絮凝特性对自絮凝颗粒酵母耐酒精能力的影响及作用机制

首次报道絮凝特性提高酵母菌耐酒精能力的现象及其机制。融合株SPSC与其两亲本粟酒裂殖酵母变异株和酿酒酵母变异株于 30℃经 18% (V/V)酒精冲击 7h的存活率分别为 52%、37%和 9%。细胞膜磷脂脂肪酸组成分析表明 ,两絮凝酵母 (融合株SPSC和粟酒裂殖酵母变异株 )的棕榈酸含量均约为非絮凝酵母 (酿酒酵母变异株 )的两倍 ,而棕榈油酸和油酸的含量明显低于后者。研究表明 ,当两絮凝酵母在培养中由于柠檬酸钠的作用 (抑制絮凝体的形成 )而以游离细胞生长存在时 ,其细胞膜磷脂棕榈酸含量显著下降 ,而棕榈油酸和油酸的含量明显增加 ,结果细胞膜磷脂脂肪酸组成特点与酿酒酵母变异株相似 ;而且实验表明 ,絮凝特性的消失伴随菌体耐酒精能力的急剧下降 ,变得与酿酒酵母变异株的水平相当。这些结果提示两絮凝酵母具有较强的耐酒精能力与其细胞膜磷脂脂肪酸组成中含有更高比例的棕榈酸有关。     

Heterologous production of dihomo-gamma-linolenic acid in Saccharomyces cerevisiae

To make dihomo-gamma-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Delta12 fatty acid desaturase, rat Delta6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15 degrees C than in those grown at 20 degrees C, and no DGLA production was observed in the cells grown at 30 degrees C. In NSD at 15 degrees C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15 degrees C, the yield of DGLA reached 2.19 microg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.     

大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间的关系

通过对大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间关系研究发现,10℃培养,对数期细胞有最大的压力抗性,随着培养温度的升高直到4 5℃,压力抗性呈下降的趋势;相反,10℃培养,稳定期的细胞对压力最敏感,随着培养温度的升高,压力抗性呈增加趋势,30～37℃时达到最大,之后到4 5℃有下降。对数期和稳定期细胞膜脂中不饱和脂肪酸的组成随温度的上升而下降,这与从全细胞中抽提的磷脂的熔点密切相关。因此,对数期细胞压力抗性随着膜流动性的增大而升高;但稳定期细胞,膜流动性与压力抗性之间不存在简单的对应变化关系     

Polyunsaturated fatty acid biosynthesis in Saccharomyces cerevisiae: expression of ethanol tolerance and the FAD2 gene from Arabidopsis thaliana.

The Arabidopsis thaliana delta-12 fatty acid desaturase gene (FAD2) was overexpressed in Saccharomyces cerevisiae by using the GAL1 promoter. S. cerevisiae harboring the FAD2 gene was capable of forming hexadecadienoyl (16:2) and linoleoyl (18:2) residues in the membrane lipid when cultured in medium containing galactose. Gas-liquid chromatography analysis of total lipids indicated that the transformed S. cerevisiae accumulated these dienoic fatty acyl residues and that they accounted for approximately 50% of the total fatty acyl residues. Phospholipid analysis of this strain indicated that the oleoyl (18:1) residue binding phosphatidylcholine (PC) was mostly converted to the 18:2 residue binding PC, whereas 50% of the palmitoleoyl (16:1) residue binding PC was converted to the 16:2 residue binding PC. A marked effect on the unsaturation of 16:1 and 18:1 was observed when S. cerevisiae harboring the FAD2 gene was cultured at 8 degrees C. To assess the ethanol tolerance of S. cerevisiae producing polyunsaturated fatty acids, the cell viability of this strain in the presence of ethanol was examined. The results indicated that S. cerevisiae cells overexpressing the FAD2 gene had greater resistance to 15% (vol/vol) ethanol than did the control cells.     

The effect of cold, acid and ethanol shocks on synthesis of membrane fatty acid, freeze-drying survival and malolactic activity of Oenococcus oeni

The effects of stress shocks on the freeze-drying viability, malolactic activity and membrane fatty acid composition of the Oenococcus oeni SD-2a cells were studied. O. oeni SD-2a cells after 2 h of stress exposure exhibited better freeze-drying viability and malolactic fermentation ability. A decrease in unsaturated fatty acids/saturated fatty acids (UFA/SFA) ratio and in the C18:1 relative concentration, and an increase in cyclopropane fatty acids (CFA) content mainly due to the increase in C19cyc11 relative concentration were observed in all stress shocked cells. There was a significant negative correlation between C19cyc11 and C18:lcis11, C16:0 in all stress shocks. The freeze-drying viability exhibited a significant positive correlation with the levels of C19cyc11 in cold and acid shocks. The only significant positive correlation between the ability of O. oeni SD-2a to conduct malic acid degradation and membrane composition existed with C14:0 in ethanol shocks. In general, freeze-drying viabilities were maximum for cells with low UFA/SFA ratio and high CFA levels, and, consequently, with low membrane fluidity. Moreover, CFA formation played a major role in protecting stress shocked cells from lyophilization. However, changes observed in membrane fatty acid composition are not enough to explain the greater freeze-drying viability of cells shocked at 8% ethanol. Thus, other mechanisms could be responsible for this increase in the bacterial resistance to lyophilization.     

Heat shock causes oxidative stress and induces a variety of cell rescue proteins in Saccharomyces cerevisiae KNU5377

In this study, we attempted to characterize the physiological response to oxidative stress by heat shock in Saccharomyces cerevisiae KNU5377 (KNU5377) that ferments at a temperature of 40 degrees C. The KNU5377 strain evidenced a very similar growth rate at 40 degrees C as was recorded under normal conditions. Unlike the laboratory strains of S. cerevisiae, the cell viability of KNU5377 was affected slightly under 2 hours of heat stress conditions at 43 degrees C. KNU5377 evidenced a time-dependent increase in hydroperoxide levels, carbonyl contents, and malondialdehyde (MDA), which increased in the expression of a variety of cell rescue proteins containing Hsp104p, Ssap, Hsp30p, Sod1p, catalase, glutathione reductase, G6PDH, thioredoxin, thioredoxin peroxidase (Tsa1p), Adhp, Aldp, trehalose and glycogen at high temperature. Pma1/2p, Hsp90p and H+-ATPase expression levels were reduced as the result of exposure to heat shock. With regard to cellular fatty acid composition, levels of unsaturated fatty acids (USFAs) were increased significantly at high temperatures (43 degrees C), and this was particularly true of oleic acid (C18:1). The results of this study indicated that oxidative stress as the result of heat shock may induce a more profound stimulation of trehalose, antioxidant enzymes, and heat shock proteins, as well as an increase in the USFAs ratios. This might contribute to cellular protective functions for the maintenance of cellular homeostasis, and may also contribute to membrane fluidity.     

Analysis of fatty acid composition of Candida species by gas-liquid chromatography using a polar column

The fatty acid composition of representative Candida species was examined by gas-liquid chromatography (GLC) using a polar column. The major fatty acids were C14:0, C16:0, C18:0 saturated, C16:1 and C18:1 monoenoic series, with or without C18 polyunsaturated acids (C18:2 and C18:3). In Torulopsis glabrata and Saccharomyces cerevisiae the C18:2 and C18:3 acids were not found, but the C10:0 and C12:0 acids were detected in S. cerevisiae. These results indicated that the Candida genus could be distinguished from Torulopsis and Saccharomyces genera by GLC analysis of fatty acids. Quantitative differences in the fatty acid composition between cells grown at high temperature (37 degrees C) and low temperature (25 degrees C) were found generally in Candida species, and the amounts of C18 polyunsaturated acids (C18:2 and C18:3) increased in the cells grown at 25 degrees C. Each Candida species showed a characteristic profile in fatty acid composition. Determination of the cellular fatty acid composition in Candida species is likely to be useful for the grouping or chemotaxonomy of newer isolates of Candida species.     

钙离子通过降低细胞膜透性提高自絮凝颗粒酵母耐酒精能力

生长阶段和冲击阶段均添加 1 6 4mmol LCa2 能显著提高自絮凝颗粒酵母于 30℃在 2 0 % (V V)酒精冲击下的存活率 ,经过 9h冲击 ,对照组的存活率为 0 ,而添加Ca2 试验组的存活率为 5 0 0 % ,表明添加适当浓度的Ca2 能显著提高菌体的耐酒精能力。通过考察Ca2 对菌体于 30℃在 15 % (V V)酒精冲击下细胞膜透性的影响发现 ,生长阶段和冲击阶段均添加 1 6 4mmol LCa2 的试验组的菌体胞外核苷酸平衡浓度和细胞膜透性系数 (P′)分别仅为对照组水平的 5 0 0 %和 2 9 3% ,表明添加适当浓度的Ca2 能显著降低受冲击菌体的细胞膜透性 ;而且 ,添加Ca2 提高存活率与添加Ca2 降低胞外核苷酸浓度和P′存在直接的对应关系。因此 ,Ca2 提高自絮凝颗粒酵母耐酒精能力是与其降低受冲击菌体的细胞膜透性密切相关的。     

Combined cold, acid, ethanol shocks in Oenococcus oeni: effects on membrane fluidity and cell viability

The effects of combined cold, acid and ethanol on the membrane physical state and on the survival of Oenococcus oeni were investigated. Membrane fluidity was monitored on intact whole O. oeni cells subjected to single and combined cold, acid and ethanol shocks by using fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Results showed that cold shocks (14 and 8 degrees C) strongly rigidified plasma membrane but did not affect cell survival. In contrast, ethanol shocks (10-14% v/v) induced instantaneous membrane fluidisation followed by rigidification and resulted in low viability. Acid shocks (pH 4.0 and pH 3.0) exerted a rigidifying effect on membrane without affecting cell viability. Whatever the shock orders, combined cold (14 degrees C) and ethanol (14% v/v) shocks resulted in strong membrane rigidification. Interestingly, O. oeni survived combined cold and ethanol shocks more efficiently than single ethanol shock. Membrane rigidification was induced by ethanol-and-acid (10% v/v - pH 3.5) shock and correlated with total cell death. In contrast, O. oeni recovered its viability when subjected to cold (8 degrees C)-then-ethanol-and-acid shock which strongly rigidified the membrane. Our results suggested a positive short-term effect of combined cold, acid and ethanol shocks on membrane fluidity and viability of O. oeni.     

Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164

The relationship among growth temperature, membrane fatty acid composition, and pressure resistance was examined in Escherichia coli NCTC 8164. The pressure resistance of exponential-phase cells was maximal in cells grown at 10 degrees C and decreased with increasing growth temperatures up to 45 degrees C. By contrast, the pressure resistance of stationary-phase cells was lowest in cells grown at 10 degrees C and increased with increasing growth temperature, reaching a maximum at 30 to 37 degrees C before decreasing at 45 degrees C. The proportion of unsaturated fatty acids in the membrane lipids decreased with increasing growth temperature in both exponential- and stationary-phase cells and correlated closely with the melting point of the phospholipids extracted from whole cells examined by differential scanning calorimetry. Therefore, in exponential-phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, there was apparently no simple relationship between membrane fluidity and pressure resistance. When exponential-phase or stationary-phase cells were pressure treated at different temperatures, resistance in both cell types increased with increasing temperatures of pressurization (between 10 and 30 degrees C). Based on the above observations, we propose that membrane fluidity affects the pressure resistance of exponential- and stationary-phase cells in a similar way, but it is the dominant factor in exponential-phase cells whereas in stationary-phase cells, its effects are superimposed on a separate but larger effect of the physiological stationary-phase response that is itself temperature dependent.     

Coupling effects of osmotic pressure and temperature on the viability of Saccharomyces cerevisiae

The osmotic tolerance of cells of Saccharomyces cerevisiae as a function of glycerol concentration and temperature has been investigated. Results show that under isothermal conditions (25 degrees C) cells are resistant (94% viability) to hyperosmotic treatment at 49.2 MPa. A thigher osmotic pressure, cell viability decreases to 25% at 99 MPa. Yeast resistance to high osmotic stress (99 Mpa) is enhanced at low temperatures (5-11 degrees C). Therefore, the temperature at which hyperosmotic pressure is achieved greatly affects cell viability. These results suggest that temperature control is a suitable means of enhancing cell survival in response to osmotic dehydration.     

Effects of triclosan on growth, viability and fatty acid synthesis of the oyster protozoan parasite Perkinsus marinus

Perkinsus marinus, a protozoan parasite of the Eastern oyster Crassostrea virginica, has severely impacted oyster populations from the Mid-Atlantic region to the Gulf of Mexico coast of North America for more than 30 yr. Although a chemotherapeutic treatment to reduce or eliminate P. marinus from infected oysters would be useful for research and hatchery operations, an effective and practical drug treatment does not currently exist. In this study, the antimicrobial drug triclosan 5-chloro-2-(2,4 dichlorophenoxy) phenol, a specific inhibitor of Fab1 (enoyl-acyl-carrier-protein reductase), an enzyme in the Type II class of fatty acid synthetases, was tested for its effects on viability, proliferation and fatty acid synthesis of in vitro-cultured P. marinus meronts. Treatment of P. marinus meront cell cultures with concentrations of > or = 2 microM triclosan at 28 degrees C (a temperature favorable for parasite proliferation) for up to 6 d stopped proliferation of the parasite. Treatment at > or = 5 microM at 28 degrees C greatly reduced the viability and fatty acid synthesis of meront cells. Oyster hemocytes treated with > or = 20 microM triclosan exhibited no significant (p < 0.05) reduction in viability relative to controls for up to 24 h at 13 degrees C. P. marinus meronts exposed to > or = 2 microM triclosan for 24 h at 13 degrees C exhibited significantly (p < 0.05) lower viability relative to controls. Exposure of P. marinus meronts to triclosan concentrations of > or = 20 microM resulted in > 50% mortality of P. marinus cells after 24 h. These results suggest that triclosan may be effective in treating P. marinus-infected oysters.     

Effect of growth temperature on membrane fatty acid composition and susceptibility to cold shock of Bacillus amyloliquefaciens.

We investigated the fatty acid composition of the membrane of Bacillus amyloliquefaciens grown at different temperatures. A decrease in growth temperature was accompanied by an increase in the ratio of branched- to straight-chain fatty acids and a marked increase in the level of unsaturation of branched-chain fatty acids. When cells of this organism grown at 30 degrees C were cold shocked, viability and ability to secrete extracellular protease were lost. Growth of this organism at lower temperatures or addition of Tween 80 to cells caused the critical temperature zone for cold shocking to be lowered significantly. These results suggest a direct correlation between membrane fluidity and the susceptibility to cold shock.     

Total fatty acid content of the plasma membrane of Saccharomyces cerevisiae is more responsible for ethanol tolerance than the degree of unsaturation

The effect of change in unsaturated fatty acid composition on ethanol tolerance in Saccharomyces cerevisiae overexpressing ScOLE1 (?9 fatty acid desaturase gene of S. cerevisiae), CaFAD2 (?12 fatty acid desaturase gene of Candida albicans), or CaFAD3 (ω3 fatty acid desaturase gene of C. albicans) was examined. ScOLE1 over-expression increased the total unsaturated fatty acid content and enhanced ethanol tolerance, compared with a control strain. In contrast, overexpression of CaFAD2 and CaFAD3, which led to production of linoleic acid (18:2) and α-linolenic acid (18:3), respectively, neither changed total unsaturated fatty acids nor enhanced ethanol tolerance. The total unsaturated fatty acid content rather than the degree of unsaturation is thus an important factor for ethanol tolerance.     

The effects of temperature and pH on the ethanol tolerance of the wine yeasts, Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata

The influences of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata were examined in the presence of ethanol concentrations between 2.5 and 15% v/v. At 15°C, the maximum concentrations of ethanol permitting the growth of S. cerevisiae, C. stellata and K. apiculata were 15%, 11% and 9%, respectively. These maximum concentrations were decreased at 10°C and 30°C. Cells of S. cerevisiae showed no loss in viability when incubated for 12 d at 10°C or 15°C in the presence of 15% ethanol but showed some loss at 30°C. Cells of C. stellata were tolerant of 12.5% ethanol at 10°C and 15°C but not at 30°C. Cells of K. apiculata were tolerant of 10–12.5% ethanol at 15°C but not at 10°C or 30°C. Sensitivity of the yeast cells to ethanol was marginally increased on decreasing the pH from 6-0 to 3–0.     

Cultured human skin fibroblasts modify their plasma membrane lipid composition and fluidity according to growth temperature suggesting homeoviscous adaptation at hypothermic (30 degrees C) but not at hyperthermic (40 degrees C) temperatures.

Mammalian cell metabolism is responding to changes in temperature. Body temperature is regulated around 37 degrees C, but temperatures of exposed skin areas may vary between 20 degrees C and 40 degrees C for extended periods of time without apparent disturbance of adequate cellular functions. Cellular membrane functions are depending from temperatures but also from their lipid environment, which is a major component of membrane fluidity. Temperature-induced changes of membrane fluidity may be counterbalanced by adaptive modification of membrane lipids. Temperature-dependent changes of whole cell- and of purified membrane lipids and possible homeoviscous adaptation of membrane fluidity have been studied in human skin fibroblasts cultured at 30 degrees C, 37 degrees C, and 40 degrees C for ten days. Membrane anisotropy was measured by polarized fluorescence spectroscopy using TMA-DPH for superficial and DPH for deeper membrane layers. Human fibroblasts were able to adapt themselves to hypothermic temperatures (30 degrees C) by modifying the fluidity of the deeper apolar regions of the plasma membranes as reported by changes of fluorescence anisotropy due to appropriate changes of their plasma membrane lipid composition. This could not be shown for the whole cells. At 40 degrees C growth temperature, adaptive changes of the membrane lipid composition, except for some changes in fatty acid compositions, were not seen. Independent from the changes of the membrane lipid composition, the fluorescence anisotropy of the more superficial membrane layers (TMA-DPH) increased in cells growing at 30 degrees C and decreased in cells growing at 40 degrees C.     

Growth and fermentation characteristics of new selected strains of Saccharomyces at high temperatures and high cell densities

Maintenance of high cell viability was the main characteristic of our new strains of thermotolerant Saccharomyces. Total sugar conversion to ethanol was observed for sugarcane juice fermentation at 38-40 degrees C in less than 10 h and without continuous aeration of the culture. Invertase activity differed among the selected strains and increased during fermentation but was not dependent on cell viability. Invertase activity of the cells and optimum temperature for growth, as well as velocity of ethanol formation, were dependent on medium composition and the type of strain used. At high sugarcane syrup concentrations, the best temperature for ethanol formation by strain 781 was 35 degrees C. Distinct differences among the velocities of ethanol production using selected strains were also observed in sugarcane syrup at 35-38 degrees C.     

Possible involvement of acetyl coenzyme A carboxylase as well as fatty acid synthetase in the temperature-controlled synthesis of fatty acids in Saccharomyces cerevisiae

Fatty acid synthetase (FAS) preparations from Saccharomyces cerevisiae cells grown at either 35 or 10 degrees C produced the same products at different temperatures and showed quite similar temperature-dependencies in Arrhenius plots, with break points at 25 degrees C. This break point does not appear to reflect a phase transition of phospholipids present in the purified FAS preparations but rather is associated with protein conformational changes. S. cerevisiae cells grown at 35 degrees C and then shifted to 10 degrees C produced fatty acids with a shorter average chain length than those fatty acids synthesized at 10 degrees C by cells already adapted to 10 degrees C (hyper response). Acetyl-CoA carboxylase activity was relatively higher in the cells grown at 35 degrees C than in the cells grown at 10 degrees C; moreover, fatty acids with longer average chain lengths were synthesized in vitro at higher malonyl-CoA concentrations, which was consistent with the difference in the average chain lengths of newly synthesized fatty acids in cells grown at 35 and 10 degrees C. However, the activity levels of acetyl-CoA carboxylase and fatty acid synthetase alone did not account for the hyper response phenomena.     

The effects of unsaturated fatty acid depletion on the proton permeability and energetic functions of yeast mitochondria

1. The fatty acid composition of the ole-1 and ole-1 petite mutants of Saccharomyces cerevisiae was manipulated by growing the organism in the presence of defined supplements of Tween 80 or by allowing cells that had first been grown in the presence of Tween 80 to deplete their unsaturated fatty acids by sequent growth in the absence of Tween 80. 2. The transition temperature of Arrhenius plots of mitochondrial ATPase (adenosine triphosphatase) increases as the unsaturated fatty acid content is lowered. 3. Cells require larger amounts of unsaturated fatty acids to grow on ethanol at lower temperatures. 4. Cells that stop growing owing to unsaturated fatty acid depletion at low temperatures are induced to grow further by raising the temperature and this results in a further depletion of unsaturated acids. This is due to a higher rate, but not a greater efficiency, of mitochondrial ATP synthesis. 5. Arrhenius plots of the passive permeability of mitochondria to protons between 4 and 37 degrees C are linear. The rate and the Arrhenius activation energy of proton entry increase greatly as the unsaturated fatty acid content is lowered. 6. Unsaturated fatty acid depletion has the same effects on the proton permeability of ole-1 petite mitochondria, indicating that the mitochondrially synthesized subunits of the ATPase are not involved in the enhanced rates of proton entry. 7. The adenylate energy charge of depleted ole-1 cells is greatly decreased by growth on ethanol medium. 8. The adenylate energy charge of isolated mitochondria is also lowered by unsaturated fatty acid depletion. 9. The results confirm that unsaturated fatty acid depletion uncouples oxidative phosphorylation in yeast both in vivo and in vitro, and is a consequence of changes in the lipid part of the membrane.