Evolutionary and biochemical characterization of a Chromochloris zofingiensis MBOAT with wax synthase and diacylglycerol acyltransferase activity

Wax synthase (WS) catalyzes the last step in wax ester biosynthesis in green plants. Two unrelated sub-families of WS, including the bifunctional acyltransferase and plant-like WS have been reported, but the latter is largely uncharacterized in microalgae. Here, we functionally characterized a putative plant-like WS (CzWS1) from the emerging model green microalga Chromochloris zofingiensis. Our results showed that plant-like WS evolved under different selection constraints in plants and microalgae, with positive selection likely contributing to functional divergence. Unlike jojoba with high amounts of wax ester in seeds and a highly active WS enzyme, C. zofingiensis has no detectable wax ester but a high abundance of WS transcripts. Co-expression analysis showed that C. zofingiensis WS has different expression correlation with lipid biosynthetic genes from jojoba, and may have a divergent function. In vitro characterization indicated that CzWS1 had diacylglycerol acyltransferase activity along with WS activity, and overexpression of CzWS1 in yeast and Chlamydomonas reinhardtii affected triacylglycerol accumulation. Moreover, biochemical and bioinformatic analyses revealed the relevance of the C-terminal region of CzWS1 in enzyme function. Taken together, our results indicated a functional divergence of plant-like WS in plants and microalgae, and the importance of its C-terminal region in specialization of enzyme function.

Automated summary

Our study reveals the functional divergence of plant-like WS in plants and microalgae, with positive selection likely contributing to functional differences in different environments. Specifically, the putative plant-like WS (CzWS1) in Chromochloris zofingiensis functions in wax ester biosynthesis but shows different expression patterns compared to jojoba WS, indicating potential divergent functions.