Endosomal sorting complex required for transport (ESCRT-III) is a membrane remodeling complex involved in a large number of cellular processes. It appears to perform an essential function in eukaryotes, since to date no eukaryotic organism completely devoid of ESCRT-III has been found. Yet, yeast cells with a deletion of all eight known ESCRT-III genes are viable. We therefore searched for new, previously undiscovered ESCRT-III like proteins in yeast. HHPred uncovered several proteins with similarity to Snf7. The similarity was mostly restricted to the α1-α2 hairpin region of Snf7. A conserved pattern of amino acids was detected in this region. One of the proteins with an ESCRT-III like sequence pattern, which strikingly resembled Snf7 in its secondary structure, was studied more closely. We named the protein encoded by ORF YPL199c Etl1 (ESCRT-three-like 1). Etl1 is palmitoylated and localizes to the plasma membrane. In contrast to other palmitoylated proteins, Etl1 does not appear to be associated with lipid rafts, since it could be easily extracted from the membrane by Triton X-100 treatment. When ETL1 was deleted in the octuple ESCRT-III deletion background, the yeast cells were still viable. So far, despite a number of experiments, a bona fide ESCRT-III function could not be demonstrated for Etl1.
All living organisms can enter a non-dividing state known as quiescence, which often functions as a survival strategy. In many cases, quiescent cells retain the ability to re-enter the cell cycle when conditions become favorable. Therefore, preserving the integrity of non-dividing cells is essential not only to prevent their deterioration, but also to ensure proper population re-establishment once growth resumes. This also applies to the genome, whose stability must be maintained during both proliferation and quiescence. For several years, we have investigated this aspect of quiescence in fission yeast, which enters a G0 state in response to nitrogen starvation. We have shown that wild-type G0 cells accumulate mutations over time, with a mutational spectrum distinct from that observed during proliferation. We are now extending this work to mutants defective in various DNA repair pathways. In doing so, we developed a robust protocol ensuring highly reproducible results when assessing mutagenesis in quiescent cells. Here, we describe this protocol in detail.
Together with other fungi, yeasts make up a significant component of the plant microbiome. As the planet warms, cacti expand their range. Cactus-associated yeasts are known to exhibit signatures of adaptation to the cactus host. Our previous isolation of a wild Saccharomyces paradoxus yeast from a cactus in a forest of oaks populated by S. cerevisiae prompted us to further explore cactus-associated fungi and look for genomic and phenotypic signatures of adaptation. Here we characterize seven yeast isolates, five from cacti and two from adjacent non-cactus plants, among which was a novel species of Coniochaeta isolated from wild grape that we name C. udwismasis. Closely-related isolates from distinct plant hosts exhibited distinct features, including differences in thermotolerance, freeze-thaw tolerance, pigmentation, and predicted septin protein complex assembly, providing new insights into possible mechanisms of cactus adaptation by the yeast microbiome.
Efficient secretion of heterologous proteins is essential for advancing yeast-based bioprocesses, yet signal peptide (SP) optimization in the thermotolerant methylotrophic yeast Ogataea polymorpha remains limited. This study integrates in-silico SP discovery, experimental validation, and bioprocess engineering to enhance secretion of the thermophilic xylanase X11P under sucrose-inducible expression. Genome-wide screening of 5184 O. polymorpha proteins using SignalP, Phobius, DeepLoc, WoLF PSORT, and ProP identified 11 high-confidence SP candidates. Comparative analysis with Komagataella phaffii endogenous proteins guided selection of seven SPs for experimental evaluation. Among these, the novel O. polymorpha α-mating factor-like peptide FUN_005010 exhibited strong secretion-promoting activity, with its prepro-sequence yielding the highest extracellular xylanase levels and outperforming the classical Saccharomyces cerevisiae α-MF. To evaluate industrial applicability, sucrose-based fermentation strategies were systematically optimized in a 5-L bioreactor. Controlled sucrose feeding and balanced C/N ratios were found to be critical for maximizing maltase (MAL) promoter-driven expression. A stepwise increasing sucrose feed combined with induction at 30°C enabled X11P titers up to 770 U/mL, representing a 15-fold improvement over shake-flask cultures. This work demonstrates that the combination of SP evaluation and optimized sucrose-inducible fed-batch operation significantly enhances X11P production in O. polymorpha. The identified FUN_005010-prepro SP and the refined process framework provide valuable tools for developing O. polymorpha as a high-performance industrial expression platform.
Summary Schizosaccharomyces pombe offers a powerful model for mitochondrial studies In vivo and in vitro methods to study mitochondrial function and homeostasis Assays to study mitochondrial metabolism, bioenergetics, and redox balance Description of biosensors for in vivo measurement of ATP, H 2 O 2 , NAD(P)H Tools to analyze morphology and respiratory supercomplexes.
The glucose repression system is a mechanism for effective energy acquisition by glucose assimilation in microorganisms. In yeast, Saccharomyces cerevisiae, which is known as a prion-like protein [GAR+], is involved in the bypass of glucose repression. It has been reported that the emergence of [GAR+] cells was promoted by lactate and acetate. Herein, we examined the promotion of [GAR+] emergence by pyruvate (both the end product of glycolysis and a precursor of the synthesis of organic acids), and we compared the results of pyruvate with those of lactate or acetate. Pyruvate, as well as other organic acids, promoted [GAR+] emergence in all S. cerevisiae strains tested, but the induction patterns of [GAR+] emergence by organic acids differed among the strains. The ability of all three organic acids to induce [GAR+] was weakened at pH values greater than the pKa value of each organic acid, indicating that the undissociated forms of organic acids may promote [GAR+] emergence.
The genus Nakaseomyces provides four species that are closely related but have different characteristics. For example, N. glabratus (formerly known as Candida glabrata) is a common human pathogen, whereas N. bracarensis and N. nivariensis have been isolated in clinical settings but are not common human pathogens. N. delphensis was isolated from fruit and there is no evidence it is pathogenic. Given the differences, we developed the clade as a molecular genetic system where we could introduce plasmids and assess transcriptional output from cloned promoters. We engineered a CRISPR/Cas9 plasmid that allows for rapid Gibson cloning of gRNAs, generated auxotrophic strains for amino acids and nucleotides, and introduced plasmids into each species. We used promoter-YFP plasmids to determine that while there are differences between the species, each species likely has intact thiamine and phosphate (THI and PHO) signal transduction pathways, and that gene expression in N. glabratus and N. bracarensis is more similar to one another than to the other two species. Finally, we determine that N. glabratus, N. bracarensis, and N. nivariensis persist in a murine macrophage for 24 h, whereas N. delphensis does not. This work describes new molecular tools for genetic manipulation in the Nakaseomyces clade and allows for evolutionary questions to be explored.
In this study, we introduce a novel approach for analysing long, repetitive genomic sequences. Our methods significantly advance research on rDNA polymorphism. First, we describe a technique for isolating high-molecular-weight DNA from individual chromosomes, enabling selective enrichment of sequencing libraries for extensive genomic regions of interest. Second, we present rDNAmine, a bioinformatic toolkit for capturing and examining large repetitive arrays in Oxford Nanopore sequencing data. This approach facilitates the study of polymorphisms within long repeats, bypassing traditional alignment-based methods and providing a more efficient and scalable solution for investigating repetitive regions. We demonstrate the effectiveness of our approach through the analysis of rDNA arrays in two yeast species, Saccharomyces cerevisiae and Candida albicans. In S. cerevisiae, rDNA arrays show limited polymorphism, while in C. albicans, we observe substantial variation in rDNA module size, with two distinct repeat populations within the array. These findings reveal species-specific differences in the structural organisation of rDNA loci, highlighting the diverse nature of tandem repeat architecture. The rDNAmine toolkit is broadly applicable to various organisms and repetitive genomic contexts, offering a versatile platform for studying repetitive sequences.
All organisms must be able to sense and respond to adverse environments, especially those that threaten cellular integrity. The age of genomics clarified the breadth and specificity of cellular stress responses, including in free-living microbes directly exposed to a changing environment. The environmental stress response (ESR) in Saccharomyces cerevisiae was among the first responses defined at the transcriptome-wide level as a common program triggered by diverse types of stress. Since its original publication over 25 years ago, many studies have explored the role, regulation, and evolution of the ESR and underlying principles of stress defense. This perspective reviews the history of the ESR, recent insights and perspectives into its purpose and regulation, and remaining questions in stress biology primed for the power of yeast experimentation.
The evolution of the yeast, Saccharomyces cerevisiae, from a genetically tractable model organism to a chassis for genome-scale engineering represents one of the most influential trajectories in eukaryotic biology. The Synthetic Yeast Genome Project (Sc2.0) embodies the current height of this trajectory, having now delivered functional synthetic versions of all 16 native yeast chromosomes and bringing the construction of the first fully synthetic eukaryotic cell within reach. Beyond its technical achievements, Sc2.0 has reshaped how eukaryotic genomes are understood and explored through iterative design-build-test-learn (DBTL) cycles, and reframed the yeast genome as a dynamic, highly modifiable system rather than a static biological blueprint. Moreover, the progress on genome engineering pipelines and synthetic biology has laid the foundations for the de novo development of modular synthetic chromosomes (neochromosomes) that operate orthogonally to the native genome. These synthetic platforms provide dedicated, large-scale genomic landing pads for refactoring genetic networks, reallocating redundancy, and introducing large, multiplexed gene assemblies, thereby extending yeast engineering toward programmable and hyper-versatile biological systems. To commemorate the 40th anniversary of the journal Yeast, this minireview celebrates the exceptional power of yeast genetics, outlining key conceptual and technological advances emerging from the Sc2.0 endeavour and beyond. Finally, we examine the cross-cutting engineering insights and the future potential of neochromosomes for the next generation of synthetic yeasts.
Yeast flocculation is a phenomenon in which yeast zymolectins bind to mannose on adjacent cells resulting in aggregation within fermentation media. Flocculation has been extensively studied and is often assumed to occur only after media sugars are depleted as these are thought to competitively bind to zymolectins. However, yeast within the brewing industry have often been observed flocculating prematurely or staying suspended after media sugars were depleted. In this study, the mechanism of yeast (Saccharomyces pastorianus) flocculation was further investigated, specifically, how flocculation ability, zymolectins, and mannose associated with yeast cells changed throughout fermentation. It was observed that yeast flocculation behavior changed throughout fermentation despite no changes in zymolectin concentrations (as measured using bound fluoroprobes) independent of media sugar concentration. However, a strong positive correlation was observed between yeast flocculation ability and mannose concentration which increased as the fermentation progressed. This implies that the changes observed in flocculation behavior during fermentation may be more dependent on cell mannose rather than media sugars or lectins. These results have implications for yeast immobilization and flocculation control schemes. This research enhances the current understanding of the yeast flocculation mechanism with applications in brewing, bioethanol production, pharmaceutical production, precision fermentation, and cell biomass industries.
Optogenetic switches are molecular systems enabling light-controlled gene expression. These systems are based on the reconstitution of chimeric Transcription Factors (TFs) including a DNA-binding Domain (DBD), a photoreceptor domain, and an Activation Domain (AD). Thus, depending on the light-mediated homodimerization or heteromerization of the chimeric TF, the optogenetic switches can be classified as single-component or two-component systems, respectively. In the budding yeast Saccharomyces cerevisiae, optogenetic switches have shown multiple applications in metabolic engineering and biotechnology. Here, we expand the repertory of optogenetic switches available in yeast, developing a collection of plasmids and yeast strains carrying single-component or two-component optogenetic switches based on the BcWCL1 protein, a blue-light photoreceptor from Botrytis cinerea that contains a LOV (Light Oxygen Voltage) domain. By dissecting the N-terminal, C-terminal, and both protein regions simultaneously, we developed 18 plasmids encoding the BcWCL1 versions fused to the Gal4-DBD and Gal4-AD. Assembled plasmids were combined and transformed into yeast to generate single-component and two-component optogenetic switches, whose blue-light response was measured as transcriptional activity of the luciferase reporter. In general, we observed that the two-component configuration increased the luciferase expression in response to blue-light compared to single-component switches. Importantly, BcWCL1 versions with deletions in the C-terminal region showed the highest luciferase expression upon blue-light stimulation with the lowest background in the dark condition, suggesting that the C-terminal region modulates the blue-light response of this protein. Altogether, we generated new single-component and two-component optogenetic switches, each of them achieving different levels of light-activated luciferase expression, enabling their potential applications in yeast biotechnology.
Dietary restriction (DR) is a robust lifespan-extending intervention across species. While budding yeast is a fundamental model for DR, results from glucose restriction (GR) often show inconsistencies. This may stem from auxotrophic markers in engineered strains, which can induce abnormal cellular states under starvation. We hypothesized that GR extends chronological lifespan (CLS) primarily by avoiding auxotrophic starvation, thereby allowing cells to better adapt to nutrient depletion. Using non-dividing survival assays for precise nutritional control, we observed that yeast survive significantly longer under carbon starvation than under auxotrophic starvation. The extent of CLS extension was diminished when auxotrophic starvation was absent. Yeast cells under auxotrophic starvation showed decreased resistance to H2O2 and increased mutation rate-phenotypes that suggest a failure to enter a robust, quiescent-like state. These findings suggest that auxotrophic starvation may bias not only CLS studies but also broader yeast studies. By reconsidering previous studies with attention to auxotrophic starvation, more meaningful conclusions could emerge. Since auxotrophic nutrients in yeast are analogous to essential amino acids in higher organisms, our findings have broader implications for understanding DR in various species.
Given the biotechnological potential of yeast-derived oils for oleochemical production, genes encoding lipid metabolism enzymes are key targets for metabolic engineering. Genetic engineering tools such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, Transcription Activator-Like Effector Nucleases (TALENs), Zinc-Finger Nucleases (ZFNs), RNA interference (RNAi), and integrative plasmids can be used to modulate fatty acid biosynthesis and optimize lipid production. Among them, the CRISPR/Cas9 system, recognized for its simplicity and efficiency, has been deployed as a tool to create oleaginous yeast strains with high lipid productivity and features suitable for application in biorefineries. Species such as Cutaneotrichosporon oleaginosus, Rhodotorula toruloides, Candida spp., and Yarrowia lipolytica have already been engineered using CRISPR/Cas9 to enhance the production of fatty acids and their derivatives. However, designing and constructing an efficient CRISPR/Cas9 platform for oleaginous yeasts faces several hurdles, including low transformation efficiency, difficulties in expressing Cas9 and sgRNAs efficiently and consistently, the lack of well-characterized promoters, limited availability of PAM sequences, and poorly understood DNA repair mechanisms. Here, we address the application of the CRISPR/Cas9 system in oleaginous yeasts, laying out the challenges to developing efficient platforms and highlighting key trends in the field. We compare and discuss alternative CRISPR-Cas9 expression strategies to provide an overview of the current landscape and support the development of new approaches.
This study aimed to isolate, identify, and evaluate yeasts originating from urban honeys as potential starters for mead production. Honey samples from urban apiaries from Poland were analyzed. A total of 47 yeast isolates were obtained and identified as belonging to eight genera: Starmerella, Zygosaccharomyces, Saccharomyces, Rhodotorula, Dothiora, Cystobasidium, Schizosaccharomyces, and Filobasidium. Among them, Starmerella magnoliae was predominant (24 isolates). Zygosaccharomyces rouxii and Z. mellis also occurred frequently. Three Saccharomyces cerevisiae strains (CMIFS 189, CMIFS 191, CMIFS 208) were selected for further trials and applied in the fermentation of trójniak-type meads (honey-to-water ratio 1:2), together with a reference mead starter, Enovini® HONEY (Browin, Poland). Physicochemical analysis showed ethanol contents of 12.22%-15.49%, with CMIFS 191 producing the lowest alcohol but the highest extract. Glycerol levels (0.62%-0.85%) were lower than literature values, while volatile acidity ranged from 0.80 to 1.27 g/L and total acidity from 2.97 to 3.45 g/L. Polyphenol levels (270-299 µg/mL) were high, and antioxidant assays (ABTS, DPPH, RP) showed strain-dependent effects. Volatile analysis revealed alcohols as the dominant group, followed by esters and aldehydes, shaping fruity and floral aroma notes. Based on sensory evaluation, CMIFS 191 showed the highest overall acceptability, whereas the Enovini® HONEY reference starter obtained the lowest sensory scores under the applied conditions. Overall, honey-derived S. cerevisiae strains showed strong potential as novel starters for mead production.
Six yeast isolates were recovered from Ipomoea flowers collected in the Cerrado biome of Tocantins, Brazil. Sequence analyses of the ITS-5.8S region and the D1/D2 domains of the large subunit (LSU) rRNA gene indicated that these isolates represent a novel species of the genus Candidozyma, phylogenetically related to Candidozyma auris and Ca. ruelliae. A phylogenomic analysis based on 2116 single-copy orthologs from Candidozyma species with available whole-genome sequences showed that the new species, represented by strain UFMG-CM-Y6065, is a sister species to Ca. ruelliae. The name Candidozyma cisalpinoae sp. nov. (MycoBank no. 861366) is proposed to accommodate the new species. The holotype is CBS16108. Sporulation or other evidence of sexual reproduction was not observed, although the genome sequence showed the presence of a functional mating type locus (MATa) and functional pheromone peptides, indicating that the species is haplontic and heterothallic. The species exhibited resistance to multiple antifungals, growth at 42°C, biofilm formation, adhesion to buccal epithelial cells, and expression of efflux pumps, traits of clinical relevance that have been reported for other species in the genus Candidozyma.
To maintain the integrity of the genome, cells have evolved a complex signalling system, termed the DNA damage response (DDR), which detects DNA damage and promotes DNA repair. To date, over 600 proteins have been identified that play an integral role in the DDR. RAD9, encoding a DDR mediator protein, was the prototypical DNA damage checkpoint gene, establishing the genetic regulation of transient cell-cycle delays upon DNA damage. Rad9, identified 38 years ago in the budding yeast Saccharomyces cerevisiae as a damage-dependent cell-cycle regulator, is now known to regulate additional responses to DNA damage including both cell-cycle recovery and repair. The Rad9 protein is extensively phosphorylated both during a normal cell cycle and following DNA damage and several of these modifications have been linked to specific Rad9 roles within the DDR. Proteins structurally and functionally related to Rad9 exist in mammalian cells (e.g., 53BP1, BRCA1, MDC1) and insights into their regulation and mechanism of action have been informed by studies in yeast. This review will discuss the cellular mechanisms governing the DDR with an emphasis on the multifaceted role of Rad9 in sensing and responding to DNA damage, and how phosphorylation events regulate its function within the DDR. As the cellular events governing the DDR are well conserved, discoveries in yeast can be extrapolated to humans and may lead to the identification of additional novel protein targets, with several DDR inhibitors currently in clinical use or showing promise in clinical trials.
The fission yeast Schizosaccharomyces pombe is a prominent model organism widely used to investigate fundamental cellular mechanisms. In addition to S. pombe, the genus Schizosaccharomyces includes six other species-S. octosporus, S. japonicus, S. cryophilus, S. osmophilus, S. lindneri, and S. versatilis. These fission yeast species share a common ancestor from which the genus diversified over more than 200 million years. This extensive evolutionary divergence provides opportunities for comparative genomics. Here, we present the Schizosaccharomyces orthogroup (SOG) resource, a web platform developed from our high-quality genome assemblies, gene annotations, and orthology assignments. Most fission yeast genes are assigned to one of over 5,000 orthogroups. The platform enables users to visualize orthogroup sequence alignments and phylogenetic trees, retrieve coding and flanking sequences, and explore the conservation of local synteny. This resource will benefit researchers focusing on individual genes as well as those investigating gene evolution at broader scales. It is freely accessible at https://www.sogweb.org.
