Molecular cloning and overexpression of DGA1, an acyl-CoA-dependent diacylglycerol acyltransferase, in the oleaginous yeast Rhodosporidiobolus fluvialis DMKU-RK253.

Triacylglycerol (TAG) is a major component of lipid storage in yeast. The acyl CoA: diacylgycerol acyltransferase (DGAT) that catalyzes the final and rate-limiting step in the production of TAG is rather interesting. Consequently, cloning and analysis of the gene-encoding TAG synthase, diacylglycerol acyltransferase gene (DGA1), of the oleaginous yeast Rhodosporidiobolus fluvialis DMKU-RK253 were undertaken. Analysis of the deduced amino acid sequence of DGA1 from R. fluvialis DMKU-RK253 (RfDGA1) showed similarity with the acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) from other organisms. The cDNA of RfDGA1 was cloned into the yeast expression vector pYES2 and heterologously overexpressed in Saccharomyces cerevisiae. One of the transformants showed a 1.6-fold increase in lipid content compared with the wild-type strain harbouring the pYES2 empty vector. Furthermore, DGA1 overexpression in R. fluvialis DMKU-RK253 resulted in a 2.5-fold increase in lipid content when compared with the wild-type strain, and no significant differences in fatty acid composition were observed between RfDGA1-overexpressed and wild-type strains. Taken together, our results supported our hypothesis that the RfDGA1 is a genetic factor that can be used for the development of a strain with improved lipid accumulation capabilities.