Temperature is one of the most important parameters to affect the duration and rate of alcoholic fermentation and final wine quality. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae, which was the case of cryotolerant yeasts Saccharomyces uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the proteomic landscape of these cryotolerant species grown at 12°C and 28°C, which we compared with the proteome of S. cerevisiae (poorly adapted at low temperature). Our results showed that the main differences among the proteomic profiling of the three Saccharomyces strains grown at 12°C and 28°C lay in translation, glycolysis and amino acid metabolism. Our data corroborate previous transcriptomic results, which suggest that S. kudriavzevii is better adapted to grow at low temperature as a result of enhanced more efficient translation. Fitter amino acid biosynthetic pathways can also be mechanisms that better explain biomass yield in cryotolerant strains. Yet even at low temperature, S. cerevisiae is the most fermentative competitive species. A higher concentration of glycolytic and alcoholic fermentation enzymes in the S. cerevisiae strain might explain such greater fermentation activity.

Interspecific hybridization has proven to be a potentially valuable technique for generating de novo lager yeast strains that possess diverse and improved traits compared to their parent strains. To further enhance the value of hybridization for strain development, it would be desirable to combine phenotypic traits from more than two parent strains, as well as remove unwanted traits from hybrids. One such trait, that has limited the industrial use of de novo lager yeast hybrids, is their inherent tendency to produce phenolic off-flavours; an undesirable trait inherited from the Saccharomyces eubayanus parent. Trait removal and the addition of traits from a third strain could be achieved through sporulation and meiotic recombination or further mating. However, interspecies hybrids tend to be sterile, which impedes this opportunity.

The inoculation of wines with autochthonous yeast allows obtaining complex wines with a peculiar microbial footprint characteristic from a wine region. Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. However, the interactions between these yeasts are not well understood, especially those regarding the availability of nutrients. The aim of the present study was to analyze the effect of nitrogen and sugar concentration on the evolution of mixed yeast populations on controlled laboratory-scale fermentations monitored by density, plate culturing, PCR-DGGE and sugar and nitrogen consumption. Furthermore, the effect of the time of inoculation of Saccharomyces cerevisiae respect the initial co-inoculation of three non-Saccharomyces yeasts was evaluated over the evolution of fermentation. Our results have shown that S. cerevisiae inoculation during the first 48 h conferred a stabilizing effect over the fermentations with non-Saccharomyces strains tested and, generally, reduced yeast diversity at the end of the fermentation. On the other hand, nitrogen limitation increased the time of fermentation and also the proportion of non-Saccharomyces yeasts at mid and final fermentation. High sugar concentration resulted in different proportions of the inoculated yeast depending on the time of S. cerevisiae inoculation. This work emphasizes the importance of the concentration of nutrients on the evolution of mixed fermentations and points to the optimal conditions for a stable fermentation in which the inoculated yeasts survived until the end.

The use of non-Saccharomyces yeast for wine making is becoming a common trend in many innovative wineries. The application is normally aimed at increasing aromas, glycerol, reducing acidity, and other improvements. This manuscript focuses on the reproduction of the native microbiota from the vineyard in the inoculum. Thus, native selected yeasts (Hanseniaspora uvarum, Metschnikowia pulcherrima, Torulaspora delbrueckii, Starmerella bacillaris species and three different strains of Saccharomyces cerevisiae) were inoculated sequentially, or only S. cerevisiae (three native strains together or one commercial) was used. Inoculations were performed both in laboratory conditions with synthetic must (400 mL) as well as in industrial conditions (2000 kg of grapes) in red winemaking in two different varieties, Grenache and Carignan. The results showed that all the inoculated S. cerevisiae strains were found at the end of the vinifications, and when non-Saccharomyces yeasts were inoculated, they were found in appreciable populations at mid-fermentation. The final wines produced could be clearly differentiated by sensory analysis and were of similar quality, in terms of sensory analysis panelists' appreciation.

Yeast cryotolerance may be advantageous for cider making, where low temperatures are usually employed. Here, we crossed the cryotolerant S. eubayanus with a S. cerevisiae wine strain and assessed the suitability of the hybrids for low-temperature cider fermentation. All strains fermented the juice to 5% ABV, but at different rates; hybrid strains outperformed S. cerevisiae, which was sensitive to low temperatures. The best hybrid fermented similarly to S. eubayanus. S. eubayanus produced sulphurous off flavours which masked a high concentration of fruity ester notes. This phenotype was absent in the hybrid strains, resulting in distinctly fruitier ciders. Aroma was assessed by an independent consumer panel, which rated the hybrid ciders as identical to the wine strain cider. Both were significantly more pleasant than the S. eubayanus cider. Interspecific hybridization can apparently be used effectively to improve low-temperature fermentation performance without compromising product quality.

Gene expression evolution occurs through changes in cis- or trans-regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus) and within species (comparing two closely related S. cerevisiae strains).

An unknown interspecies Saccharomyces hybrid, "Muri," was recently isolated from a "kveik" culture, a traditional Norwegian farmhouse brewing yeast culture (Preiss et al., 2018). Here we used whole genome sequencing to reveal the strain as an allodiploid Saccharomyces cerevisiae × Saccharomyces uvarum hybrid. Phylogenetic analysis of its sub-genomes revealed that the S. cerevisiae and S. uvarum parent strains of Muri appear to be most closely related to English ale and Central European cider and wine strains, respectively. We then performed phenotypic analysis on a number of brewing-relevant traits in a range of S. cerevisiae, S. uvarum and hybrid strains closely related to the Muri hybrid. The Muri strain possesses a range of industrially desirable phenotypic properties, including broad temperature tolerance, good ethanol tolerance, and efficient carbohydrate use, therefore making it an interesting candidate for not only brewing applications, but potentially various other industrial fermentations, such as biofuel production and distilling. We identified the two S. cerevisiae and S. uvarum strains that were genetically and phenotypically most similar to the Muri hybrid, and then attempted to reconstruct the Muri hybrid by generating de novo interspecific hybrids between these two strains. The de novo hybrids were compared with the original Muri hybrid, and many appeared phenotypically more similar to Muri than either of the parent strains. This study introduces a novel approach to studying hybrid strains and strain development by combining genomic and phenotypic analysis to identify closely related parent strains for construction of de novo hybrids.

Ubiquitination is an important post-translational modification involved in diverse biological processes. Therefore, genomewide representation of the ubiquitination system for a species is important.

All RNA species in yeast cells are subject to turnover. Work over the past 20 years has defined degradation mechanisms for messenger RNAs, transfer RNAs, ribosomal RNAs, and noncoding RNAs. In addition, numerous quality control mechanisms that target aberrant RNAs have been identified. Generally, each decay mechanism contains factors that funnel RNA substrates to abundant exo- and/or endonucleases. Key issues for future work include determining the mechanisms that control the specificity of RNA degradation and how RNA degradation processes interact with translation, RNA transport, and other cellular processes.

Mobile Genetic Elements (MGEs) are selfish DNA integrated in the genomes. Their detection is mainly based on consensus-like searches by scanning the investigated genome against the sequence of an already identified MGE. Mobilomics aims at discovering all the MGEs in a genome and understanding their dynamic behavior: The data for this kind of investigation can be provided by comparative genomics of closely related organisms. The amount of data thus involved requires a strong computational effort, which should be alleviated.

Saccharomyces pastorianus strains are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus that have been domesticated for centuries in lager beer brewing environments. As sequences and structures of S. pastorianus genomes are being resolved, molecular mechanisms and evolutionary origins of several industrially relevant phenotypes remain unknown. This study investigates how maltotriose metabolism, a key feature in brewing, may have arisen in early S. eubayanus × S. cerevisiae hybrids. To address this question, we generated a nearly complete genome assembly of Himalayan S. eubayanus strains of the Holarctic subclade. This group of strains has been proposed to be the S. eubayanus subgenome origin of current S. pastorianus strains. The Himalayan S. eubayanus genomes harbored several copies of an S. eubayanusAGT1 (SeAGT1) α-oligoglucoside transporter gene with high sequence identity to genes encountered in S. pastorianus Although Himalayan S. eubayanus strains cannot grow on maltose and maltotriose, their maltose-hydrolase and SeMALT1 and SeAGT1 maltose transporter genes complemented the corresponding null mutants of S. cerevisiae Expression, in Himalayan S. eubayanus of a functional S. cerevisiae maltose metabolism regulator gene (MALx3) enabled growth on oligoglucosides. The hypothesis that the maltotriose-positive phenotype in S. pastorianus is a result of heterosis was experimentally tested by constructing an S. cerevisiae × S. eubayanus laboratory hybrid with a complement of maltose metabolism genes that resembles that of current S. pastorianus strains. The ability of this hybrid to consume maltotriose in brewer's wort demonstrated regulatory cross talk between subgenomes and thereby validated this hypothesis. These results support experimentally the new postulated hypothesis on the evolutionary origin of an essential phenotype of lager brewing strains and valuable knowledge for industrial exploitation of laboratory-made S. pastorianus-like hybrids.IMPORTANCES. pastorianus, an S. cerevisiae × S. eubayanus hybrid, is used for production of lager beer, the most produced alcoholic beverage worldwide. It emerged by spontaneous hybridization and colonized early lager brewing processes. Despite accumulation and analysis of genome sequencing data of S. pastorianus parental genomes, the genetic blueprint of industrially relevant phenotypes remains unresolved. Assimilation of maltotriose, an abundant sugar in wort, has been postulated to be inherited from the S. cerevisiae parent. Here, we demonstrate that although Asian S. eubayanus isolates harbor a functional maltotriose transporter SeAGT1 gene, they are unable to grow on α-oligoglucosides, but expression of S. cerevisiae regulator MAL13 (ScMAL13) was sufficient to restore growth on trisaccharides. We hypothesized that the S. pastorianus maltotriose phenotype results from regulatory interaction between S. cerevisiae maltose transcription activator and the promoter of SeAGT1 We experimentally confirmed the heterotic nature of the phenotype, and thus these results provide experimental evidence of the evolutionary origin of an essential phenotype of lager brewing strains.

Naturally occurring sequence variation that affects gene expression is an important source of phenotypic differences among individuals within a species. We and others have previously shown that such regulatory variation can occur both at the same locus as the gene whose expression it affects (local regulatory variation) and elsewhere in the genome at trans-acting factors. Here we present a detailed analysis of genome-wide local regulatory variation in Saccharomyces cerevisiae. We used genetic linkage analysis to show that nearly a quarter of all yeast genes contain local regulatory variation between two divergent strains. We measured allele-specific expression in a diploid hybrid of the two strains for 77 genes showing strong self-linkage and found that in 52%-78% of these genes, local regulatory variation acts directly in cis. We also experimentally confirmed one example in which local regulatory variation in the gene AMN1 acts in trans through a feedback loop. Genome-wide sequence analysis revealed that genes subject to local regulatory variation show increased polymorphism in the promoter regions, and that some but not all of this increase is due to polymorphisms in predicted transcription factor binding sites. Increased polymorphism was also found in the 3' untranslated regions of these genes. These findings point to the importance of cis-acting variation, but also suggest that there is a diverse set of mechanisms through which local variation can affect gene expression levels.

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is the authoritative community resource for the Saccharomyces cerevisiae reference genome sequence and its annotation. In recent years, we have moved toward increased representation of sequence variation and allelic differences within S. cerevisiae. The publication of numerous additional genomes has motivated the creation of new tools for their annotation and analysis. Here we present the Variant Viewer: a dynamic open-source web application for the visualization of genomic and proteomic differences. Multiple sequence alignments have been constructed across high quality genome sequences from 11 different S. cerevisiae strains and stored in the SGD. The alignments and summaries are encoded in JSON and used to create a two-tiered dynamic view of the budding yeast pan-genome, available at http://www.yeastgenome.org/variant-viewer.

Subcellular protein localisation is essential for the mechanisms that govern cellular homeostasis. The ability to understand processes leading to this phenomenon will therefore enhance our understanding of cellular function. Here we review recent developments in this field with regard to mass spectrometry, fluorescence microscopy and computational prediction methods. We highlight relative strengths and limitations of current methodologies focussing particularly on studies in the yeast Saccharomyces cerevisiae. We further present the first cell-wide spatial proteome map of S. cerevisiae, generated using hyperLOPIT, a mass spectrometry-based protein correlation profiling technique. We compare protein subcellular localisation assignments from this map, with two published fluorescence microscopy studies and show that confidence in localisation assignment is attained using multiple orthogonal methods that provide complementary data.

Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca(2+), yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700 pN and rupture lengths of up to 600 nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force-dependent interactions to drive cell adhesion. IMPORTANCE The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends on amyloid-like β-aggregation of short amino acid sequences in the adhesins. In Candida albicans adhesin Als5, shear stress from vortex mixing can unfold part of the protein to expose aggregation-prone sequences, and then adhesins aggregate into nanodomains. We therefore tested whether shear stress from mixing can increase flocculation activity by potentiating similar protein remodeling and aggregation in the flocculins. The results demonstrate the applicability of the Als adhesin model and provide a rational framework for the enhancement or inhibition of flocculation in industrial applications.

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is an expertly curated database of literature-derived functional information for the model organism budding yeast, Saccharomyces cerevisiae. SGD constantly strives to synergize new types of experimental data and bioinformatics predictions with existing data, and to organize them into a comprehensive and up-to-date information resource. The primary mission of SGD is to facilitate research into the biology of yeast and to provide this wealth of information to advance, in many ways, research on other organisms, even those as evolutionarily distant as humans. To build such a bridge between biological kingdoms, SGD is curating data regarding yeast-human complementation, in which a human gene can successfully replace the function of a yeast gene, and/or vice versa. These data are manually curated from published literature, made available for download, and incorporated into a variety of analysis tools provided by SGD.

The architecture and regulation of Saccharomyces cerevisiae metabolic network are among the best studied owing to its widespread use in both basic research and industry. Yet, several recent studies have revealed notable limitations in explaining genotype-metabolic phenotype relations in this yeast, especially when concerning multiple genetic/environmental perturbations. Apparently unexpected genotype-phenotype relations may originate in the evolutionarily shaped cellular operating principles being hidden in common laboratory conditions. Predecessors of laboratory S. cerevisiae strains, the wild and the domesticated yeasts, have been evolutionarily shaped by highly variable environments, very distinct from laboratory conditions, and most interestingly by social life within microbial communities. Here we present a brief review of the genotypic and phenotypic peculiarities of S. cerevisiae in the context of its social lifestyle beyond laboratory environments. Accounting for this ecological context and the origin of the laboratory strains in experimental design and data analysis would be essential in improving the understanding of genotype-environment-phenotype relationships.

Synthetic biology enables the production of small molecules by recombinant microbes for pharma, food, and materials applications. The secretion of products reduces the cost of separation and purification, but it is challenging to engineer due to the limited understanding of the transporter proteins' functions. Here we describe a method for genome-wide transporter disruption that, in combination with a metabolite biosensor, enables the identification of transporters impacting the production of a given target metabolite in yeast Saccharomyces cerevisiae. We applied the method to study the transport of xenobiotic compounds, cis,cis-muconic acid (CCM), protocatechuic acid (PCA), and betaxanthins. We found 22 transporters that influenced the production of CCM or PCA. The transporter of the 12-spanner drug:H(+) antiporter (DHA1) family Tpo2p was further confirmed to import CCM and PCA in Xenopus expression assays. We also identified three transporter proteins (Qdr1p, Qdr2p, and Apl1p) involved in betaxanthins transport. In summary, the described method enables high-throughput transporter identification for small molecules in cell factories.

Transcriptional control of gene expression requires interactions between the cis-regulatory elements (CREs) controlling gene promoters. We developed a sensitive computational method to identify CRE combinations with conserved spacing that does not require genome alignments. When applied to seven sensu stricto and sensu lato Saccharomyces species, 80% of the predicted interactions displayed some evidence of combinatorial transcriptional behavior in several existing datasets including: (1) chromatin immunoprecipitation data for colocalization of transcription factors, (2) gene expression data for coexpression of predicted regulatory targets, and (3) gene ontology databases for common pathway membership of predicted regulatory targets. We tested several predicted CRE interactions with chromatin immunoprecipitation experiments in a wild-type strain and strains in which a predicted cofactor was deleted. Our experiments confirmed that transcription factor (TF) occupancy at the promoters of the CRE combination target genes depends on the predicted cofactor while occupancy of other promoters is independent of the predicted cofactor. Our method has the additional advantage of identifying regulatory differences between species. By analyzing the S. cerevisiae and S. bayanus genomes, we identified differences in combinatorial cis-regulation between the species and showed that the predicted changes in gene regulation explain several of the species-specific differences seen in gene expression datasets. In some instances, the same CRE combinations appear to regulate genes involved in distinct biological processes in the two different species. The results of this research demonstrate that (1) combinatorial cis-regulation can be inferred by multi-genome analysis and (2) combinatorial cis-regulation can explain differences in gene expression between species.

No abstract available