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.

Lipid biosynthesis in yeasts: A comparison of the lipid biosynthetic pathway between the model nonoleaginous yeast Saccharomyces cerevisiae and the model oleaginous yeast Yarrowia lipolytica.

Lipid biosynthesis and its regulation have been studied mostly in the nonoleaginous yeast Saccharomyces cerevisiae that serves as a model for eukaryotic cells. On the other hand, the yeast Yarrowia lipolytica has been put forward as a model for oleaginous microorganisms because its genetics is known and tools for its genetic manipulation are becoming increasingly available. A comparison of the lipid biosynthetic pathways that function in these two microorganisms shows many similarities in key biosynthetic and regulatory steps. An example is the enzyme phosphatidic acid phosphatase that controls the synthesis of triacylglycerol (TAG) in both yeasts. Controlling the TAG synthesis is crucial for metabolic engineering efforts that aim to increase the production of microbial lipids (i.e. single cell oils) because TAG comprises the final product of these processes. At the same time the comparison reveals fundamental differences (e.g. in the generation of acetyl-CoA for lipid biosynthesis) stemming from the oleaginous nature of Y. lipolytica. These differences warranty more studies in Y. lipolytica where the biochemistry and molecular biology of oleaginicity can be further explored.

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.

Distinct roles of the phosphatidate phosphatases lipin 1 and 2 during adipogenesis and lipid droplet biogenesis in 3T3-L1 cells.

Lipins are evolutionarily conserved Mg(2+)-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.

Transcription factor Reb1p regulates DGK1-encoded diacylglycerol kinase and lipid metabolism in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the DGK1-encoded diacylglycerol kinase catalyzes the CTP-dependent phosphorylation of diacylglycerol to form phosphatidate. This enzyme, in conjunction with PAH1-encoded phosphatidate phosphatase, controls the levels of phosphatidate and diacylglycerol for phospholipid synthesis, membrane growth, and lipid droplet formation. In this work, we showed that a functional level of diacylglycerol kinase is regulated by the Reb1p transcription factor. In the electrophoretic mobility shift assay, purified recombinant Reb1p was shown to specifically bind its consensus recognition sequence (CGGGTAA, -166 to -160) in the DGK1 promoter. Analysis of cells expressing the PDGK1-lacZ reporter gene showed that mutations (GT→TG) in the Reb1p-binding sequence caused an 8.6-fold reduction in ß-galactosidase activity. The expression of DGK1(reb1), a DGK1 allele containing the Reb1p-binding site mutation, was greatly lower than that of the wild type allele, as indicated by analyses of DGK1 mRNA, Dgk1p, and diacylglycerol kinase activity. In the presence of cerulenin, an inhibitor of de novo fatty acid synthesis, the dgk1Δ mutant expressing DGK1(reb1) exhibited a significant defect in growth as well as in the synthesis of phospholipids from triacylglycerol mobilization. Unlike DGK1, the DGK1(reb1) expressed in the dgk1Δ pah1Δ mutant did not result in the nuclear/endoplasmic reticulum membrane expansion, which occurs in cells lacking phosphatidate phosphatase activity. Taken together, these results indicate that the Reb1p-mediated regulation of diacylglycerol kinase plays a major role in its in vivo functions in lipid metabolism.

Phosphatidate phosphatase plays role in zinc-mediated regulation of phospholipid synthesis in yeast.

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated by mechanisms that control the homeostasis of the essential mineral zinc (Carman, G.M., and Han, G. S. (2007) Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion. Biochim. Biophys. Acta 1771, 322-330; Eide, D. J. (2009) Homeostatic and adaptive responses to zinc deficiency in Saccharomyces cerevisiae. J. Biol. Chem. 284, 18565-18569). The synthesis of phosphatidylcholine is balanced by the repression of CDP-diacylglycerol pathway enzymes and the induction of Kennedy pathway enzymes. PAH1-encoded phosphatidate phosphatase catalyzes the penultimate step in triacylglycerol synthesis, and the diacylglycerol generated in the reaction may also be used for phosphatidylcholine synthesis via the Kennedy pathway. In this work, we showed that the expression of PAH1-encoded phosphatidate phosphatase was induced by zinc deficiency through a mechanism that involved interaction of the Zap1p zinc-responsive transcription factor with putative upstream activating sequence zinc-responsive elements in the PAH1 promoter. The pah1Δ mutation resulted in the derepression of the CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol pathway enzyme) and loss of the zinc-mediated regulation of the enzyme. Loss of phosphatidate phosphatase also resulted in the derepression of the CKI1-encoded choline kinase (Kennedy pathway enzyme) but decreased the synthesis of phosphatidylcholine when cells were deficient of zinc. This result confirmed the role phosphatidate phosphatase plays in phosphatidylcholine synthesis via the Kennedy pathway.

Phosphatidate phosphatase activity plays key role in protection against fatty acid-induced toxicity in yeast.

The PAH1-encoded phosphatidate (PA) phosphatase in Saccharomyces cerevisiae is a pivotal enzyme that produces diacylglycerol for the synthesis of triacylglycerol (TAG) and simultaneously controls the level of PA used for phospholipid synthesis. Quantitative lipid analysis showed that the pah1Δ mutation caused a reduction in TAG mass and an elevation in the mass of phospholipids and free fatty acids, changes that were more pronounced in the stationary phase. The levels of unsaturated fatty acids in the pah1Δ mutant were unaltered, although the ratio of palmitoleic acid to oleic acid was increased with a similar change in the fatty acid composition of phospholipids. The pah1Δ mutant exhibited classic hallmarks of apoptosis in stationary phase and a marked reduction in the quantity of cytoplasmic lipid droplets. Cells lacking PA phosphatase were sensitive to exogenous fatty acids in the order of toxicity palmitoleic acid > oleic acid > palmitic acid. In contrast, the growth of wild type cells was not inhibited by fatty acid supplementation. In addition, wild type cells supplemented with palmitoleic acid exhibited an induction in PA phosphatase activity and an increase in TAG synthesis. Deletion of the DGK1-encoded diacylglycerol kinase, which counteracts PA phosphatase in controlling PA content, suppressed the defect in lipid droplet formation in the pah1Δ mutant. However, the sensitivity of the pah1Δ mutant to palmitoleic acid was not rescued by the dgk1Δ mutation. Overall, these findings indicate a key role of PA phosphatase in TAG synthesis for protection against fatty acid-induced toxicity.

DGK1-encoded diacylglycerol kinase activity is required for phospholipid synthesis during growth resumption from stationary phase in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, triacylglycerol mobilization for phospholipid synthesis occurs during growth resumption from stationary phase, and this metabolism is essential in the absence of de novo fatty acid synthesis. In this work, we provide evidence that DGK1-encoded diacylglycerol kinase activity is required to convert triacylglycerol-derived diacylglycerol to phosphatidate for phospholipid synthesis. Cells lacking diacylglycerol kinase activity (e.g. dgk1Δ mutation) failed to resume growth in the presence of the fatty acid synthesis inhibitor cerulenin. Lipid analysis data showed that dgk1Δ mutant cells did not mobilize triacylglycerol for membrane phospholipid synthesis and accumulated diacylglycerol. The dgk1Δ phenotypes were partially complemented by preventing the formation of diacylglycerol by the PAH1-encoded phosphatidate phosphatase and by channeling diacylglycerol to phosphatidylcholine via the Kennedy pathway. These observations, coupled to an inhibitory effect of dioctanoyl-diacylglycerol on the growth of wild type cells, indicated that diacylglycerol kinase also functions to alleviate diacylglycerol toxicity.

Metabolic activities of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures.

The growth of Yarrowia lipolytica on glycerol was studied in bioreactor repeated batch cultures and three distinct phases, namely biomass production phase, lipogenic phase and citric acid production phase were identified during growth cycle. In each phase, yeast cells were characterised by specific morphological and biochemical features. Though high activity of NAD(+) dependent iso-citric dehydrogenase (NAD(+)-ICDH) was detected during biomass production phase, this activity was significantly decreased afterwards inducing lipogenesis. A further drop in NAD(+)-ICDH activity to minimal levels and a decrease in glycerol kinase activity were observed during the citric acid production phase. Surprisingly, citric acid production was accompanied by storage (neutral) lipid turnover, along with remarkable biosynthesis of glycolipids, sphingolipids and phospholipids. Oleic acid was the major fatty acid in all lipid fractions and phosphatidylcholine was the main phospholipid. This study allows concluding that Y. lipolytica successfully converts glycerol via phosphorylation pathway into valuable biotechnological products, such as single cell oil and citric acid.

Fatty acid composition in lipid fractions lengthwise the mycelium of Mortierella isabellina and lipid production by solid state fermentation.

This paper investigates the correlation between mycelial age and fatty acid biosynthesis. The correlation was investigated by analyzing the lipid composition lengthwise the mycelium of the oleaginous fungus Mortierella isabellina, a potential producer of gamma-linolenic acid (GLA). Young mycelia were rich in polar lipids (glycolipids plus sphingolipids and phospholipids), while neutral lipid content increased in aged mycelia. In young mycelia, each polar lipid fraction contained almost 40% (w/w) polyunsaturated fatty acids (PUFAs), but this content decreased to less than 30% (w/w) in aged mycelia. On the other hand, PUFA content in neutral lipids fluctuated slightly with age. These results indicate that PUFA biosynthesis is favored in young, fast growing mycelia, while it decreases significantly in aged mycelia. This trend was also observed when we grew M. isabellina on pear pomace, an agro-industrial waste. Pear pomace cultures yielded significant amounts of lipid, which reached 12% (w/w) in dry fermented mass. The produced lipid was rich in GLA and the maximum GLA content in dry fermented mass was 2.9 mg/g.

Gamma-linolenic acid production by Cunninghamella echinulata growing on complex organic nitrogen sources.

Growth of two strains of Cunninghamella echinulata on various nitrogen containing raw materials (corn gluten, corn steep, whey concentrate,yeast extract and tomato waste hydrolysate) yielded important amounts of biomass containing various quantities of gamma-linolenic acid (GLA) rich cellular lipids. Especially, growth on tomato waste hydrolysate (TWH) yielded 17.6 g/l of biomass containing 39.6% oil and significant quantities of GLA corresponding to 800 mg/l GLA. Mycelium-bounded proteolytic activity was detected during early growth stages on TWH and declined thereafter, increasing the concentration of assimilable nitrogen in the medium. However, addition of glucose in the medium during the stationary phase triggered the biosynthesis of reserve lipid, since an increase of the proportion of neutral lipids from 45% to 79% in total lipids was observed, while polar lipids decreased from 35% to 12% and from 20% to 9% for glycolipids plus sphingolipids and phospholipids, respectively.

Citric acid production by Yarrowia lipolytica cultivated on olive-mill wastewater-based media.

Yarrowia lipolytica ACA-DC 50109 cultivated on olive-mill wastewater (O.M.W.)-based media, enriched with commercial-industrial glucose, presented an efficient cell growth. Parameters of growth were unaffected by the presence of O.M.Ws in the growth medium. In diluted O.M.Ws enriched with high glucose amounts (initial sugar concentration, 65 g l(-1)), a notable quantity of total citric acid was produced (28.9 g l(-1)). O.M.W.-based media had a noteworthy stimulating effect on the production of citric acid, since both final citric acid concentration and conversion yield of citric acid produced per unit of sugar consumed were higher when compared with the respective parameters obtained from trials without added O.M.W. Adaptation of the strain in O.M.W.-based media favoured the biosynthesis of cellular unsaturated fatty acids (principally of oleic and palmitoleic acids). Additionally, a non-negligible decrease of the phenolic compounds in the growth medium [up to 15% (wt/wt)], a slight decrease of the phyto-toxicity, and a remarkable decolourisation of the O.M.W. were observed. All these results suggest the potentiality of O.M.Ws utilisation in the fermentation process of citric acid production.

Lipids of Cunninghamella echinulata with emphasis to gamma-linolenic acid distribution among lipid classes.

Changes in lipid composition of the oleaginous fungus Cunninghamella echinulata were monitored during growth. Lipid fractions and individual lipid classes varied in amount, relative proportions, and fatty acid profile depending on the developmental stage. Neutral lipids (N), comprised mainly of triacylglycerol, were accumulated in the fungal mycelium during both the late exponential and the stationary growth phases with a concomitant decrease in the amount of polar lipids. While fatty acid composition of N fraction remained almost constant, individual N classes showed a noticeable alteration in gamma-linolenic acid (GLA) concentration. The glycolipid plus sphingolipid (G+S) fraction consisted mainly of monoglycosylglycerol and diglycosylglycerol. The sugar composition of G+S fraction was analyzed and showed a partial replacement of galactose for glucose as growth proceeded. Phospholipid (P) major classes were phosphatidylcholine (PC) and phosphatidylethanolamine, followed by phosphatidylinositol, phosphatidylserine, and diphosphatidylglycerol. P fatty acid composition showed significant changes with time, resulting in a considerable drop in the unsaturation index of this fraction. While in mid exponential growth phase, all P classes contained more than 20% w/w GLA of total fatty acids, and their concentration decreased to 12-17% w/w, except for the PC class where GLA concentration remained at high levels (e.g., more than 20% w/w). The constant level of GLA in PC at all growth phases suggests that PC was the major source of GLA. Sterol analysis showed that their concentration increased during growth, whereas ergosterol was the major component.