Beyond S. cerevisiae for winemaking: Fermentation-related trait diversity in the genus Saccharomyces

Saccharomyces cerevisiae is the yeast of choice for most inoculated wine fermentations worldwide. However, many other yeast species and genera display phenotypes of interest that may help address the environmental and commercial challenges the wine industry has been facing in recent years. This work aimed to provide, for the first time, a systematic phenotyping of all Saccharomyces species under winemaking conditions. For this purpose, we characterized the fermentative and metabolic properties of 92 Saccharomyces strains in synthetic grape must at two different temperatures. The fermentative potential of alternative yeasts was higher than expected, as nearly all strains were able to complete fermentation, in some cases more efficiently than commercial S. cerevisiae strains. Various species showed interesting metabolic traits, such as high glycerol, succinate and odour-active compound production, or low acetic acid production, compared to S. cerevisiae. Altogether, these results reveal that non-cerevisiae Saccharomyces yeasts are especially interesting for wine fermentation, as they may offer advantages over both S. cerevisiae and non-Saccharomyces strains. This study highlights the potential of alternative Saccharomyces species for winemaking, paving the way for further research and, potentially, for their industrial exploitation.</p

Integrative Omics Analysis Reveals a Limited Transcriptional Shock After Yeast Interspecies Hybridization

The formation of interspecific hybrids results in the coexistence of two diverged genomes within the same nucleus. It has been hypothesized that negative epistatic interactions and regulatory interferences between the two sub-genomes may elicit a so-called genomic shock involving, among other alterations, broad transcriptional changes. To assess the magnitude of this shock in hybrid yeasts, we investigated the transcriptomic differences between a newly formed Saccharomyces cerevisiae × Saccharomyces uvarum diploid hybrid and its diploid parentals, which diverged ∼20 mya. RNA sequencing (RNA-Seq) based allele-specific expression (ASE) analysis indicated that gene expression changes in the hybrid genome are limited, with only ∼1–2% of genes significantly altering their expression with respect to a non-hybrid context. In comparison, a thermal shock altered six times more genes. Furthermore, differences in the expression between orthologous genes in the two parental species tended to be diminished for the corresponding homeologous genes in the hybrid. Finally, and consistent with the RNA-Seq results, we show a limited impact of hybridization on chromatin accessibility patterns, as assessed with assay for transposase-accessible chromatin using sequencing (ATAC-Seq). Overall, our results suggest a limited genomic shock in a newly formed yeast hybrid, which may explain the high frequency of successful hybridization in these organisms

CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes

The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-Type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.</p

Restoring fertility in yeast hybrids: Breeding and quantitative genetics of beneficial traits

Hybrids between species can harbor a combination of beneficial traits from each parent and may exhibit hybrid vigor, more readily adapting to new harsher environments. Interspecies hybrids are also sterile and therefore an evolutionary dead end unless fertility is restored, usually via auto-polyploidisation events. In the Saccharomyces genus, hybrids are readily found in nature and in industrial settings, where they have adapted to severe fermentative conditions. Due to their hybrid sterility, the development of new commercial yeast strains has so far been primarily conducted via selection methods rather than via further breeding. In this study, we overcame infertility by creating tetraploid intermediates of Saccharomyces interspecies hybrids to allow continuous multigenerational breeding. We incorporated nuclear and mitochondrial genetic diversity within each parental species, allowing for quantitative genetic analysis of traits exhibited by the hybrids and for nuclear–mitochondrial interactions to be assessed. Using pooled F12 generation segregants of different hybrids with extreme phenotype distributions, we identified quantitative trait loci (QTLs) for tolerance to high and low temperatures, high sugar concentration, high ethanol concentration, and acetic acid levels. We identified QTLs that are species specific, that are shared between species, as well as hybrid specific, in which the variants do not exhibit phenotypic differences in the original parental species. Moreover, we could distinguish between mitochondria-type–dependent and –independent traits. This study tackles the complexity of the genetic interactions and traits in hybrid species, bringing hybrids into the realm of full genetic analysis of diploid species, and paves the road for the biotechnological exploitation of yeast biodiversity