Search Results (135)

The alkaline pectate lyase A from Paenibacillus barcinonensis, encoded by pelA (GenBank accession no. CAB40884), is an enzyme with high activity on pectin and potential application in sustainable industrial biotechnology. In this study, pelA was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by pelA, and two of them in conserved regions of class III pectate lyases, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both standard and glycosylation deficient strains of S. cerevisiae. The correct targeting of the recombinant fusion proteins was confirmed by Western blot analysis using Pir-specific antibodies, whilst enzymatic activity on polygalacturonic acid was demonstrated on both plate assays and colorimetric assays. Maximum activities were over two and a half times higher when the enzyme was expressed in the glycosylation deficient strain, suggesting a better adaptation of this strain to the secretion of the functional enzyme. Full article

Yeast surface display is a powerful strategy for enzyme immobilization and whole-cell biocatalysis; however, the intracellular processing of heterologous enzymes during secretion and anchoring remains poorly understood. In this study, a GH5 endoglucanase gene (eglS, 1.4 kb) from Bacillus subtilis, originally isolated from a paper mill effluent, was cloned into the pYD1 vector and expressed in Saccharomyces cerevisiae EBY100 using the Aga1–Aga2 surface display system. The recombinant strain produced clear degradation halos on carboxymethyl cellulose (CMC) plates, confirming cellulolytic activity at the whole-cell level. Zymographic analysis revealed multiple active enzyme forms depending on the cellular fraction analyzed. Intracellular extracts displayed active bands ranging from 70 to 57 kDa, consistent with immature or partially processed Aga2 fusion proteins, whereas cell wall-associated fractions showed active bands between 55 and 35 kDa, suggesting proteolytic processing during secretion and surface anchoring. The apparent specific activity of the cytoplasmic fraction was 5.33 ± 0.31 U mg⁻¹, while the cell wall-associated fraction exhibited a higher apparent specific activity (58.4 ± 10.1 U mg⁻¹). Although these values were obtained from non-purified fractions and therefore do not represent intrinsic enzymatic constants, they indicate a relative enrichment of catalytically active enzyme in the cell wall-associated fraction, consistent with functional surface display. The presence of multiple active enzyme forms and the enhanced catalytic efficiency observed in the cell wall-associated fraction suggest that the engineered yeast strain may serve as a promising whole-cell biocatalyst, with potential applications in consolidated bioprocessing (CBP) strategies for lignocellulosic biomass conversion. Full article

In Saccharomyces cerevisiae (S. cerevisiae), sister kinetochores are mono-oriented during meiosis I to ensure accurate homolog segregation, a process dependent on Hrr25 kinase activity. However, its direct interactors remain poorly defined. To address this, we performed a yeast two-hybrid (Y2H) screen using Hrr25 as bait. HRR25 was cloned into a Y2H vector and functionally validated by complementation of a temperature-sensitive hrr25-ts mutant. Screening across three reading frames identified three putative interactors: Hed1, Cyr1, and Rep1. Additional open reading frames (ORFs), including DAD1, SYS1, and YDR015C were identified but were oppositely oriented to the GAL4 activation domain. Structural modeling and phosphorylation prediction identified high-confidence Hrr25 target residues, including S70/T73 on Hed1, S323 on Rep1, and S198/S527 on Cyr1, whereas Sys1 and YDR015C lacked favorable sites. Although Dad1 was not validated as a direct interactor from Y2H, S63 was identified as a favorable phosphorylation site, and its full-length ORF in the interacting clone and known biological role supported its inclusion. Among the meiotic candidates, Hed1 may link Hrr25 activity to homologous recombination, while Dad1 represents a plausible target for regulating kinetochore–microtubule interactions. Collectively, these findings identify new candidate interactors and substrates of Hrr25 and suggest a broader role in coordinating recombination and kinetochore function during meiosis, warranting further experimental validation. Full article

γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter, is known for its physiological functions in alleviating anxiety and improving sleep. Currently, high-yielding GABA food products are mainly obtained through screening wild-type high-producing strains (e.g., Saccharomyces cerevisiae isolated from Sichuan pickles yielding 0.67 g/L) or employing co-culture systems (e.g., Enterococcus faecium and Lactiplantibacillus plantarum reaching 6.35 g/L). While effective, these methods often rely on natural screening strains or multi-microbial interactions. This study employed CRISPR-Cas9 technology to knockout the UGA1 gene in Saccharomyces cerevisiae, a key gene responsible for GABA degradation. Starting from the low higher alcohol Saccharomyces cerevisiae SY-LH, we successfully constructed the recombinant strain SY-LHU. Remarkably, this study discovered a significant upregulation of GAD1 gene expression following UGA1 knockout, which further enhanced GABA synthesis capacity. Under optimal fermentation conditions (inoculum size 4 × 10⁷ cells/mL, wort concentration 10 °P, sugar addition 60 g/L, 30 °C for 10 days, and mixing the malt broth every 48 h), the validation fermentation was performed and the GABA content in the wort beverage reached 280.36 mg/L, representing a 385.4% increase compared to the pre-optimization level. Furthermore, sensory evaluation by a trained panel yielded a mean score of 88, with no significant off-flavors detected, demonstrating the product’s high consumer acceptance. This pioneering work provides a novel and feasible technical pathway for developing functional alcoholic beverages with sleep-aiding properties. Full article

Deoxynivalenol (DON), a Group III carcinogenic mycotoxin frequently detected in cereals and animal-derived food products, poses serious health risks to animals and humans. In this study, we developed a genetically engineered Saccharomyces cerevisiae strain as a proof-of-concept platform for DON detoxification. The yeast was engineered to co-express two detoxification genes, YTDepA and YTDepB (homologs of DepA and DepB from Devosia mutans 17-2-E-8) originally identified in Youhaiella tibetensis. Concurrently, the pyrroloquinoline quinone (PQQ) biosynthesis gene cluster from Klebsiella pneumoniae was integrated to supply the essential cofactor. Gene expression was verified by qRT-PCR and Western blot. The recombinant strain demonstrated a significant 13.98% detoxification of DON after 72 h of fermentation (p < 0.05), as confirmed by HPLC–MS, while the strain expressing only the PQQ cluster showed no detoxification activity. This study establishes an integrated yeast cell factory for DON detoxification and highlights key limitations to guide future optimization efforts. Full article

Polyamines, putrescine, spermidine and spermine, are essential polycationic metabolites present in all eukaryotic cells, where they regulate fundamental processes including nucleic acid stabilization, translation, and stress responses. Spermidine synthase (SPDS), a member of the aminopropyltransferase (APT) family, catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dc-SAM) to putrescine to form spermidine. Although genomic analyses predict the presence of SPDS homologs in multiple fungal species, polyamine biosynthesis has not been experimentally characterized in the multidrug-resistant fungal pathogen Candidozyma auris. Here, we report the biochemical and functional characterization of the C. auris spermidine synthase, CauSpe3. The CauSPE3 gene complemented a Saccharomyces cerevisiae spe3Δ mutant demonstrating conserved function in vivo. Recombinant CauSpe3 was expressed in Escherichia coli, purified and analyzed using the fluorescence-based DAB-APT assay, which uses 1,2-diacetylbenzene (DAB) for polyamine detection. CauSpe3 catalyzed efficient conversion of putrescine to spermidine in the presence of dc-SAM, with K_half values of 65.5 ± 7.11 µM for putrescine and 66.9 ± 2.09 µM for dc-SAM, and V_max values of 7.1 ± 0.57 and 7.9 ± 0.12 nmol·µg⁻¹·min⁻¹, respectively. A catalytic-site mutant and heat-inactivated enzyme showed no detectable activity, and product formation was confirmed by means of thin-layer chromatography and mass spectrometry. These findings establish CauSpe3 as a functional spermidine synthase. Full article

Recombinogenic DNA damage can initiate chromosomal rearrangements that can alter gene expression or accelerate cancer progression in higher eukaryotes. Thus, there is a critical need to identify genes that suppress chromosomal rearrangements and environmental exposures that promote genetic instability. Cell cycle checkpoints modulate the cell cycle so that DNA repair occurs before the replication or segregation of damaged chromosomes. Saccharomyces cerevisiae (budding yeast) RAD9 was the first cell cycle checkpoint gene identified, which initiated intensive research studies into the mechanisms of checkpoint activation and the phenotypes of checkpoint mutants. The budding yeast Rad9 protein serves as both an adaptor and scaffold that facilitates downstream effector activation to orchestrate a DNA damage response at multiple stages of the cell cycle, which facilitates double-strand break (DSB) repair by sister chromatid recombination. However, the role of RAD9 in homologous recombination and in suppressing gross chromosomal rearrangements (GCRs) is not completely understood. In this review we discuss how RAD9 can promote genome instability resulting from aberrant DNA replication intermediates, while suppressing DSB-associated rearrangements. We also discuss possible mechanisms accounting for the synergistic increase in genomic instability in double mutants defective in both RAD9 and recombinational repair. We emphasize that while there is an overlap between checkpoint and recombinational repair pathways, RAD9 and checkpoint pathways can function independently to suppress chromosomal instability. These studies thus elucidate checkpoint mechanisms that control homologous recombination between repeated sequences. Full article

Genetic variation underlies the capacity of populations to adapt, yet what drives how this variation is generated and maintained in natural populations remains poorly understood. Fundamental processes such as mutation, ploidy, and recombination are known to shape genetic variation and adaptive potential but are typically studied in isolation and under controlled laboratory conditions. How these processes act together under varying environmental conditions to structure genetic variation across complex natural populations remains unresolved. In yeasts, these processes are dependent on reproductive mode, ploidy shifts, and environmental stressors, which jointly shape genomic stability and adaptive potential. Here, we review our current knowledge on the roles of mutation, ploidy, and recombination in adaptation in the model yeasts Saccharomyces cerevisiae and the human pathogenic Cryptococcus. We highlight heterogeneity in mutation rates, recombination, and ploidy states across strains, environments, and populations, challenging the assumption that these parameters are uniform. We argue that fluctuating environments, increasingly driven by climate change, are likely to intensify interactions among these processes to impact evolution in ways that remain difficult to predict. Integrating population genomics with ecologically realistic frameworks will be essential for understanding natural evolutionary dynamics and anticipating fungal adaptation and disease emergence. Full article

Chromosome dynamics, recombination, and nucleolar organization intersect during meiotic prophase I, yet how the recombination context influences nucleolar architecture remains unclear. We analyzed the nucleolar pool of Cdc14 in Saccharomyces cerevisiae under matched prophase I gating and a uniform, frame-based operational definition of transient two-focus episodes. In a prophase-arrest reference, Cdc14–mCherry formed a predominant single nucleolar focus with occasional, reversible two-focus episodes that Nop56–GFP placed within the nucleolar compartment (nucleolar splitting). Splitting rose sharply when interhomolog recombination was compromised and remained elevated when Spo11 catalytic activity was abolished, indicating that increased DSB formation is not required and pointing instead to the homolog engagement state as a key variable. Population checkpoint readouts did not map onto the phenotype: Hop1 phosphorylation differed strongly across genotypes, yet splitting remained high in recombination-defective and DSB-free contexts and low in the reference. Timing analyses showed that events concentrated early and declined in the reference, whereas recombination-defective and DSB-free backgrounds retained activity into later windows across thresholds. We propose that nucleolar splitting reflects a rheological response of the nucleolus to chromosome-scale forces that vary with homolog engagement, consistent with contributions from DSB-independent chromosome dynamics such as telomere clustering, telomere-led rapid prophase movements, and centromere coupling/pairing. Together, these data support the nucleolus as a mesoscale, mechanically sensitive readout of meiotic chromosome dynamics. Full article

In view of the technical bottleneck of microbial dynamic monitoring during the solid-state fermentation of traditional Baijiu, this study introduced green fluorescent protein (GFP) labeling technology into the dominant Saccharomyces cerevisiae of Jiang-flavored Baijiu to construct the chromosomal integration engineering strain named FSC01. By designing an integrated recombinant plasmid containing the GFP gene and the geneticmycin resistance gene, an engineered strain that stably expresses fluorescent proteins was obtained by electroconversion. Flow cytometry verification showed that FSC01 showed excellent linear responses in the pure microbial system (R² = 0.998) and the complex matrix of Baijiu jiupei (R² = 0.981), with a detection limit of 10² cells/mL, and the detection cycle was shortened to 10 min. Solid-state fermentation simulation experiments show that the inoculation volume of FSC01 of 10⁵ cells/kg can not only ensure the effective identification of fluorescence signals, but also does not significantly interfere with the growth and growth patterns of the original yeast (p > 0.05), which is highly consistent with the results of the traditional plate counting method. Dynamic monitoring shows that Saccharomyces cerevisiae during fermentation presents a typical succession pattern of “increase first and then decrease”, reaching a peak on the 7th day (1.2 × 10⁷ cells/g), which is positively correlated with the base alcohol yield rate (26.7%). Compared with metagenomic (72 h) and PMA-qPCR (4 h) methods, this technology breaks through the limitations of specificity and timeliness of live bacteria detection, and provides a single-cell-level dynamic analysis tool for the digitization of traditional brewing processes. In the future, it will be expanded to monitor key functional microorganisms such as lactic acid bacteria through a multi-color fluorescent labeling system, and optimized pretreatment to eliminate starch granule interference, and promote the in-depth application of synthetic biology technology in the traditional fermentation industry. Full article

The COVID-19 pandemic accelerated vaccine innovation but also exposed weaknesses in global access and manufacturing. Yeast-based platforms, particularly Saccharomyces cerevisiae and Pichia pastoris, also known as Komagataella phaffii, offer a practical complement to vector systems. These eukaryotic microorganisms combine safety, scalability, and cost-effectiveness with the ability to express complex antigens and assemble virus-like particles. Building on the success of the recombinant hepatitis B vaccine, recent advances in glycoengineering, CRISPR-based host optimization, and surface display technologies have expanded the utility of yeast-based platforms for the rapid development of vaccines. Yeast-derived SARS-CoV-2 receptor-binding domain (RBD) subunit vaccines, such as Corbevax and Abdala (CIGB-66), demonstrate that affordable, immunogenic, and thermostable products are feasible at scale. Emerging innovations in glycan humanization, thermostable formulations, and oral or mucosal delivery highlight the potential of yeast-based vaccines for decentralized manufacturing and equitable pandemic preparedness. This review summarizes recent technical and clinical progress in yeast-based vaccine research, positioning these platforms as accessible and adaptable tools for future outbreak responses and global immunization strategies. Full article

The transcriptional response to alkalinization in Saccharomyces cerevisiae, Aspergillus nidulans and Candida albicans raised the interest of the scientific community many years ago for diverse reasons, and the underlying signaling pathways have been elucidated in these organisms in detail. Within the last few years, transcriptomic data for other fungal species have become available, although in most cases little is known about the molecular basis controlling their adaptive response. The objective of this work is to provide an overview on how different fungi remodel their gene expression in response to environmental alkalinization, highlighting the similitudes and differences among them. Microbial stress-responsive promoters have been considered useful tools for biotechnological applications, such as expression of recombinant proteins of industrial interest. Recent work, emphasizing the usefulness of alkaline pH-inducible promoters for heterologous protein production, will also be discussed. Full article

Topoisomerase 1 (Top1) removes transcription-related helical torsions and thus plays an important role in preventing genome instability instigated by the formation of non-canonical DNA secondary structures. The genetically tractable Saccharomyces cerevisiae model proved effective in defining the critical function of Top1 to prevent recombination and chromosomal rearrangement at G4-forming genomic loci and studying the human cancer-associated Top1 mutants through the expression of analogous yeast mutants. We previously showed that cleavage-defective Top1 mutants strongly elevate the rate of recombination at G4 DNA, which involves binding to G4 DNA and interaction with the protein nucleolin (Nsr1 in yeast). Here, we further explored the mechanism of genome instability induced by the yeast Top1Y740* mutant, analogous to the human Top1W765Stop mutant conferring resistance to CPT. We show that yTop1Y740* elevates duplications as well as recombination specifically at G4-forming sequences. Interestingly, SUMOylation of yTop1Y740*, which does not affect the G4 DNA-binding or Nsr1-interaction by this mutant, is necessary for such elevated G4-specific genome instability. Many tumors with mutations at the C-terminal residues of Top1, particularly W765, have significantly high G4-associated mutations, underscoring the importance of further investigation into how SUMOylation affects the function of these Top1 mutants at G4 DNA. Full article

Copper contamination in the environment poses significant risks to both soil and human health, making the need for reliable monitoring methods crucial. In this study, we report the use of the EmStat Pico module as potentiostat to develop a portable electrochemical biosensor for copper detection, utilizing yeast Saccharomyces cerevisiae cells immobilized on a polydopamine (PDA)-coated screen-printed electrode (SPE). By optimizing the sensor design with a horizontal assembly and the volume reduction in the electrolyte solution, we achieved a 10-fold increase in current density with higher range of copper concentrations (0–300 µM CuSO₄) compared to traditional (or previous) vertical dipping setups. Additionally, the use of genetically engineered copper-responsive yeast cells further improved sensor performance, with the recombinant strain showing a 1.7-fold increase in current density over the wild-type strain. The biosensor demonstrated excellent reproducibility (R² > 0.95) and linearity over a broad range of copper concentrations, making it suitable for precise quantitative analysis. To further enhance portability and usability, a Bluetooth-enabled electrochemical platform was integrated with a web application for real-time data analysis, enabling on-site monitoring and providing a reliable, cost-effective tool for copper detection in real world settings. This system offers a promising solution for addressing the growing need for efficient environmental monitoring, especially in agriculture. Full article

Diacylglycerol-O-acyltransferase 1 (DGAT1, EC 2.3.1.20) is a pivotal enzyme in plant triacylglycerol (TAG) biosynthesis. Previous work identified conserved di-arginine (R) motifs (R-R, R-X-R, and R-X-X-R) in its N-terminal cytoplasmic acyl-CoA binding domain. To elucidate their functional significance, we engineered R-rich sequences in the N-termini of Tropaeolum majus and Zea mays DGAT1s. Comparative analysis with their respective non-mutant constructs showed that deleting or substituting R with glycine in the N-terminal region of DGAT1 markedly reduced lipid accumulation in both Camelina sativa seeds and Saccharomyces cerevisiae cells. Immunofluorescence imaging revealed co-localization of non-mutant and R-substituted DGAT1 with lipid droplets (LDs). However, disruption of an N-terminal di-R motif destabilizes DGAT1, alters LD organization, and impairs recombinant oleosin retention on LDs. Further evidence suggests that the di-R motif mediates DGAT1 retrieval from LDs to the endoplasmic reticulum (ER), implicating its role in dynamic LD–ER protein trafficking. These findings establish the conserved di-R motifs as important regulators of DGAT1 function and LD dynamics, offering insights for the engineering of oil content in diverse biological systems. Full article