Choowong Auesukaree

Genome-wide identification of genes involved in tolerance to various environmental stresses in Saccharomyces cerevisiae

During fermentation, yeast cells are exposed to a number of stresses — such as high alcohol concentration, high osmotic pressure, and temperature fluctuation — so some overlap of mechanisms involved in the response to these stresses has been suggested. To identify the genes required for tolerance to alcohol (ethanol, methanol, and 1-propanol), heat, osmotic stress, and oxidative stress, we performed genome-wide screening by using 4828 yeast deletion mutants. Our screens identified 95, 54, 125, 178, 42, and 30 deletion mutants sensitive to ethanol, methanol, 1-propanol, heat, NaCl, and H2O2, respectively. These deleted genes were then classified based on their cellular functions, and cross-sensitivities between stresses were determined. A large number of genes involved in vacuolar H+-ATPase (V-ATPase) function, cytoskeleton biogenesis, and cell wall integrity, were required for tolerance to alcohol, suggesting their protective role against alcohol stress. Our results revealed a partial overlap between genes required for alcohol tolerance and those required for thermotolerance. Genes involved in cell wall integrity and the actin cytoskeleton are required for both alcohol tolerance and thermotolerance, whereas the RNA polymerase II mediator complex seems to be specific to heat tolerance. However, no significant overlap of genes required for osmotic stress and oxidative stress with those required for other stresses was observed. Interestingly, although mitochondrial function is likely involved in tolerance to several stresses, it was found to be less important for thermotolerance. The genes identified in this study should be helpful for future research into the molecular mechanisms of stress response.

Highly efficient bioethanol production by a Saccharomyces cerevisiae strain with multiple stress tolerance to high temperature, acid and ethanol.

Use of super strains exhibiting tolerance to high temperature, acidity and ethanol is a promising way to make ethanol production economically feasible. We describe here the breeding and performance of such a multiple-tolerant strain of Saccharomyces cerevisiae generated by a spore-to-cell hybridization technique without recombinant DNA technology. A heterothallic strain showing a high-temperature (41°C) tolerant (Htg(+)) phenotype, a derivative from a strain isolated from nature, was crossed with a homothallic strain displaying high-ethanol productivity (Hep(+)), a stock culture at the Thailand Institute of Scientific and Technological Research. The resultant hybrid TJ14 displayed ability to rapidly utilize glucose, and produced ethanol (46.6g/l) from 10% glucose fermentation medium at high temperature (41°C). Not only ethanol productivity at 41°C but also acid tolerance (Acd(+)) was improved in TJ14 as compared with its parental strains, enabling TJ14 to grow in liquid medium even at pH 3. TJ14 maintained high ethanol productivity (46.0g/l) from 10% glucose when fermentation was done under multiple-stress conditions (41°C and pH 3.5). Furthermore, when TJ14 was subjected to a repeated-batch fermentation scheme, the growth and ethanol production of TJ14 were maintained at excellent levels over ten cycles of fermentation. Thus, the multiple-stress (Htg(+) Hep(+) Acd(+)) resistant strain TJ14 should be useful for cost-effective bioethanol production under high-temperature and acidic conditions.

Transcriptional regulation of phosphate-responsive genes in low-affinity phosphate-transporter-defective mutants in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, two systems have been shown to be involved in the active transport of inorganic phosphate (P(i)) across the plasma membrane, the high-affinity system and the low-affinity system. The high-affinity system consists of Pho84p and Pho89p. The low-affinity system has recently been shown to be composed of Pho87p, Pho90p, and Pho91p. In this study, we found that the Deltapho87Deltapho90Deltapho91 strain which shows repressed PHO5 expression under high-P(i) condition has, unlike the wild-type strain, increased levels of PHO5 expression at an intermediate P(i) concentration of 0.5mM, whereas it is not defective in terms of P(i) uptake under the same conditions. Moreover, we observed that the transcription levels of PHO84 and PHO89 are also increased in low-affinity P(i)-transporter-defective mutants, indicating that the inactivation of low-affinity P(i) transporters leads to the activation of the PHO pathway. In contrast to that of PHO5, PHO84, and PHO89, the transcription of PHO87, PHO90, and PHO91 genes is independent of P(i) concentration and Pho4p activity, and the increased expression level of these transporters does not occur when other transporters including PHO84 are inactivated. The fact that low-affinity P(i)-transporter-defective mutants exhibit a derepression of P(i)-responsive genes suggests that low-affinity transporters play a role not only in P(i) transport but also in the regulation of the P(i) signal transduction pathway.

Bioaccumulation and biosorption of Cd2+ and Zn2+ by bacteria isolated from a zinc mine in Thailand

The three bacteria, Tsukamurella paurometabola A155, Pseudomonas aeruginosa B237, and Cupriavidus taiwanensis E324, were isolated from soils collected from a zinc mine in Tak Province, Thailand. Among these bacteria, P. aeruginosa B237 and C. taiwanensis E324 were tolerant of both cadmium and zinc, while T. paurometabola A155 was highly tolerant of zinc only. Bioaccumulation experiment revealed that Cd(2+) and Zn(2+) were mainly adsorbed on the cell walls of these bacteria rather than accumulated inside the cells. During Cd(2+) and Zn(2+) biosorption, P. aeruginosa B237 and T. paurometabola A155 showed the highest removal efficiencies for Cd(2+) and Zn(2+), respectively. The maximum biosorption capacities of P. aeruginosa B237 and T. paurometabola A155 biomasses for Cd(2+) and Zn(2+) biosorptions were 16.89 and 16.75 mg g(-1), respectively, under optimal conditions. The experimental data of Cd(2+) and Zn(2+) biosorptions fitted well with Langmuir isotherm model, suggesting that Cd(2+) and Zn(2+) adsorptions occurred in a monolayer pattern on a homogeneous surface. Furthermore, the pseudo-second order and pseudo-first order kinetic models best described the biosorption kinetics of Cd(2+) and Zn(2+) adsorptions, respectively, suggesting that the Cd(2+) and Zn(2+) adsorptions took place mainly by chemisorption (Cd(2+)) and physisorption (Zn(2+)).

Isolation and characterization of lead-tolerant Ochrobactrum intermedium and its role in enhancing lead accumulation by Eucalyptus camaldulensis

In this study, the potential of rhizospheric bacteria in promoting the growth and Pb accumulation by the woody plant Eucalyptus camaldulensis under hydroponic conditions was investigated for the first time. Three Pb-tolerant bacteria were isolated from the rhizosphere of E. camaldulensis grown in Pb-contaminated soils in the Bo Ngam Pb mine, Thailand. Based on analysis of partial 16S rRNA gene sequence, the three isolates were identified as Microbacterium paraoxydans BN-2, Ochrobactrum intermedium BN-3, and Bacillus fusiformis BN-4. Among these strains, O. intermedium BN-3 showed the highest tolerance to not only Pb but also Cd and Zn. After growth in the presence of Pb, the membranes of O. intermedium BN-3 cells exhibited an increase in unsaturated fatty acid levels but a decrease in fluidity. In hydroponic studies, inoculation of O. intermedium BN-3 significantly increased the biomass and Pb accumulation by E. camaldulensis compared to the uninoculated control. The results suggested the role of the natural rhizospheric bacteria localized to the root surface of E. camaldulensis in promoting Pb accumulation and plant growth. Our results indicate that O. intermedium BN-3 and other indigenous rhizospheric bacteria have the potential to improve the efficiency of phytoremediation of Pb-contaminated sites.

CDC19 encoding pyruvate kinase is important for high-temperature tolerance in Saccharomyces cerevisiae

Use of thermotolerant strains is a promising way to reduce the cost of maintaining optimum temperatures in the fermentation process. Here we investigated genetically a Saccharomyces cerevisiae strain showing a high-temperature (41°C) growth (Htg(+)) phenotype and the result suggested that the Htg(+) phenotype of this Htg(+) strain is dominant and under the control of most probably six genes, designated HTG1 to HTG6. As compared with a Htg(-) strain, the Htg(+) strain showed a higher survival rate after exposure to heat shock at 48°C. Moreover, the Htg(+) strain exhibited a significantly high content of trehalose when cultured at high temperature and stronger resistance to Congo Red, an agent that interferes with cell wall construction. These results suggest that a strengthened cell wall in combination with increased trehalose accumulation can support growth at high temperature. The gene CDC19, encoding pyruvate kinase, was cloned as the HTG2 gene. The CDC19 allele from the Htg(+) strain possessed five base changes in its upstream region, and two base changes resulting in silent mutations in its coding region. Interestingly, the latter base changes are probably responsible for the increased pyruvate kinase activity of the Htg(+) strain. The possible mechanism leading to this increased activity and to the Htg(+) phenotype, which may lead to the activation of energy metabolism to maintain cellular homeostasis, is discussed.

Characterization and gene expression profiles of thermotolerant Saccharomyces cerevisiae isolates from Thai fruits.

For industrial applications, fermentation of ethanol at high temperature offers advantages such as reduction in cooling costs, reduced risk of microbial contamination and higher efficiency of fermentation processes including saccharification and continuous ethanol stripping. Three thermotolerant Saccharomyces cerevisiae isolates (C3723, C3751 and C3867) from Thai fruits were capable of growing and producing 38 g/L ethanol up to 41°C. Based on genetic analyses, these isolates were prototrophic and homothallic, with dominant homothallic and thermotolerant phenotypes. After short-term (30 min) and long-term (12 h) exposure at 37°C, expression levels increased for the heat stress-response genes HSP26, SSA4, HSP82, and HSP104 encoding the heat shock proteins small HSP, HSP70, HSP90 and the HSP100 family, respectively. In isolates C3723 and C3867, expression was significantly higher than that in reference isolates W303 and TISTR5606 for TPS1 encoding trehalose-6-phosphate synthase, NTH1 encoding neutral trehalase and GSY1 encoding glycogen synthase. The results suggested that continuous high expression of heat stress-response genes was important for the long-term, heat stress tolerance of these thermotolerant isolates.

Cellular mechanisms contributing to multiple stress tolerance in Saccharomyces cerevisiae strains with potential use in high-temperature ethanol fermentation

High-temperature ethanol fermentation has several benefits including a reduction in cooling cost, minimizing risk of bacterial contamination, and enabling simultaneous saccharification and fermentation. To achieve the efficient ethanol fermentation at high temperature, yeast strain that tolerates to not only high temperature but also the other stresses present during fermentation, e.g., ethanol, osmotic, and oxidative stresses, is indispensable. The C3253, C3751, and C4377 Saccharomyces cerevisiae strains, which have been previously isolated as thermotolerant yeasts, were found to be multiple stress-tolerant. In these strains, continuous expression of heat shock protein genes and intracellular trehalose accumulation were induced in response to stresses causing protein denaturation. Compared to the control strains, these multiple stress-tolerant strains displayed low intracellular reactive oxygen species levels and effective cell wall remodeling upon exposures to almost all stresses tested. In response to simultaneous multi-stress mimicking fermentation stress, cell wall remodeling and redox homeostasis seem to be the primary mechanisms required for protection against cell damage. Moreover, these strains showed better performances of ethanol production than the control strains at both optimal and high temperatures, suggesting their potential use in high-temperature ethanol fermentation.

Molecular mechanisms of the yeast adaptive response and tolerance to stresses encountered during ethanol fermentation

During ethanol fermentation, yeast cells encounter various stresses including sugar substrates-induced high osmolarity, increased ethanol concentration, oxygen metabolism-derived reactive oxygen species (ROS), and elevated temperature. To cope with these fermentation-associated stresses, appropriate adaptive responses are required to prevent stress-induced cellular dysfunctions and to acquire stress tolerances. This review will focus on the cellular effects of these stresses, molecular basis of the adaptive response to each stress, and the cellular mechanisms contributing to stress tolerance. Since a single stress can cause diverse effects, including specific and non-specific effects, both specific and general stress responses are needed for achieving comprehensive protection. For instance, the high-osmolarity glycerol (HOG) pathway and the Yap1/Skn7-mediated pathways are specifically involved in responses to osmotic and oxidative stresses, respectively. On the other hand, due to the common effect of these stresses on disturbing protein structures, the upregulation of heat shock proteins (HSPs) and trehalose is induced upon exposures to all of these stresses. A better understanding of molecular mechanisms underlying yeast tolerance to these fermentation-associated stresses is essential for improvement of yeast stress tolerance by genetic engineering approaches.

Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses

ABSTRACT During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H+-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2 and Δvma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. IMPORTANCE The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic fermentation. The results revealed that straight-chain alcohols induced cytosolic and vacuolar acidification through their membrane-permeabilizing effects. Contrary to expectations, a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress, but not in the maintenance of intracellular pH, seems to be important for protecting yeast cells against ethanol stress. These findings will expand our understanding of the mechanisms of ethanol tolerance and provide promising clues for the development of ethanol-tolerant yeast strains.