Regulation of NAD+ metabolism is interconnected with multiple nutrient-sensing pathways and cellular processes. The phosphate (Pi)-sensing (PHO) signaling pathway contributes to NAD+ degradation, and PHO-responsive genes are reciprocally regulated in a NAD+-dependent manner. In this study, we examine whether the NAD+ biosynthetic enzyme Nma1 has a direct role in modulating PHO signaling. We show that Nma1 physically interacts with Pho4, a transcription factor translocating to the nucleus to activate PHO-responsive genes during Pi depletion. Overexpression of NMA1 or its catalytically inactive variant significantly reduces Pi depletion-induced Pho4 nuclear localization and the activation of PHO-responsive genes, indicating the NAD+ synthesis activity of Nma1 is dispensable in the downregulation of PHO signaling. Interestingly, mutating the C-terminal domain of Nma1, which is required for the ATPase chaperone activity of mammalian NMNATs, fails to decrease Pho4 nuclear localization and the expression of PHO-responsive genes. Moreover, loss of Nma1 increases Pho4 nuclear localization under moderate Pi-depleted conditions, suggesting that cells lacking Nma1 are more sensitive to Pi availability alterations. These results support that Nma1 can moderate PHO activation by maintaining Pho4 in the cytoplasm. Our findings uncover a novel regulatory mechanism for PHO signaling, and this regulation may help coordinate NAD+ metabolism with Pi homeostasis.
Over the past three decades, Saccharomyces cerevisiae has gone from being the first eukaryote to have its genome fully sequenced to one of the most studied biological systems. Advances in sequencing technologies, functional genomics, and population genomics have expanded the scope of study from a single laboratory reference genome to thousands of natural isolates, and the species-wide pangenome. These advances have revealed vast genetic and structural diversity, shaped by evolution, ecology, and domestication, while large-scale experimental resources have made yeast a model organism of choice in systems biology. In this review, we trace this transition from a reference genome view to an understanding of diversity at the population level, highlighting how telomere-to-telomere assemblies, graph-pangenome, and multi-omics approaches are transforming our ability to link genomic variation to phenotype. Together, these advances place S. cerevisiae at the forefront of efforts to understand and predict genotype-phenotype relationships in eukaryotes.
In industrial yeast fermentations, population-level viability assays routinely report healthy cultures even as aged, metabolically compromised mother-cell subpopulations go undetected. Replicative age contributes to this hidden heterogeneity, but its link to a cell's metabolic competence has been difficult to measure simultaneously in the same cell by high-throughput flow cytometry. Here, we introduce a dual-parameter flow cytometry workflow combining bud-scar labeling with a recombinant His6-SUMO-mCherry-chitin-binding domain fusion protein (∼610 nm emission) and 5(6)-carboxyfluorescein diacetate (CFDA)-based viability detection (∼525 nm), providing spectral orthogonality without computational autofluorescence correction. We applied it to four industrially relevant yeasts: Saccharomyces pastorianus W-34/70, S. cerevisiae var. diastaticus BE-134, S. cerevisiae var. chevalieri LA-01, and Komagataella phaffii X33. In S. pastorianus, viability declined monotonically from 88% in daughter cells to 5% in the oldest resolved mother-cell class; longitudinal monitoring over 96 h captured a progressive widening of the age-dependent viability gradient masked at the population level. Among S. cerevisiae variants, age-dependent gradients were weaker and strain-specific, while in K. phaffii reliable discrimination required exponential growth. Replicative age is thus a strain-dependent predictor of metabolic competence rather than a universally conserved one, and the workflow offers brewers and bioprocess developers a practical tool for age-resolved fermentation monitoring and pitching-yeast assessment.
The first European isolates of Saccharomyces eubayanus were discovered in Ireland in 2022 and belong to the same clade as the S. eubayanus parent of the hybrid lager yeast Saccharomyces pastorianus. Here, we report the isolation of 10 additional Irish strains and we explore maltose metabolism. Maltose metabolism genes are clustered in subtelomeric MAL loci including maltase (MALS), maltose transporter (MALT), and regulator (MALR). Despite having intact MAL loci, the Irish strains do not grow on maltose. Two Irish S. eubayanus strains (UCD650 and UCD926) were passaged in medium containing maltose as the sole carbon source until they acquired the ability to metabolize maltose. The UCD650-derived lineage acquired an I243N substitution in MalR, and a 30-kb duplication of the MAL locus. The UCD926 parent has a similar MAL locus duplication and its evolved lineage acquired a W307L substitution in MalR. Expression of MALT and MALS was increased in the evolved isolates. Introducing the W307L mutation into UCD926 by CRISPR-Cas9 editing fully recapitulates the maltose utilization phenotype. The I243N variant alone has little effect on expression of maltose genes. We find that maltose utilization is influenced both by the copy number of MAL genes and by gain-of-function mutations in MalR.
Efficient utilization of lignocellulosic hydrolysates in yeast-based biorefineries requires simultaneous consumption of glucose and xylose, which is often limited by preferential glucose uptake. In Kluyveromyces marxianus, we kinetically characterized two native xylose transporters, KMAR_10 531 and KMAR_60 179, identifying medium- and low-affinity xylose transporters, respectively. KMAR_10 531 also mediated high-affinity glucose uptake (Km 0.28 ± 0.1 mM), limiting xylose utilization in mixed-sugar media. Guided by structural modelling, we engineered the KMAR_10 531 N325V variant, which reduced glucose affinity ∼20-fold while improving xylose affinity more than three-fold (Km reduced from 46.9 ± 9.5 to 14.9 ± 3.6 mM). Expression of KMAR_10 531 N325V in a pentose-transporter-deficient K. marxianus strain enabled simultaneous glucose-xylose co-consumption in flasks and bioreactors, overcoming the diauxic growth observed with the native transporter. In bioreactors, the engineered strain consumed ∼90% of available xylose within 45 h and produced increased biomass compared to native transporter. This study provides the first example of engineering an Hgt-like transporter for altered sugar specificity.
The publication of the Saccharomyces cerevisiae genome sequence thirty years ago marked a defining shift in modern biology, establishing yeast as the first fully sequenced eukaryotic model cell. Access to a complete reference genome has catalyzed a renaissance in experimental biology, providing the foundation for advances in functional genomics, systems biology, synthetic biology, and biotechnology. This article celebrates the achievements of the yeast sequencing project and highlights how genomics has evolved from a descriptive resource into a central enabling infrastructure for engineering biology, leading to a post-genomic era defined by genome-scale design. We chart the progression from decoding the genome to the omics revolution, highlighting both recent discoveries and persistent enigmas surrounding the 'dark matter' of the yeast genome. We then describe how access to a well-annotated reference genome has culminated in a new era of synthetic genomics exemplified by the Sc2.0 project. Finally, we explore how emerging trends in artificial intelligence and bioelectronics may shape the next thirty years of yeast genomics and engineering biology. This integration of past achievements and future trajectories reinforces the enduring role of S. cerevisiae as a primary eukaryotic model for biological discovery and biotechnological innovation.
In this work, we investigated the genomic and phenotypic basis of stress tolerance in the native Saccharomyces cerevisiae strain M6, isolated from spontaneously fermenting must at a vineyard located in Atacama Desert, through a de novo hybrid genome assembly generated using short- and long-read sequencing technologies. The assembly comprised 11.89 Mb with high completeness (99.5%), and flow cytometry analysis confirmed a diploid genome organization. Phylogenomic analyses placed M6 within the Wine/European lineage, although displaying genomic divergence relative to other wine-associated strains. Using S288C as the reference genome, comparative variant analysis was performed for M6 and four closely related strains, including Wine/European and Alpechin strains. Coding variants were identified in genes associated with osmotic sensing and signaling (SSK1, SSK2), trehalose metabolism (TPS2, NTH1). Growth assays demonstrate that M6 exhibits enhanced performance under elevated temperatures (35°C-38°C) and high salinity (up to 1.5 M NaCl) compared with commercial and laboratory strains. In addition, Biolog YT assays revealed broad carbohydrate utilization capacity, including the metabolism of maltose, galactose, and raffinose-family oligosaccharides. Together, these results provide integrated genomic and phenotypic evidence of stress resistance and metabolic flexibility in strain M6, highlighting its potential as a biotechnological resource for fermentation processes.
Non-conventional yeasts are recognized as valuable hosts for producing biofuels, pharmaceuticals, and other high-value chemicals, owing to their diverse physiological traits, ability to utilize various substrates, and greater tolerance to environmental stresses compared to conventional model yeast Saccharomyces cerevisiae. To fully optimizing metabolic flux toward desired products, effective genetic engineering tools enabling precise modulation of gene expression and coordinated control of metabolic pathways are essential. In this context, we discussed classical transcriptional regulation tools like promoters, and transcription factors, alongside innovations in synthetic biology that allow metabolic engineering in non-conventional yeasts to produce higher biofuels and other useful products, promoting the development of sustainable resources, and assisting the development of innovative bio-products. It also discussed innovative programmable technologies, such as CRISPR/Cas-mediated transcriptional activation and repression, as well as dynamic regulatory systems that can fine-tune metabolic routes and balance cellular resources. Strategies for promoter engineering, transcription factor manipulation for transcriptional regulation, and metabolic rewiring were highlight as methods to boost pathway efficiency and yields. This review concluded with current challenges and future directions, focusing on integrating synthetic biology and systems biology to create robust, controllable transcriptional frameworks for next-generation yeast cell factories.
Abstract This retrospective traces my scientific journey from my childhood in the working-class St. Johann district of Basel, to my unexpected contributions to the field of cellular signaling and nutrient sensing. Framed by early struggles with identity as the son of an Italian immigrant and a turbulent search for academic belonging, my path to scientific independence was anything but linear. I candidly recount my detours—repairing shoes, filling car batteries with acid, and hauling ladders as a field biologist—before finally finding my true calling in the quiet precision of the laboratory. The narrative highlights the often-serendipitous milestones in my career, beginning with early graduate work that challenged prevailing assumptions to establish the simple sugar trehalose as a critical cellular survival factor. It follows my transformative, though initially humbling, postdoctoral years in the United States, a period of acute professional uncertainty upon returning to Switzerland, and my gradual rise to independence. Through every triumph and setback, this story is anchored by my love and partnership with my wife, Michèle—my grounding counterpoint and the steadfast constant in a life of shifting scientific models. Scientifically, the narrative culminates in our laboratory’s collaborative successes: identifying Rim15 as the integrator of PKA and TORC1 signalling that controls the entry into cellular quiescence and uncovering the EGO/Rag GTPase complex—the vacuolar command center that activates TORC1 to drive the exit from this dormant state—as a universal blueprint for eukaryotic nutrient sensing. By reflecting on how our fundamental genetic discoveries in yeast translated into clinical interventions for human diseases, this essay illustrates the unpredictable yet immense value of basic science. More than a chronicle of academic achievement, this is a human story exploring the intense pressures of the scientific enterprise, the importance of mentorship and personal connection, and the enduring power of curiosity-driven research.
Abstract This is the story of Milan Höfer’s life and scientific legacy. Born in 1936 in the former Sudetenland and shaped by political upheaval, Höfer overcame early barriers to pursue chemistry at Charles University, Prague (Czechia), where mentorship under Arnošt Kleinzeller redirected him toward biochemistry. His research spanned calcium transport, mitochondrial energetics, proton-coupled sugar uptake, ion channels, and membrane potential measurements across diverse yeast species. After fleeing Czechoslovakia in 1968, Höfer pursued a distinguished career at the University of Bonn, training international scientists, authoring influential texts, and co-founding SMYTE, The Small Meeting on Yeast Transport and Energetics now in its 40th meeting. His work extended into biotechnology, including fungal lignite solubilization and enzyme-based bioprocessing. Through extensive international teaching and collaboration, Höfer contributed significantly to the global scientific community, leaving a lasting impact on membrane transport research and the generations of scientists he mentored.
Looking back over nearly five decades in yeast research, I often marvel, over a glass of wine, at how a single-celled organism like yeast has taken me on such a remarkable journey. We’ve travelled across four continents, using basic science and applied biotechnology, and into the heart of international collaborations that continue to redefine what is possible in biology. When I was growing up on a farm in rural South Africa, I never imagined that yeast would become both my lifelong research companion and my passport to the world of scientific discovery and innovation. Yeast taught me that impactful research should be directed toward increasing fundamental understanding in a context responsive to the applied needs of end-users, at both the level of problem selection and experimental design. Writing this Retrospective is therefore both an honour and an opportunity to reflect not only on the science and where it is leading but also on the people, places, and serendipitous moments that have shaped my career.
The human gastrointestinal (GI) microbiota has come to be recognized as a modulator of health. However, interest in fungi and their function as members of the microbiota has lagged behind interest in bacteria. Despite the lack of historical interest, fungi are prevalent in the human GI tract and have an outsized impact on host immunity. In this review, we aim to examine the associations and potential impact of yeasts on human health outcomes. This review summarizes the associations between yeasts and inflammatory bowel diseases, highlights the predictive service that yeasts may provide in cancer therapy, and explores the possibility of yeasts as therapeutic effectors. There remain significant challenges in data analysis and identifying the relevance of fungal morphology; however, the pathways for clinical translation open to yeasts in the GI tract make these challenges worth overcoming.
The yeast Saccharomyces cerevisiae coordinates growth, metabolism, and stress adaptation through signaling pathways that respond to changes in nutrient availability. Classical nutrient-sensing systems, including the cAMP-protein kinase A (PKA), Snf1/AMP-activated protein kinase, target of rapamycin complex 1 (TORC1)-Sch9, Ssy1-Ptr3-Ssy5, and general amino acid control pathways, have revealed how yeast senses and responds to extracellular carbon, nitrogen, phosphate, and amino acid levels. In addition to these established pathways, plasma-membrane nutrient transporters also function in signaling rather than solely mediating substrate uptake. These dual-function proteins, termed nutrient transceptors, couple nutrient transport or extracellular nutrient recognition to rapid intracellular responses, often activating PKA without detectable changes in cAMP levels. This review focuses on yeast nutrient transceptors, specifically Gap1, Mep2, Pho84, Sul1/Sul2, Can1, Ftr1, and Zrt1. These proteins link extracellular nutrient availability to intracellular regulatory responses, including trehalose mobilization, stress resistance, growth resumption, filamentous development, and, in some cases, TORC1-Sch9 signaling. Mechanistic insights, including transport-signaling uncoupling and potential physical association with downstream protein kinases, are also discussed. Collectively, this evidence establishes nutrient transceptors as an essential additional layer of nutrient sensing in yeast, highlighting their role in translating extracellular nutrient cues into cellular responses.
Rhodotorula toruloides is an oleaginous yeast with great potential for chemical and biofuel production, due to its ability to utilize lignocellulosic biomass and to produce high levels of carotenoids and storage lipids. However, its broader application in biotechnology has been limited by the lack of efficient genetic transformation methods. Although protoplast transformation is wildly used in fungal systems, it has remained largely unexplored in R. toruloides. In this study, we established a protoplast transformation protocol using linear DNA fragments and R. toruloides strain BOT-A2, a recently isolated strain with high lipid-producing potential. We first confirmed that BOT-A2 is a MAT A2 haploid strain. We then produced a β-1,3-glucomannanase (Man5C) that effectively digests the R. toruloides cell wall. Key parameters affecting transformation efficiency were systematically optimized, including the Man5C digestion conditions, antibiotic selection pressure, cell growth phase, protoplast yield and viability, PEG formulation, calcium ion concentration, and regeneration conditions. Using the optimized protocol, we successfully transformed BOT-A2 with three heterologous resistance cassettes (hygromycin R, bleomycin, and G418) yielding 190, 226, and 244 transformants per µg of DNA, respectively. This method provides a platform for genetic manipulation and is expected to facilitate both fundamental research and metabolic engineering in R. toruloides BOT-A2.
The Ltv1 protein has been characterized with roles in ribosome biogenesis, maintenance of rRNA stability, ATP export, osmotic stress response, and signaling activation of the Target of Rapamycin pathway. We screened ltv1Δ/ltv1Δ mutants for additional phenotypic manifestations due to loss of Ltv1. We observed growth differences consistent with Ltv1's well characterized roles, alongside evidence supporting a less-established role in reactive oxygen species response. In further characterization of cells lacking Ltv1 we documented higher levels of endogenous ROS and a greater accumulation response of reactive oxygen species to exogenous stress. Utilizing RNA-Sequencing we then determined the gene expression differences underlying the ltv1-deficiency induced oxidative stress sensitivity. This work elucidates a new significant role of Ltv1 in cellular homeostasis and protection against damage.
Metabolism underpins cellular function by supplying energy, biosynthetic precursors, and redox balance and in yeast there are thousands of metabolic reactions that are tightly coordinated through multilayered regulation. The yeast Saccharomyces cerevisiae has become a central model for studying metabolism and its regulation and following publication of its genome in 1996, this yeast became pivotal in systems biology. Systems biology integrates experimental data with mathematical modeling to analyse complex cellular networks. A major advance for metabolic analysis was the development of flux balance analysis and genome sequencing enabled reconstruction of the first genome-scale metabolic model (GEM) for yeast. This initial GEM described how hundreds of genes, reactions, and metabolites interact across compartments. Subsequent models, including Yeast8 and Yeast9, expanded the coverage and predictive power, and these models enable metabolic comparison, physiological analysis, omics integration, and design of strains that can be used for production of chemicals and biopharmaceuticals. Overall, S. cerevisiae remains a cornerstone of systems biology and biotechnology, with continued advances expected in integrative modeling and engineering applications.
Unravelling the genomic blueprint of a reference laboratory strain of the yeast Saccharomyces cerevisiae 30 years ago opened a new era in understanding yeast biology. Since then, genomics has transformed our ability to study, adapt, improve, and tailor wine yeast strains in the laboratory and manage them in the cellar. This minireview highlights key advances in wine yeast genomics, from early whole-genome sequencing of industrial S. cerevisiae strains to the recent assembly of complex non-Saccharomyces genomes, including the wine spoilage yeast Brettanomyces bruxellensis. Comparative genomics has revealed the genetic foundations of strain specific traits critical to fermentation performance, aroma production, stress tolerance, and microbial interactions in the vineyard and winery. Beyond cataloguing gene content, integrative genomic approaches have elucidated evolutionary dynamics, domestication events, and adaptation to industrial environments. These insights underpin the rational development of novel starter cultures and biotechnological interventions, fostering consistent wine quality and diversity of sensory profiles for targeted consumer markets. Looking ahead, advances in pan-genomics and functional genomics promise to deepen our understanding of metabolic networks, gene-environment interactions, and the broader ecological context of wine fermentation. Collectively, the study of wine yeast genomics not only illuminates fundamental biological principles but also provides practical tools for innovation, including pathway engineering with synthetic enzyme fusions, and the creation of purpose-built synthetic neo-chromosomes. Excitingly, S. cerevisiae, the first eukaryote to have its genome sequenced, is now poised to become the first eukaryote with an entirely synthetic genome ̶ the Sc2.0 project ̶ heralding a bold future for yeast genomics.
Bioethanol is a pivotal sustainable alternative to fossil fuels. Saccharomyces cerevisiae is the primary microorganism for its industrial production. Enhancing the fermentative performance of S. cerevisiae is crucial for sustainable bioethanol production. To systematically enhance the fermentative capacity of yeast, this study employed targeted genetic engineering to overexpress MSS11-a principal transcription factor within the mitogen-activated protein kinase (MAPK) signaling cascade. The resultant recombinant strain, Eng-1, demonstrated a marked 23.9% enhancement in final ethanol titer (18.94 g/l vs. 15.29 g/l) and a commensurate 23.8% improvement in yield coefficient (0.473 vs. 0.382 g ethanol per g glucose) relative to the isogenic control strain Eng-0 under microaerobic batch fermentation. This phenotype was consistently validated in a distinct genetic background (JT139 derivative), underscoring the generalizable function of MSS11. Transcriptomic analysis revealed that MSS11 orchestrates a dual regulatory mechanism: it upregulates pyruvate-supplying pathway genes to enhance carbon flux toward ethanol, while concurrently activating a suite of genes involved in oxidative, osmotic, and ethanol stress tolerance to improve cellular robustness. These findings establish MSS11 as a novel and effective metabolic engineering target for yeast, and they elucidate its dual regulatory mechanism in coordinately enhancing carbon conversion and cellular robustness, thereby providing a strategic framework for constructing high-performance microbial cell factories for advanced biofuel production.
Disruptions in cellular homeostasis and proteostasis are central to many human diseases, yet direct mechanistic investigation in human systems remains constrained by biological complexity and ethical limitations. Therefore, researchers have turned to the use of model systems that allow the more efficient dissection of fundamental cellular processes. The unicellular yeast Saccharomyces cerevisiae has emerged as a powerful eukaryotic model for studying disorders driven by defects in homeostasis and proteostasis. The relevant processes are highly conserved in yeast, enabling precise genetic manipulation and real-time analysis of mechanisms that are difficult to study in mammalian systems. Yeast models have been deployed to study prion propagation, lysosomal enzyme trafficking, and mitochondrial dysfunction. Yeast also provides a versatile platform for drug discovery, particularly through the use of the yeast two-hybrid system and high-throughput screens. Despite an inability to recapitulate the full complexity of multicellular organisms, yeast remains an invaluable tool for investigating human diseases and for the development of therapeutics. This review highlights how yeast has uniquely advanced the understanding of human diseases including those associated with prions, lysosomal proteins, and mitochondria and can be combined with the utility of yeast in drug discovery-collectively establishing yeast as a model for studying human disorders.
Completion of the Saccharomyces cerevisiae genome sequence three decades ago marked a defining moment in eukaryotic genomics. Beyond a technical milestone, it established a shared reference that transformed how yeast biology is studied, interpreted, and extended across disciplines. This Editorial revisits the yeast genome sequencing project with a focus on the scientific culture that enabled it: an extraordinarily collaborative community willing to coordinate effort, share resources, and collectively tackle biological complexity. That consilient culture proved essential in converting a static DNA sequence into a dynamic framework for discovery, enabling systematic exploration of gene function, cellular organization, and genome-scale biology. As this Special Collection celebrates the 30th anniversary of the yeast genome sequence, we reflect on how shared infrastructure, and collective ambition turned the genome sequences of three related lab strains (S288c and its derivatives FY1679 and AB972) into a lasting platform for innovation. Apart from enabling the field of population genomics and access to the vast genetic diversity of the species, these foundations have facilitated the synthesis and assembly of all 16 chromosomes of the same laboratory strain of S. cerevisiae, bringing the Sc2.0 project within reach of creating the first eukaryotic cell with a fully synthetic genome. This transition-from genome reading to genome writing-positions yeasts as powerful systems for iterative design-build-test-learn cycles and for reimagining genomes of other Saccharomyces and non-Saccharomyces strains (including those used in industry) as highly modifiable biological platforms.
