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.     

Distribution of trehalose between the cells and the rehydration medium in dehydrated Saccharomyces cerevisiae

The work was concerned with studying the balance of trehalose distribution between the rehydration medium and Saccharomyces cerevisiae cells grown in a chemically defined medium and dehydrated using the convective technique. A direct linear correlation between the viability of populations and the overall residual trehalose content in the cells and in the medium after the rehydration of dry yeast cells was shown to be most important. An inverse correlation was established between the viability of yeast cells and the amount of trehalose mobilised by the cells in the process of rehydration.     

Uptake of trehalose by Saccharomyces cerevisiae.

Trehalose, a storage sugar of baker's yeast, is known not to be metabolized when added to a cell suspension in water or a growth medium and to support growth only after a lag of about 10 h. However, it was transported into cells by at least two transport systems, the uptake being active, with a pH optimum at 5.5. There was no stoicheiometry with the shift of protons into cells observed at high trehalose concentrations. Trehalose remained intact in cells and was not appreciably lost to a trehalose-free medium. The uptake systems were present directly after growth on glucose, then decayed with a half-life of about 25 min but could be reactivated by aerobic incubation with trehalose, maltose, alpha-methyl-D-glucoside, glucose or ethanol. The uptake systems thus induced were different as revealed by competition experiments. At least one of the systems for trehalose uptake showed cooperative kinetics. Comparative anaysis with other disaccharides indicated the existence in Saccharomyces cerevisiae, after induction with trehalose, of at least four systems for the uptake of alpha-methyl-D-glucoside, four systems for maltose, together with the two for trehalose, variously shared by the sugars, the total of alpha-glucoside-transporting systems being five.     

Role of thioredoxin peroxidase in aging of stationary cultures of Saccharomyces cerevisiae

A soluble protein from Saccharomyces cerevisiae acts as a peroxidase but requires a NADPH-dependent thioredoxin system and was named thioredoxin peroxidase (TPx). The role of TPx in aging of stationary cultures of S. cerevisiae was investigated in a wild-type strain and a mutant yeast strain in which the tsa gene that encodes TPx was disrupted by homologous recombination. The occurrence of oxidative stress during aging of stationary cultures of the yeast has been proposed. Comparison of 5-day-old (young) stationary cultures of S. cerevisiae and of cultures aged for 3 months (old) revealed decreased viability, increased generation of reactive oxygen species, modulation of cellular redox status, and increased cellular oxidative damage reflected by increased protein carbonyl content and lipid peroxidation. The magnitude of this stress was augmented in yeast mutant lacking TPx. These results suggest that TPx may play a direct role in cellular defense against aging of stationary cultures presumably, functioning as an antioxidant enzyme.     

The effect of trehalose on the fermentation performance of aged cells of Saccharomyces cerevisiae

The fermentation process offers a wide variety of stressors for yeast, such as temperature, aging, and ethanol. To evaluate a possible beneficial effect of trehalose on ethanol production, we used mutant strains of Saccharomyces cerevisiae possessing different deficiencies in the metabolism of this disaccharide: in synthesis, tps1; in transport, agt1; and in degradation, ath1 and nth1. According to our results, the tps1 mutant, the only strain tested unable to synthesize trehalose, showed the lowest fermentation yield, indicating that this sugar is important to improve ethanol production. At the end of the first fermentation cycle, only the strains deficient in transport and degradation maintained a significant level of the initial trehalose. The agt1, ath1, and nth1 strains showed the highest survival rates and the highest proportions of non-petites. Accumulation of petites during fermentation has been correlated to low ethanol production. When recycled back for a subsequent fermentation, those mutant strains produced the highest ethanol yields, suggesting that trehalose is required for improving fermentative capacity and longevity of yeasts, as well as their ability to withstand stressful industrial conditions. Finally, according to our results, the mechanism by which trehalose improves ethanol production seems to involve mainly protection against protein oxidation.     

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.     

Replicative and chronological aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has directly or indirectly contributed to the identification of arguably more mammalian genes that affect aging than any other model organism. Aging in yeast is assayed primarily by measurement of replicative or chronological life span. Here, we review the genes and mechanisms implicated in these two aging model systems and key remaining issues that need to be addressed for their optimization. Because of its well-characterized genome that is remarkably amenable to genetic manipulation and high-throughput screening procedures, S. cerevisiae will continue to serve as a leading model organism for studying pathways relevant to human aging and disease.     

Stress co-tolerance and trehalose content in baking strains of Saccharomyces cerevisiae

Fourteen wild-type baking strains of Saccharomyces cerevisiae were grown in batch culture to true stationary phase (exogenous carbon source exhausted) and tested for their trehalose content and their tolerance to heat (52°C for 4.5 min), ethanol (20% v/v for 30 min), H₂O₂ (0.3 M for 60 min), rapid freezing (−196°C for 20 min, cooling rate 200°C min⁻¹), slow freezing (−20°C for 24 h, cooling rate 3°C min⁻¹), salt (growth in 1.5 M NaCl agar) or acetic acid (growth in 0.4% w/v acetic acid agar) stresses. Stress tolerance among the strains was highly variable and up to 1000-fold differences existed between strains for some types of stress. Compared with previously published reports, all strains were tolerant to H₂O₂ stress. Correlation analysis of stress tolerance results demonstrated relationships between tolerance to H₂O₂ and tolerance to all stresses except ethanol. This may imply that oxidative processes are associated with a wide variety of cellular stresses and also indicate that the general robustness associated with industrial yeast may be a result of their oxidative stress tolerance. In addition, H₂O₂ tolerance might be a suitable marker for the general assessment of stress tolerance in yeast strains. Trehalose content failed to correlate with tolerance to any stress except acetic acid. This may indicate that the contribution of trehalose to tolerance to other stresses is either small or inconsistent and that trehalose may not be used as a general predictor of stress tolerance in true stationary phase yeast. Received 10 October 1995/ Accepted in revised form 10 September 1996     

Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae.

Heat shock resulted in rapid accumulation of large amounts of trehalose in Saccharomyces cerevisiae. In cultures growing exponentially on glucose, the trehalose content of the cells increased from 0.01 to 1 g/g of protein within 1 h after the incubation temperature was shifted from 27 to 40 degrees C. When the temperature was readjusted to 27 degrees C, the accumulated trehalose was rapidly degraded. In parallel, the activity of the trehalose-phosphate synthase, the key enzyme of trehalose biosynthesis, increased about sixfold during the heat shock and declined to the normal level after readjustment of the temperature. Surprisingly, the activity of neutral trehalase, the key enzyme of trehalose degradation, also increased about threefold during the heat shock and remained almost constant during recovery of the cells at 27 degrees C. In pulse-labeling experiments with [14C]glucose, trehalose was found to be turned over rapidly in heat-shocked cells, indicating that both anabolic and catabolic enzymes of trehalose metabolism were active in vivo. Possible functions of the heat-induced accumulation of trehalose and its rapid turnover in an apparently futile cycle during heat shock are discussed.     

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.     

Two distinct pathways for trehalose assimilation in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.     

Autonomous oscillations in Saccharomyces cerevisiae during batch cultures on trehalose

We report that autonomous oscillations, which usually happen in aerobic glucose-limited continuous cultures of yeast at low dilution rate, were also observed in trehalose discontinuous cultures of Saccharomyces cerevisiae. This unexpected oscillatory behaviour was therefore examined using fast Fourier transformation of online gas measurements. This robust mathematical analysis underlined the existence of two types of oscillation. The first was found to be linked to the cell cycle because (a) the periodicity corresponded to a fraction of the generation time and (b) the oscillations were accompanied by a transient increase in the budding index, mobilization of storage carbohydrates, and fermentative activity. Moreover, these oscillations occurred in a range of specific growth rates between 0.04 and 0.15 h(-1). All these criteria were consistent with the cell-cycle-related metabolic oscillations observed in the same range of growth rates in glucose-limited continuous cultures. The second type were short-period respiratory oscillations, independent of the specific growth rate. Both types of oscillation were found to take place consecutively and/or simultaneously during batch culture on trehalose. In addition, mobilization of intracellular trehalose emerged as a key parameter for the sustainability of these autonomous oscillations as they were no longer observed in a mutant defective in neutral trehalase activity. We propose that batch culture on trehalose may be an excellent device for further investigation of the molecular mechanisms that underlie autonomous oscillations in yeast.     

压力对酵母菌及其海藻糖生成的影响

酵母菌海藻糖是其在培养条件发生“恶劣”变化时生成的一种应激代谢产物。当压力在0．5．1．0MPa时,酵母菌海藻糖含量为6．5mg／g,较对照提高27％。确定压力提高酵母海藻糖的最适条件为：采用复合培养基,菌体前培养时间20h,压力1．0MPa、加压温度34℃,pH6．0,升降压速度为0．10MPa/min,加压培养3h,此时,酵母海藻糖含量达到11mg／g。     

Regulation of trehalose metabolism in Saccharomyces cerevisiae mutants during temperature shifts

Temperature shifts from 23 degrees C to 36 degrees C resulted in trehalose accumulation in Saccharomyces independently of genetic lesions in the cAMP-protein kinase cascade. In parallel, trehalose 6-phosphate synthase activity increased about 3-fold in all strains; the increase could be inhibited by cycloheximide, suggesting that protein synthesis was required. Heat shock treatment after the temperature shift led to a drastic increase in trehalose activity, and deactivation of the biosynthetic enzyme with a consequent drop in trehalose. Up to now no definite correlation between acquisition of thermotolerance and trehalose accumulation has been made.     

酿酒酵母衰老机制研究进展

酿酒酵母衰老机制的研究对解析高等真核生物衰老的分子机制具有重要意义。酿酒酵母有两种衰老形式：时序衰老（chronologicalaging）和复制衰老（replicative aging）。酿酒酵母衰老研究中通常使用的寿命定义有两种：世代寿命和时序寿命。前者是指单个酿酒酵母细胞在死亡之前的分裂次数;后者是指一定数量的酵母细胞在后二次生长和稳定期的存活时间。本文分别综述了这两种衰老形式的分子机制及两者的相同点和不同点。     

酿酒酵母细胞衰老机理的研究进展

酿酒酵母的细胞衰老研究作为生命科学领域的前沿课题,对解析高等真核生物衰老的分子机制具有重要意义。迄今为止,在酵母中已经确立的衰老模式有两种,即复制型衰老和时序型衰老。细胞衰老的影响因子较多,涉及到很多过程,所以研究起来非常复杂。综述了两种细胞衰老机制的研究进展。     

Internal trehalose protects endocytosis from inhibition by ethanol in Saccharomyces cerevisiae

Endocytosis in Saccharomyces cerevisiae is inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following. (i) The appearance of ethanol resistance was accompanied by trehalose accumulation. (ii) Mutant cells unable to synthesize trehalose also were unable to develop resistance. (iii) Mutant cells that accumulated trehalose during growth on sugars were resistant to ethanol even under this nonstressing condition. (iv) Mutant cells unable to synthesize Hsp104 were able to develop resistance. We conclude that trehalose is the major factor in the protection of endocytosis from ethanol. Our results suggest another important physiological role for trehalose in yeast.     

Effect of hydrostatic pressure on the morphology and ultrastructure of wild-type and trehalose synthase mutant cells of Saccharomyces cerevisiae

AIMS: Saccharomyces cerevisiae was used for studying the physiological effects of hydrostatic pressure. METHODS AND RESULTS: The effects of hydrostatic pressure on the ultrastructure of wild-type and trehalose-6-phosphate synthase (tps1) mutant cells were investigated by transmission electron microscopy. Pressure induced several morphological changes in wild-type and tps1 cells, the latter showing greater structural alterations. When the cells were submitted to a preheat treatment they both acquired resistance to the pressure treatment. CONCLUSION: As the tps1 mutant was 1000-fold more barosensitive than its parental strain, it showed greater structural alterations compared with the wild-type. Microscopic images of the yeast cells suggested that hydrostatic pressure induced changes in the cytoskeleton and therefore, on the cell wall and in the dynamics of the organelles. SIGNIFICANCE AND IMPACT OF THE STUDY: This work presents the effects of hydrostatic pressure on the morphology of yeast cells and confirms the importance of several different factors in the protection of cells against stress.