Characterization of phosphatidic acid phosphatase activity in the oleaginous yeast<i>Yarrowia lipolytica</i>and its role in lipid biosynthesis

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The PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the Mg 2+ -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

Section: Resultssupporting

confidence: 90%

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

Ukey

Carmon

Hardman

et al. 2020

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“…Also in S. cerevisiae , PA phosphatase activity is regulated in a growth‐dependent manner, being higher in stationary phase (Pascual et al, ). In contrast, Y. lipolytica presents a different regulation where PA phosphatase activity is higher in the exponential phase and decreases as cells progress to stationary phase (Hardman et al, ).…”

Section: Discussionmentioning

confidence: 99%

“…That is because the substrate PA localizes in the membrane fraction, which means that only the membrane‐associated activity is physiologically relevant. In Yarrowia lipolytica , the Pah1 homologue localizes in the cytosol (Bredeweg et al, ), but the PA phosphatase activity is much higher in the membrane fraction (Hardman, McFalls, & Fakas, ).…”

Section: Introductionmentioning

confidence: 99%

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

Hardman

Ukey

Fakas

2018

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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.

“…A large body of investigation has focused upon understanding and engineering of carbon metabolism in Y. lipolytica for the purpose of increasing lipid and citrate production, including elucidating the specific pathways, maximizing the availability of reducing equivalents, reducing the amount of lipolysis, and eliminating the diversion of carbon into storage as glycogen (Beopoulos, Cescut, et al, ; Bhutada et al, ; Coelho, Amaral, & Belo, ; Dulermo et al, ; Dulermo et al, ; Dulermo et al, ; Fakas, ; Fickers, Marty, & Nicaud, ; Fickers, Nicaud, Gaillardin, Destain, & Thonart, ; Haddouche et al, ; Hardman, McFalls, & Fakas, ; Loira, Dulermo, Nicaud, & Sherman, ; Papanikolaou et al, ; Trebulle, Nicaud, Leplat, & Elati, ). Yet, despite the central role of nitrogen starvation in the process, the examination of nitrogen metabolic pathways and their influence on the extent of citrate and/or lipid accumulation has received comparatively less attention.…”

Section: Introductionmentioning

confidence: 99%

Glutamate dehydrogenases in the oleaginous yeast Yarrowia lipolytica

Trotter

Juco

et al. 2019

Glutamate dehydrogenases (GDHs) are fundamental to cellular nitrogen and energy balance. Yet little is known about these enzymes in the oleaginous yeast Yarrowia lipolytica. The YALI0F17820g and YALI0E09603g genes, encoding potential GDH enzymes in this organism, were examined. Heterologous expression in gdh‐null Saccharomyces cerevisiae and examination of Y. lipolytica strains carrying gene deletions demonstrate that YALI0F17820g (ylGDH1) encodes a NADP‐dependent GDH whereas YALI0E09603g (ylGDH2) encodes a NAD‐dependent GDH enzyme. The activity encoded by these two genes accounts for all measurable GDH activity in Y. lipolytica. Levels of the two enzyme activities are comparable during logarithmic growth on rich medium, but the NADP‐ylGDH1p enzyme activity is most highly expressed in stationary and nitrogen starved cells by threefold to 12‐fold. Replacement of ammonia with glutamate causes a decrease in NADP‐ylGdh1p activity, whereas NAD‐ylGdh2p activity is increased. When glutamate is both carbon and nitrogen sources, the activity of NAD‐ylGDH2p becomes dominant up to 18‐fold compared with that of NADP‐ylGDH1p. Gene deletion followed by growth on different carbon and nitrogen sources shows that NADP‐ylGdh1p is required for efficient nitrogen assimilation whereas NAD‐ylGdh2p plays a role in nitrogen and carbon utilization from glutamate. Overexpression experiments demonstrate that ylGDH1 and ylGDH2 are not interchangeable. These studies provide a vital basis for future consideration of how these enzymes function to facilitate energy and nitrogen homeostasis in Y. lipolytica.