A mutant GH3 family ß-glucosidase from Oenococcus oeni exhibits superior adaptation to wine stresses and potential for improving wine aroma and phenolic profiles.

In this study, we conducted a comprehensive investigation into a GH3 family ß-glucosidase (BGL) from the wild-type strain of Oenococcus oeni and its mutated counterpart from the acid-tolerant mutant strain. Our analysis revealed the mutant BGL's remarkable capacity to adapt to wine-related stress conditions, including heightened tolerance to low pH, elevated ethanol concentrations, and metal ions. Additionally, the mutant BGL exhibited superior hydrolytic activity towards various substrates. Through de novo modeling, we identified specific amino acid mutations responsible for its resilience to low pH and high ethanol environments. In simulated wine conditions, the mutant BGL outperformed both wild-type and commercial BGLs, efficiently releasing terpene and phenolic aglycones from glycosides in wine grapes. These findings not only expand our understanding of O. oeni BGLs but also highlight their potential in enhancing wine production. The mutant BGL's enhanced adaptation to wine stress conditions opens promising avenue for improving wine quality and flavor.

Alcoholic fermentation drives the selection of Oenococcus oeni strains in wine but not in cider.

Oenococcus oeni is the predominant lactic acid bacteria species in wine and cider, where it performs the malolactic fermentation (MLF). The O. oeni strains analyzed to date form four major genetic lineages named phylogroups A, B, C and D. Most of the strains isolated from wine, cider, or kombucha belong to phylogroups A, B + C, and D, respectively, although B and C strains were also detected in wine. This study was performed to better understand the distribution of the phylogroups in wine and cider. Their population dynamics were determined by qPCR all through wine and cider productions, and the behavior of the strains was analyzed in synthetic wines and ciders. Phylogroups A, B and C were all represented in grape must and throughout the alcoholic fermentation, but on the transition to MLF, only phylogroup A remained at high levels in all wine productions. In the case of cider, phylogroups A, B and C were detected in stable levels during the process. When they were tested in synthetic wine and cider, all phylogroups performed MLF, but with different survival rates depending on the ethanol content. In this sense, ethanol and fermentation kinetics are the main agent that drives the selection of phylogroup A strains in wine, while B and C strains dominates in cider containing less ethanol.

Malolactic fermentation of lactic acid bacteria isolated from southern Brazilian red wine.

The objective was to isolate lactic acid bacteria (LAB) from southern Brazil's wines and investigate their potential as starter cultures for malolactic fermentation (MLF) in Merlot (ME) and Cabernet Sauvignon (CS) wines through the fermentative capacity. The LAB were isolated from CS, ME, and Pinot Noir (PN) wines in the 2016 and 2017 harvests and evaluated for morphological (color and shape of the colonies), genetic, fermentative (increase in pH, acidity reduction, preservation of anthocyanins, decarboxylation of L-malic acid, yield of L-lactic acid, and content of reduced sugars), and sensory characteristics. Four strains were identified as Oenococcus oeni [CS(16)3B1, ME(16)1A1, ME(17)26, and PN(17)65], one as Lactiplantibacillus plantarum [PN(17)75], and one as Paucilactobacillus suebicus [CS(17)5]. Isolates were evaluated in the MLF and compared to a commercial strain (O. oeni), as well as a control (without inoculation and spontaneous MLF), and standard (without MLF). CS(16)3B1 and ME(17)26 isolates finished the MLF for CS and ME wines, respectively, after 35 days, similar to the commercial strain, and CS(17)5 and ME(16)1A1 isolates ended the MLF in 45 days. In the sensory analysis, ME wines with isolated strains received better scores for flavor and overall quality than the control. Compared to the commercial strain, CS(16)3B1 isolate obtained the highest scores for buttery flavor and taste persistence. CS(17)5 isolate received the higher scores for a fruity flavor and overall quality and the lowest for a buttery flavor. The native LAB displayed MLF potential, regardless of the year and grape species from which they were isolated.

Protein content of the Oenococcus oeni extracellular vesicles-enriched fraction.

Malolactic fermentation is essential for the quality of red wines and some other wine styles. Spontaneous malolactic fermentation is often driven by Oenococcus oeni, and commercial starters for this purpose are also often of this species. The increasing number of microbial species and inoculation strategies in winemaking has prompted a growing interest in microbial interactions during wine fermentation. Among other interaction mechanisms, extracellular vesicles have been hypothesized to play a role in this context. Extracellular vesicles have already been described and analysed for several wine yeast species. In this work, the production of extracellular vesicles by O. oeni is reported for the first time. The protein content of these extracellular vesicles is also characterised. It shows differences and similarities with the recently described protein content of Lactiplantibacillus plantarum, a bacterial species also capable of performing malolactic fermentation of wine (and used sometimes as an alternative starter). This work further contributes to the development of the field of extracellular vesicles in food biotechnology.

Comparative functional analysis of malate metabolism genes in Oenococcus oeni and Lactiplantibacillus plantarum at low pH and their roles in acid stress response.

Oenococcus oeni and Lactiplantibacillus plantarum are major wine-associated lactic acid bacteria that positively influence wine by carrying out malolactic fermentation. O. oeni is the most widely used commercial starter in winemaking because of its fast and efficient malate metabolism capacity under harsh wine conditions. To date, very little is known about the specific molecular mechanism underlying the differences in malate metabolism between O. oeni and L. plantarum under harsh wine conditions. Therefore, in this study, the functions of genes encoding malic enzyme (ME) and malolactic enzyme (MLE) under acid stress in O. oeni and L. plantarum, previously described to have the ability to direct malate metabolism, were comparatively verified through genetic manipulation in L. plantarum. Results showed that the MLE was the only enzyme responsible for direct malate metabolism under acid stress in O. oeni and L. plantarum. In addition, the MLEs in O. oeni and L. plantarum were positively related to acid tolerance by metabolizing malate and increasing the medium pH. Furthermore, the MLE in O. oeni exhibited significantly higher malate metabolism activity than that in L. plantarum under acid stress.

Genomic Analysis of an Excellent Wine-Making Strain Oenococcus oeni SD-2a.

Oenococcus oeni is an important microorganism in wine-making-related engineering, and it improves wine quality and stability through malolactic fermentation. Although the genomes of more than 200 O. oeni strains have been sequenced, only a few include completed genome maps. Here, the genome sequence of O. oeni SD-2a, isolated from Shandong, China, has been determined. It is a fully assembled genome sequence of this strain. The complete genome is 1,989,703 bp with a G+C content of 37.8% without a plasmid. The genome includes almost all the essential genes involved in central metabolic pathways and the stress genes reported in other O. oeni strains. Some natural competence-related genes, like comEA, comEC, comFA, comG operon, and comFC, suggest that O. oeni SD-2a may have natural transformation potential. A comparative genomics analysis revealed 730 gene clusters in O. oeni SD-2a homologous to those in four other lactic acid bacteria species (O. oeni PSU-1, O. oeni CRBO-11381, Lactiplantibacillus plantarum UNQLp11, and Pediococcus pentosaceus KCCM40703). A collinearity analysis showed poor collinearity between O. oeni SD-2a and O. oeni PSU-1, indicating great differences in their evolutionary histories. The results provide general knowledge of O. oeni SD-2a and lay the foundation for specific gene function analyses.

Assessment of the lysogenic status in the lactic acid bacterium O. oeni during the spontaneous malolactic fermentation of red wines.

After alcoholic fermentation, most wines undergo malolactic fermentation (MLF), driven by the lactic acid bacterium Oenococcus oeni, which improves their organoleptic properties and microbiological stability. Prophages were recently shown to be notably diverse and widely disseminated in O. oeni genomes. Such in silico predictions confirmed previous cultivation-based approaches which showed frequent lysis of strains upon treatment with the inducing agent mitomycin C. Both strategies used to assess lysogeny in the species were so far applied to a number of strains collected from distinct countries, wineries, cepages and fermentation processes. Results may not therefore be representative of the lysogenic population in natural communities driving the MLF during winemaking. Here we report the prevalence of lysogeny during winemaking in three wineries in the Bordeaux area. The dominant LAB population was collected in 11 red wines upon completion of MLF. Using VNTR and prophage typing analyses, our data confirm the presence of lysogens in the population driving the spontaneous MLF in all tested wines, although lysogeny rates varied across wineries. Higher prevalence of lysogeny was associated to a reduced diversity in VNTR profiles, the dominance of a few prophage-types and presence of some bacterial genetic backgrounds that were particularly prone to lysogenization.

Molecular adaptation response of Oenococcus oeni in non-Saccharomyces fermented wines: A comparative multi-omics approach.

Oenococcus oeni is the main agent responsible for malolactic fermentation (MLF) in wine. This usually takes place in red wines after alcoholic fermentation (AF) carried out by Saccharomyces cerevisiae. In recent years, there is an increasing interest in using non-Saccharomyces yeast, usually in combination with S. cerevisiae, to improve wine quality. Current studies report a stimulatory effect of non-Saccharomyces on MLF, generally related to a decrease in the inhibitor compounds found in wine. In this work, we followed a comparative multi-omics approach, including transcriptomic and proteomic analysis, to study the molecular adaptation of O. oeni in wines fermented with Torulaspora delbrueckii and Metschnikowia pulcherrima, two of the most frequently used non-Saccharomyces, in sequential inoculation with S. cerevisiae. We compared the results to the adaptation of O. oeni in S. cerevisiae wine to determine the main changes arising from the use of non-Saccharomyces. The duration of MLF was shortened when using non-Saccharomyces, to half the time with T. delbrueckii and to a quarter with M. pulcherrima. In this work, we observed for the first time how O. oeni responds at molecular level to the changes brought about by non-Saccharomyces. We showed a differential adaptation of O. oeni in the wines studied. In this regard, the main molecular functions affected were amino acid and carbohydrate transport and metabolism, from which peptide metabolism appeared as a key feature under wine-like conditions. We also showed that the abundance of Hsp20, a well-known stress protein, depended on the duration time. Thus, the use of non-Saccharomyces reduced the abundance of Hsp20, which could mean a less stressful wine-like condition for O. oeni.

Integrative multiomics analysis of the acid stress response of Oenococcus oeni mutants at different growth stages.

BACKGROUND: Acid stress is one of the most important environmental stresses that adversely affect the growth of lactic acid bacteria (LAB), such as Oenococcus oeni which was isolated from grape-berries and mainly used in wine fermentation. The aim of this paper is to comprehensively characterize the mechanisms of acid stress regulation in O. oeni and to provide a viable theoretical basis for breed and improvement of existing LAB. METHOD: First, six O. oeni mutants with acid-sensitive (strains b2, a1, c2) and acid-tolerant (strains b1, a3, c1) phenotypes were screened from three wild-type O. oeni, and then their genome (sequencing), transcriptome and metabolome (LC-MS/MS) were examined. RESULTS: A total of 459 genes were identified with one or more intragenic single nucleotide polymorphisms (SNPs) in these mutants, and were extensively involved in metabolism and cellular functions with a high mutation rates in purine (46%) and pyrimidine (48%) metabolic pathways. There were 210 mutated genes that cause significant changes in expression levels. In addition, 446 differentially accumulated metabolites were detected, and they were consistently detected at relatively high levels in the acid-tolerant O. oeni mutant. The levels of intracellular differentially expressed genes and differential metabolites changed with increasing culture time. CONCLUSION: The integrative pathways analysis showed that the intracellular response associated with acid regulation differed significantly between acid-sensitive and acid-tolerant O. oeni mutants, and also changed at different growth stages.

Phage Encounters Recorded in CRISPR Arrays in the Genus Oenococcus.

The Oenococcus genus comprises four recognized species, and members have been found in different types of beverages, including wine, kefir, cider and kombucha. In this work, we implemented two complementary strategies to assess whether oenococcal hosts of different species and habitats were connected through their bacteriophages. First, we investigated the diversity of CRISPR-Cas systems using a genome-mining approach, and CRISPR-endowed strains were identified in three species. A census of the spacers from the four identified CRISPR-Cas loci showed that each spacer space was mostly dominated by species-specific sequences. Yet, we characterized a limited records of potentially recent and also ancient infections between O. kitaharae and O. sicerae and phages of O. oeni, suggesting that some related phages have interacted in diverse ways with their Oenococcus hosts over evolutionary time. Second, phage-host interaction analyses were performed experimentally with a diversified panel of phages and strains. None of the tested phages could infect strains across the species barrier. Yet, some infections occurred between phages and hosts from distinct beverages in the O. oeni species.

Analysis of Transcriptomic Response to SO2 by Oenococcus oeni Growing in Continuous Culture.

To successfully complete malolactic fermentation (MLF), Oenococcus oeni must overcome wine stress conditions of low pH, high ethanol, and the presence of SO2. Failure to complete MLF may result in detrimental effects to the quality and stability of the resulting wines. Research efforts to date have focused on elucidating the mechanisms and genetic features that confer the ability to withstand low pH and high ethanol concentrations on O. oeni; however, the responses to SO2 stress are less well defined. This study focused on characterizing the transcriptional response of O. oeni to SO2 challenge during cultivation in a continuous system at wine-like pH (3.5). This experimental design allowed the precise discrimination of transcriptional changes linked to SO2 stress from responses associated with growth stage and cultivation parameters. Differential gene expression analysis revealed major transcriptional changes following SO2 exposure and suggested that this compound primarily interacts with intracellular proteins, DNA, and the cell envelope of O. oeni. The molecular chaperone hsp20, which has a demonstrated function in the heat, ethanol, and acid stress response, was highly upregulated, confirming its additional role in the response of this species to SO2 stress. This work also reports the first nanopore-based complete genome assemblies for O. oeni. IMPORTANCE Malolactic fermentation is an indispensable step in the elaboration of most wines and is generally performed by Oenococcus oeni, a Gram-positive heterofermentative lactic acid bacterium species. While O. oeni is tolerant to many of the wine stresses, including low pH and high ethanol concentrations, it has high sensitivity to SO2, an antiseptic and antioxidant compound regularly used in winemaking. Understanding the physiological changes induced in O. oeni by SO2 stress is essential for the development of more robust starter cultures and methods for their use. This study describes the main transcriptional changes induced by SO2 stress in the wine bacterium O. oeni and provides foundational understanding on how this compound interacts with the cellular components and the induced protective mechanisms of this species.

Metagenomic and phytochemical analyses of kefir water and its subchronic toxicity study in BALB/c mice.

BACKGROUND: In recent years, researchers are interested in the discovery of active compounds from traditional remedies and natural sources, as they reveal higher therapeutic efficacies and improved toxicological profiles. Among the various traditional treatments that have been widely studied and explored for their potential therapeutic benefits, kefir, a fermented beverage, demonstrates a broad spectrum of pharmacological properties, including antioxidant, anti-inflammation, and healing activities. These health-promoting properties of kefir vary among the kefir cultures found at the different part of the world as different media and culture conditions are used for kefir maintenance and fermentation. METHODS: This study investigated the microbial composition and readily found bioactive compounds in water kefir fermented in Malaysia using 16S rRNA microbiome and UHPLC sequencing approaches. The toxicity effects of the kefir water administration in BALB/c mice were analysed based on the mice survival, body weight index, biochemistry profile, and histopathological changes. The antioxidant activities were evaluated using SOD, FRAP, and NO assays. RESULTS: The 16S rRNA amplicon sequencing revealed the most abundant species found in the water kefir was Lactobacillus hilgardii followed by Lactobacillus harbinensis, Acetobacter lovaniensis, Lactobacillus satsumensis, Acetobacter tropicalis, Lactobacillus zeae, and Oenococcus oeni. The UHPLC screening showed flavonoid and phenolic acid derivatives as the most important bioactive compounds present in kefir water which has been responsible for its antioxidant activities. Subchronic toxicity study showed no toxicological signs, behavioural changes, or adverse effects by administrating 10 mL/kg/day and 2.5 mL/kg/day kefir water to the mice. Antioxidants assays demonstrated enhanced SOD and FRAP activities and reduced NO level, especially in the brain and kidney samples. CONCLUSIONS: This study will help to intensify the knowledge on the water kefir microbial composition, available phytochemicals and its toxicological and antioxidant effects on BALB/c mice since there are very limited studies on the water kefir grain fermented in Malaysia.

Altered Metabolic Strategies: Elaborate Mechanisms Adopted by Oenococcus oeni in Response to Acid Stress.

Oenococcus oeni plays a key role in inducing malolactic fermentation in wine. Acid stress is often encountered under wine conditions. However, the lack of systematic studies of acid resistance mechanisms limits the downstream fermentation applications. In this study, the acid responses of O. oeni were investigated by combining transcriptome, metabolome, and genome-scale metabolic modeling approaches. Metabolite profiling highlighted the decreased abundance of nucleotides under acid stress. The gene-metabolite bipartite network showed negative correlations between nucleotides and genes involved in ribosome assembly, translation, and post-translational processes, suggesting that stringent response could be activated under acid stress. Genome-scale metabolic modeling revealed marked flux rerouting, including reallocation of pyruvate, attenuation of glycolysis, utilization of carbon sources other than glucose, and enhancement of nucleotide salvage and the arginine deiminase pathway. This study provided novel insights into the acid responses of O. oeni, which will be useful for designing strategies to address acid stress in wine malolactic fermentation.

Influences of acid and ethanol stresses on Oenococcus oeni SD-2a and its proteomic and transcriptional responses.

BACKGROUND: During winemaking, malolactic fermentation (MLF) is usually induced by Oenococcus oeni owing to its high resistance to wine stress factors. To ensure a controlled and efficient MLF process, starter cultures are inoculated in wine. In previous studies, O. oeni strains with sub-lethal acid or ethanol stresses showed higher freeze-drying vitality and better MLF performance. To explore the mechanisms involved, influences of acid and ethanol stresses on O. oeni SD-2a were investigated in this study to gain a better understanding of the cross-protection responses. RESULTS: The results showed that acid and ethanol stresses both caused damage to cell membranes and decreased cellular adenosine triphosphate concentration. At the same time, acid stress increased the uptake of glutathione, while ethanol stress led to cell depolarization. The results of comparative proteomic analysis highlighted that heat shock protein was induced with almost all acid and ethanol stresses. In addition, the expression of stress-relevant genes (hsp20, clpP, trxA, ctsR, recO, usp) increased greatly with ethanol and acid stress treatments. Finally, the viability of O. oeni was improved with acid and ethanol pretreatments after freeze-drying. CONCLUSIONS: This study demonstrated that acid and ethanol stresses had mixed influences on O. oeni SD-2a. Some physiological and molecular changes would contribute to a more stress-tolerant state of O. oeni, thereby improving the viability of lyophilized cells. © 2020 Society of Chemical Industry.

Assessment of ß-D-glucosidase activity and bgl gene expression of Oenococcus oeni SD-2a.

Glycosidases enhance flavor during wine-making by mediating the enzymatic release of aroma molecules. In order to better understand the aroma enhancement potential of Oenococcus oeni SD-2a, ß-D-glucosidase (ßG) activities in the culture supernatant, whole cells, and disrupted cell lysate were assessed at mid log, late log and stationary growth phase. The enzymatic activity was also compared further from cell cultures with 5 different carbon sources (glucose, cellobiose, arbutin, glucose and cellobiose, glucose and arbutin) at late log phase. Correspondingly, expression levels of 3 bgl genes, OEOE-0224, OEOE-1210, and OEOE-1569 were investigated from cell cultures of the 3 growth phases, and the 5 cell cultures with different carbon sources. Finally, the volatile aroma compounds released by O. oeni SD-2a in synthetic wines with natural glycosides were evaluated by GC-MS. Results showed ßG of O. oeni SD-2a was not extracellular enzyme, and the location of it didn't change with the change of growth phase and carbon source studied. ßG activities in the whole cells and disrupted cell lysate were similar and constant at the 3 growth phases. As for the carbon sources, ßG activities of whole cells and disrupted lysate were positively affected by cellobiose. While arbutin displayed positive and negative effect on ßG activity of whole cells and disrupted lysate, respectively. It is probably that bgl genes OEOE-0224 and OEOE-1210 were related to ßG activity of SD-2a whole cells, while OEOE-1569 was responsible for ßG activity of disrupted lysate. More kinds of volatile compounds and higher total concentration were released by SD-2a in synthetic wine compared with control. Thus, SD-2a showed a great potential for flavor enhancement under wine-like conditions. This study provides more information for further study of ßG activity from O. oeni SD-2a.

Identification and Characterization of the Small Heat Shock Protein Hsp20 from Oenococcus oeni SD-2a.

Oenococcus oeni can exert its function in hostile wine conditions during the malolactic fermentation process. Therefore, it is an important microbial resource for exploring resistance genes. Hsp20 is an important small heat shock protein from O. oeni. The conserved consensus motif "A-x-x-x-x-G-x-L" of Hsp20 announced its role as a member of the small heat shock protein family. The hsp20 gene from O. oeni SD-2a was cloned to create the recombinant plasmid pTriEx-Hsp20. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the Hsp20 protein was induced by isopropyl-ß-D-thiogalactoside (IPTG). The hsp20 gene from O. oeni SD-2a was successfully expressed, and a 20-kDa fusion protein was identified by SDS-PAGE. The purified Hsp20 protein was obtained using Ni-affinity chromatography. Additionally, BL21(DE3)/Hsp20 and BL21(DE3)/Ctrl were treated at high temperatures of 42 and 52 °C, at pH values of 2.0-12.0, under oxidative shock with 0.1% (v/v) and 0.2% (v/v) H2O2, and under an osmotic shock of 430 and 860 mM NaCl to compare the effects of heterologous expression of the Hsp20 protein from O. oeni SD-2a for stress resistance. Notably, Hsp20 overexpression showed enhanced resistance than the control strain did when confronted with different elevated stress conditions. The results demonstrated heterologous expression of the hsp20 gene from O. oeni SD-2a significantly improved the resistance of the host E. coli bacteria against stress conditions.

The influence of different strains of Oenococcus oeni malolactic bacteria on profile of organic acids and phenolic compounds of red wine cultivars Rondo and Regent growing in a cold region.

Wines produced from grapes cultivated in cool climate areas are characterized by high levels of organic acids. One method to correct this is malolactic fermentation (MLF). The aim of this study was to determine the effectiveness of different strains of Oenococcus oeni bacteria (Viniflora CH11, Viniflora CH16, Viniflora CH35, Viniflora Oenos, SIHA LACT Oeno) during the biological acidity reduction process. Red wine from Rondo and Regent cultivars was obtained by ethanol fermentation of the pulp, at 20 °C for 14 days. The profile of organic acids was examined with a particular focus on changes in the content of l-malic and l-lactic acids. Additionally, the impact on profile and quantity of phenolic compounds and antioxidant capacity was measured. The results showed that MLF had a positive influence on content of organic acids through the reduction of l-malic acid content with a simultaneous increase of the amount of l-lactic acid. The best effect was obtained with the CH11 and CH35 bacterial strains. The biological acidity reduction process had no significant (P > 0.05) impact on phenolic content or antioxidant capacity. However, the wine making process (ethanol fermentation, maturation) contributed to the reduction of polyphenols and in consequence lower antioxidant capacity of the final tested wines. PRACTICAL APPLICATION: The present study provides useful information on the impact of different Oenococcus oeni bacterial strains on MLF in red wines, reduction of l-malic to l-lactic acid, and stability of phenolic compounds during MLF and the maturation period. Also, this article provides information about phenolic compounds and antioxidant capacity during malolactic fermentation and maturity of red wines made from hybrids of Vitis vinifera such as Rondo and Regent cultivars.

The metagenome-assembled genome of Candidatus Oenococcus aquikefiri from water kefir represents the species Oenococcus sicerae.

Recently, a metagenomic study of a water kefir fermentation ecosystem enabled the reconstruction of a metagenome-assembled genome (MAG) of an Oenococcus species that was different from the three species of this genus known so far. Therefore, the name Candidatus Oenococcus aquikefiri was proposed for this novel Oenococcus species. In the meantime, however, a fourth member of the genus, Oenococcus sicerae, isolated from French cider, was reported. The comparison of its genome sequence with the Candidatus O. aquikefiri MAG showed an average nucleotide identity (ANI) value of 98.53%. In addition, the 16S rRNA and pheS genes of the two species were 99.4% and 99.9% identical, respectively. As the presence of O. sicerae in a water kefir metagenome was also revealed by metagenomic recruitment plotting, it can be stated that Candidatus O. aquikefiri and O. sicerae belong to the same species. Intraspecies variations include the presence or absence of a citrate lyase operon and components of various phosphotransferase (PTS) transport systems.

Heterologous expression of the puuE from Oenococcus oeni SD-2a in Lactobacillus plantarum WCFS1 improves ethanol tolerance.

Oenococcus oeni is the main bacteria extensively used in malolactic fermentation due to its high tolerance against stress factors in wine production. Among these, ethanol is one of the main challenges to O. oeni, and its ethanol tolerance mechanism remains unclear. In this study, the puuE gene related to ethanol tolerance from O. oeni SD-2a was heterologously expressed in Lactobacillus plantarum WCFS1. Results showed that the recombinant strain (W-pMG36epuuE) exhibited better growth performance and survival rate compared to the control strain (W-pMG36e) under ethanol-stress conditions. In addition, it was found that the activities of superoxide dismutase and the concentration of glutathione of W-pMG36epuuE were significantly higher than those of W-pMG36e. This resulted in the decrease of intracellular reactive oxygen species (ROS) accumulation (10.34% lower than control). Moreover, heterologous expression of puuE in WCFS1 exhibited improved activities of two ATPases in membrane, increasing the cell membrane integrity (37.67% higher than control). These results revealed the role of the puuE gene in improving ethanol tolerance in O. oeni by decreasing ROS accumulation and enhancing cell membrane integrity.