Yeast最新文献

Introducing plasmids into yeast is a critical step for many phenotypic assays and genetic engineering applications. However, it is often challenging for applications that involve large pools of variants because the population structure can be easily altered by traditional methods such as chemical transformation. In this study, we introduce drug-marked plasmids into a heterogeneous yeast population using both transformation and cytoduction (mating without nuclear fusion). Using a highly diverse barcoded yeast collection, we quantify the efficiency of both methods. We demonstrate that for cytoduction, but not transformation, nearly all the genotypes in the initial pool were detected in the final pool, with a high correlation to their initial frequencies. Finally, we map QTL that impact both cytoduction and transformation. Overall, we demonstrate the efficiency of cytoduction as a means of introducing plasmids into yeast. This is significant because it provides a means of manipulating diverse yeast populations, such as pools constructed for bulk segregant analysis, deep mutational scanning, large-scale gene editing, or populations from long-term evolution experiments.

How could quercetin exert a pro-survival phenotype (antioxidant) and simultaneously be toxic for eukaryotic cells? The redox capacity of quercetin may explain its antioxidant and toxic effects, based on the idea that quercetin impairs the electron transport chain, affecting ATP production and forming quercetin-derived free radicals. Herein, we provide evidence that quercetin supplementation: (1) depolarizes the mitochondrial membrane and augments the ADP/ATP ratio; (2) increases superoxide anion cellular levels; (3) changes the cellular response to H₂O₂ challenge associated with the antioxidant cellular response; and (4) sensitizes the cellular response to lipoperoxidation challenge. These events suggest that the quercetin pro-oxidant effect is related to mitochondrial respiration dysfunction and could induce cellular antioxidant response.

The phyllosphere is a crucial interface for plant-environment interactions, hosting a diverse microbial community, including yeasts. This community affects the host's fitness and can act as a plant resilience booster. Nonetheless, abiotic factors can have a significant impact on the microbial community. Therefore, this work aims to investigate the potential effects of rain and drought on the taxonomic and functional diversity of epiphytic yeasts associated with Bromelia laciniosa leaves in the Caatinga, a tropical dryland in South America. A total of 262 isolates were obtained. Based on their D1/D2 region of the LSU gene rRNA sequences, the isolates were identified as belonging to 76 species of yeasts and yeast-like fungi, including 53 Basidiomycetes and 23 Ascomycetes. Furthermore, 23 species (ca. 30% of the total) are possible new species. Most of the variables related to rainfall and drought did not affect the yeast taxonomic diversity. Furthermore, the impact of rain and drought on the community composition differs between functional and taxonomic diversities, which may suggest a decoupling between these dimensions. The functional and taxonomic structure of the yeast community in the Caatinga is complex, and rain and drought alone are not the absolute factors governing its dynamics. Additionally, the functional traits may provide valuable insights into the behavior of the yeast community in bromeliads and help predict the effects of dry-wet cycles on the leaf-inhabiting yeast community, as well as potential impacts on the host.

In eukaryotes, oxygen consumption is mainly driven by the respiratory activity of mitochondria, which generates most of the cellular energy that sustains life. This parameter provides direct information about mitochondrial activity of all aerobic biological systems. Using the Seahorse analyzer instrument, we show here that deletion of the oca3/emc2 gene (oca3Δ) encoding the Emc2 subunit of the ER membrane complex (EMC), a conserved chaperone/insertase that aids membrane protein biogenesis in the ER, severely affects oxygen consumption rates and quiescence survival in Schizosaccharomyces pombe yeast cells. Remarkably, the respiratory defect of the oca3Δ mutation (EMC dysfunction) is rescued synergistically by disruption of ergosterol biosynthesis (erg5Δ) and the action of the membrane fluidizing agent tween 20, suggesting a direct role of membrane fluidity and sterol composition in mitochondrial respiration in the fission yeast.

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.

Hanseniaspora uvarum is consistently observed as the dominant non-Saccharomyces species in spontaneous grape juice fermentations. However, the physiological mechanisms and physicochemical variables influencing the prevalence of H. uvarum over other non-Saccharomyces species remain unclear. We tested the factors contributing to H. uvarum dominance by inoculating a chemically diverse set of grape juices with a mock community whose composition was based on a previously published comprehensive microbial survey of commercial spontaneous fermentations. The diverse composition of these grape juices appeared to have minimal impact on the overall microbial dynamics of fermentation, with H. uvarum consistently emerging as the dominant non-Saccharomyces species in nearly all conditions tested. Flow cytometry analysis confirmed that H. uvarum has a faster growth rate than Saccharomyces cerevisiae and several other Hanseniaspora species. Moreover, its growth was not affected by the presence of S. cerevisiae. H. uvarum negatively affected the growth of S. cerevisiae, with significant implications for fermentation performance and sugar consumption. Our study suggests that the fast growth rate of H. uvarum enables it to dominate the grape juice environment quickly during early fermentation stages. This physiological advantage may be critical to the outcome of spontaneous fermentations, as evidenced by its direct impact on S. cerevisiae and fermentation performance.

The Lithium-PEG method for transforming yeast cells is a standard procedure used in most yeast laboratories. After several optimizations, this method can yield up to 10⁶ transformants per µg of plasmid. Some applications, such as library screening or complex transformations, necessitate maximizing transformation yield. Here, we demonstrate that the addition of a sorbitol solution serves as an osmo-protectant during and after heat shock, resulting in up to a tenfold increase in transformation efficiency. This optimization requires only one additional pipetting step compared to the original protocol, making it practical for routine use.

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.