Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type 1 plant diacylglycerol acyltransferase
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Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol to produce triacylglycerol (TAG). This enzyme, which is critical to numerous facets of oilseed development, has been highlighted as a genetic engineering target to increase storage lipid production in microorganisms designed for biofuel applications. Here, four transcriptionally active DGAT1 genes were identified and characterized from the oil crop Brassica napus. Overexpression of each BnaDGAT1 in Saccharomyces cerevisiae increased TAG biosynthesis. Further studies showed that adding an N-terminal tag could mask the deleterious influence of the DGATs’ native N-terminal sequences, resulting in increased in vivo accumulation of the polypeptides and an increase of up to about 150-fold in in vitro enzyme activity. The levels of TAG and total lipid fatty acids in S. cerevisiae producing the N-terminally tagged BnaDGAT1.b at 72 h were 53 and 28 % higher than those in cultures producing untagged BnaA.DGAT1.b, respectively. These modified DGATs catalyzed the synthesis of up to 453 mg fatty acid/L by this time point. The results will be of benefit in the biochemical analysis of recombinant DGAT1 produced through heterologous expression in yeast and offer a new approach to increase storage lipid content in yeast for industrial applications.
KeywordsYeast DGAT Storage lipid synthesis Brassica napus Biofuel
The authors thank Drs. G. Séguin-Swartz and G. Rakow for providing B. napus line DH12075. RJW acknowledges the support provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada, Alberta Enterprise and Advanced Education, Alberta Innovates Bio Solutions, and the Canada Research Chairs Program. MSG is a recipient of the NSERC Graham Bell Canada Graduate Scholarship, the Alberta Innovates Graduate Student NSERC Top-up Award, and the President’s Doctoral Prize of Distinction.
Conflict of interest
The authors have no conflicts of interests to declare.
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