Cloning Of Genes Involved In EPA Biosynthesis Of Pythium Splendens And Construction Of Transgenic Strains

Eicosapentaenoic acid (EPA), which belongs to the ω-3polyunsaturated fatty acids,has many important physiological functions, such as lowering blood lipid level, preventingatherosclerosis and cardiovascular disease, anti-cancer, anti-inflammatory, etc. Theresearch and application of EPA has attracted great attention in the worldwide andproduction of EPA by microorganisms has been the hot area of research. In this paper, thegenes involved in EPA biosynthesis of Pythium splendens RBB-5were cloned and thetransgenic strains were constructed. The results were as follows:(1) Δ6desaturase gene and Δ17desaturase gene were cloned from P. splendens. Thenucleotide sequences shared the highest identities of86%and99%with that of genes everreported. Three histidine conserved domains (HXXXH, HXXHH and Q/HXHH) werefound in deduced amino acid sequences of genes, and a domain of cytochrome b5existedin amino acid sequence of Δ6desaturase gene. The evolution of the two genes wasanalyzed in the research as well. In addition, Δ12desaturase gene was cloned fromMortierella alpina.(2) Δ6desaturase gene was cloned into pPIC3.5K vector and transferred into Pichiapastoris GS115for function identificatin. The result indicated that Δ6desaturase gene wassuccessefully expressed in yeast. Δ6desaturase could convert LA of yeast into GLA, andthe output of GLA accounted for1.3%of the total fatty acids, but it could not convertALA into STA. The result of substrate selectivity showed that Δ6desaturase had apreference for the substrate of LA.(3) The cloned three genes were constructed into expression vector and transferredinto P. splendens through Agrobacterium tumefaciens-mediated transformation,repectively. Hygromycin B was applied to screen the transformed strains. The results ofmicroexamination of GFP and PCR detection showed that the genes have been integratedinto the genome of P. splendens. Analysis of fatty acids from transgenic strains showedthat a general incresse of EPA yield was achieved and the highest increase was24.6%.The transgenic strains obtained could be used for the analysis of the relevance betweengenes expression and biosynthesis of fatty acids. The research results laid the groundwork for the construction of EPA high-yieding genetic engineering strains as well.