Details

Autor(en) / Beteiligte

• Chen, Guanqun
• Xu, Yang
• Siloto, Rodrigo M. P.
• Caldo, Kristian Mark P.
• Vanhercke, Thomas
• Tahchy, Anna El
• Niesner, Nathalie
• Chen, Yongyan
• Mietkiewska, Elzbieta
• Weselake, Randall J.

Titel

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

Ist Teil von

• The Plant journal : for cell and molecular biology, 2017-10, Vol.92 (2), p.167-177

Ort / Verlag

England: Blackwell Publishing Ltd

Erscheinungsjahr

2017

Quelle

Wiley-Blackwell Journals

Beschreibungen/Notizen

• Summary Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the acyl‐CoA‐dependent biosynthesis of triacylglycerol, the predominant component of seed oil. In some oil crops, including Brassica napus, the level of DGAT1 activity can have a substantial effect on triacylglycerol production. Structure–function insights into DGAT1, 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 DGAT1 (BnaDGAT1) 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 BnaDGAT1 activity, and 67% of these residues were conserved among plant DGAT1s. The identified amino acid residue substitution sites occur throughout the BnaDGAT1 polypeptide, with 89% of the substitutions located outside the putative substrate binding or active sites. In addition, cDNAs 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 BnaDGAT1. Overall, the results provide novel insights into amino acid residues underlying plant DGAT1 function and performance‐enhanced BnaDGAT1 variants for increasing vegetable oil production. Significance Statement Plant diacylglycerol acyltransferase 1 (DGAT1) variants with increased activity were generated through random mutagenesis and used to gain insight into amino acid residues critical for enzyme activity and to increase the triacylglycerol content of vegetative tissue. The results shed new light on structure‐function in plant DGAT1 and the engineering of designer enzyme variants for increasing the oil content of plant biomass.