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 DGAT2s. 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 DGAT2s and two parental DGAT2s 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.