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

Abstract

SUMMARYDiacylglycerol 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 membranebound 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.

Diacylglycerol Acyltransferase 1 Is Regulated by Its N-Terminal Domain in Response to Allosteric Effectors

“…Briefly, 100 mL of yeast culture was added with 5 mL of 0.1 mg mL 21 dye solution (Nile Red dissolved in methanol). Fluorescence emission was measured before and after the addition of dye solution, and the change in fluorescence units over optical density was used as an indicator of neutral lipid content (Chen et al, 2017). Three technical replicates were performed for each BnaDGAT1 construct.…”

Properties and Biotechnological Applications of Acyl‐CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae

“…Tobacco ( N. tabacum and N. benthamiana ) has served as the most common platform for producing oil in vegetative tissues given its ability to produce high biomass. DGAT1 has been used to boost oil in leaf or/and stem of tobacco through overexpression of DGAT1 alone (Andrianov et al, ; Wu et al, ) or in combination with one or more cDNA encoding proteins/enzymes such as acyl‐CoA:monoacylglycerol acyltransferase, WRI, oleosin, cysteine‐oleosin, and thioesterase (Chen et al, ; El Tahchy et al, ; Kelly et al, ; Petrie et al, ; Vanhercke et al, , ; Winichayakul et al, ). The latter multigene strategies have proven to be more effective in green tissues for enhancing the carbon flux into TAG at multiple metabolic levels, including upregulation of fatty‐acid biosynthesis (“Push”; e.g.…”

“…Pioneering work on this involved the development of a yeast H1246‐based high‐throughput system for selection of high‐performance enzyme variants (Siloto et al, ). Many improved BnaDGAT1 variants were generated using the aforementioned method and the two most promising ones were used to increase the oil content of tobacco leaves (Chen et al, ). Kinetic analysis indicated that one of the BnaDGAT1 variants exhibited apparent decreased substrate inhibition at concentrations of acyl‐CoA beyond 5 μM (Xu et al, ).…”

“…Kinetic analysis indicated that one of the BnaDGAT1 variants exhibited apparent decreased substrate inhibition at concentrations of acyl‐CoA beyond 5 μM (Xu et al, ). The possible role of the ninth and tenth predicted TMD in enzyme regulation was also identified as a considerable number of beneficial mutations were localized near and within this region (Chen et al, ). A similar yeast‐based high‐throughput system also identified Corylus americana and G. max DGAT1 variants with improved kinetic properties (Roesler et al, ).…”

“…Thus far, our great progress has been made in probing the properties and regulation of DGAT1 and exploring the biotechnological uses of DGAT1. In addition to overexpression of cDNA encoding the wild‐type DGAT1, there have been recent successes in the manipulation of oil production using high‐performance enzyme variants generated via directed evolution (Chen et al, ; Roesler et al, ). Recent advances in genome‐editing techniques, such as, CRISPR (Belhaj et al, ) and targeting‐induced local lesions in genomes (TILLING; Till et al, ), open up new perspectives on improving the enzyme action in planta .…”