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.

Genetic bases for the metabolism of the DMS precursor S-methylmethionine by Saccharomyces cerevisiae.

Dimethyl sulfide (DMS) is a sulfur containing volatile that enhances general fruity aroma and imparts aromatic notes in wine. The most important precursor of DMS is S-methylmethionine (SMM), which is synthesized by grapes and can be metabolized by the yeast S. cerevisiae during wine fermentation. Precursor molecules left after fermentation are chemically converted to DMS during wine maturation, meaning that wine DMS levels are determined by the amount of remaining precursors at bottling. To elucidate SMM metabolism in yeast we performed quantitative trait locus (QTL) mapping using a population of 130 F2-segregants obtained from a cross between two wine yeast strains, and we detected one major QTL explaining almost 30% of trait variation. Within the QTL, gene YLL058W and SMM transporter gene MMP1 were found to influence SMM metabolism, from which MMP1 has the bigger impact. We identified and characterized a variant coding for a truncated transporter with superior SMM preserving attributes. A population analysis with 85 yeast strains from different origins revealed a significant association of the variant to flor strains and minor occurrence in cheese and wine strains. These results will help selecting and improving S. cerevisiae strains for the production of wine and other fermented foods containing DMS such as cheese or beer.

Yeast Plasma Membrane Fungal Oligopeptide Transporters Display Distinct Substrate Preferences despite Their High Sequence Identity.

Fungal Oligopeptide Transporters (Fot) Fot1, Fot2 and Fot3 have been found in Saccharomyces cerevisiae wine strains, but not in strains from other environments. In the S. cerevisiae wine strain EC1118, Fot1 and Fot2 are responsible for a broader range of oligopeptide utilization in comparison with strains not containing any Fot. This leads to better fermentation efficiency and an increased production of desirable organoleptic compounds in wine. Despite the benefits associated with Fot activity in S. cerevisiae within the wine environment, little is known about this family of transporters in yeast. The presence of Fot1, Fot2 and Fot3 in S. cerevisiae wine strains is due to horizontal gene transfer from the yeast Torulaspora microellipsoides, which harbors Fot2Tm, FotX and FotY proteins. Sequence analyses revealed that Fot family members have a high sequence identity in these yeast species. In this work, we aimed to further characterize the different Fot family members in terms of subcellular localization, gene expression in enological fermentation and substrate specificity. Using CRISPR/Cas9, we constructed S. cerevisiae wine strains containing each different Fot as the sole oligopeptide transporter to analyze their oligopeptide preferences by phenotype microarrays. The results of oligopeptide consumption show that Fot counterparts have different di-/tripeptide specificities, suggesting that punctual sequence divergence between FOT genes can be crucial for substrate recognition, binding and transport activity. FOT gene expression levels in different S. cerevisiae wine strains during enological fermentation, together with predicted binding motifs for transcriptional regulators in nitrogen metabolism, indicate that these transporters may be under the control of the Nitrogen Catabolite Repression (NCR) system. Finally, we demonstrated that Fot1 is located in the yeast plasma membrane. This work contributes to a better understanding of this family of oligopeptide transporters, which have demonstrated a key role in the utilization of oligopeptides by S. cerevisiae in enological fermentation.

Aborting meiosis allows recombination in sterile diploid yeast hybrids.

Hybrids between diverged lineages contain novel genetic combinations but an impaired meiosis often makes them evolutionary dead ends. Here, we explore to what extent an aborted meiosis followed by a return-to-growth (RTG) promotes recombination across a panel of 20 Saccharomyces cerevisiae and S. paradoxus diploid hybrids with different genomic structures and levels of sterility. Genome analyses of 275 clones reveal that RTG promotes recombination and generates extensive regions of loss-of-heterozygosity in sterile hybrids with either a defective meiosis or a heavily rearranged karyotype, whereas RTG recombination is reduced by high sequence divergence between parental subgenomes. The RTG recombination preferentially arises in regions with low local heterozygosity and near meiotic recombination hotspots. The loss-of-heterozygosity has a profound impact on sexual and asexual fitness, and enables genetic mapping of phenotypic differences in sterile lineages where linkage analysis would fail. We propose that RTG gives sterile yeast hybrids access to a natural route for genome recombination and adaptation.

The yeast mating-type switching endonuclease HO is a domesticated member of an unorthodox homing genetic element family.

The mating-type switching endonuclease HO plays a central role in the natural life cycle of Saccharomyces cerevisiae, but its evolutionary origin is unknown. HO is a recent addition to yeast genomes, present in only a few genera close to Saccharomyces. Here we show that HO is structurally and phylogenetically related to a family of unorthodox homing genetic elements found in Torulaspora and Lachancea yeasts. These WHO elements home into the aldolase gene FBA1, replacing its 3' end each time they integrate. They resemble inteins but they operate by a different mechanism that does not require protein splicing. We show that a WHO protein cleaves Torulaspora delbrueckii FBA1 efficiently and in an allele-specific manner, leading to DNA repair by gene conversion or NHEJ. The DNA rearrangement steps during WHO element homing are very similar to those during mating-type switching, and indicate that HO is a domesticated WHO-like element.

In the same way as a sperm from a male and an egg from a female join together to form an embryo in most animals, yeast cells have two sexes that coordinate how they reproduce. These are called "mating types" and, rather than male or female, an individual yeast cell can either be mating type "a" or "alpha". Every yeast cell contains the genes for both mating types, and each cell's mating type is determined by which of those genes it has active. Only one mating type gene can be 'on' at a time, but some yeast species can swap mating type on demand by switching the corresponding genes 'on' or 'off'. This switch is unusual. Rather than simply activate one of the genes it already has, the yeast cell keeps an inactive version of each mating type gene tucked away, makes a copy of the gene it wants to be active and pastes that copy into a different location in its genome. To do all of this yeast need another gene called HO. This gene codes for an enzyme that cuts the DNA at the location of the active mating type gene. This makes an opening that allows the cell to replace the 'a' gene with the 'alpha' gene, or vice versa. This system allows yeast cells to continue mating even if all the cells in a colony start off as the same mating type. But, cutting into the DNA is risky, and can damage the health of the cell. So, why did yeast cells evolve a system that could cause them harm? To find out where the HO gene came from, Coughlan et al. searched through all the available genomes from yeast species for other genes with similar sequences and identified a cluster which they nicknamed "weird HO" genes, or WHO genes for short. Testing these genes revealed that they also code for enzymes that make cuts in the yeast genome, but the way the cell repairs the cuts is different. The WHO genes are jumping genes. When the enzyme encoded by a WHO gene makes a cut in the genome, the yeast cell copies the gene into the gap, allowing the gene to 'jump' from one part of the genome to another. It is possible that this was the starting point for the evolution of the HO gene. Changes to a WHO gene could have allowed it to cut into the mating type region of the yeast genome, giving the yeast an opportunity to 'domesticate' it. Over time, the yeast cell stopped the WHO gene from jumping into the gap and started using the cut to change its mating type. Understanding how cells adapt genes for different purposes is a key question in evolutionary biology. There are many other examples of domesticated jumping genes in other organisms, including in the human immune system. Understanding the evolution of HO not only sheds light on how yeast mating type switching evolved, but on how other species might harness and adapt their genes.

QTL mapping of modelled metabolic fluxes reveals gene variants impacting yeast central carbon metabolism.

The yeast Saccharomyces cerevisiae is an attractive industrial microorganism for the production of foods and beverages as well as for various bulk and fine chemicals, such as biofuels or fragrances. Building blocks for these biosyntheses are intermediates of yeast central carbon metabolism (CCM), whose intracellular availability depends on balanced single reactions that form metabolic fluxes. Therefore, efficient product biosynthesis is influenced by the distribution of these fluxes. We recently demonstrated great variations in CCM fluxes between yeast strains of different origins. However, we have limited understanding of flux modulation and the genetic basis of flux variations. In this study, we investigated the potential of quantitative trait locus (QTL) mapping to elucidate genetic variations responsible for differences in metabolic flux distributions (fQTL). Intracellular metabolic fluxes were estimated by constraint-based modelling and used as quantitative phenotypes, and differences in fluxes were linked to genomic variations. Using this approach, we detected four fQTLs that influence metabolic pathways. The molecular dissection of these QTLs revealed two allelic gene variants, PDB1 and VID30, contributing to flux distribution. The elucidation of genetic determinants influencing metabolic fluxes, as reported here for the first time, creates new opportunities for the development of strains with optimized metabolite profiles for various applications.

Lipids modulate acetic acid and thiol final concentrations in wine during fermentation by Saccharomyces cerevisiae × Saccharomyces kudriavzevii hybrids.

Saccharomyces cerevisiae × Saccharomyces kudriavzevii hybrids are typically used for white wine fermentation because of their cryotolerance. One group of these hybrids presents a unique ability to release thiol varietal aroma products as well as excessive amounts of acetic acid under specific conditions, which is detrimental for wine organoleptic quality. The aim of this work is to better assess the effects of lipids, sugar concentrations and temperature on the production of acetic acid and thiols during wine fermentation. To this end, we used a Box-Behnken experimental design and response surface modeling on the production of acetic acid and thiols in S. cerevisiae × S. kudriavzevii hybrids from the Eg8 family during fermentation of a synthetic must. We showed that these hybrids produced lower levels of acetic acid when the initial lipid concentration was increased, whereas they produced greater levels when the initial sugar concentration was high. Moreover, we found that lipids had a positive impact on the final concentrations of 4-methyl-4-mercaptopentan-2-one and 3-mercaptohexan-1-ol (3MH), giving box tree and citrus flavors, respectively. The increase of 3MH was concomitant with a decrease of 3-mercaptohexyl acetate (3MHA) characterized by a passion fruit aroma, indicating that lipid addition reduces the rate of 3MH acetylation into 3MHA. These results highlight the key role of lipid management in acetic acid metabolism and thiol release by S. cerevisiae × S. kudriavzevii hybrids and underline its technological interest in alcoholic fermentation to avoid the overproduction of volatile acidity while favoring the release of volatile thiols.

Genome Sequence of Torulaspora microellipsoides CLIB 830T.

We report here the genome sequence of the ascomycetous yeast Torulaspora microellipsoides CLIB 830T A reference genome for this species, which has been found as a donor of genetic material in wine strains of Saccharomyces cerevisiae, will undoubtedly give clues to our understanding of horizontal transfer mechanisms between species in the wine environment.

Specific Phenotypic Traits of Starmerella bacillaris Related to Nitrogen Source Consumption and Central Carbon Metabolite Production during Wine Fermentation.

Over the last few years, the potential of non-Saccharomyces yeasts to improve the sensory quality of wine has been well recognized. In particular, the use of Starmerella bacillaris in mixed fermentations with Saccharomyces cerevisiae was reported as an appropriate way to enhance glycerol formation and reduce ethanol production. However, during sequential fermentation, many factors, such as the inoculation timing, strain combination, and physical and biochemical interactions, can affect yeast growth, the fermentation process, and/or metabolite synthesis. Among them, the availability of yeast-assimilable nitrogen (YAN), due to its role in the control of growth and fermentation, has been identified as a key parameter. Consequently, a comprehensive understanding of the metabolic specificities and the nitrogen requirements would be valuable to better exploit the potential of Starm. bacillaris during wine fermentation. In this study, marked differences in the consumption of the total and individual nitrogen sources were registered between the two species, while the two Starm. bacillaris strains generally behaved uniformly. Starm. bacillaris strains are differentiated by their preferential uptake of ammonium compared with amino acids that are poorly assimilated or even produced (alanine). Otherwise, the non-Saccharomyces yeast exhibits low activity through the acetaldehyde pathway, which triggers an important redistribution of fluxes through the central carbon metabolic network. In particular, the formation of metabolites deriving from the two glycolytic intermediates glyceraldehyde-3-phosphate and pyruvate is substantially increased during fermentations by Starm. bacillaris This knowledge will be useful to better control the fermentation process in mixed fermentation with Starm. bacillaris and S. cerevisiaeIMPORTANCE Mixed fermentations using a controlled inoculation of Starmerella bacillaris and Saccharomyces cerevisiae starter cultures represent a feasible way to modulate wine composition that takes advantage of both the phenotypic specificities of the non-Saccharomyces strain and the ability of S. cerevisiae to complete wine fermentation. However, according to the composition of grape juices, the consumption by Starm. bacillaris of nutrients, in particular of nitrogen sources, during the first stages of the process may result in depletions that further limit the growth of S. cerevisiae and lead to stuck or sluggish fermentations. Consequently, understanding the preferences of non-Saccharomyces yeasts for the nitrogen sources available in grape must together with their phenotypic specificities is essential for an efficient implementation of sequential wine fermentations with Starm. bacillaris and S. cerevisiae species. The results of our study demonstrate a clear preference for ammonium compared to amino acids for the non-Saccharomyces species. This finding underlines the importance of nitrogen sources, which modulate the functional characteristics of inoculated yeast strains to better control the fermentation process and product quality.

Adaptation of S. cerevisiae to Fermented Food Environments Reveals Remarkable Genome Plasticity and the Footprints of Domestication.

The budding yeast Saccharomyces cerevisiae can be found in the wild and is also frequently associated with human activities. Despite recent insights into the phylogeny of this species, much is still unknown about how evolutionary processes related to anthropogenic niches have shaped the genomes and phenotypes of S. cerevisiae. To address this question, we performed population-level sequencing of 82 S. cerevisiae strains from wine, flor, rum, dairy products, bakeries, and the natural environment (oak trees). These genomic data enabled us to delineate specific genetic groups corresponding to the different ecological niches and revealed high genome content variation across the groups. Most of these strains, compared with the reference genome, possessed additional genetic elements acquired by introgression or horizontal transfer, several of which were population-specific. In addition, several genomic regions in each population showed evidence of nonneutral evolution, as shown by high differentiation, or of selective sweeps including genes with key functions in these environments (e.g., amino acid transport for wine yeast). Linking genetics to lifestyle differences and metabolite traits has enabled us to elucidate the genetic basis of several niche-specific population traits, such as growth on galactose for cheese strains. These data indicate that yeast has been subjected to various divergent selective pressures depending on its niche, requiring the development of customized genomes for better survival in these environments. These striking genome dynamics associated with local adaptation and domestication reveal the remarkable plasticity of the S. cerevisiae genome, revealing this species to be an amazing complex of specialized populations.