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Permanent link (DOI): https://doi.org/10.7939/R34B2XJ6J

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

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Author or creator
Chen, Guanqun
Xu, Yang
Siloto, Rodrigo M. P.
Caldo, Kristian Mark P.
Vanhercke, Thomas
El Tahchy, Anna
Niesner, Nathalie
Chen, Yongyan
Mietkiewska, Elzbieta
Weselake, Randall J.
Additional contributors
Subject/Keyword
triacylglycerol biosynthesis
diacylglycerol acyltransferase
directed evolution
topology
leaf oil production
Brassica napus
Nicotiana benthamiana
Type of item
Journal Article (Published)
Language
English
Place
Time
Description
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.
Date created
2017/09/10
DOI
doi:10.7939/R34B2XJ6J
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Attribution-NonCommercial 4.0 International
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File title: High‐performance variants of plant diacylglycerol acyltransferase 1 generated by directed evolution provide insights into structure function
File title: High‐performance variants of plant diacylglycerol acyltransferase 1 generated by directed evolution provide insights into structure function
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