ERA-NET: Model-guided evolution for balanced attenuation of wine ethanol content by developing non-GMO yeast strains and communities

Rising temperatures in European wine-producing regions are having a negative impact on this key economic sector. Climate change causes an imbalance between the technological and phenolic maturity of wine grapes and, therefore, an increase in alcohol in wines. This trend is of great concern for the European wine sector since it has a negative impact on wine quality, becomes an obstacle to international trade, and undermines the compatibility of moderate wine consumption with a healthy lifestyle. Since the alcohol content in wine is the direct result of the action of yeast cells on grape sugar, this project (CoolWine) aims to reduce the efficiency of yeast in converting sugar into ethanol. The researchers involved in the project have developed new yeast strains and new fermentation procedures so that less sugar is converted into alcohol during wine fermentation (but allowing full sugar consumption to maintain wine quality). This is typically implemented by genetic improvement of wine yeasts; and by using respiratory metabolism as a clean way to ensure sugar consumption while avoiding the production of ethanol or any other compounds that would eventually put wine quality at risk. The consortium takes advantage of the recent developments in Systems Biology in order to reach the right metabolic features without resorting to GMOs. A powerful tool for the non-GMO genetic improvement of wine yeasts is experimental evolution. Further, the combination of microbial metabolic pathways resulting from the development of microbial consortia is also expected to result in lower ethanol yield during alcoholic fermentation. In CoolWine, the design of yeast combinations and the conditions for experimental evolution have been based on Systems Biology analysis supported by yeast metabolic models. Characterization of the Core CoolWine Collection (a wine yeast strains collection gathered ad hoc for CoolWine) provided some interesting wine yeast strains. One strain of S. cerevisiae showed a moderate ethanol reduction in aerated fermentations (to an extent close to the objectives of the CoolWine). This allows developing one-step fermentation procedures tuned for low alcohol production (the use of other yeast species would require two-step processes). The procedure has been scaled up to 2000 liters and further volume increases are on the way. In addition, several non-Saccharomyces strains show very low ethanol yield under aerobic conditions. Mixed starter cultures with these and S. cerevisiae are now in the process of being optimized before scaling up the process. Agreements are currently underway to scale up the processes with the first interesting strains obtained in the project. CoolWine researchers have also developed a powerful pipeline for fungal genome-scale metabolic model reconstruction. Those are helping dissect the aerobic and anaerobic metabolism in grape must of different wine yeast species. They have also used massive parallel experimental evolution to genetically improve strains of several wine yeast species; and are currently analyzing over 8000 yeast evolved populations in order to identify useful strains for lower ethanol winemaking. In addition, by experimental evolution in liquid medium, researchers managed to reduce volatile acidity of some S. cerevisiae strains under the conditions required for alcohol level reduction. Furthermore, applied ethicists and anthropologists involved in the project have been working on the views of CoolWine objectives from the society. A social impact assessment (SIA) has been conducted to identify stakeholders and map the potential impact of the biotechnological intervention in the community –understanding it from a sociological and anthropological point of view. The SIA has been focused on how ideas and attitudes on wine culture and wine consumption are constructed; and the response of consumers, non-consumers, producers, distributors, wine experts, and health professionals towards CoolWine. Finally, CoolWine was philosophically analysed as part of a climate resilience strategy, understanding wine making as an important test case for how to create transparent technological strategies to meet the challenges of climate change.

One approach to the valorisation of the results of CoolWine has relied on scientific publications (SCI and pre-prints) and patent applications. Many articles are already published, but some additional publications and patents are in the pipeline. The first patent has already been object of a licence agreement with an external company. This agreement, in turn, has prompted the development of a research contract, with the same company, for scaling up and commercially developing aerobic fermentation with selected yeast strains for alcohol level reduction in wines. Finally, CoolWine researchers are found to participate in initiatives to communicate the activity of the project and their labs to the general society. Two videos have already been published, one in the framework of the Science Week, and a second one “An ethnographic gaze applied to CoolWine”, based on our contribution in the European Biotechnology and Society Online Seminar Series 2020.

Biotechnological Problem: Increasing temperature in the European wine producing regions is having a negative impact on this key sector. Climate change results in a lack of balance between technological and phenolic ripening of wine grapes and, as a consequence, alcohol increase in wines. This trend is of great concern for the European wine industry because it has a negative impact on wine quality, becomes a hurdle for international trade, and jeopardizes compatibility of moderate wine consumption with a healthy lifestyle. CoolWine Strategy: We propose a two-track strategy to reduce ethanol yield during wine fermentation (Figure1). Track 1: model-guided adaptive laboratory evolution of wine yeasts. Track 2: model-guided assembly of improved communities including S. cerevisiae as well as alternative yeast species. Scientific Approach: CSIC partner has previously successfully used microbial consortia and oxygenated fermentations to reduce ethanol content of wines. Currently, several companies and research groups are also trying to follow this path, thus endorsing the technological and commercial validity of the approach. Although the current results are encouraging, we have also identified some bottlenecks (e.g. increased acetate production that is harmful for wine quality). In order to overcome these hindrances, we will tackle both applied and basic scientific challenges. For developing improved wine yeasts through model-guided adaptive laboratory evolution (ALE), currently available metabolic models and computational tools will be improved in order to account for data concerning respiro-fementative balance and acetic acid production. Computational models will be further informed by experimental data from different yeast mutants. In terms of the community models, we will identify metabolic pathways to be positively or negatively selected for in each species during ALE. This will allow us to develop microbial consortia suitable for alcohol level reduction.