Heterologous expression of the Oenococcus oeni two-component signal transduction response regulator in the Lactiplantibacillus plantarum WCFS1 strain enhances acid stress tolerance.

BACKGROUND: Oenococcus oeni is a commercial wine-fermenting bacterial strain, owing to its high efficiency of malolactic fermentation and stress tolerance. The present study explored the function of key genes in O. oeni to enhance stress resistance by heterologous expression of these genes in another species. RESULTS: The orf00404 gene that encodes a two-component signal transduction response regulator in O. oeni was heterologously expressed in Lactiplantibacillus plantarum WCFS1. The expression of orf00404 significantly enhanced the growth rate of the recombinant strain under acid stress. At 60 h, 72 h, and 108 h of culture at pH 4.0, the recombinant strain had 1562, 641, and 748 differentially expressed genes compared to the control strain, respectively. At all three time points, 20 genes were upregulated in the recombinant strain, including the lamA-D operon-coding genes of the quorum-sensing two component signal transduction system and the spx5 RNA polymerase-binding protein coding gene, which may help adaptation to acid stress. In addition, 47 genes were downregulated in the recombinant strain at all three time points, including the hsp1 heat shock protein-coding gene, the trxA1 thioredoxin-coding gene, and the dinP, mutY, umuC, and uvrB DNA damage repair-related protein-coding genes, potentially indicating that the recombinant strain was less susceptible to stress and had less DNA damage than the control strain in acid stress conditions. The recombinant strain had higher membrane fluidity, permeability, and integrity at an early stage of logarithmic growth (72 h), suggesting that it had a more complete and active cell membrane state at this stage. The intracellular ATP content was significantly reduced in the recombinant strain at the beginning of logarithmic growth (60 h), implying that the recombinant strain consumed more energy at this stage to resist acid stress and growth. CONCLUSIONS: These results indicated that the recombinant strain enhances acid stress tolerance by regulating a gene expression pattern, increasing ATP consumption, and enhancing cell membrane fluidity, membrane permeability, and membrane integrity at specific growth stages. Thus, the recombinant strain may have potential application in the microbial biotechnology industry.

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.

Biochemical and Structural Characterization of a Novel Psychrophilic Laccase (Multicopper Oxidase) Discovered from Oenococcus oeni 229 (ENOLAB 4002).

Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K4[Fe(CN6)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments.

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.

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.

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.

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.

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.

The Expansion of Inosine at the Wobble Position of tRNAs, and Its Role in the Evolution of Proteomes.

The modification of adenosine to inosine at the first position of transfer RNA (tRNA) anticodons (I34) is widespread among bacteria and eukaryotes. In bacteria, the modification is found in tRNAArg and is catalyzed by tRNA adenosine deaminase A, a homodimeric enzyme. In eukaryotes, I34 is introduced in up to eight different tRNAs by the heterodimeric adenosine deaminase acting on tRNA. This substrate expansion significantly influenced the evolution of eukaryotic genomes in terms of codon usage and tRNA gene composition. However, the selective advantages driving this process remain unclear. Here, we have studied the evolution of I34, tRNA adenosine deaminase A, adenosine deaminase acting on tRNA, and their relevant codons in a large set of bacterial and eukaryotic species. We show that a functional expansion of I34 to tRNAs other than tRNAArg also occurred within bacteria, in a process likely initiated by the emergence of unmodified A34-containing tRNAs. In eukaryotes, we report on a large variability in the use of I34 in protists, in contrast to a more uniform presence in fungi, plans, and animals. Our data support that the eukaryotic expansion of I34-tRNAs was driven by the improvement brought by these tRNAs to the synthesis of proteins highly enriched in certain amino acids.

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 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.

Expanding the biodiversity of Oenococcus oeni through comparative genomics of apple cider and kombucha strains.

BACKGROUND: Oenococcus oeni is a lactic acid bacteria species adapted to the low pH, ethanol-rich environments of wine and cider fermentation, where it performs the crucial role of malolactic fermentation. It has a small genome and has lost the mutS-mutL DNA mismatch repair genes, making it a hypermutable and highly specialized species. Two main lineages of strains, named groups A and B, have been described to date, as well as other subgroups correlated to different types of wines or regions. A third group "C" has also been hypothesized based on sequence analysis, but it remains controversial. In this study we have elucidated the species population structure by sequencing 14 genomes of new strains isolated from cider and kombucha and performing comparative genomics analyses. RESULTS: Sequence-based phylogenetic trees confirmed a population structure of 4 clades: The previously identified A and B, a third group "C" consisting of the new cider strains and a small subgroup of wine strains previously attributed to group B, and a fourth group "D" exclusively represented by kombucha strains. A pair of complete genomes from group C and D were compared to the circularized O. oeni PSU-1 strain reference genome and no genomic rearrangements were found. Phylogenetic trees, K-means clustering and pangenome gene clusters evidenced the existence of smaller, specialized subgroups of strains. Using the pangenome, genomic differences in stress resistance and biosynthetic pathways were found to uniquely distinguish the C and D clades. CONCLUSIONS: The obtained results, including the additional cider and kombucha strains, firmly established the O. oeni population structure. Group C does not appear as fully domesticated as group A to wine, but showed several unique patterns which may be due to ongoing specialization to the cider environment. Group D was shown to be the most divergent member of O. oeni to date, appearing as the closest to a pre-domestication state of the species.

Lysogeny in the Lactic Acid Bacterium Oenococcus oeni Is Responsible for Modified Colony Morphology on Red Grape Juice Agar.

Oenococcus oeni is the lactic acid bacterium (LAB) that most commonly drives malolactic fermentation in wine. Although oenococcal prophages are highly prevalent, their implications on bacterial fitness have remained unexplored and more research is required in this field. An important step toward achieving this goal is the ability to produce isogenic pairs of strains that differ only by the lysogenic presence of a given prophage, allowing further comparisons of different phenotypic traits. A novel protocol for the rapid isolation of lysogens is presented. Bacteria were first picked from the center of turbid plaques produced by temperate oenophages on a sensitive nonlysogenic host. When streaked onto an agar medium containing red grape juice (RGJ), cells segregated into white and red colonies. PCR amplifications with phage-specific primers demonstrated that only lysogens underwent white-red morphotypic switching. The method proved successful for various oenophages irrespective of their genomic content and attachment site used for site-specific recombination in the bacterial chromosome. The color switch was also observed when a sensitive nonlysogenic strain was infected with an exogenously provided lytic phage, suggesting that intracolonial lysis triggers the change. Last, lysogens also produced red colonies on white grape juice agar supplemented with polyphenolic compounds. We posit that spontaneous prophage excision produces cell lysis events in lysogenic colonies growing on RGJ agar, which, in turn, foster interactions between lysed materials and polyphenolic compounds to yield colonies easily distinguishable by their red color. Furthermore, the technique was used successfully with other species of LAB.IMPORTANCE The presence of white and red colonies on red grape juice (RGJ) agar during enumeration of Oenococcus oeni in wine samples is frequently observed by stakeholders in the wine industry. Our study brings an explanation for this intriguing phenomenon and establishes a link between the white-red color switch and the lysogenic state of O. oeni It also provides a simple and inexpensive method to distinguish between lysogenic and nonlysogenic derivatives in O. oeni with a minimum of expended time and effort. Noteworthy, the protocol could be adapted to two other species of LAB, namely, Leuconostoc citreum and Lactobacillus plantarum It could be an effective tool to provide genetic, ecological, and functional insights into lysogeny and aid in improving biotechnological processes involving members of the lactic acid bacterium (LAB) family.

Distribution of Oenococcus oeni populations in natural habitats.

Oenococcus oeni is the lactic acid bacteria species most commonly encountered in wine, where it develops after the alcoholic fermentation and achieves the malolactic fermentation that is needed to improve the quality of most wines. O. oeni is abundant in the oenological environment as well as in apple cider and kombucha, whereas it is a minor species in the natural environment. Numerous studies have shown that there is a great diversity of strains in each wine region and in each product or type of wine. Recently, genomic studies have shed new light on the species diversity, population structure, and environmental distribution. They revealed that O. oeni has unique genomic features that have contributed to its fast evolution and adaptation to the enological environment. They have also unveiled the phylogenetic diversity and genomic properties of strains that develop in different regions or different products. This review explores the distribution of O. oeni and the diversity of strains in natural habitats.

Increased ethanol tolerance associated with the pntAB locus of Oenococcus oeni and Lactobacillus buchneri.

Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that was absent in almost all other lactic acid bacteria (LAB) genomes. Our hypothesis is that the pntAB locus contributes to the ethanol tolerance trait of these two distinct ethanol-tolerant organisms. The pntAB locus, consisting of the pntA and pntB genes, codes for NADP(H) transhydrogenase subunits. This membrane-bound transhydrogenase catalyzes the reduction of NADP+ and is known as an important enzyme in maintaining cellular redox balance. In this study, the transhydrogenase operon from L. buchneri NRRL B-30929 and O. oeni PSU-1 were cloned and analyzed. The LbpntB shared 71.0% identity with the O. oeni (OopntB). The entire pntAB locus was expressed in Lactococcus lactis ssp. lactis IL1403 resulting in an increased tolerance to ethanol (6%), butanol (1.8%) and isopropanol (1.8%) when compared to the control strain. However, the recombinant E. coli cells carrying the entire pntAB locus did not show any improved ethanol tolerance. Independent expression of OopntB and LbpntB in recombinant E. coli BL21(DE3)pLysS host demonstrated higher tolerance to ethanol when compared with a control E. coli BL21(DE3)pLysS strain carrying pET28b vector. Ethanol tolerance comparison of E. coli strains carrying LbpntB and OopntB showed that LbpntB conferred higher ethanol tolerance (4.5%) and resulted in greater biomass, while the OopntB conferred lower ethanol tolerance (4.0%) resulted lower biomass. Therefore, the pntB gene from L. buchneri is a better choice in generating higher ethanol tolerance. This is the first study to uncover the role of pntAB locus on ethanol tolerance.

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.

Adaptation of two groups of Oenococcus oeni strains to red and white wines: the role of acidity and phenolic compounds.

AIMS: Oenococcus oeni is the lactic acid bacteria species which is the most adapted to wine. Recently, two groups of strains that form two genetic lineages were described in red and white Burgundy wines. The aim of this study was to analyse the phenotypes of these strains in order to determine how they have adapted specifically to either red or white wine. METHODS AND RESULTS: Four strains from each group were tested in grape must and in wines to evaluate their tolerance to pH and to phenolic compound content. White wine strains proved to be the most tolerant to low pH, both in grape must and in wine, whereas they were inhibited by the presence of grape tannins in wine. Red wine strains were more sensitive to acidity, but very resistant to phenolic compounds. CONCLUSIONS: The results suggest that pH and phenolic compounds drive strain selection at several stages of wine production. SIGNIFICANCE AND IMPACT OF THE STUDY: Although it is well known that O. oeni is well adapted to wine, this study shows that strains of some genetic lineages within this species have evolved to adapt better than others to specific types of wines.

Ethanol stress in Oenococcus oeni: transcriptional response and complex physiological mechanisms.

Oenococcus oeni is the dominant species able to cope with a hostile environment of wines, comprising cumulative effects of low pH, high ethanol and SO2 content, nonoptimal growth temperatures and growth inhibitory compounds. Ethanol tolerance is a crucial feature for the activity of O. oeni cells in wine because ethanol acts as a disordering agent of its cell membrane and negatively affects metabolic activity; it damages the membrane integrity, decreases cell viability and, as other stress conditions, delays the start of malolactic fermentation with a consequent alteration of wine quality. The cell wall, cytoplasmic membrane and metabolic pathways are the main sites involved in physiological changes aimed to ensure an adequate adaptive response to ethanol stress and to face the oxidative damage caused by increasing production of reactive oxygen species. Improving our understanding of the cellular impact of ethanol toxicity and how the cell responds to ethanol stress can facilitate the development of strategies to enhance microbial ethanol tolerance; this allows to perform a multidisciplinary endeavour requiring not only an ecological study of the spontaneous process but also the characterization of useful technological and physiological features of the predominant strains in order to select those with the highest potential for industrial applications.

Application of directed evolution to develop ethanol tolerant Oenococcus oeni for more efficient malolactic fermentation.

Malolactic fermentation (MLF) is an important step in winemaking, which can be notoriously unreliable due to the fastidious nature of Oenococcus oeni. This study aimed to use directed evolution (DE) to produce a more robust strain of O. oeni having the ability to withstand high ethanol concentrations. DE involves an organism mutating and potentially adapting to a high stress environment over the course of extended cultivation. A continuous culture of O. oeni was established and exposed to progressively increasing ethanol content such that after approximately 330 generations, an isolate from this culture was able to complete MLF in high ethanol content medium earlier than its parent. The ethanol tolerance of a single isolate, A90, was tested to confirm the phenotype and its fermentation performance in wine. In order to investigate the genotypic differences in the evolved strain that led to the ethanol tolerance phenotype, the relative expression of a number of known stress response genes was compared between SB3 and A90. Notably, there was increase in hsp18 expression in 20% (v/v) ethanol by both strains with A90 exhibiting a higher degree of expression. This study is the first to use directed evolution for O. oeni strain improvement and confirms that this technique can be used successfully for the development of new candidate strains for the wine industry. This study also adds to the current knowledge on the genetic basis of ethanol tolerance in this bacterium.

Directed evolution of Oenococcus oeni strains for more efficient malolactic fermentation in a multi-stressor wine environment.

High concentrations of ethanol, low pH, the presence of sulfur dioxide and some polyphenols have been reported to inhibit Oenococcus oeni growth, thereby negatively affecting malolactic fermentation (MLF) of wine. In order to generate superior O. oeni strains that can conduct more efficient MLF, despite these multiple stressors, a continuous culture approach was designed to directly evolve an existing ethanol tolerant O. oeni strain, A90. The strain was grown for ∼350 generations in a red wine-like environment with increasing levels of stressors. Three strains were selected from screening experiments based on their completion of fermentation in a synthetic wine/wine blend with 15.1% (v/v) ethanol, 26 mg/L SO2 at pH 3.35 within 160 h, while the parent strain fermented no more than two thirds of l-malic acid in this medium. These superior strains also fermented faster and/or had a larger population in four different wines. A reduced or equivalent amount of the undesirable volatile, acetic acid, was produced by the optimised strains compared to a commercial strain in Mouvedre and Merlot wines. These findings demonstrate the feasibility of using directed evolution as a tool to generate more efficient MLF starters tailored for wines with multiple stressors.