Yeast

Quiescence is a conserved cellular state wherein cells cease proliferation and remain poised to re-enter the cell cycle when conditions are appropriate. Budding yeast is a powerful model for studying cellular quiescence. In this work, we demonstrate that the pH of the YPD media strongly affects quiescence entry efficiency in Saccharomyces cerevisiae. Adjusting media pH to 5.5 significantly improves quiescence entry efficiency compared to unadjusted YPD media. Thermotolerance of the produced quiescence yeast are similar, suggesting the media pH influences the quantity of quiescent cells more than quality of quiescence reached.

Meiosis is required for the formation of gametes in all sexually reproducing species and the process is well conserved across the tree of life. However, meiosis is sensitive to a variety of external factors, which can impact chromosome pairing, recombination, and fertility. For example, the optimal temperature for successful meiosis varies between species of plants and animals. This suggests that meiosis is temperature sensitive, and that natural selection may act on variation in meiotic success as organisms adapt to different environmental conditions. To understand how temperature alters the successful completion of meiosis, we utilized two species of the budding yeast Saccharomyces with different temperature preferences: thermotolerant Saccharomyces cerevisiae and cold tolerant Saccharomyces uvarum. We surveyed three metrics of meiosis: sporulation efficiency, spore viability, and recombination rate in multiple strains of each species. As per our predictions, the proportion of cells that complete meiosis and form spores is temperature sensitive, with thermotolerant S. cerevisiae having a higher temperature threshold for successful meiosis than cold tolerant S. uvarum. We confirmed previous observations that S. cerevisiae recombination rate varies between strains and across genomic regions, and add new results that S. uvarum has higher recombination rates than S. cerevisiae. We find that temperature significantly influences recombination rate plasticity in S. cerevisiae and S. uvarum, in agreement with studies in animals and plants. Overall, these results suggest that meiotic thermal sensitivity is associated with organismal thermal tolerance, and may even result in temporal reproductive isolation as populations diverge in thermal profiles.

Hybrid vigor, or heterosis, is widely exploited in yeast strain improvement. Yet, how ploidy and genetic background jointly shape heterosis across industrially relevant stresses remains unclear. Here, we generated 1023 Saccharomyces cerevisiae intraspecific hybrids derived from 18 genetically diverse parents, using two different approaches, yielding sets of hybrids with variable ploidy for the same parental combinations. High-throughput growth assays in media with five stress conditions (14% ethanol, 1.5 M NaCl, 0.15 M lactic acid, 0.05 M acetic acid, 0.05 M HMF) revealed extensive heterosis across 6138 hybrid-condition combinations. Most combinations displayed mid-parent heterosis and over a third exceeded the best parent, with the strongest gains during growth in the presence of 14% ethanol and 1.5 M NaCl. Increasing ploidy was generally associated with reduced growth and reduced best-parent heterosis, whereas greater predicted hybrid heterozygosity or genetic distance between parents was positively associated with heterosis in the presence of 14% ethanol and 1.5 M NaCl. Domestication status also affected these trends, as crosses between two domesticated strains tended to perform better in the presence of ethanol and NaCl, while crosses between two wild strains grew best in control conditions and in the presence of acetic acid. Together, these results demonstrate condition-dependent contributions of ploidy and parentage to heterosis and provide targeted breeding strategies for the improvement of stress-tolerance in industrial yeasts.

Even though it is a well-accepted fact that the energy metabolism of yeast is likely to impact all cellular activities, surprising little is known about the ATP homeostasis of particular yeast strains that are commonly used in cell biological studies. Therefore, we determined key parameters such as oxygen consumption and fermentation rates of the lab strain SEY6210. Our data indicated that even at high glucose concentrations, SEY6210 produces 30-50% of cellular ATP from oxidative phosphorylation. Loss of respiration, either by disrupting ATP synthase function or by growth in anaerobic conditions, was not fully compensated by fermentation and as a result affected energy intensive processes such as the maintenance of the plasma membrane proton gradient and the associated import of nutrients.

Features of the natural life cycle of the budding yeast Saccharomyces cerevisiae were crucial to its domestication as a laboratory experimental model, especially the ability to maintain stable haploid clones and cross them at will to combine alleles via meiosis. Stable haploidy results from mutations in HO, which encodes an endonuclease required for haploid-specific mating-type switching. Previous studies found an unexpected diversity of HO alleles among natural isolates within a small geographic area. We developed a hands-on field and laboratory activity for middle school students in Denver, Colorado, USA to isolate wild yeast from oak bark, identify species via DNA sequencing, and sequence HO from S. cerevisiae isolates. We find limited HO diversity in North American oak isolates, pointing to efficient, continuous dispersal across the continent. By contrast, we isolated the "dairy yeast", Kluyveromyces lactis, from a tree <10 m away and found that it represents a new population distinct from an oak population in an adjacent state, pointing to high genetic diversity. The outreach activity partnered middle school, high school, and university students in making scientific discoveries and can be adapted to other locations and natural yeast habitats. Indeed, a pilot sampling activity in southeast Texas yielded S. cerevisiae oak isolates with a new allele of HO and, from a nearby prickly pear cactus, a heat-tolerant isolate of Saccharomyces paradoxus.

Scientific advances have emboldened human efforts toward exploring the potential of extinct, extant, or future life on Mars. An important aspect of this endeavor is understanding how an organism adapts to stress-inducing environmental conditions on Mars, such as radiation, shock waves, extreme temperatures, and chaotropic stress due to higher levels of perchlorates. A conserved approach used by organisms across evolutionary scales to adapt and overcome stress conditions is the assembly of ribonucleoprotein (RNP) condensates. In this study, we employ a multidisciplinary approach to understand yeast survivability and adaptation under Mars-like stress conditions, specifically shock waves and perchlorate, by focusing on RNP condensates. Our study reveals that yeast survives 5.6 M intensity shock waves. Exposure to either shock waves or sodium perchlorate induces the formation of P-bodies, a conserved stress-induced condensate. Yeast mutants defective in P-body assembly show defective growth in response to perchlorate stress. Transcriptome analysis, followed by validation, identified several relevant transcripts whose levels are perturbed in response to Mars-like conditions. Finally, identification of several transcripts whose abundance is altered in the P-body assembly mutant upon stress highlights a new connection between response to Martian stress conditions and RNP condensates. This study, a first of its kind, highlights the importance of RNP condensates in understanding the impact of Martian conditions on life in general. This study paves the way for using RNP condensates as a biomarker for assessing the health of life forms during space explorations.

Even in the absence of mutagenic factors, spontaneous errors in DNA replication generate genetic diversity in microbial populations. The spontaneous mutation rate is not immutable but may be conditionally elevated. However, it remains unresolved whether apparently non-stressful nutritional or genotypic conditions affect the spontaneous mutation rate. Here, we determined the spontaneous mutation rate of haploid Saccharomyces cerevisiae CEN.PK113-7D in three nutritional and three genotypic conditions. The nutritional and genotypic conditions influenced the specific growth rate of the S. cerevisiae population. Thus, we extended the established fluctuation assay for spontaneous mutation rate determination with CAN1 as a reporter gene to populations with different generation times. We applied the method to determine the spontaneous mutations rates in wild type S. cerevisiae grown on glucose and ammonium, raffinose and ammonium, and glucose and L-tryptophan as sole carbon and nitrogen sources. Alike we determined the spontaneous mutation rates of two engineered S. cerevisiae strains (i.e., representing genotypic conditions different from wild type) on glucose and ammonium medium. The engineered strains had two to three heterologous or variant genes integrated into the genome, common to simple heterologous small molecule production host strains. In all alternative nutritional and genotypic conditions, the spontaneous mutation rate of S. cerevisiae was reduced compared to wild type growing on glucose and ammonium medium. Spontaneous mutation rate is fundamentally relevant for evolvability of strains, but it may also influence the performance robustness of microbial populations in applications such as food or beverage fermentation or biotechnological chemical production. Our novel findings are important for biotechnological processes using microbial cells, often engineered and cultivated in unnatural chemical environments.

Species diversity is a commonly stated contributor to the fate of an invader, and thus community resistance, in both microbial and non-microbial communities. Termed the "diversity-invasion hypothesis", a positive relationship between diversity and resistance to invasion is observed when an introduced species exhibits lower levels of survival in resident communities with higher species richness. The diversity-invasion hypothesis is an attractive perspective with convincing theory and examples, yet an "invasion paradox" of contrasting results means that a positive role of diversity against invasion is still not a certainty and under debate. In this study we investigated the relationship between resistance to invasion and resident community species richness versus species identity (i.e., keystone species). Using synthetic communities comprised of combinations of four wine yeasts (Saccharomyces cerevisiae, Lachancea thermotolerans, Torulaspora delbrueckii, Starmerella bacillaris), we tracked over 21 days the presence of introduced Brettanomyces bruxellensis spoilage yeast and Lactiplantibacillus plantarum lactic acid bacteria to ask the following: 1. Does yeast community species richness impact the establishment of B. bruxellensis yeast and L. plantarum bacteria during wine fermentation? 2. How does yeast species identity influence such establishment? We found that species identity rather than richness drove the prevention of establishment of B. bruxellensis and L. plantarum, with S. cerevisiae playing a critical keystone species role. Aside from spoilage prevention by S. cerevisiae, the four resident yeast species demonstrated a strict dominance ranking of competitive exclusion regardless of background community composition. Our research lends evidence against the commonly predicted positive relationship between species richness and resistance to invasion. Furthermore, as spontaneously fermented natural wines and diverse starter cultures gain popularity, our findings support a remaining importance of S. cerevisiae in preventing B. bruxellensis spoilage..

With the sustained popularity of hazy IPAs, brewers have explored multiple approaches to maximizing stable haze that will remain in suspension throughout the shelf life of the beer. Our recent investigations into yeast-dependent haze have uncovered specific brewing yeast strains that promote the formation of haze in heavily dry-hopped beer styles. These brewing strains have been termed "haze-positive" and furthermore, the timing of dry hop additions has been found to be another key factor in producing this stable haze. Classical genetics have identified YIL169C (herein referred to as HZY1) as both necessary and sufficient for the haze-positive phenotype in the yeast strain most widely used for Hazy IPAs. HZY1 encodes a candidate glycoprotein and our recent findings suggest it is localized to the cell wall through a GPI anchor. Surprisingly, using long-read sequencing data we uncovered extensive genetic variation in HZY1 across brewing strains. The haze-positive phenotype correlates with an expansion in the N-terminal serine-rich region. We propose that the Hzy1 glycoprotein is a critical component to yeast-dependent colloidal haze and the genetic variation in this locus contributes the range of haze phenotypes observed across industrial brewing strains.

The impact of the genetic background on the lipidome of yeast strains remains underexplored. This study systematically compares the lipidomes of five commonly used laboratory yeast strains: BY4741, W303, D273-10B, RM11-1a, and CEN.PK2-1c. Shotgun lipidomics reveals significant variations in lipid class and acyl chain composition down to the level of molecular species. Notably, the most abundant lipid class differed between the strains: phosphatidylinositol (PI) lipids are predominant in BY4741, while phosphatidylethanolamine (PE) lipids are in D273. Ergosterol esters, which are the storage form of the major yeast sterol ergosterol, are at higher levels in all strains other than BY4741, correlating with a low gene expression of lipid metabolic enzymes Hmg1 and Are2 in BY4741. Despite these lipidomic differences, transcriptomic analysis did not show significant changes in most genes related to lipid metabolism, suggesting post-transcriptional modifications, protein abundance, and metabolic flux as potential regulatory mechanisms. This study underscores the complexity of lipidome regulation and the need for further investigation into the underlying mechanisms.

Norwegian kveik are a recently described family of domesticated Saccharomyces cerevisiae brewing yeasts used by farmhouse brewers in western Norway for generations to produce traditional Norwegian farmhouse ale. Kveik ale yeasts have been domesticated by farmhouse brewers through serial repitching of the yeast in warm wort (>30{degrees}C) punctuated by long periods of dry storage. Kveik yeasts are alcohol tolerant, flocculant, capable of utilizing maltose/maltotriose, phenolic off flavour negative, and exhibit elevated thermotolerance when compared to other modern brewers yeasts belonging to the Beer 1 clade. However, the optimal fermentation and growth temperatures (Topt) for kveik ale yeasts and the influence of fermentation temperature of the production of flavour-active metabolites like fusel alcohols and sulfur compounds (H2S, SO2) are not known. Here we show that kveik ale yeasts have an elevated optimal fermentation temperature (Topt) when compared to commercial American Ale yeast (SafAle US-05) and that they produce fewer off-flavours at high temperatures (>30{degrees}C) when compared to commercial American Ale yeasts. The tested kveik yeasts show significantly higher maximum fermentation rates than American Ale yeast not only at elevated temperatures (>30{degrees}C), but also at typical ale fermentation temperatures (20{degrees}C-25{degrees}C). Finally, we demonstrate that kveik ale yeasts are heterogeneous in their Topt and that they attenuate standard wort robustly above their Topt unlike our control American Ale yeast which showed very poor apparent attenuation in our standard wort at temperatures >> Topt. Our results provide further support that kveik yeasts may possess favourable fermentation kinetics and sensory properties compared to American Ale yeasts. The observations here provide a roadmap for brewers to fine tune their commercial fermentations using kveik ale yeasts for optimal performance and/or flavour impact.

Quantitative genetics requires large datasets of diverse phenotyped-genotyped strains from the same species. A special need is for such archived biological material and computerized data in sexually reproducing individuals from a species. Here we leverage sexual mating among close to 100 diverse natural isolates of the yeast S. cerevisiae that form about 4,000 hybrids combinations in several ecologically relevant growth conditions. In a first genetic study of this new resource we focus on fitness measurements and its modes of inheritance as a quantitative trait from parents to offspring hybrids. We employ genomic barcoding of all strains and a barcode recombination technique to follow hybrids of each successful mate combination. For all parents, and separately for all offspring hybrids we measure fitness under each condition. We focus on the inheritance of fitness, the ultimate evolutionary trait, and its inheritance as a quantitative trait upon sexual mating. Predicting hybrid fitness given parental parameters is a major challenge as it is likely multi-factorial. We find that hybrids fitness in fermentable carbon source correlates positively, yet modestly, with parental fitness, while on non-fermentable carbon, hybrid fitness shows no detectable correlation with parental fitness. Instead, the non-fermentable condition, hybrid fitness increases sharply with genetic distance between their parents, suggesting that outbreeding maximizes fitness irrespective of parental fitness at that condition. The number of minor alleles in the genome of each hybrid, analogous to polygenic risk score in classical genetics, negatively correlates with fitness in both conditions. Fitness inheritance can be explained by either a dominance or a co-dominance modes of inheritance, in the non-fermentable and fermentable conditions respectively. Our newly suggested biological resource and data provide new foundations for a quantitative research in genetics and evolution upon sexual mating. Furthermore, our barcoded strains and mating tracking method provide an important research resource for the yeast community.

The fission yeast Schizosaccharomyces pombe is a single-celled eukaryote that can be cultured as a haploid or as a diploid. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our two laboratories encountered independently sudden-onset, major impediments to such research. Spore suspensions and vegetative cells no longer plated effectively on minimal media. By systematically analyzing multiple different media components from multiple different suppliers, we identified the source of the problem. Specific lots of agar, from different suppliers, were toxic. Interestingly, the inhibitory effect was attenuated on rich media. Consequently, quality control checks that use only rich media can provide false assurances on the quality of the agar. Lastly, we describe likely sources of the toxicity and we provide specific guidance for quality control measures that should be applied by all vendors as preconditions for their sale of agar. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/597796v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@41d7c1org.highwire.dtl.DTLVardef@bbec24org.highwire.dtl.DTLVardef@18c6f76org.highwire.dtl.DTLVardef@e9b707_HPS_FORMAT_FIGEXP M_FIG C_FIG Take-awayO_LISporadically, batches of agar from different suppliers strongly inhibit the plating efficiency of S. pombe spores and vegetative cells on minimal media. C_LIO_LIQuality control checks that are not quantitative or that use only rich media can provide false assurances on the quality of the agar. C_LIO_LIVendors should conduct rigorous, thorough, organism-specific tests for potential toxicity of each lot of agar as a pre-condition for its sale. C_LI

How genomic differences contribute to phenotypic differences across species is a major question in biology. The recently characterized genomes, isolation environments, and qualitative patterns of growth on 122 sources and conditions of 1,154 strains from 1,049 fungal species (nearly all known) in the subphylum Saccharomycotina provide a powerful, yet complex, dataset for addressing this question. In recent years, machine learning has been successfully used in diverse analyses of biological big data. Using a random forest classification algorithm trained on these genomic, metabolic, and/or environmental data, we predicted growth on several carbon sources and conditions with high accuracy from presence/absence patterns of genes and of growth in other conditions. Known structural genes involved in assimilation of these sources were important features contributing to prediction accuracy, whereas isolation environmental data were poor predictors. By further examining growth on galactose, we found that it can be predicted with high accuracy from either genomic (92.6%) or growth data in 120 other conditions (83.3%) but not from isolation environment data (65.7%). When we combined genomic and growth data, we noted that prediction accuracy was even higher (93.4%) and that, after the GALactose utilization genes, the most important feature for predicting growth on galactose was growth on galactitol. These data raised the hypothesis that several species in two orders, Serinales and Pichiales (containing Candida auris and the genus Ogataea, respectively), have an alternative galactose utilization pathway because they lack the GAL genes. Growth and biochemical assays of several of these species confirmed that they utilize galactose through an oxidoreductive D-galactose pathway, rather than the canonical GAL pathway. We conclude that machine learning is a powerful tool for investigating the evolution of the yeast genotype-phenotype map and that it can help uncover novel biology, even in well-studied traits.

Leavening dough is one of the most widespread applications of fermentative yeast and the most common practice for the general public to come into contact with microbial cultures. Saccharomyces cerevisiae is the typical species used for dough making, but the evolutionary origin of strains isolated from dough is mixed. Here, using 49 newly sequenced and 183 previously described isolates from the bakery environment, we show that the traditional strains used in Europe for sourdough making are more closely related to Chinese Mantou sourdough lineages than to commercially used baker yeast strains. Surprisingly, the expansion of these traditional European strains into other human-associated niches, including the human body, has been very limited. This is in stark contrast to the Mixed-origin clade commercial baking yeasts, which consists of only a few globally distributed clonal lineages that dominate the yeast market and recurrently colonize sourdoughs and human hosts. These clonal lineages consistently maintain their ploidy, unique heterozygous chromosomal rearrangements, and stable aneuploidies in addition to several diverse structural variants. In addition to the previously known diploid and tetraploid groups of commercial isolates, we describe a widely distributed stable triploid aneuploid clonal lineage. We show that company practices and global trade help the distribution of these clonal clusters and that these yeasts are characterized by their exclusively mitotic reproduction.

Quantifying post-stress survival in yeasts is crucial for biological, biomedical, and industrial research. Traditional methods like Colony Forming Unit (CFU) assays are labor-intensive and time-consuming. In this study, we systematically characterize a two-dye (SYTO 9 / Propidium Iodide) LIVE/DEAD assay coupled with flow cytometry to rapidly and scalably quantify post-stress survival in diverse yeast species. By optimizing staining buffer, dye concentrations and staining time, we minimized artifacts and improved reproducibility. Notably, we identify an "Intermediate" population containing damaged cells with enhanced SYTO 9 uptake but little propidium iodide (PI) accumulation under sublethal stress, providing finer gradations of cellular damage compared to CFU or PI staining alone. We demonstrate the assays applicability across Candida glabrata, Saccharomyces cerevisiae, and Candida albicans after hydrogen peroxide treatment and in C. glabrata after Amphotericin B exposure. While CFU is more sensitive at lower stress levels, the SYTO 9/PI staining effectively distinguishes sublethal from lethal doses, offering a valuable alternative for rapid, high-throughput survival quantification.

Purine auxotrophy is a typical marker for many laboratory yeast strains. Supplementation of additional purine source (like adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We tested purine starvation effects in budding yeast Saccharomyces cerevisiae ade8 knockout. We explored effects brought by purine starvation in cellular, central carbon metabolism and in the global transcriptome level. We observed that cells cultivated in purine depleted media became significantly more tolerant to severe thermal, oxidative and desiccation stresses when compared to the cells cultivated in media with all necessary supplements. When starved for purine, cells stop their cell cycle in G1 or G0 state; intracellular concentration of ATP, ADP and AMP decreases, but adenylate charge remains stable. Intracellular RNA concentration decreases and massive downregulation of ribosomal RNA occurs. We think that purine auxotrophic starvation in a way mimics "natural" nitrogen or carbon starvations and therefore initiates elements of a transcriptional program typical for stationary phase cells (cell cycle arrest, increased stress resistance). Therefore our results demonstrate that organised metabolic response is initiated not only via "natural starvations", but also when starving for metabolic intermediates, like purines.

Global public health is increasingly threatened by the fast emergence of antibiotic resistance, and novel types of antibiotics are urgently needed. Metazoans have evolved their own antimicrobial mechanism, such as human group IIA secreted phospholipase A (sPLA2), which can efficiently inhibit the growth of gram-positive bacteria, but with much lower efficiency toward gram-negative bacteria. Here, we verified the antibacterial activity of a plant lipase, PLIP1 from Arabidopsis thaliana, against the gram-negative bacteria Escherichia coli, which belongs to the WHO priority 1 (critical) pathogen Enterobacteriaceae family. We also explored the potential of evolving PLIP1 as a more potent antimicrobial agent towards E. coli. Our results imply the possibility of using plant lipases as a potential antimicrobial and shed light on the future exploration of plant enzymes for novel and more efficient antibacterial agents.

In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas cutting combined with homologous recombination of a repair template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains, and rely on relatively low efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an E. coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo, which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic (HIS3, URA3) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a {beta}-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide for how to use this system.

Budding yeast employs a variety of survival strategies in response to starvation, including pseudohyphal development, invasive growth, and sporulation. Here we report an innate pathway of "viability resurgence in quiescent time" (VRQT) by aged cultures to preserve and expand the population survivorship. Without additional nutrients, a few stationary-phase cells synchronously enter mitosis, increasing the population viability that does not likely result from mutations. VRQT is a population density-dependent collective behavior that can be triggered by quorum sensing aromatic alcohols. Genetic analyses demonstrate that VRQT is independent of many canonical pathways for cell growth, development, or stress responses. This community survival program allows yeast to proactively extend vitality amidst a common nutritional crisis. Wild strains isolated from clinical samples exhibit VRQT, suggesting that this survival mechanism must be considered when treating human infections.