The future of sustainable industrial biotechnology relies on microbial hosts that can thrive where conventional strains fail. Although Saccharomyces cerevisiae remains the workhorse of industrial production, its intrinsic metabolic constraints and susceptibility to multiple stresses underscore the need for alternative yeast chassis. Here, we present a comprehensive characterization of 79 yeast isolates from the Brazilian Yeast Collection (BRYC), sourced from diverse ecosystems across six Brazilian biomes. Strains were evaluated for growth under industrially relevant conditions, including alternative carbon sources, inhibitory compounds from biomass pretreatment, elevated temperatures, and high ethanol concentrations. Phenotyping was carried out in both solid and liquid media, allowing comparison of trait analyses across cultivation conditions, and revealing how growth environment influences phenotypic expression. Performance was further assessed in raw and hydrolyzed sugarcane and agave biomass. This high-throughput approach identified multiple non- Saccharomyces yeasts with superior traits compared to S. cerevisiae controls. Pichia kudriavzevii (BRYC98) exhibited exceptional multi-stress tolerance, including thermotolerance and resistance to aldehydes, ethanol, and acetic acid. Candida flosculorum (BRYC21), a poorly characterized native yeast species, exhibited remarkable versatility in substrate utilization. Debaryomyces hansenii strains showed the best growth in saponin-rich agave leaf juice - the most challenging substrate tested-, while Wickerhamomyces anomalus strains consistently outperformed controls in sugarcane molasses and hydrolysate. Overall, around 68% of the non-conventional strains exhibited higher robustness across the 15 tested conditions for both maximum biomass and specific growth rate compared to S. cerevisiae. These findings expand the portfolio of stress-resilient, metabolically versatile yeasts and provide a foundation for their development as alternative industrial chassis.

Candida albicans is a prevalent opportunistic fungal pathogen that typically resides as a commensal in multiple niches in the human body. C. albicans exhibits substantial phenotypic and genotypic diversity, driven by standard mutational events and genomic mechanisms such as loss of heterozygosity, aneuploidy, and a parasexual cycle. For the past 60 years, efforts have been made to characterize intrapopulation diversity to identify C. albicans relatedness groups. The methods used for strain delineation have transitioned from low-resolution phenotypic typing methods to more robust sequence-based approaches, including multilocus sequence typing (MLST) and, more recently, whole-genome sequencing (WGS). MLST provided the first widely adopted framework for phylogenetic classification, distinguishing genetically distinct clusters among C. albicans isolates. However, in recent years, WGS has offered improved resolution, revealing evidence of gene flow and recombination. These methodological advances have also enhanced our understanding of population structure and associated traits, including antifungal resistance and virulence. This review traces the development of methods used to characterize genetic phenotypic and diversity in C. albicans, outlines current common practices in describing its population structure, and highlights opportunities for greater consistency in how phylogenetic clusters are named and defined.

Objectives: This study aimed to evaluate the effects of 5-butyl-2-pyridinecarboxylic acid on growth inhibition, biofilm formation, biofilm destabilization, and cellular integrity of four different Candida species. Materials and Methods: The anti- Candida activity of 5-butyl-2-pyridinecarboxylic acid was assessed using disk diffusion and microdilution assays to determine minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Growth kinetics were monitored through growth curve and time-kill assays, while synergistic effects with standard antifungal drugs were evaluated using fractional inhibitory concentration (FIC) indices. Cellular damage was analyzed by scanning electron microscopy (SEM) and lactate dehydrogenase (LDH) release assays. Biofilm inhibition and destabilization were determined using microtiter plate assays with crystal violet staining. A molecular docking study predicted the potential target of action. Results: The compound exhibited potent inhibitory activity, with MIC and MFC values ranging from 0.129 ± 0.013 to 0.258 ± 0.012 mg/ml. At concentrations between 0.128 and 0.258 mg/ml, it induced rapid viability loss and complete lethality within 15 hours. SEM and LDH assays revealed significant cell wall disruption and increased permeability. Time-kill kinetics confirmed potent fungicidal activity. The compound also showed synergistic effects with clotrimazole and luliconazole against C. albicans and C. glabrata, while partial synergy was observed against C. parapsilosis. Moderate antibiofilm activity was also recorded compared to usnic acid. 5-butyl-2-pyridine carboxylic acid was predicted as a potential target of Candida ergosterol biosynthetic enzyme 14α-demethylase (CYP51). Conclusions: This study highlights the promising anti- Candida potential of 5-butyl-2-pyridine carboxylic acid. By disrupting cell walls, increasing membrane permeability, exhibiting synergism with conventional antifungals, and being predicted as an inhibitor against 14α-demethylase (CYP51), the compound represents a potential lead candidate. Further safety and mechanistic studies are needed to explore its application in antifungal drug development.

The mating-type locus of the miso and soy sauce yeast Zygosaccharomyces sp. contains a gene encoding the transcription factor Mat a2 (hereafter ZygoMat a2), which has a DNA-binding domain containing a high mobility group (HMG)-box not found in the baker’s yeast Saccharomyces cerevisiae. To investigate the role of ZygoMat a2 in mating-type a expression, we constructed mutant strains of ZygoMAT a2 using CRISPR-Cas9. The resulting mutants showed reduced expression of ZygoSTE6, which is specific to mating-type a, and did not mate, suggesting that ZygoMat a2 regulates the expression of mating-type a in Zygosaccharomyces sp. Expression of ZygoSTE6 was observed when the HMG-box of ZygoMat a2 was exchanged with that of Mat1-Mc, which regulates mating-type expression in Schizosaccharomyces pombe, indicating that ZygoMat a2 recognizes its target DNA sequence via the HMG-box. Substitution of the predicted upstream activation sequence (UAS) in ZygoSTE6 with the UAS of a mating-type a-specific gene from another yeast species led to mating-type a-specific gene expression in transformed Zygosaccharomyces sp. yeast, indicating that ZygoMat a2 can recognize the UAS of mating-type a-specific genes of other species. We speculated that the flexibility in the binding sequences of ZygoMat a2 is due to synergistic or cooperative binding with the DNA-binding protein ZygoMcm1. This hypothesis was supported by replacement of the UAS of ZygoSTE6 with the complete palindrome sequence, P(PAL), and by the results of protein and DNA structure prediction using AlphaFold. Collectively, our study indicates that ZygoMat a2 regulates mating-type a-specific gene expression via its HMG-box, which shows flexible recognition of the binding sequence of other HMG-box transcription factors.

The past decades have witnessed a rapid increase in the development of resistance to widely used antifungal drugs. Interestingly, interference with iron homeostasis of Candida albicans has been reported to lead to increases fluconazole susceptibility. In yeasts, excess iron is stored in the vacuole, from where it can be released into the cytoplasm during time of scarcity. Genes involved in the transport of iron from the vacuole include FET99 and FTH2. FET99 encodes a multicopper oxidase responsible for the uptake and release of iron stored in the vacuole, while FTH2 encodes a vacuolar iron permease. Importantly, the overall status of the membrane lipids serves as important determinant of drug susceptibility and the relationship between lipid metabolism and iron homeostasis may influence the pathogen’s susceptibility to certain drugs. To explore this interaction, fluconazole sensitivity of Δ/Δ fet99 and Δ/Δ fth2 planktonic cells and developing biofilms was evaluated. These deletions caused a decrease in susceptibility. The lipid droplet, ergosterol content, lipidome and mitochondrial membrane potential of both mutants were also evaluated. It was found that the lipid droplet content was decreased in the mutant biofilms compared to their parental strain. In addition, the mitochondrial membrane, cell membrane and neutral lipidomes of the mutants were significantly influenced, possibly contributing to the decrease susceptibility by limiting fluconazole uptake. This work provides additional information regarding the interplay between lipid metabolism, iron homeostasis and fluconazole resistance in C. albicans, which may be exploited as novel drug targets.

We have investigated the interplay between glycolytic oscillations and intracellular K + concentration in the yeast S. cerevisiae. Intracellular K + concentration was measured using the fluorophore PBFI. We found that K + is an essential ion for the occurrence of glycolytic oscillations and that intracellular K + concentration oscillates synchronously with other variables such as NADH, intracellular ATP and mitochondrial membrane potential. We also investigated if glycolysis and intracellular K + concentration oscillate in a number of yeast strains with mutations in K + transporters in the plasma membrane, mitochondrial membrane and in the vacuolar membrane. Most of these strains are still capable of showing glycolytic oscillations, but two strains are not: (i) a strain with a deletion in the mitochondrial Mdm38p K +/H + transporter and (ii) a strain with deletion of the late endosomal Nhx1p K +/H + (Na +/H +) transporter. In these two mutant strains intracellular K + concentration seems to be low, indicating that the two transporters may be involved in transport of K + into the cytosol. In the strain Mdm38p Δ oscillations in glycolysis could be restored by addition of the K +/H + exchange ionophore nigericin. Furthermore, in two non-oscillating mutant strain with a defective V-ATPase and deletion of the Arp1p protein the intracellular K + is relatively high, suggesting that the V-ATPase is essential for transport of K + out of the cytosol and that the cytoskeleton may be involved in binding K + to reduce the concentration of free ion in the cytosol. Analyses of the time series of oscillations of NADH, ATP, mitochondrial membrane potential and potassium concentration using data-driven modeling corroborate the conjecture that K + ion is essential for the emergence of oscillations and support the experimental findings using mutant strains.

In this study, we introduce a novel approach for the high-throughput quantitative analysis of mitotic defects in the fission yeast Schizosaccharomyces pombe using Imaging Flow Cytometry (IFC). Fission yeast is a valuable model organism for cell cycle research, and the ‘cut’ phenotype, indicative of mitotic catastrophe, has been instrumental in discovering cell cycle regulators. Traditional fluorescence microscopy methods for quantifying ‘cut’ events suffer from subjectivity and limited throughput. Our IFC pipeline overcomes these limitations by automating the detection of ‘cut’ cells based on the unique characteristic of daughter cell nuclei becoming trapped in the cell wall during aberrant mitosis. We demonstrate the pipeline’s effectiveness using wild-type and mutant strains, with results validated against manual scoring. Our study establishes IFC as a powerful tool for investigating mitotic fidelity in fission yeast, with implications for advancing cell biology research.

Transcription presents challenges to genome stability both directly, by altering genome topology and exposing single-stranded DNA to chemical insults and nucleases, and indirectly by introducing potential obstacles to the DNA replication machinery. Such obstacles include the RNA polymerase holoenzyme itself, DNA bound regulatory factors, G-quadruplexes and RNA::DNA hybrid structures known as R-loops. Here we review the detrimental impacts of transcription on genome stability in budding yeast, as well as the mitigating effects of transcription-coupled DNA repair and of systems that maintain DNA replication fork processivity and integrity. We conclude that the impacts of transcription on DNA replication by the replisome must be very mild with little direct mutagenic outcome under normal conditions. However, transcription can significantly impair the fidelity of replication fork rescue mechanisms, particularly Break Induced Replication (BIR), which is used to restart collapsed replication forks when other means fail. This leads to de novo mutations, structural variation and extrachromosomal circular DNA formation that contribute to genetic heterogeneity. By re-analysing published datasets, we show that different classes of genes have different interactions with the replisome, and that highly transcribed environment dependant genes in S. cerevisiae tend to be located close to replication origins. We have previously implicated interactions between replication origins and BIR forks in adaptive transcription-induced copy number variation events, which indicates that environment dependent genes are preferentially located in genomic environments more prone to instability, particularly under replication stress.

Atacama is the most hyper-arid Desert in the world. In this study, we describe a novel species, Nakazawaea atacamensis f. a., sp. nov., isolated from plant samples in the Atacama Desert of Chile. In total, three isolates of N. atacamensis were obtained from independent Neltuma chilensis bark samples (synonym Prosopis chilensis, Algarrobo). The novel species was delineated based on morphological, physiological, biochemical, and molecular characteristics. A neighbour-joining analysis using the sequences of the D1/D2 domains of LSU rRNA revealed that N. atacamensis sp. nov. clustered with Nakazawaea pomicola. The sequence of N. atacamensis differed from closely related species by 1.3% to 5.2% in the D1/D2 domains. A phylogenomic analysis based on single nucleotide polymorphism’s data confirms that the novel species belongs to the genus Nakazawaea, and placed N. atacamensis closer to N. peltata. Phenotypic comparisons demonstrated that N. atacamensis sp. nov. exhibited distinct carbon assimilation patterns compared to its related species. Genome sequencing of the ATA-11A-B T strain revealed a genome size of approximately 12.4 Mbp, similar to other Nakazawaea species, with 5,116 protein-coding genes annotated using InterProScan. In addition, N. atacamensis exhibited the capacity to ferment synthetic wine must, representing a potential new yeast for mono or co-culture wine fermentations. This comprehensive study expands our understanding of the genus Nakazawaea and highlights the ecological and industrial potential of these yeasts in fermentation processes. The holotype of N. atacamensis sp. nov. is CBS 18375 T. The Mycobank number is MB 849680.

The yeast Saccharomyces cerevisiae and most eukaryotes carry two 5’→3’ exoribonuclease paralogues that are very similar. In yeast, they are called Xrn1, which shuttles between the nucleus and cytoplasm and executes major cytoplasmic mRNA decay, and Rat1, which carries a strong nuclear localization sequence (NLS) and localizes in the nucleus. Xrn1 is 40% homologous to Rat1 but has an extra ~500 amino acids C-terminal extension. In the cytoplasm, Xrn1 can degrade decapped mRNAs during the last round of translation by ribosomes “co-translational mRNA decay”. The division of labor between the two enzymes is still enigmatic and can serve as a paradigm for division of labor of many other paralogues. Here we show that Rat1 is capable of functioning in cytoplasmic mRNA decay, provided that Rat1 remains cytoplasmic due to its NLS disruption (cRat1). This indicates that the actual segregation of the two paralogues plays roles in their specific functions. However, segregation is not sufficient for fully complementing Xrn1 function. Specifically, cRat1 can only partially recover cell volume, mRNA stability, proliferation rate, 5’→3’ decay alterations that characterize xrn1Δ cells. In particular, co-translational decay is only little complemented by cRat1. Adding the Xrn1 C-terminal domain to Rat1 does not improve the phenotypes indicating that lack of C-terminal is not the reason for the partial complementation. Collectively, it seems that during evolution the two paralogues acquire unique features that make the division of work beneficial.

While flocculation has demonstrated its efficacy in enhancing yeast robustness and ethanol production, its potential application for lactic acid fermentation remains largely unexplored. Our study examined the differences between flocculating and nonflocculating Saccharomyces cerevisiae strains in terms of their metabolic dynamics when incorporating an exogenous lactic acid pathway, across varying cell densities and in the presence of lignocellulose-derived byproducts. Comparative gene expression profiles revealed that cultivating a nonflocculant strain at higher cell density yielded a substantial upregulation of genes associated with glycolysis, energy metabolism, and other key pathways, resulting in elevated levels of fermentation products. Meanwhile, the flocculating strain displayed an inherent ability to sustain high glycolytic activity regardless of the cell density. Moreover, our investigation revealed a significant reduction in glycolytic activity under chemical stress, potentially attributable to diminished ATP supply during the energy investment phase. Conversely, the formation of flocs in the flocculating strain conferred protection against toxic chemicals present in the medium, fostering more stable lactic acid production levels. Additionally, the distinct flocculation traits observed between the two examined strains may be attributed to variations in the nucleotide sequences of the flocculin genes and their regulators. This study uncovers the potential of flocculation for enhanced lactic acid production in yeast, offering insights into metabolic mechanisms and potential gene targets for strain improvement.

Ntšhe, a Setswana name for sweet reeds, a sweet sorghum variety ( Sorghum bicolor (L)) is a commercial crop consumed as a delicacy in enormous quantities in Botswana and in southern Africa at large. Pre and post-harvest losses due to infestation by larval stages of stem borer moths, Chilo partellus often lead to severe financial losses as consumers condemn the worm-infested sweet reeds as unpalatable, “ Mo gase ntšhe tota tota” (this is not a sweet reed). Valorisation of condemned sweet reeds is one attractive route to reduce economic losses. Here, we took advantage of our understanding of yeast-insect interactions to isolate yeasts associated with larval stages of the stem borer moths and investigated their potential for production of an alcoholic sweet reed beverage. We isolated 33 yeast strains representing from the galleries and frass as well as from the guts of the larval moths. Assessment of their ability to ferment the simplest sugar, glucose, resulted in identification of 14 strains belonging to Hanseniaspora and Candida genera. These strains were further assessed for their capacity to ferment by calculating the rate of accumulation of carbon dioxide and ethanol when grown in the principal sugars found in sweet sorghum juice as sole carbon sources as well in sweet sorghum juice. In addition, as an industrially relevant trait, we tested the potential of the strains to tolerate brewing/fermentation-associated stresses. Furthermore, we assessed the aromatic complexity of the produced beverage. Our results suggest that non-conventional yeasts associated with the larval moths have potential for valorization of condemned sweet sorghum stalks to produce a sweet sorghum beverage.