Ethanol Stress Research Articles

Extension of Chronological Life in <i>Saccharomyces cerevisiae</i> under Ethanol Stress by Thermally Processed Rice <i>Koji</i> Extracts

The effect of thermally processed rice koji extracts on survival of yeast Saccharomyces cerevisiae was examined in comparison with non-heated koji extract. In chronological life span (CLS) tests on high-sugar fermentation, the survivals of the yeast cells grown with heated koji extracts were higher than non-heated koji extract. Heat treatment of the extracts by autoclaving led to a loss in most of amino acids due to the Maillard reaction, although histidine contents slightly increased. In glucose-arginine mixtures, arginine was partly converted to histidine by autoclaving and the addition of histidine prolonged the CLS of yeast cells. The yeast cells grown with the non-heated extracts were more resistant to oxidative stress whereas the antioxidant activities were lower than those of the heated extracts. The yeast cells grown with the heated extracts were more tolerant to ethanol and had a higher reduction capacity in the late stationary phase when the cells were incubated in the presence of ethanol. Maillard reaction products elevated the levels of reactive oxygen species to yeast cells grown under ethanol stress in the late stationary phase. These results suggest that thermally processed koji extracts can act as a protectant against ethanol stress during the late stationary phase of yeast growth and extend the CLS due to the increase of histidine contents by autoclaving.

Concomitant stress potentiates the preference for, and consumption of, ethanol induced by chronic pre-exposure to ethanol.

Ethanol abuse is linked to several acute and chronic injuries that can lead to health problems. Ethanol addiction is one of the most severe diseases linked to the abuse of this drug. Symptoms of ethanol addiction include compulsive substance intake and withdrawal syndrome. Stress exposure has an important role in addictive behavior for many drugs of abuse (including ethanol), but the consequences of stress and ethanol in the organism when these factors are concomitant results in a complex interaction. We investigated the effects of concomitant, chronic administration of ethanol and stress exposure on the withdrawal and consumption of, as well as the preference for, ethanol in mice. Male Swiss mice (30–35 g, 8-10 per group) were exposed to an ethanol liquid diet as the only source of food for 15 days. In the final 5 days, they were exposed to forced swimming stress. Twelve hours after removal of the ethanol liquid diet, animals were evaluated for ethanol withdrawal by measuring anxiety-related behaviors and locomotor activity. Twenty-four hours after evaluation of ethanol withdrawal, they were evaluated for voluntary consumption of ethanol in a “three-bottle choice” paradigm. Mice exposed to chronic consumption of ethanol had decreased locomotor activity during withdrawal. Contrary to our expectations, a concomitant forced swimming stress did not aggravate ethanol withdrawal. Nevertheless, simultaneous ethanol administration and stress exposure increased voluntary consumption of ethanol, mainly solutions containing high concentrations of ethanol. These results showed that stressful situations during ethanol intake may aggravate specific addiction-related behaviors.

Gastro protective and H+, K+-ATPase/H. pylori inhibitory properties of pectic polysaccharides from potato

Polysaccharide is one among the important classes of biological polymers that is reported to exhibit disease preventive properties. The present study describes the isolation of galactans and confirmation of the same by sugar analysis and determination of anti-ulcer effect of Potato Galactan Polysaccharide (PGP). Data indicated that PGP possessed sugar composition of rhamnose (2%), arabinose (3%), mannose (3%), galactose (94%) and uronic acid (17%) confirming that PGP thus isolated is a galactan. PGP exhibited potent H+, K+-ATPase inhibitory activity (IC50 420μg/mL) in vitro as opposed to lansoprazole, a known antiulcer drug with IC50 19.3μg/mL. The antiulcer potency of PGP was evaluated in ethanol stress induced gastric ulcer model in vivo. About 84% reduction in ulcer index; enhanced mucosal recovery, normalization of H+, K+-ATPase, antioxidant and antioxidant enzymes substantiated the antiulcer potentials of PGP. Mucosal recovery could be attributed to cytoprotective and DNA protective ability of PGP that can help in mucosal layer regeneration. Further, PGP was also effective in inhibiting Helicobacter pylori as per growth inhibition assay followed by scanning electron microscopic studies suggesting that PGP is effective in curbing the growth of H. pylori, which is responsible for ∼70% of gastric ulcer/cancer incidences.

Interaction between handling induced stress and anxiolytic effects of ethanol in zebrafish: A behavioral and neurochemical analysis

Stress is often considered an important factor in the development of alcohol addiction. In rodents, various types of stressors have been shown to potentiate the effects of alcohol on behavioral responses, and to increase consumption of this substance. However, few have investigated the interaction between stress and alcohol in zebrafish. In the current study we present a repeated handling stress paradigm we developed for zebrafish, and examine whether stress alters alcohol induced behavioral and neurochemical responses. Our results show that repeated handling of zebrafish conducted for 2 consecutive days is sufficient to increase anxiety-like behavioral responses quantified 24h post-stressor. Repeatedly handled zebrafish also exhibited a reduction in the levels of serotonin’s metabolite, 5-hydroxyindole acetic acid (quantified by high precision liquid chromatography) compared to unhandled controls. A 60-min acute exposure to 1% ethanol was found to significantly increase locomotor activity and decrease anxiety-like behavioral responses in stressed zebrafish but not in controls. Furthermore, unhandled control zebrafish exhibited a significant increase in whole-brain dopamine levels following exposure to ethanol but the increase was not observed in repeatedly handled fish. Our findings suggest that ethanol induced locomotor activity and anxiolysis is potentiated by handling stress and may be partially mediated by changes in dopaminergic and serotonergic activity. Overall, we demonstrate the validity of our repeated handling stressor paradigm for zebrafish, which can be used to investigate the interaction between stress and ethanol.

Glutamatergic transmission in the central nucleus of the amygdala is selectively altered in Marchigian Sardinian alcohol-preferring rats: Alcohol and CRF effects

The CRF system of the central nucleus of the amygdala (CeA) is important for the processing of anxiety, stress, and effects of acute and chronic ethanol. We previously reported that ethanol decreases evoked glutamate transmission in the CeA of Sprague Dawley rats and that ethanol dependence alters glutamate release in the CeA. Here, we examined the effects of ethanol, CRF and a CRF1 receptor antagonist on spontaneous and evoked glutamatergic transmission in CeA neurons from Wistar and Marchigian Sardinian Preferring (msP) rats, a rodent line genetically selected for excessive alcohol drinking and characterized by heightened activity of the CRF1 system. Basal spontaneous and evoked glutamate transmission in CeA neurons from msP rats was increased compared to Wistar rats. Ethanol had divergent effects, either increasing or decreasing spontaneous glutamate release in the CeA of Wistar rats. This bidirectional effect was retained in msP rats, but the magnitude of the ethanol-induced increase in glutamate release was significantly smaller. The inhibitory effect of ethanol on evoked glutamatergic transmission was similar in both strains. CRF also either increased or decreased spontaneous glutamate release in CeA neurons of Wistar rats, however, in msP rats CRF only increased glutamate release. The inhibitory effect of CRF on evoked glutamatergic transmission was also lost in neurons from msP rats. A CRF1 antagonist produced only minor effects on spontaneous glutamate transmission, which were consistent across strains, and no effects on evoked glutamate transmission. These results demonstrate that the genetically altered CRF system of msP rats results in alterations in spontaneous and stimulated glutamate signaling in the CeA that may contribute to both the anxiety and drinking behavioral phenotypes.

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C18:1) but lower levels of hexadecanoic (C16:0) and palmitelaidic (C16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.

Evaluation of the safety, gastroprotective activity and mechanism of action of standardised leaves infusion extract of Copaifera malmei Harms

Ethnopharmacological relevanceCopaifera malmei Harms (Fabaceae) is a plant that occurs in the central region of Brazil, where the plant's leaves infusion is popularly used to treat gastric ulcer and inflammatory diseases. This study was aimed to investigate the gastroprotective activity and mode of action of the plants' leaves infusion in order to establish the scientific basis for such usage, and to assess its potential as a source of an anti-ulcer agent. Materials and methodsLeaves infusion extract of the plant (SIECm) was prepared, freeze dried and lyophilised. Its qualitative and quantitative phytochemical constituents were investigated using TLC and HPLC techniques. The safety profile was evaluated on CHO-k1 epithelial cells viability using the Alamar blue assay, and by acute toxicity test in mice. The gastroprotection and anti-ulcer efficacy of the SIECm (25, 100 and 400mg/kg, p.o.) were tested using acute (acidified ethanol, piroxicam and water restrain stress), and chronic (acetic acid) experimental ulcer models. The plausible mode of action of the SIECm was assessed using gastric secretion, gastric barrier mucus, nitric oxide, and its antioxidant (myeloperoxidase and catalase) effects in mice and rats. The histopathological analyses of the ulcerated tissues as well as the extract’s activity on Helicobacter pylori were also investigated. ResultsPhytochemical tests indicated the presence of mainly phytosterols, phenolics and flavonoids. The SIECm exhibited no cytotoxic effects on the CHO-k1 cells, and no oral acute toxicity in mice. It prevented against the acute induced ulcerations by enhancing gastroprotection through gastric mucus production, NO modulation, antioxidant, reduced gastric secretion and enhanced chronic ulcers healing process, as shown by reduction/prevention of epithelial and vascular damage, in addition to reduction in leucocyte infiltration. The SIECm however did not exhibit activity against H. pylori. ConclusionThe SIECm is safe, contain useful phytochemicals and exhibited significant gastroprotective/anti-ulcer effects. The results justify its folkloric usage, and provided scientific evidence of its potential as a source of new phytodrug to treat gastric ulcers.

Quorum-Sensing Kinetics in Saccharomyces cerevisiae: A Symphony of ARO Genes and Aromatic Alcohols.

The kinetics of quorum sensing in Saccharomyces cerevisiae were studied using a mini-fermentation platform. The quorum-sensing molecules were monitored using our previous HPLC approach that is here supported by quantitative real-time PCR analysis of the quorum-sensing genes. We thus initially confirm correlations between peak production rates of the monitored quorum-sensing molecules 2-phenylethanol, tryptophol, and tyrosol and peak expression of the genes responsible for their synthesis: ARO8, ARO9, and ARO10. This confirms the accuracy of our previously implemented kinetic model, thus favoring its use in further studies in this field. We also show that the quorum-sensing kinetics are precisely dependent on the population growth phase and that tyrosol production is also regulated by cell concentration, which has not been reported previously. Additionally, we show that during wine fermentation, ethanol stress reduces the production of 2-phenylethanol, tryptophol, and tyrosol, which opens new challenges in the control of wine fermentation.

Phenotypic convergence in bacterial adaptive evolution to ethanol stress.

BackgroundBacterial cells have a remarkable ability to adapt to environmental changes, a phenomenon known as adaptive evolution. During adaptive evolution, phenotype and genotype dynamically changes; however, the relationship between these changes and associated constraints is yet to be fully elucidated.ResultsIn this study, we analyzed phenotypic and genotypic changes in Escherichia coli cells during adaptive evolution to ethanol stress. Phenotypic changes were quantified by transcriptome and metabolome analyses and were similar among independently evolved ethanol tolerant populations, which indicate the existence of evolutionary constraints in the dynamics of adaptive evolution. Furthermore, the contribution of identified mutations in one of the tolerant strains was evaluated using site-directed mutagenesis. The result demonstrated that the introduction of all identified mutations cannot fully explain the observed tolerance in the tolerant strain.ConclusionsThe results demonstrated that the convergence of adaptive phenotypic changes and diverse genotypic changes, which suggested that the phenotype–genotype mapping is complex. The integration of transcriptome and genome data provides a quantitative understanding of evolutionary constraints.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0454-6) contains supplementary material, which is available to authorized users.

Metabolomic approach for improving ethanol stress tolerance in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is widely used for brewing and ethanol production. The ethanol sensitivity of yeast cells is still a serious problem during ethanol fermentation, and a variety of genetic approaches (e.g., random mutant screening under selective pressure of ethanol) have been developed to improve ethanol tolerance. In this study, we developed a strategy for improving ethanol tolerance of yeast cells based on metabolomics as a high-resolution quantitative phenotypic analysis. We performed gas chromatography-mass spectrometry analysis to identify and quantify 36 compounds on 14 mutant strains including knockout strains for transcription factor and metabolic enzyme genes. A strong relation between metabolome of these mutants and their ethanol tolerance was observed. Data mining of the metabolomic analysis showed that several compounds (such as trehalose, valine, inositol and proline) contributed highly to ethanol tolerance. Our approach successfully detected well-known ethanol stress related metabolites such as trehalose and proline thus, to further prove our strategy, we focused on valine and inositol as the most promising target metabolites in our study. Our results show that simultaneous deletion of LEU4 and LEU9 (leading to accumulation of valine) or INM1 and INM2 (leading to reduction of inositol) significantly enhanced ethanol tolerance. This study shows the potential of the metabolomic approach to identify target genes for strain improvement of S. cerevisiae with higher ethanol tolerance.

Changes and roles of membrane compositions in the adaptation of Saccharomyces cerevisiae to ethanol.

Bioethanol fermentation by Saccharomyces cerevisiae is often stressed by the accumulation of ethanol. Cell membrane is the first assaulting target of ethanol. Ethanol-adapted S. cerevisiae strains provide opportunity to shed light on membrane functions in the ethanol tolerance. This study aimed at clarifying the roles of cell membrane in the ethanol tolerance of S. cerevisiae through comparing membrane components between S. cerevisiae parental strain and ethanol-adapted strains. A directed evolutionary engineering was performed to obtain the ethanol-adapted S. cerevisiae strains. The parental, ethanol-adapted M5 and M10 strains were selected to be compared the percentage of viable cells after exposing to ethanol stress and cell membrane compositions (i.e., ergosterol, trehalose, and fatty acids). Compared with the parental strain, M5 or M10 strain had higher survival rate in the presence of 10% v/v ethanol. Compared with that in the parental strain, contents of trehalose, ergosterol, and fatty acids increased about 15.7, 12.1, and 29.3%, respectively, in M5 strain, and about 47.5, 107.8, and 61.5%, respectively, in M10 strain. Moreover, expression differences of genes involved in fatty acids metabolisms among the parental, M5 and M10 strains were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and results demonstrated that M5 or M10 strain had higher expression of ACC1 and OLE1 than the parental strain. These results indicated that although being exposed to step-wise increased ethanol, S. cerevisiae cells might remodel membrane components or structure to adapt to the ethanol stress.

Assessment of Gastroprotective Potential of <i>Delonix regia</i> (Boj Ex Hook) Raf against Ethanol and Cold Restrain Stress-Induced Ulcer in Rats

Purpose: To assess the gastroprotective potential of the stem bark ethanol extract of Delonix regia (EDR) on ethanol and cold restrain stress-induced ulcer in experimental rats.Methods: EDR (100, 200 and 400 mg/kg doses, orally) was evaluated on ethanol and cold restrain stress-induced ulcer in experimental rats. In ethanol induced ulcer model, ulcer index, percent protection, reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO), cytokines and nitric oxide (NO) levels in stomach tissue were evaluated. In the cold restrain stress model, ulcer index, percent protection, and GSH levels were evaluated. 2,2-Diphenyl-1-picryl hydrazyl (DPPH) radical scavenging assay of EDR was also performed.Results: EDR caused a significant (p < 0.05-0.001) decreased ulcer index in ethanol (61.33-76.00%) and cold restrain stress (47.34-84.28 %) models. The EDR caused a significant (p < 0.05 - 0.001) increase in SOD (0.20 - 0.27 U/mg protein, CAT (200 - 270 μmole H2O2/mg of protein/minute), GSH (1.63 - 1.17 μg/mg protein) and reduction in nitric oxide (NO) level, pro-inflammatory cytokine (TNF-α and IL-6) levels and inhibition in neutrophil accumulation (p < 0.001) in ethanol-induced model. EDR exhibited significant antioxidant activity with IC50 value of 45.23 ± 3.23 μg/ml.Conclusion: The results suggest that EDR has gastroprotective effect in the two ulcer models and this may be due to its antioxidant effect.Keywords: Delonix regia, Fabaceae, Ethanol-induced ulcer, Cold restrain stress-induced ulcer, Gastroprotective, Pro-inflammatory cytokines

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

Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with 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 the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. 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.

The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA

One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136–S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136–S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress.

Effect of Ethanol, Sulfur Dioxide and Glucose on the Growth of Wine Spoilage Yeasts Using Response Surface Methodology.

Response surface methodology (RSM) was used to study the effect of three factors, sulfur dioxide, ethanol and glucose, on the growth of wine spoilage yeast species, Zygosaccharomyces bailii, Schizosaccharomyces pombe, Saccharomycodes ludwigii and Saccharomyces cerevisiae. Seventeen central composite rotatable design (CCRD) trials were designed for each test yeast using realistic concentrations of the factors (variables) in premium red wine. Polynomial regression equations were fitted to experimental data points, and the growth inhibitory conditions of these three variables were determined. The overall results showed Sa. ludwigii as the most resistant species growing under high ethanol/free sulfur dioxide concentrations, i.e., 15% (v/v)/20 mg L-1, 14% (v/v)/32 mg L-1 and 12.5% (v/v)/40 mg L-1, whereas other yeasts did not survive under the same levels of ethanol/free sulfur dioxide concentrations. The inhibitory effect of ethanol was primarily observed during longer incubation periods, compared with sulfur dioxide, which showed an immediate effect. In some CCRD trials, Sa. ludwigii and S. cerevisiae showed growth recovery after a short death period under the exposure of 20–32 mg L-1 sulfur dioxide in the presence of 11% (v/v) or more ethanol. However, Sc. pombe and Z. bailii did not show such growth recovery under similar conditions. Up to 10 g L-1 of glucose did not prevent cell death under the sulfur dioxide or ethanol stress. This observation demonstrates that the sugar levels commonly used in wine to sweeten the mouthfeel do not increase wine susceptibility to spoilage yeasts, contrary to the anecdotal evidence.

Transcriptomic and proteomic analysis of Oenococcus oeni PSU-1 response to ethanol shock

The correct development of malolactic fermentation depends on the capacity of Oenococcus oeni to survive under harsh wine conditions. The presence of ethanol is one of the most stressful factors affecting O. oeni performance. In this study, the effect of ethanol addition (12% vol/vol) on O. oeni PSU-1 has been evaluated using a transcriptomic and proteomic approach. Transcriptomic analysis revealed that the main functional categories of the genes affected by ethanol were metabolite transport and cell wall and membrane biogenesis. It was also observed that some genes were over-expressed in response to ethanol stress (for example, the heat shock protein Hsp20 and a dipeptidase). Proteomic analysis showed that several proteins are affected by the presence of ethanol. Functions related to protein synthesis and stability are the main target of ethanol damage. In some cases the decrease in protein concentration could be due to the relocation of cytosolic proteins in the membrane, as a protective mechanism. The omic approach used to study the response of O. oeni to ethanol highlights the importance of the cell membrane in the global stress response and opens the door to future studies on this issue.

Identification of a transporter Slr0982 involved in ethanol tolerance in cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria have been engineered to produce ethanol through recent synthetic biology efforts. However, one major challenge to the cyanobacterial systems for high-efficiency ethanol production is their low tolerance to the ethanol toxicity. With a major goal to identify novel transporters involved in ethanol tolerance, we constructed gene knockout mutants for 58 transporter-encoding genes of Synechocystis sp. PCC 6803 and screened their tolerance change under ethanol stress. The efforts allowed discovery of a mutant of slr0982 gene encoding an ATP-binding cassette transporter which grew poorly in BG11 medium supplemented with 1.5% (v/v) ethanol when compared with the wild type, and the growth loss could be recovered by complementing slr0982 in the Δslr0982 mutant, suggesting that slr0982 is involved in ethanol tolerance in Synechocystis. To decipher the tolerance mechanism involved, a comparative metabolomic and network-based analysis of the wild type and the ethanol-sensitive Δslr0982 mutant was performed. The analysis allowed the identification of four metabolic modules related to slr0982 deletion in the Δslr0982 mutant, among which metabolites like sucrose and L-pyroglutamic acid which might be involved in ethanol tolerance, were found important for slr0982 deletion in the Δslr0982 mutant. This study reports on the first transporter related to ethanol tolerance in Synechocystis, which could be a useful target for further tolerance engineering. In addition, metabolomic and network analysis provides important findings for better understanding of the tolerance mechanism to ethanol stress in Synechocystis.

Complementing DIGE proteomics and DNA subarray analyses to shed light on Oenococcus oeni adaptation to ethanol in wine-simulated conditions

Direct addition of Oenococcus oeni starters into wine can cause viability problems. In the present study, the influence of ethanol in wine-simulated conditions on O. oeni has been evaluated by complementing microarray techniques and DIGE proteomics. Two different ethanol concentrations were studied. In 12% ethanol, pyrimidine anabolism was stimulated, but in 8% ethanol some energy-consuming biosynthetic pathways were limited. The most significant result was the stress response induced by alcohol that concerned both the cell-envelope and specific stress proteins. Interestingly, 8% and 12% ethanol triggered different stress responses: in mild ethanol stress (8%), chaperones with prevalent refolding activity (like HSP20) were over-expressed, whereas at higher alcohol concentration (12%), together with HSP20 and the refolding DNAJ/K, also chaperones having proteolytic activity (like ClpP) were induced. Furthermore the stress response repressor HrcA was downregulated only at 12% ethanol, suggesting that it controls stress pathways, which are different from those active at 8% alcohol. This result confirms that the HrcA system is operative in O. oeni where the CtrS system is prevalent. Biological significanceThe use of malolactic starter cultures has become widespread to control the MLF process and to prevent off-flavors. There is significant interest in understanding the molecular mechanisms that O. oeni uses to adapt to harsh wine conditions. The overall results highlight that the alcohol-induced stress response involves not only biosynthesis of stress proteins but also envelope-linked mechanisms. From a practical point of view this research underlines the importance of starters acclimation to induce responses that would allow better adaptation to the wine. As a consequence, a well adapted starter can complete malolactic fermentation and improve the final wine quality.

Direct ethanol production from starch using a natural isolate, Scheffersomyces shehatae: Toward consolidated bioprocessing.

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a one-step process, is a promising strategy for cost-effective ethanol production from starchy biomass. To gain insights into starch-based ethanol production using CBP, an extensive screening was undertaken to identify naturally occurring yeasts that produce ethanol without the addition of any amylases. Three yeast strains were capable of producing a significant amount of ethanol. Quantitative assays revealed that Scheffersomyces shehatae JCM 18690 was the strain showing the highest ethanol production ability. This strain was able to utilize starch directly, and the ethanol concentration reached 9.21 g/L. We attribute the ethanol-producing ability of this strain to the high levels of glucoamylase activity, fermentation potential and ethanol stress tolerance. This study strongly suggests the possibility of starch-based ethanol production by consolidated bioprocessing using natural yeasts such as S. shehatae JCM 18690.

Induction of autophagy by zinc during acute ethanol intoxication in mice

Recently, autophagy has emerged as an important player in the defense against ethanol toxicity. The potential role of zinc in the modulation of autophagy during ethanol stress in humans and animal models has not been examined previously. In this study, the effect of zinc deficiency, adequacy and excess on hepatic autophagic flux, liver injury, lipids accumulation and metallothionein expression was examined in C57BL/6 mice acutely exposed to ethanol. Mice were fed diets containing three different levels of zinc: low, adequate and excess for 30 days. After this, mice were given ethanol or water at a total accumulative dosage of 4.5‐g/kg body weight. Mice were sacrificed at 24h after the first gavage. Hepatic autophagic flux was estimated by measuring LC3II/LC3I ratio and Beclin1, p62/ SQSTM1 and Lamp2 levels. The dietary treatments did not affect food intake and body weight. Low zinc intake caused inhibition of autophagic flux in mice exposed or not to ethanol. In addition, zinc deficient mice exhibited increased liver injury and accumulation of lipids in liver. Conversely, high zinc intake was associated with increased autophagic flux and decreased accumulation of lipids in liver in mice exposed to ethanol. Lastly, ethanol consumption increased the expression of metallothionein mRNA in liver. The results of this study suggest that adequate zinc intake is critical to maintain autophagy. The protective effects of zinc against ethanol toxicity could be mediated by autophagy.Supported by NIH 1R03AA022451‐01