Abstract
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.
Highlights
Acyl-CoA reductase (FAR)[11,12]
Given the absence of obvious organellar targeting signals, the most likely location of EgWSDs, excluding EgWSD5 and EgWSD6, is likely to be the ER membrane, which is the main place of wax ester synthesis in E. gracilis[7]
As for EgWSD5 and EgWSD6, prediction of their hydrophobicity varied depending on programs used, indicating they will be of ambiguous protein with relation to localization
Summary
Acyl-CoA reductase (FAR)[11,12]. wax esters are produced by the esterification of fatty acyl-CoA and fatty alcohol, catalyzed by wax ester synthase (WS) or acyl-CoA:fatty alcohol acyltransferase[13]. Enzymes exhibiting activity of wax ester synthesis have been identified and characterized from a wide range of organisms, and have been classified into two main groups. The second group comprises bifunctional enzymes with both WS and acyl-CoA:diacylglycerol acyltransferase (DGAT) activities, which was first identified and characterized from Acinetobacter calcoaceticus[17]. Teerawanichpan and Qiu[13] identified WS in E. gracilis, that showed significant similarity to jojoba WS, and confirmed that recombinant WS has the ability to produce myristyl myristate and other wax ester molecules through heterologous co-expression systems in yeast cells with E. gracilis FAR. We characterize the putative WSDs using heterologous expression systems in yeast cells, and evaluated their physiological contributions toward wax ester synthesis in E. gracilis cells using gene silencing method. We show that the WSDs are involved in wax ester formation in vivo under anaerobic conditions, and discuss their physiological significance
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