Klonierung Docosahexaenoyl-CoA-spezifischer Acyltransferase-Gene
The most dominating lipids composing mammalian cell membranes are long chain polyunsaturated fatty acids (LCPUFA). They are essential for the correct function of e.g. the nervous and ocular systems as well as for reproduction. Additionally they are precursors for the formation of different eicosanoids. The biosynthesis rate of these fatty acids in mammals is usually low and insufficient to cover their needs. They must, therefore, be provided from diet. Transgenic oilseeds accumulating LCPUFA represent an attractive sustainable source to support human nutrition. Generation of these transgenic lines should be achieved by cloning the gene encoding for lysophosphatidic acid transferase (LPAAT), an enzyme that specifically introduces LCPUFA to the second position of the glycerol backbone in seed oils. Analysis of lipidic composition of different organisms led to the identification of two relevant strains; the bacterium S. handedai and the eukaryotic microorganism Thraustochytrium. A comparative enzymatic analysis of membrane fractions from both organisms showed that LPAAT from S. hanedai was more active on C20-and C22-fatty acids, whereas Thraustochytrium-LPAAT had a preference for C22-fatty acid. To clone the LCPUFA-specific LPAAT genes, genomic libraries from S. hanedai and cDNA expression libraries from Thraustochytrium were constructed and screened. Gene sequences from both organisms were identified and compared to the gene bank database. Both gene sequences displayed high homology to bacterial LPAATs. Functional S. hanedai-LPAAT enzyme showing pronounced specificity to LCPUFAs was successfully expressed in E. coli. Considering the sequence similarity between the two genes, it was assumed that Thraustochytrium-LPAAT enzyme would display similar specificity, but unlike S. hanedai-LPAAT enzyme production of Thraustochytrium-LPAAT in E. coli and S. cerevisiae proved to be difficult since expression levels were below detection limits and no enzymatic activity could be measured. The expression levels were improved by fusing the LPAAT gene to glutathione-S-transferase (GST). However the fusion partner prevented the formation of catalytically active conformation of the enzyme leading to production of inactive protein. Expression studies carried out in different organisms, insect cells were found out to be best suited for the expression of Thraustochytrium-LPAAT gene.
info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, Acyltransferasen, sehr langkettige mehrfach ungesättigte Fettsäuren
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