Tommaso Bonciani

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.

Fast method for identifying inter- and intra-species Saccharomyces hybrids in extensive genetic improvement programs based on yeast breeding

The present work proposes a two‐step molecular strategy to select inter‐ and intra‐species Saccharomyces hybrids obtained by spore‐to‐spore mating, one of the most used methods for generating improved hybrids from homothallic wine yeasts.

High-glutathione producing yeasts obtained by genetic improvement strategies: a focus on adaptive evolution approaches for novel wine strains

Glutathione (GSH) is the most abundant non-protein thiol in living organisms. Due to its important antioxidant role, it is widely used in medicine, as a food additive, and in the cosmetic industry. Recently, GSH has received growing attention in winemaking because of its ability to control oxidative spoilage damage and to protect various aromatic compounds. Indeed, GSH concentration in wine is highly variable and several factors are involved in its regulation, ranging from grape must to yeast fermentation activity. This short review aims at highlighting the common genetic strategies, useful for obtaining wine yeasts with enhanced GSH production, paying particular attention to the adaptive evolution approaches. Moreover, other strategies, such as random mutagenesis, metabolic engineering and hybridization have been briefly reviewed with a stress on both their strengths and weaknesses in terms of actual feasibility and acceptance by wine consumers.

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.

Qualitative and quantitative screening of the β-glucosidase activity in Saccharomyces cerevisiae and Saccharomyces uvarum strains isolated from refrigerated must

The aim of the present work was to screen a pool of 75 yeasts belonging to the species Saccharomyces cerevisiae and Saccharomyces uvarum in order to select the strains endowed with β‐glucosidase activity. The first screening was a qualitative assay based on chromogenic substrates (arbutin and esculin). The second screening was the quantitative evaluation of the β‐glucosidase activity via a p‐nitrophenyl‐β‐d‐glucopyranoside assay. The measurement was performed on three different cell preparations, including the extracellular compartment, the cell lysates and the whole cells. This study pointed out the high frequency of β‐glucosidase activity in S. uvarum strains. In particular, we retrieved three promising S. uvarum strains, CRY14, VA42 and GRAS14, featuring a high enzymatic activity, exploitable for winemaking.