Functional characterization of enzymes involved in polyunsaturated fatty acid metabolism

Benefit description

The enzyme function is determined by expressing the open reading frame (ORF) of the gene of interest in yeast Saccharomyces cerevisiae. Briefly, the ORF is amplified by PCR with primers containing restriction sites for further cloning into the yeast expression vector pYES2. The purified plasmids containing the putative ORFs are then transformed into S. cerevisiae competent cells. One single recombinant yeast colony is grown in S. cerevisiae minimal medium-uracil broth to produce a bulk culture required to run the functional assay. In order to assess the role of the enzyme in the biosynthesis of polyunsaturated fatty acids (PUFA), individual flasks of transgenic yeast are supplemented with FA substrates. The assay is completed with the fatty acid analysis of the yeast culture (price not included) assessing the conversion of the substrates in the corresponding products.