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

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

Métabolisme lipidique et cycle du glyoxylate chez la levure Yarrowia lipolytica

The yeast Yarrowia lipolytica is an oleaginous yeast which is able to grow on hydrophobic substrates and C2 compounds as a sole carbo source. The first part of this study deals with the localization of the two proteins Lro1p and Dga1p involved in the final step of triglycerides synthesis. These proteins were shown to be localized in the cytoplasmic membrane and at the surface of the lipid bodies for Lro1p and at the lipid bodies surface for Dga1p. The second part of this study helps to decipher about the function of the glyoxylate cycle in yeast Y. lipolytica. The first objective was to understand the function of the malate dehydrogenase gene in the yeast Y. lipolytica. Unlike in the yeast S. cerevisiae which has three genes encoding a malate dehydrogenase (cytoplasmic, mitochondrial and peroxysomale), Y. lipolytica has only two genes. The first gene YALI0D16753g encodes the mitochondrial malate dehydrogenase and the second gene YALI0E14190g is subject to alternatives splicing. Indeed, depends on the splicing of the intron at the 3' splicing site, the gene YALI0E14190g codes for a cytoplasmic malate dehydrogenase (C-terminal sequence PAN) or a malate dehydrogenase targeted into peroxisome (C-terminal sequence AKI). In the third part, we analyzed the other genes of the glyoxylate cycle. The disruption of the gene ICL1 gene (YALI0C16885g) results in the inability of the mutant to growth on oleic acid and C2 compounds (ethanol, acetate). However, the deletion of the others genes of the glyoxylate cycle namely YALI0E15708g (equivalent of MLS1) and YALI0E02684g (equivalent of CIT2) had no impact on growth on media requiring the involvement of the glyoxylate cycle.La levure Yarrowia lipolytica est une levure oléagineuse capable de croître sur les substrats hydrophobes et les composés en C2 comme seul source de carbonne. La première partie de notre étude a permis de déterminer la localisation des protéines Lro1p et Dga1p impliquées dans la dernière étape de la synthèse des triglycérides. Ces protéines sont localisées dans la membrane cytoplasmique et à la surface des corps lipidique pour Lro1p et à la surface des corps lipidique pour Lro1p et à la surface des corps lipidiques pour dga1p. La deuxième partie de cette étude a permis d'avoir une idée plus précise du fonctionnement du cycle du glyoxylate chez la levure Y. lipolytica. Le premier objectif de cette deuxième partie de notre étude était de comprendre le fonctionnement du gène de la malate déshydrogénase chez cette levure. Contrairement à la levure S. cerevisiae qui possède trois gènes codant pour une malate déshydrogénaze, Y. lipolytica ne possède que deux gènes. Le premier gène YALI0D16753g code une malate déshydrogénase mitochondriale et le second gène YALI0E14190g présente une particularité d'épissage alternatif. En effet, le gène YALI0E14190g, en fonction de l'épissage, code une malate déshydrogénase cytoplasmique (séquence C-terminale PAN) ou une malate déshydrogénase adressée aux peroxysomes (séquence C-terminale AKI). Dans une troisième partie, nous nous sommes intéressés aux autres gènes du cycle du glyoxylate. La disruption du gène ICL1 a entrainé une incapacité de croissance du mutant sur acide oléique et sur les composés en C2 (éthanol, acétate). Néanmoins la suppression MLS et CIT2 n'a pas eu d'impact lors de la croissance sur les milieux nécessitant l'implication cycle du glyoxylate