Lipid content of Saccharomyces cerevisiae strains with different degrees of ethanol tolerance

Summary We analysed the fatty acid and sterol compositions of various Saccharomyces cerevisiae strains with ethanol tolerance varying from 4% to 12% (v/v) ethanol and at different concentrations of ethanol. The results we obtained agree with the existence of a relationship between membrane fluidity and ethanol tolerance but they do not support a direct role of unsaturated fatty acids in this tolerance. On the other hand, they support the importance of ergosterol in this phenomenon.     

The sensitivity of a commercial lager yeast to novel inhibitors of unsaturated fatty acid and ergosterol biosynthesis

Summary Cyclopropenoid fatty acids inhibited fatty acid desaturation in a lager strain ofSaccharomyces uvarum. N,N-dimethylazasqualene prevented ergosterol biosynthesis in the same yeast. Inhibitions took place under conditions favourable for ergosterol and unsaturated fatty acid formation, each independently of the other.     

Thermostability of Pseudomonas hydrogenothermophila Cytochrome c-552

The alcohol-fermenting yeast Torulaspora delbrueckii No. 3110 was less tolerant to high temperature than Saccharomyces cerevisiae IFO 0224 as measured by alcohol fermentation during mild agitation: at 40°C, ethanol production of the two yeasts was 0.8 and 5.2 wt% respectively. The No. 3110 cells had much unsaturated fatty acid (C18:2) and little ergosterol, which suggests that the low tolerance might be caused by high membrane fluidity. Two types of miconazole-resistant mutants were isolated and characterized. Strain M47 had less unsaturated fatty acid and was found to be more temperature tolerant than No. 3110. Strain M59 was defective in ergosterol synthesis and was less temperature tolerant than No. 3110. These results indicate the importance of membrane rigidity in temperature tolerance.

M59 aaccumulated much less trehalose than No. 3110 did. Addition of trehalose to the permeabilized cell system of M59 restored the temperature sensitivity, but not when the trehalase inhibitor deoxynojirimycin was also added, which suggests that the accumulation and metabolism of trehalose is important for the expression of temperature tolerance.     

THE BIOGENESIS OF MITOCHONDRIA : III. The Lipid Composition of Aerobically and Anaerobically Grown Saccharomyces cerevisiae as Related to the Membrane Systems of the Cells

The growth conditions known to influence the occurrence of mitochondrial profiles and other cell membrane systems in anaerobic cells of S. cerevisiae have been examined, and the effect of the several growth media on the lipid composition of the organism has been determined. The anaerobic cell type containing neither detectable mitochondrial profiles nor the large cell vacuole may be obtained by the culture of the organism on growth-limiting levels of the lipids, ergosterol, and unsaturated fatty acids. Under these conditions, the organism has a high content of short-chain saturated fatty acids (10:0, 12:0), phosphatidyl choline, and squalene, compared with aerobically grown cells, and it is especially low in phosphatidyl ethanolamine and the glycerol phosphatides (phosphatidyl glycerol + cardiolipin). The high levels of unsaturated fatty acids normally found in the phospholipids of the aerobic cells are largely replaced by the short-chain saturated acids, even though the phospholipid fraction contains virtually all of the small amounts of unsaturated fatty acid present in the anaerobic cells. Such anaerobic cells may contain as little as 0.12 mg of ergosterol per g dry weight of cells while the aerobic cells contain about 6 mg of ergosterol per g dry weight. Anaerobic cell types containing mitochondrial profiles can be obtained by the culture of the organism in the presence of excess quantities of ergosterol and unsaturated fatty acids. Such cells have increased levels of total phospholipid, ergosterol, and unsaturated fatty acids, although these compounds do not reach the levels found in aerobic cells. The level of ergosterol in anaerobic cells is markedly influenced by the nature of the carbohydrate in the medium; those cells grown on galactose media supplemented with ergosterol and unsaturated fatty acids have well defined mitochondrial profiles and an ergosterol content (2 mg per g dry weight of cells) three times that of equivalent glucose-grown cells which have poorly defined organelle profiles. Anaerobic cells which are low in ergosterol synthesize increased amounts of squalene.     

Effect of altered sterol levels on the transport of amino acids and membrane structure ofMicrosporum gypseum

Ergosterol and cholesterol supplementation resulted in a significant increase (1·5-fold) in the sterol content while phospholipid remained unaffected inMicrosporum gypseum. The levels of phosphatidylethanolamine and phosphatidylcholine increased in ergosterol supplemented cells. However, a decrease in phosphatidylcholine and an increase in phosphatidylethanolamine was observed in cholesterol grown cells. The ratio of unsaturated to saturated fatty acids decreased on ergosterol/cholesterol supplementation. The uptake of amino acids (lysine, glycine and aspartic acid) decreased in sterol supplemented cells. Studies with fluorescent probe l-anilinonaphthalene-8-sulfonate showed structural changes in membrane organisation as evident by increased number of binding sites in such cells.     

Effect of sterol side chains on growth and membrane fatty acid composition of Saccharomyces cerevisiae.

Saccharomyces cerevisiae GL7 cells require exogenous sterol and unsaturated fatty acid for growth. When grown in the presence of cholesterol or 7-dehydrocholesterol, the cells incorporated less saturated fatty acid into phospholipids than cells grown with ergosterol, stigmasterol, or beta-sitosterol as the sterol source. This lower saturated fatty acid content was most pronounced in phosphatidylethanolamine, slightly less so in phosphatidylcholine, and least evident in phosphatidylserine and phosphatidylinositol. Growing the cells with the various sterols did not affect the ratios of individual phospholipids. The ability of strain GL7 to use 7-dehydrocholesterol as the only sterol supplement for growth was dependent upon the nature of the unsaturated fatty acids added to the growth medium. In the presence of linoleic, linolenic, or a mixture of palmitoleic and oleic acids, excellent growth was observed with either ergosterol, cholesterol, or 7-dehydrocholesterol. However, when the medium was supplemented with either oleic or petroselenic acid, the cells grew more slowly (oleic) or much more poorly (petroselenic) with 7-dehydrocholesterol than with ergosterol. A specific relationship between sterol structure and membrane fatty acid composition in yeast cells is implied.     

Fatty acid and complex lipid composition of a Saccharomyces cerevisiae mutant unable to synthesize delta-aminolevulinic acid.

The lipid composition of a Saccharomyces cerevisiae mutant (GL 1–38) lacking δ-aminolevulinic acid synthase (EC 2.3.1.37) was investigated. This mutant is unable to synthesize heme compounds and, as a consequence, cannot make unsaturated fatty acids or ergosterol. The mutant cells were grown (i) in medium supplemented with δ-aminolevulinic acid or (ii) in medium supplemented with Tween 80 (as a source of oleate) and ergosterol. After growth in the presence of δ-aminolevulinic acid, the fatty acid composition of total lipids and mitochondrial lipids was the same as that of the corresponding wild-type strain. After growth in the presence of Tween 80 and ergosterol, the mutant cells contained increased levels of oleate and greatly decreased levels of palmitoleate. The ratio of unsaturated to saturated fatty acids in these cells was still close to that of the wild type but much lower than that of the medium. The sphingolipids accounted for 5.2% of the lipid phosphate in the wild type and, after growth in Tween 80 and ergosterol, for 12.7% in the mutant. Changes in other phospholipids were too small to be considered significant.     

Lipid composition of the plasma-membrane of the halotolerant alga,Dunaliella salina

The major lipids of the isolated plasma-membrane of the halotolerant alga Dunaliella salina are diacylglyceroltrimethylhomoserine (DGTS, 23.5%), sterol peroxides (7-dehydroporiferasterol peroxide and ergosterol peroxide, 22%), phosphatidylcholine (13%) and phosphatidylethanolamine (11%). Free sterols comprised 5% of the lipids and contained predominantly 7-dehydroporiferasterol and ergosterol. The major fatty acids of the plasma-membrane were palmitic (31%), oleic (13%), linoleic (20%) and γ-linolenic (17%) acids. In constrast to the whole cells, the plasma-membrane contained less (11%) α-linolenic acid and no 16-carbon unsaturated fatty acids. Sterol peroxides were identified by ¹H-NMR and ¹³C-NMR spectroscopy, mass spectrometry, and by comparison on thin-layer chromatography to the product of ergosterol photooxygenation. We believe that this is the first report on the occurrence of sterol peroxides as major constituents of a biological membrane. It is suggested that they may play a role in the unusual membrane-permeability properties of the plasma-membrane of Dunaliella.     

Evidence for a functional association of DNA synthesis with the membrane in mitochondria of Saccharomyces cerevisiae

We have studied the effect of membrane fatty acid composition on replicative DNA synthetic activity in mitochondria isolated from Saccharomyces cerevisiae. Cells containing different levels of membrane unsaturated fatty acids were obtained by growth of a fatty acid desaturase mutant of Saccharomyces cerevisiae in glucose-limited chemostat cultures supplemented with various concentrations of Tween 80. Arrhenius plots of DNA synthetic activity in isolated mitochondria show a discrete discontinuity at specific temperature which are dependent on the membrane unsaturated fatty acid content of the mitochondria. This indicates a functional association of DNA replication with the mitochondrial membrane in Saccharomyces cerevisiae.     

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin.Following supplementation with saturated fatty acids of longer than 15 carbons (100 μM) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth.Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35–41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72–82% after addition of unsaturated fatty acids.A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids.     

Impact of UV-B Radiation on Thylakoid Membrane and Fatty Acid Profile of <Emphasis Type="Italic">Spirulina platensis</Emphasis>

Ultraviolet-B (UV-B) radiation to thylakoid membrane and fatty acid profile has been investigated in cyanobacterium, Spirulina platensis. The thylakoid membrane was isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells followed by differential density gradient centrifugation and morphological variations were examined. UV radiation distorted the membrane on the outer side with reduced chlorophyll a (chl a) content compared to its untreated counterpart. Liquid chromatography-mass spectrometry (LC-MS) was used for characterization of chl a of the thylakoid membrane. UV-B exposure resulted in alterations in the pigment-protein complexes 47 kDa and 43 kDa. Furthermore, 94 kDa and 20 kDa protein appeared in UV-B-exposed thylakoid membrane of S. platensis. The composition of fatty acid in response to UV-B radiation was detected by gas chromatography–mass spectrometry having 23.5% saturated fatty acid (SFA), 76.4% monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA). In contrast to its UV-B-untreated counterpart, SFA was 46.6%, and MUFA and PUFA were 53.3%. Our findings suggest that UV-B radiation not only affects membrane morphology and its protein profile but also reduces saturated fatty acid and increases unsaturated fatty acids in S. platensis.     

Synthesis of trans unsaturated fatty acids in Pseudomonas putida P8 by direct isomerization of the double bond of lipids

The phospholipids of Pseudomonas putida P8 contain monounsaturated fatty acids in the cis and trans configuration. Cells of this phenol-degrading bacterium change the proportions of these isomers in response to the addition or elimination of a membrane active compound such as 4-chlorophenol. This study undoubtedly reveals that the cis unsaturated fatty acids are directly converted into trans isomers without involvement of de novo synthesis of fatty acids. Oleic acid, which cannot be synthesized by this bacterium, was incorporated as a cis unsaturated fatty acid marker in the membrane lipids of growing cells. The conversion of this fatty acid into the corresponding trans isomer was demonstrated by gas chromatographic-mass spectrometric analysis and use of ¹⁴C-labeled oleic acid. Separation and isolation of the cellular membranes showed that the fatty acid isomerase is located in the cytoplasmic membrane of P. putida P8.Abbreviation 4-CP 4-chlorophenol     

乙醇胁迫处理对酒酒球菌SD-2a生理特性的影响

为制备高效的葡萄酒乳酸菌发酵剂,以酒酒球菌SD-2a为试验材料,研究了5%、10%不同浓度的乙醇胁迫处理对菌体生长、细胞内MLE活性、冻干存活率及细胞膜脂肪酸组分的影响。试验结果表明乙醇胁迫处理明显降低了菌体的生长速率和生物产量。与对照相比,5%乙醇胁迫处理降低了菌体的冻干存活率,而10%乙醇处理却显著增加了菌体的冻干存活率。细胞膜脂肪酸组分的测定结果表明,前者细胞膜U/S比值为1.12,比对照降低了26.3%,而后者细胞膜U/S比值为2.29,比对照增加了50.6%。故认为酒酒球菌SD-2a为适应不同浓度的乙醇胁迫环境,在细胞膜脂肪酸水平上所采用的机制不同,且菌体的冻干存活率与其细胞膜脂肪酸组分密切相关。     

Prolonged Culture of the Voracious Flagellate Peranema in Antioxidant-Containing Media*

SYNOPSIS. Peranema trichophorum remained vigorous at least a year in milk-lecithin media and 3 months in a nearly defined autoclavable biphasic agar medium fortified with the fat-soluble antioxidant butylated hydroxytoluene. The only undefined substance in the present “defined” medium is crude soybean lecithin; 0.001% lecithin suffices in the presence of a mixture of long-chain saturated and unsaturated fatty acids. Linoleic acid may be indispensable. Histidine is an absolute requirement as well as a favored substrate. Cholesterol, not ergosterol, satisfied the sterol requirement. Voracity is retained in these media as shown by engorgement on plastic latex particles.     

BIOGENESIS OF MITOCHONDRIA : XIII. The Isolation of Mitochondrial Structures from Anaerobically Grown Saccharomyces cerevisiae

Morphologically intact structures have been isolated from anaerobically grown yeast cells which have many of the properties of yeast mitochondria. The structures are about 0.5 µ in diameter and contain malate dehydrogenase, succinate dehydrogenase, oligomycin-sensitive ATPase, and DNA of buoyant density 1.683 g/cc, characteristic of yeast mitochondria. The morphology of the structures is critically dependent on their lipid composition. When isolated from cells grown anaerobically in the presence of supplements of unsaturated fatty acid and ergosterol, their unsaturated fatty acid content is similar to that of mitochondria from aerobically grown cells. These lipid-complete structures consist pre-dominantly of double-membrane vesicles enclosing a dense matrix which contains a folded inner membrane system bordering electron-transparent regions which are somewhat different from the cristae of functional mitochondria. In contrast, the structures from cells grown without lipid supplements are much simpler in morphology; they have a dense granular matrix surrounded by a double membrane but have no obvious folded inner membrane system within the matrix. The lipid-depleted structures are very fragile and are only isolated in intact form from protoplasts that have been prefixed with glutaraldehyde     

A lipid-mediated quality control process in the Golgi apparatus in yeast

When heme biosynthesis is disrupted, the yeast Saccharomyces cerevisiae becomes unable to synthesize its major sterol, ergosterol, and desaturate fatty acids. We took advantage of this physiological peculiarity to evaluate the consequences of ergosterol and/or unsaturated fatty acid (UFA) depletions on the biogenesis of a model polytopic plasma membrane protein, the uracil permease Fur4p. We show that under UFA shortage, which results in low amounts of diunsaturated phospholipid species, and under ergosterol depletion, Fur4p is prematurely routed from the Golgi apparatus to the vacuolar lumen in a process that requires the ubiquitin ligase Rsp5p. Interestingly, this diversion is not correlated to Fur4p exclusion from detergent-resistant membranes. In an independent set of experiments, we show that Fur4p targeting to the plasma membrane depends on phosphatidylethanolamine amounts and more specifically on the propensity of this phospholipid to form a hexagonal phase. In light of recent literature, we propose a model in which ergosterol and diunsaturated phospholipid species maintain optimal membrane curvature for Fur4p to evade the Golgi quality control process and to be properly delivered to its normal destination.     

Regulation by heme of sterol uptake in Saccharomyces cerevisiae

The leaky heme mutants G204, G216, and G214 are shown to accumulate exogenous sterols. Unlike hem mutants which have complete blocks in the heme pathway, these strains do not require ergosterol, methionine, or unsaturated fatty acids for growth. The addition of aminolevulinic acid to the growth medium inhibited sterol uptake in G204 96% but had only a slight effect on sterol uptake by strains G214 and G216. Sterol uptake in all three strains was inhibited 83-94% when cells were grown in the presence of hematin. Sterol analysis of these strains grown in the presence and absence of either aminolevulinic acid or hematin revealed that saturation of the cell membrane with ergosterol was not responsible for the dramatic decrease in sterol uptake. These results suggest that sterol uptake by yeast cells is controlled by heme, and explain the non-viability of yeast strains that are heme competent and auxotrophic for sterols.     

蝉拟青霉菌丝体与天然蝉花中化学成分的比较分析

以天然蝉花Cordyceps cicadae做对比,对一株蝉拟青霉Paecilomyces cicadae菌株发酵菌丝体的化学成分进行了分析,包括粗成分、生物活性成分、无机元素、氨基酸、脂肪酸等。结果表明:蝉拟青霉菌丝体中虫草多糖的含量为33.2mg/g,甘露醇的含量为78.9mg/g,麦角甾醇的含量为0.6256mg/g,腺苷的含量为1.0620mg/g,钙、铁、锌、硒和锰微量元素的含量分别为5187.59μg/g、207.23μg/g、41.93μg/g、0.087μg/g和28.24μg/g,18种氨基酸齐全,不饱和脂肪酸是优势脂肪酸,以亚麻油酸、油酸为主,其相对含量分别为53.91%和20.63%,与天然蝉花相比,发酵菌丝体中虫草多糖、甘露醇、麦角甾醇、腺苷、氨基酸总量、必需氨基酸总量和不饱和脂肪酸的含量明显高于后者,其它化学成分和天然蝉花中相应成分基本一致。     

Influence of stressful fermentation conditions on neutral lipids of a Saccharomyces cerevisiae brewing strain

The neutral lipid fraction of the aerobically grown starter yeast culture of a Saccharomyces cerevisiae brewing strain, and three-first recycled yeast generations exposed to multiple stress factors during beer fermentation was studied. No pronounced changes in the cellular neutral lipid content between the non-stressed starter and stressed recycled cells were found. However, it was found that recycled yeast generations modulate their neutral lipid composition during fermentation. The ergosterol content was increased at the expense of steryl esters (SEs) and squalene, which resulted in a higher ergosterol/SEs molar ratio and a slightly higher ergosterol/squalene molar ratio. In addition, the proportion of unsaturated fatty acids, mainly palmitoleic acid increased in the neutral lipid fraction of the stressed recycled yeast generations. These results suggest that some specific neutral lipid species and fatty acids stored in the neutral lipid fraction are involved in the adaptive response of the brewer’s yeast to stressful fermentation conditions. The striking finding was a high squalene content in the neutral lipid fraction of both the starter yeast culture and recycled yeast generations (22.4 vs. 19–20%, respectively), implying a possible biotechnological exploitation of this biologically active molecule from the yeast biomass.