Method for increasing the content of polyunsaturated long-chained fatty acids in transgenic organisms

#This application is a national stage application (under 35 U.S.C. 371) of PCT/EP2005/007754 filed Jul. 15, 2005, which claims benefit of German application 10 2004 034 442.6 filed Jul. 16, 2004 and German application 10 2004 060 340.5 filed Dec. 15, 2004.

#The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Sequence_Listing—13477—00006. The size of the text file is 603 KB, and the text file was created on Mar. 29, 2010.

#The present invention relates to a method for increasing the content of polyunsaturated long-chain fatty acids in an organism by introducing into said organism nucleic acids coding for polypeptides or proteins having a phospholipase, ketoacyl-CoA reductase and/or dehydratase activity. Advantageously, said enzymes originate from Ostreococcus or Thraustochytrium.

#FIG. 1 shows different synthesis pathways for the biosynthesis of DHA (docosahexaenoic acid).

#FIG. 2 shows gas chromatogram of the fatty acid extract from flax seed transformed with the construct pGPTV-USP_PSE1_d6Des(Pt)_d5Des(Pt).

#FIG. 3 shows sequence alignment of phospholipase A2 from H. sapiens (SEQ ID NO: 146) and the Ostreococcus sequence PLA2(Ot) (SEQ ID NO: 2).

#There are still a number of limiting steps in the fatty acid biosynthesis which impair the increase of the content of polyunsaturated long-chain fatty acids. It could thus be shown in transgenic plants, as have, for example, been described in DE 10 219 203, that the elongation, i.e. the chain extension, from C18 to C20 fatty acids is such a limiting step (FIG. 2). FIG. 2 shows the gas chromatogram of the fatty acid extract from flaxseed, transformed with the construct pGPTV-USP_PSE1_d6Des(Pt)_d5Des(Pt), according to the descriptions from DE 10 219 203. The newly formed products from the activities of the genes are marked with arrows. The synthesis of the final products arachidonic acid (ARA) and eicosapentaenoic acid (EPA) proceeds via γ-linolenic acid (g18:3) or stearidonic acid (18:4) (first step, see also FIG. 1). In the second step, the elongation to form the intermediate products 20:3n-6 and 20:4n-3 is performed (elongation step). In the last step, the intermediate products are then reacted to form ARA and EPA. A strong decrease in the product quantities from the first to the second step can be observed. FIG. 2 shows that after the first step in the aerobic LCPUFA synthesis, the amount of product is drastically reduced by the desaturation of linoleic or linolenic acid (see FIG. 1). This may indicate that the conversion of the elongation of Δ6-desaturated fatty acids is effected to an insufficient extent. Herein, the conversion rate is significantly lower than could be shown in yeast experiments in which the Δ6-desaturated fatty acids had been fed (Zank et al. 2002, Plant Journal 31:255-268).

#Thus, the problem was posed to provide further genes or enzymes suitable for the synthesis of LCPUFAs, in particular genes exhibiting a phospholipase A2, ketoacyl-CoA reductase and/or dehydratase activity, to produce polyunsaturated fatty acids and to further optimize the biosynthesis of fatty acids in oils and/or lipids by said genes or enzymes.

#It was a further problem to develop a method for producing oils or lipids having a high content of unsaturated fatty acids, advantageously of polyunsaturated fatty acids, in an organism, advantageously in an eukaryotic organism, preferably in a plant or a microorganism. Said problem was solved by the method according to the present invention for producing oils or lipids having a high content of unsaturated fatty acids in transgenic organisms. Said method is characterized in that it comprises the following procedural steps: a) introducing at least one nucleic acid sequence coding for a phospholipase A2 activity into the organism, or b) introducing at least one nucleic acid sequence coding for a ketoacyl-CoA reductase activity into the organism, or c) introducing at least one nucleic acid sequence coding for a dehydratase activity into the organism, and d) cultivating and harvesting the transgenic organism.