A multi-phase approach to select new wine yeast strains with enhanced fermentative fitness and glutathione production

The genetic improvement of winemaking yeasts is a virtually infinite process, as the design of new strains must always cope with varied and ever-evolving production contexts. Good wine yeasts must feature both good primary traits, which are related to the overall fermentative fitness of the strain, and secondary traits, which provide accessory features augmenting its technological value. In this context, the superiority of “blind,” genetic improvement techniques, as those based on the direct selection of the desired phenotype without prior knowledge of the genotype, was widely proven. Blind techniques such as adaptive evolution strategies were implemented for the enhancement of many traits of interest in the winemaking field. However, these strategies usually focus on single traits: this possibly leads to genetic tradeoff phenomena, where the selection of enhanced secondary traits might lead to sub-optimal primary fermentation traits. To circumvent this phenomenon, we applied a multi-step and strongly directed genetic improvement strategy aimed at combining a strong fermentative aptitude (primary trait) with an enhanced production of glutathione (secondary trait). We exploited the random genetic recombination associated to a library of 69 monosporic clones of strain UMCC 855 (Saccharomyces cerevisiae) to search for new candidates possessing both traits. This was achieved by consecutively applying three directional selective criteria: molybdate resistance (1), fermentative aptitude (2), and glutathione production (3). The strategy brought to the selection of strain 21T2-D58, which produces a high concentration of glutathione, comparable to that of other glutathione high-producers, still with a much greater fermentative aptitude

Improved wine yeasts by direct mating and selection under stressful fermentative conditions

Hybridization of yeasts allows whole-genome modifications that can be exploited to obtain global improvements in industrial traits, such as those involved in the winemaking industry. In our work we applied direct mating to achieve the construction of hybrids and we subsequently applied these hybrids in fermentation trials under stressful conditions, in order to select hybrid strains with improved technological traits. Five hybrids, obtained from six parental strains by direct spore conjugation, were validated through PCR amplification of highly variable minisatellite-containing genes; the validation phase also revealed three meiotic derivative strains, characterized by contracted number of repeats. Analysis of the mating-type locus in the homozygous spore progeny of parental strains provided useful insights into the understanding of hybridization yields and unveiled some irregularities in yeast autodiploidization mechanism. The fermentative trials were followed by chemical analysis; afterwards principal component analysis allowed the metabolic footprinting of wine yeasts and the selection of the two best industrial candidates, which display superior phenotypes in fermentative fitness and secondary metabolite production, respectively

Genetic variation and expression changes associated with molybdate resistance from a glutathione producing wine strain of Saccharomyces cerevisiae.

Glutathione (GSH) production during wine fermentation is a desirable trait as it can limit must and wine oxidation and protect various aromatic compounds. UMCC 2581 is a Saccharomyces cerevisiae wine strain with enhanced GSH content at the end of wine fermentation. This strain was previously derived by selection for molybdate resistance following a sexual cycle of UMCC 855 using an evolution-based strategy. In this study, we examined genetic and gene expression changes associated with the derivation of UMCC 2581. For genetic analysis we sporulated the diploid UMCC 855 parental strain and found four phenotype classes of segregants related to molybdate resistance, demonstrating the presence of segregating variation from the parental strain. Using bulk segregant analysis we mapped molybdate traits to two loci. By sequencing both the parental and evolved strain genomes we identified candidate mutations within the two regions as well as an extra copy of chromosome 1 in UMCC 2581. Combining the mapped loci with gene expression profiles of the evolved and parental strains we identified a number of candidate genes with genetic and/or gene expression changes that could underlie molybdate resistance and increased GSH levels. Our results provide insight into the genetic basis of GSH production relevant to winemaking and highlight the value of enhancing wine strains using existing variation present in wine strains