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.

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Distribution of Oenococcus oeni populations in natural habitats

The effect of ethanol on the metabolism of Oenococcus oeni, the bacterium responsible for the malolactic fermentation of wine, is still scarcely understood. Here, we characterized the global metabolic response in O. oeni to increasing ethanol contents, ranging from 0 to 12% (v/v). We first optimized a wine-like, defined culture medium, MaxOeno, to allow sufficient bacterial growth to be able to quantitate different metabolites in batch cultures of O. oeni. Then, taking advantage of the recently reconstructed genome-scale metabolic model iSM454 for O. oeni PSU-1 and the resulting experimental data, we determined the redistribution of intracellular metabolic fluxes, under the different ethanol conditions. Four growth phases were clearly identified during the batch cultivation of O. oeni PSU-1 strain, according to the temporal consumption of malic and citric acids, sugar and aminoacids uptake, and biosynthesis rates of metabolic products – biomass, erythritol, mannitol and acetic acid, among others. We showed that, under increasing ethanol conditions, O. oeni favors anabolic reactions related with cell maintenance, as the requirements of NAD(P)+ and ATP increased with ethanol content. Specifically, cultures containing 9 and 12% ethanol required 10 and 17 times more NGAM (non-growth associated maintenance ATP) during phase I, respectively, than cultures without ethanol. Malolactic fermentation and citric acid consumption are vital at high ethanol concentrations, as they are the main source for proton extrusion, allowing higher ATP production by F0F1-ATPase, the main route of ATP synthesis under these conditions. Mannitol and erythritol synthesis are the main sources of NAD(P)+, countervailing for 51-57% of its usage, as predicted by the model. Finally, cysteine shows the fastest specific consumption rate among the amino acids, confirming its key role for bacterial survival under ethanol stress. As a whole, this study provides a global insight into how ethanol content exerts a differential physiological response in O. oeni PSU-1 strain. It will help to design better strategies of nutrient addition to achieve a successful malolactic fermentation of wine.

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Mapping the Physiological Response of Oenococcus oeni to Ethanol Stress Using an Extended Genome-Scale Metabolic Model.

Oenococcus oeni is the bacterium most often associated with spontaneous malolactic fermentation (MLF) of wine During MLF, malic acid is transformed into lactic acid and several metabolites are modified, modulating wine’s total acidity and improving its sensory properties Previous works have suggested that certain genetic groups of O oeni strains are associated to different kinds of products In the present study we have spotted two groups of strains isolated mainly from Burgundy wines, one associated to red wines and the other to white wines Sequencing 14 genomes of red and white wine strains revealed that they share a common ancestor that probably colonised two different substrates –red and white wine-associated environments–, diverging over time and disseminating to various regions Their capacity to perform MLF and modify the volatile profile of wine was determined by fermenting a chardonnay wine and analysing its volatile fraction with a non-targeted metabolomics approach by GC-MS The strains had a different impact on the volatile composition depending on their group of origin These results show for the first time a correspondence between the product of origin of the strains and the volatile profile of the wines they produce Furthermore, the genetic features that might be implied in these different phenotypes are examined

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Two different Oenococcus oeni lineages are associated to either red or white wines in Burgundy: genomics and metabolomics insights

Oenococcus oeni is the lactic acid bacterium that most commonly drives malolactic fermentation (MLF) in wine. Though the importance of MLF in terms of wine microbial stability and sensory improvement is well established, it remains a winemaking step not so easy to control. O. oeni displays many adaptation tools to resist the harsh wine conditions which explain its natural dominance at this stage of winemaking. Previous findings showed that capsular polysaccharides and endogenous produced dextran increased the survival rate and the conservation time of malolactic starters. In this paper, we showed that exopolysaccharides specific production rates were increased in the presence of single stressors relevant to wine (pH, ethanol). The transcription of the associated genes was investigated in distinct O. oeni strains. The conditions in which eps genes and EPS synthesis were most stimulated were then evaluated for the production of freeze dried malolactic starters, for acclimation procedures and for MLF efficiency. Sensory analysis tests on the resulting wines were finally performed.

Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance.

Brettanomyces bruxellensis phenotypic diversity, tolerance to wine stress and wine spoilage ability

Oenococcus oeni is the bacterial species that drives malolactic fermentation in most wines. Several studies have described a high intraspecific diversity regarding carbohydrate degradation abilities but the link between the phenotypes and the genes and metabolic pathways has been poorly described. A collection of 41 strains whose genomic sequences were available and representative of the species genomic diversity was analyzed for growth on 18 carbohydrates relevant in wine. The most frequently used substrates (more than 75% of the strains) were glucose, trehalose, ribose, cellobiose, mannose and melibiose. Fructose and L-arabinose were used by about half the strains studied, sucrose, maltose, xylose, galactose and raffinose were used by less than 25% of the strains and lactose, L-sorbose, L-rhamnose, sorbitol and mannitol were not used by any of the studied strains. To identify genes and pathways associated with carbohydrate catabolic abilities, gene-trait matching and a careful analysis of gene mutations and putative complementation phenomena were performed. For most consumed sugars, we were able to propose putatively associated metabolic pathways. Most associated genes belong to the core genome. O. oeni appears as a highly specialized species, ideally suited to fermented fruit juice and more specifically to wine for a subgroup of strains.

https://hal.archives-ouvertes.fr/hal-01607032/document

Alice Cibrario

Papers

Carbohydrate metabolism in Oenococcus oeni : a genomic insight