The role of trehalose in dehydration resistance of Saccharomyces cerevisiae

Abstract High levels of intracellular trehalose in stationary-phase cells of Saccharomyces cerevisiae or cells incubated in the absence of a nitrogen source were found to increase the resistance of the cells to dehydration. Exponential-phase cells showed negligible dehydration resistance. When stationary-phase cells were inoculated into fresh medium, trehalose was rapidly broken down, and this was correlated with a rapid loss of dehydration resistance. It appeared that a minimum internal concentration of 120 mM trehalose was required before there was a significant increase in dehydration resistance. Exogenous trehalose increased the dehydration resistance of S. cerevisiae : this effect was most marked for stationary-phase cells, where almost 100% survival was obtained at trehalose concentrations of 500 mM and above while maximum survival for exponential cells was less than 10%, even at 1000 mM external trehalose.     

Involvement of the compatible solutes trehalose and sucrose in the response to salt stress of a cyanobacterial Scytonema species isolated from desert soils

The response to moderate salt stress of a Scytonema species isolated from a soil crust in the arid region of central Australia was studied. An increase in intracellular trehalose and sucrose concentrations was detected by NMR and HPLC analysis following salt stress, maximal amounts being produced by exposure to 150 mM NaCl after 48 h. When the organism was subsequently returned to normal growth conditions, the cellular concentrations of these solutes decreased. The biosynthesis of trehalose and sucrose was studied and found, in both cases, to involve both sugar phosphate synthase and phosphatase enzymes. The combined synthase activities and the individual phosphatase activities in cell extracts were increased by salt stress. Trehalose phosphorylase was the only catabolic enzyme detected for trehalose; neither trehalase nor phosphotrehalase activities could be detected. This is the first report of trehalose phosphorylase activity in cyanobacteria. Both trehalose and sucrose phosphorylase activities increased in salt-stressed cells, whereas the activity of invertase did not change.     

Regulation of trehalose metabolism by Adox and AdoMet in Saccharomyces cerevisiae

Effect of a potent methylation inhibitor oxidized adenosine (Adox), and a universal methyl group donor S-adenosyl-L-methionine (AdoMet) on trehalose metabolism was studied in two haploids of S. cerevisiae of mating types MATalpha, met3 (6460 -8D) and MATa, leu2, ura3, his4 (8534 -10A). Trehalose level decreased in presence of Adox in both strains. Both neutral trehalase (NT) and trehalose-6-phosphate (tre-6-p) synthase activities increased in presence of Adox in -8D strain. Decrease in trehalose level in -8D thus could not be explained in the light of increased tre-6-p synthase activity; however, it could be correlated with increased NT activity. In strain -10A, NT activity was reduced in presence of Adox while tre-6-p synthase activity increased. Enzyme activity profiles in -10A thus do not explain the reduced trehalose level on Adox treatment. Effect of AdoMet was not very prominent in either strain, though in -8D a small increase in trehalose level was seen on treatment. Intracellular AdoMet level of untreated cells of -10A was seen to be almost six times higher than that of -8D. Further, AdoMet treatment caused increase in its level compared to untreated cells, suggesting AdoMet uptake. No effect of either compound was seen on acid trehalase (AT) activity in any strain. The results suggest that there was a possible effect of demethylation on trehalose metabolism (particularly in the synthetic direction) in both strains, though effect of methylation was not very prominent, the reason for which is not very clear.     

Effect of growth conditions and trehalose content on cryotolerance of bakers' yeast in frozen doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56 degrees C min. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (mu = 0.088 h compared with 0.117 h) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20 degrees C instead of 30 degrees C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Heterologous expression of alcohol oxidase in Saccharomyces cerevisiae: properties of the enzyme and implications for microbody development

Alcohol oxidase (AO) expressed in transformed oleic acid-grown Saccharomyces cerevisiae, accumulated into microbodies to up to 8% of the total protein content of the organelles. This led to a small increase in volume fraction of the organelles, but not in their number. Most of the AO protein was present in large aggregates in the cytosol. The AO synthesized was inactive, irrespective of its subcellular localization and did not contain FAD. When the same AO gene was expressed in fused protoplasts of transformed S. cerevisiae and Hansenula polymorpha, the enzyme was properly assembled and activated in H. polymorpha microbodies.     

外源海藻糖对小麦幼苗耐盐性的影响

以盐敏感小麦品种鲁麦15为材料,分别用完全Hoagland营养液、150mmol／L NaCl和150mmol／L NaCl 10mmol／L海藻糖处理小麦幼苗,测定小麦幼苗生长、离子含量、根系质膜H^ -ATPase、SOD活性、MDA含量等指标,旨在探讨外源海藻糖在抗盐性中的作用。结果表明：外源海藻糖可明显缓解盐胁迫对小麦幼苗生长的抑制作用;明显提高NaCl胁迫条件下小麦幼苗叶片中K^ 的含量,降低Na^ 的含量,降低其Na^ ／K^ ;提高NaCl胁迫条件下小麦幼苗SOD活性,降低MDA的含量,降低细胞质膜透性,缓解根系质膜H^ -ATPase活性抑制。以上结果表叫外源海藻糖可能通过增加活性氧清除能力、缓解质膜伤害、维持胞质离子稳态提高植物抗盐性。     

Osmotic adjustment and the accumulation of organic solutes in whole cells and protoplasts of Saccharomyces cerevisiae

In the presence of a suitable carbon source, whole cells and protoplasts of Saccharomyces cerevisiae synthesized glycerol as a compatible organic solute in response to increased external osmotic pressure. Boyle-van't Hoff plots showed that protoplasts, and non-turgid cells, exhibited a linear relationship between volume and the external osmotic pressure (i.e. they behaved as near-ideal osmometers), and that both protoplasts and cells have a component which is not osmotically responsive--the non-osmotic volume (NOV). Glycerol levels in whole cells and protoplasts were elevated by increased external osmotic pressure over a similar time-scale to the period of exponential cell growth, reaching a maximum value at 6-12 h and declining thereafter. This suggests that the restoration of turgor pressure in whole cells was not the sole regulator of glycerol accumulation. Stationary phase whole cells had negligible levels of intracellular glycerol after growth in a medium of raised osmotic pressure. However, intracellular trehalose synthesis in these cells began earlier and reached a higher maximum level than in basal medium. Once exponential growth had stopped, cell turgor and internal osmotic pressure decreased somewhat. These new, lower values may be determined by the extent of trehalose accumulation in stationary phase cells.     

Germination of Saccharomyces cerevisiae ascospores without trehalose mobilization as revealed by in vivo 13C nuclear magnetic resonance spectroscopy.

Saccharomyces cerevisiae ascospores germinate in the presence of acetate without any detectable trehalose degradation, as revealed by high-resolution nuclear magnetic resonance spectroscopy and by a standard colorimetric assay. The results presented here substantiate the hypothesis that in S. cerevisiae trehalose supplies energy during dormancy of the spores and not during the germination process.     

海藻糖与热激蛋白在酿酒酵母耐受乙醇胁迫中的作用

在燃料乙醇发酵生产过程中,酿酒酵母经常会受到高浓度乙醇的胁迫,导致乙醇转化率和产量降低。面对高浓度乙醇的胁迫,酿酒酵母也具有应对胁迫的应激机制。在对这种应激机制进行了解的基础上,如能提高酿酒酵母对乙醇的耐受性,对于燃料乙醇生产具有重要意义。在高浓度乙醇胁迫下,酿酒酵母细胞会产生一系列保护性物质,如海藻糖、热激蛋白、脯氨酸等,这些物质能够提高酿酒酵母细胞对乙醇的耐受性。海藻糖作为一种重要的碳源、能量贮藏物质,不仅能稳定细胞膜、蛋白质和核酸等大分子物质,还可增强酿酒酵母对高浓度乙醇的耐受性。此外,酿酒酵母还可以产生大量的热激蛋白,增强酿酒酵母的抗逆性。从海藻糖和热激蛋白在乙醇胁迫下对酿酒酵母细胞保护作用的研究方面进行了综述,并对存在的问题进行了讨论与展望。     

Fed-batch cultivation of baker's yeast followed by nitrogen or carbon starvation: effects on fermentative capacity and content of trehalose and glycogen

An industrial strain of Saccharomyces cerevisiae (DGI 342) was cultivated in fed-batch cultivations at a specific growth rate of 0.2 h(-1). The yeast was then exposed to carbon or nitrogen starvation for up to 8 h, to study the effect of starvation on fermentative capacity and content of protein, trehalose and glycogen. Nitrogen starvation triggered the accumulation of trehalose and glycogen. After 8 h of starvation, the content of trehalose and glycogen was increased 4-fold and 2-fold, respectively. Carbon starvation resulted in a partial conversion of glycogen into trehalose. The trehalose content increased from 45 to 64 mg (g dry-weight)(-1), whereas the glycogen content in the same period was reduced from 55 to 5 mg (g dry-weight)(-1). Glycogen was consumed faster than trehalose during storage of the starved yeast for 1 month. Nitrogen starvation resulted in a decrease in the protein content of the yeast cells, and the fermentative capacity per gram dry-weight decreased by 40%. The protein content in the carbon-starved yeast increased as a result of starvation due to the fact that the content of glycogen was reduced. The fermentative capacity per gram dry-weight was, however, unaltered.     

Differential importance of trehalose in stress resistance in fermenting and nonfermenting Saccharomyces cerevisiae cells.

The trehalose content in laboratory and industrial baker's yeast is widely believed to be a major determinant of stress resistance. Fresh and dried baker's yeast is cultured to obtain a trehalose content of more than 10% of the dry weight. Initiation of fermentation, e.g., during dough preparation, is associated with a rapid loss of stress resistance and a rapid mobilization of trehalose. Using specific Saccharomyces cerevisiae mutants affected in trehalose metabolism, we confirm the correlation between trehalose content and stress resistance but only in the absence of fermentation. We demonstrate that both phenomena can be dissociated clearly once the cells initiate fermentation. This was accomplished both for cells with moderate trehalose levels grown under laboratory conditions and for cells with trehalose contents higher than 10% obtained under pilot-scale conditions. Retention of a high trehalose level during fermentation also does not prevent the loss of fermentation capacity during preparation of frozen doughs. Although higher trehalose levels are always correlated with higher stress resistance before the addition of fermentable sugar, our results show that the initiation of fermentation causes the disappearance of any other factor(s) required for the maintenance of stress resistance, even in the presence of a high trehalose content.     

Extracellular trehalose utilization by Saccharomyces cerevisiae

Two haploid strains of Saccharomyces cerevisiae viz. MATalpha and MATa were grown in glucose and trehalose medium and growth patterns were compared. Both strains show similar growth, except for an extended lag phase in trehalose grown cells. In both trehalose grown strains increase in activities of both extracellular trehalase activities and simultaneous decrease in extracellular trehalose level was seen. This coincided with a sharp increase in extracellular glucose level and beginning of log phase of growth. Alcohol production was also observed. Secreted trehalase activity was detected, in addition to periplasmic activity. It appeared that extracellular trehalose was hydrolyzed into glucose by extracellular trehalase activity. This glucose was utilized by the cells for growth. The alcohol formation was due to the fermentation of glucose. Addition of extracellular trehalase caused reduction in the lag phase when grown in trehalose medium, supporting our hypothesis of extracellular utilization of trehalose.     

Expression of bacterial mtlD in Saccharomyces cerevisiae results in mannitol synthesis and protects a glycerol-defective mutant from high-salt and oxidative stress.

Polyols, or polyhydroxy alcohols, are produced by many fungi. Saccharomyces cerevisiae produces large amounts of glycerol, and several fungi that cause serious human infections produce D-arabinitol and mannitol. Glycerol functions as an intracellular osmolyte in S. cerevisiae, but the functions of D-arabinitol and mannitol in pathogenic fungi are not yet known. To investigate the functions of mannitol, we constructed a new mannitol biosynthetic pathway in S. cerevisiae. S. cerevisiae transformed with multicopy plasmids encoding the mannitol-1-phosphate dehydrogenase of Escherichia coli produced mannitol, whereas S. cerevisiae transformed with control plasmids did not. Although mannitol production had no obvious phenotypic effects in wild-type S. cerevisiae, it restored the ability of a glycerol-defective, osmosensitive osg1-1 mutant to grow in the presence of high NaCl concentrations. Moreover, osg1-1 mutants producing mannitol were more resistant to killing by oxidants produced by a cell-free H2O2-FeSO4-NaI system than were controls. These results indicate that mannitol can (i) function as an intracellular osmolyte in S. cerevisiae, (ii) substitute for glycerol as the principal intracellular osmolyte in S. cerevisiae, and (iii) protect S. cerevisiae from oxidative damage by scavenging toxic oxygen intermediates.     

Sterol content, fatty acid composition of phospholipids, and permeability of labeled ethylene glycols in relation to salt-tolerance of yeasts

Two yeasts, the salt-tolerant Debaryomyces hansenii and the non-tolerant Saccharomyces cerevisiae were grown in basal media (4 m M NaCl) and also a high salinities that produced a similar salt stress in the two species in terms of growth rate reduction (i.e., 1.4 M NaCl for S. cerevisae and 2.7 M NaCl for D. hansenii ). A study was made of the sterol content, the fatty acid composition of the phospholipids, and the permeation of a series of tritiated ethylene glycols of graded molecular weights. On the basis of cell dry weight the amount of total and free sterols increased in both species when cultured at high salinity. Irrespective of growth medium salinity, the molar ratio of free sterols to phospholipids was higher in D. hansenii than in S. cerevisiae . Increased salinity produced only minor changes in the fatty acid composition of the phospholipids in D. hansenii , whereas in S. cerevisiae there was a marked decrease of linolenic acid with a concomitant increase of linoleic acid.
In both yeasts there was an energy linked component in the uptake of ethylene glycol, which component could be inhibited by sodium azide and N -ethylmaleimide. The passive permeability for ethylene-, diethylene- and triethylene glycol increased for both species at increased salinity. This increase was more pronounced for S. cerevisiae than for D. hansenii . Polyethylene glycol of M , 200 as well as higher polyethylene glycols appeared to be excluded or very slowly admitted by the yeasts.     

Trehalose promotes the survival of Saccharomyces cerevisiae during lethal ethanol stress, but does not influence growth under sublethal ethanol stress

Trehalose is known to protect cells from various environmental assaults; however, its role in the ethanol tolerance of Saccharomyces cerevisiae remains controversial. Many previous studies report correlations between trehalose levels and ethanol tolerance across a variety of strains, yet variations in genetic background make it difficult to separate the impact of trehalose from other stress response factors. In the current study, investigations were conducted on the ethanol tolerance of S. cerevisiae BY4742 and BY4742 deletion strains, tsl1 Δ and nth1 Δ, across a range of ethanol concentrations. It was found that trehalose does play a role in ethanol tolerance at lethal ethanol concentrations, but not at sublethal ethanol concentrations; differences of 20–40% in the intracellular trehalose concentration did not provide any growth advantage for cells incubated in the presence of sublethal ethanol concentrations. It was speculated that the ethanol concentration-dependent nature of the trehalose effect supports a mechanism for trehalose in protecting cellular proteins from the damaging effects of ethanol.     

酿酒酵母海藻糖合成酶基因的克隆和在大肠村菌中的表达

用PCR方法克隆了1．5kb的酿酒母Sacchromyces cerevisiae海藻糖合成酶基因TPSI,将该片段连接到pUC19载体,通过转化分别引入海藻糖合成酶基因缺失和缺陷的大肠杆菌Escherichia coli FF4169 和FF4050,对转化株的质粒DNA酶切分析表明均含有1．5kb PCR克隆片段,生长曲线实验证明,带有克隆片段的转化株在含0．5mol/L NaCl的高渗透压基础培养基中生长良好;用高效液相色谱（HPLC）结合蒸发散射（ELSD）技术测定细胞内海藻糖实验证明转化株能够合成海藻糖。     

Induction of acetic acid tolerance and trehalose accumulation by added and produced ethanol in Saccharomyces cerevisiae

Both added (49.6 g/l) and produced ethanol (46.2 g/l) caused an increase in the acetic acid tolerance of Saccharomyces cerevisiaegrown in an anaerobic chemostat; added ethanol, however, to a less extent than produced ethanol. The ethanol induced acetic acid tolerance of the cells was linked with an accumulation of trehalose within the cells. These results indicate that trehalose plays a role in the ethanol induced acetic acid tolerance of S. cerevisiae.