Abstract
During winemaking, the yeast Saccharomyces cerevisiae plays a major role in converting the sugars of the grape must into ethanol through alcoholic fermentation, with a yield approaching 92% of the theoretical maximum. On the other hand, increasing ethanol yield during fermentation is of commercial and technological interest to the Lallemand company. The aim of this thesis was to overcome the limitations of the central metabolism of S. cerevisiae in order to develop new strains with increased ethanol yield. Two non-GMO approaches were used: adaptive laboratory evolution and random mutagenesis using ultraviolet (UV) light as mutagenic agent.Firstly, we confirmed the small but significant variability of ethanol yield in S. cerevisiae and identified links between their genetic origin and the production of other metabolites. A protocol for measuring primary metabolic yields under standard oenological fermentation conditions was also established. The experimental evolution targeted different parts of glycolysis via two different strategies: furfural toxicity linked to its aldehyde chemical function and the use of a glucose analogue, 2-deoxyglucose (2-DG). Only yeast populations resistant to 2-DG were obtained and then analysed for their fermentation phenotype. UV mutagenesis was used to generate a collection of over 500 mutants, which were characterised in fermentation. Around ten mutants were selected for in-depth study in fermentation, and two stood out for their high ethanol production in grape must. The genomic study of these mutants revealed the loss of a copy of chromosome XIV and identified candidate genes explaining their phenotype. In particular, the SSK2 gene, involved in the regulation of glycerol synthesis, has a nucleotide polymorphism affecting the sequence of its protein. In addition, we demonstrated the importance of the mitochondrial respiratory chain in anaerobic fermentation by studying mutants with different levels of succinate production, a metabolite of industrial interest. The absence of the respiratory chain reduces productions of succinate, ethanol and lactate, and increases productions of glycerol, acetate and α-ketoglutarate, with minimal impact on fermentation performance. These phenotypes were linked to the disruption of FAD cofactor regeneration during succinate production and in interaction with the respiratory chain. In conclusion, this thesis has provided promising strains while contributing to a better understanding of the metabolism of S. cerevisiae in fermentation.