Search Results (92)

In this study, we investigated the impact of two native strains of Starmerella bacillaris, used both in pure culture and in a co-inoculation with Saccharomyces cerevisiae, on the volatile profile of a chemically defined fermented model must. The focus of this study was the production of monoterpenes and sesquiterpenes and their potential sensory contributions. Geraniol and linalool were detected in all fermentations with Starmerella bacillaris, in ranges of 26.7–43.9 µg/L and 34.3–41.3 µg/L, respectively, independent of the inoculation strategy used. Both strains produced concentrations above their respective odour thresholds of 20 µg/L and 25.5 µg/L. Odour activity value (OAV) analysis confirmed that fermentations with Starmerella bacillaris, particularly under co-inoculation conditions, generated the highest OAVs for these monoterpenes. Citronellol was only detected in mixed fermentations, while nerolidol and farnesol isomers were produced in variable amounts, depending on the strain and inoculation strategy, at concentrations below the odour threshold. These findings demonstrate the ability of Starmerella bacillaris to facilitate de novo biosynthesis of linalool, geraniol, and sesquiterpenes during alcoholic fermentation—in the case of linalool and geraniol, at concentrations exceeding their respective odour thresholds—highlighting the biotechnological potential of these native strains to enhance aroma in wines, particularly those made from neutral grape varieties. Full article

This study explores the production of anhydrous ethanol from discarded fruits, aiming to determine optimal fermentation conditions and evaluate the feasibility of a green separation technology. Fermentation experiments were performed using juices from Psidium guajava (S1), Carica paapaya (S2), and mucilage residues of Coffea arabica (S3). All fermentations were carried out at a pH of 4.5 for 7 days in 1 L bioreactors. A full 2² factorial design was applied to evaluate the effects of two variables: yeast type (commercial Saccharomyces cerevisiae [CY] vs. native yeast [NY]) and temperature (21 °C vs. 30 °C). Higher ethanol concentrations were achieved with CY at 30 °C, yielding 6.79% ethanol for S3. A multi-criteria matrix prioritized coffee residues due to their high ethanol yield, biomass availability, and economic viability. The ethanol was dehydrated using a packed-bed bioadsorption system with crushed corn, which increased purity from 6.7% v/v to 98.9% v/v in two stages, while avoiding azeotropic limitations. Energy analysis revealed low specific consumption (3.68 MJ/kg), outperforming conventional distillation. The results of this study, obtained at operating temperatures of 30 °C and 21 °C, a pH of 4.5, and an operating time of 7 days in a 1L bioreactor, demonstrate ethanol concentrations of 6.79%, confirming the technical feasibility of using agricultural waste as a raw material and validating the efficiency of a bioadsorption-based dehydration system. These findings address the current gap in integrating green ethanol separation with low-cost agricultural residues and highlight a sustainable alternative for decentralized bioethanol production. Full article

Histone 3 lysine 4 methylation (H3K4me) is an evolutionarily conserved epigenetic marker associated with transcriptional activation, playing a crucial role in growth and development. In yeast, all forms of H3K4 methylation are catalyzed by the COMPASS complex. However, purifying endogenous COMPASS remains challenging due to its low abundance, compositional complexity, and structural instability, resulting in low yield, poor purity, and heterogeneity in isolated complexes. These technical limitations have impeded the structural elucidation of the intact COMPASS complex and contributed to inconsistencies in reported in vitro enzymatic activity, thereby limiting a comprehensive understanding of its functions. Here, we present an optimized tandem affinity purification strategy that enables the high-yield isolation of native COMPASS from Saccharomyces cerevisiae with >99% purity and intact subunit composition, as validated by biochemical analyses. Using recombinant nucleosomes as substrates, we systematically characterized its catalytic properties and found that endogenously purified COMPASS exhibited strict dependence on H2B ubiquitination for catalyzing H3K4 methylation. This work establishes an efficient purification strategy for future structural and functional studies of COMPASS and provides critical insights into its catalytic properties. Full article

All traditional mezcal producers use artisan methods to produce mezcal. The low technological development in the elaboration processes results in low yield and high residual sugar concentration. First, this work optimized the concentration of initial sugars and yeast-assimilable nitrogen (YAN) in Agave durangensis juice fermentation at the laboratory level. A yield near 0.49 g EtOH/g sugar and a productivity of 1.54 g EtOH/L*h was obtained with an initial sugar concentration of 120 g/L and a YAN concentration of 0.227 g/L. Only Saccharomyces cerevisiae was found after 24 h of incubation at laboratory level, using MALDI-TOF identification. Agave durangensis heads crushed by the artisan process were used to test the inoculant performance. A mezcal yield of 11.6 kg agave/L of mezcal was obtained using the S cerevisiae inoculant and nitrogen addition, which was significantly different (p < 0.05) from other treatments. The population dynamics during fermentation were analyzed through isolation and identification using MALDI-TOF. Several yeast species (Pichia kluyveri, Torulaspora delbrueckii, Zygosaccharomyces bailii, and Saccharomyces cerevisiae) were found at the beginning of fermentation. Nonetheless, only S. cerevisiae was found at the end of fermentation. The implantation of the inoculant used was confirmed through the comparative analysis of amplification patterns of the GTG5 microsatellite of the strains identified as S. cerevisiae, finding that the inoculated strain proportion was greater than 80% of the yeast population. A technological alternative to increase the efficiency of the process is combining the addition of YAN and the inoculation of the native S. cerevisiae, which was isolated from artisan alcoholic fermentation of agave to produce mezcal. Full article

The introduction of heterologous pathways into microbial cell compartments offers several potential advantages, including increasing enzyme concentrations and reducing competition with native pathways, making this approach attractive for producing complex metabolites like fatty acids and fatty alcohols. However, measuring subcellular concentrations of these metabolites remains technically challenging. Here, we explored 3-hydroxypropionic acid (3-HP), readily quantifiable and sharing the same precursors—acetyl-CoA, NADPH, and ATP—with the above-mentioned products, as a reporter metabolite for peroxisomal engineering in the yeast Komagataella phaffii. To this end, the malonyl-CoA reductase pathway for 3-HP production was targeted into the peroxisome of K. phaffii using the PTS1-tagging system, and further tested with different carbon sources. Thereafter, we used compartmentalized 3-HP production as a reporter system to showcase the impact of different strategies aimed at enhancing the peroxisomal NADPH pool. Co-overexpression of genes encoding a NADPH-dependent redox shuttle from Saccharomyces cerevisiae (IDP2/IDP3) significantly increased 3-HP yields across all substrates, whereas peroxisomal targeting of the S. cerevisiae NADH kinase Pos5 failed to improve 3-HP production. This study highlights the potential of using peroxisomal 3-HP production as a biosensor for evaluating peroxisomal acetyl-CoA and NAPDH availability by simply quantifying 3-HP, demonstrating its potential for peroxisome-based metabolic engineering in yeast. Full article

Copper is an essential micronutrient and the ability to scavenge tightly bound or trace levels of copper ions at the host–pathogen interface is vital for fungal proliferation in animal hosts. Recent studies suggest that trace metal ion acquisition is critical for the establishment and propagation of Pseudogymnoascus destructans, the fungal pathogen responsible for white-nose syndrome (WNS), on their bat host. However, little is known about these metal acquisition pathways in P. destructans. In this study, we report the characterization of the P. destructans high-affinity copper transporter VC83_00191 (PdCTR1a), which is implicated as a virulence factor associated with the WNS disease state. Using Saccharomyces cerevisiae as a recombinant expression host, we find that PdCTR1a can efficiently traffic Cu ions into the yeast cytoplasm. Complementary studies in the native P. destructans fungus provide evidence that PdCTR1a transcripts and protein levels are dictated by Cu-bioavailability in the growth media. Our study demonstrates that PdCTR1a is a functional high-affinity copper transporter and is relevant to Cu homeostasis pathways in P. destructans. Full article

Yeast-yeast interactions have been studied mainly using wine yeasts. However, studies are rare for native yeasts from agave juice fermentation. Therefore, this work used strains isolated from the alcoholic fermentation of agave to study the survival of non-Saccharomyces yeasts co-cultivated with Saccharomyces cerevisiae in media of different nutritional qualities. First, the feasibility of using simple and low-cost culture media was demonstrated to study the interactions between Saccharomyces cerevisiae and non-Saccharomyces yeasts. The results presented here demonstrated the antagonistic effect exerted by S. cerevisiae on Torulaspora delbrueckii, which showed a more significant loss of viability. However, the nutritional composition of the culture medium also influences this effect. It was clear that a nutritionally rich medium improved the survival of non-Saccharomyces yeasts. Lastly, the change in the survival of non-Saccharomyces yeasts also entails a variation in the concentration and diversity of minor volatile compounds produced during fermentation. This was observed in the variety and relative abundance of compounds belonging to the most numerous chemical families, such as alcohols, esters, and terpenes. Full article

The yeast Saccharomyces cerevisiae ensures successful fermentation in winemaking, although the persistent use of commercial strains lead to the loss of aroma complexity of wines. Hence, the research of indigenous S. cerevisiae with proper oenological features and well adapted to specific wine-growing areas become of great interest for winemakers. Here, 206 pure cultures of S. cerevisiae were isolated from two wineries during a two-year sampling campaign and bio-typed through interdelta sequences analyses with the aim to evaluate the occurrence and persistence of the S. cerevisiae wild population linked to each winery. Both wineries belong to the same Verdicchio DOC wine area (Castelli di Jesi), and never used commercial yeasts during fermentation. Results showed 19 different biotypes with a specific population of S. cerevisiae in each winery, without cross-contamination with each other and with commercial starter strains. Moreover, inside each winery a persistence of some dominant biotypes was observed over time (three biotypes in winery 1; 95% of isolates in the two years and one biotype in winery 2; 20% of isolates in the two years), indicating a sort of “winery-effect”. The evaluation of S. cerevisiae populations for the oenological characters by microfermentations showed a proper and well distinct aromatic imprinting on the resulted wines supporting the concept of “winery effect”. Full article

The Brazilian Cerrado region has a rich plant diversity, with fruits that have peculiar and unique sensory characteristics. For these reasons, using these fruits for biotechnological production is a promising alternative, mainly to protect this biome from deforestation and degradation. The production of fermented acetic acid is an option to add value to native fruits and offer the market beverages with better nutritional quality and bioactive compounds. This work aimed to characterize fruits and to develop cagaita (Eugenia dysenterica DC.) acetic fermented beverage. The fruits were subjected to physical-chemical analyses in the first part. Subsequently, different treatments for fermentation were tested using two types of enzymes (amylase and pectinase), two subspecies of Saccharomyces cerevisiae yeast (UFLA CA11 and thermoresistant LNF Angel), and the chaptalization of the must with sucrose (16 °Brix). Alcoholic fermentation was carried out in an incubator with temperature control at 34 ± 1 °C. The pH, total soluble solids, titratable acidity, alcohol content, and density of the fermented products were monitored daily. The chaptalized must with amylase addition and thermoresistant yeast had the best performance during alcoholic fermentation, demonstrating that thermoresistant yeast is an economically advantageous and efficient alternative for the cagaita juice fermentation process. Subsequently, acetic fermentation was carried out using the slow method. Heat-resistant yeast without added enzymes was used to produce cagaita acetic fermented beverages within the parameters of the Brazilian legislation. Furthermore, phenolic compounds and antioxidant activity in the final product were observed. The work demonstrated the possibility of using cagaita fruits in biotechnological processes to produce new food products. Full article

In view of climate change and the increasingly antagonistic wine market, the exploitation of native genetic resources is revisited in relation to sustainable wine production. ‘Sideritis’ is a late-ripening Greek grape variety, which is quite promising for counteracting wine quality issues associated with the annual temperature rise. The aim of this study was to improve the quality and to enhance the aroma of ‘Sideritis’ wine through the use of native yeasts. To improve vinification, Hanseniaspora opuntiae L1 was used along with Saccharomyces cerevisiae W7 in mixed fermentations (SQ). The addition of H. οpuntiae significantly altered the chemical profile of the wine compared to the single-inoculated fermentations with W7 (IS). H. opuntiae increased all the acetate esters, except for hexyl acetate and (Z)-3-hexen-1-ol acetate. The concentration of 2-phenylethyl acetate, which imparts flowery and sweet notes, exhibited a 2.6-fold increase in SQ as compared to IS wines. SQ also showed higher levels in several ethyl esters, including ethyl butyrate, ethyl heptanoate and ethyl 7-octenoate, which are associated with fruity notes compared to IS. H. opuntiae produced citronellol, a terpene associated with rose and green notes, and increased the overall acceptance of the wine. Present results are thus quite promising for improving ‘Sideritis’ wine quality towards a sustainable wine production in Greece in view of global warming. Full article

Terpenes are diverse specialized metabolites naturally found within plants and have important roles in inter-species communication, adaptation and interaction with the environment. Their industrial applications span a broad range, including fragrances, flavors, cosmetics, natural colorants to agrochemicals and therapeutics, yet formal chemical synthesis is economically challenging due to structural complexities. Engineering terpene biosynthesis could represent an alternative in microbial biotechnological workhorses, such as Saccharomyces cerevisiae or Escherichi coli, utilizing sugars or complex media as feedstocks. Host species that metabolize renewable and affordable carbon sources may offer unique sustainable biotechnological alternatives. Methylotrophs are bacteria with the capacity to utilize one-carbon feedstocks, such as methanol or formate. They colonize the phyllosphere (above-ground area) of plants, and many accumulate abundant carotenoid pigments. Methylotrophs have the capacity to take up and use a subset of the rare earth elements known as lanthanides. These metals can enhance one-carbon (methylotrophic) metabolism. Here, we investigated whether manipulating the metabolism enables and enhances terpene production. A carotenoid-deficient mutant potentially liberates carbon, which may contribute to bioproduct accumulation. To test this hypothesis, terpene-producing bacterial strains regulated by two distinct promoters were generated. Wildtype Methylobacterium extorquens, ∆Meta1_3665, a methylotrophic mutant lacking the carotenoid pathway, and an E. coli strain were transformed with an exogenous terpene pathway and grown both in the presence and absence of lanthanides. The extraction, and the comparison of analytical profiles, provided evidence that engineered cultured M. extorquens under control of a native, inducible methylotrophic promoter can yield the sesquiterpene patchoulol when supplemented with lanthanide. In contrast, using a moderate-strength constitutive promoter failed to give production. We demonstrated colonization of the phyllosphere with the engineered strains, supporting the future engineering of selected species of the plant microbiome and with promising implications for the synthetic biology of small molecules. Full article

Saccharomyces cerevisiae var. boulardii (Sb) is currently receiving significant attention as a synthetic probiotic platform due to its ease of manipulation and inherent effectiveness in promoting digestive health. A comprehensive exploration of Sb and other S. cerevisiae strains (Sc) would shed light on the refinement and expansion of their therapeutic applications. This review aims to provide a thorough overview of Saccharomyces yeasts from their native health benefits to recent breakthroughs in the engineering of Saccharomyces yeasts as synthetic therapeutic platforms. Molecular typing and phenotypic assessments have uncovered notable distinctions, including the superior thermotolerance and acid tolerance exhibited by Sb, which are crucial attributes for probiotic functions. Moreover, parabiotic and prebiotic functionalities originating from yeast cell wall oligosaccharides have emerged as pivotal factors influencing the health benefits associated with Sb and Sc. Consequently, it has become imperative to select an appropriate yeast strain based on a comprehensive understanding of its actual action in the gastrointestinal tract and the origins of the targeted advantages. Overall, this review underscores the significance of unbiased and detailed comparative studies for the judicious selection of strains. Full article

Two strains of Saccharomyces cerevisiae (Sc01 and Sc02) and one strain of Wickerhamomyces anomalus (Wa) were isolated from organic Verdejo spontaneous fermentations and used for the development of experimental winemaking. Sc01 and Sc02 represented 52.7% of the population of the Saccharomyces strains isolated throughout the fermentation process. W. anomalus appeared as the predominant species among the non-Saccharomyces yeasts. Wa turned out to be the strain of this species with the shortest lag phase and positive enzymatic activities, and it was selected for white wine production. Fermentations with unique inoculation of S. cerevisiae strains were compared with sequential inoculation with W. anomalus. The results showed that the sequential inoculations did not affect the fermentation kinetics or physicochemical characteristics of the wines compared with the unique inoculations. However, this study identified a significant impact on the aromatic profiles of the produced wines due to the sequential inoculations. This modification resulted in a similar new aromatic profile in both sequential inoculations, demonstrating common characteristics related to the contribution of W. anomalus. In general, the sequential fermentations were mainly characterized by lower levels of acetate esters and an increase in ethyl acetate levels, whereas lower levels of ethyl octanoate and ethyl dec-9-enoate were detected. Propan-1-ol and butan-1-ol showed an increase in the sequential fermentations, while 4-methylpentan-1-ol and 2-phenylethanol were found in lower concentrations. These results highlight the great influence that the presence of specific strains of native non-Saccharomyces yeasts exerts on the characteristics of elaborate wines. Full article

Increased human population and the rapid decline of fossil fuels resulted in a global tendency to look for alternative fuel sources. Environmental concerns about fossil fuel combustion led to a sharp move towards renewable and environmentally friendly biofuels. Ethanol has been the primary fossil fuel alternative due to its low carbon emission rates, high octane content and comparatively facile microbial production processes. In parallel to the increased use of bioethanol in various fields such as transportation, heating and power generation, improvements in ethanol production processes turned out to be a global hot topic. Ethanol is by far the leading yeast output amongst a broad spectrum of bio-based industries. Thus, as a well-known platform microorganism and native ethanol producer, baker’s yeast Saccharomyces cerevisiae has been the primary subject of interest for both academic and industrial perspectives in terms of enhanced ethanol production processes. Metabolic engineering strategies have been primarily adopted for direct manipulation of genes of interest responsible in mainstreams of ethanol metabolism. To overcome limitations of rational metabolic engineering, an alternative bottom-up strategy called inverse metabolic engineering has been widely used. In this context, evolutionary engineering, also known as adaptive laboratory evolution (ALE), which is based on random mutagenesis and systematic selection, is a powerful strategy to improve bioethanol production of S. cerevisiae. In this review, we focus on key examples of metabolic and evolutionary engineering for improved first- and second-generation S. cerevisiae bioethanol production processes. We delve into the current state of the field and show that metabolic and evolutionary engineering strategies are intertwined and many metabolically engineered strains for bioethanol production can be further improved by powerful evolutionary engineering strategies. We also discuss potential future directions that involve recent advancements in directed genome evolution, including CRISPR-Cas9 technology. Full article

Fermentation is a critical step in the production of coffee when following standard wet processing, one of the most common methods used to remove the mucilage layer from coffee cherries. During this step, the de-pulped coffee cherries undergo fermentation with native yeast that modifies the flavor profile of the resultant coffee. This study aimed to ferment green coffee beans using commercial yeast strains from beer and wine prized for their ability to produce specific flavors, and subsequently evaluate the aroma and flavor of the coffee using coffee consumers. Four Saccharomyces cerevisiae strains were used: Belgian Ale, Sourvisiae, 71 B, and Tropical IPA, along with one non-Saccharomyces, Toluraspora delbrueckii (Biodiva), and a non-inoculated control sample. The green coffee beans underwent a controlled wet fermentation for 72 h, followed by roasting, grinding, and brewing. Results showed that flavor profiles varied broadly by yeast strain, suggesting that producing novel flavors in coffee through fermentation is feasible and that these flavors survive the roasting process; however, higher liking scores were still reported for the control sample compared to the fermented samples. Biodiva, a strain used in wine to produce esters and fruity flavors, resulted in coffee with highly fruity notes, and all strains were rated more floral than the control, while the sample fermented with Sourvisiae yeast used in the brewing of sour ales resulted in coffee that was both perceived as more sour and had the lowest pH, likely due to the degree of lactic acid this strain is engineered to produce. Further, there were significant color differences between the samples. In conclusion, fermenting green coffee beans with brewing and winemaking yeast strains strongly impacted the flavor and aroma of the resultant coffee; however, evaluating larger panels of strains or optimizing strain performance may yield flavor profiles more suitable for coffee. Full article