Analysis of the stress resistance of commercial wine yeast strains

Alcoholic fermentation is an essential step in wine production that is usually conducted by yeasts belonging to the species Saccharomyces cerevisiae. The ability to carry out vinification is largely influenced by the response of yeast cells to the stress conditions that affect them during this process. In this work, we present a systematic analysis of the resistance of 14 commercial S. cerevisiae wine yeast strains to heat shock, ethanol, oxidative, osmotic and glucose starvation stresses. Significant differences were found between these yeast strains under certain severe conditions, Vitilevure Pris Mouse and Lalvin T73 being the most resistant strains, while Fermiblanc arom SM102 and UCLM S235 were the most sensitive ones. Induction of the expression of the HSP12 and HSP104 genes was analyzed. These genes are reported to be involved in the tolerance to several stress conditions in laboratory yeast strains. Our results indicate that each commercial strain shows a unique pattern of gene expression, and no clear correlation between the induction levels of either gene and stress resistance under the conditions tested was found. However, the increase in mRNA levels in both genes under heat shock indicates that the molecular mechanisms involved in the regulation of their expression by stress function in all of the strains.     

Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts

Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation.     

Cadmium-induced synthesis of HSP70 and a role of glutathione in Euglena gracilis

The effect of cadmium-induced oxidative stress, with or without glutathione supplementation, was investigated in the single cell eukaryotic phytoflagellate, Euglena gracilis strains Z and its achlorophyllous mutant SMZ as experimental models. Both these strains actively synthesize thiols to prevent or resist cadmium toxicity. The content of glutathione, as a representative antioxidant, was also examined in both strains. Exposure to cadmium induced heat-shock protein 70 (HSP70) synthesis in both strains of E. gracilis. Glutathione supplementation also induced HSP70. Overall, these results indicate that glutathione was closely linked to the induction of stress-related proteins. The sensitivity to cadmium-stress was higher in strain Z than SMZ. The results suggest that chloroplasts may have a role in the regulation of HSP70 expression. The relationship between HSP70 and GSH levels is still far from understood, and further research may shed light upon their up-regulation in the presence of Cd.     

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.     

Analysis of adaptation to high ethanol concentration in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using DNA microarray

In industrial process, yeast cells are exposed to ethanol stress that affects the cell growth and the productivity. Thus, investigating the intracellular state of yeast cells under high ethanol concentration is important. In this study, using DNA microarray analysis, we performed comprehensive expression profiling of two strains of Saccharomyces cerevisiae, i.e., the ethanol-adapted strain that shows active growth under the ethanol stress condition and its parental strain used as the control. By comparing the expression profiles of these two strains under the ethanol stress condition, we found that the genes related to ribosomal proteins were highly up-regulated in the ethanol-adapted strain. Further, genes related to ATP synthesis in mitochondria were suggested to be important for growth under ethanol stress. We expect that the results will provide a better understanding of ethanol tolerance of yeast. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cadmium-induced synthesis of HSP70 and a role of glutathione in Euglena gracilis

Abstract

The effect of cadmium-induced oxidative stress, with or without glutathione supplementation, was investigated in the single cell eukaryotic phytoflagellate, Euglena gracilis strains Z and its achlorophyllous mutant SMZ as experimental models. Both these strains actively synthesize thiols to prevent or resist cadmium toxicity. The content of glutathione, as a representative antioxidant, was also examined in both strains. Exposure to cadmium induced heat-shock protein 70 (HSP70) synthesis in both strains of E. gracilis. Glutathione supplementation also induced HSP70. Overall, these results indicate that glutathione was closely linked to the induction of stress-related proteins. The sensitivity to cadmium-stress was higher in strain Z than SMZ. The results suggest that chloroplasts may have a role in the regulation of HSP70 expression. The relationship between HSP70 and GSH levels is still far from understood, and further research may shed light upon their up-regulation in the presence of Cd.     

Changes in protein composition ofSaccharomyces brewing strains in response to heat shock and ethanol stress

Summary Heat shock and ethanol stress of brewing yeast strains resulted in the induction of a set of proteins referred to as heat shock proteins (HSPs). At least six strongly induced HSPs were identified in a lager brewing strain and four HSPs in an ale brewing strain. Four of these HSPs with molecular masses of approximately 70, 38, 26 and 23 kDa were also identified in two laboratory strains ofSaccharomyces cerevisiae. The appearance of HSPs correlated with increased survival of strains at elevated temperatures and high concentrations of ethanol. These results suggest that HSPs may play a role in the ethanol and thermotolerance of yeasts. The properties of these proteins and membrane fatty acids in relation to heat and ethanol shock are being investigated.     

Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae.

This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates. Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol. Therefore we constructed mutants in two parental strains (YPH499 and W303-1a). Some differences appeared in the growth characteristics of mutants obtained from these two parental strains. For these experiments we used ethanol, pyruvate or lactate as substrates. Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway. The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain. The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent. To understand these differences, we measured the enzyme content of these different strains. We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase. Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation. Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient. However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation.     

Selection and adaptation of Saccharomyces cerevisae to increased ethanol tolerance and production

A total of 24 yeast strains were tested for their capacity to produce ethanol, and of these, 8 were characterized by the best ethanol yields (73.11-8 1.78%). The most active mutant Saccharomyce s cerevisiae ER-A, resistant to ethanol stress, was characterized by high resistance to acidic (pH 1.0 and 2.0), oxidative (1 and 2% of H2O2), and high temperature (45 and 52 degrees C) stresses. During cultivation under all stress conditions, the mutants showed a considerably increased viability ranging widely from about 1.04 to 3.94-fold in comparison with the parent strain S. cerevisiae ER. At an initial sucrose concentration of 150 g/l in basal medium A containing yeast extract and mineral salts, at 300C and within 72 h, the most active strain, S. cerevisiae ER-A, reached an ethanol concentration of 80 g/1, ethanol productivity of 1.1 g/Il/h, and an ethanol yield (% of theoretical) of 99.13. Those values were significantly higher in comparison with parent strain (ethanol concentration 71 g/1 and productivity of 0,99 g/l/h). The present study seems to confirm the high effectiveness of selection of ethanol-resistant yeast strains by adaptation to high ethanol concentrations, for increased ethanol production.     

Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis

During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.     

Redox interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in mixed culture under enological conditions

Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.     

小菜蛾热休克蛋白基因的鉴定及其表达模式分析

热休克蛋白(heat shock protein, HSP)在昆虫应对外界胁迫刺激时起着重要作用。为了系统研究小菜蛾Plutella xylostella HSP基因家族, 根据家蚕的HSP蛋白序列, 采用本地Blast程序对小菜蛾全基因组数据库进行同源序列检索, 从小菜蛾基因组数据库中鉴定了25个HSP基因, 包括2个HSP90、 8个HSP70和15个sHSP（small heat shock protein, sHSP）基因。小菜蛾、 家蚕Bombyx mori、 黑腹果蝇Drosophila melanogaster和赤拟谷盗Tribolium castaneum的HSP系统进化分析显示, 昆虫的小分子量热休克蛋白sHSP具有很强的种属特异性, HSP70家族的保守性比sHSP强。小菜蛾HSP基因表达模式分析显示, 与敏感品系对比, 抗性品系（抗毒死蜱和抗氟虫氰品系）中HSP基因具有不同的表达模式。小菜蛾1, 2和3龄幼虫HSP基因表达模式较为接近, 而与4龄幼虫中的表达模式相差较大; 4龄幼虫和蛹中的表达模式相近; 雌成虫和雄成虫中的表达模式显著不同, 与果蝇精子形成有关的两个热休克蛋白HSP23和HSP27基因［分别为CCG003980.1 (Px23.5)和CCG005412.2 (Px27.5)］, 在小菜蛾雄成虫中的表达量显著高于雌成虫。研究结果表明小菜蛾HSP基因不仅在杀虫剂抗性、 发育分化, 甚至在生殖上均可能起着重要的作用。本研究为深入研究小菜蛾HSP与生长发育、 抗逆行为的相互关系奠定了基础。     

Heat shock and light activation of aChlamydomonas HSP70 gene are mediated by independent regulatory pathways

Induction ofHSP70 heat shock genes by light has been demonstrated inChlamydomonas. Our aim was to establish whether this induction by light is mediated by the heat stress sensing pathway or by an independent signal chain. Inhibitors of cytoplasmic protein synthesis revealed an initial difference. Cycloheximide and other inhibitors of protein synthesis preventedHSP70A induction upon illumination but not during heat stress. Analysis ofHSP70A induction in cells that had differentiated into gametes revealed a second difference. While heat shock resulted in elevatedHSP70A mRNA levels, light was no longer able to serve as an inducer in gametes. To identify the regulatory sequences that mediate the response of theHSP70A gene to either heat stress or light we introduced a series of progressive 5 truncations into its promoter sequence. Analyses of the levels of mRNA transcribed from these deletion constructs showed that in most of them the responses to heat shock and light were similar, suggesting that light induction is mediated by a light-activated heat shock factor. However, we show that theHSP70A promoter also containscis-acting sequences involved in light induction that do not participate in induction by heat stress. Together, these results provide evidence for a regulation ofHSP70A gene expression by light through a heat shock-independent signal pathway.     

Three different proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity from Alcaligenes eutrophus

The existence of three different proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity was confirmed in Alicaligenes eutrophus. The fermentative alcohol dehydrogenase, which also exhibits acetaldehyde dehydrogenase activity, is one of these proteins. The other two proteins were purified from A. eutrophus N9A mutant AS4 grown on ethanol applying chromatography on DEAE-Sephacel and triazine-dye affinity media. Acetaldehyde dehydrogenase II, which amounts to about 14% of the total soluble protein in cells grown on ethanol, was purified to homogeneity. The relative molecular masses of the native enzyme and of the subunits were 195,000 or 56,000, respectively. This enzyme exhibits a high affinity for acetaldehyde (Km = 4 microM). Acetaldehyde dehydrogenase I amounts only to less than 1% of the total soluble protein. The relative molecular masses of the native enzyme and of the subunits were 185,000 and 52,000, respectively. This enzyme exhibits a low affinity for acetaldehyde (Km = 2.6 mM). Antibodies raised against acetaldehyde dehydrogenase II did not react with acetaldehyde dehydrogenase I. Two different strains, A. eutrophus N9A mutant AS1, which represents a different mutant type and can utilize both ethanol or 2,3-butanediol, and the type strain of A. eutrophus (TF93), which can utilize ethanol, form two acetaldehyde dehydrogenases during growth on ethanol, too. As in AS4, one of these enzymes from each strain amounted to a substantial portion of the total soluble protein in the cells. These major acetaldehyde dehydrogenases were purified from both strains; they resemble acetaldehyde dehydrogenase II isolated from AS4 in all relevant properties. Antibodies against the enzyme isolated from AS4 gave identical cross-reactions with the enzymes isolated from AS1 and TF93.