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Yeast optimisation has been crucial in improving the quality and efficiency of beer production, one of the world's most widely consumed beverages. In this context, rare mating hybridisation is a promising technique for yeast optimization to generate novel and improved non-GMO strains. The limitation of this technique is the lack of knowledge and comparable data on yeast strains hybridisable to Saccharomyces cerevisiae, probably the most important yeast species in beer production. Yeast from the genera Saccharomyces, Naumovozyma, Nakaseomyces and Kazachstania have been described to be able to form hybrids with S. cerevisiae. In the present study, 242 yeast strains were analysed under brewing conditions, including Saccharomyces species (S. cerevisiae, S. kudriavzevii, S. uvarum, S. eubayanus, S. paradoxus, S. mikatae, S. jurei and S. arboricola) and non-Saccharomyces species (Naumovozyma, Nakaseomyces and Kazaschtania), representing the full genetic variability (species and subpopulations) described up to the start of the study. The fermentation profile was analysed by monitoring weight loss during fermentation to determine kinetic parameters and CO 2 production. Metabolic analysis was performed to determine the concentration of sugars (maltotriose, maltose and glucose), alcohols (ethanol, glycerol and 2,3-butanediol) and organic acids (malic acid, succinic acid and acetic acid). Maltose and maltotriose are the predominant sugars in beer wort. The ability to consume these sugars determines the characteristics of the final product. Dataset comparisons were then made at species, subpopulation and isolation source level. The results obtained in this study demonstrate the great phenotypic variability that exists within the genus Saccharomyces and within each species of this genus, which could be useful in the generation of optimised brewing hybrids. Yeasts with different fermentative capacities and fermentative behaviours can be found under brewing conditions. S. cerevisiae, S. uvarum and S. eubayanus are the species that contain strains with similar fermentation performance to commercial strains., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Species delineation in microorganisms is challenging due to the limited markers available for accurate species assignment. Here, we applied an integrative taxonomy approach, combining extensive sampling, whole-genome sequence-based classification, phenotypic profiling, and assessment of interspecific reproductive isolation. Our work reveals the presence of a distinct Saccharomyces lineage in Nothofagus forests of coastal Patagonia. This lineage, designated Saccharomyces chiloensis sp. nov., exhibits 7% genetic divergence from its sister species S. uvarum, as revealed by whole-genome sequencing and population analyses. The South America-C (SA-C) coastal Patagonia population forms a unique clade closely related to a previously described divergent S. uvarum population from Oceania (AUS, found in Australia and New Zealand). Our species reclassification is supported by a low Ortho Average Nucleotide Identity (OANI) of 93% in SA-C and AUS relative to S. uvarum, which falls below the suggested species delineation threshold of 95%, indicating an independent evolutionary lineage. Hybrid spore viability assessment provided compelling evidence that SA-C and AUS are reproductively isolated from S. uvarum. In addition, we found unique structural variants between S. chiloensis sp. nov. lineages, including large-scale chromosomal translocations and inversions, together with a distinct phenotypic profile, emphasizing their intraspecies genetic distinctiveness. We suggest that S. chiloensis sp. nov diverged from S. uvarum in allopatry due to glaciation, followed by post-glacial dispersal, resulting in distinct lineages on opposite sides of the Pacific Ocean. The discovery of S. chiloensis sp. nov. illustrates the uniqueness of Patagonia's coastal biodiversity and underscores the importance of adopting an integrative taxonomic approach in species delineation to unveil cryptic microbial species. The holotype of S. chiloensis sp. nov. is CBS 18620T., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Peña 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.)

This study aimed to investigate how parental genomes contribute to yeast hybrid metabolism using a metabolomic approach. Previous studies have explored central carbon and nitrogen metabolism in Saccharomyces species during wine fermentation, but this study analyses the metabolomes of Saccharomyces hybrids for the first time. We evaluated the oenological performance and intra- and extracellular metabolomes, and we compared the strains according to nutrient consumption and production of the main fermentative by-products. Surprisingly, no common pattern was observed for hybrid genome influence; each strain behaved differently during wine fermentation. However, this study suggests that the genome of the S. cerevisiae species may play a more relevant role in fermentative metabolism. Variations in biomass/nitrogen ratios were also noted, potentially linked to S. kudriavzevii and S. uvarum genome contributions. These results open up possibilities for further research using different "omics" approaches to comprehend better metabolic regulation in hybrid strains with genomes from different species., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Lager brewing first occurred in Bavaria in the 15th century, associated with restrictions of brewing to colder months. The lager yeast, Saccharomyces pastorianus, is cold tolerant. It is a hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus, and has been found only in industrial settings. Natural isolates of S. eubayanus were first discovered in Patagonia 11 years ago. They have since been isolated from China, Tibet, New Zealand, and North America, but not from Europe. Here, we describe the first European strains UCD646 and UCD650, isolated from a wooded area on a university campus in Dublin, Ireland. We generated complete chromosome level assemblies of both genomes using long- and short-read sequencing. The UCD isolates belong to the Holarctic clade. Genome analysis shows that isolates similar to the Irish strains contributed to the S. eubayanus component of S. pastorianus, but isolates from Tibet made a larger contribution., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

With the current COVID-19 pandemic, we all realized how important interactions are. Interactions are everywhere. At the cellular level, protein interactions play a key role and their ensemble, also called interactome, is often referred as the basic building blocks of life. Given its importance, the maintenance of the integrity of the interactome is a real challenge in the cell. Many events during evolution can disrupt interactomes and potentially result in different characteristics for the organisms. However, the molecular underpinnings of changes in interactions at the cellular level are still largely unexplored. Among the perturbations, hybridization puts in contact two different interactomes, which may lead to many changes in the protein interaction network of the hybrid, including gains and losses of interactions. We recently investigated the fate of the interactomes after hybridization between yeast species using a comparative proteomics approach. A large-scale conservation of the interactions was observed in hybrids, but we also noticed the presence of proteostasis-related changes. This suggests that, despite a general robustness, small differences may accumulate in hybrids and perturb their protein physiology. Here, we summarize our work with a broader perspective on the importance of interactions., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Populations of microbes are constantly evolving heterogeneity that selection acts upon, yet heterogeneity is nontrivial to assess methodologically. The necessary practice of isolating single-cell colonies and thus subclone lineages for establishing, transferring, and using a strain results in single-cell bottlenecks with a generally neglected effect on the characteristics of the strain itself. Here, we present evidence that various subclone lineages for industrial yeasts sequenced for recent genomic studies show considerable differences, ranging from loss of heterozygosity to aneuploidies. Subsequently, we assessed whether phenotypic heterogeneity is also observable in industrial yeast, by individually testing subclone lineages obtained from products. Phenotyping of industrial yeast samples and their newly isolated subclones showed that single-cell bottlenecks during isolation can indeed considerably influence the observable phenotype. Next, we decoupled fitness distributions on the level of individual cells from clonal interference by plating single-cell colonies and quantifying colony area distributions. We describe and apply an approach using statistical modeling to compare the heterogeneity in phenotypes across samples and subclone lineages. One strain was further used to show how individual subclonal lineages are remarkably different not just in phenotype but also in the level of heterogeneity in phenotype. With these observations, we call attention to the fact that choosing an initial clonal lineage from an industrial yeast strain may vastly influence downstream performances and observations on karyotype, on phenotype, and also on heterogeneity., (© 2021 The Authors. Yeast published by John Wiley & Sons Ltd.)

A collection of 33 Saccharomyces yeasts were used for wine fermentation with a sole nitrogen source: ammonium and four individual aroma-inducing amino acids. The fermentation performance and chemical wine composition were evaluated. The most valuable nitrogen sources were valine as a fermentation promoter on non-cerevisiae strains, phenylalanine as fruity aromas enhancer whereas the ethanol yield was lessened by leucine and isoleucine. S. cerevisiae SC03 and S. kudriavzevii SK02 strains showed to be the greatest producers of fruity ethyl esters while S. kudriavzevii strains SK06 and SK07 by shortening the fermentation duration. S. uvarum strains produced the greatest succinic acid amounts and, together with S. eubayanus, they reached the highest production of 2-phenylethanol and its acetate ester; whereas S. kudriavzevii strains were found to be positively related to high glycerol production., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Hybridization and polyploidization are powerful mechanisms of speciation. Hybrid speciation often coincides with whole-genome duplication (WGD) in eukaryotes. This suggests that WGD may allow hybrids to thrive by increasing fitness, restoring fertility and/or increasing access to adaptive mutations. Alternatively, it has been suggested that hybridization itself may trigger WGD. Testing these models requires quantifying the rate of WGD in hybrids without the confounding effect of natural selection. Here we show, by measuring the spontaneous rate of WGD of more than 1300 yeast crosses evolved under relaxed selection, that some genotypes or combinations of genotypes are more prone to WGD, including some hybrids between closely related species. We also find that higher WGD rate correlates with higher genomic instability and that WGD increases fertility and genetic variability. These results provide evidence that hybridization itself can promote WGD, which in turn facilitates the evolution of hybrids.

We have previously isolated heterothallic haploid strains from original homothallic diploids of two yeast species in the family Saccharomycetaceae. In this study, heterothallic haploid strains were isolated from an original homothallic diploid of Saccharomyces kudriavzevii type strain, followed by investigation of sexual interactions among these yeast strains, in addition to S. cerevisiae laboratory strains. It has been shown that prezygotic reproductive isolation was observed between Kazachstania naganishii and S. cerevisiae with α-factor mating pheromones representing crossaction with each other beyond the genus boundary. Using heterothallic strains, postzygotic reproductive isolation system was shown to reside in the genus Saccharomyces by mass mating and cell-cell contact experiments. In mass mating experiments, crossaction of α-factor and a-factor mating pheromones and sexual agglutination effectively occurred beyond species boundaries among S. kudriavzevii, S. paradoxus, and S. cerevisiae. When the fates of cell-cell pairs from these Saccharomyces yeast species were systematically chased one by one, interspecific F1 hybrids were effectively produced, while sporulations were partially prohibited, with spore germination perfectly blocked in the hybrids. These results indicated that postzygotic reproductive isolation definitively resides among these Saccharomyces yeast species and that disorder of chromosome organization had to some extent occurred in interspecific F1 hybrids., (© 2021 John Wiley & Sons, Ltd.)

Sterols are essential components of the yeast membrane and their synthesis requires oxygen. Yet, Saccharomyces cerevisiae has developed the ability to take up sterols from the medium under anaerobiosis. Here we investigated sterol uptake efficiency and the expression of genes related to sterol import in Saccharomyces and non-Saccharomyces wine yeast species fermenting under anaerobic conditions. The sterol uptake efficiency of 39 strains was evaluated by flow cytometry (with 25-NBD Cholesterol, a fluorescent cholesterol probe introduced in the medium) and we found an important discrepancy between Saccharomyces and non-Saccharomyces wine yeast species that we correlated to a lower final cell population and a lower fermentation rate. A high uptake of sterol was observed in the various Saccharomyces strains. Spot tests performed on 13 of these strains confirmed the differences between Saccharomyces and non-Saccharomyces strains, suggesting that the presence of the sterol uptake transporters AUS1 and PDR11 could cause these discrepancies. Indeed, we could not find any homologue to these genes in the genome of Hanseniaspora uvarum, H. guillermondii, Lachancea thermotolerans, Torulaspora delbreueckii, Metschnikowia pulcherrima, or Starmarella bacillaris species. The specialization of sterol import function for post genome-duplication species may have favored growth under anaerobiosis., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition., Competing Interests: The authors have declared that no competing interests exist.

Recently, the increase in microbreweries and the consequent production of craft beers have reached exponential growth. The interest in non-conventional yeasts for innovation and a unique selling feature in beer fermentation is increasing. This work studied the autochthonous Saccharomyces and non-Saccharomyces yeasts, isolated from various food sources, with the ability to modify and improve the fermentative and aromatic profiles during alcoholic fermentation. The ability to ferment maltose and produce desirable aroma compounds were considered as the key characters for the screening selection. A synthetic beer wort was developed for this purpose, to simulate beer wort composition. A total of forty-seven yeast strains belonging to different genera were analysed according to their fermentation profile, volatile compounds production and sensory analysis. Three native strains of Saccharomyces cerevisiae, Zygoascus meyerae and Pichia anomala were selected to evaluate their aromatic profile in single and mixed fermentations. The strains produced 4-vinylguaiacol, β-phenylethyl alcohol, and isoamyl alcohol at levels significantly above the sensory threshold, making them interesting for wheat and blond craft beer styles. The native Hanseniaspora vineae was also included in a co-fermentation treatment, resulting in a promising yeast to produce fruity beers., (Copyright © 2020. Published by Elsevier B.V.)

Genome introgressions drive evolution across the animal 1 , plant 2 and fungal 3 kingdoms. Introgressions initiate from archaic admixtures followed by repeated backcrossing to one parental species. However, how introgressions arise in reproductively isolated species, such as yeast 4 , has remained unclear. Here we identify a clonal descendant of the ancestral yeast hybrid that founded the extant Saccharomyces cerevisiae Alpechin lineage 5 , which carries abundant Saccharomyces paradoxus introgressions. We show that this clonal descendant, hereafter defined as a 'living ancestor', retained the ancestral genome structure of the first-generation hybrid with contiguous S. cerevisiae and S. paradoxus subgenomes. The ancestral first-generation hybrid underwent catastrophic genomic instability through more than a hundred mitotic recombination events, mainly manifesting as homozygous genome blocks generated by loss of heterozygosity. These homozygous sequence blocks rescue hybrid fertility by restoring meiotic recombination and are the direct origins of the introgressions present in the Alpechin lineage. We suggest a plausible route for introgression evolution through the reconstruction of extinct stages and propose that genome instability allows hybrids to overcome reproductive isolation and enables introgressions to emerge.

In this work the potential of comparative transcriptomics was explored of Saccharomyces (S.) cerevisiae and S. pastorianus for their discrimination. This way an alternative should be demonstrated to comparative genomics, which can be difficult as a result of their aneuoploid genomes composed of mosaics of the parental genomes. Strains were selected according to their application in beer brewing, i.e. top and bottom fermenting yeasts. Comparative transcriptomics was performed for four strains each of commercially available S. cerevisiae (top fermenting) and Saccharomyces pastorianus (bottom fermenting) brewing yeasts grown at two different temperatures to mid-exponential growth phase. A non-reference based approach was chosen in the form of alignment against a de novo assembled brewery-associated pan transcriptome to exclude bias introduced by manual selection of reference genomes. The result is an analysis workflow for self-contained comparative transcriptomics of Saccharomyces yeasts including, but not limited to, the analysis of core and accessory gene expression, functional analysis and metabolic classification. The functionality of this workflow is demonstrated along the principal differentiation of accessory transcriptomes of S. cerevisiae versus S. pastorianus strains. Hence, this work provides a concept enabling studies under different brewing conditions., Competing Interests: The authors have declared that no competing interests exist.

Rationale: Seventeen different dried yeast strains, including twelve strains of Saccharomyces cerevisiae and five strains of S. pastorianus, were analyzed using direct analysis in real time (DART) time-of-flight mass spectrometry. The resulting mass spectra were used for rapid species and strain differentiation based upon small-molecule metabolomic profiles., Methods: Yeast strains purchased from local shops were suspended in a 1:1 water-methanol solution. Solutions were sampled by dipping the sealed end of a melting point capillary into each vial. Six replicates were measured in positive-ion and negative-ion mode for each strain using an automated linear rail with the DART source operated with helium gas and a gas heater temperature of 350°C. Averaged and centroided mass spectra were exported for analysis with chemometric software., Results: Negative-ion DART mass spectra exhibited less chemical background and more distinctive components than positive-ion DART mass spectra. An on-line search of the Yeast Metabolome Database provided candidate metabolites for selection as features for chemometric analysis. Negative-ion DART mass spectra could distinguish both species and all strains. The DART analysis was also able to identify potential metabolomic differences between top-fermenting and bottom-fermenting yeast, between beer and baking yeast, and between red wine and champagne yeast., Conclusions: All strains could be distinguished by their negative-ion DART mass spectra with 97.7% validation accuracy. Clear differences were observed between dry and liquid forms and Saccharomyces strains with different applications to baking or beverage fermentation. Possible differences in metabolite profiles were suggested, but not confirmed, by accurate mass data., (© 2020 John Wiley & Sons, Ltd.)

The genus Saccharomyces is an evolutionary paradox. On the one hand, it is composed of at least eight clearly phylogenetically delineated species; these species are reproductively isolated from each other, and hybrids usually cannot complete their sexual life cycles. On the other hand, Saccharomyces species have a long evolutionary history of hybridization, which has phenotypic consequences for adaptation and domestication. A variety of cellular, ecological, and evolutionary mechanisms are responsible for this partial reproductive isolation among Saccharomyces species. These mechanisms have caused the evolution of diverse Saccharomyces species and hybrids, which occupy a variety of wild and domesticated habitats. In this article, we introduce readers to the mechanisms isolating Saccharomyces species, the circumstances in which reproductive isolation mechanisms are effective and ineffective, and the evolutionary consequences of partial reproductive isolation. We discuss both the evolutionary history of the genus Saccharomyces and the human history of taxonomists and biologists struggling with species concepts in this fascinating genus.

In this study, we presented the first metabolome time course analysis performed among a set of S. uvarum, S. kudriavzevii and S. cerevisiae strains under winemaking conditions. Extracellular and intracellular metabolites, as well as physiological parameters of yeast cells, were monitored along the process to find evidence of different metabolic strategies among species to perform alcoholic fermentation. A thorough inspection of time trends revealed several differences in utilization or accumulation of fermentation by-products. We confirmed the ability of S. uvarum and S. kudriavzevii strains to produce higher amounts of glycerol, succinate or some fusel alcohols and their corresponding esters. We also reported differences in the yields of less common fermentative by-products involved in redox homeostasis, namely 2,3 butanediol and erythritol. 2,3 butanediol yield was higher in must ferment with cryophilic strains and erythritol, a pentose phosphate pathway derivative, was particularly overproduced by S. uvarum strains. Contrary to S. cerevisiae, a singular production-consumption rate of acetate was also observed in S. uvarum and S. kudriavzevii fermentations. Since acetate is a precursor for acetyl-CoA production which is involved in the biosynthesis of membrane lipids, cryophilc strains might take advantage of extracellular acetate to remodel cell membrane as ethanol content increased during fermentation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chemical and sensory properties of Torrontés Riojano sparkling wines, prepared using second fermentation with Saccharomyces strains EC1118, bayanus C12 and IFI473I, were explored. All sparkling wines showed high levels of several volatile ethyl esters and terpenes associated to fruity and floral aromas. The sensory profiles showed significant differences for the floral aroma descriptor among EC1118, bayanus C12 and IFI473I and for bubble persistence for strain bayanus C12. Our results suggest that the sensory properties of these sparkling wines could be dependent on the chemical and organoleptic properties of the base wine more than the yeast strain used for second fermentation.

Many species are able to hybridize, but the sterility of these hybrids effectively prevents gene flow between the species, reproductively isolating them and allowing them to evolve independently. Yeast hybrids formed by Saccharomyces cerevisiae and Saccharomyces paradoxus parents are viable and able to grow by mitosis, but they are sexually sterile because most of the gametes they make by meiosis are inviable. The genomes of these two species are so diverged that they cannot recombine properly during meiosis, so they fail to segregate efficiently. Thus most hybrid gametes are inviable because they lack essential chromosomes. Recent work shows that chromosome mis-segregation explains nearly all observed hybrid sterility-genetic incompatibilities have only a small sterilising effect, and there are no significant sterilising incompatibilities in chromosome arrangement or number between the species. It is interesting that chromosomes from these species have diverged so much in sequence without changing in configuration, even though large chromosomal changes occur quite frequently, and sometimes beneficially, in evolving yeast populations.

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., Competing Interests: the authors have declared that no competing interests exist.

Saccharomyces cerevisiae is the most extensively studied yeast and, over the last century, provided insights on the physiology, genetics, cellular biology and molecular mechanisms of eukaryotes. More recently, the increase in the discovery of wild strains, species and hybrids of the genus Saccharomyces has shifted the attention towards studies on genome evolution, ecology and biogeography, with the yeast becoming a model system for population genomic studies. The genus currently comprises eight species, some of clear industrial importance, while others are confined to natural environments, such as wild forests devoid from human domestication activities. To date, numerous studies showed that some Saccharomyces species form genetically diverged populations that are structured by geography, ecology or domestication activity and that the yeast species can also hybridize readily both in natural and domesticated environments. Much emphasis is now placed on the evolutionary process that drives phenotypic diversity between species, hybrids and populations to allow adaptation to different niches. Here, we provide an update of the biodiversity, ecology and population structure of the Saccharomyces species, and recapitulate the current knowledge on the natural history of Saccharomyces genus., (© FEMS 2020.)

Khanthuli peat swamp forest (PSF) is one of a few fertile peat swamp forests that remain in Thailand. It is composed of primary PSF and some areas which have been degraded to secondary PSF due to drought, wildfires and land conversion, which have resulted in a decrease in peat layers and change in the species of the plant community. In this study, diversity of yeasts in peat from both primary and secondary PSF areas of the Khanthuli PSF was determined based on culture-dependent approaches, using dilution plate and enrichment techniques. A total of 66 yeast isolates were identified by the analysis of sequence similarity of the D1/D2 region of the large subunit rRNA gene or the combined analysis of sequence of the D1/D2 region and internal transcribed spacer region and confirmed by phylogenetic analysis of the D1/D2 region to belong to 22 known yeast species and six potential new species in the genera Candida (Kurtzmaniella, Lodderomyces, Ogataea, Pichia and Yamadazyma clades), Clavispora, Cyberlindnera, Galactomyces, Hanseniaspora, Metschnikowia, Saturnispora, Schwanniomyces, Cryptotrichosporon, Pichia, Curvibasidium, Papiliotrema, Rhodotorula, and Saitozyma. The most prevalent yeasts in the primary PSF were Cyberlindnera subsufficiens and Galactomyces candidus, while Saitozyma podzolica was the most frequently found in peat from the secondary PSF. Common yeast species in both, primary and secondary PSF, were Cy. subsufficiens, G. candidus and Rhodotorula mucilaginosa.

Lack of the prezygotic barrier in the Saccharomyces genus facilitates the construction of artificial interspecific hybrids among different Saccharomyces species. Hybrids that maintain the interesting features of parental strains have been applied in industry for many beneficial purposes. Two of the most important problems faced by wine makers is nitrogen deficiency in grape must and low-temperature fermentation. In our study, hybrids were constructed by using selected low nitrogen-demanding cryotolerant S. eubayanus, S. uvarum strains and S. cerevisiae. The fermentation capacity of the hybrid strains was tested under four conditions by combining two temperatures, 12 °C and 28 °C, and two nitrogen concentrations, 60 mg/L and 300 mg/L. The hybrid strains obtained combined characters of both parental strains and conferred better fermentation rates under low-temperature or low-nitrogen conditions. The hybrid strains also produced larger amounts of acetate esters and higher alcohols, which increase aroma intensity and complexity in wine. Nitrogen sources were more rapidly consumed by the hybrid strains, which allows greater competition ability under nitrogen-deficiency conditions. Therefore, the interspecific hybridisation between low nitrogen-demanding cryotolerant strains and S. cerevisiae is a potential solution for low-temperature or low-nitrogen fermentations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The 26S proteasome is a multisubunit ATP-dependent protease complex and is necessary for the normal function of the eukaryotic cell and its survival in stress. Twenty years ago, we, in collaboration with German researchers, were the first to experimentally describe a system for coordinated regulation of proteasomal gene expression in the yeast Saccharomyces cerevisiae. This system consists of the ScRpn4 transcription factor and its binding site, called PACE. Based on the results of a bioinformatics search in the first sequenced yeast genomes, Rpn4-like proteins and PACE-like elements were postulated for other species of the class Saccharomycetes. We experimentally characterized Rpn4-like proteins in the biotechnologically significant yeast species Komagataella pfaffii (Pichia pastoris), Yarrowia lipolytica, and Debaryomyces hansenii and the opportunistic yeast Candida glabrata. As ample information accumulates for the genome sequences of new yeast species and strains, the question arises as to how diverse the regulatory system of proteasomal genes is in terms of structure and likely mechanisms of function. In this work, a bioinformatics search for Rpn4-like proteins and PACE-like elements was conducted in 3111 strains belonging to 427 yeast species of the class Saccharomycetes. It was shown that only the DNA-binding domain is conserved among Rpn4-like proteins, in accordance with conservation of PACE elements. Certain systems were found to contain more than one Rpn4-like protein with structural differences in the DNA-binding domain or to include an autoregulation of the genes for Rpn4-like proteins. Given that Rpn4-like proteins and proteasomes play a role in the cell response to stress, the diversity of systems for the regulation of proteasomal genes was assumed to corresponds to adaptation of organisms to their living environments.

An increasing number of infections originating from probiotic use are reported worldwide, with the majority of such cases caused by the yeast Saccharomyces 'boulardii', a subtype of S. cerevisiae. Reliably linking infectious cases to probiotic products requires unequivocal genotyping data, however, these techniques are often time-consuming and difficult to implement in routine diagnostics. This leads to a widespread lack of genetic data regarding the origin of Saccharomyces infections. We propose a quick and reliable PCR-based protocol for the identification of S. 'boulardii' based on a combined analysis of interdelta fingerprinting and microsatellite typing. By applying various typing methods and our proposed method to the clinical yeast collection of a Hungarian hospital we show that probiotic origin is common among clinical Saccharomyces, and that the new multiplex method enables rapid and unequivocal identification of probiotic yeast infections. This method can be applied for the identification of yeast infection sources, helping decisions on probiotic use., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The sequencing of over a thousand Saccharomyces cerevisiae genomes revealed a complex pangenome. Over one third of the discovered genes are not present in the S. cerevisiae core genome but instead are often restricted to a subset of yeast isolates and thus may be important for adaptation to specific environmental niches. We refer to these genes as "pan-genes," being part of the pangenome but not the core genome. Here, we describe the evolutionary journey and characterisation of a novel pan-gene, originally named hypothetical (HYPO) open-reading frame. Phylogenetic analysis reveals that HYPO has been predominantly retained in S. cerevisiae strains associated with brewing but has been repeatedly lost in most other fungal species during evolution. There is also evidence that HYPO was horizontally transferred at least once, from S. cerevisiae to Saccharomyces paradoxus. The phylogenetic analysis of HYPO exemplifies the complexity and intricacy of evolutionary trajectories of genes within the S. cerevisiae pangenome. To examine possible functions for Hypo, we overexpressed a HYPO-GFP fusion protein in both S. cerevisiae and Saccharomyces pastorianus. The protein localised to the plasma membrane where it accumulated initially in distinct foci. Time-lapse fluorescent imaging revealed that when cells are grown in wort, Hypo-gfp fluorescence spreads throughout the membrane during cell growth. The overexpression of Hypo-gfp in S. cerevisiae or S. pastorianus strains did not significantly alter cell growth in medium-containing glucose, maltose, maltotriose, or wort at different concentrations., (© 2019 John Wiley & Sons, Ltd.)

The species of the genus Saccharomyces are commonly inhabiting tree bark and the surrounding soil, but their abundance have likely been underestimated due to biases in culturing methods. Metagenomic studies have so far been unable to detect Saccharomyces species in wild environments. Here, we sequenced the mycobiome of soils surrounding different trees at various altitudes in the Italian Alps. To survey for yeasts species belonging to Saccharomyces genus rather than other fungal species, we performed a selectivity step involving the isolation of the internal transcribed spacer (ITS) region that is specific to this yeast group. Reads mapping to Saccharomyces species were detected in all soil samples, including reads for S. mikatae and for S. eubayanus. ITS1 alignment of the S. cerevisiae, S. paradoxus and S. kudriavzevii sequences showed up to three base pair polymorphisms with other known strains, indicating possible new lineages. Basidiomycetous fungi were still the dominant species, compared to the Ascomycota, but the selectivity step allowed for the first time the detection and study of the biodiversity of the Saccharomyces species in their natural environment., (© 2018 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The selection of yeasts with low nitrogen requirement is a current need in winemaking. In this work, we analysed nitrogen requirements of strains belonging to the cryotolerant species S. uvarum, S. eubayanus and S. kudriavzevii, in order to evaluate their potential for conducting the fermentation of low nitrogen content grape musts. Our result demonstrated that S. eubayanus is the species less influenced by the increasing nitrogen concentrations in both growth and fermentation conditions. Strains showing the best behaviours, S. eubayanus NPCC 1285 and S. uvarum NPCC 1317, were selected to be tested in mixed cultures with S. cerevisiae T73 at different temperatures (12 °C, 20 °C and 28 °C) in synthetic grape must with different nitrogen concentrations (60, 140 and 300 mg/L YAN). The cryotolerant strains dominated the fermentations carried out at 12 °C while S. cerevisiae prevailed at 28 °C independently from the nitrogen concentration. At intermediate temperature, 20 °C, S. eubayanus mono and mixed cultures showed the best fermentative behaviour especially with low and intermediate nitrogen concentration. In summary, cryotolerant Saccharomyces species, particularly S. eubayanus, could be interesting tools to avoid fermentations stucks caused by low nitrogen content in grape musts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The aim of this study was to characterize the yeast consortium isolated from Grignolino grapes in a newly planted vineyard in Piedmont (Italy) via analysis of the intra-vineyard yeast distribution of grape samples from single rows. A two-phase approach allowed the identification of culturable yeasts present on grape skins and, through an enriching procedure via grape fermentation, the isolation of low frequency non-Saccharomyces and Saccharomyces spp. fermentative species, including S. paradoxus, which is highly unusual during grape fermentation, along with the intra-specific characterization of S. cerevisiae isolates. Culture-based molecular techniques revealed a grape yeast microbiota formed by (in order of abundance) Hanseniaspora uvarum, the yeast-like fungus Aerobasidium pullulans, Candida zemplinina, Pichia kluyveri, Candida californica, Curvibasidium cygneicollum, Meyerozima caribbica, Rhodotorula babjevae, Metschnikowia pulcherrima and Cryptococcus flavescens. Technological properties of isolated Saccharomyces spp. strains were analysed, identifying strains, including S. paradoxus, potentially suitable as an ecotypical starter for territorial wines., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Today's beer market is challenged by a decreasing consumption of traditional beer styles and an increasing consumption of specialty beers. In particular, lager-type beers (pilsner), characterized by their refreshing and unique aroma and taste, yet very uniform, struggle with their sales. The development of novel variants of the common lager yeast, the interspecific hybrid Saccharomyces pastorianus, has been proposed as a possible solution to address the need of product diversification in lager beers. Previous efforts to generate new lager yeasts through hybridization of the ancestral parental species (S. cerevisiae and S. eubayanus) yielded strains with an aromatic profile distinct from the natural biodiversity. Unfortunately, next to the desired properties, these novel yeasts also inherited unwanted characteristics. Most notably is their phenolic off-flavor (POF) production, which hampers their direct application in the industrial production processes. Here, we describe a CRISPR-based gene editing strategy that allows the systematic and meticulous introduction of a natural occurring mutation in the FDC1 gene of genetically complex industrial S. cerevisiae strains, S. eubayanus yeasts and interspecific hybrids. The resulting cisgenic POF- variants show great potential for industrial application and diversifying the current lager beer portfolio., Competing Interests: The authors have declared that no competing interests exist.

The yeast Lachancea thermotolerans (previously Kluyveromyces thermotolerans) is a species of large, yet underexplored, oenological potential. This study delivers comprehensive oenological phenomes of 94 L. thermotolerans strains obtained from diverse ecological niches worldwide, classified in nine genetic groups based on their pre-determined microsatellite genotypes. The strains and the genetic groups were compared for their alcoholic fermentation performance, production of primary and secondary metabolites and pH modulation in Chardonnay grape juice fermentations. The common oenological features of L. thermotolerans strains were their glucophilic character, relatively extensive fermentation ability, low production of acetic acid and the formation of lactic acid, which significantly affected the pH of the wines. An untargeted analysis of volatile compounds, used for the first time in a population-scale phenotyping of a non-Saccharomyces yeast, revealed that 58 out of 90 volatiles were affected at an L. thermotolerans strain level. Besides the remarkable extent of intra-specific diversity, our results confirmed the distinct phenotypic performance of L. thermotolerans genetic groups. Together, these observations provide further support for the occurrence of domestication events and allopatric differentiation in L. thermotolerans population.

Saccharomyces eubayanus is the close relative of the Lager-brewing yeast and was firstly found in North Patagonia associated with Nothofagus trees. In recent years additional strains were found in North America, Asia and New Zealand, and genomic analyses showed the existence of two main populations of this yeast, both of them present in Patagonia. Here, we performed the most comprehensive study of S. eubayanus in Patagonia natural environments (400 samples) and confirmed that this region has the highest isolation success rate for this species described worldwide (more than 10-fold). The genetic characterization of 200 isolates (COX2, DCR1, intFR) revealed five geographically structured subpopulations. We hypothesized that marine ingressions and glaciations, which shaped the Patagonian landscape, contributed on population differentiation. The first large screening of fermentation performance of 60 wild S. eubayanus strains indicated which subpopulations would be more suitable for beer production., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Wine fermentation has not significantly changed since ancient times and the most traditional aspects are seen by the market as elements that uplift wine nuances and quality. In recent years, new trends have emerged from the sector in line with consumer preferences, and due to the effects of global climate change on grape ripening. In the first cases, the consumers are looking for wines with less ethanol and fruitier aromas and in the second cases the wineries want to reduce the wine alcohol levels and/or astringency. New yeast starters of alternative Saccharomyces species and their hybrids can help to solve some problems that wineries face. In this article we review several physiological and genetic aspects of S. uvarum and S. kudriavzevii and the hybrids, which are especially relevant during the winemaking process, such as their good fermentative capabilities at low temperatures, resulting in wines with lower alcohol and higher glycerol amounts.

Aims: Selection projects aiming at the identification of new Saccharomyces strains are always on going as the use of the suitable yeast can strongly improve fermented food production, particularly winemaking. They are mainly targeted on Saccharomyces cerevisiae, but other species in the Saccharomyces genus are of interest. For this reason, more and more efficient molecular techniques for yeast identification able to accelerate yeast selection process are always needed. Among the Saccharomyces genus, four yeasts are widespread in natural environments: S. cerevisiae; S. uvarum; S. kudriavzevii and S. paradoxus. Therefore, among the Saccharomyces species, their discrimination is of great interest., Methods and Results: A two-step protocol is proposed. Firstly the Saccharomyces genus identification is achieved by multiplex PCR analysis. Then, the Saccharomyces species is determined by a new method based on high-resolution melting analysis (HRMA)., Conclusions: For HRMA two primer pairs have been proposed. The first was able to achieve the simultaneous identification of the four widespread Saccharomyces species, the second was used for the unambiguous discrimination of S. cerevisiae within its taxonomical genus., Significance and Impact of the Study: This assay allowed an easy, rapid and simultaneous discrimination of S. cerevisiae, S. uvarum and S. paradoxus during yeast selection programs., (© 2018 The Society for Applied Microbiology.)

Beer, a beverage consumed throughout the world, is mainly derived from cereals. In this study, fungal and mycotoxin contamination, as well as the physicochemical properties of maize-based opaque beer (umqombothi) obtained from the Gauteng province of South Africa, was investigated. The mean water activity, pH and total titratable acidity of the analysed beer samples were 0.91, 3.76 and 1.20% lactic acid, respectively. The investigation revealed Aspergillus, Penicillium, Phoma and Saccharomyces as the predominant fungal genera with a mean fungal load of 3.66 × 10 5  CFU/mL. Among the mycotoxigenic fungal species recovered, Aspergillus flavus had the highest incidence of 26%. Previously unreported strains such as P. chrysogenum strain AD25, A. sydowii strain AD 22 and A. tritici strain AD 11 were found. Furthermore, mycotoxin quantitative analysis via liquid chromatography-tandem mass spectrophotometry showed that deoxynivalenol was the dominant mycotoxin occurring in 84% of the samples. This was followed by enniatin B that occurred in 75% of samples ranging from 12 to 44 μg/L and fumonisin B 1 (FB 1 ) (incidence of 53% at a maximum level of 182 μg/L). Generally, there was low occurrence aflatoxins, whereas T-2, HT-2, nivalenol, zearalenone, 3- and 15-acetyl-deoxynivalenol were not detected. All the samples analysed had safe levels of mycotoxins tested but were contaminated by at least two mycotoxins that could pose some additive or synergistic health effects among consumers. On average: a 60 kg adult consuming 1-6 L/day of the beer was exposed to FB 1  + FB 2 at an estimated 2.20-13.20 μg/kg body weight/day. These values were far above the maximum tolerable daily intake of 2 μg/kg bw/day established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The study demonstrates that consumption of umqombothi can significantly enhance dietary exposure to multiple mycotoxins among consumers, and therefore accentuates the need for strategies aimed at reducing toxigenic fungal colonization and mycotoxin contamination in the beer processing chain., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Saccharomyces species, which are mostly used in the food and beverage industries, are known to differ in their fermentation efficiency and tolerance of adverse fermentation conditions. However, the basis of their difference has not been fully elucidated, although their genomes have been sequenced and analyzed. Five strains of four Saccharomyces species (S. cerevisiae, S. kudriavzevii, S. bayanus, and S. paradoxus), when grown in parallel in laboratory conditions, exhibit very similar basic physiological parameters such as membrane potential, intracellular pH, and the degree to which they are able to quickly activate their Pma1 H + -ATPase upon glucose addition. On the other hand, they differ in their ability to proliferate in media with a very low concentration of potassium, in their osmotolerance and tolerance to toxic cations and cationic drugs in a growth-medium specific manner, and in their capacity to survive anhydrobiosis. Overall, S. cerevisiae (T73 more than FL100) and S. paradoxus are the most robust, and S. kudriavzevii the most sensitive species. Our results suggest that the difference in stress survival is based on their ability to quickly accommodate their cell size and metabolism to changing environmental conditions and to adjust their portfolio of available detoxifying transporters.

Yeasts have been used for food and beverage fermentations for thousands of years. Today, numerous different strains are available for each specific fermentation process. However, the nature and extent of the phenotypic and genetic diversity and specific adaptations to industrial niches have only begun to be elucidated recently. In Saccharomyces, domestication is most pronounced in beer strains, likely because they continuously live in their industrial niche, allowing only limited genetic admixture with wild stocks and minimal contact with natural environments. As a result, beer yeast genomes show complex patterns of domestication and divergence, making both ale (S. cerevisiae) and lager (S. pastorianus) producing strains ideal models to study domestication and, more generally, genetic mechanisms underlying swift adaptation to new niches., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Most studies describing the human gut microbiome in healthy and diseased states have emphasized the bacterial component, but the fungal microbiome (i.e., the mycobiome) is beginning to gain recognition as a fundamental part of our microbiome. To date, human gut mycobiome studies have primarily been disease centric or in small cohorts of healthy individuals. To contribute to existing knowledge of the human mycobiome, we investigated the gut mycobiome of the Human Microbiome Project (HMP) cohort by sequencing the Internal Transcribed Spacer 2 (ITS2) region as well as the 18S rRNA gene., Results: Three hundred seventeen HMP stool samples were analyzed by ITS2 sequencing. Fecal fungal diversity was significantly lower in comparison to bacterial diversity. Yeast dominated the samples, comprising eight of the top 15 most abundant genera. Specifically, fungal communities were characterized by a high prevalence of Saccharomyces, Malassezia, and Candida, with S. cerevisiae, M. restricta, and C. albicans operational taxonomic units (OTUs) present in 96.8, 88.3, and 80.8% of samples, respectively. There was a high degree of inter- and intra-volunteer variability in fungal communities. However, S. cerevisiae, M. restricta, and C. albicans OTUs were found in 92.2, 78.3, and 63.6% of volunteers, respectively, in all samples donated over an approximately 1-year period. Metagenomic and 18S rRNA gene sequencing data agreed with ITS2 results; however, ITS2 sequencing provided greater resolution of the relatively low abundance mycobiome constituents., Conclusions: Compared to bacterial communities, the human gut mycobiome is low in diversity and dominated by yeast including Saccharomyces, Malassezia, and Candida. Both inter- and intra-volunteer variability in the HMP cohort were high, revealing that unlike bacterial communities, an individual's mycobiome is no more similar to itself over time than to another person's. Nonetheless, several fungal species persisted across a majority of samples, evidence that a core gut mycobiome may exist. ITS2 sequencing data provided greater resolution of the mycobiome membership compared to metagenomic and 18S rRNA gene sequencing data, suggesting that it is a more sensitive method for studying the mycobiome of stool samples.

Exploring the evolutionary patterns of mitochondrial genomes is important for our understanding of the Saccharomyces sensu stricto (SSS) group, which is a model system for genomic evolution and ecological analysis. In this study, we first obtained the complete mitochondrial sequences of two important species, Saccharomyces mikatae and Saccharomyces kudriavzevii. We then compared the mitochondrial genomes in the SSS group with those of close relatives, and found that the non-coding regions evolved rapidly, including dramatic expansion of intergenic regions, fast evolution of introns and almost 20-fold higher rearrangement rates than those of the nuclear genomes. However, the coding regions, and especially the protein-coding genes, are more conserved than those in the nuclear genomes of the SSS group. The different evolutionary patterns of coding and non-coding regions in the mitochondrial and nuclear genomes may be related to the origin of the aerobic fermentation lifestyle in this group. Our analysis thus provides novel insights into the evolution of mitochondrial genomes.

Brewing yeasts of the genus Saccharomyces are either available from yeast distributor centers or from breweries employing their own "in-house strains". During the last years, the classification and characterization of yeasts of the genus Saccharomyces was achieved by using biochemical and DNA-based methods. The current lack of fast, cost-effective and simple methods to classify brewing yeasts to a beer type, may be closed by Matrix Assisted Laser Desorption/Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) upon establishment of a database based on sub-proteome spectra from reference strains of brewing yeasts. In this study an extendable "brewing yeast" spectra database was established including 52 brewing yeast strains of the most important types of bottom- and top-fermenting strains as well as beer-spoiling S. cerevisiae var. diastaticus strains. 1560 single spectra, prepared with a standardized sample preparation method, were finally compared against the established database and investigated by bioinformatic analyses for similarities and distinctions. A 100% separation between bottom-, top-fermenting and S. cerevisiae var. diastaticus strains was achieved. Differentiation between Alt and Kölsch strains was not achieved because of the high similarity of their protein patterns. Whereas the Ale strains show a high degree of dissimilarity with regard to their sub-proteome. These results were supported by MDS and DAPC analysis of all recorded spectra. Within five clusters of beer types that were distinguished, and the wheat beer (WB) cluster has a clear separation from other groups. With the establishment of this MALDI-TOF MS spectra database proof of concept is provided of the discriminatory power of this technique to classify brewing yeasts into different major beer types in a rapid, easy way, and focus brewing trails accordingly. It can be extended to yeasts for specialty beer types and other applications including wine making or baking.

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.

The process from yeast isolation to their use in laboratory experiments is lengthy. Historically, Saccharomyces strains were easily obtained by sampling alcoholic fermentation processes or other substrates associated with human activity in which Saccharomyces was heavily enriched. In contrast, wild Saccharomyces yeasts are found in complex microbial communities and small population sizes, making isolation challenging. We have overcome this problem by enriching yeast on media favoring the growth of Saccharomyces over other microorganisms. The isolation process is usually followed by molecular characterization that allows the strain identification. Finally, yeast isolated from domestic or wild environments need to be genetically manipulated before they can be used in laboratory experiments., (© 2017 Cold Spring Harbor Laboratory Press.)

Two strains, D5088T and D5095, representing a novel yeast species belonging to the genus Saccharomyces were isolated from oak tree bark and surrounding soil located at an altitude of 1000 m above sea level in Saint Auban, France. Sequence analyses of the internal transcribed spacer (ITS) region and 26S rRNA D1/D2 domains indicated that the two strains were most closely related to Saccharomyces mikatae and Saccharomyces paradoxus. Genetic hybridization analyses showed that both strains are reproductively isolated from all other Saccharomyces species and, therefore, represent a distinct biological species. The species name Saccharomyces jurei sp. nov. is proposed to accommodate these two strains, with D5088T (=CBS 14759T=NCYC 3947T) designated as the type strain.

Gene regulation is a ubiquitous mechanism by which organisms respond to their environment. While organisms are often found to be adapted to the environments they experience, the role of gene regulation in environmental adaptation is not often known. In this study, we examine divergence in cis-regulatory effects between two Saccharomycesspecies, S. cerevisiaeand S. uvarum, that have substantially diverged in their thermal growth profile. We measured allele specific expression (ASE) in the species' hybrid at three temperatures, the highest of which is lethal to S. uvarumbut not the hybrid or S. cerevisiae. We find that S. uvarumalleles can be expressed at the same level as S. cerevisiaealleles at high temperature and most cis-acting differences in gene expression are not dependent on temperature. While a small set of 136 genes show temperature-dependent ASE, we find no indication that signatures of directional cis-regulatory evolution are associated with temperature. Within promoter regions we find binding sites enriched upstream of temperature responsive genes, but only weak correlations between binding site and expression divergence. Our results indicate that temperature divergence between S. cerevisiaeand S. uvarumhas not caused widespread divergence in cis-regulatory activity, but point to a small subset of genes where the species' alleles show differences in magnitude or opposite responses to temperature. The difficulty of explaining divergence in cis-regulatory sequences with models of transcription factor binding sites and nucleosome positioning highlights the importance of identifying mutations that underlie cis-regulatory divergence between species., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The use of the nuclear DNA reassociation technique has led taxonomists to consider Saccharomyces uvarum a synonym of S. bayanus. The latter, however, is not a species but a hybrid harbouring S. eubayanus (Seu) and S. uvarum (Su) subgenomes with a minor DNA contribution from S. cerevisiae (Sc). To recognize genetically pure lines of S. uvarum and putative interspecies hybrids among so-called S. bayanus strains present in public culture collections, we propose the use of four markers that were defined from the S. bayanus CBS 380T composite genome, namely SeuNTS2 (rDNA), ScMAL31, MTY1 and SuMEL1. Saccharomyces carlsbergensis CBS 1513 was found to be similar to S. bayanus except that it carries the SeuMEL1 allele. Different marker combinations revealed that among 33 strains examined only a few were similar to CBS 380T, but many pure S. uvarum lines and putative Su/Seu-related hybrids occurred. Our results demonstrated that these hybrids were erroneously considered authentic S. bayanus and therefore the varietal state 'Saccharomyces bayanus var. uvarum comb. nov. Naumov' is not valid. Our markers constitute a tool to get insights into the genomic makeup of Saccharomyces interspecies hybrids. We also make a proposal to name those hybrids that may also be applicable to other fungal hybrids., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The fungal kingdom displays an extraordinary diversity of lifestyles, developmental processes, and ecological niches. The MAPK (mitogen-activated protein kinase) cascade consists of interlinked MAPKKK, MAPKK, and MAPK, and collectively such cascades play pivotal roles in cellular regulation in fungi. However, the mechanism by which evolutionarily conserved MAPK cascades regulate diverse output responses in fungi remains unknown. Here we identified the full complement of MAPK cascade components from 231 fungal species encompassing 9 fungal phyla. Using the largest data set to date, we found that MAPK family members could have two ancestors, while MAPKK and MAPKKK family members could have only one ancestor. The current MAPK, MAPKK, and MAPKKK subfamilies resulted from duplications and subsequent subfunctionalization during the emergence of the fungal kingdom. However, the gene structure diversification and gene expansion and loss have resulted in significant diversity in fungal MAPK cascades, correlating with the evolution of fungal species and lifestyles. In particular, a distinct evolutionary trajectory of MAPK cascades was identified in single-celled fungi in the Saccharomycetes. All MAPK, MAPKK, and MAPKKK subfamilies expanded in the Saccharomycetes; genes encoding MAPK cascade components have a similar exon-intron structure in this class that differs from those in other fungi., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Apple chicha is a fresh low alcoholic beverage elaborated by aboriginal communities of Andean Patagonia (Argentina and Chile). In the present work, we identified the yeast microbiota associated with this fermentation, and characterized genetically those belonging to the genus Saccharomyces. Both Saccharomyces cerevisiae and S. uvarum were found in the analyzed fermentations. Phylogenetic and population structure analyses based on genes sequence analysis were carried out for both S. cerevisiae and S. uvarum strains obtained in this study and a set of additional strains from diverse origins. The results demonstrate that S. cerevisiae strains from apple chicha belong to the big group of wine/European strains of this species, while S. uvarum strains were included in the Holartic population of this species. Additionally, some S. uvarum strains from chichas evidenced as an admixture of both pure Holartic and pure South American populations. Our results suggest that Holartic strains could have been introduced in South America together with the domestication of apple trees by Mapuche communities. This Holartic population suffered admixis with the naturally present South American population of this species, originating strains bearing genetic features from the two populations, detectable in both chichas and natural habitats., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Saccharomyces yeast species are currently the most important yeasts involved in industrial-scale food fermentations. However, there are hundreds of other yeast species poorly studied that are highly promising for flavour development, some of which have also been identified in traditional food fermentations. This work explores natural yeast biodiversity in terms of aroma formation, with a particular focus on aromas relevant for industrial fermentations such as wine and beer. Several non-Saccharomyces species produce important aroma compounds such as fusel alcohols derived from the Ehrlich pathway, acetate esters and ethyl esters in significantly higher quantities than the well-known Saccharomyces species. These species are Starmera caribaea, Hanseniaspora guilliermondii, Galactomyces geotrichum, Saccharomycopsis vini and Ambrosiozyma monospora. Certain species revealed a strain-dependent flavour profile while other species were very homogenous in their flavour profiles. Finally, characterization of a selected number of yeast species using valine or leucine as sole nitrogen sources indicates that the mechanisms of regulation of the expression of the Ehrlich pathway exist amongst non-conventional yeast species., (Copyright © 2016. Published by Elsevier Ltd.)

In the past, the galactose-negative (Gal(-)) phenotype was a key physiological character used to distinguish Saccharomyces bayanus from S. cerevisiae In this work, we investigated the inactivation of GAL gene networks in S. bayanus, which is an S. uvarum/S. eubayanus hybrid, and in S. cerevisiae wine strains erroneously labelled 'S. bayanus'. We made an inventory of their GAL genes using genomes that were either available publicly, re-sequenced by us, or assembled from public data and completed with targeted sequencing. In the S. eubayanus/S. uvarum CBS 380(T) hybrid, the GAL/MEL network is composed of genes from both parents: from S. uvarum, an otherwise complete set that lacks GAL4, and from S. eubayanus, a truncated version of GAL4 and an additional copy of GAL3 and GAL80 Similarly, two different truncated GAL4 alleles were found in S. cerevisiae wine strains EC1118 and LalvinQA23. The lack of GAL4 activity in these strains was corrected by introducing a full-length copy of S. cerevisiae GAL4 on a CEN4/ARS plasmid. Transformation with this plasmid restored galactose utilisation in Gal(-) strains, and melibiose fermentation in strain CBS 380(T) The melibiose fermentation phenotype, formerly regarded as characteristic of S. uvarum, turned out to be widespread among Saccharomyces species., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Transmissible self-assembled fibrous cross-β polymer infectious proteins (prions) cause neurodegenerative diseases in mammals and control non-Mendelian heritable traits in yeast. Cross-species prion transmission is frequently impaired, due to sequence differences in prion-forming proteins. Recent studies of prion species barrier on the model of closely related yeast species show that colocalization of divergent proteins is not sufficient for the cross-species prion transmission, and that an identity of specific amino acid sequences and a type of prion conformational variant (strain) play a major role in the control of transmission specificity. In contrast, chemical compounds primarily influence transmission specificity via favoring certain strain conformations, while the species origin of the host cell has only a relatively minor input. Strain alterations may occur during cross-species prion conversion in some combinations. The model is discussed which suggests that different recipient proteins can acquire different spectra of prion strain conformations, which could be either compatible or incompatible with a particular donor strain.