The ability of the yeast Saccharomyces cerevisiae to adapt to the changing environment of industrial processes lies in the activation and coordination of many molecular pathways. The most relevant ones are nutrient signaling pathways because they control growth and stress response mechanisms as a result of nutrient availability or scarcity and, therefore, leave an ample margin to improve yeast biotechnological performance. A standardized grape juice fermentation assay allowed the analysis of mutants for different elements of many nutrient signaling pathways under different conditions (low/high nitrogen and different oxygenation levels) to allow genetic-environment interactions to be analyzed. The results indicate that the cAMP-dependent PKA pathway is the most relevant regardless of fermentation conditions, while mutations on TOR pathways display an effect that depends on nitrogen availability. The production of metabolites of interest, such as glycerol, acetic acid and pyruvate, is controlled in a coordinated manner by the contribution of several components of different pathways. Ras GTPase Ras2, a stimulator of cAMP production, is a key factor for achieving fermentation, and is also relevant for sensing nitrogen availability. Increasing cAMP concentrations by deleting an enzyme used for its degradation, phosphodiesterase Pde2, proved a good way to increase fermentation kinetics, and offered keys for biotechnological improvement. Surprisingly glucose repression protein kinase Snf1 and Nitrogen Catabolite Repression transcription factor Gln3 are relevant in fermentation, even in the absence of starvation. Gln3 proved essential for respiration in several genetic backgrounds, and its presence is required to achieve full glucose de-repression. Therefore, most pathways sense different types of nutrients and only their coordinated action can ensure successful wine fermentation.
Peroxiredoxins are a family of peroxide-degrading enzymes for challenging oxidative stress. They receive their reducing power from redox-controlling proteins called thioredoxins, and these, in turn, from thioredoxin reductase. The main cytosolic peroxiredoxin is Tsa1, a moonlighting protein that also acts as protein chaperone a redox switch controlling some metabolic events. Gene deletion of peroxiredoxins in wine yeasts indicate that TSA1, thioredoxins and thioredoxin reductase TRR1 are required for normal growth in medium with glucose and sucrose as carbon sources. TSA1 gene deletion also diminishes growth in molasses, both in flasks and bioreactors. The TSA1 mutation brings about an expected change in redox parameters but, interestingly, it also triggers a variety of metabolic changes. It influences trehalose accumulation, lowering it in first molasses growth stages, but increasing it at the end of batch growth, when respiratory metabolism is set up. Glycogen accumulation at the entry of the stationary phase also increases in the tsa1Δ mutant. The mutation reduces fermentative capacity in grape juice, but the vinification profile does not significantly change. However, acetic acid and acetaldehyde production decrease when TSA1 is absent. Hence, TSA1 plays a role in the regulation of metabolic reactions leading to the production of such relevant enological molecules.
Modern enology relies on the use of selected yeasts, both Saccharomyces and non-conventional, as starters to achieve reliable fermentations. That allows the selection of the right strain for each process and also the improvement of such strain, by traditional methods or approaches involving genetic manipulation. Genetic engineering allows deletion, overexpression and point mutation of endogenous yeast genes with known interesting features in winemaking and the introduction of foreign and novel activities. Besides, it is a powerful tool to understand the molecular mechanisms behind the desirable traits of a good wine strain, as those directed mutations reveal phenotypes of interest. The genetic editing technology called CRISPR-Cas9 allows a fast, easy and non-invasive manipulation of industrial strains that renders cells with no traces of foreign genetic material. Genetic manipulation of non-Saccharomyces wine yeasts has been less common, but those new technologies together with the increasing knowledge on the genome of such strains opens a promising field of yeast improvement.
Wine strains of Saccharomyces cerevisiae have to adapt their metabolism to the changing conditions during their biotechnological use, from the aerobic growth in sucrose-rich molasses for biomass propagation to the anaerobic fermentation of monosaccharides of grape juice during winemaking. Yeast have molecular mechanisms that favor the use of preferred carbon and nitrogen sources to achieve such adaptation. By using specific inhibitors, it was determined that commercial strains offer a wide variety of glucose repression profiles. Transcription factor Gln3 has been involved in glucose and nitrogen repression. Deletion of GLN3 in two commercial wine strains produced different mutant phenotypes and only one of them displayed higher glucose repression and was unable to grow using a respiratory carbon source. Therefore, the role of this transcription factor contributes to the variety of phenotypic behaviors seen in wine strains. This variability is also reflected in the impact of GLN3 deletion in fermentation, although the mutants are always more tolerant to inhibition of the nutrient signaling complex TORC1 by rapamycin, both in laboratory medium and in grape juice fermentation. Therefore, most aspects of nitrogen catabolite repression controlled by TORC1 are conserved in winemaking conditions.
The bulk of grape juice fermentation is carried out by the yeast Saccharomyces cerevisiae, but non-Saccharomyces yeasts can modulate many sensorial aspects of the final products in ways not well understood. In this study, some of such non-conventional yeasts were screened as mixed starter cultures in a fermentation defined medium in both simultaneous and sequential inoculations. One strain of Starmerella bacillaris and another of Zygosaccharomyces bailii were chosen by their distinct phenotypic footprint and their ability to reduce ethanol levels at the end of fermentation, particularly during simultaneous vinification. S. bacillaris losses viability strongly at the end of mixed fermentation, while Z. bailii remains viable until the end of vinification. Interestingly, for most non-Saccharomyces yeasts, simultaneous inoculation helps for survival at the end of fermentation compared to sequential inoculation. S. cerevisiae viability was unchanged by the presence of the either yeast. Characterization of both strains indicates that S. bacillaris behavior is overall more different from S. cerevisiae than Z. bailii. S. bacillaris has a less strict glucose repression mechanism and molecular markers like catabolite repression kinase Snf1 is quite different in size. Besides, S. cerevisiae transcriptome changes to a bigger degree in the presence of S. bacillaris than when inoculated with Z. bailii. S. bacillaris induces the translation machinery and repress vesicular transport. Both non-Saccharomyces yeast induce S. cerevisiae glycolytic genes, and that may be related to ethanol lowering, but there are specific aspects of carbon-related mechanisms between strains: Z. bailii presence increases the stress-related polysaccharides trehalose and glycogen while S. bacillaris induces gluconeogenesis genes.
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