RESUMEN
This study aimed to assess the impact of Saccharomyces cerevisiae and different non-Saccharomyces cerevisiae (Zygosaccharomyces bailii, Hanseniaspora opuntiae and Zygosaccharomyces rouxii) on the volatile compounds and sensory properties of low-alcohol pear beverages fermented from three varieties of pear juices (Korla, Laiyang and Binzhou). Results showed that all three pear juices were favorable matrices for yeasts growth. Non-Saccharomyces cerevisiae exhibited a higher capacity for acetate ester production compared to Saccharomyces cerevisiae, resulting in a significant enhancement in sensory complexity of the beverages. PCA and sensory analysis demonstrated that pear varieties exerted a stronger influence on the crucial volatile components and aroma characteristics of the fermented beverages compared to the yeast species. CA results showed different yeast strains exhibited suitability for the fermentation of specific pear juice varieties.
Asunto(s)
Fermentación , Odorantes , Pyrus , Saccharomyces cerevisiae , Compuestos Orgánicos Volátiles , Compuestos Orgánicos Volátiles/metabolismo , Compuestos Orgánicos Volátiles/análisis , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Pyrus/microbiología , Pyrus/química , Odorantes/análisis , Jugos de Frutas y Vegetales/análisis , Jugos de Frutas y Vegetales/microbiología , Gusto , Humanos , Zygosaccharomyces/metabolismo , Zygosaccharomyces/crecimiento & desarrollo , Hanseniaspora/metabolismo , Hanseniaspora/crecimiento & desarrollo , Frutas/microbiología , Frutas/química , SaccharomycetalesRESUMEN
Yeast assimilable nitrogen (YAN) is one of the important factors affecting yeast growth and metabolism. However, the nitrogen requirement of indigenous commercial S. cerevisiae NX11424 is unclear. In this study, metabolomics was used to analyze the metabolite profiles of the yeast strain NX11424 under high (433 mg/L) and low (55 mg/L) YAN concentrations. It was found that yeast biomass exhibited different trends under different YAN conditions and was generally positively correlated with the initial YAN concentration, while changes of key biomarkers of yeast strain NX11424 at different stages of fermentation showed a similar trend under high and low YAN concentrations. The YAN concentration affected the metabolite levels of the yeast strain NX11424, which resulted in the significant difference in the levels of pyruvic acid, α-oxoglutarate, palmitoleic acid, proline, butane-2,3-diol, citrulline, ornithine, galactinol, citramalic acid, tryptophan, alanine, phosphate and phenylethanol, mainly involving pathways such as central carbon metabolism, amino acid metabolism, fatty acid metabolism, purine metabolism, and energy metabolism. Yeast strain NX11424 could utilize proline to produce protein under a low YAN level. The intracellular level of citrulline and ornithine under high YAN concentration was higher than that under low YAN level. Yeast strain NX11424 is more suitable for fermentation at lower YAN level. The results obtained here will help to rational utilize of YAN by S. cerevisiae NX11424, and is conducive to precise control of the alcohol fermentation and improve wine quality.
Asunto(s)
Fermentación , Metabolómica , Nitrógeno , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Nitrógeno/metabolismo , Vino/análisis , Vino/microbiología , Biomasa , Aminoácidos/metabolismoRESUMEN
Fino Sherry wine undergoes biological aging carried out by a velum of flor yeast within a traditional dynamic system known as "criaderas and solera". The complex microbiota of biofilm-forming Saccharomyces cerevisiae strains play a crucial role in shaping the distinctive organoleptic profile of these types of wines. For this reason, the aim of this study is to analyze the changes produced by different flor yeast strains in the volatilome and the aminogram of different wines from the criaderas and solera system during biological aging in the laboratory, simulating a flor yeast velum condition at different stages of the system. Results suggest that each strain metabolizes wine differently, finding that depending on the wine, some strains are better suited for the process than others. In addition, it is found that the content of biogenic amines in Fino Sherry wines, previously attributed to malolactic bacteria, varies according to the yeast strain metabolizing the wine, suggesting that flor yeast could be used to modify biogenic amines content during biological aging. Results indicate that the use of selected flor yeast starters in biological aging may be of interest to modulate some parameters during Fino Sherry wine aging.
Asunto(s)
Fermentación , Saccharomyces cerevisiae , Compuestos Orgánicos Volátiles , Vino , Vino/análisis , Vino/microbiología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Compuestos Orgánicos Volátiles/metabolismo , Compuestos Orgánicos Volátiles/análisis , Compuestos de Nitrógeno/metabolismo , Aminas Biogénicas/metabolismo , Aminas Biogénicas/análisisRESUMEN
Environmental conditions significantly impact the metabolism of Saccharomyces cerevisiae, a Crabtree-positive yeast that maintains a fermentative metabolism in high-sugar environments even in the presence of oxygen. Although the introduction of oxygen has been reported to induce alterations in yeast metabolism, knowledge of the mechanisms behind these metabolic adaptations in relation to redox cofactor metabolism and their implications in the context of wine fermentation remains limited. This study aimed to compare the intracellular redox cofactor levels, the cofactor ratios, and primary metabolite production in S. cerevisiae under aerobic and anaerobic conditions in synthetic grape juice. The molecular mechanisms underlying these metabolic differences were explored using a transcriptomic approach. Aerobic conditions resulted in an enhanced fermentation rate and biomass yield. Total NADP(H) levels were threefold higher during aerobiosis, while a decline in the total levels of NAD(H) was observed. However, there were stark differences in the ratio of NAD+/NADH between the treatments. Despite few changes in the differential expression of genes involved in redox cofactor metabolism, anaerobiosis resulted in an increased expression of genes involved in lipid biosynthesis pathways, while the presence of oxygen increased the expression of genes associated with thiamine, methionine, and sulfur metabolism. The production of fermentation by-products was linked with differences in the redox metabolism in each treatment. This study provides valuable insights that may help steer the production of metabolites of industrial interest during alcoholic fermentation (including winemaking) by using oxygen as a lever of redox metabolism.
Asunto(s)
Fermentación , Oxidación-Reducción , Oxígeno , Saccharomyces cerevisiae , Vino , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Oxígeno/metabolismo , Vino/microbiología , Vino/análisis , Anaerobiosis , Vitis/microbiología , Vitis/metabolismo , NAD/metabolismo , Etanol/metabolismo , NADP/metabolismo , Aerobiosis , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Coenzimas/metabolismoRESUMEN
The fermentative model yeast Saccharomyces cerevisiae has been extensively used to study the genetic basis of stress response and homeostasis. In this study, we performed quantitative trait loci (QTL) analysis of the high-temperature fermentation trait of the progeny from the mating of the S. cerevisiae natural isolate BCC39850 (haploid#17) and the laboratory strain CEN.PK2-1C. A single QTL on chromosome X was identified, encompassing six candidate genes (GEA1, PTK2, NTA1, NPA3, IRT1, and IML1). The functions of these candidates were tested by reverse genetic experiments. Deletion mutants of PTK2, NTA1, and IML1 showed growth defects at 42 °C. The PTK2 knock-out mutant also showed significantly reduced ethanol production and plasma membrane H+ ATPase activity and increased sensitivity to acetic acid, ethanol, amphotericin B (AMB), and ß-1,3-glucanase treatment. The CRISPR-Cas9 system was used to construct knock-in mutants by replacement of PTK2, NTA1, IML1, and NPA3 genes with BCC39850 alleles. The PTK2 and NTA1 knock-in mutants showed increased growth and ethanol production titers at 42 °C. These findings suggest an important role for the PTK2 serine/threonine protein kinase in regulating plasma membrane H+ ATPase activity and the NTA1 N-terminal amidase in protein degradation via the ubiquitin-proteasome system machinery, which affects tolerance to heat stress in S. cerevisiae.
Asunto(s)
Etanol , Fermentación , Calor , Sitios de Carácter Cuantitativo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Etanol/metabolismoRESUMEN
In scenarios where yeast and bacterial cells coexist, it is of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared, mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries, and analyzed using bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter Counter (at 1:1 and 100:1 ratios). We observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter Counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (p > .05). In contrast, the Coulter Counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (p < .05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasn't possible due to an overlap between yeast cell debris and bacterial cells. We conclude that each method has limitations, advantages, and disadvantages. ONE-SENTENCE SUMMARY: This study compares methods for simultaneously quantifying yeast and bacterial cells in a mixed sample, highlighting that in different cell proportions, some methods cannot quantify both cell types and present distinct advantages and limitations regarding time, cost, and precision.
Asunto(s)
Microbiología Industrial , Saccharomyces cerevisiae , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/citología , Microbiología Industrial/métodos , Citometría de Flujo/métodos , Recuento de Colonia Microbiana/métodos , Carga Bacteriana/métodos , Saccharum/microbiología , Microscopía/métodosRESUMEN
In their natural environments, microorganisms mainly operate at suboptimal growth conditions with fluctuations in nutrient abundance. The resulting cellular adaptation is subject to conflicting tasks: growth or survival maximisation. Here, we study this adaptation by systematically measuring the impact of a nitrogen downshift to 24 nitrogen sources on cellular metabolism at the single-cell level. Saccharomyces lineages grown in rich media and exposed to a nitrogen downshift gradually differentiate to form two subpopulations of different cell sizes where one favours growth while the other favours viability with an extended chronological lifespan. This differentiation is asymmetrical with daughter cells representing the new differentiated state with increased viability. We characterise the metabolic response of the subpopulations using RNA sequencing, metabolic biosensors and a transcription factor-tagged GFP library coupled to high-throughput microscopy, imaging more than 800,000 cells. We find that the subpopulation with increased viability is associated with a dormant quiescent state displaying differences in MAPK signalling. Depending on the identity of the nitrogen source present, differentiation into the quiescent state can be actively maintained, attenuated, or aborted. These results establish amino acids as important signalling molecules for the formation of genetically identical subpopulations, involved in chronological lifespan and growth rate determination.
Asunto(s)
Aminoácidos , Nitrógeno , Fenotipo , Saccharomyces cerevisiae , Aminoácidos/metabolismo , Nitrógeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de la Célula Individual , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Multiple researchers are reporting toxic agar, but the ultimate culprit remains unclear.
Asunto(s)
Agar , Saccharomyces cerevisiae , Schizosaccharomyces , Agar/provisión & distribución , Agar/toxicidad , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/crecimiento & desarrollo , Laboratorios , Algas Marinas/química , Algas Marinas/metabolismoRESUMEN
The life cycle of most non-conventional yeasts, such as Torulaspora delbrueckii (Td), is not as well-understood as that of Saccharomyces cerevisiae (Sc). Td is generally assumed to be haploid, which detracts from some biotechnological properties compared to diploid Sc strains. We analyzed the life cycle of several Td wine strains and found that they were mainly diploid during exponential growth in rich medium. However, most cells became haploid in stationary phase, as observed for Sc haploid heterothallic strains. When transferred and incubated in nutrient-deficient media, these haploid cells became polymorphic, enlarged, and transitioned to diploid or polyploid states. The increased ploidy, that mainly results from supernumerary mitosis without cytokinesis, was followed by sporulation. A similar response was observed in yeasts that remained alive during the second fermentation of base wine for sparkling wine making, or during growth in ethanol-supplemented medium. This response was not observed in the Sc yeast populations under any of the experimental conditions assayed, which suggests that it is a specific adaptation of Td to the stressful fermentation conditions. This response allows Td yeasts to remain alive and metabolically active longer during wine fermentation. Consequently, we designed procedures to increase the cell size and ploidy of haploid Td strains. Td inocula with increased ploidy showed enhanced fermentation efficiency compared to haploid inocula of the same strains.
Asunto(s)
Fermentación , Ploidias , Torulaspora , Vino , Vino/microbiología , Torulaspora/genética , Torulaspora/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Haploidia , Microbiología de Alimentos , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismoRESUMEN
Saccharomyces cerevisiae CCMA 0159 is reported as a promising biocontrol agent against ochratoxin A (OTA)-producing fungi in coffee. Coffea arabica and Coffea canephora (var. Conilon or Robusta) are the most widely consumed coffee species around the world, cultivated in tropical and subtropical regions, each exhibiting distinct physicochemical and sensory characteristics. The objective of this study was to compare the growth and OTA production by Aspergillus carbonarius, A. ochraceus, and A. westerdijkiae in C. arabica and C. canephora, along with assessing the efficiency of S. cerevisiae CCMA 0159 in biocontrolling ochratoxigenic fungi in both coffee varieties. A. carbonarius exhibited a higher growth rate and OTA production in both coffee varieties, with C. canephora showing particular susceptibility. Conversely, A. ochraceus and A. westerdijkiae demonstrated lower growth and OTA production. S. cerevisiae was effective in biocontrolling the fungal isolates, inhibiting over 80 % of A. carbonarius growth in both coffee varieties. Among the mechanisms of action of the biological control agent, the production of volatile organic compounds stands out. The results of this study confirm the significant potential of S. cerevisiae CCMA 0159 as a biocontrol agent against Aspergillus for application in coffee-producing areas.
Asunto(s)
Aspergillus , Coffea , Ocratoxinas , Saccharomyces cerevisiae , Ocratoxinas/biosíntesis , Aspergillus/crecimiento & desarrollo , Aspergillus/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Coffea/microbiología , Contaminación de Alimentos/prevención & control , Contaminación de Alimentos/análisis , Café/microbiología , Agentes de Control Biológico , Microbiología de AlimentosRESUMEN
Ginseng, a cornerstone of traditional herbal medicine in Asia, garnered significant attention for its therapeutic potential. Central to its pharmacological effects are ginsenosides, the primary active metabolites, many of which fall within the dammarane-type and share protopanaxadiol as a common precursor. Challenges in extracting protopanaxadiol and ginsenosides from ginseng arise due to their low concentrations in the roots. Emerging solutions involve leveraging microbial cell factories employing genetically engineered yeasts. Here, we optimized the fermentation conditions via the Design of Experiment, realizing 1.2 g/L protopanaxadiol in simple shake flask cultivations. Extrapolating the optimized setup to complex ginsenosides, like compound K, achieved 7.3-fold (0.22 g/L) titer improvements. Our adaptable fermentation conditions enable the production of high-value products, such as sustainable triterpenoids synthesis. Through synthetic biology, microbial engineering, and formulation studies, we pave the way for a scalable and sustainable production of bioactive compounds from ginseng.
Asunto(s)
Fermentación , Ginsenósidos , Triterpenos , Ginsenósidos/biosíntesis , Ginsenósidos/metabolismo , Triterpenos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Panax/metabolismo , Panax/crecimiento & desarrollo , Panax/química , Ingeniería Metabólica , SapogeninasRESUMEN
Aquaporin (AQP) channels found in all domains of life are transmembrane proteins which mediate passive transport of water, glycerol, signaling molecules, metabolites, and charged solutes. Discovery of new classes of ion-conducting AQP channels has been slow, likely reflecting time- and labor-intensive methods required for traditional electrophysiology. Work here defines a sensitive mass-throughput system for detecting AQP ion channels, identified by rescue of cell growth in the K+-transport-defective yeast strain CY162 following genetic complementation with heterologously expressed cation-permeable channels, using the well characterized human AQP1 channel for proof of concept. Results showed AQP1 conferred transmembrane permeability to cations which rescued survival in CY162 yeast. Comprehensive testing showed that growth response properties fully recapitulated AQP1 pharmacological agonist and antagonist profiles for activation, inhibition, dose-dependence, and structure-function relationships, demonstrating validity of the yeast screening tool for AQP channel identification and drug discovery efforts. This method also provided new information on divalent cation blockers of AQP1, pH sensitivity of antagonists, and ion permeability of human AQP6. Site-directed mutagenesis of AQP1 channel regulatory domains confirmed that yeast growth rescue was mediated by the introduced channels. Optical monitoring with a lithium-sensitive photoswitchable probe in living cells independently demonstrated monovalent cation permeability of AQP1 channels in yeast plasma membrane. Ion channel properties of AQP1 expressed in yeast were consistent with those of AQP1 expressed in Xenopus laevis oocyte and K+-transport defective Escherichia coli. Outcomes here establish a powerful new approach for efficient screening of phylogenetically diverse AQPs for yet untested functions as cation channels.
Asunto(s)
Acuaporina 1 , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Acuaporina 1/genética , Acuaporina 1/metabolismo , Animales , Acuaporinas/genética , Acuaporinas/metabolismo , Xenopus laevis , Descubrimiento de Drogas/métodos , Permeabilidad de la Membrana Celular , Oocitos/metabolismo , Potasio/metabolismoRESUMEN
Cell size and growth are intimately related across the evolutionary scale, but whether cell size is important to attain maximal growth or fitness is still an open question. We show that growth rate is a non-monotonic function of cell volume, with maximal values around the critical size of wild-type yeast cells. The transcriptome of yeast and mouse cells undergoes a relative inversion in response to cell size, which we associate theoretically and experimentally with the necessary genome-wide diversity in RNA polymerase II affinity for promoters. Although highly expressed genes impose strong negative effects on fitness when the DNA/mass ratio is reduced, transcriptomic alterations mimicking the relative inversion by cell size strongly restrain cell growth. In all, our data indicate that cells set the critical size to obtain a properly balanced transcriptome and, as a result, maximize growth and fitness during proliferation.
Asunto(s)
Tamaño de la Célula , ARN Polimerasa II , Saccharomyces cerevisiae , Transcriptoma , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Animales , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Ratones , Regulación Fúngica de la Expresión Génica , Regiones Promotoras Genéticas , Proliferación Celular , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Hyperosmotic stress tolerance is crucial for Saccharomyces cerevisiae in producing value-added products from renewable feedstock. The limited understanding of its tolerance mechanism has impeded the application of these microbial cell factories. Previous studies have shown that Med3 plays a role in hyperosmotic stress in S. cerevisiae. However, the specific function of Med3 in hyperosmotic stress tolerance remains unclear. In this study, we showed that the deletion of the mediator Med3 impairs S. cerevisiae growth under hyperosmotic stress. Phenotypic analyses and yeast two-hybrid assays revealed that Med3 interacts with the transcription factor Stb5 to regulate the expression of the genes gnd1 and ald6, which are involved in NADPH production under hyperosmotic stress conditions. The deletion of med3 resulted in a decrease in intracellular NADPH content, leading to increased oxidative stress and elevated levels of intracellular reactive oxygen species under hyperosmotic stress, thereby impacting bud formation. These findings highlight the significant role of Med3 as a regulator in maintaining NADPH generation and redox homeostasis in S. cerevisiae during hyperosmotic stress.IMPORTANCEHyperosmotic stress tolerance in the host strain is a significant challenge for fermentation performance in industrial production. In this study, we showed that the S. cerevisiae mediator Med3 is essential for yeast growth under hyperosmotic conditions. Med3 interacts with the transcription factor Stb5 to regulate the expression of genes involved in the NADPH-generation system during hyperosmotic stress. Adequate NADPH ensures the timely removal of excess reactive oxygen species and supports bud formation under these conditions. This work highlights the crucial role of Med3 as a regulator in maintaining NADPH generation and redox homeostasis in S. cerevisiae during hyperosmotic stress.
Asunto(s)
NADP , Presión Osmótica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , NADP/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Regulación Fúngica de la Expresión Génica , Estrés Oxidativo , Complejo Mediador/metabolismo , Complejo Mediador/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The fitness effects of new mutations determine key properties of evolutionary processes. Beneficial mutations drive evolution, yet selection is also shaped by the frequency of small-effect deleterious mutations, whose combined effect can burden otherwise adaptive lineages and alter evolutionary trajectories and outcomes in clonally evolving organisms such as viruses, microbes, and tumors. The small effect sizes of these important mutations have made accurate measurements of their rates difficult. In microbes, assessing the effect of mutations on growth can be especially instructive, as this complex phenotype is closely linked to fitness in clonally evolving organisms. Here, we perform high-throughput time-lapse microscopy on cells from mutation-accumulation strains to precisely infer the distribution of mutational effects on growth rate in the budding yeast, Saccharomyces cerevisiae. We show that mutational effects on growth rate are overwhelmingly negative, highly skewed towards very small effect sizes, and frequent enough to suggest that deleterious hitchhikers may impose a significant burden on evolving lineages. By using lines that accumulated mutations in either wild-type or slippage repair-defective backgrounds, we further disentangle the effects of 2 common types of mutations, single-nucleotide substitutions and simple sequence repeat indels, and show that they have distinct effects on yeast growth rate. Although the average effect of a simple sequence repeat mutation is very small (approximately 0.3%), many do alter growth rate, implying that this class of frequent mutations has an important evolutionary impact.
Asunto(s)
Aptitud Genética , Repeticiones de Microsatélite , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Repeticiones de Microsatélite/genética , Mutación/genética , Acumulación de MutacionesRESUMEN
The pieddecuve (PdC) technique involves using a portion of grape must to undergo spontaneous fermentation, which is then used to inoculate a larger volume of must. This allows for promoting autochthonous yeasts present in the must, which can respect the typicality of the resulting wine. However, the real impact of this practice on the yeast population has not been properly evaluated. In this study, we examined the effects of sulphur dioxide (SO2), temperature, ethanol supplementation, and time on the dynamics and selection of yeasts during spontaneous fermentation to be used as PdC. The experimentation was conducted in a synthetic medium and sterile must using a multi-species yeast consortium and in un-inoculated natural grape must. Saccharomyces cerevisiae dominated both the PdC and fermentations inoculated with commercial wine yeast, displaying similar population growth regardless of the tested conditions. However, using 40 mg/L of SO2 and 1% (v/v) ethanol during spontaneous fermentation of Muscat of Alexandria must allowed the non-Saccharomyces to be dominant during the first stages, regardless of the temperature tested. These findings suggest that it is possible to apply the studied parameters to modulate the yeast population during spontaneous fermentation while confirming the effectiveness of the PdC methodology in controlling alcoholic fermentation.
Asunto(s)
Etanol , Fermentación , Saccharomyces cerevisiae , Dióxido de Azufre , Vitis , Vino , Levaduras , Vitis/microbiología , Vino/microbiología , Vino/análisis , Etanol/metabolismo , Dióxido de Azufre/farmacología , Dióxido de Azufre/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Levaduras/metabolismo , Temperatura , Estrés FisiológicoRESUMEN
In recent years, the boom of the craft beer industry refocused the biotech interest from ethanol production to diversification of beer aroma profiles. This study analyses the fermentative phenotype of a collection of non-conventional yeasts and examines their role in creating new flavours, particularly through co-fermentation with industrial Saccharomyces cerevisiae. High-throughput solid and liquid media fitness screening compared the ability of eight Saccharomyces and four non-Saccharomyces yeast strains to grow in wort. We determined the volatile profile of these yeast strains and found that Hanseniaspora vineae displayed a particularly high production of the desirable aroma compounds ethyl acetate and 2-phenethyl acetate. Given that H. vineae on its own can't ferment maltose and maltotriose, we carried out mixed wort co-fermentations with a S. cerevisiae brewing strain at different ratios. The two yeast strains were able to co-exist throughout the experiment, regardless of their initial inoculum, and the increase in the production of the esters observed in the H. vineae monoculture was maintained, alongside with a high ethanol production. Moreover, different inoculum ratios yielded different aroma profiles: the 50/50 S. cerevisiae/H. vineae ratio produced a more balanced profile, while the 10/90 ratio generated stronger floral aromas. Our findings show the potential of using different yeasts and different inoculum combinations to tailor the final aroma, thus offering new possibilities for a broader range of beer flavours and styles.
Asunto(s)
Cerveza , Fermentación , Hanseniaspora , Odorantes , Saccharomyces cerevisiae , Cerveza/microbiología , Cerveza/análisis , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Hanseniaspora/metabolismo , Hanseniaspora/crecimiento & desarrollo , Odorantes/análisis , Compuestos Orgánicos Volátiles/metabolismo , Compuestos Orgánicos Volátiles/análisis , Compuestos Orgánicos Volátiles/química , Etanol/metabolismo , Aromatizantes/metabolismo , Aromatizantes/química , Acetatos/metabolismo , Técnicas de Cocultivo , Alcohol Feniletílico/análogos & derivadosRESUMEN
This work presents a low-cost, open-source turbidimeter, the 'Erlenmeter', designed to monitor the growth of microorganisms in batch cultures. It is easy to build, based exclusively on inexpensive off-the-shelf electronic components and 3D-printed parts. The Erlenmeter allows measuring the optical density of cultures on standard Erlenmeyer flasks without the need to open the flasks to collect aliquots, ensuring speed, minimal use of consumables, and elimination of the risk of contamination. These features make it particularly well-suited not just for routine research assays but also for experimental teaching. Here we illustrate the use of the Erlenmeter turbidimeter to record the growth of the microalga Phaeodactylum tricornutum, of the bacterium Escherichia coli, and of the yeast Saccharomyces cerevisiae, model organisms that are widely used in research and teaching. The Erlenmeter allows a detailed characterization of the growth curves of all organisms, confirming its usefulness for studying microbial populations dynamics both for research purposes and in classroom settings.
Asunto(s)
Escherichia coli , Nefelometría y Turbidimetría , Saccharomyces cerevisiae , Escherichia coli/crecimiento & desarrollo , Escherichia coli/aislamiento & purificación , Saccharomyces cerevisiae/crecimiento & desarrollo , Nefelometría y Turbidimetría/instrumentación , Nefelometría y Turbidimetría/métodos , Microalgas/crecimiento & desarrollo , FenotipoRESUMEN
Membrane potential is a useful marker for antimicrobial susceptibility testing (AST) due to its fundamental roles in cell function. However, the difficulties associated with measuring the membrane potential in microbes restrict its broad application. In this study, we present bioelectrical AST (BeAST) using the model fungus Saccharomyces cerevisiae. Using fluorescent indicators [DiBAC4(3), ThT, and TMRM], we measured plasma and mitochondrial membrane-potential dynamics upon electric stimulation. We find that a 2.5 second electric stimulation induces hyperpolarization of plasma membrane lasting 20 minutes in vital S. cerevisiae, but depolarization in inhibited cells. The numerical simulation of FitzHugh-Nagumo model successfully recapitulates vitality-dependent dynamics. The model also suggests that the magnitude of plasma-membrane potential dynamics (PMD) correlates with the degree of inhibition. To test this prediction and to examine if BeAST can be used for assessing novel anti-fungal compounds, we treat cells with biogenic silver nanoparticles (bioAgNPs) synthesized using orange fruit flavonoids and Fusarium oxysporum. Comparing BeAST with optical density assay alongside various stressors, we show that PMD correlates with the magnitude of growth inhibitions. These results suggest that BeAST holds promise for screening anti-fungal compounds, offering a valuable approach to tackling antimicrobial resistance. IMPORTANCE: Rapid assessment of the efficacy of antimicrobials is important for optimizing treatments, avoiding misuse and facilitating the screening of new antimicrobials. The need for rapid antimicrobial susceptibility testing (AST) is growing with the rise of antimicrobial resistance. Here, we present bioelectrical AST (BeAST). Combining time-lapse microscopy and mathematical modeling, we show that electrically induced membrane potential dynamics of yeast cells correspond to the strength of growth inhibition. Furthermore, we demonstrate the utility of BeAST for assessing antimicrobial activities of novel compounds using biogenic silver nanoparticles.
Asunto(s)
Antifúngicos , Potenciales de la Membrana , Pruebas de Sensibilidad Microbiana , Saccharomyces cerevisiae , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Antifúngicos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Fusarium/efectos de los fármacos , Fusarium/crecimiento & desarrollo , Membrana Celular/efectos de los fármacosRESUMEN
The effectivity of utilization of exogenous sterols in the yeast Saccharomyces cerevisiae exposed to hypoxic stress is dependent on the sterol structure. The highly imported sterols include animal cholesterol or plant sitosterol, while ergosterol, typical of yeasts, is imported to a lesser extent. An elevated utilization of non-yeast sterols is associated with their high esterification and relocalization to lipid droplets (LDs). Here we present data showing that LDs and sterol esterification play a critical role in the regulation of the accumulation of non-yeast sterols in membranes. Failure to form LDs during anaerobic growth in media supplemented with cholesterol or sitosterol resulted in an extremely long lag phase, in contrast to normal growth in media with ergosterol or plant stigmasterol. Moreover, in hem1∆, which mimics anaerobiosis, neither cholesterol nor sitosterol supported the growth in an LD-less background. The incorporation of non-ergosterol sterols into the membranes affected fundamental membrane characteristics such as relative membrane potential, permeability, tolerance to osmotic stress and the formation of membrane domains. Our findings reveal that LDs assume an important role in scenarios wherein cells are dependent on the utilization of exogenous lipids, particularly under anoxia. Given the diverse lipid structures present in yeast niches, LDs fulfil a protective role, mitigating the risk of excessive accumulation of potentially toxic steroids and fatty acids in the membranes. Finally, we present a novel function for sterols in a model eukaryotic cell - alleviation of the lipotoxicity of unsaturated fatty acids.