Characterization of Type-2 Diacylglycerol Acyltransferases in the Green Microalga Chromochloris zofingiensis.

Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step of the acyl-CoA-dependent TAG biosynthesis and thus is a key target for manipulating oil production in microalgae. The microalga Chromochloris zofingiensis can accumulate substantial amounts of triacylglycerol (TAG) and represents a promising source of algal lipids. In this study, C. zofingiensis DGAT2s (CzDGAT2s) were characterized with in silico, in vivo (yeast), and in vitro assays. Putative CzDGAT2s were identified, and their functional motifs and evolutionary relationship with other DGAT2s were analyzed. When CzDGAT2s were individually expressed in a TAG-deficient Saccharomyces cerevisiae strain, only CzDGAT2C could restore the TAG biosynthesis. Further in vitro assays indicated that CzDGAT2C displayed typical DGAT activity, which was fitted to the Michaelis-Menten equation, and N- and C-terminals were important for the enzyme activity. In addition, membrane yeast two-hybrid assay revealed a possible DGAT2 activity modulation via the formation of homodimer/heterodimer among different DGAT2 isoforms.