Project description:The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 × 10(5) cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production.
Project description: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.
Project description:Saccharomyces cerevisiae is a common yeast with several applications, among which the most ancient is winemaking. Because individuals belonging to this species show a wide genetic and phenotypic variability, the possibility to identify the strains driving fermentation is pivotal when aiming at stable and palatable products. Metagenomic sequencing is increasingly used to decipher the fungal populations present in complex samples such as musts. However, it does not provide information at the strain level. Microsatellites are commonly used to describe the genotype of single strains. Here we developed a population-level microsatellite profiling approach, SID (Saccharomyces cerevisiae IDentifier), to identify the strains present in complex environmental samples. We optimized and assessed the performances of the analytical procedure on patterns generated in silico by computationally pooling Saccharomyces cerevisiae microsatellite profiles, and on samples obtained by pooling DNA of different strains, proving its ability to characterize real samples of grape wine fermentations. SID showed clear differences among S. cerevisiae populations in grape fermentation samples, identifying strains that are likely composing the populations and highlighting the impact of the inoculation of selected exogenous strains on natural strains. This tool can be successfully exploited to identify S. cerevisiae strains in any kind of complex samples.
Project description:The present study analyzes the lack of culturability of different non-Saccharomyces strains due to interaction with Saccharomyces cerevisiae during alcoholic fermentation. Interaction was followed in mixed fermentations with 1:1 inoculation of S. cerevisiae and ten non-Saccharomyces strains. Starmerella bacillaris, and Torulaspora delbrueckii indicated longer coexistence in mixed fermentations compared with Hanseniaspora uvarum and Metschnikowia pulcherrima. Strain differences in culturability and nutrient consumption (glucose, alanine, ammonium, arginine, or glutamine) were found within each species in mixed fermentation with S. cerevisiae. The interaction was further analyzed using cell-free supernatant from S. cerevisiae and synthetic media mimicking both single fermentations with S. cerevisiae and using mixed fermentations with the corresponding non-Saccharomyces species. Cell-free S. cerevisiae supernatants induced faster culturability loss than synthetic media corresponding to the same fermentation stage. This demonstrated that some metabolites produced by S. cerevisiae played the main role in the decreased culturability of the other non-Saccharomyces yeasts. However, changes in the concentrations of main metabolites had also an effect. Culturability differences were observed among species and strains in culture assays and thus showed distinct tolerance to S. cerevisiae metabolites and fermentation environment. Viability kit and recovery analyses on non-culturable cells verified the existence of viable but not-culturable status. These findings are discussed in the context of interaction between non-Saccharomyces and S. cerevisiae.
Project description:Ten years have passed since the genome of Saccharomyces cerevisiae-more precisely, the S288c strain-was completely sequenced. However, experimental work in yeast is commonly performed using strains that are of unknown genetic relationship to S288c. Here, we characterized the nucleotide-level similarity between S288c and seven commonly used lab strains (A364A, W303, FL100, CEN.PK, summation 1278b, SK1 and BY4716) using 25mer oligonucleotide microarrays that provide complete and redundant coverage of the approximately 12 Mb Saccharomyces cerevisiae genome. Using these data, we assessed the frequency and distribution of nucleotide variation in comparison to the sequenced reference genome. These data allow us to infer the relationships between experimentally important strains of yeast and provide insight for experimental designs that are sensitive to sequence variation. We propose a rational approach for near complete sequencing of strains related to the reference using these data and directed re-sequencing. These data and new visualization tools are accessible online in a new resource: the Yeast SNPs Browser (YSB; http://gbrowse.princeton.edu/cgi-bin/gbrowse/yeast_strains_snps) that is available to all researchers.
Project description:Fermentation of xylose is a fundamental requirement for the efficient production of ethanol from lignocellulosic biomass sources. Although they aggressively ferment hexoses, it has long been thought that native Saccharomyces cerevisiae strains cannot grow fermentatively or non-fermentatively on xylose. Population surveys have uncovered a few naturally occurring strains that are weakly xylose-positive, and some S. cerevisiae have been genetically engineered to ferment xylose, but no strain, either natural or engineered, has yet been reported to ferment xylose as efficiently as glucose. Here, we used a medium-throughput screen to identify Saccharomyces strains that can increase in optical density when xylose is presented as the sole carbon source. We identified 38 strains that have this xylose utilization phenotype, including strains of S. cerevisiae, other sensu stricto members, and hybrids between them. All the S. cerevisiae xylose-utilizing strains we identified are wine yeasts, and for those that could produce meiotic progeny, the xylose phenotype segregates as a single gene trait. We mapped this gene by Bulk Segregant Analysis (BSA) using tiling microarrays and high-throughput sequencing. The gene is a putative xylitol dehydrogenase, which we name XDH1, and is located in the subtelomeric region of the right end of chromosome XV in a region not present in the S288c reference genome. We further characterized the xylose phenotype by performing gene expression microarrays and by genetically dissecting the endogenous Saccharomyces xylose pathway. We have demonstrated that natural S. cerevisiae yeasts are capable of utilizing xylose as the sole carbon source, characterized the genetic basis for this trait as well as the endogenous xylose utilization pathway, and demonstrated the feasibility of BSA using high-throughput sequencing.
Project description:The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea that wine making strains have been domesticated for wine production. In this study, we demonstrate that humans can distinguish between wines produced using wine strains and wild strains of S. cerevisiae as well as its sibling species, Saccharomyces paradoxus. Wine strains produced wine with fruity and floral characteristics, whereas wild strains produced wine with earthy and sulfurous characteristics. The differences that we observe between wine and wild strains provides further evidence that wine strains have evolved phenotypes that are distinct from their wild ancestors and relevant to their use in wine production.
Project description: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).
Project description:Recently, a new type of hybrid resulting from the hybridization between Saccharomyces cerevisiae and Saccharomyces kudriavzevii was described. These strains exhibit physiological properties of potential biotechnological interest. A preliminary characterization of these hybrids showed a trend to reduce the S. kudriavzevii fraction of the hybrid genome. We characterized the genomic constitution of several wine S. cerevisiae x S. kudriavzevii strains by using a combined approach based on the restriction fragment length polymorphism analysis of gene regions, comparative genome hybridizations with S. cerevisiae DNA arrays, ploidy analysis, and gene dose determination by quantitative real-time PCR. The high similarity in the genome structures of the S. cerevisiae x S. kudriavzevii hybrids under study indicates that they originated from a single hybridization event. After hybridization, the hybrid genome underwent extensive chromosomal rearrangements, including chromosome losses and the generation of chimeric chromosomes by the nonreciprocal recombination between homeologous chromosomes. These nonreciprocal recombinations between homeologous chromosomes occurred in highly conserved regions, such as Ty long terminal repeats (LTRs), rRNA regions, and conserved protein-coding genes. This study supports the hypothesis that chimeric chromosomes may have been generated by a mechanism similar to the recombination-mediated chromosome loss acting during meiosis in Saccharomyces hybrids. As a result of the selective processes acting during fermentation, hybrid genomes maintained the S. cerevisiae genome but reduced the S. kudriavzevii fraction.
Project description: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.