title: Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type 1 plant diacylglycerol acyltransferase.
abstract: 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.
authors.affiliation: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Greer
authors.forename: Martin
authors.initials: M
authors.lastname: Truksa
authors.forename: Wei
authors.initials: W
authors.lastname: Deng
authors.forename: Shiu-Cheung
authors.initials: SC
authors.lastname: Lung
authors.forename: Guanqun
authors.initials: G
authors.lastname: Chen
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Weselake
mesh_terms: D029688:Brassica napus / Q000201:enzymology / Q000235:genetics; D051048:Diacylglycerol O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D015870:Gene Expression; D060847:Metabolic Engineering; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014280:Triglycerides / Q000378:metabolism*
chemical_list: D005227:Fatty Acids; D011994:Recombinant Proteins; D014280:Triglycerides; D051048:Diacylglycerol O-Acyltransferase