Yeast最新文献

Multiple isoforms of bovine intestinal alkaline phosphatase (BIAP) have been identified, among which type Ⅱ (BIAP Ⅱ) exhibits the highest specific activity. While Komagataella phaffii has been successfully employed for the secretory expression of recombinant BIAP Ⅱ, substantial proteolytic degradation during the secretion and expression processes has been observed, leading to reduced protein yield and challenging purification procedures. Our investigation demonstrates that the proteolytic cleavage of BIAP Ⅱ is predominantly mediated by secretory pathway proteases, particularly the aspartic protease yapsin (Yps), with Yps1 playing a crucial role. Genetic disruption of the YPS1 gene resulted in a remarkable 2.5-fold increase in BIAP Ⅱ production yield compared to the parental strain, accompanied by significantly reduced proteolytic degradation. Through detailed analysis, we have identified the Yps1 cleavage site within the BIAP Ⅱ peptide chain, located between Lys₁₃₇ and Lys₁₃₈. To further minimize BIAP Ⅱ proteolysis, we developed a YPS multigene-deficient engineered strain using CRISPR/Cas9-mediated triple gene editing technology. Additionally, we have established a novel dual-color quantitative PCR (DC-qPCR) method that enables rapid and precise determination of target gene dosage, thereby enhancing screening efficiency while reducing experimental errors associated with repeated sample processing. The strategies and methodologies developed in this study may serve as a valuable reference for optimizing the expression of various secretory heterologous proteins in Komagataella phaffii.

The target of rapamycin complex 1 (TORC1) protein kinase plays an important role in regulating various cellular activities in response to nutrient availability. In this study, an autophagy-related protein 8 (atg8) mutant of Saccharomyces cerevisiae was highly sensitive to cellular processes in which TORC1 activity was inhibited by rapamycin treatment or by a mutated allele of KOG1 which encodes a subunit of TORC1. Atg8 exhibits both lipidation-dependent and -independent activities, each involving distinct factors. Lipidation of Atg8 is necessary for autophagy and functions with autophagy-related proteins like Atg7, whereas the lipidation-independent activities of Atg8 require Hfl1. The atg7Δhfl1Δ double mutant exhibited defects for the impaired TORC1 activities, suggesting that both lipidation-dependent and -independent functions of Atg8 are required for survival during impaired TORC1 activity. Moreover, atg8Δ and atg7Δhfl1Δ mutants exhibited sensitivity to metal ion Zn²⁺ during low-dose rapamycin treatment. The results suggest that Atg8-mediated functions and TORC1 signaling events play an important role in cell growth, possibly by maintaining vacuole integrity.

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.

Tda1p is a protein kinase in Saccharomyces cerevisiae. Here we investigate the function of TDA1 during the diauxic shift using transcriptomics. We compared the gene expression in the deletion mutant tda1∆ and the reference strain (BY4741) during both the aerobic fermentation phase (log phase), and the respiratory phase (post-diauxic shift phase, PDS) in three separate independent experiments. We found: Differential gene expression analysis showed that compared to the reference strain, the tda1∆ mutant exhibited an upregulation of the glucose repressed hexose transporter HXT6 during the log phase, and upregulation of mitochondrial proteins and genes related to mitochondrial translation during the PDS phase. Gene set enrichment analysis showed an enrichment in mitochondrial translation in the PDS phase for the deletion mutant tda1∆, but not for the reference strain. Transcription factor analysis showed that the enrichment of Mig1p repressed genes was not statistically significant in TDA1 deletion mutants for neither log-phase nor PDS-phase. This conflicted with the previously suggested model that argued for an interaction between Tda1p and Mig1p. Instead, transcription factor analysis showed an enrichment of genes regulated by the HAP-complex, which regulates mitochondrial translation, during the PDS-phase in the tda1∆ mutant. The combined evidence from this study indicates that Tda1p does not participate in Mig1p-mediated glucose repression. Instead, we propose that it is involved in the regulation of mitochondrial translation by repressing the expression of HAP complex subunits.

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.

Two yeast strains belonging to the ascomycetous yeast genus Saccharomycopsis were isolated from soil collected from a forest in Wufeng Tujia Autonomous County, Yichang, Hubei province, China. Phylogenetic analyzes of the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit rRNA gene showed that they closely related to S. fermentans and S. babjevae but differed from S. fermentans by 17 (3.09%, 15 substitutions and two gaps) and 30 (4.85%, 22 substitutions and eight gaps) mismatches, and from S. babjevae by 13 (2.39%, eight substitutions and five gaps) and 21 (3.46%, 14 substitutions and seven gaps) mismatches in the D1/D2 domain and ITS region, respectively. A phylogenomic analysis based on 1260 single-copy orthologs confirmed the close relationship of the new Chinese strains with S. fermentans and S. babjevae. The whole genome average nucleotide identity (ANI) values of the new strains with the two species are 85.7% and 86.9%, respectively. The results suggest that the two strains represent a novel species, for which the name Saccharomycopsis yichangensis sp. nov. (holotype strain CGMCC 2.7390) is proposed. The Fungal Names number is FN 572295. The novel yeast is homothallic and produces asci containing four spheroidal ascospores with an equatorial or subequatorial ledge. This species can prey on cells of Jamesozyma jinghongensis, Meyerozyma carpophila and Saccharomyces cerevisiae through invasive infection pegs.

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.

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.