The Yarrowia lipolytica PAH1 homologue contributes but is not required for triacylglycerol biosynthesis during growth on glucose.

The PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the Mg2+ -dependent dephosphorylation of phosphatidate to produce diacylglycerol, which can be acylated to form triacylglycerol (TAG). In the model oleaginous yeast Yarrowia lipolytica, TAG is the major lipid produced, and its biosynthesis requires a continuous supply of diacylglycerol, which can be provided by the PAP reaction. However, the regulation of Pah1 has not been studied in detail in Y. lipolytica, and thus its contribution to the biosynthesis of TAG in this yeast is not well understood. In this work, we examined the regulation of the PAH1-mediated PAP activity and Pah1 abundance and localization in cells growing on glucose. We found that Pah1 abundance and localization were regulated in a growth-dependent manner, yet the loss of Pah1 did not have a major effect on PAP activity. We also examined the effects of the Y. lipolytica pah1Δ mutation on cell physiology and lipid biosynthesis. The lack of Pah1 in the pah1Δ mutant resulted in a moderate decrease in TAG levels and an increase in phospholipid levels. These results showed that Pah1 contributed to TAG biosynthesis in Y. lipolytica but also suggested the presence of other activities in the pah1Δ mutant that compensate for the loss of Pah1. Also, the levels of linoleic acid were elevated in pah1Δ cells with a concomitant decrease in the oleic acid levels suggesting that the pah1Δ mutation affected the biosynthesis of fatty acids.

Phosphatidate phosphatase activity is induced during lipogenesis in the oleaginous yeast Yarrowia lipolytica.

Phosphatidate (PA) phosphatase dephosphorylates the membrane phospholipid PA to diacylglycerol (DAG) that can be used for the synthesis of the storage lipid triacylglycerol (TAG). In Yarrowia lipolytica, TAG biosynthesis is induced during the lipogenic phase, which results in the accumulation of this lipid in cells. The accumulation of TAG during lipogenesis requires the supply of DAG, but the source of this DAG is not known in Y. lipolytica. In this study, the regulation of PA phosphatase during lipogenesis and its contribution to TAG biosynthesis was examined in Y. lipolytica. Lipogenesis was triggered by growing cells in high-glucose media, whereas control cultures were grown in low-glucose media. PA phosphatase activity increased in a time-dependent manner as high-glucose cells progressed in the lipogenic phase. In contrast, the activity decreased in low-glucose cells that did not accumulate lipids. An analysis of the subcellular localization of the PA phosphatase activity showed that the membrane-associated activity increased during lipogenesis. The significance of this increase can be explained by the fact that only the membrane-associated PA phosphatase activity is responsible for the production of DAG. Taken together, these results indicate that PA phosphatase is involved in TAG biosynthesis during lipogenesis in Y. lipolytica.

Characterization of phosphatidic acid phosphatase activity in the oleaginous yeast Yarrowia lipolytica and its role in lipid biosynthesis.

Phosphatidic acid phosphatase (PAP) catalyses the committed step of triacylglycerol (TAG) biosynthesis and thus regulates the amounts of TAG produced by the cell. TAG is the target of biotechnological processes developed for the production of food lipids or biofuels. These processes are using oleaginous microorganisms like the yeast Yarrowia lipolytica as the TAG producers. Thus manipulating key enzymatic activities like PAP in Y. lipolytica could drive lipid biosynthesis towards TAG production and increase TAG yields. In this study, PAP activity in Y. lipolytica was characterized in detail and its role in lipid biosynthesis was addressed. PAP activity increased 2.5-fold with the addition of Mg2+ (1 mm) in the assay mixture, which means that most of the PAP activity was due to Mg2+ -dependent PAP enzymes (e.g. Pah1, App1). In contrast, N-ethylmaleimide (NEM) potently inhibited PAP activity, indicating the presence of NEM-sensitive PAP enzymes (e.g. App1, Lpp1). Localization studies revealed that the majority of PAP activity resides in the membrane fraction, while the cytosolic fraction harbours only a small amount of activity. PAP activity was regulated in a growth-dependent manner, being induced at the early exponential phase and declining thereafter. PAP activity did not correlate with TAG synthesis, which increased as cells progressed from the exponential phase to the early stationary phase. In stationary phase, TAG was mobilized with the concomitant synthesis of sterols and sterol esters. These results provide the first insights into the role of PAP in lipid biosynthesis by Y. lipolytica. Copyright © 2016 John Wiley & Sons, Ltd.