FEMS yeast research最新文献

Optogenetics is an empowering technology that uses light-responsive proteins to control biological processes. Because of its genetic tractability, abundance of genetic tools, and robust culturing conditions, Saccharomyces cerevisiae has served for many years as an ideal platform in which to study, develop, and apply a wide range of optogenetic systems. In many instances, yeast has been used as a steppingstone in which to characterize and optimize optogenetic tools to later be deployed in higher eukaryotes. More recently, however, optogenetic tools have been developed and deployed in yeast specifically for biotechnological applications, including in nonconventional yeasts. In this review, we summarize various optogenetic systems responding to different wavelengths of light that have been demonstrated in diverse yeast species. We then describe various applications of these optogenetic tools in yeast, particularly in metabolic engineering and recombinant protein production. Finally, we discuss emerging applications in yeast cybergenetics-the interfacing of yeast and computers for closed-loop controls of yeast bioprocesses-and the potential impact of optogenetics in other future biotechnological applications.

Yeast is a widely utilized chassis for heterologous protein production, with Komagataella phaffii well-established as a prominent nonconventional yeast in this field. Despite its widespread recognition, there remains considerable potential to further optimize these cell factories to meet high production demands in a cost-effective and sustainable manner. Understanding the cellular response to the challenges of heterologous protein production can equip genetic engineers with crucial knowledge to develop enhanced strategies for constructing more efficient cell factories. In this study, we explore the molecular response of various K. phaffii strains that produce either the human insulin precursor or Mambalgin-1, examining changes in transcription and changes in intra- and extracellular protein levels. Our findings provide valuable insights into the molecular mechanisms that regulate the behaviour of K. phaffii production strains under the stress of producing different heterologous proteins. We believe that these results will serve as a foundation for identifying new genetic targets to improve strain robustness and productivity. In conclusion, we present new cellular and molecular insights into the response of K. phaffii cell factories to the challenges of burdensome heterologous protein production and our findings point to different engineering strategies for improved cell factory performance.

The human fungal pathogen Candida glabrata is closely related to the budding yeast Saccharomyces cerevisiae. The sexual cycle in S. cerevisiae has been extensively characterized. Haploid cells 'a' and alpha secrete pheromones involved in mating of the opposite cell type leading to the formation of a diploid cell. Under harsh conditions, diploid cells undergo meiosis for the formation of four haploid spores. In C. glabrata, cells are also found as 'a' and alpha and this organism possesses most S. cerevisiae homologous genes involved in meiosis and mating. However, mating has never been observed in C. glabrata. In S. cerevisiae, the non-essential UME6 gene is involved in controlling the expression of meiotic genes. We have previously shown that Zcf11, a putative homolog of Ume6, is encoded by an essential gene but its function is unknown. Here, we show that the expression of UME6 in C. glabrata can partially complement a Zcf11 knock-down and that these factors recognize the same DNA sequence. Importantly, expression profiling using a Zcf11 knock-down strain revealed that this factor is a negative regulator of meiotic genes expression as well as some genes involved in mating. Thus, regulation of the expression of meiotic genes is functional in this organism reinforcing the view that C. glabrata may have a sexual cycle under specific conditions.

Polyethylene terephthalate (PET) is one of the most used polymers in the packaging industry; enzymatic recycling is emerging as a sustainable strategy to deal with waste PET, producing the virgin monomers terephthalic acid and ethylene glycol (EG). These monomers can be feedstocks for further microbial transformations. While EG metabolism has been uncovered in bacteria, in yeast the pathway for the oxidation to glycolic acid (GA) has only been proposed, but never experimentally elucidated. In this work, we investigated in Saccharomyces cerevisiae the potential contribution to this metabolism of two endogenous genes, YLL056C (a putative alcohol dehydrogenase) and GOR1 (glyoxylate reductase). Secondly, the possible role of alcohol dehydrogenases (ADHs) was considered, too. Finally, two heterologous genes (gox0313 from Gluconobacter oxydans and AOX1 from Komagataella phaffii) were expressed with the intent to push EG oxidation toward GA. Our main findings revealed that (i) Gor1, Yll056c, and ADHs are not involved in EG oxidation and (ii) the bottleneck of the catabolism is the first step in the pathway, due to the endogenous mechanisms for aldehyde detoxification. Multiomics studies are required to completely elucidate the pathway for EG catabolism, while further engineering directed toward relieving the bottleneck is needed to fully unleash the potential of yeasts for the upcycling of EG to GA.

Komagataella phaffii has gained recognition as a versatile platform for recombinant protein production, with applications covering biopharmaceuticals, industrial enzymes, food additives, etc. Its advantages include high-level protein expression, moderate post-translational modifications, high-density cultivation, and cost-effective methanol utilization. Nevertheless, it still faces challenges for the improvement of production efficiency and extension of applicability. This review highlights the key strategies used to facilitate productivity in K. phaffii, including systematic advances in genetic manipulation tools, transcriptional and translational regulation, protein folding and secretion optimization. Glycosylation engineering is also concerned as it enables humanized glycosylation profiles for the use in therapeutic proteins and functional food additivities. Omics technologies and genome-scale metabolic models provide new insights into cellular metabolism, enhancing recombinant protein expression. High-throughput screening technologies are also emphasized as crucial for constructing high-expression strains and accelerating strain optimization. With advancements in gene-editing, synthetic and systems biology tools, the K. phaffii expression platform has been significantly improved for fundamental research and industrial use. Future innovations aim to fully harness K. phaffii as a next-generation cell factory, providing efficient, scalable, and cost-effective solutions for diverse applications. It continues to hold promise as a key driver in the field of biotechnology.

Quorum sensing (QS) is a known mechanism by which microbial populations adjust gene expression and coordinate community-wide social behaviors based on the proximate population density. This regulatory system has garnered significant interest in both scientific research and the food industry. However, a central question remains whether industrial strains of Saccharomyces cerevisiae, the yeast species predominantly utilized in brewing, employ quorum signalling mechanisms similar to those observed in laboratory strains and other fungi. Despite the potential relevance of microbial social behavior regulators to brewing practices, studies examining QS in Saccharomyces spp. are limited. In this investigation, three industrial brewing strains of S. cerevisiae were cultivated on SLAD (nitrogen-restrictive) and SHAD (nitrogen-sufficient) agar media supplemented with 200 μM of the aromatic alcohol 2-phenylethanol (2-PE) over 72 h at 24°C. Subsequent analyses of the harvested biomass included proteomic, lipidomic, and metabolomic assessments. Results indicated that two of the industrial strains showed minimal differences in their profiles upon exposure to 2-PE, while the third strain exhibited significant differences. These findings imply that the impact of the QS molecule 2-PE on the proteome, lipidome, and metabolome of industrial S. cerevisiae may be strain-specific rather than universally applicable to the species.

Food microorganisms have been employed for centuries for the processing of fermented foods, leading to adapted populations with phenotypic traits of interest. The yeast Monosporozyma unispora (formerly Kazachstania unispora) has been identified in a wide range of fermented foods and beverages. Here, we studied the genetic and phenotypic diversity of a collection of 53 strains primarily derived from cheese, kefir, and sourdough. The 12.7-Mb genome of the type strain CLIB 234T was sequenced and assembled into near-complete chromosomes and annotated at the structural and functional levels, with 5639 coding sequences predicted. Comparison of the pangenome and core genome revealed minimal differences. From the complete yeast collection, we gathered genetic data (diversity, phylogeny, and population structure) and phenotypic data (growth capacity on solid media). Population genomic analyses revealed a low level of nucleotide diversity and strong population structure, with the presence of two major clades corresponding to ecological origins (cheese and kefir vs. plant derivatives). A high prevalence of extensive loss of heterozygosity and a slow linkage disequilibrium decay suggested a predominantly clonal mode of reproduction. Phenotypic analyses revealed growth variation under stress conditions, including high salinity and low pH, but no definitive link between phenotypic traits and environmental adaptation was established.

Yeast shares a longer than 10 000-year history with humans in food fermentation by producing various volatile flavor compounds that contribute to the final taste and aroma of foods. Yeast-associated volatile flavor compounds include esters, benzenoids, sulfur compounds, and phenolic derivatives, which enhance the sensory complexity of fermented foods and beverages. Genome-scale technologies have advanced and transformed our understanding of the genetic and evolutionary drivers of volatile flavor diversity. The conventional approach to aroma enrichment and flavor balancing through single-strain optimization has been redefined through yeast cofermentation strategies, such as the pairing of Saccharomyces cerevisiae with nonconventional yeast species. This minireview summarizes the latest genomic insights into volatile flavor compound formation through ester, benzenoid, sulfur, and phenolic pathways in various yeast species and highlights the shaping of the next generation of food fermentation innovation via cofermentation combined with omics analysis, followed by a future perspective on synthetic biology for industrial applicability.

Fungal β-1,3-glucan synthase (Fks) plays a central role in synthesizing β-1,3-glucan, the main structural polysaccharide of fungal cell walls, and serves as a key target for antifungal drugs, such as echinocandins and ibrexafungerp. Recent cryo-electron microscopy (cryo-EM) studies have revealed the architecture of the Fks1 and Fks1-Rho1 complex and provided new insights into its catalytic and regulatory mechanisms. This review summarizes current understanding of Fks, including its domain organization, transmembrane topology, conformational dynamics, and evolutionary comparison with structurally resolved glycosyltransferases (GTs), including bacterial cellulose synthase (BcsA), plant cellulose synthase (CesA), and other eukaryotic GTs. Through comparison of publicly available cryo-EM structures of Fks in both the apo-state and Rho1-bound state, a working mechanism of the activated Fks has been discussed. In addition, we present a potential gating model of β-glucan translocation and drug-inhibition by integrating literature with structure-based analyses. This review provides a structure-based functional model of fungal β-1,3-glucan synthase and the putative binding mechanism of its inhibitor, aiming to support future antifungal drug discovery.

Unlike glucose, the sub-optimal xylose utilization in recombinant Saccharomyces cerevisiae strains may stem from an unusual signaling response that is not adapted to detecting xylose as a fermentable substrate. We hypothesize that the membrane receptor Snf3p, known for sensing extracellular low glucose levels, may contribute to xylose recognition. To test this, we explored the effect of SNF3 inactivation and overexpression by measuring the response of the HXT2p-GFP biosensor integrated into S. cerevisiae strains with heterogeneous xylose assimilation and metabolism capacities. We showed that the absence of SNF3 effectively reduced HXT2p induction, while its overexpression improved signaling in the presence of xylose, suggesting the involvement of the receptor in the extracellular detection of this sugar. Although we attempted to engineer a xylose sensing system based on a chimeric receptor, its integration did not lead to considerable improvements in signal activation, indicating the need for further investigation. Finally, we showed that triggering the Snf3p pathway impacted xylose metabolism, with altered receptor levels prompting shifts in both biomass production and metabolite accumulation. Our findings suggest that understanding xylose sensing and its metabolic connection is essential for promoting more efficient xylose utilization in S. cerevisiae, a key step toward optimizing industrial bioprocesses.