Impact of metabolism regulation by nutrients and redox state on the technological performance of wine yeasts

The wine industry is currently facing challenges such as climate change and the demand for wines with lower alcohol content. These factors have driven the development of biotechnological strategies to improve the performance and adaptability of Saccharomyces cerevisiae, the main yeast used in wine fermentation. During its use in oenology, S. cerevisiae, commonly commercialized as active dry yeast, is subjected to multiple stress conditions and must undergo metabolic adaptations across three key industrial stages: biomass propagation, dehydration, and wine fermentation. Oxidative stress is one of these adverse conditions and can significantly compromise yeast viability and performance during its industrial application. This doctoral thesis investigated the molecular mechanisms underlying the adaptation of wine strains of S. cerevisiae to industrial environments. Two main objectives were addressed: (1) the characterization of the cytosolic peroxiredoxin Tsa1 as a redox regulator of metabolism, exploring its influence on acetic acid and trehalose metabolism; and (2) the study of the retrograde (RTG) pathway as a target to reprogram yeast metabolism and reduce ethanol production during vinification. The results showed that Tsa1 regulates acetic acid metabolism depending on metabolic state and genetic background, under both laboratory and industrial conditions. This regulation is exerted through modulation of aldehyde dehydrogenase activity, especially that of Ald6, also contributing to the maintenance of extracellular pH homeostasis. Moreover, during yeast biomass propagation in molasses, Tsa1 was shown to regulate the enzymatic activities involved in trehalose synthesis and degradation, and its deletion led to an increase in intracellular trehalose accumulation and improved cell viability after dehydration. Additionally, it was demonstrated that the molecular mechanisms underlying the impact of Tsa1 on trehalose metabolism differ between laboratory and wine strains of S. cerevisiae: in laboratory strains, its function is mediated by the PKA pathway, whereas in wine strains it acts independently, possibly through direct regulation of the enzymes involved in the synthesis and degradation of this disaccharide. In parallel, it was found that deletion of MKS1, a repressor of the RTG pathway, allowed for the improvement of the fermentative profile of several wine strains, achieving a reduction in ethanol and acetic acid production, along with an increase in glycerol concentration. To generate non-genetically modified strains with similar phenotypes, an adaptive laboratory evolution (ALE) strategy was implemented, using the toxic lysine analogue (AEC) as selective pressure. The evolved strains exhibited high tolerance to AEC, hyperactivation of the RTG pathway, and a fermentative profile similar to that of previously characterized mks1Δ mutants. Genomic analysis of selected evolved strains revealed mutations in the RTG2 and LYS20/21 genes, and functional analysis demonstrated that the identified RTG2 variants were primarily responsible for the observed metabolic changes. Finally, the phenotype of the evolved strain eTAE 29l was validated in pilot-scale fermentations, confirming its capacity to reduce ethanol and volatile acidity levels, increase glycerol production and maintain sensory quality. These results led to a patent application to protect both the evolved strains and the generation method, offering a non-transgenic strategy aligned with current demands in the wine sector.

This work was made possible through the initial funding of a predoctoral contract from the Generalitat Valenciana (ACIF/2020/122), followed by a predoctoral contract from the Universitat de València’s Atracció de Talent 2020 program