Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

Thermotolerance and trehalose accumulation induced by heat shock in yeast cells of Candida albicans

Candida albicans yeast cells growing exponentially on glucose are extremely sensitive to severe heat shock treatments (52.5°C for 5 min). When these cultures were subjected to a mild temperature preincubation (42°C), they became thermotolerant and displayed higher resistance to further heat stress. The intracellular content of trehalose was very low in exponential cells, but underwent a marked increase upon non-lethal heat exposure. The accumulation of trehalose is likely due to heat-induced activation of the trehalose-6-phosphate synthase complex, whereas the external trehalase remained practically unmodified. After a temperature reversion shift (from 42°C to 28°C), the pool of trehalose was rapidly mobilized without any concomitant change in trehalase activity. These results support an important role of trehalose in the mechanism of acquired thermotolerance in C. albicans and seem to exclude the external trehalase as a key enzyme in this process.     

Thermal stress responses in Antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR

Living organisms have some common and unique strategies to response to thermal stress. However, the amount of data on thermal stress response of certain organism is still lacking, especially psychrophilic yeast from the extreme habitat. Therefore, it is not known whether psychrophilic yeast shares the common responses of other organisms when exposed to thermal stresses. In this work, the cold shock and heat shock responses in Antarctic psychrophilic yeast Glaciozyma antarctica PI12 which had an optimal growth temperature of 12 °C were determined. The expression levels of 14 thermal stress-related genes were measured using real-time quantitative PCR (qPCR) when the yeast cells were exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions. The expression profiles of the 14 genes at these three temperatures varied indicating that these genes had their specific roles to ensure the survival of the yeast. Under cold shock condition, the afp4 and fad genes were over-expressed possibly as a way for the G. antarctica PI12 to avoid ice crystallization in the cell and to maintain the membrane fluidity. Under the heat shock condition, hsp70 was significantly up-regulated possibly to ensure the proteins fold properly. Among the six oxidative stress-related genes, MnSOD and prx were up-regulated under cold shock and heat shock, respectively, possibly to reduce the negative effects caused by oxidative stress. Interestingly, it was found that the trehalase gene, nth1 that plays a role in degrading excess trehalose, was down-regulated under the heat shock condition possibly as an alternative way to accumulate trehalose in the cells to protecting them from being damaged.     

Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica

The response to heat stress in six yeast species isolated from Antarctica was examined. The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20°C, and the other psychrotrophic, capable of growth at temperatures above 20°C. In addition to species-specific heat shock protein (hsp) profiles, a heat shock (15°C–25°C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M. frigida, and M. gelida, but not evident in Leucosporidium antarcticum. Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90. Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined. In the psychrotrophic yeast, Leucosporidium fellii and L. scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed. In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted. Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species. A 10 Celsius degree increase in temperature above the growth temperature (15°C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance. On the other hand, in psychrotrophic yeast grown at 25°C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance. Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation. It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles. Received: August 7, 1997 / Accepted: October 22, 1997     

Physiological implication of intracellular trehalose and mannitol changes in response of entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis> to thermal stress

To explore possible role of intracellular trehalose accumulation in fungal tolerance to summer-like thermal stress, 3-day colonies of Beauveria bassiana grown on a glucose-free medium at 25°C were separately exposed to 35, 37.5 and 40°C for 1–18 h, respectively. Trehalose accumulation in stressed mycelia increased from initial 4.2 to 88.3, 74.7 and 65.5 mg g⁻¹ biomass after 6-h stress at 35, 37.5 and 40°C, respectively, while intracellular mannitol level generally declined with higher temperatures and longer stress time. The stress-enhanced trehalose level was significantly correlated to decreased trehalase activity (r ² = 0.73) and mannitol content (r ² = 0.38), which was inversely correlated to the activity of mannitol dehydrogenase (r ² = 0.41) or mannitol 1-phosphate dehydrogenase (r ² = 0.30) under the stresses. All stressed cultures were successfully recovered at 25°C but their vigor depended on stressful temperature, time length and the interaction of both (r ² = 0.98). The highest level of 6-h trehalose accumulation at 35°C was found enhancing the tolerance of the stressed cultures to the greater stress of 48°C. The results suggest that the trehalose accumulation result partially from metabolized mannitol and contribute to the fungal thermotolerance. Trehalase also contributed to the thermotolerance by hydrolyzing accumulated trehalose under the conditions of thermal stress and recovery.     

Thermal Destabilization of Stem Bromelain by Trehalose

Trehalose, a naturally occurring osmolyte, is considered as a universal protein stabilizer. We investigated the effect of the disaccharides, trehalose and sucrose, on the thermal stability and conformation of bromelain. To our surprise, bromelain in the presence of 1 M trehalose/sucrose was destabilized under thermal stress. The average Tm values as determined by UV spectroscopy and CD spectropolarimetry decreased by 5° and 7°C for bromelain in 1 M sucrose or trehalose solutions, respectively. The enzyme was also found to inactivate faster at 60°C in the presence of these osmolytes. The tertiary and secondary structure of bromelain undergoes small changes in the presence of sucrose/trehalose. Studies on the binding of these osmolytes with the native and the heat denatured enzyme revealed that sucrose/trehalose lead to preferential hydration of the denatured bromelain as compared to the native one, hence stabilizing more the denatured conformation. This is perhaps the first report on the destabilization of a protein by trehalose.     

Trehalose accumulation from cassava starch and release by a highly thermosensitive and permeable mutant of <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis>

Highly thermosensitive and permeable mutants are the mutants from which intracellular contents can be released when they are incubated both in low osmolarity water and at non-permissive temperature (usually 37°C). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named A11-b was obtained from Saccharomycopsis fibuligera A11-12, a trehalose overproducer in which the acid protease gene has been disrupted. Of the total trehalose, 73.8% was released from the mutant cells suspended in distilled water after they had been treated at 37°C overnight. However, only 10.0% of the total trehalose was released from the cells of S. fibuligera A11-12 treated under the same conditions. The cell volume of the mutant cells suspended in distilled water and treated at 37°C overnight was much bigger than that of S. fibuligera A11-12 treated under the same conditions. The cell growth and trehalose accumulation of the mutant were almost the same as those of S. fibuligera A11-12 during the cultivation at the flask level and in a 5-l fermentor. Both could accumulate around 28.0% (w/w) trehalose from cassava starch. After purification, the trehalose crystal from the aqueous extract of the mutant was obtained.     

Acquisition of thermotolerant yeast Saccharomyces cerevisiae by breeding via stepwise adaptation

A thermotolerant Saccharomyces cerevisiae yeast strain, YK60‐1, was bred from a parental strain, MT8‐1, via stepwise adaptation. YK60‐1 grew at 40°C, a temperature at which MT8‐1 could not grow at all. YK60‐1 exhibited faster growth than MT8‐1 at 30°C. To investigate the mechanisms how MT8‐1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress‐responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60‐1. Furthermore, nontargeting metabolome analysis showed that YK60‐1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8‐1. In conclusion, S. cerevisiae MT8‐1 acquired thermotolerance by induction of specific stress‐responsive genes and enhanced intracellular trehalose levels. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1116–1123, 2013     

Continuous cultivation of bakers' yeast: Change in cell composition at different dilution rates and effect of heat stress on trehalose level

The cell composition of bakers' yeast in a continuous culture was determined for different dilution rates. Also, the cellular response to heat stress in terms of trehalose, RNA, glycogen and protein was determined at a specified dilution rate of 0.1/h. The amount of storage saccharides, trehalose and glycogen, was found to decrease whereas the amount of RNA and protein increased with increasing dilution rates. As the dilution rate was increased from 0.1 to 0.4/h at 0.05 intervals the steady-state trehalose content decreased from 33 to 8.6 mg/g biomass, and glycogen content from 150 to 93 mg/g biomass. On the other hand, the protein content increased from 420 to 530 mg/g biomass and the RNA content from 93 to 113 mg/g biomass. Heat stress was applied by increasing the medium temperature from 30 to 36, 38 or 40°C at constant dilution rates. The highest amount of trehalose accumulation, 108 mg/g biomass, was achieved when heat stress at 38°C was applied. The protein content, on the other hand, decreased from 350 to 325 mg/g biomass at the end of the experiment.     

Preservation of frozen yeast cells by trehalose.

Two different methods commonly used to preserve intact yeast cells-freezing and freeze-drying-were compared. Different yeast cells submitted to these treatments were stored for 28 days and cell viability assessed during this period. Intact yeast cells showed to be less tolerant to freeze-drying than to freezing. The rate of survival for both treatments could be enhanced by exogenous trehalose (10%) added during freezing and freeze-drying treatments or by a combination of two procedures: a pre-exposure of cells to 40 degrees C for 60 min and addition of trehalose. A maximum survival level of 71.5 +/- 6.3% after freezing could be achieved at the end of a storage period of 28 days, whereas only 25.0 +/- 1.4% showed the ability to tolerate freeze-drying treatment, if both low-temperature treatments were preceded by a heat exposure and addition of trehalose to yeast cells. Increased survival ability was also obtained when the pre-exposure treatment of yeast cells was performed at 10 degrees C for 3 h and trehalose was added: these treatments enhanced cell survival following freezing from 20.5 +/- 7. 7% to 60.0 +/- 3.5%. Although both mild cold and heat shock treatments could enhance cell tolerance to low temperature, only the heat treatment was able to increase the accumulation of intracellular trehalose whereas, during cold shock exposure, the intracellular amount of trehalose remained unaltered. Intracellular trehalose levels seemed not to be the only factor contributing to cell tolerance against freezing and freeze-drying treatments; however, the protection that this sugar confers to cells can be exerted only if it is to be found on both sides of the plasma membrane.     

Biochemical characterization of neutral trehalase activity in Saccharomyces boulardii

Lyophilized cells of the non-pathogenic yeast Saccharomyces boulardii are used in many countries for the treatment of several types of diarrhoea and other gastrointestinal diseases. Although the cells must be viable, their mechanism of action is unknown. The disaccharide trehalose is a protectant against several forms of environmental stress in yeast and is involved in maintaining cell viability. There is no information on the enzymes involved in degradation of trehalose in S. boulardii. The aim of the present study was to characterize trehalase activity in this yeast. Cells of S. boulardii grown in glucose exhibited neutral trehalase activity only in the exponential phase. Acidic trehalase was not detected in glucose medium. Cells grown in trehalose exhibited acid and neutral trehalase activities at all growth stages, particularly in the exponential phase. The optimum pH and temperature values for neutral trehalase activity were determined as 6.5 and 30 °C respectively, the half-life being approximately 3 min at 45 °C. The relative molecular mass of neutral trehalase is 80 kDa and the K _m 6.4 mM (±0.6). Neutral trehalase activity at pH 6.5 was weakly inhibited by 5 mM EDTA and strongly inhibited by ATP, as well as the divalent ions Cu⁺⁺, Fe⁺⁺ and Zn⁺⁺. Enzyme activity was stimulated by Mg⁺⁺ and Ca⁺⁺ only in the absence of cAMP. The presence of cAMP with no ion additions increased activity by 40%. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anticancer drugs as inducers of thermotolerance in yeast

Yeast cell viability was evaluated microscopically following exposure to heat shock for 30 min at 53°C. The cells were previously grown in the presence of potential stressors (anticancer drugs;e.g., 5-fluorouracil, methotrexate, cisplatin, bleomycin, mitomycin-C and camptothecin-11). The induction of thermotolerance was documented by significantly increased viability after heat shock. This effect, which was reversed by cycloheximide, was comparable to that observed following exposure to a mild heat stress. These data demonstrate that pretreatment with sub-toxic concentrations of some of the clinically used antineoplastic agents conferres thermotolerance to yeast, possibly through the synthesis of protein components.     

Enhanced thermotolerance for ethanol fermentation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain by overexpression of the gene coding for trehalose-6-phosphate synthase

The effect of overexpression of the trehalose-6-phosphate (T6P) synthase gene (TPS1) on ethanol fermentation of Saccharomyces cerevisiae has been studied at 30 and 38°C. The activity of T6P synthase and the accumulation of trehalose during ethanol fermentation were significantly improved by overexpression of TPS1, and especially at 38°C. Ethanol produced by transformants with and without TPS1 gene overexpression at 38°C was approx. 60 and 37 g/l, respectively. The fermentation efficiency of transformants with TPS1 gene overexpression at 38°C was similar to that at 30°C. The critical growth temperature was increased from 36 to 42°C by TPS1 gene overexpression. These results indicated that overexpression of the TPS1 gene had a beneficial effect on the fermentation capacity of the title yeast strain at high temperatures.     

Histamine modulates the cellular stress response in yeast

The cellular stress response is a universal protective reaction to adverse environmental or microenvironmental conditions, such as heat and drugs, associated in part with the highly conserved heat shock proteins (HSPs). Histamine is a key inflammatory mediator derived from l-histidine that governs vital cellular processes beyond inflammation, while recent evidence implies additional actions in both prokaryotes and eukaryotes. This study explored the possible role of histamine in the heat shock response in yeast, an established experimental model for the pharmacological investigation of the cellular stress response. The response was evaluated by determining growth and viability of post-logarithmic phase grown yeast cultures after heat shock at 53°C for 30 min. Thermal preconditioning at 37°C for 2 h served as a positive control. The effect of histamine was investigated following long-term administration through the post-logarithmic phase of growth or short-term administration for 2 h prior to heat shock. Short-term treatment with 1 mM histamine resulted in de novo protein synthesis-dependent acquisition of thermotolerance, while lower doses or long-term administration of histamine failed to induce the heat-resistant phenotype. Preliminary investigation of HSP104, HSP70 and HSP60 expression by western blotting showed an increase of these proteins after thermal preconditioning. However, a differential HSP and tubulin expression appeared to underlie the response of yeast cells to histamine. In conclusion, histamine was capable of inducing the adaptive phenotype, while the contribution of HSPs and tubulin and the potential implications remain largely elusive.     

Production and Properties of Alkaline Dextranase from a Newly Isolated Brevibacterium

About 500 strains of dextranase producing microorganisms were examined in detail for pH- activity and enzyme stability. A gram positive bacterium identified as belonging to the genus Brevibacterium was found to produce alkaline dextranase. Maximal dextranase synthesis was obtained when grown aerobically at 26°C for 3 days in a medium containing 1 % dextran, 2% ethanol, 1 % polypeptone and 0.05 % yeast extract together with trace amounts of inorganic salts.

Brevibacterium dextranase had an optimum pH of 8.0 for activity at 37°C and an optimal temperature at 53°C at pH 7.5. The enzyme was quite stable over the range of pH 5.0 to 10.5 on 24 hr incubation at 37°C, especially on alkaline pH. The enzyme was also heat stable at 60°C for 10 min.     

The effect of calnexin deletion on the expression level of PDI in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under heat stress conditions

We cultured calnexin-disrupted and wild-type Saccharomyces cerevisiae strains under conditions of heat stress. The growth rate of the calnexin-disrupted yeast was almost the same as that of the wild-type yeast under those conditions. However, the induced mRNA level of the molecular chaperone PDI in the ER was clearly higher in calnexin-disrupted S. cerevisiae relative to the wild type at 37°C, despite being almost the same in the two strains under normal conditions. The western blotting analysis for PDI protein expression in the ER yielded results that show a parallel in their mRNA levels in the two strains. We suggest that PDI may interact with calnexin under heat stress conditions, and that the induction of PDI in the ER can recover part of the function of calnexin in calnexin-disrupted yeast, and result in the same growth rate as in wild-type yeast.     

Heat stress hardening of oriental armyworms is induced by a transient elevation of reactive oxygen species during sublethal stress

Pre‐exposure to mild heat stress enhances the thermotolerance of insects. Stress hardening is a beneficial physiological plasticity, but the mechanism underlying it remains elusive. Here we report that reactive oxygen species (ROS) concentrations were quickly and transiently elevated in the armyworms, Mythimna separata, by exposing them to 40°C, but not other tested temperatures. Larvae exposed to 40°C had subsequently elevated antioxidant activity and the highest survival of all tested heating conditions. The elevation of ROS after lethal heating at 44°C for 1 h was approximately twofold compared to heating at 40°C. Injection of an optimal amount of hydrogen peroxide (H₂O₂) similarly caused sequential elevation of ROS and antioxidant activity in the test larval hemolymph, which led to significantly enhanced survival after lethal heat stress. The H₂O₂‐induced thermotolerance was abolished by coinjection of potent antioxidants such as ascorbic acid or N‐acetylcysteine. Both preheating at 40°C and H₂O₂ injection enhanced expression of genes encoding superoxide dismutase 1, catalase, and heat shock protein 70 in the fat body of test larvae, indicating the adequate heat stress induced a transient elevation of ROS, followed by upregulation of antioxidant activity. We infer that thermal stress hardening is induced by a small timely ROS elevation that triggers a reduction–oxidation signaling mechanism.     

Role of Hsp70 synthesis in the fate of the insulin-receptor complex after heat shock in cultured fetal hepatocytes

The influence of a mild heat shock on the fate of the insulin-receptor complex was studied in cultured fetal rat hepatocytes whose insulin glycogenic response is sensitive to heat [Zachayus and Plas (1995): J Cell Physiol 162:330–340]. After exposure from 15 min to 2 hr at 42.5°C, the amount of ¹²⁵I-insulin associated with cells at 37°C was progressively decreased (by 35% after 1 hr), while the release of ¹²⁵I-insulin degradation products into the medium was also inhibited (by 75%), more than expected from the decrease in insulin binding. Heat shock did not affect the insulin-induced internalization of cell surface insulin receptors but progressively suppressed the recycling at 37°C of receptors previously internalized at 42.5°C in the presence of insulin. When compared to the inhibitory effects of chloroquine on insulin degradation and insulin receptor recycling, which were immediate (within 15 min), those of heat shock developed within 1 hr of heating. The protein level of insulin receptors was not modified after heat shock and during recovery at 37°C, while that of Hsp72/73 exhibited a transitory accumulation inversely correlated with variations in insulin binding, as assayed by Western immunoblotting from whole cell extracts. Coimmunoprecipitation experiments revealed a heat shock-stimulated association of Hsp72/73 with the insulin receptor. Affinity labeling showed an interaction between ¹²⁵I-insulin and Hsp72/73 in control cells, which was inhibited by heat shock. These results suggest that increased Hsp72/73 synthesis interfered with insulin degradation and prevented the recycling of the insulin receptor and its further thermal damage via a possible chaperone-like action in fetal hepatocytes submitted to heat stress. © 1996 Wiley-Liss, Inc.