Wax Ester Synthase/Diacylglycerol Acyltransferase Isoenzymes Play a Pivotal Role in Wax Ester Biosynthesis in Euglena gracilis

Wax ester fermentation is a unique energy gaining pathway for a unicellular phytoflagellated protozoan, Euglena gracilis, to survive under anaerobiosis. Wax esters produced in E. gracilis are composed of saturated fatty acids and alcohols, which are the major constituents of myristic acid and myristyl alcohol. Thus, wax esters can be promising alternative biofuels. Here, we report the identification and characterization of wax ester synthase/diacylglycerol acyltrasferase (WSD) isoenzymes as the terminal enzymes of wax ester production in E. gracilis. Among six possible Euglena WSD orthologs predicted by BLASTX search, gene expression analysis and in vivo evaluation for enzyme activity with yeast expressing individual recombinant WSDs indicated that two of them (EgWSD2 and EgWSD5) predominantly function as wax ester synthase. Furthermore, experiments with gene silencing demonstrated a pivotal role of both EgWSD2 and EgWSD5 in wax ester synthesis, as evidenced by remarkably reduced wax ester contents in EgWSD2/5-double knockdown E. gracilis cells treated with anaerobic conditions. Interestingly, the decreased ability to produce wax ester did not affect adaptation of E. gracilis to anaerobiosis. Lipid profile analysis suggested allocation of metabolites to other compounds including triacylglycerol instead of wax esters.