scholarly journals Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

2015 ◽  
Vol 14 (9) ◽  
pp. 884-897 ◽  
Author(s):  
Steve Swinnen ◽  
Annelies Goovaerts ◽  
Kristien Schaerlaekens ◽  
Françoise Dumortier ◽  
Pieter Verdyck ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Tolerance ◽  
Stress Factors ◽  
Causative Mutation ◽  
Ethanol Stress ◽  
Limiting Factor ◽  
Ethanol Sensitivity ◽  
Content Type ◽  
High Ethanol ◽  
Very High

ABSTRACTVery high ethanol tolerance is a distinctive trait of the yeastSaccharomyces cerevisiaewith notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained theura3Δ0mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage forLYS2,LEU2,HIS3, andMET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.

scholarly journals Membrane Fluidity of Saccharomyces cerevisiae from Huangjiu (Chinese Rice Wine) Is Variably Regulated by OLE1 To Offset the Disruptive Effect of Ethanol

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Yijin Yang ◽  
Yongjun Xia ◽  
Wuyao Hu ◽  
Leren Tao ◽  
Li Ni ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Membrane Fluidity ◽  
Ethanol Tolerance ◽  
Acid Metabolism ◽  
Ethanol Stress ◽  
Disruptive Effect ◽  
Rice Wine ◽  
Chinese Rice Wine ◽  
High Ethanol

ABSTRACT An evolution and resequencing strategy was used to research the genetic basis of Saccharomyces cerevisiae BR20 (with 18 vol% ethanol tolerance) and the evolved strain F23 (with 25 vol% ethanol tolerance). Whole-genome sequencing and RNA sequencing (RNA-seq) indicated that the enhanced ethanol tolerance under 10 vol% ethanol could be attributed to amino acid metabolism, whereas 18 vol% ethanol tolerance was due to fatty acid metabolism. Ultrastructural analysis indicated that F23 exhibited better membrane integrity than did BR20 under ethanol stress. At low concentrations (<5 vol%), the partition of ethanol into the membrane increased the membrane fluidity, which had little effect on cell growth. However, the toxic effects of medium and high ethanol concentrations (5 to 20 vol%) tended to decrease the membrane fluidity. Under high ethanol stress (>10 vol%), the highly tolerant strain was able to maintain a relatively constant fluidity by increasing the content of unsaturated fatty acid (UFA), whereas less-tolerant strains show a continuous decrease in fluidity and UFA content. OLE1, which was identified as the only gene with a differential single-nucleotide polymorphism (SNP) mutation site related to fatty acid metabolism, was significantly changed in response to ethanol. The role of OLE1 in membrane fluidity was positively validated in its overexpressed transformants. Therefore, OLE1 lowered the rate of decline in membrane fluidity and thus enabled the yeast to better fight the deleterious effects of ethanol. IMPORTANCE Yeasts with superior ethanol tolerance are desirable for winemakers and wine industries. In our previous work, strain F23 was evolved with superior ethanol tolerance and fermentation activity to improve the flavor profiles of Chinese rice wine. Therefore, exploring the genomic variations and ethanol tolerance mechanism of strain F23 could contribute to an understanding of its effect on the flavor characteristics in the resulting Chinese rice wine. The cellular membrane plays a vital role in the ethanol tolerance of yeasts; however, how the membrane is regulated to fight the toxic effect of ethanol remains to be elucidated. This study suggests that the membrane fluidity is variably regulated by OLE1 to offset the disruptive effect of ethanol. Current work will help develop more ethanol-tolerant yeast strains for wine industries and contribute to a deep understanding of its high flavor-producing ability.

scholarly journals Association of Constitutive Hyperphosphorylation of Hsf1p with a Defective Ethanol Stress Response in Saccharomyces cerevisiae Sake Yeast Strains

2011 ◽  
Vol 78 (2) ◽  
pp. 385-392 ◽  
Author(s):  
Chiemi Noguchi ◽  
Daisuke Watanabe ◽  
Yan Zhou ◽  
Takeshi Akao ◽  
Hitoshi Shimoi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Response ◽  
Heat Shock ◽  
Laboratory Strain ◽  
Underlying Mechanism ◽  
Ethanol Stress ◽  
Heat Shock Element ◽  
Content Type ◽  
Yeast Strains ◽  
Acute Ethanol

ABSTRACTModern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p inSaccharomyces cerevisiaesake yeast. The HSE-lacZactivity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. SinceHSF1allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a Δppt1mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entirePPT1gene locus. We confirmed that the expression of laboratory yeast-derived functionalPPT1recovered the HSE-mediated stress response of sake yeast. In addition, deletion ofPPT1in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of thePPT1gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains.

scholarly journals Peculiar H+Homeostasis of Saccharomyces cerevisiae during the Late Stages of Wine Fermentation

2012 ◽  
Vol 78 (17) ◽  
pp. 6302-6308 ◽  
Author(s):  
Tiago Viana ◽  
Maria C. Loureiro-Dias ◽  
Virgílio Loureiro ◽  
Catarina Prista
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intracellular Ph ◽  
Stationary Phases ◽  
Physiological Parameter ◽  
Late Stationary Phase ◽  
Glucose Fermentation ◽  
Content Type ◽  
Wine Strain ◽  
Late Stages ◽  
High Ethanol

ABSTRACTIntracellular pH (pHin) is a tightly regulated physiological parameter, which controls cell performance in all living systems. The purpose of this work was to evaluate if and how H+homeostasis is accomplished by an industrial wine strain ofSaccharomyces cerevisiaewhile fermenting real must under the harsh winery conditions prevalent in the late stages of the fermentation process, in particular low pH and high ethanol concentrations and temperature. Cells grown at 15, 25, and 30°C were harvested in exponential and early and late stationary phases. Intracellular pH remained in the range of 6.0 to 6.4, decreasing significantly only by the end of glucose fermentation, in particular at lower temperatures (pHin5.2 at 15°C), although the cells remained viable and metabolically active. The cell capability of extruding H+via H+-ATPase and of keeping H+out by means of an impermeable membrane were evaluated as potential mechanisms of H+homeostasis. At 30°C, H+efflux was higher in all stages. The most striking observation was that cells in late stationary phase became almost impermeable to H+. Even when these cells were challenged with high ethanol concentrations (up to 20%) added in the assay, their permeability to H+remained very low, being almost undetectable at 15°C. Comparatively, ethanol significantly increased the H+permeability of cells in exponential phase. Understanding the molecular and physiological events underlying yeast H+homeostasis at late stages of fermentations may contribute to the development of more robust strains suitable to efficiently produce a high-quality wine.

scholarly journals Mitochondrial Superoxide Dismutase and Yap1p Act as a Signaling Module Contributing to Ethanol Tolerance of the Yeast Saccharomyces cerevisiae

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Anna N. Zyrina ◽  
Ekaterina A. Smirnova ◽  
Olga V. Markova ◽  
Fedor F. Severin ◽  
Dmitry A. Knorre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hydrogen Peroxide ◽  
Superoxide Dismutase ◽  
Ethanol Tolerance ◽  
Yeast Cells ◽  
Ethanol Stress ◽  
Mitochondrial Enzymes ◽  
Mitochondrial Superoxide ◽  
Mitochondrial Superoxide Dismutase

ABSTRACT There are two superoxide dismutases in the yeast Saccharomyces cerevisiae—cytoplasmic and mitochondrial enzymes. Inactivation of the cytoplasmic enzyme, Sod1p, renders the cells sensitive to a variety of stresses, while inactivation of the mitochondrial isoform, Sod2p, typically has a weaker effect. One exception is ethanol-induced stress. Here we studied the role of Sod2p in ethanol tolerance of yeast. First, we found that repression of SOD2 prevents ethanol-induced relocalization of yeast hydrogen peroxide-sensing transcription factor Yap1p, one of the key stress resistance proteins. In agreement with this, the levels of Trx2p and Gsh1p, proteins encoded by Yap1 target genes, were decreased in the absence of Sod2p. Analysis of the ethanol sensitivities of the cells lacking Sod2p, Yap1p, or both indicated that the two proteins act in the same pathway. Moreover, preconditioning with hydrogen peroxide restored the ethanol resistance of yeast cells with repressed SOD2. Interestingly, we found that mitochondrion-to-nucleus signaling by Rtg proteins antagonizes Yap1p activation. Together, our data suggest that hydrogen peroxide produced by Sod2p activates Yap1p and thus plays a signaling role in ethanol tolerance. IMPORTANCE Baker's yeast harbors multiple systems that ensure tolerance to high concentrations of ethanol. Still, the role of mitochondria under severe ethanol stress in yeast is not completely clear. Our study revealed a signaling function of mitochondria which contributes significantly to the ethanol tolerance of yeast cells. We found that mitochondrial superoxide dismutase Sod2p and cytoplasmic hydrogen peroxide sensor Yap1p act together as a module of the mitochondrion-to-nucleus signaling pathway. We also report cross talk between this pathway and the conventional retrograde signaling cascade activated by dysfunctional mitochondria.

Aeration strategy: a need for very high ethanol performance in Saccharomyces cerevisiae fed-batch process

2004 ◽  
Vol 63 (5) ◽  
pp. 537-542 ◽  
Author(s):  
S. Alfenore ◽  
X. Cameleyre ◽  
L. Benbadis ◽  
C. Bideaux ◽  
J.-L. Uribelarrea ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Batch Process ◽  
Fed Batch ◽  
High Ethanol ◽  
Very High

scholarly journals Improvement of Ethanol Tolerance by Inactive Protoplast Fusion in Saccharomyces cerevisiae

2020 ◽  
Vol 2020 ◽  
pp. 1-10 ◽  
Author(s):  
Yi Xin ◽  
Mei Yang ◽  
Hua Yin ◽  
Jianming Yang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protoplast Fusion ◽  
Ethanol Tolerance ◽  
Assimilation Rate ◽  
Biotechnological Applications ◽  
Capital Costs ◽  
Fermentation Efficiency ◽  
N Assimilation ◽  
Beer Production ◽  
High Ethanol

Saccharomyces cerevisiae is a typical fermentation yeast in beer production. Improving ethanol tolerance of S. cerevisiae will increase fermentation efficiency, thereby reducing capital costs. Here, we found that S. cerevisiae strain L exhibited a higher ethanol tolerance (14%, v/v) than the fermentative strain Q (10%, v/v). In order to enhance the strain Q ethanol tolerance but preserve its fermentation property, protoplast fusion was performed with haploids from strain Q and L. The fusant Q/L-f2 with 14% ethanol tolerance was obtained. Meanwhile, the fermentation properties (flocculability, SO2 production, α-N assimilation rate, GSH production, etc.) of Q/L-f2 were similar to those of strain Q. Therefore, our works established a series of high ethanol-tolerant strains in beer production. Moreover, this demonstration of inactivated protoplast fusion in industrial S. cerevisiae strain opens many doors for yeast-based biotechnological applications.

scholarly journals Examining the Role of Membrane Lipid Composition in Determining the Ethanol Tolerance of Saccharomyces cerevisiae

2014 ◽  
Vol 80 (10) ◽  
pp. 2966-2972 ◽  
Author(s):  
Clark M. Henderson ◽  
David E. Block
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Membrane ◽  
Lipid Bilayers ◽  
Lipid Composition ◽  
Ethanol Tolerance ◽  
Yeast Cells ◽  
Alcohol Tolerance ◽  
Membrane Composition ◽  
Content Type ◽  
Modern Mass

ABSTRACTYeast (Saccharomyces cerevisiae) has an innate ability to withstand high levels of ethanol that would prove lethal to or severely impair the physiology of other organisms. Significant efforts have been undertaken to elucidate the biochemical and biophysical mechanisms of how ethanol interacts with lipid bilayers and cellular membranes. This research has implicated the yeast cellular membrane as the primary target of the toxic effects of ethanol. Analysis of model membrane systems exposed to ethanol has demonstrated ethanol's perturbing effect on lipid bilayers, and altering the lipid composition of these model bilayers can mitigate the effect of ethanol. In addition, cell membrane composition has been correlated with the ethanol tolerance of yeast cells. However, the physical phenomena behind this correlation are likely to be complex. Previous work based on often divergent experimental conditions and time-consuming low-resolution methodologies that limit large-scale analysis of yeast fermentations has fallen short of revealing shared mechanisms of alcohol tolerance inSaccharomyces cerevisiae. Lipidomics, a modern mass spectrometry-based approach to analyze the complex physiological regulation of lipid composition in yeast and other organisms, has helped to uncover potential mechanisms for alcohol tolerance in yeast. Recent experimental work utilizing lipidomics methodologies has provided a more detailed molecular picture of the relationship between lipid composition and ethanol tolerance. While it has become clear that the yeast cell membrane composition affects its ability to tolerate ethanol, the molecular mechanisms of yeast alcohol tolerance remain to be elucidated.

scholarly journals Independent Mechanisms for Acquired Salt Tolerance versus Growth Resumption Induced by Mild Ethanol Pretreatment inSaccharomyces cerevisiae

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Elizabeth A. McDaniel ◽  
Tara N. Stuecker ◽  
Manasa Veluvolu ◽  
Audrey P. Gasch ◽  
Jeffrey A. Lewis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Resistance ◽  
Natural Variation ◽  
Lethal Dose ◽  
Cross Protection ◽  
High Salt ◽  
Ethanol Stress ◽  
Content Type ◽  
Ethanol Pretreatment ◽  
Acquired Stress Resistance

ABSTRACTAll living organisms must recognize and respond to various environmental stresses throughout their lifetime. In natural environments, cells frequently encounter fluctuating concentrations of different stressors that can occur in combination or sequentially. Thus, the ability to anticipate an impending stress is likely ecologically relevant. One possible mechanism for anticipating future stress is acquired stress resistance, where cells preexposed to a mild sublethal dose of stress gain the ability to survive an otherwise lethal dose of stress. We have been leveraging wild strains ofSaccharomyces cerevisiaeto investigate natural variation in the yeast ethanol stress response and its role in acquired stress resistance. Here, we report that a wild vineyard isolate possesses ethanol-induced cross protection against severe concentrations of salt. Because this phenotype correlates with ethanol-dependent induction of theENAgenes, which encode sodium efflux pumps already associated with salt resistance, we hypothesized that variation inENAexpression was responsible for differences in acquired salt tolerance across strains. Surprisingly, we found that theENAgenes were completely dispensable for ethanol-induced survival of high salt concentrations in the wild vineyard strain. Instead, theENAgenes were necessary for the ability to resume growth on high concentrations of salt following a mild ethanol pretreatment. Surprisingly, this growth acclimation phenotype was also shared by the lab yeast strain despite lack ofENAinduction under this condition. This study underscores that cross protection can affect both viability and growth through distinct mechanisms, both of which likely confer fitness effects that are ecologically relevant.IMPORTANCEMicrobes in nature frequently experience “boom or bust” cycles of environmental stress. Thus, microbes that can anticipate the onset of stress would have an advantage. One way that microbes anticipate future stress is through acquired stress resistance, where cells exposed to a mild dose of one stress gain the ability to survive an otherwise lethal dose of a subsequent stress. In the budding yeastSaccharomyces cerevisiae, certain stressors can cross protect against high salt concentrations, though the mechanisms governing this acquired stress resistance are not well understood. In this study, we took advantage of wild yeast strains to understand the mechanism underlying ethanol-induced cross protection against high salt concentrations. We found that mild ethanol stress allows cells to resume growth on high salt, which involves a novel role for a well-studied salt transporter. Overall, this discovery highlights how leveraging natural variation can provide new insights into well-studied stress defense mechanisms.

scholarly journals Acquired Resistance to Severe Ethanol Stress on Protein Quality Control in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Masashi Yoshida ◽  
Sae Kato ◽  
Shizu Fukuda ◽  
Shingo Izawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Resistance ◽  
Ethanol Tolerance ◽  
Protein Quality ◽  
Yeast Cells ◽  
Ethanol Stress ◽  
Mild Stress ◽  
Insoluble Protein ◽  
Protein Levels ◽  
Brewing Process

Acute severe ethanol stress (10% v/v) damages proteins and causes the intracellular accumulation of insoluble proteins in Saccharomyces cerevisiae. On the other hand, a pretreatment with mild stress increases tolerance to subsequent severe stress, which is called acquired stress resistance. It currently remains unclear whether the accumulation of insoluble proteins under severe ethanol stress may be mitigated by increasing protein quality control (PQC) activity in cells pretreated with mild stress. In the present study, we examined the induction of resistance to severe ethanol stress in PQC, and confirmed that a pretreatment with 6% (v/v) ethanol or mild thermal stress at 37°C significantly reduced insoluble protein levels and the aggregation of Lsg1, which is prone to denaturation and aggregation by stress, in yeast cells under 10% (v/v) ethanol stress. The induction of this stress resistance required the new synthesis of proteins; the expression of proteins comprising the bi-chaperone system (Hsp104, Ssa3, and Fes1), Sis1, and Hsp42 was up-regulated during the pretreatment and maintained under subsequent severe ethanol stress. Since the pretreated cells of deficient mutants in the bi-chaperone system (fes1Δhsp104Δ and ssa2Δssa3Δssa4Δ) failed to sufficiently reduce insoluble protein levels and Lsg1 aggregation, the enhanced activity of the bi-chaperone system appears to be important for the induction of adequate stress resistance. In contrast, the importance of proteasomes and aggregases (Btn2 and Hsp42) in the induction of stress resistance has not been confirmed. These results provide further insights into the PQC activity of yeast cells under severe ethanol stress, including the brewing process. IMPORTANCE Although the budding yeast S. cerevisiae, which is used in the production of alcoholic beverages and bioethanol, is highly tolerant of ethanol, high concentrations of ethanol are also stressful to the yeast and cause various adverse effects, including protein denaturation. A pretreatment with mild stress improves the ethanol tolerance of yeast cells; however, it currently remains unclear whether it increases PQC activity and reduces the levels of denatured proteins. In the present study, we found that a pre-treatment with mild ethanol up-regulated the expression of proteins involved in PQC and mitigated the accumulation of insoluble proteins, even under severe ethanol stress. These results provide novel insights into ethanol tolerance and the adaptive capacity of yeast. They may also contribute to research on the physiology of yeast cells during the brewing process, in which the concentration of ethanol gradually increases.

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