Synthesis Of Sterol Esters Research Articles

Abolishing storage lipids induces protein misfolding and stress responses in Yarrowia lipolytica.

Yarrowia lipolytica naturally saves excess carbon as storage lipids. Engineering efforts allow redirecting the high precursor flux required for lipid synthesis toward added-value chemicals such as polyketides, flavonoids, and terpenoids. To redirect precursor flux from storage lipids to other products, four genes involved in triacylglycerol and sterol ester synthesis (DGA1, DGA2, LRO1, and ARE1) can be deleted. To elucidate the effect of the deletions on cell physiology and regulation, we performed chemostat cultivations under carbon and nitrogen limitations, followed by transcriptome analysis. We found that storage lipid-free cells show an enrichment of the unfolded protein response, and several biological processes related to protein refolding and degradation are enriched. Additionally, storage lipid-free cells show an altered lipid class distribution with an abundance of potentially cytotoxic free fatty acids under nitrogen limitation. Our findings not only highlight the importance of lipid metabolism on cell physiology and proteostasis, but can also aid the development of improved chassy strains of Y. lipolytica for commodity chemical production.

Production of β-carotene in Saccharomyces cerevisiae through altering yeast lipid metabolism.

Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals. However, as a non-oleaginous yeast, S. cerevisiae has a limited production capacity for lipophilic compounds, such as β-carotene. To increase its accumulation of β-carotene, we engineered different lipid metabolic pathways in a β-carotene producing strain and investigated the relationship between lipid components and the accumulation of β-carotene. We found that overexpression of sterol ester synthesis genes ARE1 and ARE2 increased β-carotene yield by 1.5-fold. Deletion of phosphatidate phosphatase (PAP) genes (PAH1, DPP1, and LPP1) also increased β-carotene yield by twofold. Combining these two strategies resulted in a 2.4-fold improvement in β-carotene production compared with the starting strain. These results demonstrated that regulating lipid metabolism pathways is important for β-carotene accumulation in S. cerevisiae, and may also shed insights to the accumulation of other lipophilic compounds in yeast.

Nanoscale Investigation into the Cellular Response of Glioblastoma Cells Exposed to Protons.

Exposure to ionizing radiation can induce cellular defense mechanisms including cell activation and rapid proliferation prior to metastasis and in extreme cases can result in cell death. Herewith we apply infrared nano- and microspectroscopy combined with multidimensional data analysis to characterize the effect of ionizing radiation on single glioblastoma nuclei isolated from cells treated with 10 Gy of X-rays or 1 and 10 Gy of protons. We observed chromatin fragmentation related to the formation of apoptotic bodies following X-ray exposure. Following proton irradiation we detected evidence of a DNA conformational change (B-DNA to A-DNA transition) related to DNA repair and accompanied by an increase in protein content related to the synthesis of peptide enzymes involved in DNA repair. We also show that proton exposure can increase cholesterol and sterol ester synthesis, which are important lipids involved in the metastatic process changing the fluidity of the cellular membrane in preparation for rapid proliferation.

Optimization and modeling for the synthesis of sterol esters from deodorizer distillate by lipase-catalyzed esterification.

In this paper, cotton seed oil deodorizer distillate (CSODD), was recovered to obtain fatty acid sterol ester (FASE), which is one of the biological activated substances added as human therapeutic to lower cholesterol. Esterification reactions were carried out using Candida rugosa lipase as a catalyst, and the conversion of phytosterol was optimized using response surface methodology. The highest conversion (90.8 ± 0.4%) was reached at 0.84 wt% enzyme load, 1:25 solvent/CSODD mass ratio, and 44.2°C after 12 H reaction. A kinetic model based on the reaction rate equation was developed to describe the reaction process. The activation energy of the reaction was calculated to be 56.9 kJ/mol and the derived kinetic parameters provided indispensable basics for further study. The optimization and kinetic research of synthesizing FASE from deodorizer distillate provided necessary information for the industrial applications in the near future. Experimental results showed that the proposed process is a promising alternative to recycle sterol esters from vegetable oil deodorizer distillates in a mild, efficient, and environmental friendly method.

TORC1 inhibition induces lipid droplet replenishment in yeast.

Lipid droplets (LDs) are intracellular structures that regulate neutral lipid homeostasis. In mammals, LD synthesis is inhibited by rapamycin, a known inhibitor of the mTORC1 pathway. In Saccharomyces cerevisiae, LD dynamics are modulated by the growth phase; however, the regulatory pathways involved are unknown. Therefore, we decided to study the role of the TORC1 pathway on LD metabolism in S. cerevisiae. Interestingly, rapamycin treatment resulted in a fast LD replenishment and growth inhibition. The discovery that osmotic stress (1 M sorbitol) also induced LD synthesis but not growth inhibition suggested that the induction of LDs in yeast is not a secondary response to reduced growth. The induction of LDs by rapamycin was due to increased triacylglycerol but not sterol ester synthesis. Induction was dependent on the TOR downstream effectors, the PP2A-related phosphatase Sit4p and the regulatory protein Tap42p. The TORC1-controlled transcriptional activators Gln3p, Gat1p, Rtg1p, and Rtg3p, but not Msn2p and Msn4p, were required for full induction of LDs by rapamycin. Furthermore, we show that the deletion of Gln3p and Gat1p transcription factors, which are activated in response to nitrogen availability, led to abnormal LD dynamics. These results reveal that the TORC1 pathway is involved in neutral lipid homeostasis in yeast.

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.

Synthesis of fatty acid sterol esters using cholesterol esterase from Trichoderma sp. AS59

We recently reported the characterization of novel cholesterol esterase (EC. 3.1.1.13) from Trichoderma sp. and preliminary work on sterol ester synthesis. In the present study, we further examined the enzyme ability to synthesize cholesterol esters from cholesterol and free fatty acids of various chain lengths, and compared the fatty acid specificity in synthesis with that in hydrolysis. The enzyme catalyzed the synthesis of medium- and long-chain fatty acid cholesterol esters, but failed to synthesize short-chain fatty acid esters. The fatty acid specificities in the synthesis and hydrolysis of cholesterol esters were entirely different from each other. Unlike other lipolytic enzymes, the enzyme was largely independent of water content in the synthesis of cholesterol oleate, and it achieved near-complete esterification in the presence of an equimolar excess of oleic acid. Of additional interest is the finding that the addition of n-hexane markedly enhanced the esterification activities on all the medium- and long-chain saturated fatty acids used. Based on these findings, we attempted to synthesize stigmasterol stearate as a food additive to lower cholesterol levels in blood plasma, and found that the enzyme catalyzed effective synthesis of the ester without the need of dehydration during the reaction, indicating the potential utility of the enzyme in the food industry.

Cellular Sterol Ester Synthesis in Plants Is Performed by an Enzyme (Phospholipid:Sterol Acyltransferase) Different from the Yeast and Mammalian Acyl-CoA:Sterol Acyltransferases

A gene encoding a sterol ester-synthesizing enzyme was identified in Arabidopsis. The cDNA of the Arabidopsis gene At1g04010 (AtPSAT) was overexpressed in Arabidopsis behind the cauliflower mosaic virus 35S promoter. Microsomal membranes from the leaves of overexpresser lines catalyzed the transacylation of acyl groups from phosphatidylethanolamine to sterols. This activity correlated with the expression level of the AtPSAT gene, thus demonstrating that this gene encodes a phospholipid:sterol acyltransferase (PSAT). Properties of the AtPSAT were examined in microsomal fractions from the tissues of an overexpresser. The enzyme did not utilize neutral lipids, had the highest activity with phosphatidylethanolamine, had a 5-fold preference for the sn-2 position, and utilized both saturated and unsaturated fatty acids. Various sterols and sterol intermediates, including triterpenic precursors, were acylated by the PSAT, whereas other triterpenes were not. Sterol selectivity studies showed that the enzyme is activated by end product sterols and that sterol intermediates are preferentially acylated by the activated enzyme. This indicates that PSAT both regulates the pool of free sterols as well as limits the amount of free sterol intermediates in the membranes. Two T-DNA insertion mutants in the AtPSAT gene, with strongly reduced (but still measurable) levels of sterol esters in their tissues, had no detectable PSAT activity in the microsomal fractions, suggesting that Arabidopsis possess other enzyme(s) capable of acylating sterols. The AtPSAT is the only intracellular enzyme found so far that catalyzes an acyl-CoA-independent sterol ester formation. Thus, PSAT has a similar physiological function in plant cells as the unrelated acyl-CoA:sterol acyltransferase has in animal cells.

An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Sacchoromyces cerevisiae

The major route for the synthesis of triacylglycerol (TAG) in yeast as well as in all TAG-accumulating organisms has been suggested to occur via the acylation of diacylglycerol (DAG) by acyl-CoA:diacylglycerol acyltransferase (DAGAT). Genes encoding DAGAT have been identified in both plant and animal tissues. These genes show strong sequence similarities to genes encoding acyl-CoA:cholesterol acyltransferase (ACAT). So far no Saccharomyces cerevisiae DAGAT gene has been published; however, two ACAT-like genes, ARE1 and ARE2, are present in the yeast genome. Both these genes have been suggested to be involved in the synthesis of sterol esters. We have now shown that the ARE1 gene in yeast also is involved in the synthesis of TAG, whereas the ARE2 gene is more specifically involved in the synthesis of sterol esters.

Specific high affinity binding and degradation of high density lipoproteins by isolated epithelial cells of human small intestine

The interaction of high density lipoproteins (HDL) with isolated epithelial cells of human small intestine (enterocytes) was studied. 125I-HDL 3 (density = 1,125 to 1,126 g/cm 3) exhibit a high-affinity (Kd = 8.3 × 10 −8. Bmax = 886 ng/mg cell protein), saturable and reversible binding to isolated enterocytes. In the presence of excess unlabeled HDL 3, the cell surface-bound 125I-HDL 3 are released into the medium nondegraded. Treatment of cells with pronase does not affect 125I-HDL 3 binding. The binding is accompanied with internalization and degradation of 125I-HDL 3. Chloroquine inhibits the degradation and increases 125I-HDL 3 uptake. A threefold excess of HDL 3 and HDL 2 inhibits the binding and degradation of 125I-HDL 3 by 60%, whereas a 20-fold excess of low density lipoproteins (LDL), only by 20%. HDL 3 (20 to 1,000 μg/mL) stimulates the synthesis of sterols and inhibits sterol ester synthesis in enterocytes. The obtained results make it possible to assume that epithelial cells of the small intestine may participate in the catabolism of HDL in human organism.

Biochemistry of ungerminated and germinated spores of the vesicular-arbuscular mycorrhizal fungus, Glomus caledonius: changes in neutral and polar lipids.

Neutral and polar spore lipids of the vesicular-arbuscular (VA) endophyte Glomus caledonius, were identified and quantitatively determined during spore germination, germ tube growth, and germ tube senescence. There are no previous reports detailing the spore lipid components of any member of the Endogenaceae, which is in the Zygomycotina. The fungus contained 45 to 72% total lipid depending upon its stage of growth. The concentration of neutral lipids decreased during germination while the polar lipids increased. Triacylglycerides were the most abundant neutral lipid, and lesser amounts of diacylglycerides, monoacylglycerides, free fatty acids, bound fatty acids, hydrocarbons, and sterols. The major fatty acids identified by gas--liquid chromatography and mass spectrometry were 16:1, 16:0, and 18:1. The minor fatty acids identified were n-3 and n-6 polyunsaturates. The n-3 polyunsaturated fatty acids have not been reported before in Zygomycetes. The fatty acid composition of the individual lipid classes was examined. The major phospholipids were phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, with smaller amounts of diphosphatidylglycerol and phosphatidic acid. The free sterol fraction was in greater quantity than sterol esters during germination and germ tube elongation. The capacity to synthesize sterols was demonstrated. Approximate net rates of change in the different lipid components were calculated. During spore germination and early germ tube growth, there was a net synthesis of lipids, with a large production of free fatty acids, in the germinating spore. Later in the growth period there was a net degradation of lipid, characterized by a large conversion of free fatty acids to unidentified compounds. During this period net free sterol synthesis ceased and sterol ester synthesis continued using the existing free sterol.

Measurement in vitro of the esterification of yeast sterols.

A highly reproducible and rapid procedure is described for isolating free sterols and sterol esters from cell-free extracts of yeast. Using this technique, the sterol esterifying enzyme has been studied. Sterol ester hydrolase has also been demonstrated. In agreement with whole cell experiments, the enzyme activity for sterol ester synthesis has been found to increase on entrance of the culture into the stationary phase of growth.

Yeast sterol esters and their relationship to the growth of yeast.

Variation in the percentage of sterols esterified to long-chain fatty acids during cellular growth has been examined. Under all conditions, a constant percentage of sterol esters was maintained during exponential growth. This maintenance level was found to vary with different growth conditions. A sharp increase in the rate of esterification was observed upon entry of the culture into the stationary growth phase. The minor cellular sterol components were found to accumulate after this period of rapid sterol ester synthesis, with a relative decrease in the size of the ergosterol pool. Evidence is presented that sterol esters of ergosterol precursors are unable to be metabolized to ergosterol. Once esterified, the fatty acids do not appear to be scavenged during starvation conditions.

Purification and properties of aortic cholesteryl ester hydrolase.

The enzyme(s) present in acetonedried powder of rat and rabbit aortas, which catalyzes the synthesis and hydrolysis of cholesteryl ester, was purified partially by acid precipitation, acetone fractionation, O-(diethylaminoethyl) cellulose chromatography, and Sephadex G-100 filtration. The synthetic activity was purified by 120-fold (rat) and 140-fold (rabbit). Purification of hydrolytic activity was 90-fold (rat) and 103-fold (rabbit). Cholesteryl ester hydrolase activity was separated from nonspecific esterase by column chromatography. Both synthetic and the hydrolytic activities are apparently the functions of one enzyme. The mol wt of the enzyme was estimated to be 140,000 dalton as determined by Sephadex G-200 gel filtration. The extracts of the acetone-dried powders of aortas of both species contained an inhibitor of synthetic activity. The inhibitor was nondialyzable and was precipitated at pH 5.7. Both activities were found to be fairly nonspecifc with regard to sterol and fatty acids. With oleic acid, the relative rates of sterol ester synthesis were: cholesterol, 100; cholestanol, 94; desmosterol, 35; coprostanol, 24; ergosterol, 20; and beta-sitosterol, 19. Epicholesterol was not esterified. Oleic acid was most active in cholesteryl ester synthesis, the relative rates being: oleic greater than linoleic greater than arachidonic greater than palmitic greater than stearic greater than butyric. The rate of hydrolysis was maximum with cholesteryl linoleate followed by oleate, linolenate, palmitate, stearate, and laurate in decreasing order.

Changes in Neutral Lipid Constituents during Differentiation of the Cellular Slime Mold, Dictyostelium discoideum

Abstract The total lipid content of differentiating Dictyostelium discoideum was 100 to 125 mg per g dry weight; of this, about 95 mg per g was polar lipid (mainly phospholipid) and 25 mg per g was neutral lipid in the early stages. During culmination and maturation of the sorocarp, polar lipid declined to 75 mg per g, while neutral lipid increased to 40 mg per g. Per 1010 cells, polar lipid declined from 135 to 75 mg and neutral lipid increased from 33 to 45 mg. The neutral lipid fraction was resolved into its major components and the level of each in milligrams per g dry weight was determined at 10 different stages of slime mold differentiation. Stigmast-22-en-3β-ol remained near 10 to 11 during the early stages, rose to 15 at the beginning of migration, and then declined back to about 12; stigmastenol C16-C18 esters began a prolonged rise from 1 to 2 during early aggregation, ultimately reaching 15 in 3-day sorocarps; ubiquinone declined from 0.5 to 0.2 to 0.3 during aggregation where it remained until culmination, when it rose rapidly to a maximum of 1 to 2 in mature sorocarps; free fatty acids increased to a maximum of 7 during early aggregation and then fell sharply to less than 2 during migration; and triglycerides and diglycerides declined from initial levels of 2 to 4 to about 1 during the early stages. Approximate balances among the different lipid components were calculated from the changes in quantities. During the period prior to early aggregation, a net synthesis of lipid (phospholipid, sterol, and sterol ester) occurred from unidentified precursors. During aggregation metabolism shifted toward a net degradation, characterized by a rapid conversion of free fatty acids to unidentified products. This net degradation continued during the migration-preculmination period, although phospholipid replaced the nearly exhausted triglyceride and free fatty acid reserves as the major source of lipid. During the later periods net sterol synthesis ceased, but sterol ester synthesis continued at the expense of preformed free sterol.

Compartmental Utilization of Carboxyl-14C-Tripalmitin by Tissue Homogenate of Pine Seeds

A tissue homogenate of megagemetophyte of germinating seeds of Jeffrey pine (Pinus Jefferii Grev. and Balf.) was incubated with sonication-dispersed and albumin-carried (14)C-tripalmitin in order to elucidate the sequential and quantitative role of cellular organelles in utilizing lipid reserve in seeds. After 5 minutes at 30 C, 25% of the tracer was localized in the fat body fraction, 9% in the pellet containing mitochondria and glyoxysomes, 14% in the supernatant, and 2% was found as CO(2). Radioactivity increased with time of incubation in the latter three fractions indicating the forward direction of utilization. Fat bodies contained mainly lipases and hydrolyzed the tracer to palmitate with diglyceride and monoglyceride as intermediates. About two-thirds of the palmitate had left the fat bodies in 5 minutes and entered the pellet fraction within which the tracer was distributed 1:2 in mitochondria and glyoxysomes, respectively. Longer incubation reduced the ratio to 1:3 while both organelles acquired more radioactive intermediates. Labeled acetyl-CoA and intermediate of beta-oxidation were found in both organelle-containing fractions. The supernatant fraction contained radioactive diglycerides, monoglycerides, palmitate, sterol esters, and phospholipids, indicating lipase activity and direct utilization of fatty acid for the synthesis of sterol esters and polar lipids.

Changes in the lipid composition and fine structure of Saccharomyces cerevisiae during ascus formation.

Eighty to ninety percent of vegetative cells of Saccharomyces cerevisiae DCL 740 incubated in KCl-acetate medium form asci, the majority of which are four-spored. Ascospores are visible in asci after about 24 hr, and spore formation is complete after about 48 hr. The dry weight of the cells increases by about 75% during 48 hr of incubation, while the lipid content of the cells increases by a factor of four. The increase in lipid content is attributed mainly to an increased synthesis of sterol esters and triacylglycerols and to a lesser extent of phospholipids. The phospholipid and sterol compositions do not change appreciably, but there is a marked increase in the proportion of unsaturated fatty acid residues in ascan lipids. Uniformly labeled (14)C-acetate is incorporated mainly into sterol esters and triacylglycerols and phospholipids. Pulse-labeling by adding acetate-U-(14)C to sporulating cultures and harvesting after a further 6 hr of incubation reveal two main periods of acetate incorporation, namely between 0 and 18 hr, and between 24 and 30 hr. Electron micrographs of thin sections through developing asci show that the principal changes in fine structure occur between 18 and 24 hr and include the appearance of numerous electron-transparent vesicles which become aligned around the meiotic nucleus, and the laying down of extensive endoplasmic reticulum membranes. Changes in fine structure are discussed in relation to the alterations in lipid content and composition of asci.

Lipid composition of Saccharomyces cerevisiae as influenced by growth temperature

The lipid compositions of Saccharomyces cerevisiae N.C.Y.C. 366 grown in batch culture at 30 °C and 15 °C were compared. The compositions of lipids from cells grown in a chemostat, under glucose limitation and at constant dissolved oxygen tension at two rates at 30 °C and one rate at 15 °C, were also examined to discover whether the growth temperature-induced changes in the lipid composition of batch-grown cells were caused solely by the change in temperature or whether they were conditioned partially or wholly by the lowering of the growth rate that accompanies a decrease in the growth temperature of batch cultures. Lowering the growth temperature of batch cultures was accompanied by an increase in the contents of total lipids, total fatty acids, triacylglycerols and phospholipid (mainly as phosphatidylcholine), but had little or no effect on the contents of diacylglycerols, free fatty acids, sterols or sterol esters. Analyses of lipids from chemostat-grown cells suggested that the increased synthesis of triacylglycerols and phospholipid was caused mainly by lowering of the growth temperature, but that the increased content of phosphatidylcholine could be attributed mainly to a decrease in growth rate. Lowering the rate at which cells were grown continuously at 30 °C caused an increased synthesis of sterol esters whereas lowering the temperature at which cells were grown at a fixed rate was accompanied by a decreased content of both total and esterified sterols. Extracts of all cells examined contained two major sterols, Δ 5,7,22,24,(28)-ergostatetraene-3β-ol and ergosterol. There was little change in either the fatty acid composition or the degree of unsaturation of the fatty acids in batch-grown or chemostat-grown cells following a change in incubation temperature.

Cholesterol esterase — A polymeric enzyme

A direct interaction of cholic acid and pancreatic cholesterol esterase has been shown using labeled bile salt of high specific activity and Sephadex G-50 chromatography. This interaction results in the formation of a protein of M.W. 400,000 which appears to be a hexamer of the protein in pancreatic juice (M.W. 65,000 – 69,000). Rechromatography of the polymer on Sephadex G-200 results in dissociation to the monomeric form. Evidence is provided, suggesting that the polymeric form of cholesterol esterase represents the active enzyme responsible for sterol ester synthesis and hydrolysis.

The effect of cultural conditions on the ergosterol ester components of yeast

The ergosterol ester fraction of Saccharomyces cerevisiae has been analyzed following a variety of incubation conditions. Free fatty acids were obtained after saponification of the sterol ester fraction and were analyzed gas chromatographically. Cells grown under anaerobic conditions showed a maximal rate of synthesis of sterol esters at 48 hours. Under aerobic incubation conditions, maximum sterol esterification was seen at 12 hours. Esters of C 16 and C 18 fatty acids are the predominant ones, followed by those of C 14 acids. The rates of synthesis of free sterol and sterol esters follow similar patterns.