JNK Signaling Positively Regulates Acute Ethanol Tolerance in C. elegans.

Alcohol use disorder (AUD) is a chronic neurobehavioral condition characterized by a cycle of tolerance development, increased consumption, and reinstated craving and seeking behaviors during withdrawal. Understanding the intricate mechanisms of AUD necessitates reliable animal models reflecting its key features. Caenorhabditis elegans (C. elegans), with its conserved nervous system and genetic tractability, has emerged as a valuable model organism to study AUD. Here, we employ an ethanol vapor exposure model in Caenorhabditis elegans, recapitulating AUD features while maintaining high-throughput scalability. We demonstrate that ethanol vapor exposure induces intoxication-like behaviors, acute tolerance, and ethanol preference, akin to mammalian AUD traits. Leveraging this model, we elucidate the conserved role of c-jun N-terminal kinase (JNK) signaling in mediating acute ethanol tolerance. Mutants lacking JNK signaling components exhibit impaired tolerance development, highlighting JNK's positive regulation. Furthermore, we detect ethanol-induced JNK activation in C. elegans. Our findings underscore the utility of C. elegans with ethanol vapor exposure for studying AUD and offer novel insights into the molecular mechanisms underlying acute ethanol tolerance through JNK signaling.

Histidine modified Fe3O4 nanoparticles improving the ethanol yield and tolerance of Saccharomyces cerevisiae.

Saccharomyces cerevisiae, the primary microorganism involved in ethanol production, is hindered by the accumulation of ethanol, leading to reduced ethanol production. In this study, we employed histidine-modified Fe3O4 nanoparticles (His-Fe3O4) for the first time, to the best of our knowledge, as a method to enhance ethanol yield during the S. cerevisiae fermentation process. The results demonstrated that exposing S. cerevisiae cells to Fe3O4 nanoparticles (Fe3O4 NPs) led to increased cell proliferation and glucose consumption. Moreover, the introduction of His-Fe3O4 significantly boosted ethanol content by 17.3% (p < 0.05) during fermentation. Subsequent findings indicated that the increase in ethanol content was associated with enhanced ethanol tolerance and improved electron transport efficiency. This study provided evidence for the positive effects of His-Fe3O4 on S. cerevisiae cells and proposed a straightforward approach to enhance ethanol production in S. cerevisiae fermentation. The mediation of improved ethanol tolerance offers significant potential in the fermentation and bioenergy sectors.

Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks.

BACKGROUND: The selection of Saccharomyces cerevisiae strains with higher alcohol tolerance can potentially increase the industrial production of ethanol fuel. However, the design of selection protocols to obtain bioethanol yeasts with higher alcohol tolerance poses the challenge of improving industrial strains that are already robust to high ethanol levels. Furthermore, yeasts subjected to mutagenesis and selection, or laboratory evolution, often present adaptation trade-offs wherein higher stress tolerance is attained at the expense of growth and fermentation performance. Although these undesirable side effects are often associated with acute selection regimes, the utility of using harsh ethanol treatments to obtain robust ethanologenic yeasts still has not been fully investigated. RESULTS: We conducted an adaptive laboratory evolution by challenging four populations (P1-P4) of the Brazilian bioethanol yeast, Saccharomyces cerevisiae PE-2_H4, through 68-82 cycles of 2-h ethanol shocks (19-30% v/v) and outgrowths. Colonies isolated from the final evolved populations (P1c-P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c-P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting from BUD3 disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutations cyr1A1474T/usv1Δ conferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. The cyr1A1474T/usv1Δ strain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production. CONCLUSIONS: Our study combined genomic, mutational, and fitness analyses to understand the genetic underpinnings of yeast evolution to ethanol shocks. Although fitness analyses revealed that most evolved mutations impose a cost for cell propagation, combination of key mutations cyr1A1474T/usv1Δ endowed yeasts with higher tolerance for growth in the presence of ethanol. Moreover, alleles selected for acute stress survival comprising the P1c genotype conferred stress tolerance and optimal performance under conditions simulating the Brazilian industrial ethanol production.

The joint action of yeast eisosomes and membraneless organelles in response to ethanol stress.

Elevated ethanol concentrations in yeast affect the plasma membrane. The plasma membrane in yeast has many lipid-protein complexes, such as Pma1 (MCP), Can1 (MCC), and the eisosome complex. We investigated the response of eisosomes, MCPs, and membraneless structures to ethanol stress. We found a correlation between ethanol stress and proton flux with quick acidification of the medium. Moreover, ethanol stress influences the symporter expression in stressed cells. We also suggest that acute stress from ethanol leads to increases in eisosome size and SG number: we hypothesized that eisosomes may protect APC symporters and accumulate an mRNA decay protein in ethanol-stressed cells. Our findings suggest that the joint action of these factors may provide a protective effect on cells under ethanol stress.

Investigating human-derived lactic acid bacteria for alcohol resistance.

BACKGROUND: Excessive alcohol consumption has been consistently linked to serious adverse health effects, particularly affecting the liver. One natural defense against the detrimental impacts of alcohol is provided by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which detoxify harmful alcohol metabolites. Recent studies have shown that certain probiotic strains, notably Lactobacillus spp., possess alcohol resistance and can produce these critical enzymes. Incorporating these probiotics into alcoholic beverages represents a pioneering approach that can potentially mitigate the negative health effects of alcohol while meeting evolving consumer preferences for functional and health-centric products. RESULTS: Five lactic acid bacteria (LAB) isolates were identified: Lactobacillus paracasei Alc1, Lacticaseibacillus rhamnosus AA, Pediococcus acidilactici Alc3, Lactobacillus paracasei Alc4, and Pediococcus acidilactici Alc5. Assessment of their alcohol tolerance, safety, adhesion ability, and immunomodulatory effects identified L. rhamnosus AA as the most promising alcohol-tolerant probiotic strain. This strain also showed high production of ADH and ALDH. Whole genome sequencing analysis revealed that the L. rhamnosus AA genome contained both the adh (encoding for ADH) and the adhE (encoding for ALDH) genes. CONCLUSIONS: L. rhamnosus AA, a novel probiotic candidate, showed notable alcohol resistance and the capability to produce enzymes essential for alcohol metabolism. This strain is a highly promising candidate for integration into commercial alcoholic beverages upon completion of comprehensive safety and functionality evaluations.

Characterization of a Novel Hyperthermophilic GH1 ß-Glucosidase from Acidilobus sp. and Its Application in the Hydrolysis of Soybean Isoflavone Glycosides.

A putative ß-glucosidase gene, BglAc, was amplified from Acidilobus sp. through metagenome database sampling from a hot spring in Yellowstone National Park. BglAc is composed of 485 amino acid residues and bioinformatics analysis showed that it belongs to the GH1 family of ß-glucosidases. The gene was successfully expressed in Escherichia coli with a molecular weight of approximately 55.3 kDa. The purified recombinant enzyme showed the maximum activity using p-nitrophenyl-ß-D-glucopyranoside (pNPG) as the substrate at optimal pH 5.0 and 100 °C. BglAc exhibited extraordinary thermostability, and its half-life at 90 °C was 6 h. The specific activity, Km, Vmax, and Kcat/Km of BglAc toward pNPG were 357.62 U mg-1, 3.41 mM, 474.0 µmol min-1·mg-1, and 122.7 s-1mM-1. BglAc exhibited the characteristic of glucose tolerance, and the inhibition constant Ki was 180.0 mM. Furthermore, a significant ethanol tolerance was observed, retaining 96% relative activity at 10% ethanol, and even 78% at 20% ethanol, suggesting BglAc as a promising enzyme for cellulose saccharification. BglAc also had a strong ability to convert the major soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their corresponding aglycones. Overall, BglAc was actually a new ß-glucosidase with excellent thermostability, ethanol tolerance, and glycoside hydrolysis ability, indicating its wide prospects for applications in the food industry, animal feed, and lignocellulosic biomass degradation.

Characterization of Autochthonous Lactic Acid Bacteria Isolated from a Traditional Ethiopian Beverage, Tella.

This study aimed to isolate lactic acid bacteria (LAB) from a traditional Ethiopian fermented product, Tella, and evaluate their functional properties. Of forty-three isolates, seven LAB were screened and identified as Pediococcus pentosaceus, Latilactobacillus curvatus, Leuconostoc mesenteroides, and Lactiplantibacillus plantarum species. The isolates were tested for their alcohol tolerance, acid and bile resistance, auto-aggregation, co-aggregation, hydrophobicity, antibacterial activity, and antibiotic susceptibility. LAB isolates, specifically P. pentosaceus TAA01, L. mesenteroides TDB22, and L. plantarum TDM41, showed a higher degree of alcohol tolerance in 8% and 10% (w/v) ethanol concentrations. Additionally, these three isolates displayed survival rates >85% in both acidic pH and bile environments. Among the isolates, L. plantarum TDM41 demonstrated the highest auto-aggregation, co-aggregation, and hydrophobicity with (44.9 ± 1.7)%, (41.4 ± 0.2)%, and (52.1 ± 0.1)% values, respectively. The cell-free supernatant of the isolates exhibited antibacterial activity against foodborne pathogens of Escherichia coli, Salmonella Enteritidis, and Staphylococcus aureus. Each isolate exhibited various levels of resistance and susceptibility to seven antibiotics and resistance was observed against four of the antibiotics tested. After performing a principal component analysis, Pediococcus pentosaceus TAA01, L. mesenteroides TDB22, and L. plantarum TDM41 were selected as the most promising ethanol-tolerant probiotic isolates.

[Expression and characterization of mesophilic GH1 ß-glucosidase CdBglA from acidophilic Cuniculiplasma divulgatum].

ß-glucosidase has important applications in food, pharmaceutics, biomass conversion and other fields, exploring ß-glucosidase with strong adaptability and excellent properties thus has received extensive interest. In this study, a novel glucosidase from the GH1 family derived from Cuniculiplasma divulgatum was cloned, expressed, and characterized, aiming to find a better ß-glucosidase. The amino acid sequences of GH1 family glucosidase derived from C. divulgatum were obtained from the NCBI database, and a recombinant plasmid pET-30a(+)-CdBglA was constructed. The recombinant protein was induced to express in Escherichia coli BL21(DE3). The enzymatic properties of the purified CdBglA were studied. The molecular weight of the recombinant CdBglA was 56.0 kDa. The optimum pH and temperature were 5.5 and 55 ℃, respectively. The enzyme showed good pH stability, 92.33% of the initial activity could be retained when treated under pH 5.5-11.0 for 1 h. When pNPG was used as a substrate, the kinetic parameters Km, Vmax and Kcat/Km were 0.81 mmol, 291.99 µmol/(mg·min), and 387.50 s-1 mmol-1, respectively. 90.33% of the initial enzyme activity could be retained when CdBglA was placed with various heavy metal ions at a final concentration of 5 mmol/L. The enzyme activity was increased by 28.67% under 15% ethanol solution, remained unchanged under 20% ethanol, and 43.68% of the enzyme activity could still be retained under 30% ethanol. The enzyme has an obvious activation effect at 0-1.5 mol/L NaCl and can tolerate 0.8 mol/L glucose. In conclusion, CdBglA is an acidic and mesophilic enzyme with broad pH stability and strong tolerance to most metal ions, organic solvents, NaCl and glucose. These characteristics may facilitate future theoretical research and industrial production.

From Saccharomyces cerevisiae to Ethanol: Unlocking the Power of Evolutionary Engineering in Metabolic Engineering Applications.

Increased human population and the rapid decline of fossil fuels resulted in a global tendency to look for alternative fuel sources. Environmental concerns about fossil fuel combustion led to a sharp move towards renewable and environmentally friendly biofuels. Ethanol has been the primary fossil fuel alternative due to its low carbon emission rates, high octane content and comparatively facile microbial production processes. In parallel to the increased use of bioethanol in various fields such as transportation, heating and power generation, improvements in ethanol production processes turned out to be a global hot topic. Ethanol is by far the leading yeast output amongst a broad spectrum of bio-based industries. Thus, as a well-known platform microorganism and native ethanol producer, baker's yeast Saccharomyces cerevisiae has been the primary subject of interest for both academic and industrial perspectives in terms of enhanced ethanol production processes. Metabolic engineering strategies have been primarily adopted for direct manipulation of genes of interest responsible in mainstreams of ethanol metabolism. To overcome limitations of rational metabolic engineering, an alternative bottom-up strategy called inverse metabolic engineering has been widely used. In this context, evolutionary engineering, also known as adaptive laboratory evolution (ALE), which is based on random mutagenesis and systematic selection, is a powerful strategy to improve bioethanol production of S. cerevisiae. In this review, we focus on key examples of metabolic and evolutionary engineering for improved first- and second-generation S. cerevisiae bioethanol production processes. We delve into the current state of the field and show that metabolic and evolutionary engineering strategies are intertwined and many metabolically engineered strains for bioethanol production can be further improved by powerful evolutionary engineering strategies. We also discuss potential future directions that involve recent advancements in directed genome evolution, including CRISPR-Cas9 technology.

Selection of Ethanol Tolerant Strains of Candida albicans by Repeated Ethanol Exposure Results in Strains with Reduced Susceptibility to Fluconazole.

Candida albicans is a commensal yeast that has important impacts on host metabolism and immune function, and can establish life-threatening infections in immunocompromised individuals. Previously, C. albicans colonization has been shown to contribute to the progression and severity of alcoholic liver disease. However, relatively little is known about how C. albicans responds to changing environmental conditions in the GI tract of individuals with alcohol use disorder, namely repeated exposure to ethanol. In this study, we repeatedly exposed C. albicans to high concentrations (10% vol/vol) of ethanol-a concentration that can be observed in the upper GI tract of humans following consumption of alcohol. Following this repeated exposure protocol, ethanol small colony (Esc) variants of C. albicans isolated from these populations exhibited increased ethanol tolerance, altered transcriptional responses to ethanol, and cross-resistance/tolerance to the frontline antifungal fluconazole. These Esc strains exhibited chromosomal copy number variations and carried polymorphisms in genes previously associated with the acquisition of fluconazole resistance during human infection. This study identifies a selective pressure that can result in evolution of fluconazole tolerance and resistance without previous exposure to the drug.

Screening and oenological property analysis of ethanol-tolerant non-Saccharomyces yeasts isolated from Rosa roxburghii Tratt.

Ethanol tolerance is crucial for the oenological yeasts. Rosa roxburghii Tratt, a Rosaceae plant native to China, is rich in nutritional and medicinal ingredients. In this study, ethanol-tolerant non-Saccharomyces yeasts were screened, and their oenological properties were further evaluated. Three ethanol-tolerant yeast strains (designated as C6, F112, and F15), which could tolerate 12% (v/v) ethanol treatment, were isolated from R. roxburghii, and identified as Candida tropicalis, Pichia guilliermondii, and Wickerhamomyces anomalus, respectively. The winemaking condition tolerances of these ethanol-tolerant yeast strains were similar to those of Saccharomyces cerevisiae X16. However, their growth, sugar metabolic performance and sulphureted hydrogen activities, were different. The ß-glucosidase production ability of strain W. anomalus F15 was lower than that of S. cerevisiae X16, and strains of C. tropicalis C6 and P. guilliermondii F112 were similar to S. cerevisiae X16. Electronic sensory properties of the R. roxburghii wines fermented using ethanol-tolerant yeasts together with S. cerevisiae showed no significant differences. However, the mixed inoculation of the ethanol-tolerant yeast strains with S. cerevisiae could regulate the volatile aroma characteristics of the fermented R. roxburghii wine, enriching and enhancing the aroma flavor. Therefore, the selected ethanol-tolerant yeasts have the potential for application in the production of unique R. roxburghii wine.

Improving ethanol tolerance of ethyl carbamate hydrolase by diphasic high pressure molecular dynamic simulations.

Ethyl carbamate (EC) is mainly found in fermented foods and fermented alcoholic beverages, which could cause carcinogenic potential to humans. Reducing EC is one of the key research priorities to address security of fermented foods. Enzymatic degradation of EC with EC hydrolase in food is the most reliable and efficient method. However, poor tolerance to ethanol severely hinders application of EC hydrolase. In this study, the mutants of EC hydrolase were screened by diphasic high pressure molecular dynamic simulations (dHP-MD). The best variant with remarkable improvement in specific activity and was H68A/K70R/S325N, whose specific activity was approximately 3.42-fold higher than WT, and relative enzyme activity under 20% (v/v) was 5.02-fold higher than WT. Moreover, the triple mutant increased its stability by acquiring more hydration shell and forming extra hydrogen bonds. Furthermore, the ability of degrading EC of the immobilized triple mutant was both detected in mock wine and under certain reaction conditions. The stability of immobilized triple mutant and WT were both improved, and immobilized triple mutant degraded nearly twice as much EC as that of immobilized WT. Overall, dHP-MD was proved to effectively improve enzyme activity and ethanol tolerance for extent application at industrial scale.

Biochemical characteristics of Myceliophthora thermophila recombinant ß-glucosidase (MtBgl3c) applicable in cellulose bioconversion.

The GH3 ß-glucosidase gene of Myceliophthora thermophila (MtBgl3c) has been cloned and heterologously expressed in E. coli for the first time. This study highlights the important characteristics of recombinant MtBgl3c (rMtBgl3c) which make it a promising candidate in industrial applications. Optimization of the production of rMtBgl3c led to 28,000 U L-1. On purification, it has a molecular mass of ∼100 kDa. It is a broad substrate specific thermostable enzyme that exhibits pH and temperature optima at 5.0 and 55 °C, respectively. The amino acid residues Asp287 and Glu514 act as nucleophile and catalytic acid/base, respectively in the enzyme catalysis. Its low Km value (1.28 mM) indicates a high substrate affinity as compared to those previously reported. The rMtBgl3c displays a synergistic action with the commercial enzyme cocktail in the saccharification of sugarcane bagasse suggesting its utility in the cellulose bioconversion. Tolerance to solvents, detergents as well as glucose make this enzyme applicable in wine, detergent, paper and textile industries too.

Evolving Robust and Interpretable Enzymes for the Bioethanol Industry.

Obtaining a robust and applicable enzyme for bioethanol production is a dream for biorefinery engineers. Herein, we describe a general method to evolve an all-round and interpretable enzyme that can be directly employed in the bioethanol industry. By integrating the transferable protein evolution strategy InSiReP 2.0 (In Silico guided Recombination Process), enzymatic characterization for actual production, and computational molecular understanding, the model cellulase PvCel5A (endoglucanase II Cel5A from Penicillium verruculosum) was successfully evolved to overcome the remaining challenges of low ethanol and temperature tolerance, which primarily limited biomass transformation and bioethanol yield. Remarkably, application of the PvCel5A variants in both first- and second-generation bioethanol production processes (i. Conventional corn ethanol fermentation combined with the in situ pretreatment process; ii. cellulosic ethanol fermentation process) resulted in a 5.7-10.1 % increase in the ethanol yield, which was unlikely to be achieved by other optimization techniques.

Expression and characterization of mesophilic GH1 β-glucosidase CdBglA from acidophilic Cuniculiplasma divulgatum / 生物工程学报

β-glucosidase has important applications in food, pharmaceutics, biomass conversion and other fields, exploring β-glucosidase with strong adaptability and excellent properties thus has received extensive interest. In this study, a novel glucosidase from the GH1 family derived from Cuniculiplasma divulgatum was cloned, expressed, and characterized, aiming to find a better β-glucosidase. The amino acid sequences of GH1 family glucosidase derived from C. divulgatum were obtained from the NCBI database, and a recombinant plasmid pET-30a(+)-CdBglA was constructed. The recombinant protein was induced to express in Escherichia coli BL21(DE3). The enzymatic properties of the purified CdBglA were studied. The molecular weight of the recombinant CdBglA was 56.0 kDa. The optimum pH and temperature were 5.5 and 55 ℃, respectively. The enzyme showed good pH stability, 92.33% of the initial activity could be retained when treated under pH 5.5-11.0 for 1 h. When pNPG was used as a substrate, the kinetic parameters Km, Vmax and Kcat/Km were 0.81 mmol, 291.99 μmol/(mg·min), and 387.50 s-1 mmol-1, respectively. 90.33% of the initial enzyme activity could be retained when CdBglA was placed with various heavy metal ions at a final concentration of 5 mmol/L. The enzyme activity was increased by 28.67% under 15% ethanol solution, remained unchanged under 20% ethanol, and 43.68% of the enzyme activity could still be retained under 30% ethanol. The enzyme has an obvious activation effect at 0-1.5 mol/L NaCl and can tolerate 0.8 mol/L glucose. In conclusion, CdBglA is an acidic and mesophilic enzyme with broad pH stability and strong tolerance to most metal ions, organic solvents, NaCl and glucose. These characteristics may facilitate future theoretical research and industrial production.

Ethanol tolerance of Clostridium thermocellum: the role of chaotropicity, temperature and pathway thermodynamics on growth and fermentative capacity.

BACKGROUND: Clostridium thermocellum is a promising candidate for consolidated bioprocessing of lignocellulosic biomass to ethanol. The low ethanol tolerance of this microorganism is one of the remaining obstacles to industrial implementation. Ethanol inhibition can be caused by end-product inhibition and/or chaotropic-induced stress resulting in increased membrane fluidization and disruption of macromolecules. The highly reversible glycolysis of C. thermocellum might be especially sensitive to end-product inhibition. The chaotropic effect of ethanol is known to increase with temperature. This study explores the relative contributions of these two aspects to investigate and possibly mitigate ethanol-induced stress in growing and non-growing C. thermocellum cultures. RESULTS: To separate chaotropic from thermodynamic effects of ethanol toxicity, a non-ethanol producing strain AVM062 (Pclo1313_2638::ldh* ∆adhE) was constructed by deleting the bifunctional acetaldehyde/alcohol dehydrogenase gene, adhE, in a lactate-overproducing strain. Exogenously added ethanol lowered the growth rate of both wild-type and the non-ethanol producing mutant. The mutant strain grew quicker than the wild-type at 50 and 55 °C for ethanol concentrations ≥ 10 g L-1 and was able to reach higher maximum OD600 at all ethanol concentrations and temperatures. For the wild-type, the maximum OD600 and relative growth rates were higher at 45 and 50 °C, compared to 55 °C, for ethanol concentrations ≥ 15 g L-1. For the mutant strain, no positive effect on growth was observed at lower temperatures. Growth-arrested cells of the wild-type demonstrated improved fermentative capacity over time in the presence of ethanol concentrations up to 40 g L-1 at 45 and 50 °C compared to 55 °C. CONCLUSION: Positive effects of temperature on ethanol tolerance were limited to wild-type C. thermocellum and are likely related to mechanisms involved in the ethanol-formation pathway and redox cofactor balancing. Lowering the cultivation temperature provides an attractive strategy to improve growth and fermentative capacity at high ethanol titres in high-cellulose loading batch cultivations. Finally, non-ethanol producing strains are useful platform strains to study the effects of chaotropicity and thermodynamics related to ethanol toxicity and allow for deeper understanding of growth and/or fermentation cessation under industrially relevant conditions.

Comparative genomic analysis of Lacticaseibacillus paracasei SMN-LBK from koumiss.

Lacticaseibacillus paracasei SMN-LBK, which was isolated in Xinjiang, has been shown to be a probiotic strain and used as the auxiliary starter for dairy fermentation. Comparative genomic analysis was performed to investigate the metabolic preference and ethanol tolerance mechanisms of L. paracasei SMN-LBK. The results of comparative genomics showed that L. paracasei strains had high conservation and genetic diversity. SMN-LBK encoded various genes related to carbohydrate and amino acid metabolism pathways, which endow this strain with good fermentation potential. In addition, 6 CRISPR sequences and 8 cas proteins were found in SMN-LBK, and these could play vital roles in the immune system. Furthermore, a unique cluster of potential secondary metabolism genes related to bacteriocins was detected in the genome of SMN-LBK, and this could be important for the preservation of fermented foods. Multiple genes related to alcohol tolerance were also identified. In conclusion, our study explained the traits that were previously demonstrated for SMN-LBK as phenotypes and provided a theoretical basis for the application of SMN-LBK in the food industry.

The characterisation of Wickerhamomyces anomalus M15, a highly tolerant yeast for bioethanol production using seaweed derived medium.

Advanced generation biofuels have potential for replacing fossil fuels as society moves forward into a net-zero carbon future. Marine biomass is a promising source of fermentable sugars for fermentative bioethanol production; however the medium derived from seaweed hydrolysis contains various inhibitors, such as salts that affected ethanol fermentation efficiency. In this study the stress tolerance of a marine yeast, Wickerhamomyces anomalus M15 was characterised. Specific growth rate analysis results showed that Wickerhamomyces anomalus M15 could tolerate up to 600 g/L glucose, 150 g/L xylose and 250 g/L ethanol, respectively. Using simulated concentrated seaweed hydrolysates, W. anomalus M15's bioethanol production potential using macroalgae derived feedstocks was assessed, in which 5.8, 45.0, and 19.9 g/L ethanol was produced from brown (Laminaria digitata), green (Ulva linza) and red seaweed (Porphyra umbilicalis) based media. The fermentation of actual Ulva spp. hydrolysate harvested from United Kingdom shores resulted in a relatively low ethanol concentration (15.5 g/L) due to challenges that arose from concentrating the seaweed hydrolysate. However, fed-batch fermentation using simulated concentrated green seaweed hydrolysate achieved a concentration of 73 g/L ethanol in fermentations using both seawater and reverse osmosis water. Further fermentations conducted with an adaptive strain W. anomalus M15-500A showed improved bioethanol production of 92.7 g/L ethanol from 200 g/L glucose and reduced lag time from 93 h to 24 h in fermentation with an initial glucose concentration of 500 g/L. The results indicated that strains W. anomalus M15 and W. anomalus M15-500A have great potential for industrial bioethanol production using marine biomass derived feedstocks. It also suggested that if a concentrated high sugar content seaweed hydrolysate could be obtained, the bioethanol concentration could achieve 90 g/L or above, exceeding the minimum industrial production threshold.

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.

Isolation and Characterization of High-Ethanol-Tolerance Lactic Acid Bacteria from Australian Wine.

Lactic acid bacteria are very important in winemaking. In this study, 108 lactic acid bacteria isolates were obtained from high-ethanol-content (~17% (v/v)) Grenache wines during uninoculated malolactic fermentation (MLF). The 16S rRNA and species-specific PCR showed that 104 of these were Oenococcusoeni, three were Lactobacillus hilgardii, and one was Staphylococcus pasteuri. AFLP of HindIII and MseI digests of the genomic DNA of the O. oeni strains was developed for the first time to discriminate the strains. The results showed that the method was a suitable technique for discriminating the O. oeni strains. Based on the cluster analysis, nine O. oeni strains were chosen for inclusion in an ethanol tolerance assay involving monitoring of optical density (ABS600nm) and viable plating. Several O. oeni strains (G63, G46, G71, G39) survived and grew well in MRS-AJ with 17% (v/v) ethanol, while the commercial O. oeni reference strain did not. Strain G63 could also survive and grow for 168 h after inoculation in MRS-AJ medium with 19% (v/v) ethanol. These results suggest that O. oeni G63, G46, G71, and G39 could potentially be used as MLF starters for high-ethanol-content wines. All three L. hilgardii strains could survive and grow in MRS-AJ with 19% (v/v) ethanol, perhaps also indicating their suitability as next-generation MLF starter cultures.