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Hybridization between Saccharomyces cerevisiae and Saccharomyces eubayanus resulted in the emergence of S. pastorianus , a crucial yeast for lager fermentation. However, our understanding of hybridization success and hybrid vigor between these two species remains limited due to the scarcity of S. eubayanus parental strains. Here, we explore hybridization success and the impact of hybridization on fermentation performance and volatile compound profiles in newly formed lager hybrids. By selecting parental candidates spanning a diverse array of lineages from both species, we reveal that the Beer and PB-2 lineages exhibit high rates of hybridization success in S. cerevisiae and S. eubayanus , respectively. Polyploid hybrids were generated through a spontaneous diploid hybridization technique (rare-mating), revealing a prevalence of triploids and diploids over tetraploids. Despite the absence of heterosis in fermentative capacity, hybrids displayed phenotypic variability, notably influenced by maltotriose consumption. Interestingly, ploidy levels did not significantly correlate with fermentative capacity, although triploids exhibited greater phenotypic variability. The S. cerevisiae parental lineages primarily influenced volatile compound profiles, with significant differences in aroma production. Interestingly, hybrids emerging from the Beer S. cerevisiae parental lineages exhibited a volatile compound profile resembling the corresponding S. eubayanus parent. This pattern may result from the dominant inheritance of the S. eubayanus aroma profile, as suggested by the over-expression of genes related to alcohol metabolism and acetate synthesis in hybrids including the Beer S. cerevisiae lineage. Our findings suggest complex interactions between parental lineages and hybridization outcomes, highlighting the potential for creating yeasts with distinct brewing traits through hybridization strategies., Importance: Our study investigates the principles of lager yeast hybridization between Saccharomyces cerevisiae and Saccharomyces eubayanus . This process gave rise to the lager yeast Saccharomyces pastorianus . By examining how these novel hybrids perform during fermentation and the aromas they produce, we uncover the genetic bases of brewing trait inheritance. We successfully generated polyploid hybrids using diverse strains and lineages from both parent species, predominantly triploids and diploids. Although these hybrids did not show improved fermentation capacity, they exhibited varied traits, especially in utilizing maltotriose, a key sugar in brewing. Remarkably, the aroma profiles of these hybrids were primarily influenced by the S. cerevisiae parent, with Beer lineage hybrids adopting aroma characteristics from their S. eubayanus parent. These insights reveal the complex genetic interactions in hybrid yeasts, opening new possibilities for crafting unique brewing yeasts with desirable traits., Competing Interests: The authors declare no conflict of interest.

Lager yeasts are limited to a few strains worldwide, imposing restrictions on flavour and aroma diversity and hindering our understanding of the complex evolutionary mechanisms during yeast domestication. The recent finding of diverse S. eubayanus lineages from Patagonia offers potential for generating new lager yeasts with different flavour profiles. Here, we leverage the natural genetic diversity of S. eubayanus and expand the lager yeast repertoire by including three distinct Patagonian S. eubayanus lineages. We used experimental evolution and selection on desirable traits to enhance the fermentation profiles of novel S. cerevisiae x S. eubayanus hybrids. Our analyses reveal an intricate interplay of pre-existing diversity, selection on species-specific mitochondria, de-novo mutations, and gene copy variations in sugar metabolism genes, resulting in high ethanol production and unique aroma profiles. Hybrids with S. eubayanus mitochondria exhibited greater evolutionary potential and superior fitness post-evolution, analogous to commercial lager hybrids. Using genome-wide screens of the parental subgenomes, we identified genetic changes in IRA2, IMA1, and MALX genes that influence maltose metabolism, and increase glycolytic flux and sugar consumption in the evolved hybrids. Functional validation and transcriptome analyses confirmed increased maltose-related gene expression, influencing greater maltotriose consumption in evolved hybrids. This study demonstrates the potential for generating industrially viable lager yeast hybrids from wild Patagonian strains. Our hybridization, evolution, and mitochondrial selection approach produced hybrids with high fermentation capacity and expands lager beer brewing options., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Molinet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The study of natural variation can untap novel alleles with immense value for biotechnological applications. Saccharomyces eubayanus Patagonian isolates exhibit differences in the diauxic shift between glucose and maltose, representing a suitable model to study their natural genetic variation for novel strains for brewing. However, little is known about the genetic variants and chromatin regulators responsible for these differences. Here, we show how genome-wide chromatin accessibility and gene expression differences underlie distinct diauxic shift profiles in S. eubayanus. We identified two strains with a rapid diauxic shift between glucose and maltose (CL467.1 and CBS12357) and one strain with a remarkably low fermentation efficiency and longer lag phase during diauxic shift (QC18). This is associated in the QC18 strain with lower transcriptional activity and chromatin accessibility of specific genes of maltose metabolism and higher expression levels of glucose transporters. These differences are governed by the HAP complex, which differentially regulates gene expression depending on the genetic background. We found in the QC18 strain a contrasting phenotype to those phenotypes described in S. cerevisiae, where hap4Δ , hap5Δ , and cin5Δ knockouts significantly improved the QC18 growth rate in the glucose-maltose shift. The most profound effects were found between CIN5 allelic variants, suggesting that Cin5p could strongly activate a repressor of the diauxic shift in the QC18 strain but not necessarily in the other strains. The differences between strains could originate from the tree host from which the strains were obtained, which might determine the sugar source preference and the brewing potential of the strain. IMPORTANCE The diauxic shift has been studied in budding yeast under laboratory conditions; however, few studies have addressed the diauxic shift between carbon sources under fermentative conditions. Here, we study the transcriptional and chromatin structure differences that explain the natural variation in fermentative capacity and efficiency during diauxic shift of natural isolates of S. eubayanus. Our results show how natural genetic variants in transcription factors impact sugar consumption preferences between strains. These variants have different effects depending on the genetic background, with a contrasting phenotype to those phenotypes previously described in S. cerevisiae. Our study shows how relatively simple genetic/molecular modifications/editing in the lab can facilitate the study of natural variations of microorganisms for the brewing industry.

Most organisms belonging to the Saccharomycotina subphylum have high genetic diversity and a vast repertoire of metabolisms and lifestyles. Lachancea cidri is an ideal yeast model for exploring the interplay between genetics, ecological function and evolution. Lachancea cidri diverged from the Saccharomyces lineage before the whole-genome duplication and is distributed across the South Hemisphere, displaying an important ecological success. We applied phylogenomics to investigate the genetic variation of L. cidri isolates obtained from Australia and South America. Our approach revealed the presence of two main lineages according to their geographic distribution (Aus and SoAm). Estimation of the divergence time suggests that SoAm and Aus lineages diverged near the last glacial maximum event during the Pleistocene (64-8 KYA). Interestingly, we found that the French reference strain is closely related to the Australian strains, with a recent divergence (405-51 YA), likely associated to human movements. Additionally, we identified different lineages within the South American population, revealing that Patagonia contains a similar genetic diversity comparable to that of other lineages in S. cerevisiae. These findings support the idea of a Pleistocene-dated divergence between South Hemisphere lineages, where the Nothofagus and Araucaria ecological niches likely favoured the extensive distribution of L. cidri in Patagonia., (© 2022 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., (© 2022. The Author(s).)

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.