Experimental evolution of the wine-associated bacterium Oenococcus oeni in extreme environments: applications to the selection of improved malolactic starters – EVOLOWINE

Our objectives are to highlight the evolutionary routes that allow the hypermutator O. oeni to adapt to the drastic and dynamic wine environment. We will provide a better assessment of the current starters commercialized by the industrial partner, with a focus on lysogeny, and deliver solid data about the diversity of O. oeni in unexplored wine products which will lead to open access results and may have commercial applications for partner 2. Next, we will speed up the adaptation of strains (including current commercial starters) to specific wine types in which their implantation is currently problematic: white wine with low pH, and red wine with high alcohol/PC content. This will be possible by the design of ALE studies in O. oeni under wine production conditions.
To meet such objectives, our work plan includes three tasks: ? provide an accurate and exhaustive assess to the population diversity of O. oeni, with two directions: expand genome sequencing data collection of strains from specific and so far uncovered wine types, and make a transition from short- to long-read sequencing technologies (Task 1) ? design of evolution experiments to better understand the interactions between O. oeni and its bacteriophages (Task 2) and ? design of ALE studies of selected strains (including lysogens) in conditions relevant to two specific wine-producing processes (Task.3).

Task 1: 403 strains belonging to the O. oeni species have been collected from specific and so far unexplored wines, including hybrid varieties. VNTR typing resulted in 90 distinct profiles. A selection of 50 souches has been made and their sequencing is under progress
Task 2: co-evolution experiments with O. oeni and the strictly lytic phage virulent Krappator 27 have been realized over 5 months including repeated transferts each week. Strains and phages have been isolated at every step. Their phenotypes are currently assessed and will help understanding arm-race in O. oeni.
Task 3: the composition and origin of the wines to be used for ALE studies have been chosen in april 2022. Those wines have been prepared by our industrail partner in september 2022 (alcoholic fermentation) and sent to Bordeaux where its has been pasteurized.

The project raises interesting fundamental questions about life and evolution in extreme environments. A comprehensive description of the O. oeni species diversity and adaptation to wine will be provided through comparative genomic analysis and ALE studies. This will give important clues on the molecular mechanisms involved in genome evolution, and the underlying mechanisms which confer superiority in wine to O. oeni strains. The world of wine is approached in an innovative way, which also highlights (i) the peculiarity of the making process (hypermutator status of the model bacterium studied, multi-stressor situation, spatio-temporal dynamics of both constraints and microorganisms, and contribution of man to domestication of microorganisms) and (ii) the potential of wine as a model ecosystem to address major scientific questions. Evolowine is expected to provide results for the whole scientific community, through research papers in high ranking journals, with priority given to open access journals (such as OENO One, an initiative by ISVV and U. Bordeaux; oeno-one.eu). We anticipate that the scope of the work may also find an echo within the Lactic Acid Bacteria community, as (i) the presence of related Oenococcus species has been recently reported in other foods with increasing consumption, such as water kefirs, and also (ii) considering the recent re-evaluation of the genetic relatedness and phylogeny of the O. oeni species within the present genus Lactobacillus and its sister taxa in the Lactobacillaceae and Leuconostocaceae, which are of industrial relevance.

two papers are submitted

Stuck malolactic fermentations are feared as they depreciate wine quality. They are due to the capricious nature of the lactic acid bacterium Oenococus oeni, as well as to complex wine compositions which are difficult to ferment. Such problematic wine compositions are intrinsically associated to specific appelations and wine styles (low pH in white wines), while others result from climatic changes (high ethanol and polyphenolic contents in red wines). Such issues are more and more frequent and represent a concern for the whole sector. Suppliers of commercial starters, as their use is progressively expanding, can not currently provide concerned winemakers with robust direct vat inoculation starters and solve the issues related to FML in difficult wines. Suppliers are committed to developing solutions and strains tailored precisely to the individual requirements of their customers, and more globally are urged to improve their selection schemes. EVOLOWINE is at the frontier between fundamental and translational research and aims at tackling these issues. The objectives are to understand the evolutionary routes that allow this peculiar bacterium which has an hypermutator phenotype, to adapt to drastic wine environment. Adaptive laboratory evolution studies will be conducted in order to provide robust starters for difficult wines. The 48-months project involves two academic research teams from Bordeaux (EA 4577 INRA Oenologie) and Grenoble (TIMC-IMAG, CNRS), and Lallemand SAS (Oenology Division) in Toulouse, a company that sells malolactic starters.