Co-expression of thraustochytrium sp. fjn-10 δ5-eiongase and δ4-desaturase in saccharomyces cerevisiae

Docosahexaenoic acid (DHA C22:6n-3), a typical long chain polyunsaturated fatty acids (PUFAs) has many positive effects on diseases. δ5- elongase gene (elo5, 860 bp) and δ4-desaturase gene (Jad4, 1 600 bp) were amplified by PCR using plasmid pYTFD5 and pYFAD4 as templates, respectively. elo5-fad4 fusion gene amplified by overlap extension PCR was digested by Hind III and Sph I and subcloned into the yeast-Escherichia coli shuttle vector pYES2.0. The recombinant plasmid pYEL05-FAD4 containing target gene was transformed into Saccharomyces cerevisiae strain INVScl and the recombinant yeast cells were selected on agar synthetic medium lacking uracil. Expression of the fusion gene in transformant was induced by the addition of galactose to 2% (w/V). The yeast culture medium was supplemented with exogenous fatty acid substrate, eicosapentaenoic acid. Total fatty acids were extracted from the induced cells and subjected to methyl-esterification. The resultant fatty acid methyl esters were analyzed by GC detection. A novel peak corresponding to DHA (docosahexaenoic acid, C22:6n-3) methyl ester standards which was absent in the cell transformed with empty vector was detected with the same retention time and mass analysis. These results indicated that the protein encoded by elo5-fad4 could specifically catalyze EPA to DHA.