A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds

Abstract

Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45–50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions.

Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type 1 plant diacylglycerol acyltransferase

“…To initially characterize the products of these genes, we expressed their CDSs in S. cerevisiae, a model organism commonly used for plant lipid research (Liu et al 2011;Sandager et al 2002;Siloto et al 2009a;Turchetto-Zolet et al 2011;Yu et al 2008;Zhang et al 2013). This initial characterization, however, was hindered by the large differences observed in BnaDGAT1 isoforms' accumulation when produced in this species.…”

An Improved Variant of Soybean Type 1 Diacylglycerol Acyltransferase Increases the Oil Content and Decreases the Soluble Carbohydrate Content of Soybeans

“…An alternative hypothesis for the higher 18:1 content considers that DAG and PC can be interconverted by choline phosphotransferase (Slack et al, 1985). A kinetically improved DGAT variant would be more likely to pull DAG toward TAG synthesis and away from PCbased desaturation reactions, even in the presence of a strong soybean FAD3 desaturase activity, as discussed previously (Zhang et al, 2013). The increased incorporation of saturated fatty acids into TAG, on the other hand, may be caused by increased flux through a distinct route of TAG biosynthesis with preference for nascent saturated acyl-CoAs (Bates et al, 2009).…”

A Specialized Diacylglycerol Acyltransferase Contributes to the Extreme Medium-Chain Fatty Acid Content of Cuphea Seed Oil

“…Cells were harvested by centrifugation, washed twice with 0.1% NaHCO 3 , freeze-dried, and used for fatty acid and TAG analysis. Lipids were extracted from cell pellets as described in Bligh and Dyer (1959) and Zhang et al (2013). Neutral lipids including TAG were resolved on Silica Gel 60 (Merck) TLC plates using a solvent consisting of 70:30:1 v/v) heptane:ethyl ether:acetic acid and detected with iodine vapor.…”

“…Total lipids were extracted for analysis of TAG, DAG, and PC fatty acid composition (Bligh and Dyer, 1959;Zhang et al, 2013). Camelina seeds (30 mg) in 13 3 100-mm glass tubes were ground using a TH Homogenizer (Omni) in 3 mL methanol:chloroform (2:1 v/v).…”