Ethanol Stress Research Articles

Hsp from Lactobacillus plantarum Expression in Lactococcus lactis MG1363

Small heat shock proteins are protective proteins produced by organisms under thermal stress. They are widely present in living organisms. Here, Hsp18, Hsp18.55 and Hsp19.5 genes were cloned from Lactobacillus plantarum and heterologous expressed in Lactococcus lactis, and their potential functions under ethanol stress were investigated. The results showed that the recombinant strain over expressing Hsp19.5 gene had stronger stress resistance, which provided a basis for further study of the survival ability of other microorganisms under ethanol stress.

Predicting stress response and improved protein overproduction in Bacillus subtilis

Bacillus subtilis is a well-characterized microorganism and a model for the study of Gram-positive bacteria. The bacterium can produce proteins at high densities and yields, which has made it valuable for industrial bioproduction. Like other cell factories, metabolic modeling of B. subtilis has discovered ways to optimize its metabolism toward various applications. The first genome-scale metabolic model (M-model) of B. subtilis was published more than a decade ago and has been applied extensively to understand metabolism, to predict growth phenotypes, and served as a template to reconstruct models for other Gram-positive bacteria. However, M-models are ill-suited to simulate the production and secretion of proteins as well as their proteomic response to stress. Thus, a new generation of metabolic models, known as metabolism and gene expression models (ME-models), has been initiated. Here, we describe the reconstruction and validation of a ME model of B. subtilis, iJT964-ME. This model achieved higher performance scores on the prediction of gene essentiality as compared to the M-model. We successfully validated the model by integrating physiological and omics data associated with gene expression responses to ethanol and salt stress. The model further identified the mechanism by which tryptophan synthesis is upregulated under ethanol stress. Further, we employed iJT964-ME to predict amylase production rates under two different growth conditions. We analyzed these flux distributions and identified key metabolic pathways that permitted the increase in amylase production. Models like iJT964-ME enable the study of proteomic response to stress and the illustrate the potential for optimizing protein production in bacteria.

Comparative Genomics Analysis Provides New Insights into High Ethanol Tolerance of Lactiplantibacillus pentosus LTJ12, a Novel Strain Isolated from Chinese Baijiu.

Lactic acid bacteria have received a significant amount of attention due to their probiotic characteristics. The species Lactiplantibacillus plantarum and Lactiplantibacillus pentosus are genotypically closely related, and their phenotypes are so similar that they are easily confused and mistaken. In the previous study, an ethanol-resistant strain, LTJ12, isolated from the fermented grains of soy sauce aroma type baijiu in North China, was originally identified as L. plantarum through a 16S rRNA sequence analysis. Here, the genome of strain LTJ12 was further sequenced using PacBio and Illumina sequencing technology to obtain a better understanding of the metabolic pathway underlying its resistance to ethanol stress. The results showed that the genome of strain LTJ12 was composed of one circular chromosome and three circular plasmids. The genome size is 3,512,307 bp with a GC content of 46.37%, and the number of predicted coding genes is 3248. Moreover, by comparing the coding genes with the GO (Gene Ontology), COG (Cluster of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases, the functional annotation of the genome and an assessment of the metabolic pathways were performed, with the results showing that strain LTJ12 has multiple genes that may be related to alcohol metabolism and probiotic-related genes. Antibiotic resistance gene analysis showed that there were few potential safety hazards. Further, after conducting the comparative genomics analysis, it was found that strain LTJ12 is L. pentosus but not L. plantarum, but it has more functional genes than other L. pentosus strains that are mainly related to carbohydrate transport and metabolism, transcription, replication, recombination and repair, signal transduction mechanisms, defense mechanisms and cell wall/membrane/envelope biogenesis. These unique functional genes, such as gene 2754 (encodes alcohol dehydrogenase), gene 3093 (encodes gamma-D-glutamyl-meso-diaminopimelate peptidase) and some others may enhance the ethanol tolerance and alcohol metabolism of the strain. Taken together, L. pentosus LTJ12 might be a potentially safe probiotic with a high ethanol tolerance and alcohol metabolism. The findings of this study will also shed light on the accurate identification and rational application of the Lactiplantibacillus species.

Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis.

Zymomonas mobilis is a promising microorganism for industrial bioethanol production. However, ethanol produced during fermentation is toxic to Z. mobilis and affects its growth and bioethanol production. Although several reports demonstrated that the RNA-binding protein Hfq in Z. mobilis contributes to the tolerance against multiple lignocellulosic hydrolysate inhibitors, the role of Hfq on ethanol tolerance has not been investigated. In this study, hfq in Z. mobilis was either deleted or overexpressed and their effects on cell growth and ethanol tolerance were examined. Our results demonstrated that hfq overexpression improved ethanol tolerance of Z. mobilis, which is probably due to energy saving by downregulating flagellar biosynthesis and heat stress response proteins, as well as reducing the reactive oxygen species induced by ethanol stress via upregulating the sulfate assimilation and cysteine biosynthesis. To explore proteins potentially interacted with Hfq, the TEV protease mediated Yeast Endoplasmic Reticulum Sequestration Screening system (YESS) was established in Z. mobilis. YESS results suggested that Hfq may modulate the cytoplasmic heat shock response by interacting with the heat shock proteins DnaK and DnaJ to deal with the ethanol inhibition. This study thus not only revealed the underlying mechanism of enhanced ethanol tolerance by hfq overexpression, but also provided an alternative approach to investigate protein-protein interactions in Z. mobilis.

Protective effects of thiamine on Wickerhamomyces anomalus against ethanol stress.

Wickerhamomyces anomalus (W. anomalus) is widely reported in the brewing industry and has positive effects on the aromatic profiles of wines because of its unique physiological characteristics and metabolic features. However, the accumulation of ethanol during fermentation inhibits the growth of W. anomalus. Thiamine is involved in the response against various abiotic stresses in microorganisms. Therefore, we used transcriptomic and metabolomic analyses to study the effect of thiamine on ethanol-stressed W. anomalus. The results indicate that thiamine could alleviate the inhibitory effect of ethanol stress on the survival of W. anomalus. Differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) caused by the thiamine intervention were identified as oxidative phosphorylation through integrated transcriptomic and metabolomic analyses. In addition, ethanol treatment decreased the content of intracellular adenosine triphosphate (ATP), while thiamine partially alleviated this phenomenon. The present comprehensive transcriptional overview and metabolomic analysis provide insights about the mechanisms of thiamine protection on W. anomalus under ethanol stress and promote the potential applications of W. anomalus in the fermentation industry.

Small protein Cgl2215 enhances phenolic tolerance by promoting MytA activity in Corynebacterium glutamicum

Corynebacterium glutamicum is a promising chassis microorganism for the bioconversion of lignocellulosic biomass owing to its good tolerance and degradation of the inhibitors generated in lignocellulosic pretreatments. Among the identified proteins encoded by genes within the C. glutamicum genome, nearly 400 are still functionally unknown. Based on previous transcriptome analysis, we found that the hypothetical protein gene cgl2215 was highly upregulated in response to phenol, ferulic acid, and vanillin stress. The cgl2215 deletion mutant was shown to be more sensitive than the parental strain to phenolic compounds as well as other environmental factors such as heat, ethanol, and oxidative stresses. Cgl2215 interacts with C. glutamicum mycoloyltransferase A (MytA) and enhances its in vitro esterase activity. Sensitivity assays of the ΔmytA and Δcgl2215ΔmytA mutants in response to phenolic stress established that the role of Cgl2215 in phenolic tolerance was mediated by MytA. Furthermore, transmission electron microscopy (TEM) results showed that cgl2215 and mytA deletion both led to defects in the cell envelope structure of C. glutamicum, especially in the outer layer (OL) and electron-transparent layer (ETL). Collectively, these results indicate that Cgl2215 can enhance MytA activity and affect the cell envelope structure by directly interacting with MytA, thus playing an important role in resisting phenolic and other environmental stresses.

Effects on Cell Membrane Integrity of Pichia anomala by the Accumulating Excessive Reactive Oxygen Species under Ethanol Stress.

Ethanol stress to yeast is well recognized and exists widely during the brewing process of alcohol products. Pichia anomala is an important ester-producing yeast in the brewing process of Chinese Baijiu and other alcohol products. Therefore, it is of great significance for the alcohol products brewing industry to explore the effects of ethanol stress on the growth metabolism of P. anomala. In this study, the effects of ethanol stress on the growth, esters production ability, cell membrane integrity and reactive oxygen species (ROS) metabolism of P. anomala NCU003 were studied. Our results showed that ethanol stress could inhibit the growth, reduce the ability of non-ethyl ester compounds production and destroy the cell morphology of P. anomala NCU003. The results also showed that 9% ethanol stress produced excessive ROS and then increased the activities of antioxidant enzymes (superoxide dismutase, catalase, aseorbateperoxidase and glutathione reductase) compared to the control group. However, these increased antioxidant enzyme activities could not prevent the damage caused by ROS to P. anomala NCU003. Of note, correlation results indicated that high content of ROS could promote the accumulation of malondialdehyde content, resulting in destruction of the integrity of the cell membrane and leading to the leakage of intracellular nutrients (soluble sugar and protein) and electrolytes. These results indicated that the growth and the non-ethyl ester compounds production ability of P. anomala could be inhibited under ethanol stress by accumulating excessive ROS and the destruction of cell membrane integrity in P. anomala.

Bacillus subtilis Stressosome Sensor Protein Sequences Govern the Ability To Distinguish among Environmental Stressors and Elicit Different σB Response Profiles.

Bacteria use a variety of systems to sense stress and mount an appropriate response to ensure fitness and survival. Bacillus subtilis uses stressosomes-cytoplasmic multiprotein complexes-to sense environmental stressors and enact the general stress response by activating the alternative sigma factor σB. Each stressosome includes 40 RsbR proteins, representing four paralogous (RsbRA, RsbRB, RsbRC, and RsbRD) putative stress sensors. Population-level analyses suggested that the RsbR paralogs are largely redundant, while our prior work using microfluidics-coupled fluorescence microscopy uncovered differences among the RsbR paralogs' σB response profiles with respect to timing and intensity when facing an identical stressor. Here, we use a similar approach to address the question of whether the σB responses mediated by each paralog differ in the presence of different environmental stressors: can they distinguish among stressors? Wild-type cells (with all four paralogs) and RsbRA-only cells activate σB with characteristic transient response timing irrespective of stressor but show various response magnitudes. However, cells with other individual RsbR paralogs show distinct timing and magnitude in their responses to ethanol, salt, oxidative, and acid stress, implying that RsbR proteins can distinguish among stressors. Experiments with hybrid fusion proteins comprising the N-terminal half of one paralog and the C-terminal half of another argue that the N-terminal identity influences response magnitude and that determinants in both halves of RsbRA are important for its stereotypical transient σB response timing. IMPORTANCE Bacterial survival depends on appropriate responses to diverse stressors. The general stress-response system in the environmental model bacterium Bacillus subtilis is constantly poised for an immediate response and uses unusual stress-sensing protein complexes called stressosomes. Stressosomes typically contain four different types of putative sensing protein. We asked whether each type of sensor has a distinct role in mediating response dynamics to different environmental stressors. We find that one sensor type always mediates a transient response, while the others show distinct response magnitude and timing to different stressors. We also find that a transient response is exceptional, as several engineered hybrid proteins did not show strong transient responses. Our work reveals functional distinctions among subunits of the stressosome complex and represents a step toward understanding how the general stress response of B. subtilis ensures its survival in natural environmental settings.

Analysis of the ethanol stress response mechanism in Wickerhamomyces anomalus based on transcriptomics and metabolomics approaches

BackgroundWickerhamomyces anomalus (W. anomalus) is a kind of non-Saccharomyces yeast that has a variety of unique physiological characteristics and metabolic features and is widely used in many fields, such as food preservation, biomass energy, and aquaculture feed protein production. However, the mechanism of W. anomalus response to ethanol stress is still unclear, which greatly limits its application in the production of ethanol beverages and ethanol fuels. Therefore, we checked the effects of ethanol stress on the morphology, the growth, and differentially expressed genes (DEGs) and metabolites (DEMs) of W. anomalus.ResultsHigh concentrations of ethanol (9% ethanol and 12% ethanol) remarkably inhibited the growth of W. anomalus. Energy metabolism, amino acid metabolism, fatty acids metabolism, and nucleic acid metabolism were significantly influenced when exposing to 9% ethanol and 12% ethanolstress, which maybe universal for W. anomalus to response to different concentrations of ethanol stressl Furthermore, extracellular addition of aspartate, glutamate, and arginine significantly abated ethanol damage and improved the survival rate of W. anomalus.ConclusionsThe results obtained in this study provide insights into the mechanisms involved in W. anomalus response to ethanol stress. Therefore, new strategies can be realized to improve the ethanol tolerance of W. anomalus through metabolic engineering.

Influence of ergosterol and phytosterols on wine alcoholic fermentation with Saccharomyces cerevisiae strains

Sterols are a fraction of the eukaryotic lipidome that is essential for the maintenance of cell membrane integrity and its good functionality. During alcoholic fermentation, they enhance yeast growth, metabolism and viability, as well as resistance to high sugar content and ethanol stress. Grape musts clarified in excess lead to the loss of solid particles rich in sterols, resulting in sluggish and stuck fermentations. Two sterol sources can help Saccharomyces cerevisiae yeasts to adapt to fermentation stress conditions: ergosterol (synthesized by yeast under aerobic conditions) and phytosterols (plant sterols imported by yeast cells from grape musts under anaerobiosis). Little is known about the physiological impact of phytosterols assimilation in comparison with ergosterol and the influence of sterol type on fermentation kinetics parameters. Moreover, studies to date have analyzed a limited number of yeast strains. Thus, the aim of this work was to compare the performances of a set of Saccharomyces cerevisiae wine strains that represent the diversity of industrial wine yeast, fermenting with phytosterols or ergosterol under two conditions: sterol limitation (sterol starvation) and high sugar content (the most common stress during fermentation). Results indicated that yeast cell viability was negatively impacted by both stressful conditions, resulting in sluggish and stuck fermentations. This study revealed the huge phenotype diversity of the S. cerevisiae strains tested, in particular in terms of cell viability. Indeed, strains with better viability maintenance completed fermentation earlier. Interestingly, we showed for the first time that sterol type differently affects a wide variety of phenotype, such as viability, biomass, fermentation kinetics parameters and biosynthesis of carbon central metabolism (CCM) metabolites. Ergosterol allowed preserving more viable cells at the end of fermentation and, as a consequence, a better completion of fermentation in both conditions tested, even if phytosterols also enabled the completion of alcoholic fermentation for almost all strains. These results highlighted the essential role of sterols during wine alcoholic fermentation to ensure yeast growth and avoid sluggish or stuck fermentations. Finally, this study emphasizes the importance of taking into account sterol types available during wine fermentation.

Acquired resistance to severe ethanol stress-induced inhibition of proteasomal proteolysis in Saccharomyces cerevisiae

BackgroundAlthough the budding yeast, Saccharomyces cerevisiae, produces ethanol via alcoholic fermentation, high-concentration ethanol is harmful to yeast cells. Severe ethanol stress (> 9% v/v) inhibits protein synthesis and increases the level of intracellular protein aggregates. However, its effect on proteolysis in yeast cells remains largely unknown. MethodsWe examined the effects of ethanol on proteasomal proteolysis in yeast cells through the cycloheximide-chase analysis of short-lived proteins. We also assayed protein degradation in the auxin-inducible degron system and the ubiquitin-independent degradation of Spe1 under ethanol stress conditions. ResultsWe demonstrated that severe ethanol stress strongly inhibited the degradation of the short-lived proteins Rim101 and Gic2. Severe ethanol stress also inhibited protein degradation in the auxin-inducible degron system (Paf1-AID*-6FLAG) and the ubiquitin-independent degradation of Spe1. Proteasomal degradation of these proteins, which was inhibited by severe ethanol stress, resumed rapidly once the ethanol was removed. These results suggested that proteasomal proteolysis in yeast cells is reversibly inhibited by severe ethanol stress. Furthermore, yeast cells pretreated with mild ethanol stress (6% v/v) showed proteasomal proteolysis even with 10% (v/v) ethanol, indicating that yeast cells acquired resistance to proteasome inhibition caused by severe ethanol stress. However, yeast cells failed to acquire sufficient resistance to severe ethanol stress-induced proteasome inhibition when new protein synthesis was blocked with cycloheximide during pretreatment, or when Rpn4 was lost. Conclusions and general significanceOur results provide novel insights into the adverse effects of severe ethanol stress on proteasomal proteolysis and ethanol adaptability in yeast.

Wine Yeast Cells Acquire Resistance to Severe Ethanol Stress and Suppress Insoluble Protein Accumulation during Alcoholic Fermentation.

ABSTRACTUnder laboratory conditions, acute 10% (vol/vol) ethanol stress causes protein denaturation and accumulation of insoluble proteins in yeast cells. However, yeast cells can acquire resistance to severe ethanol stress by pretreatment with mild ethanol stress (6% vol/vol) and mitigate insoluble protein accumulation under subsequent exposure to 10% (vol/vol) ethanol. On the other hand, protein quality control (PQC) of yeast cells during winemaking remains poorly understood. Ethanol concentrations in the grape must increase gradually, rather than acutely, to more than 10% (vol/vol) during the winemaking process. Gradual increases in ethanol evoke two possibilities for yeast PQC under high ethanol concentrations in the must: suppression of insoluble protein accumulation through the acquisition of resistance or the accumulation of denatured insoluble proteins. We examined these two possibilities by conducting alcoholic fermentation tests at 15°C that mimic white winemaking using synthetic grape must (SGM). The results obtained revealed the negligible accumulation of insoluble proteins in wine yeast cells throughout the fermentation process. Furthermore, wine yeast cells in fermenting SGM did not accumulate insoluble proteins when transferred to synthetic defined (SD) medium containing 10% (vol/vol) ethanol. Conversely, yeast cells cultured in SD medium accumulated insoluble proteins when transferred to fermented SGM containing 9.8% (vol/vol) ethanol. Thus, wine yeast cells acquire resistance to the cellular impact of severe ethanol stress during fermentation and mitigate the accumulation of insoluble proteins. This study provides novel insights into the PQC and robustness of wine yeast during winemaking.IMPORTANCE Winemaking is a dynamic and complex process in which ethanol concentrations gradually increase to reach >10% (vol/vol) through alcoholic fermentation. However, there is little information on protein damage in wine yeast during winemaking. We investigated the insoluble protein levels of wine yeast under laboratory conditions in SD medium and during fermentation in SGM. Under laboratory conditions, wine yeast cells, as well as laboratory strain cells, accumulated insoluble proteins under acute 10% (vol/vol) ethanol stress, and this accumulation was suppressed by pretreatment with 6% (vol/vol) ethanol. During the fermentation process, insoluble protein levels were maintained at low levels in wine yeast even when the SGM ethanol concentration exceeded 10% (vol/vol). These results indicate that the progression of wine yeast through fermentation in SGM results in stress tolerance, similar to the pretreatment of cells with mild ethanol stress. These findings further the understanding of yeast cell physiology during winemaking.

Screening and transcriptomic analysis of the ethanol-tolerant mutant Saccharomyces cerevisiae YN81 for high-gravity brewing.

Ethanol stress is one of the major limiting factors for high-gravity brewing. Breeding of yeast strain with high ethanol tolerance, and revealing the ethanol tolerance mechanism of Saccharomyces cerevisiae is of great significance to the production of high-gravity beer. In this study, the mutant YN81 was obtained by ultraviolet-diethyl sulfate (UV-DES) cooperative mutagenesis from parental strain CS31 used in high-gravity craft beer brewing. The ethanol tolerance experiment results showed that cell growth and viability of YN81 were significantly greater than that of CS31 under ethanol stress. The ethanol tolerance mechanisms of YN81 were studied through observation of cell morphology, intracellular trehalose content, and transcriptomic analysis. Results from scanning electron microscope (SEM) showed alcohol toxicity caused significant changes in the cell morphology of CS31, while the cell morphology of YN81 changed slightly, indicating the cell morphology of CS31 got worse (the formation of hole and cell wrinkle). In addition, compared with ethanol-free stress, the trehalose content of YN81 and CS31 increased dramatically under ethanol stress, but there was no significant difference between YN81 and CS31, whether with or without ethanol stress. GO functional annotation analysis showed that under alcohol stress, the number of membrane-associated genes in YN81 was higher than that without alcohol stress, as well as CS31, while membrane-associated genes in YN81 were expressed more than CS31 under alcohol stress. KEGG functional enrichment analysis showed unsaturated fatty acid synthesis pathways and amino acid metabolic pathways were involved in ethanol tolerance of YN81. The mutant YN81 and its ethanol tolerance mechanism provide an optimal strain and theoretical basis for high-gravity craft beer brewing.

High-temperature ethanol fermentation from pineapple waste hydrolysate and gene expression analysis of thermotolerant yeast Saccharomyces cerevisiae

High-temperature ethanol fermentation by thermotolerant yeast is considered a promising technology for ethanol production, especially in tropical and subtropical regions. In this study, optimization conditions for high-temperature ethanol fermentation of pineapple waste hydrolysate (PWH) using a newly isolated thermotolerant yeast, Saccharomyces cerevisiae HG1.1, and the expression of genes during ethanol fermentation at 40 °C were carried out. Three independent variables, including cell concentration, pH, and yeast extract, positively affected ethanol production from PWH at 40 °C. The optimum levels of these significant factors evaluated using response surface methodology (RSM) based on central composite design (CCD) were a cell concentration of 8.0 × 107 cells/mL, a pH of 5.5, and a yeast extract concentration of 4.95 g/L, yielding a maximum ethanol concentration of 36.85 g/L and productivity of 3.07 g/L. Gene expression analysis during high-temperature ethanol fermentation using RT–qPCR revealed that the acquisition of thermotolerance ability and ethanol fermentation efficiency of S. cerevisiae HG1.1 are associated with genes responsible for growth and ethanol stress, oxidative stress, acetic acid stress, DNA repair, the pyruvate-to-tricarboxylic acid (TCA) pathway, and the pyruvate-to-ethanol pathway.

Ethanol at Subinhibitory Concentrations Enhances Biofilm Formation in Salmonella Enteritidis.

The survival of Salmonella Enteritidis in the food chain is relevant to its biofilm formation capacity, which is influenced by suboptimal environmental conditions. Here, biofilm formation pattern of this bacterium was assessed in the presence of ethanol at sub-minimal inhibitory concentrations (sub-MICs) by microtiter plate assays, cell characteristic analyses, and gene expression tests. It was observed that ethanol at subinhibitory concentrations (1/4 MIC, 2.5%; 1/2 MIC, 5.0%) was able to stimulate biofilm formation in S. Enteritidis. The OD595 value (optical density at 595 nm) used to quantify biofilm production was increased from 0.14 in control groups to 0.36 and 0.63 under 2.5% and 5.0% ethanol stresses, respectively. Ethanol was also shown to reduce bacterial swimming motility and enhance cell auto-aggregation ability. However, other cell characteristics such as swarming activity, initial attachment and cell surface hydrophobicity were not remarkedly impacted by ethanol. Reverse transcription quantitative real-time PCR (RT-qPCR) analysis further revealed that the luxS gene belonging to a quorum-sensing system was upregulated by 2.49- and 10.08-fold in the presence of 2.5% and 5.0% ethanol, respectively. The relative expression level of other biofilm-related genes (adrA, csgB, csgD, and sdiA) and sRNAs (ArcZ, CsrB, OxyS, and SroC) did not obviously change. Taken together, these findings suggest that decrease in swimming motility and increase in cell auto-aggregation and quorum sensing may result in the enhancement of biofilm formation by S. Enteritidis under sublethal ethanol stress.

Effects of inoculation timing and mixed fermentation with Pichia fermentans on Oenococcus oeni viability, fermentation duration and aroma production during wine malolactic fermentation

There has been a growing interest in developing co-inoculum of Oenococcus oeni and Saccharomyces cerevisiae/non-Saccharomyces for simultaneous malolactic fermentation (MLF) and alcoholic fermentation (AF) of wines. This study sought to elucidate the effects of two crucial factors (inoculation timing and paired yeast) on the fermentation performance and aroma production of Merlot wine. O. oeni used for MLF was concurrently or sequentially inoculated with two yeast cultures (i.e., single S. cerevisiae and mixed S. cerevisiae /Pichia fermentans H5Y-28) used for AF. Inoculation timing determined the overall vinification duration, and conditioned the production of principle higher alcohols, terpene and O. oeni-mediated volatiles. In contrast, paired yeast improved O. oeni viability, and showed significant effect on aromatic esters and volatile fatty acids. Possibly due to lower ethanol stress, co-inoculum allowed O. oeni to initiate MLF during AF, resulting in 45% reduction of total fermentation time. Meanwhile, O. oeni growth was stimulated by P. fermentans, with 1.7-fold of the maximum population higher than that in co-fermentation without P. fermentans. Such stimulation of O. oeni growth also occurred in sequential fermentation where P. fermentans had been replaced by S. cerevisiae. Only in sequential inoculum, P. fermentans induced high levels of 3-methylbutyl acetate, ethyl 3-methylbutanoate, ethyl hexanoate and ethyl octanoate, which may result in enhanced fresh fruity trait of wines. These findings suggested a positive effect of P. fermentans H5Y-28 on O. oeni and MLF. This work provides an alternative approach to improve wine MLF and aroma outcomes using friendly non-Saccharomyces yeast with appropriate inoculation strategy.

OMICs-Based Strategies to Explore Stress Tolerance Mechanisms of Saccharomyces cerevisiae for Efficient Fuel Ethanol Production

Efficient biotransformation of lignocellulosic biomass to second-generation (2G) bioethanol requires promising strains harboring built-in resistance against limitations imposed by pretreated lignocellulose-derived compounds. Ethanol fermentation and stress tolerance of yeast cells are almost simultaneously exposed to sequence variations and multiple inhibitory factors during the phases of proliferation, metabolism, and productivity. Several studies have extensively concentrated on identification or characterization of genes which confer resistance to various stresses and yeast tolerance enhancement through genetic breeding. However, the investigation of individual genes is inadequate to explain the global molecular mechanism. Herewith, “OMICs-approaches,” including genomics, transcriptomics, proteomics, and metabolomics, which are comprehensively aimed at comparative, functional profiling of the whole metabolic network, have elucidated complex cellular reactions under stressful conditions. This review briefly discusses the research progress in the field of multi-OMICs with a special focus on stress-responsive factors in frequently used S. cerevisiae. It also highlights how to promote metabolic-engineered strains for increased tolerance and higher production yield, which should be deeply exploited to achieve robustness during the lignocellulose-to-ethanol conversion process.

Positive effect of ethanol-induced Lactococcus lactis on alcohol metabolism in mice

It is well known that exposure to environmental stresses could enhance the adaptability of bacteria and up-regulate the expression of a variety of oxidative stress-related genes and antioxidant enzymes. It is unclear whether the adaptability of microorganisms formed naturally in special environments could transfer to other organisms. The study aimed to evaluate the effects of untreated and ethanol-induced Lactococcus lactis intracellular extracts (U-IE and E-IE) on alcohol metabolism in mice. The positive effects of E-IE on alcohol metabolism in mice were revealed by the enhanced latency of loss of righting reflex (LORR), the reduced duration of LORR, the decrease of blood alcohol concentration, as well as the elevation of alcohol dehydrogenase (ADH) activities in the stomach and liver tissues. Furthermore, the potential benefits of E-IE on the liver were evaluated by biochemical parameters including the activities of serum transaminase, the levels of antioxidant enzymes, and the pathological changes of liver tissue. The present work put forward a new point that appropriate ethanol stress could enhance the intracellular ADH activity of L. lactis, and its intracellular extracts could continue to enhance alcohol metabolism in mice.

LncRNAs of Saccharomyces cerevisiae bypass the cell cycle arrest imposed by ethanol stress

Ethanol alters many subsystems of Saccharomyces cerevisiae, including the cell cycle. Two ethanol-responsive lncRNAs in yeast interact with cell cycle proteins, and here, we investigated the role of these RNAs in cell cycle. Our network dynamic modeling showed that higher and lower ethanol-tolerant strains undergo cell cycle arrest in mitosis and G1 phases, respectively, during ethanol stress. The higher population rebound of the lower ethanol-tolerant phenotype after stress relief responds to the late phase arrest. We found that the lncRNA lnc9136 of SEY6210 (a lower ethanol-tolerant strain) induces cells to skip mitosis arrest. Simulating an overexpression of lnc9136 and analyzing CRISPR–Cas9 mutants lacking this lncRNA suggest that lnc9136 induces a regular cell cycle even under ethanol stress, indirectly regulating Swe1p and Clb1/2 by binding to Gin4p and Hsl1p. Notably, lnc10883 of BY4742 (a higher ethanol-tolerant strain) does not prevent G1 arrest in this strain under ethanol stress. However, lnc19883 circumvents DNA and spindle damage checkpoints, maintaining a functional cell cycle by interacting with Mec1p or Bub1p even in the presence of DNA/spindle damage. Overall, we present the first evidence of direct roles for lncRNAs in regulating yeast cell cycle proteins, the dynamics of this system in different ethanol-tolerant phenotypes, and a new yeast cell cycle model.

Sensitivity and Resilience to Predator Stress-Enhanced Ethanol Drinking Is Associated With Sex-Dependent Differences in Stress-Regulating Systems.

Stress can increase ethanol drinking, and evidence confirms an association between post-traumatic stress disorder (PTSD) and the development of alcohol use disorder (AUD). Exposure to predator odor is considered a traumatic stressor, and predator stress (PS) has been used extensively as an animal model of PTSD. Our prior work determined that repeated exposure to intermittent PS significantly increased anxiety-related behavior, corticosterone levels, and neuronal activation in the hippocampus and prefrontal cortex in naïve male and female C57BL/6J mice. Intermittent PS exposure also increased subsequent ethanol drinking in a subgroup of animals, with heterogeneity of responses as seen with comorbid PTSD and AUD. The present studies built upon this prior work and began to characterize “sensitivity” and “resilience” to PS-enhanced drinking. Ethanol drinking was measured during baseline, intermittent PS exposure, and post-stress; mice were euthanized after 24-h abstinence. Calculation of median and interquartile ranges identified “sensitive” (>20% increase in drinking over baseline) and “resilient” (no change or decrease in drinking from baseline) subgroups. Intermittent PS significantly increased subsequent ethanol intake in 24% of male (↑60%) and in 20% of female (↑71%) C57BL/6J mice in the “sensitive” subgroup. Plasma corticosterone levels were increased significantly after PS in both sexes, but levels were lower in the “sensitive” vs. “resilient” subgroups. In representative mice from “sensitive” and “resilient” subgroups, prefrontal cortex and hippocampus were analyzed by Western Blotting for levels of corticotropin releasing factor (CRF) receptor 1, CRF receptor 2, CRF binding protein, and glucocorticoid receptor, vs. separate naïve age-matched mice. In prefrontal cortex, CRF receptor 1, CRF receptor 2, CRF binding protein, and glucocorticoid receptor levels were significantly higher in “sensitive” vs. naïve and “resilient” mice only in females. In hippocampus, CRF receptor 1, CRF receptor 2 and glucocorticoid receptor levels were significantly lower in “resilient” vs. naïve and “sensitive” mice across both sexes. These results indicate that sex strongly influences the effects of ethanol drinking and stress on proteins regulating stress and anxiety responses. They further suggest that targeting the CRF system and glucocorticoid receptors in AUD needs to consider the comorbidity of PTSD with AUD and sex of treated individuals.