Yeast最新文献

Rax1 and Rax2 proteins provide the spatial landmark signal during the bipolar budding of Saccharomyces cerevisiae for the proper assembly of the new bud. The nonconventional yeast Yarrowia lipolytica also undergoes bipolar budding, and its genome encodes YlRax1 (YALI0E10329) and YlRax2 (YALI0A04609), the orthologs of Rax1 and Rax2, respectively. In this study, we explored the roles of YlRax1 and YlRax2 in the bipolar budding of Y. lipolytica. Deletion of YlRax1 and YlRax2 caused a proportion of Y. lipolytica cells to exhibit unipolar budding. Furthermore, our results revealed that YlRax1 and YlRax2 were localized at the mother-bud neck as well as the previous division sites, and their localization to the division sites was persistent through multiple cell cycles. Moreover, our study demonstrated that the 100 amino acids at the N-terminal of YlRax1 are not essential for its function. In contrast, the transmembrane domains at the C-terminal of YlRax1 and YlRax2 are essential for their normal function, as the truncated protein fragments with deleted TM domains could not restore the normal functioning of the corresponding strains with knocked-out YlRax1 or YlRax2. These results indicate that YlRax1 and YlRax2 are involved in partially regulating bipolar budding and that these two proteins are interdependent in localization and function. Furthermore, our results indicate that there will be novel landmark proteins regulating its bipolar budding in Y. lipolytica.

Agave spirits have gained global recognition and hold a central position within the cultural heritage of Mexico. Traditional distilleries, characterized by open fermentations driven by local microbial communities, persist despite the rise of industrial-scale counterparts. In this review, we explore the environmental conditions and production practices that make the must of cooked agave stems a unique habitat for colonizing microorganisms. Additionally, we review selected studies that have characterized yeast species within these communities, with a focus on their metabolic traits and genomic features. Over 50 fungal species, predominantly Saccharomycetales and few Basidiomycetes, along with a similar number of lactic and acetic acid bacteria, have been identified. Despite variations in the chemical composition of the agave substrates and diversity of cultural practices associated with each traditional fermentation process, yeast species such as Saccharomyces cerevisiae, Kluyveromyces marxianus, Torulaspora delbrueckii, and several Pichia species have been consistently isolated across all agave spirit-producing regions. Importantly, cooked agave must is rich in fermentable sugars, yet it also contains inhibitory compounds that influence the proliferation dynamics of the microbial community. We discuss some of the genetic traits that may enable yeasts to flourish in this challenging environment and how human practices may shape microbial diversity by promoting the selection of microbes that are well-adapted to agave fermentation environments. The increasing demand for agave spirits, combined with concerns about the preservation of natural resources and cultural practices associated with their production, underscores the need to deepen our understanding of all key players, including the yeast communities involved.

Saccharomyces cerevisiae (S. cerevisiae) provides an array of cost-effective and time-efficient methods for diverse genome modifications. Among these techniques, site-directed mutagenesis of target genes is a powerful strategy to elucidate intricate structure-function relationships and create specific mutations. While various PCR-based and CRISPR/Cas9-based methods have been developed for introducing point mutations into the S. cerevisiae genome, they often involve multiple steps. In this study, we presented a rapid and effective site-directed mutagenesis strategy using one-step multi-mismatch PCR, termed Yeast Genome Mutagenesis with Multi-mismatch PCR (YGMMP). YGMMP incorporated multiple synonymous mutations proximal to the target point mutations, along with a selection marker cassette and flanking homologous sequences, into the gene segment spanning from the desired mutation to the gene's terminus through overlap PCR. The resulting PCR product was introduced into yeast cells to facilitate the selection of target variants. As a proof of concept, we applied YGMMP to generate an ADE2 mutant. The results demonstrated that the introduction of five and nine synonymous mutations, in addition to the desired single-point mutation, yielded mutagenesis efficiencies of approximately 20% and 30%, respectively. This rapid, straightforward, and efficient method has the potential to greatly simplify site-specific modifications within the S. cerevisiae genome.

There is currently much interest in the Zygosaccharomyces sp. used to produce fermented foods. Here we have used sequencing and PCR to explore differences in the genomic structures of various haploid and allodiploid Zygosaccharomyces sp. strains isolated from miso. In haploid strains, internal transcribed spacer (ITS) sequences had high identity with the ITS sequences of the type strain Z. rouxii CBS732 (92%-100%). In allodiploid strains, some ITS sequences showed high identity (92%-100%), while others showed relatively low identity (69%-83%) with CBS732. By sequencing multiple ITS regions, it might be possible to predict whether a yeast strain is haploid or allodiploid. We also explored the mating-type like loci (MTLs) of these strains. Allodiploid natural hybrid strains commonly had a P-subgenome sequence inserted in the right arm of the active MAT locus, but the length of the insert differed by strain. A 36-kbp P-subgenome sequence was also inserted into the left arm region of the surrounding MTL in the miso strain MG101. It is likely that loss of heterozygosity occurs around MTLs with homologous sequences. Last, we sequenced the whole genome of yeast strain NBRC1877, which was isolated from Japanese miso 60 years ago. The draft sequence identified chromosomes with a different structure from those of Z. rouxii CBS732. Further comparisons revealed that these chromosomes exist in other Zygosaccharomyces sp. allodiploid yeast strains and may have been formed by reciprocal translocation between tRNA genes during the process of evolution.

Yeast strain development has been essential for improving efficiency, flavour diversity, and quality of beer fermentation. Such efforts often rely on laborious in vitro screening experiments. However, with the increasing availability of large-scale 'omics' data sets, it may be possible to replace or complement such experiments with in silico screening. Compared to more traditional in vitro screening, this has several benefits, including lower costs, more rapid results and possibility to include more strains. Here, we briefly review the genetics associated with various desirable and undesirable traits in brewing yeast, and demonstrate how recent genomics, transcriptomics, and proteomics data sets derived from the 1011 yeast genomes project can be exploited for identifying strains with potentially desirable phenotypes. The discussed phenotypes are related to fermentation performance, formation of desirable flavours, and mitigation of off-flavours. Finally, we perform wort fermentations with five strains from diverse backgrounds, with diverse predicted phenotypes, to validate the in silico predictions. Most predicted phenotypes correlated well with the measured phenotypes, including formation of desirable compounds like isoamyl acetate and ethyl octanoate, as well as formation of undesirable compounds like 4-vinyl guaiacol, diacetyl, and ethanethiol. Together, the results indicate that utilising large 'omics' data sets can be a very useful tool for both strain selection and development for beer fermentation, and naturally other food and beverage fermentations as well. We hope this can inspire and yield improved and more diverse brewing strains to the industry.

Biodiversity gaps in microorganisms, such as yeasts, blur our understanding of microbial diversity, introducing biases in their biogeography, ecology, and taxonomy. The genus Carlosrosaea is a potential plant growth booster, yet it is still a little-known yeast group. Considering that databases like GBIF and GenBank are powerful tools for exploring biodiversity data, we aimed to map the geographic distribution, ecological patterns, and taxonomic potential of the genus Carlosrosaea. We found 176 records of the genus, with about 70% associated with plant material, mostly leaves. Furthermore, 55% of the records pertained to the tropical region and only 12% to the temperate. The data indicates the existence of more than a dozen possible new species of the genus, cataloged yet undescribed. This study advances our understanding of the geographic, ecological, and taxonomic aspects of Carlosrosaea. It also highlights how public databases and literature reviews provide accessible ways to analyze information about microbial groups with limited data.

Meiotic recombination is a powerful source of haplotypic diversity, and thus plays an important role in the dynamics of short-term adaptation. However, high-throughput quantitative measurement of recombination parameters is challenging because of the large size of offspring to be genotyped. One of the most efficient approaches for large-scale recombination measurement is to study the segregation of fluorescent markers in gametes. Applying this to yeast spores by flow cytometry has already been proved to be highly efficient, but manual analyses of distributions of signal intensities is time-consuming and produces nonperfectly reproducible results. Such analyses are required to identify events corresponding to spores and to assign each of them to a genotypic class depending on their fluorescence intensity. The CAYSS package automatically reproduces the manual process that we've been developing to analyze yeast recombination for years, including Maximum-Likelihood estimation of fluorescence extinction (Raffoux et al. 2018a). When comparing the results of manual versus CAYSS automatic analyses of the same cytometry data, recombination rates and interference were on average very similar, with less than 3% differences on average and strong correlations (R² > 0.9). In conclusion, as compared to manual analysis, CAYSS allows to save a lot of human time and produces totally reproducible results.

Killer yeasts, such as the K1 killer strain of S. cerevisiae, express a secreted anti-competitive toxin whose production and propagation require the presence of two vertically-transmitted dsRNA viruses. In sensitive cells lacking killer virus infection, toxin binding to the cell wall results in ion pore formation, disruption of osmotic homeostasis, and cell death. However, the exact mechanism(s) of K1 toxin killing activity, how killer yeasts are immune to their own toxin, and which factors could influence adaptation and resistance to K1 toxin within formerly sensitive populations are still unknown. Here, we describe the state of knowledge about K1 killer toxin, including current models of toxin processing and killing activity, and a summary of known modifiers of K1 toxin immunity and resistance. In addition, we discuss two key signaling pathways, HOG (high osmolarity glycerol) and CWI (cell wall integrity), whose involvement in an adaptive response to K1 killer toxin in sensitive cells has been previously documented but requires further study. As both host-virus and sensitive-killer competition have been documented in killer systems like K1, further characterization of K1 killer yeasts may provide a useful model system for study of both intracellular genetic conflict and counter-adaptation between competing sensitive and killer populations.

The relationship between oral and gastric yeasts and their role in the colonization of Helicobacter pylori in the stomach was studied. Four groups of 221, 7, 44, and 10 patients were used for the isolation of H. pylori and oral and gastric yeasts. In Group 1, gastric biopsies were used for the isolation of H. pylori and yeast, rapid urease test (RUT), staining with Gram's and hematoxylin & eosin (H&E), and immunohistochemistry (IHC) methods. In the other three groups, DNAs extracted from H. pylori and yeasts were used for the amplification of H. pylori-specific genes. Wet mounts of yeasts in Group 2 were examined to observe intracellular bacteria and released EVs. Among 221 patients, 65 (29.3%) had oral yeast, 35 (15.8%) H. pylori, and 31 (14%) gastric yeast. Culture of oral yeasts showed a significant correlation with the detection of H. pylori by IHC (10.3%), Gram stain (9%), RUT (6.3%), H&E (4.9%), and culture (4%) (p < 0.05). Gram-stained biopsies showed the occurrence of yeast and H. pylori, and the release of EVs from yeast. Detection of similar H. pylori genes in oral and gastric yeasts from patients in Group 2 showed their common source. Oral yeasts in Groups 3 and 4 also carried H. pylori genes. Wet mount preparations of yeasts showed intracellular bacteria inside the yeast vacuole and the release of EVs that could carry H. pylori. Oral yeast protects its intracellular H. pylori and releases it inside EVs to safely reach gastric mucosa. Yeast, as the environmental reservoir of H. pylori, plays a crucial role in bacterial reinfection after successful eradication.

Common Saccharomyces cerevisiae lab yeast strains derived from S288C have meiotic defects and therefore are poor sporulators. Here, we developed a plasmid system containing corrected alleles of the MKT1 and RME1 genes to rescue the meiotic defects and show that standard BY4741 and BY4742 strains containing the plasmid display faster and more efficient sporulation. The plasmid, pSPObooster, can be maintained as an episome and easily cured or stably integrated into the genome at a single locus. We demonstrate the use of pSPObooster in low- and high-throughput yeast genetic manipulations and show that it can expedite both procedures without impacting strain behavior.