The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae

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.

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The ethanol stress response and ethanol tolerance ofSaccharomyces cerevisiae

Journal of Applied Microbiology ◽

10.1111/j.1365-2672.2009.04657.x ◽

2010 ◽

Cited By ~ 49

Author(s):

D. Stanley ◽

A. Bandara ◽

S. Fraser ◽

P.J. Chambers ◽

G.A. Stanley

Keyword(s):

Stress Response ◽

Ethanol Tolerance ◽

Ethanol Stress

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

Applied and Environmental Microbiology ◽

10.1128/aem.02759-16 ◽

2016 ◽

Vol 83 (3) ◽

Cited By ~ 6

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.

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

Eukaryotic Cell ◽

10.1128/ec.00053-15 ◽

2015 ◽

Vol 14 (9) ◽

pp. 884-897 ◽

Cited By ~ 13

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01620-19 ◽

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02353-20 ◽

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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Oenological Potential of Autochthonous Saccharomyces cerevisiae Yeast Strains from the Greek Varieties of Agiorgitiko and Moschofilero

Beverages ◽

10.3390/beverages7020027 ◽

2021 ◽

Vol 7 (2) ◽

pp. 27

Author(s):

Dimitrios Kontogiannatos ◽

Vicky Troianou ◽

Maria Dimopoulou ◽

Polydefkis Hatzopoulos ◽

Yorgos Kotseridis

Keyword(s):

Saccharomyces Cerevisiae ◽

Alcoholic Fermentation ◽

Ethanol Tolerance ◽

Random Amplified Polymorphic Dna ◽

Yeast Strains ◽

Rapd Pcr ◽

Autochthonous Yeast ◽

Polymerase Chain ◽

Grape Varieties ◽

H2s Production

Nemea and Mantinia are famous wine regions in Greece known for two indigenous grape varieties, Agiorgitiko and Moschofilero, which produce high quality PDO wines. In the present study, indigenous Saccharomyces cerevisiae yeast strains were isolated and identified from spontaneous alcoholic fermentation of Agiorgitiko and Moschofilero musts in order to evaluate their oenological potential. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) recovered the presence of five distinct profiles from a total of 430 yeast isolates. The five obtained strains were evaluated at microvinifications trials and tested for basic oenological and biochemical parameters including sulphur dioxide and ethanol tolerance as well as H2S production in sterile grape must. The selected autochthonous yeast strains named, Soi2 (Agiorgitiko wine) and L2M (Moschofilero wine), were evaluated also in industrial (4000L) fermentations to assess their sensorial and oenological characteristics. The volatile compounds of the produced wines were determined by GC-FID. Our results demonstrated the feasibility of using Soi2 and L2M strains in industrial fermentations for Agiorgitiko and Moschofilero grape musts, respectively.

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The stress response to loss of signal recognition particle function in Saccharomyces cerevisiae.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)43829-x ◽

1994 ◽

Vol 269 (48) ◽

pp. 30412-30418

Author(s):

C E Arnold ◽

K D Wittrup

Keyword(s):

Saccharomyces Cerevisiae ◽

Stress Response ◽

Signal Recognition Particle ◽

Signal Recognition

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VTC4 Polyphosphate Polymerase Knockout Increases Stress Resistance of Saccharomyces cerevisiae Cells

Biology ◽

10.3390/biology10060487 ◽

2021 ◽

Vol 10 (6) ◽

pp. 487

Author(s):

Alexander Tomashevsky ◽

Ekaterina Kulakovskaya ◽

Ludmila Trilisenko ◽

Ivan V. Kulakovskiy ◽

Tatiana Kulakovskaya ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Heavy Metal ◽

Stress Response ◽

Stress Resistance ◽

Alkaline Stress ◽

Inorganic Polyphosphate ◽

Divalent Metal Transporter ◽

Metal Transporter ◽

Elevated Expression ◽

Chaperone Complex

Inorganic polyphosphate (polyP) is an important factor of alkaline, heavy metal, and oxidative stress resistance in microbial cells. In yeast, polyP is synthesized by Vtc4, a subunit of the vacuole transporter chaperone complex. Here, we report reduced but reliably detectable amounts of acid-soluble and acid-insoluble polyPs in the Δvtc4 strain of Saccharomyces cerevisiae, reaching 10% and 20% of the respective levels of the wild-type strain. The Δvtc4 strain has decreased resistance to alkaline stress but, unexpectedly, increased resistance to oxidation and heavy metal excess. We suggest that increased resistance is achieved through elevated expression of DDR2, which is implicated in stress response, and reduced expression of PHO84 encoding a phosphate and divalent metal transporter. The decreased Mg2+-dependent phosphate accumulation in Δvtc4 cells is consistent with reduced expression of PHO84. We discuss a possible role that polyP level plays in cellular signaling of stress response mobilization in yeast.

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