cell Reports 5: 81-84). The plants obtained can in are bred and crossed in the usual way. Two or more Generations should preferably be cultivated to ensure that genomic integration is stable and inheritable is.

Once a transformed plant cell has been made, a whole plant using the skilled person known methods can be obtained. Here you go as an example from callus cultures. From these still undifferentiated Cell masses can cause sprout and root formation in a known manner be induced. The sprouts obtained can be planted out and be bred. Appropriate procedures 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 Genet 89: 525-533).

The effectiveness of expression of the transgenic expressed Nucleic acids can, for example, in vitro Shoot meristem propagation using one of those described above Selection methods are determined. One can also in type and amount altered expression of a desaturase or a proOIL nucleic acid sequence and its effect on the CLA and / or fatty acid Biosynthetic performance on test plants tested in greenhouse experiments become.

From the transgenic organisms, the preferred ones are selected which is improved compared to the untransformed wild type Have CLA production. An improved CLA production means, for example, within the scope of the present invention artificially acquired ability to increase biosynthesis at least one compound from the group of the CLA whose esters such as for example CoA ester or glyceride ester, in the transgenic Organisms compared to the non-genetically modified Starting organism. The CLA production is in the transgenic Organism compared to the non-genetically modified Starting organism preferably by 10%, particularly preferably by 50%, very particularly preferably increased by 100%. Can improve also an advantageously changed qualitative composition of the CLA mixture means d. H. one versus that Parent organism increased relative proportion of (9Z, 11E) -CLA and / or (10E, 12Z) -CLA.

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

Another object of the invention relates to the use of Transgenic organisms according to the invention described above and the cells, cell cultures, parts derived from them - such as Example with transgenic plant organisms roots, leaves etc. -, 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. The use for the production of CLA is particularly preferred contained lipids, preferably triglycerides.

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

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 extracted 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. to 50 ° C. 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 carried out 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 be, for example, in the turbidity of moving with polar solvents such as acetone or chloroform and subsequent Filtration or centrifugation can be removed. Another one too Cleaning over columns is possible.

To obtain the free fatty acids from the triglycerides these 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, according to the above-mentioned methods according to the invention were produced and their use in the production of food, Animal feed, cosmetics or pharmaceuticals. For this, these are the Food, animal feed, cosmetics or pharmaceuticals added in usual amounts.

sequences

• 1. SEQ ID NO: 1: Nucleic acid sequence coding for an acyl-CoA E11 desaturase from Epiphyas postvittana.
• 2. SEQ ID NO: 2: protein sequence coding for an acyl-CoA E11 desaturase from Epiphyas postvittana.
• 3. SEQ ID NO: 3: Nucleic acid sequence coding for an acyl-CoA Z / E11 desaturase from Ostrinia nubilalis.
• 4. SEQ ID NO: 4: protein sequence coding for an acyl-CoA Z / E11 desaturase from Ostrinia nubilalis.
• 5. SEQ ID NO: 5: Nucleic acid sequence coding for an acyl-CoA Z / E11 desaturase from Ostrinia furnacalis.
• 6. SEQ ID NO: 6: protein sequence coding for an acyl-CoA Z / E11 desaturase from Ostrinia furnacalis.
• 7. SEQ ID NO: 7: Nucleic acid sequence coding for an acyl-CoA Δ11 desaturase from Helicoverpa zea.
• 8. SEQ ID NO: 8: protein sequence coding for an acyl-CoA Δ11 desaturase from Helicoverpa zea.
• 9. SEQ ID NO: 9: Nucleic acid sequence coding for an acyl-CoA Δ11 desaturase from Trichoplusia ni.
• 10. SEQ ID NO: 10: protein sequence coding for an acyl-CoA Δ11 desaturase from Trichoplusia ni.
• 11. SEQ ID NO: 11: Nucleic acid sequence coding for an acyl CoA Δ11 desaturase from Argyrotaenia velutinana.
• 12. SEQ ID NO: 12: protein sequence coding for an acyl-CoA Δ11 desaturase from Argyrotaenia velutinana.
• 13. SEQ ID NO: 13: nucleic acid sequence coding for an acyl CoA Z10 desaturase from Planotortrix octo.
• 14. SEQ ID NO: 14: protein sequence coding for an acyl-CoA Z10 Desaturase from Planotortrix octo.
• 15. SEQ ID NO: 15: 5'-ATYACHGCCGGKKMYCAYMG-3 '
• 16. SEQ ID NO: 16: 5'-GGRAABDYGTGRTGGWAGTT-3 '
• 17. SEQ ID NO: 17: 5'-CCCCAYCRNCTSTGGWCNCA-3 '
• 18. SEQ ID NO: 18: 5'-CCCTCTAGARTGRRWARTTRTGRWA-3 '
• 19. SEQ ID NO: 19: 5'-TAATACGACTCACTATAG-3 '
• 20. SEQ ID NO: 20: 5'-ACATAACTAATTACATGAT-3 '

Examples

The invention is illustrated in the following exemplary embodiments Reference to the accompanying figures explained in more detail. there Abbreviations with the following meaning are used:

General methods

The carried out in the context of the present invention Cloning steps such as B. restriction splits, Agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids on nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E.coli cells, cultivation of the bacteria and sequence analysis of recombinant DNA are as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Chemical synthesis of oligonucleotides can, for example, in a known manner, according to the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pp. 896-897). The under the Present invention performed cloning steps such. B. Restriction cleavage, 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 was carried out as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6 described. Sequencing recombinant DNA molecules were made using a laser fluorescence DNA sequencer Licor (sales by MWG Biotech, Ebersbach) after the Sanger's method (Sanger et al. (1977) Proc Natl Acad Sci 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 softies are kept separately in terrariums until the finished insects hatch. The pheromone glands can be isolated about 1 to 2 days after the insects hatch.

Example 2 Isolation of desaturase or conjugase cDNAs using degenerate primers

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 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. 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 to amplify the conserved central region of Lepidoptera desaturases / conjugases and are used in the following PCR protocol:
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)
10 µl 5 × Advantage 2 reaction buffer (CLONTECH)
1 µl 50 × Advantage 2 DNA Polymerase mix (CLONTECH)
H2O add. 50 µl

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

The PCR was carried out under the following cycle conditions:
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

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

Due to the sequence information obtained, gene-specific Primers are derived and used to amplify 5'- and 3 'areas are used (SMART RACE cDNA Amplification Kit (CLONTECH)). These PCR products are also included in the TOPO TA PCR 2.1 Vector (INVITROGEN) ligated and then in TOP 10 competent cells (INVITROGEN) transformed. Become positive colonies amplified again and the plasmid DNA purified (QIAGEN Plasmid Mini Kit) and then sequenced.

The complete sequence can be made from the fragments generated in this way a desaturase / conjugase using standard cloning techniques composed and for characterization for example in the pYES2 vector (INVITROGEN) can be transferred for yeast expression. Saccharomyces INVSc1 (INVITROGEN) are used with the appropriate pYES2 expression vectors using a modified PEG / lithium acetate protocol transformed (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 for further breeding and functional Expression selected.

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 at 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% (v / v) Tergitol NP-40 with the corresponding substrates, such as stearic acid, palmitic acid or myristic acid, were concentrated to a final concentration of 0.003% (wt. / 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 of culture were harvested by centrifugation (1000 × g, 10 min. 4 ° C.) and washed once with 100 mM NaHCO3, pH 8.0, in order to remove residual medium and fatty acids. To produce the fatty acid methyl esters (FAMES), the cell sediments are snap frozen in N2 liq. and lyophilized under N2 gas at 30 ^° 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 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 Packard 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 following fatty acids are preferably used as standard: c9-16: 1, t9-16: 1, 18: 0, c9-18: 1, t9-18: 1, c11-18: 1, t11-18: 1, c9 , c12-18: 2, t9, t12-18: 2, c9, t11-18: 2, t10, c12-18: 2. (The nomenclature of the standards corresponds to that customary for fatty acids and initially specifies the configuration (c = cis or t = trans) and position of the double bond or chain length of the fatty acid.)

Further feeding experiments with various others Fatty acids (e.g. lauric acid, trans-vaccenic acid, cis-vaccenic acid, E10 octadecenoic acid or Z10 octadecenoic acid) can be used more detailed confirmation of the substrate selectivity of this Desaturase / conjugase can be performed.

Example 4 Expression cloning from desaturase / conjugase Lepidoptera in Saccharomyces cerevisiae a) Preparation of the cDNA library

The pheromone glands become from the abdomen of adult moths isolated 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 around 30 mg of fresh tissue can be around 60 to 80 µg of total RNA can be isolated. About 5 µg of total RNA is used used to create a cDNA library. For this, can Take the SMART cDNA Library Construction Kit (CLONTECH) as an example Manufacturer information can be used. The isolated double-stranded cDNA is ultimately in the linearized pYES2 (INVITROGEN) Yeast expression vector ligated. For this purpose, the Multiple cloning site of the pTriplEx2 vector (CLONTECH) inserted. Then in the modified Vekor Double-stranded cDNA cloned after digestion with SfiIA and SfiIB.

b) yeast transformation

About 1.5 µg plasmid DNA is with the Saccharomyces cerevisiae EASY COMP Transformation kit (INVITROGEN) in INVSc1 (INVITROGEN) transformed according to the manufacturer's instructions. Two 50 µl batches are made on selection medium in a large square petri dish (245 × 245 mm) plated out and incubated for 3 days at 30 ° C.

c) Cultivation of the yeast in microtiter plates

Individual colonies are picked in with a picking robot Microtiter plates (MTP) transferred. The preculture takes place in 200 µl medium [1 × CSM-Ura; 1 × YNB without amino acids and sugar; 0.5% raffinose; 5% glycerol; 40 mg / 1 adenine sulfate; 0.5% ammonium sulfate] for 72 Hours at 30 ° C and 250 rpm. By encore of an average volume from the preculture becomes one OD600 set from 0.2 in the main culture. The main culture takes place in 1 ml medium [1 × CSM-Ura; 1 × YNB without amino acids and Sugar; 0.5% raffinose; 2% galactose; 0.2% Tergitol NP-40; 40 mg / l adenine sulfate; 0.5% ammonium sulfate; 0.3 mM Fatty acid substrate] for 2 to 3 weeks at 16 ° C and 250 rpm, until an OD600 of 3 to 4 is reached instead.

d) fatty acid analysis

The yeast cells are sedimented (1000 × g, 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 lyophilized under N2 gas at 30 ^° 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 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. Further feeding experiments with various other fatty acids (e.g. lauric acid, trans-vaccenic acid, cis-vaccenic acid, E10 octadecenoic acid or Z10 octadecenoic acid) can be carried out to confirm the substrate selectivity of this desaturase or conjugase in more detail.

e) plasmid preparation of the positive yeast

Single clones which have tested positive will be re-run over 24 hours at 28 ° C. in 10 ml minimal medium [1 × CSM-Ura; 1 × YNB w / o AA, sugars; 2% glucose; 40 mg / l adenine sulfate] attracted and with an OD600 larger than 3 by centrifugation sedimented. The yeast pellet is placed in 400 µl for 20 minutes at 37 ° C SCE buffer [1.2 M sorbitol; 0.1 M Na citrate, pH 7.0; 10 mM EDTA; 1 mg / ml Lyticase] incubated. Then 400 ul STE buffer [2% SDS; 50 mM Tris / HCl, pH 8.0; 10 mM EDTA] for cell lysis added and incubated for 10 minutes at room temperature. to Precipitation of the cell protein is added to 200 ul 5 M sodium acetate and Put on ice for 30 minutes. After centrifugation, the Transfer the supernatant and precipitate with 2.5 times the volume of ethanol. The sedimented pellet is washed with 70% ethanol and ultimately taken up in sterile water. The sequence of the DESATU RASE / KONJUGASE can optionally be performed by sequencing Use of vector-specific primers (SEQ ID NO: 12 and 13) be determined.

Example 5 Manipulation of vegetable fatty acid biosynthesis a) Generation of DNA constructs for the expression of Desaturases / conjugases from Lepidoptera in transgenic plants

For the production of chimeric DNA constructs for generation transgenic A.thaliana or B.napus plants that the To express desaturases / conjugases from Lepidoptera, the vector pBinAR used (Höfgen and Willmitzer (1990) Plant Sci 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 DESAT / KONJ was cloned into this vector transgenic A.thaliana or Brassica napus are produced Plants.

b) Production of transgenic Arabidopis thaliana plants

Wild-type Arabidopsis thaliana plants (Columbia) are grown with the Agrabacterium tumefaciens strain (GV3101 [pMP90]) based a modified vacuum infiltration 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 Sci Paris 1144 (2): 204-212). The Agrobacterium tumefaciens used Cells had previously been transformed with the plasmids.

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

c) Production of transgenic Brassica napus plants

The production of transgenic rape plants is based on one Protocol from Bade JB and Damm B (in Gene Transfer to Plants, Potrykus I and Spangenberg G (eds.) Springer Lab Manual, Springer Verlag, 1995, 30-38), in which the composition of the used media and buffer is specified.

The transformations are carried out with the Agrobacterium tumefaciens Strain GV3101 [pMP90]. The plasmids are used for transformation pBinAR-TkTP / Vit E-AT used. Brassica napus var. Westar are surface sterilized with 70% ethanol (v / v), 10 Washed for minutes at 55 ° C in water, in 1% hypochlorite solution (25% v / v Teepol, 0.1% v / v Tween 20) and incubated for 20 minutes washed six times with sterile water for 20 minutes each. The seeds are dried on filter paper for three days and 10 to 15 seeds in a glass flask with 15 ml germination medium for germination brought. From several seedlings (approx. 10 cm tall) the Roots and apices removed and the remaining hypocotyls in 6 mm long pieces cut. The approx. 600 won in this way Explants are washed for 30 minutes with 50 ml of basal medium and in transferred a 300 ml flask. After adding 100 ml The callus induction medium is the cultures for 24 hours at 100 rpm incubated.

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 Luria Broth medium without kanamycin for 4 hours at 29 ° C. up to an OD ₆₀₀ 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 OD ₆₀₀ of 0.3 by adding further basal medium.

The callus induction medium also becomes from the rape explants sterile pipettes removed, 50 ml agrobacterium solution added, mixed gently and incubated for 20 min. The Agrobacteria suspension is removed, the rape explants for 1 min washed with 50 ml callus induction medium and then 100 ml callus induction medium added. The co-cultivation is on a rotary shaker at 100 rpm for 24 h carried out. The co-cultivation is done by taking away the Callus induction medium stopped and the explants twice for each 1 min with 25 ml and twice for 60 min with 100 ml each Wash medium washed at 100 rpm. The washing medium with the Explants are transferred to 15 cm petri dishes and the medium removed with sterile pipettes.

20 to 30 explants in 90 mm are used for regeneration Petri dishes transferred with 25 ml sprout induction medium Kanamycin included. The Petri dishes are made with 2 layers of leucopor closed and at 25 ° C and 2000 lux with photoperiods of Incubated 16 hours light / 8 hours dark. Every 12 days the developing calli on fresh petri dishes Shoot induction medium implemented. All further steps to Regeneration of whole plants is carried out as by Bade, J. B and Damm, B. (in: Gene Transfer to Plants, Potrykus I and Spangenberg G (eds.) Springer Lab Manual, Springer Verlag, 1995, 30-38) described.

d) 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 Packard 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. SEQUENCE LISTING

Claims (8)

1. A process for the preparation of trigylcerides comprising unsaturated fatty acids, 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 have a double bond at the position C8, C9, C10, C11 or C12 can produce. 3. The method according to any one of claims 1 or 2, characterized characterized in that the fatty acid desaturase specifically a cis or trans double bond in fatty acids, fatty acid CoA esters or other fatty acid derivatives with a chain length of Fatty acid of 16 or 18 carbon atoms generated. 4. The method according to any one of claims 1 to 3, characterized characterized in that the fatty acid desaturase has a homology of described at least 65% to one of the fatty acid desaturases 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 characterized that the unsaturated fatty acids conjugated Include linoleic acid. 7. Triglycerides produced by a method according to one of the Claims 1 to 6. 8. Use of triglycerides according to claim 7 for the preparation of food, feed, cosmetics or fine chemicals.