Yeast最新文献

A novel Saccharomycotina yeast strain, yHQL494, was isolated from the rose hip of the wild rose Rosa californica from Castle Crags State Park, California, USA. Phylogenetic analyses of both whole genome data and the sequences from the D1/D2 region of the large ribosomal subunit (LSU) rRNA gene placed strain yHQL494 within the genus Lachancea and grouped it into a clade with Lachancea lanzarotensis and Lachancea meyersii. Taxogenomic analyses were conducted on publicly available genome sequences to gain a deeper insight into the carbon and nitrogen gene-trait associations across the Lachancea clade. The results of these analyses were found to be consistent across Lachancea species. Growth assays and microscopic analyses were conducted to determine the physiological characteristics of strain yHQL494, including the presence of hyphae or pseudohyphae, ascospore formation, fermentation abilities, and assimilation of carbon and nitrogen compounds. Based on the phenotypic and genomic characteristics of the strain yHQL494^T (=NRRL Y-64858^T, =CBS 18,574^T), we propose a new species, Lachancea rosae sp. nov. f.a.

The methylotrophic yeast Komagataella phaffii (formerly known as Pichia pastoris) is an essential host for biotechnological production. Here, we present the complete and annotated genome sequence of CBS 2612 T = NRRL Y-7556T, the type strain of K. phaffii. CBS 2612 has a genome length of 9,387,549 bp with 5412 predicted and 5389 annotated genes, of which 144 are tRNA genes including the previously missing tRNAs for tryptophan, tyrosine, and serine, and 34 rRNA genes. In total, 4 SNPs were found compared to the biotechnologically most commonly used strain, CBS 7435. Additionally, 34 lncRNA candidates could be identified, including candidates that affect telomere-regulation and flocculin genes.

Polyphosphate (polyP) is an intriguing polymer with diverse biological and industrial applications. Chemical polyP production is energy-intensive and limited in chain length at large-scale production. Alternatively, biological production offers a sustainable solution. Recent research endeavors highlighted Saccharomyces cerevisiae as a promising organism for polyP hyperaccumulation, achieving up to 28% (w/w) polyP (as KPO₃). P_i starvation and P_i feeding are essential for this hyperaccumulation phenotype. Prior research demonstrated that trace elements and vitamins increase polyP production in S. cerevisiae when added to the cultivation medium during P_i starvation. However, the role of trace elements and vitamins in enhancing polyP accumulation remained unclear. This study identified inositol and zinc to drive polyP accumulation across various laboratory and industrial S. cerevisiae strains. Moreover, these components influence the energy metabolism of yeasts. Our findings suggest that zinc boosts the phosphate-responsive signal transduction (PHO) pathway during P_i starvation. The influence of inositol on polyP hyperaccumulation remains elusive, as it does not influence the PHO pathway directly. These findings add to the ever-growing understanding of polyP metabolism in S. cerevisiae and provide further targets for optimizing biological polyP production.

The primary challenge in tarhana production is the occurrence of spontaneous fermentation, which leads to non-standardized products. Thus, we investigated the effects of backslopping, a traditional method for inoculating fermented foods, on the yeast and volatile aroma compound diversity of tarhana dough. Backslopping fermentations were conducted at different temperatures (25°C and 30°C), pHs (3.70 and 4.00), and inoculation rates (5%, 10%, and 15%). The results revealed that the fermentation temperature and pH significantly influenced the diversity of yeast species and the volatile compound profile of the tarhana dough. However, despite some variations in the PCR-DGGE profiles, the metagenomic analysis revealed that the inoculation rate had minimal effect on yeast diversity, with species diversity remaining relatively constant over the cycles. Kazachstania humilis, Kazachstania bulderi, and Pichia kluyveri were the most prevalent yeast species across all experimental conditions. Pichia membranifaciens was exclusively detected in doughs fermented at 25°C and pH 4.00, whereas Saccharomyces cerevisiae was observed only in doughs fermented at 30°C. Tarhana doughs had a wide range of volatile compounds, the most abundant of which were terpenes and terpenoids, followed by esters, alcohols, aldehydes, and phenols. Doughs fermented at 25°C and pH 3.70 were differentiated from other groups, particularly for their content of esters (e.g., ethyl acetate, ethyl lactate, ethyl decanoate, and ethyl octanoate) and alcohols (e.g., ethyl alcohol, isobutyl alcohol, benzyl alcohol). This study highlights the direct influence of backslopping on yeast diversity and its indirect impact on the aroma profile of tarhana dough, providing insights into the optimization of fermentation conditions for improved product standardization.

The thermotolerant yeast Ogataea polymorpha TBRC 4839 is a promising host for heterologous protein expression using sucrose and molasses as low-cost carbon sources, making it suitable for industrial applications. This study analyzed the genome and transcriptome of O. polymorpha under sucrose-induced conditions. The nuclear genome of strain TBRC 4839 measures 8.9 Mbp with a GC content of 47.87%, consistent with other Ogataea species. The genome encodes 5184 protein-coding genes, comparable to related strains. Additionally, the mitochondrial genome spans 49.4 Kbp and has a low GC content of approximately 20%. Transcriptomic analysis revealed that sucrose induction triggers a metabolic shift characterized by increased carbohydrate metabolism and decreased amino acid biosynthesis, stress signaling, and cell division, enabling efficient energy utilization in sucrose-rich environments. Among the identified genes with up-regulated expression, five were notable: FUN_000066 (hypothetical protein), FUN_001144 (maltose permease), FUN_001145 (maltase), FUN_002060 (mitochondrial NAD-dependent malic enzyme), and FUN_002263 (hypothetical protein). The promoter efficiency was evaluated by expressing the fungal xylanase gene under sucrose-inducing conditions using these promoters. The maltase (MAL) promoter exhibited the highest xylanase production efficiency, outperforming other promoters. Furthermore, the MAL promoter proved effective for xylanase production when molasses was used as the carbon source. These findings underscore the potential of O. polymorpha TBRC 4839 and the MAL promoter for industrial protein production.

Dynamic downregulation of the endogenous farnesyl pyrophosphate (FPP) synthase (Erg20p) is crucial to engineer heterologous monoterpene production in the yeast Saccharomyces cerevisiae. FPP downstream metabolite geranylgeranyl pyrophosphate (GGPP) can induce the degradation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase 2 (Hmg2p) through its N-terminal GGPP-sensing endoplasmic reticulum transmembrane domain (Hmg2p^N) in S. cerevisiae. Here, we investigate the use of Hmg2p^N to regulate Erg20p, aiming to restrict FPP synthesis and redirect metabolic flux to monoterpene production. While using the ERG1 promoter to regulate ERG20 transcription improved monoterpene limonene by ~10-fold, combinatory fusion of Hmg2p^N to Erg20p N-terminus further improved limonene production by 40% to 0.52 g L^-1 in synthetic minimal media. This approach yielded 0.5 g L^-1 geraniol in batch cultivation, comparable to levels achieved using the N-end-rule degron K3K15 or an auxin-inducible degron to regulate Erg20p. In rich complex media, this approach was superior, leading to 2.1 g L^-1 geraniol production in semi-fed batch cultivation. In summary, the Hmg2p^N domain is an efficient tool to constrain FPP synthesis for improved monoterpene production in S. cerevisiae.

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.