High‐performance variants of plant diacylglycerol acyltransferase 1 generated by directed evolution provide insights into structure function

Summary Diacylglycerol acyltransferase 1 ( DGAT 1) catalyzes the acyl‐CoA‐dependent biosynthesis of triacylglycerol, the predominant component of seed oil. In some oil crops, including Brassica napus , the level of DGAT 1 activity can have a substantial effect on triacylglycerol production. Structure–function insights into DGAT 1, however, remain limited because of the lack of a three‐dimensional detailed structure for this membrane‐bound enzyme. In this study, the amino acid residues governing B. napus DGAT 1 (Bna DGAT 1) activity were investigated via directed evolution, targeted mutagenesis, in vitro enzymatic assay, topological analysis, and transient expression of cDNA encoding selected enzyme variants in Nicotiana benthamiana . Directed evolution revealed that numerous amino acid residues were associated with increased Bna DGAT 1 activity, and 67% of these residues were conserved among plant DGAT 1s. The identified amino acid residue substitution sites occur throughout the Bn aDGAT 1 polypeptide, with 89% of the substitutions located outside the putative substrate binding or active sites. In addition, cDNA s encoding variants I447F or L441P were transiently overexpressed in N. benthamiana leaves, resulting in 33.2 or 70.5% higher triacylglycerol content, respectively, compared with native Bna DGAT 1. Overall, the results provide novel insights into amino acid residues underlying plant DGAT 1 function and performance‐enhanced Bna DGAT 1 variants for increasing vegetable oil production.