Hybridization and spore dissection of native wine yeasts for improvement of ethanol resistance and osmotolerance.

Global warming has a significant impact on different viticultural parameters, including grape maturation. An increment of photosynthetic activity generates a rapid accumulation of sugars in the berry, followed by a dehydration process which leads to a higher concentration of soluble solids. This effect is exacerbated by current viticultural practices which favor the harvest of very mature grapes to obtain wines with sweet tannins. Considering the initial hyperosmotic stress conditions and the high ethanol concentration of the produced wine, fermentation of grape musts with high sugar content could be problematic for yeast starters. In the present study, we were able to obtain by classical hybridization and spore dissection methods one hybrid and one monosporic wine yeast strain with a combined ethanol and osmotolerant phenotype. The improved yeasts were tested in vinification trials with high sugar concentration and displayed excellent fermentation performance. Importantly, the obtained wines also showed good organoleptic properties during sensory analysis. Based on our results, we believed our improved hybrid and monosporic strains can be considered good alternatives to be used as yeast starters for fermentations with high sugar content.

Mutagenesis, screening and isolation of Brettanomyces bruxellensis mutants with reduced 4-ethylphenol production.

The use of non-conventional yeast species to obtain interesting flavors and aromas has become a new trend in the fermented beverages industry. Among such species, Brettanomyces bruxellensis (B. bruxellensis) has been reported as capable of producing desirable or at least singular aromas in fermented beverages like beer and wine. However, this yeast can also produce an aromatic defect by producing high concentrations of phenolic compounds like, 4-ethylguaiacol and particularly 4-ethylphenol (4-EP). In the present study, we designed a mutant screening method to isolate B. bruxellensis mutants with reduced 4-EP production. More than 1000 mutants were screened with our olfactory screening method, and after further sensory and chemical analysis we were able to select a B. bruxellensis mutant strain with a significant reduction of 4-EP production (more than threefold) and less phenolic perception. Notably, the selected strain also showed higher diversity and concentration of ethyl esters, the most important group of odor active compounds produced by yeasts. Based on these results, we consider that our selected mutant strain is a good candidate to be tested as a non-conventional yeast starter (pure or in co-inoculation) to obtain wines and beers with novel aromatic properties.

Construction of low-ethanol-wine yeasts through partial deletion of the Saccharomyces cerevisiae PDC2 gene.

We propose an alternative GMO based strategy to obtain Saccharomyces cerevisiae mutant strains with a slight reduction in their ability to produce ethanol, but with a moderate impact on the yeast metabolism. Through homologous recombination, two truncated Pdc2p proteins Pdc2pΔ344 and Pdc2pΔ519 were obtained and transformed into haploid and diploid lab yeast strains. In the pdc2Δ344 mutants the DNA-binding and transactivation site of the protein remain intact, whereas in pdc2Δ519 only the DNA-binding site is conserved. Compared to the control, the diploid BY4743pdc2Δ519 mutant strain reduced up to 7.4% the total ethanol content in lab scale-vinifications. The residual sugar and volatile acidity was not significantly affected by this ethanol reduction. Remarkably, we got a much higher ethanol reduction of 10 and 15% when the pdc2Δ519 mutation was tested in a native and a commercial wine yeast strain against their respective controls. Our results demonstrate that the insertion of the pdc2Δ519 mutation in wine yeast strains can reduce the ethanol concentration up to 1.89% (v/v) without affecting the fermentation performance. In contrast to non-GMO based strategies, our approach permits the insertion of the pdc2Δ519 mutation in any locally selected wine strain, making possible to produce quality wines with regional characteristics and lower alcohol content. Thus, we consider our work a valuable contribution to the problem of high ethanol concentration in wine.