A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This ⌬gut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to ␤-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal ␤-oxidation. Additional deletion of the POX1 to POX6 genes in the ⌬gut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.

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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.

Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal ␤-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (⌬pox2 ⌬pox3 ⌬pox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.The yeast Yarrowia lipolytica can grow on alkanes or fatty acids as the sole carbon source (for a review, see reference 5). Alkanes must be converted to fatty acids before they can serve as substrates for different metabolic pathways. In mammalian cells, the degradation of fatty acids occurs through ␤-oxidation in mitochondria and peroxisomes. In yeasts, the enzymes in this pathway are present only in peroxisomes. Fatty acid degradation is a multiple-step process requiring four different enzymatic activities. The first step is catalyzed by an acyl-coenzyme A (CoA) oxidase (Aoxp). Saccharomyces cerevisiae contains only one Aox (28), but Y. lipolytica contains five Aox isoenzymes with different substrate specificities and different activity levels ( Fig. 1) (27,28). Aox3p is specific for short-chain acyl-CoAs (17), Aox2p preferentially oxidizes long-chain acylCoAs (18), and the Aox4p and Aox5p activities do not appear to be sensitive to the length of the aliphatic chain of the acyl-CoA (29). Previous studies revealed that the mutants MTLY36 (⌬pox2 ⌬pox3 ⌬pox5) and MTLY37 (⌬pox2 ⌬pox3 ⌬pox4 ⌬pox5) either had a growth defect or did not grow at all, respectively, when cultivated on oleic acid minimal medium (28).Fatty acids can also be utilized as storage molecules when they are incorporated into triacylglycerols. All eukaryotic organisms and some gram-positive bacteria store triacylglycerols in intracellular compartments that are variously termed lipid particles, lipid droplets (LDs), lipid bodies (LBs), oil bodies, oleosomes, or spherosomes (in plants). The structure of these lipid-rich compartments is similar in all cell types and is rather simple: LBs consist of a hydrophobic core formed from neutral lipids, mainly triacylglycerols and/or steryl esters...