RESUMEN
BACKGROUND: Over the last two decades, hybridization has been a powerful tool used to construct superior yeast for brewing and winemaking. Novel hybrids were primarily constructed using at least one Saccharomyces cerevisiae parent. However, little is known about hybrids used for other purposes, such as targeted flavor production, for example, 2-phenylethanol (2-PE). 2-PE, an aromatic compound widely utilised in the food, cosmetic, and pharmaceutical industries, presents challenges in biotechnological production due to its toxic nature. Consequently, to enhance productivity and tolerance to 2-PE, various strategies such as mutagenesis and genetic engineering are extensively explored to improved yeast strains. While biotechnological efforts have predominantly focused on S. cerevisiae for 2-PE production, other Saccharomyces species and their hybrids remain insufficiently described. RESULTS: To address this gap, in this study, we analysed a new interspecies yeast hybrid, II/6, derived from S. uvarum and S. kudriavzevii parents, in terms of 2-PE bioconversion and resistance to its high concentration, comparing it with the parental strains. Two known media for 2-PE biotransformation and three different temperatures were used during this study to determine optimal conditions. In 72 h batch cultures, the II/6 hybrid achieved a maximum of 2.36 ± 0.03 g/L 2-PE, which was 2-20 times higher than the productivity of the parental strains. Our interest lay not only in determining whether the hybrid improved in productivity but also in assessing whether its susceptibility to high 2-PE titers was also mitigated. The results showed that the hybrid exhibited significantly greater resistance to the toxic product than the original strains. CONCLUSIONS: The conducted experiments have confirmed that hybridization is a promising method for modifying yeast strains. As a result, both 2-PE production yield and tolerance to its inhibitory effects can be increased. Furthermore, this strategy allows for the acquisition of non-GMO strains, alleviating concerns related to additional legislative requirements or consumer acceptance issues for producers. The findings obtained have the potential to contribute to the development of practical solutions in the future.
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Alcohol Feniletílico , Saccharomyces , Alcohol Feniletílico/metabolismo , Alcohol Feniletílico/análogos & derivados , Saccharomyces/genética , Saccharomyces/metabolismo , Fermentación , Hibridación Genética , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , PichiaRESUMEN
Beer brewing is a well-known process that still faces great challenges, such as the total consumption of sugars present in the fermentation media. Lager-style beer, a major worldwide beer type, is elaborated by Saccharomyces pastorianus (Sp) yeast, which must ferment high maltotriose content worts, but its consumption represents a notable problem, especially among Sp strains belonging to group I. Factors, such as fermentation conditions, presence of maltotriose transporters, transporter copy number variation, and genetic regulation variations contribute to this issue. We assess the factors affecting fermentation in two Sp yeast strains: SpIB1, with limited maltotriose uptake, and SpIB2, known for efficient maltotriose transport. Here, SpIB2 transported significantly more maltose (28%) and maltotriose (32%) compared with SpIB1. Furthermore, SpIB2 expressed all MAL transporters (ScMALx1, SeMALx1, ScAGT1, SeAGT1, MTT1, and MPHx) on the first day of fermentation, whereas SpIB1 only exhibited ScMalx1, ScAGT1, and MPH2/3 genes. Some SpIB2 transporters had polymorphic transmembrane domains (TMD) resembling MTT1, accompanied by higher expression of these transporters and its positive regulator genes, such as MAL63. These findings suggest that, in addition to the factors mentioned above, positive regulators of Mal transporters contribute significantly to phenotypic diversity in maltose and maltotriose consumption among the studied lager yeast strains.IMPORTANCEBeer, the third most popular beverage globally with a 90% market share in the alcoholic beverage industry, relies on Saccharomyces pastorianus (Sp) strains for lager beer production. These strains exhibit phenotypic diversity in maltotriose consumption, a crucial process for the acceptable organoleptic profile in lager beer. This diversity ranges from Sp group II strains with a notable maltotriose-consuming ability to Sp group I strains with limited capacity. Our study highlights that differential gene expression of maltose and maltotriose transporters and its upstream trans-elements, such as MAL gene-positive regulators, adds complexity to this variation. This insight can contribute to a more comprehensive analysis needed to the development of controlled and efficient biotechnological processes in the beer brewing industry.
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Cerveza , Fermentación , Proteínas Fúngicas , Maltosa , Saccharomyces , Trisacáridos , Maltosa/metabolismo , Trisacáridos/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Cerveza/microbiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Transporte Biológico , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Regulación Fúngica de la Expresión GénicaRESUMEN
Lager yeasts are limited to a few strains worldwide, imposing restrictions on flavour and aroma diversity and hindering our understanding of the complex evolutionary mechanisms during yeast domestication. The recent finding of diverse S. eubayanus lineages from Patagonia offers potential for generating new lager yeasts with different flavour profiles. Here, we leverage the natural genetic diversity of S. eubayanus and expand the lager yeast repertoire by including three distinct Patagonian S. eubayanus lineages. We used experimental evolution and selection on desirable traits to enhance the fermentation profiles of novel S. cerevisiae x S. eubayanus hybrids. Our analyses reveal an intricate interplay of pre-existing diversity, selection on species-specific mitochondria, de-novo mutations, and gene copy variations in sugar metabolism genes, resulting in high ethanol production and unique aroma profiles. Hybrids with S. eubayanus mitochondria exhibited greater evolutionary potential and superior fitness post-evolution, analogous to commercial lager hybrids. Using genome-wide screens of the parental subgenomes, we identified genetic changes in IRA2, IMA1, and MALX genes that influence maltose metabolism, and increase glycolytic flux and sugar consumption in the evolved hybrids. Functional validation and transcriptome analyses confirmed increased maltose-related gene expression, influencing greater maltotriose consumption in evolved hybrids. This study demonstrates the potential for generating industrially viable lager yeast hybrids from wild Patagonian strains. Our hybridization, evolution, and mitochondrial selection approach produced hybrids with high fermentation capacity and expands lager beer brewing options.
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Cerveza , Fermentación , Hibridación Genética , Saccharomyces cerevisiae , Cerveza/microbiología , Fermentación/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Etanol/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Genoma Fúngico , Evolución Molecular , Variación Genética , Maltosa/metabolismo , MutaciónRESUMEN
Species of Saccharomyces genus have played an irreplaceable role in alcoholic beverage and baking industry for centuries. S. cerevisiae has also become an organism of choice for industrial production of alcohol and other valuable chemicals and a model organism shaping the rise of modern genetics and genomics in the past few decades. Today´s brewing industry faces challenges of decreasing consumption of traditional beer styles and increasing consumer demand for new styles, flavors and aromas. The number of currently used brewer's strains and their genetic diversity is yet limited and implementation of more genetic and phenotypic variation is seen as a solution to cope with the market challenges. This requires modification of current production strains or introduction of novel strains from other settings, e.g. industrial or wild habitats into the brewing industry. Due to legal regulation in many countries and negative customer perception of GMO organisms, the production of food and beverages requires non-GMO production organisms, whose development can be difficult and time-consuming. Here, we apply FIND-IT (Fast Identification of Nucleotide variants by DigITal PCR), an ultrafast genome-mining method, for isolation of novel yeast variants with varying flavor profiles. The FIND-IT method uses combination of random mutagenesis, droplet digital PCR with probes that target a specific desired mutation and a sub-isolation of the mutant clone. Such an approach allows the targeted identification and isolation of specific mutant strains with eliminated production of certain flavor and off-flavors and/or changes in the strain metabolism. We demonstrate that the technology is useful for the identification of loss-of function or gain of function mutations in unrelated industrial and wild strains differing in ploidy. Where no other phenotypic selection exists, this technology serves together with standard breeding techniques as a modern tool facilitating a modification of (brewer's) yeast strains leading to diversification of the product portfolio.
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Cerveza , Ingeniería Metabólica , Saccharomyces , Cerveza/microbiología , Saccharomyces/genética , Saccharomyces/metabolismo , Aromatizantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
In silico tools such as genome-scale metabolic models have shown to be powerful for metabolic engineering of microorganisms. Saccharomyces pastorianus is a complex aneuploid hybrid between the mesophilic Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. This species is of biotechnological importance because it is the primary yeast used in lager beer fermentation and is also a key model for studying the evolution of hybrid genomes, including expression pattern of ortholog genes, composition of protein complexes, and phenotypic plasticity. Here, we created the iSP_1513 GSMM for S. pastorianus CBS1513 to allow top-down computational approaches to predict the evolution of metabolic pathways and to aid strain optimization in production processes. The iSP_1513 comprises 4,062 reactions, 1,808 alleles, and 2,747 metabolites, and takes into account the functional redundancy in the gene-protein-reaction rule caused by the presence of orthologous genes. Moreover, a universal algorithm to constrain GSMM reactions using transcriptome data was developed as a python library and enabled the integration of temperature as parameter. Essentiality data sets, growth data on various carbohydrates and volatile metabolites secretion were used to validate the model and showed the potential of media engineering to improve specific flavor compounds. The iSP_1513 also highlighted the different contributions of the parental sub-genomes to the oxidative and non-oxidative parts of the pentose phosphate pathway. Overall, the iSP_1513 GSMM represent an important step toward understanding the metabolic capabilities, evolutionary trajectories, and adaptation potential of S. pastorianus in different industrial settings. IMPORTANCE: Genome-scale metabolic models (GSMM) have been successfully applied to predict cellular behavior and design cell factories in several model organisms, but no models to date are currently available for hybrid species due to their more complex genetics and general lack of molecular data. In this study, we generated a bespoke GSMM, iSP_1513, for this industrial aneuploid hybrid Saccharomyces pastorianus, which takes into account the aneuploidy and functional redundancy from orthologous parental alleles. This model will (i) help understand the metabolic capabilities and adaptive potential of S. pastorianus (domestication processes), (ii) aid top-down predictions for strain development (industrial biotechnology), and (iii) allow predictions of evolutionary trajectories of metabolic pathways in aneuploid hybrids (evolutionary genetics).
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Genoma Fúngico , Redes y Vías Metabólicas , Saccharomyces , Saccharomyces/genética , Saccharomyces/metabolismo , Redes y Vías Metabólicas/genética , Genoma Fúngico/genética , Modelos Biológicos , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Evolución Molecular , Microbiología Industrial/métodosRESUMEN
2-Phenylethanol (2-PE) is a highly valuable aromatic alcohol utilized in fragrance, cosmetics and food industries. Due to the toxic by-products from chemical synthesis and the low productivity of the extraction method, bioproduction of 2-PE by yeast is considered promising. In this study, a wild-type Saccharomyces bayanus L1 strain producing 2-PE was isolated from soy sauce mash. Transcriptional analysis showed that 2-PE was synthesized via the Ehrlich pathway and Shikimate pathway in S. bayanus L1. By improving the fermentation conditions in shaking flasks, the maximum 2-PE titer reached 4.2 g/L with a productivity of 0.058 g/L/h within 72 h. In fed-batch fermentation, S. bayanus L1 strain produced 6.5 g/L of 2-PE within 60 h, achieving a productivity of 0.108 g/L/h. These findings suggest that S. bayanus L1 strain is an efficient 2-PE producer, paving the way for highly efficient 2-PE production.
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Fermentación , Alcohol Feniletílico , Saccharomyces , Alcohol Feniletílico/metabolismo , Saccharomyces/metabolismo , Saccharomyces/genética , Alimentos de SojaRESUMEN
A novel millifluidic process introduces age-based fractionation of S. pastorianus var. carlsbergensis yeast culture through magnetophoresis. Saccharomyces yeast is a model organism for aging research used in various industries. Traditional age-based cell separation methods were labor-intensive, but techniques like magnetic labeling have eased the process by being non-invasive and scalable. Our approach introduces an age-specific fractionation using a 3D-printed millfluidic chip in a two-step process, ensuring efficient cell deflection in the magnetic field and counteracting magnetic induced convection. Among various channel designs, the pinch-shaped channel proved most effective for age differentiation based on magnetically labeled bud scar numbers. Metabolomic analyses revealed changes in certain amino acids and increased NAD+ levels, suggesting metabolic shifts in aging cells. Gene expression studies further underlined these age-related metabolic changes. This innovative platform offers a high-throughput, non-invasive method for age-specific yeast cell fractionation, with potential applications in industries ranging from food and beverages to pharmaceuticals.
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Metabolómica , Saccharomyces/metabolismo , Técnicas Analíticas Microfluídicas/instrumentación , Saccharomyces cerevisiae/metabolismo , Dispositivos Laboratorio en un ChipRESUMEN
This study aimed to investigate how parental genomes contribute to yeast hybrid metabolism using a metabolomic approach. Previous studies have explored central carbon and nitrogen metabolism in Saccharomyces species during wine fermentation, but this study analyses the metabolomes of Saccharomyces hybrids for the first time. We evaluated the oenological performance and intra- and extracellular metabolomes, and we compared the strains according to nutrient consumption and production of the main fermentative by-products. Surprisingly, no common pattern was observed for hybrid genome influence; each strain behaved differently during wine fermentation. However, this study suggests that the genome of the S. cerevisiae species may play a more relevant role in fermentative metabolism. Variations in biomass/nitrogen ratios were also noted, potentially linked to S. kudriavzevii and S. uvarum genome contributions. These results open up possibilities for further research using different "omics" approaches to comprehend better metabolic regulation in hybrid strains with genomes from different species.
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Fermentación , Nitrógeno , Saccharomyces , Vino , Vino/microbiología , Vino/análisis , Saccharomyces/genética , Saccharomyces/metabolismo , Saccharomyces/clasificación , Nitrógeno/metabolismo , Metaboloma , Carbono/metabolismo , Hibridación GenéticaRESUMEN
The utilization of the glycated amino acids formyline and pyrraline as well as their peptide-bound derivatives by 14 Saccharomyces yeasts, including 6 beer yeasts (bottom and top fermenting), one wine yeast, 6â strains isolated from natural habitats and one laboratory reference yeast strain (wild type) was investigated. All yeasts were able to metabolize glycated amino acids via the Ehrlich pathway to the corresponding Ehrlich metabolites. While formyline and small amounts of pyrraline entered the yeast cells via passive diffusion, the amounts of dipeptide-bound MRPs, especially the dipeptides glycated at the C-terminus, decreased much faster, indicating an uptake into the yeast cells. Furthermore, the glycation-mediated hydrophobization in general leads to an faster degradation rate compared to the native lysine dipeptides. While the utilization of free formyline is yeast-specific, the amounts of (glycated) dipeptides decreased faster in the presence of brewer's yeasts, which also showed a higher formation rate of Ehrlich metabolites compared to naturally isolated strains. Due to rapid uptake of alanyl dipeptides, it can be assumed that the Ehrlich enzyme system of naturally isolated yeasts is overloaded and the intracellularly released MRP is primarily excreted from the cell. This indicates adaptation of technologically used yeasts to (glycated) dipeptides as a nitrogen source.
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Dipéptidos , Norleucina , Dipéptidos/metabolismo , Dipéptidos/química , Norleucina/metabolismo , Norleucina/análogos & derivados , Norleucina/química , Saccharomyces/metabolismo , Saccharomyces cerevisiae/metabolismo , Glicosilación , PirrolesRESUMEN
In recent years, the presence of molecules derived from aromatic amino acids in wines has been increasingly demonstrated to have a significant influence on wine quality and stability. In addition, interactions between different yeast species have been observed to influence these final properties. In this study, a screening of 81 yeast strains from different environments was carried out to establish a consortium that would promote the improvement of indolic compound levels in wine. Two strains, Saccharomyces uvarum and Saccharomyces eubayanus, with robust fermentative capacity were selected to be combined with a Saccharomyces cerevisiae strain with a predisposition towards the production of indolic compounds. Fermentation dynamics were studied in pure cultures, co-inoculations and sequential inoculations, analysing strain interactions and end-of-fermentation characteristics. Fermentations showing significant interactions were further analyzed for the resulting indolic compounds and aroma profile, with the aim of observing potential interactions and synergies resulting from the combination of different strains in the final wine. Sequential inoculation of S. cerevisiae after S. uvarum or S. eubayanus was observed to increase indolic compound levels, particularly serotonin and 3-indoleacetic acid. This study is the first to demonstrate how the formation of microbial consortia can serve as a useful strategy to enhance compounds with interesting properties in wine, paving the way for future studies and combinations.
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Saccharomyces , Vino , Vino/análisis , Saccharomyces cerevisiae/metabolismo , Triptófano/análisis , Triptófano/metabolismo , Fermentación , Saccharomyces/metabolismoRESUMEN
About half of the 1.62 billion cases of anemia are because of poor diet and iron deficiency. Currently, the use of iron-enriched yeasts can be used as the most effective and possible way to prevent and treat anemia due to the ability of biotransformation of mineral compounds into the organic form. In this research, for the first time, Saccharomyces (S.) boulardii was used for iron enrichment with the aim that the probiotic properties of yeast provide a potential iron supplement besides improving the bioavailability of iron. Also, due to its higher resistance than other Saccharomyces strains against stresses, it can protect iron against processing temperatures and stomach acidic-enzymatic conditions. So, the effect of three important variables, including concentration of iron, molasses and KH2PO4 on the growth and biotransformation of yeast was investigated by the Box-Behnken design (BBD). The best conditions occurred in 3 g/l KH2PO4, 20 g/l molasses and 12 mg/l FeSO4 with the highest biotransformation 27 mg Fe/g dry cell weight (DCW) and 6 g/l biomass weight. Such yeast can improve fermented products, provide potential supplement, and restore the lost iron of bread, which is a useful iron source, even for vegetarians-vegans and play an important role in manage with anemia. It is recommended that in future researches, attention should be paid to increasing the iron enrichment of yeast through permeabilizing the membrane and overcoming the structural barrier of the cell wall.
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Anemia , Probióticos , Saccharomyces boulardii , Saccharomyces , Saccharomyces cerevisiae/metabolismo , Hierro/metabolismo , Saccharomyces/metabolismo , Probióticos/metabolismoRESUMEN
BACKGROUND: Selenium is an important nutritional supplement that mainly exists naturally in soil as inorganic selenium. Saccharomyces cerevisiae cells are excellent medium for converting inorganic selenium in nature into organic selenium. RESULTS: Under the co-stimulation of sodium selenite (Na2SeO3) and potassium selenite (K2SeO3), the activity of selenophosphate synthetase (SPS) was improved up to about five folds more than conventional Na2SeO3 group with the total selenite salts content of 30 mg/L. Transcriptome analysis first revealed that due to the sharing pathway between sodium ion (Na+) and potassium ion (K+), the K+ largely regulates the metabolisms of amino acid and glutathione under the accumulation of selenite salt. Furthermore, K+ could improve the tolerance performance and selenium-biotransformation yields of Saccharomyces cerevisiae cells under Na2SeO3 salt stimulation. CONCLUSION: The important role of K+ in regulating the intracellular selenium accumulation especially in terms of amino acid metabolism and glutathione, suggested a new direction for the development of selenium-enrichment supplements with Saccharomyces cerevisiae cell factory. © 2024 Society of Chemical Industry.
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Saccharomyces , Selenio , Selenio/metabolismo , Saccharomyces/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Selenito de Sodio/metabolismo , Ácido Selenioso/metabolismo , Glutatión/metabolismo , Sodio/metabolismo , Aminoácidos/metabolismo , Potasio/metabolismoRESUMEN
Yeast flocculation and viability are critical factors in beer production. Adequate flocculation of yeast at the end of fermentation helps to reduce off-flavors and cell separation, while high viability is beneficial for yeast reuse. In this study, we used comparative genomics to analyze the genome information on Saccharomyces pastorianus W01, and its spontaneous mutant W02 with appropriate weakened flocculation ability (better off-flavor reduction performance) and unwanted decreased viability, to investigate the effect of different gene expressions on yeast flocculation or/and viability. Our results indicate that knockout of CNE1, CIN5, SIN3, HP-3, YPR170W-B, and SCEPF1_0274000100 and overexpression of CNE1 and ALD2 significantly decreased the flocculation ability of W01, while knockout of EPL1 increased the flocculation ability of W01. Meanwhile, knockout of CIN5, YPR170W-B, OST5, SFT1, SCEPF1_0274000100, and EPL1 and overexpression of SWC3, ALD2, and HP-2 decreased the viability of W01. CIN5, EPL1, SCEPF1_0274000100, ALD2, and YPR170W-B have all been shown to affect yeast flocculation ability and viability.
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Saccharomyces cerevisiae , Saccharomyces , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Floculación , Saccharomyces/genética , Saccharomyces/metabolismo , Genómica , Cerveza/análisis , FermentaciónRESUMEN
Selection of the appropriate yeast strain is one of the most crucial steps in a brewery. Traditionally, yeast strain's abilities during beer fermentation are described according to brewer's experiences. Hence, these descriptions could be inaccurate and strictly based on sensory experiences. In this study, lager beers fermented by four traditional bottom-fermented yeast strains were characterized in detail by sensomic approach. The obtained results revealed that yeast strains can influence most of the sensory-related components in beer, not only esters and higher alcohols, but also carbonyls, amino acids, saccharides, fatty acids, heterocyclic compounds, hop oils, and other hop-related components. By comparison of chemical and sensory characteristics of each studied beer, the theoretical importance of sensory interactions on beer flavor perception was also revealed as the general conception that the beers with similar flavors have also similar chemical profiles (and vice versa) was seemed as not valid.
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Saccharomyces cerevisiae , Saccharomyces , Saccharomyces cerevisiae/metabolismo , Cerveza/análisis , Saccharomyces/metabolismo , FermentaciónRESUMEN
Ribonucleic acid (RNA) and its degradation products are important biomolecules widely used in the food and pharmaceutical industries for their flavoring and nutritional functions. In this study, we used a genome-scale metabolic network model (GSMM) to explore genetic targets for nucleic acid synthesis in a Saccharomyces pastorianus strain (G03). Yeast 8.5.0 was used as the base model, which accurately predicted G03's growth. Using OptForce, we found that overexpression of ARO8 and ATP1 among six different strategies increased the RNA content of G03 by 58.0% and 74.8%, respectively. We also identified new metabolic targets for improved RNA production using a modified GSMM called TissueModel, constructed using the GIMME transcriptome constraint tool to remove low-expressed reactions in the model. After running OptKnock, the RNA content of G03-â³BNA1 and G03-â³PMA1 increased by 44.6% and 39.8%, respectively, compared to G03. We suggest that ATP1, ARO8, BNA1, and PMA1 regulate cell fitness, which affects RNA content. This study is the first to identify strategies for RNA overproduction using GSMM and to report that regulation of ATP1, ARO8, BNA1, and PMA1 can increase RNA content in S. pastorianus. These findings also provide valuable knowledge on model reconstruction for S. pastorianus.
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ARN , Saccharomyces , ARN/metabolismo , Genoma Fúngico/genética , Saccharomyces/genética , Saccharomyces/metabolismo , Saccharomyces cerevisiae/genética , Redes y Vías Metabólicas , FermentaciónRESUMEN
Erythritol is produced in yeasts via the reduction of erythrose into erythritol by erythrose reductases (ERs). However, the genes codifying for the ERs involved in this reaction have not been described in any Saccharomyces species yet. In our laboratory, we recently showed that, during alcoholic fermentation, erythritol is differentially produced by Saccharomyces cerevisiae and S. uvarum species, the latter being the largest producer. In this study, by using BLAST analysis and phylogenetic approaches the genes GRE3, GCY1, YPR1, ARA1 and YJR096W were identified as putative ERs in Saccharomyces cerevisiae Then, these genes were knocked out in our S. uvarum strain (BMV58) with higher erythritol biosynthesis compared to control S. cerevisiae wine strain, to evaluate their impact on erythritol synthesis and global metabolism. Among the mutants, the single deletion of GRE3 markedly impacts erythritol production, although ΔYPR1ΔGCY1ΔGRE3 was the combination that most decreased erythritol synthesis. Consistent with the increased production of fermentative by-products involved in redox balance in the Saccharomyces uvarum strain BMV58, erythritol synthesis increases at higher sugar concentrations, hinting it might be a response to osmotic stress. However, the expression of GRE3 in the S. uvarum strain was found to peak just before the start of the stationary phase, being consistent with the observation that erythritol increases at the start of the stationary phase, when there is low sugar in the medium and nitrogen sources are depleted. This suggests that GRE3 plays its primary function to help the yeast cells to maintain the redox balance during the last phases of fermentation.
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Eritritol , Saccharomyces , Eritritol/metabolismo , Fermentación , Homeostasis , Osmorregulación , Oxidación-Reducción , Filogenia , Saccharomyces/genética , Saccharomyces/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Azúcares/metabolismo , Aldehído Reductasa/genética , Aldehído Reductasa/metabolismoRESUMEN
The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.
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Probióticos , Saccharomyces boulardii , Saccharomyces , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces boulardii/genética , Saccharomyces boulardii/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Probióticos/metabolismo , Endopeptidasas/metabolismoRESUMEN
Saccharomyces cerevisiae is the yeast of choice for most inoculated wine fermentations worldwide. However, many other yeast species and genera display phenotypes of interest that may help address the environmental and commercial challenges the wine industry has been facing in recent years. This work aimed to provide, for the first time, a systematic phenotyping of all Saccharomyces species under winemaking conditions. For this purpose, we characterized the fermentative and metabolic properties of 92 Saccharomyces strains in synthetic grape must at two different temperatures. The fermentative potential of alternative yeasts was higher than expected, as nearly all strains were able to complete fermentation, in some cases more efficiently than commercial S. cerevisiae strains. Various species showed interesting metabolic traits, such as high glycerol, succinate and odour-active compound production, or low acetic acid production, compared to S. cerevisiae. Altogether, these results reveal that non-cerevisiae Saccharomyces yeasts are especially interesting for wine fermentation, as they may offer advantages over both S. cerevisiae and non-Saccharomyces strains. This study highlights the potential of alternative Saccharomyces species for winemaking, paving the way for further research and, potentially, for their industrial exploitation.
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Saccharomyces , Vitis , Vino , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fermentación , Saccharomyces/genética , Saccharomyces/metabolismo , Vino/análisis , Vitis/metabolismo , Ácido Acético/metabolismo , FenotipoRESUMEN
Bone destruction is a hallmark of chronic inflammation, and bone-resorbing osteoclasts arising under such a condition differ from steady-state ones. However, osteoclast diversity remains poorly explored. Here, we combined transcriptomic profiling, differentiation assays and in vivo analysis in mouse to decipher specific traits for inflammatory and steady-state osteoclasts. We identified and validated the pattern-recognition receptors (PRR) Tlr2, Dectin-1, and Mincle, all involved in yeast recognition as major regulators of inflammatory osteoclasts. We showed that administration of the yeast probiotic Saccharomyces boulardii CNCM I-745 (Sb) in vivo reduced bone loss in ovariectomized but not sham mice by reducing inflammatory osteoclastogenesis. This beneficial impact of Sb is mediated by the regulation of the inflammatory environment required for the generation of inflammatory osteoclasts. We also showed that Sb derivatives as well as agonists of Tlr2, Dectin-1, and Mincle specifically inhibited directly the differentiation of inflammatory but not steady-state osteoclasts in vitro. These findings demonstrate a preferential use of the PRR-associated costimulatory differentiation pathway by inflammatory osteoclasts, thus enabling their specific inhibition, which opens new therapeutic perspectives for inflammatory bone loss.