Improved Characterization of Polyunsaturated Fatty Acids Desaturases and Elongases by Co-Expression in Saccharomyces cerevisiae with a Protozoan Acyl-CoA Synthetase

Saccharomyces cerevisiae is a valuable host for the expression and characterization of eukaryotic enzymes involved in polyunsaturated fatty acid (PUFA) biosynthesis, such as elongases and desaturases. The yeast allows a correct subcellular localization of these proteins, provides electron donors required by desaturases and is unable to synthesize PUFA that could interfere in the enzymes characterization. Unfortunately, S. cerevisiae incorporates very long chain PUFAs inefficiently, which could interfere in the characterization of enzymes using these substrates. Acyl-CoA synthetases (ACS) are involved in fatty acids uptake, and catalyze the synthesis of the corresponding CoA thioesters. ACS provides the substrates for elongases, acyl-CoA desaturases and acyl transferases. Transferases are required to synthesize phospholipids which in turn, are substrates for acyl-lipid desaturases. Expression in yeast of Trypanosoma brucei ACS1 notably improves the uptake of a wide variety of PUFA. Co-expression of ACS1 with Elo5 elongase from Leishmania major or Des4 desaturase from T. brucei showes, respectively, 2- and 5.6-fold increases in the uptake of the PUFA substrates and 2.4- and 3.5-fold increases in substrate conversion. It also allows to produce significant amount of Des4 desaturase product for further analysis, whereas it is obtained in trace amounts when the enzyme is expressed alone.