Directed evolution of diacylglycerol acyltransferases 2 promotes lipids and triglyceride accumulation

Triacylglycerol (TAG) is a major component of plant-neutral lipids. Diacylglycerol acyltransferase 2 ( DGAT2 ) plays an important role in plant oil accumulation by catalyzing the final step of the Kennedy pathway. In this study, ten DGAT2 sequences were originating from different oil crops into the TAG-deficient yeast strain H1246, to compare their enzyme activity of oil synthesis and filter out potential amino acid residue sites for directed evolution. Based on the synthesis efficiency of total lipids, TAGs, and the topology models of these DGAT2s , five possible amino acid sites were identified that may affect the synthesis of total lipids and TAGs. In the H1246 yeast expression system, HaDGAT2 significantly increased the total oil and TAG content; however, ClDGAT2 was weak in synthesizing both oil and TAG. Thus, building on HaDGAT2 and ClDGAT2 , these amino acid substitutions were created by point-to-point mutating and substantially affected the oil or TAG synthesis ability of DGAT2 s. Among the five amino acid substitutions, mutations at residue (3) successfully make HaDGAT2 less capable of synthesizing lipids and TAG, and ClDGAT2 more capable of synthesizing total lipids and TAG. Except mutations at residue (2), all residue mutations contributed to a weaker ability of fatty acid synthesis. In addition, ten mutant DGAT2 s and two parental DGAT2 s were overexpressed in tobacco leaves to reveal their lipid synthesis function. This approach helped us to authenticate the significance of these loci. In varying degrees, those mutations enhanced the ability of ClDGAT2 to synthesize lipids, attenuated the ability of HaDGAT2 to synthesize lipids, and altered preference for fatty acids in tobacco. Key message Comparison of gene structure and homology of ten DGAT2 s from different plants. DGAT2 genes from ten plants all enable yeast H1246 to produce lipids, but there are differences in the efficiency of synthesizing lipids. A total of five sites on the conserved structural domain of DGAT2s were identified, which may affect the DGAT2 s’ability to synthesize lipids. Mutations at S259A of HaDGAT2 can make HaDGAT2 less capable of synthesizing lipids and mutations at A204S of ClDGAT2 can make ClDGAT2 more capable of synthesizing lipids.