Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts

Abstract

Triacylglycerols (TAG) and steryl esters (SE) are the principal storage lipids in all eukaryotic cells. In yeasts, these storage lipids accumulate within special organelles known as lipid bodies (LB). In the lipid accumulation-oriented metabolism of the oleaginous yeast Yarrowia lipolytica, storage lipids are mostly found in the form of TAG, and only small amounts of SE accumulate. We report here the identification of a new DAG acyltransferase gene, DGA2, homologous to the ARE genes of Saccharomyces cerevisiae. This gene encodes a member of the type 1 acyl-CoA:diacylglycerol acyltransferase family (DGAT1), which has not previously been identified in yeasts, but is commonly found in mammals and plants. Unlike the Are proteins in S. cerevisiae, Dga2p makes a major contribution to TAG synthesis via an acyl-CoA-dependent mechanism and is not involved in SE synthesis. This enzyme appears to affect the size and morphology of LB, suggesting a direct role of storage lipid proteins in LB formation. We report that the Are1p of Y. lipolytica was essential for sterol esterification, as deletion of the encoding gene (ARE1) completely abolished SE synthesis. Unlike its homologs in yeasts, YlARE1 has no DAG acyltransferase activity. We also reconsider the role and function of all four acyltransferase enzymes involved in the final step of neutral lipid synthesis in this oleaginous yeast.

Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production

“…1a). Specifically, AMPDp, ACLp, and MAEp overexpression were investigated for their potential to increase acetyl-CoA and NADPH supply (ACCp overexpression has not been reported to significantly improve lipogenesis and was excluded 13 ) and DGA1p and DGA2p (acyl-CoA:diacylglycerol acyltransferases isozymes I and II) were included for their potential in catalysing the ultimate step in triglyceride synthesis 34 . These overexpression targets were multiplexed with deletions that served to reduce fatty-acid catabolism by reducing one or both of b-oxidation (via mfe1 deletion) 14,15 and peroxisome biogenesis (via pex10 deletion) 25,35 (Table 1).…”

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

“…This yeast platform has its own native P450 system that catalyzes terminal oxidation of alkanes and fatty acids [36]. Moreover, this platform possesses an endogenous redox partner (cytochrome P450 reductase), has the capability for large free fatty acid accumulation [111], and can efficiently generates NADPH cofactor mainly through the pentose phosphate pathway (PPP) [112,113]. This pathway is not tightly regulated by nitrogen concentration [114].…”

Strain Design of Ashbya gossypii for Single-Cell Oil Production

“…Recent studies have described the metabolic engineering of lipid biosynthesis in both oleaginous and nonoleaginous microorganisms, with rates of lipid accumulation not exceeding 60% of cell dry weight (12,14,15,39,40). Such modifications include increasing intracellular citrate levels, the overexpression of ACL genes, the overexpression of genes to enhance TAG biosynthesis, and the abolition of the beta-oxidation pathway and other competing pathways (12,14,15,22,23,40).…”

“…For lipid accumulation analyses, the carbon source of the MA2 medium was either 8% glucose or 1% glucose plus 2% oleic acid (Sigma), previously emulsified by sonication in the presence of 0.02% Tween 40 (22). Cultures with oil were centrifuged at 10,000 ϫ g for 10 min, and the resulting cell pellet was washed three times with equal volumes of SB solution (9 g/liter NaCl in 0.5% bovine serum albumin [BSA]) (23). A. gossypii transformation, sporulation conditions, and spore isolation have been described elsewhere (24).…”