A Saccharomyces eubayanus haploid resource for research studies.

Since its identification, Saccharomyces eubayanus has been recognized as the missing parent of the lager hybrid, S. pastorianus. This wild yeast has never been isolated from fermentation environments, thus representing an interesting candidate for evolutionary, ecological and genetic studies. However, it is imperative to develop additional molecular genetics tools to ease manipulation and thus facilitate future studies. With this in mind, we generated a collection of stable haploid strains representative of three main lineages described in S. eubayanus (PB-1, PB-2 and PB-3), by deleting the HO gene using CRISPR-Cas9 and tetrad micromanipulation. Phenotypic characterization under different conditions demonstrated that the haploid derivates were extremely similar to their parental strains. Genomic analysis in three strains highlighted a likely low frequency of off-targets, and sequencing of a single tetrad evidenced no structural variants in any of the haploid spores. Finally, we demonstrate the utilization of the haploid set by challenging the strains under mass-mating conditions. In this way, we found that S. eubayanus under liquid conditions has a preference to remain in a haploid state, unlike S. cerevisiae that mates rapidly. This haploid resource is a novel set of strains for future yeast molecular genetics studies.

An Out-of-Patagonia migration explains the worldwide diversity and distribution of Saccharomyces eubayanus lineages.

Population-level sampling and whole-genome sequences of different individuals allow one to identify signatures of hybridization, gene flow and potential molecular mechanisms of environmental responses. Here, we report the isolation of 160 Saccharomyces eubayanus strains, the cryotolerant ancestor of lager yeast, from ten sampling sites in Patagonia along 2,000 km of Nothofagus forests. Frequency of S. eubayanus isolates was higher towards southern and colder regions, demonstrating the cryotolerant nature of the species. We sequenced the genome of 82 strains and, together with 23 available genomes, performed a comprehensive phylogenetic analysis. Our results revealed the presence of five different lineages together with dozens of admixed strains. Various analytical methods reveal evidence of gene flow and historical admixture between lineages from Patagonia and Holarctic regions, suggesting the co-occurrence of these ancestral populations. Analysis of the genetic contribution to the admixed genomes revealed a Patagonian genetic origin of the admixed strains, even for those located in the North Hemisphere. Overall, the Patagonian lineages, particularly the southern populations, showed a greater global genetic diversity compared to Holarctic and Chinese lineages, in agreement with a higher abundance in Patagonia. Thus, our results are consistent with a likely colonization of the species from peripheral glacial refugia from South Patagonia. Furthermore, fermentative capacity and maltose consumption resulted negatively correlated with latitude, indicating better fermentative performance in northern populations. Our genome analysis, together with previous reports in the sister species S. uvarum suggests that a S. eubayanus ancestor was adapted to the harsh environmental conditions of Patagonia, a region that provides the ecological conditions for the diversification of these ancestral lineages.

Molecular profiling of beer wort fermentation diversity across natural Saccharomyces eubayanus isolates.

The utilization of S. eubayanus has recently become a topic of interest due to the novel organoleptic properties imparted to beer. However, the utilization of S. eubayanus in brewing requires the comprehension of the mechanisms that underlie fermentative differences generated from its natural genetic variability. Here, we evaluated fermentation performance and volatile compound production in ten genetically distinct S. eubayanus strains in a brewing fermentative context. The evaluated strains showed a broad phenotypic spectrum, some of them exhibiting a high fermentation capacity and high levels of volatile esters and/or higher alcohols. Subsequently, we obtained molecular profiles by generating 'end-to-end' genome assemblies, as well as metabolome and transcriptome profiling of two Patagonian isolates exhibiting significant differences in beer aroma profiles. These strains showed clear differences in concentrations of intracellular metabolites, including amino acids, such as valine, leucine and isoleucine, likely impacting the production of 2-methylpropanol and 3-methylbutanol. These differences in the production of volatile compounds are attributed to gene expression variation, where the most profound differentiation is attributed to genes involved in assimilatory sulfate reduction, which in turn validates phenotypic differences in H2 S production. This study lays a solid foundation for future research to improve fermentation performance and select strains for new lager styles based on aroma and metabolic profiles.

Distinct Transcriptional Changes in Response to Patulin Underlie Toxin Biosorption Differences in Saccharomyces Cerevisiae.

Patulin (4-hydroxy-4H-furo[3,2c]pyran-2[6H]-one) is a mycotoxin produced by a suite of fungi species. Patulin is toxic to humans and is a sporadic contaminant in products that were made from fungi-infected fruits. The baker yeast Saccharomyces cerevisiae (S. cerevisiae) has been shown to decrease patulin levels likely by converting it to the less harmful E-ascladiol, yet this capacity is dependent on the strain utilized. In this study we show that four representative strains of different S. cerevisiae lineages differ in their ability to tolerate and decrease patulin levels in solution, demonstrating that some strains are better suitable for patulin biocontrol. Indeed, we tested the biocontrol capacities of the best patulin-reducer strain (WE) in contaminated apple juice and demonstrated their potential role as an efficient natural biocontrol solution. To investigate the mechanisms behind the differences between strains, we explored transcriptomic changes of the top (WE strain) and worst (WA strain) patulin-biocontroller strains after being exposed to this toxin. Large and significant gene expression differences were found between these two strains, the majority of which represented genes associated with protein biosynthesis, cell wall composition and redox homeostasis. Interestingly, the WE isolate exhibited an overrepresentation of up-regulated genes involved in membrane components, suggesting an active role of the membrane towards patulin detoxification. In contrast, WA upregulated genes were associated with RNA metabolism and ribosome biogenesis, suggesting a patulin impact upon transcription and translation activity. These results suggest that different genotypes of S. cerevisiae encounter different stresses from patulin toxicity and that different rates of detoxification of this toxin might be related with the plasma membrane composition. Altogether, our data demonstrates the different molecular mechanisms in S. cerevisiae strains withstanding patulin exposure and opens new avenues for the selection of new patulin biocontroller strains.