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Saccharomyces cerevisiae, also known as brewer's yeast, is widely used in food and beverage fermentations. In this study, newly isolated S. cerevisiae strains, NIYL33997 and NIYL33999, were evaluated for fermentation performance in bread and rice hydrolysate medium compared to commercial yeasts. In bread fermentation, NIYL33999 produced higher levels of phenylethyl alcohol and ethyl acetate, corresponding to 2.1- and 3.0-fold increases over Saf-instant yeast. NIYL33997 showed the highest ethyl octanoate production. During fermentation in rice hydrolysate medium, NIYL33999 produced the highest concentrations of aroma compounds. Specifically, isoamyl alcohol, phenylethyl alcohol, and ethyl acetate levels increased by approximately 1.2-, 1.9-, and 1.3-fold, respectively, compared with those produced by the Songcheon yeast. NIYL33997 exhibited similar increases with lower ester production. Principal component analysis revealed clear differentiation of aroma patterns among the strains. In conclusion, S. cerevisiae NIYL33997 and NIYL33999 exhibit distinct aroma profiles, with NIYL33999 demonstrating potential as a starter for fermented foods.

Human N-glycoproteins represent a market worth hundreds of billions of dollars, yet their production in yeast is often limited by misfolding and degradation. However, few strategies have addressed this limitation by targeting differences between human and yeast N-glycan-dependent protein quality control (QC), including the absence of the UGGT-mediated reglucosylation-refolding cycle and the simpler glycoprotein degradation pathway in yeast. Here, we engineered the glycoprotein QC system of Kluyveromyces marxianus by introducing key human components and modifying native pathways. Human UGGT1 or UGGT2 enhanced soluble and secretory glycoprotein production in an activity-dependent manner, with further improvements achieved by co-expressing the human cochaperone SEP15 and reducing native glucosidase II trimming. Human EDEM2 delayed endoplasmic reticulum-associated degradation and increased secretion. Combining these strategies enhanced the production of diverse N-glycoproteins, including Fc, γ-glutamyl hydrolase, fungal xylanase, and Fc-fusion therapeutics, by up to ∼12-fold, demonstrating an effective strategy for engineering human-like glycoprotein QC in yeast to improve glycoprotein production.

Although lifespan has long been the focus of ageing research, preventing functional decline late in life is a more pressing societal need. Here, we investigate the basis of senescence and declining fitness during replicative ageing in budding yeast, and describe a metabolic perturbation that preserves late-life fitness even on an unrestricted glucose diet. We show that senescence can be prevented by constitutive activation of AMPK, though only for approximately half the ageing population, and use genetic and functional assays to link this heterogeneous response with differences in cytosolic acetyl coenzyme A (Acetyl-CoA) metabolism. In one class of ageing cell, AMPK activity maintains fitness late in life through pathways that transport cytosolic Acetyl-CoA into mitochondria, but AMPK also inhibits fatty acid synthesis which leads to lipid starvation in the other class of ageing cell. Therefore, AMPK activity has both positive and negative effects, but we show that constitutive AMPK activity uncoupled from fatty acid synthesis inhibition (the A2A mutant) suppresses senescence and maintains fitness in both classes of ageing cell. Our findings support a model in which lipid starvation and excess Acetyl-CoA availability are major drivers of senescence in replicatively aged wild-type yeast. This work shows that ageing is not intrinsically associated with declining fitness, at least in yeast, and that re-engineering highly conserved metabolic pathways allows fitness to be preserved very late in life.

This study evaluated the dual-functional potential of Trichosporonales yeasts for integrating textile dye remediation with lipid production within a wastewater biorefinery framework. Five strains from the genera Apiotrichum and Cutaneotrichosporon were screened for their ability to decolorize Reactive Black 5 while accumulating intracellular lipids. Among them, Apiotrichum domesticum JCM9580 showed the best overall performance due to its high lipid productivity, efficient dye removal, and stable yeast-like morphology, representing an operational advantage over filamentous growth forms. Results revealed an inverse relationship between lipid biosynthesis and dye decolorization, regulated by a classical C/N metabolic switch. High C/N ratios activated the lipogenic program, converting the cell into a strong sink for reducing equivalents and limiting decolorization, whereas low C/N conditions suppressed lipogenesis, preserving reducing power for rapid and nearly complete dye removal. UV-is, FTIR, and GC-S analyses indicated that reductive azo bond cleavage is an early step during decolorization, although the complete biodegradation pathway remains unresolved. In addition, Reactive Black 5 exposure increased fatty acid polyunsaturation, improving biodiesel cold-flow properties. Notably, A. domesticum achieved a cold filter plugging point of -3.86 °C. Phytotoxicity assays showed that untreated RB5 completely inhibited germination (0% GI), whereas yeast-treated samples reached GI values of 0.28-0.30 (20% dilution), representing a substantial detoxification; further dilution to 10% eliminated all inhibitory effects. Overall, these findings establish a basis for future bioprocess optimization within wastewater biorefinery systems.

Pododermatitis (bumblefoot) is a chronic, debilitating disease of the plantar surface of the foot that affects birds of prey kept in captivity worldwide. Although bacterial pathogens, especially Staphylococcus aureus, are most commonly considered as causative agents, the contribution of opportunistic yeasts to chronic, non-healing footpad lesions remains poorly characterized. Keratinophilic yeasts may sustain the disease process by degrading keratin in superficial tissues, impairing wound healing and, owing to their thermotolerance and minimal nutritional requirements, persisting in the environment of the bird's enclosure. In this study, three captive steppe eagles (Aquila nipalensis) from a single aviary in Kazakhstan, all presenting with chronic pododermatitis unresponsive to antibacterial treatment, were investigated by integrated mycological, biochemical and molecular approaches. The yeast isolates were recovered from the deep footpad lesions and identified to species level by sequencing of the ITS1-5.8S-ITS2 rDNA region. All these isolates were assigned to Candida metapsilosis, and phylogenetic analysis confirmed their close clustering with reference C. metapsilosis sequences. Phenotypic characterization showed that all isolates were thermotolerant (growth at 8-37 °C), expressed strong urease and keratinolytic activity (the latter confirmed in vitro by the hair perforation test), high saccharolytic activity and selective, weak proteolytic activity. Disk diffusion screening showed susceptibility to azoles (ketoconazole, clotrimazole, fluconazole) and reduced susceptibility to polyenes (nystatin, amphotericin B). To our knowledge, this is the first report of C. metapsilosis isolated from chronic pododermatitis lesions in captive steppe eagles. Combined with the documented in vitro virulence-associated traits and the resolution of the lesions following targeted antifungal therapy, our findings support a contributory etiological role of C. metapsilosis as an opportunistic pathogen in raptor pododermatitis in immunocompromised birds maintained under suboptimal husbandry. Mycological work-up, including molecular identification, is therefore warranted in cases of chronic, non-resolving pododermatitis in captive birds of prey.

Ribosomes stall when they encounter problematic codons or cellular stress that perturbs translation. Stalled ribosomes can lead to the formation of ribosome collisions, also known as disomes, that engage cellular surveillance and stress signaling pathways. How many disomes form during basal conditions and how disome levels change under stress remain poorly understood. Here, we used spike-in normalized Ribo-seq and Disome-seq to quantify transcriptome-wide disome levels. Applying this approach in yeast and human cells, we found that disomes comprise approximately 2-10% of translating ribosomes under basal conditions. A high-resolution Disome-seq experiment in human cells identified reproducible disome-forming sites that contribute to the basal level of disome formation in the cell. Exposure of yeast cells to methyl methanesulfonate and human cells to anisomycin increased disome abundance up to four-fold and changed the distribution of collisions in a stress-specific and context-dependent manner. Overall, these data provide a quantitative, transcriptome-wide framework for measuring disome levels and reveal how translational stress reshapes the landscape of ribosome collisions in cells.

Human lactoferrin (hLF) is a multifunctional glycoprotein of the transferrin family derived from milk and mucosal secretions, which exhibits antibacterial, anti-tumor, and immunomodulatory functions, and is an important component of infant formula. Conventional methods for lactoferrin expression are often inefficient, primarily due to inadequate protein synthesis capabilities and poor stability within microbial hosts. Herein, a Komagataella phaffii yeast strain capable of high-level secretory expression of hLF was constructed by reprogramming the endoplasmic reticulum (ER) and vacuole using CRISPR/Cas9 technology. A dual-expression cassette containing the AOX1 promoter, an α-secretion signal peptide, the hLF gene, and a terminator was integrated into three different sites of the K. phaffii genome. The stepwise strategy combining expansion of the ER membrane involved in protein synthesis with knockout of vacuolar proteases further enhanced hLF production. Subsequently, 0.1g/L FeCl₃ was added to the medium to reduce the toxicity of hLF and improve its stability. After high-density cultivation of K. phaffii through optimization of cultivation conditions in shake flasks and a 5L bioreactor, the secretory intact hLF titer reached 2214mg/L, representing a 76.3-fold increase achieved through these engineering strategies. In addition, antibacterial experiments demonstrated that this secretory hLF had a significant inhibitory effect on Escherichia coli, Staphylococcus aureus, and yeast. Overall, the developed K. phaffii protein expression platform enabled efficient production of lactoferrin, demonstrating its potential for expressing other lactoproteins.

The 26S proteasome is the hub for regulated protein turnover in eukaryotic cells. Degradation of proteins by the Ubiquitin-Proteasome System plays critical roles in every aspect of cell biology, such as the regulation of gene transcription, the quality control of translation and protein folding, and protein transport across membranes. While mRNA levels and protein abundances can be readily measured with a robust set of established tools, only a few methodologies exist to identify proteins that are degraded by the proteasome rather than the lysosome as the second major pathway for turnover. Here, we sought to address this by using genetic code expansion to introduce a photo-crosslinkable unnatural amino acid into the yeast 26S proteasome and capture cellular protein substrates as they translocate through the proteasomal ATPase motor. In vitro biochemical experiments confirmed that these modified proteasomes are functional, which allowed us to introduce them into live yeast cells for in vivo crosslinking and the identification of enriched ATP-dependent substrates by mass spectrometry. These experiments revealed a very diverse pool of proteasomal substrates that markedly changed upon cell exposure to endoplasmic reticulum stress. Together, our results represent an exciting avenue for probing the landscape of proteasomal substrates and its changes in response to various cellular conditions and stresses.

Hypoxia adaptation can partially improve and restore the composition, structure, and function of blood vessels, thereby enhancing the prognosis of related diseases such as stroke. However, the underlying molecular mechanisms of hypoxia adaptation remain elusive. In this study, multi-omics analysis was performed across yeast, zebrafish, and mouse, revealing that sphingolipid metabolism is crucial for hypoxic adaptation. We found that the expression of the key protein ceramide synthase 2 (CerS2) in mammals and its yeast ortholog very-long-chain ceramide synthase LAC1 was up-regulated. CerS2 and LAC1 are key enzymes that promote hypoxic angiogenesis by increasing the synthesis of C24:1, and this function is highly conserved across species and throughout evolution. CerS2 and LAC1 biosynthesize the long-chain unsaturated ceramide C24:1, which, via its double-bond structure, specifically binds to ROCK2 to activate HIF-1α, thereby up-regulating the expression of vascular endothelial growth factor (VEGF) and increasing AKT phosphorylation. Consequently, this process significantly enhances angiogenesis and improves blood flow perfusion in a mouse model of ischemic stroke. This study deepens our understanding of the function of unsaturated ceramides and reveals the unique mechanism of C24:1 in angiogenesis and protection under hypoxic conditions.

Strains of food-derived microbes can become facultative pathogens in susceptible human hosts. Surprisingly, we previously isolated Debaryomyces hansenii, a yeast common in fermented foods, from Crohn disease (CD) ulcers, raising questions about its strain-specific traits that influence host interactions. Here, we further developed the genetic tractability of D. hansenii and identified a single adhesin, Hil1, as a major determinant of colony morphology, biofilm formation, and immune targeting in CD patients. We used Agrobacterium tumefaciens-mediated transformation to perform a forward genetic screen in a food-derived reference strain. We isolated mutants that converted from a wrinkled, biofilm-forming phenotype to a smooth, non-adherent phenotype characteristic of CD patient isolates. Mapping of multiple insertion sites showed a disrupted subtelomeric Hyr/Iff-like adhesin gene, herein referred to as HIL1. CRISPR-Cas9-mediated deletion of HIL1 recapitulated the mutant phenotype, demonstrating that HIL1 was necessary for biofilm formation and high cell-surface hydrophobicity phenotypes. To contextualize these findings, we performed comparative genomics on a D. hansenii strain collection to assess allelic variation in the number of HIL1 tandem repeats. Longer alleles in food strains correlated with increased biofilm formation, while CD-isolated strains contained shorter HIL1 alleles and reduced binding to surfaces. Serology profiling showed that HIL1 was a direct antigenic target of circulating immunoglobulin G (IgG) in CD patients. Together, these results suggest Hil1 is a key, strain-variable adhesin shaping fungal surface properties and host immune recognition. This work establishes D. hansenii as a genetically tractable system and shows how adhesin polymorphisms may influence fungal behavior in food and disease contexts. Debaryomyces hansenii is a yeast that is common in food and is generally recognized as safe for human consumption, though recently it has been identified within diseased regions of the intestine in Crohn disease patients. A current need is to determine the genetic and phenotypic differences between safe food isolates and isolates from human Crohn disease patient ulcers. Here, we used a loss-of-function genetic screen and identified HIL1, an adhesin that we found mediates cellular adhesion in many food strains but not in patient strains. We identified circulating HIL1-reactive antibodies in patients with Crohn disease, indicating that food strains can be a target of host immune responses through Hil1.

Mercury (Hg) is a highly toxic heavy metal that poses a significant threat to crop productivity. Although bHLH transcription factors are known to regulate plant stress responses, their functions in maize (Zea mays) under Hg stress remain largely unexplored. Here, we identify ZmbHLH164, a maize bHLH gene transcriptionally induced by Hg exposure, as a key regulator of Hg tolerance. Overexpression of ZmbHLH164 significantly reduced Hg accumulation in roots and root‑to‑shoot translocation, thereby enhancing Hg tolerance in maize seedlings. Conversely, knockdown of ZmbHLH164 led to increased Hg accumulation and translocation, with a concomitant loss of tolerance. Mechanistically, using DAP‑seq in combination with yeast one‑hybrid and dual‑luciferase reporter assays, we show that ZmbHLH164 directly binds to the promoter of the catalase gene ZmCAT1 and activates its transcription. This activation enhances CAT activity, promotes H₂O₂ scavenging, and sustains reactive oxygen species (ROS) homeostasis. In parallel, DAP‑seq profiling and yeast one‑hybrid assays revealed that ZmbHLH164 also directly associates with the promoters of multiple metal transporter genes, and qRT‑PCR analysis confirmed that their expression is suppressed in ZmbHLH164‑overexpressing lines, correlating with reduced cellular Hg uptake and root‑to‑shoot translocation. Together, these findings establish that ZmbHLH164 confers Hg tolerance through a dual mechanism-activating ROS detoxification while restricting Hg transport. This regulatory module not only provides mechanistic insights into plant Hg tolerance but also highlights ZmbHLH164 as a promising target for breeding low‑Hg‑accumulating crops to enhance food and environmental safety.