Bioactive volatiles of brewer's yeasts: Antifungal action of compounds produced during wort fermentation on Aspergillus sp.

Previous investigations proved the potential of Saccharomyces cerevisiae MBELGA62 and Pichia kudriavzevii MBELGA61 as suitable biocontrolling agents against Aspergillus sp. through the production of soluble and volatile bioactive antifungal compounds. The present study delves into those finding by means of the identification of the volatile compounds produced by brewer's strains that demonstrated fungistatic and fungicidal effects against Aspergillus flavus and A. parasiticus when cultured in brewer's wort agar plates. Traditional brewer's yeasts such as S. cerevisiae MBELGA62 and Saccharomyces pastorianus SAFS235 synthetize volatiles that fully inhibited mycelial development for up to 9 days at 30 °C. The non-conventional brewer's strains P. kudriavzevii MBELGA61 and Meyerozyma guilliermondii MUS122 increased the lag phase by >100% and significantly reduced the fungal growth rate by 27.5-43.0% and 15.4-31.4%, respectively. In this context, 2-phenylethanol, 2-phenylethyl acetate and benzyl alcohol were identified as the main antifungal agents involved in Aspergillus sp.'s inhibition.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 19: Suitability of taxonomic units notified to EFSA until September 2023.

The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. The QPS approach is based on an assessment of published data for each taxonomic unit (TU), with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a TU are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 71 microorganisms notified to EFSA between April and September 2023 (30 as feed additives, 22 as food enzymes or additives, 7 as novel foods and 12 from plant protection products [PPP]), 61 were not evaluated because: 26 were filamentous fungi, 1 was Enterococcus faecium, 5 were Escherichia coli, 1 was a bacteriophage (all excluded from the QPS evaluation) and 28 were TUs that already have a QPS status. The other 10 notifications belonged to 9 TUs which were evaluated for a possible QPS status: Ensifer adhaerens and Heyndrickxia faecalis did not get the QPS recommendation due to the limited body of knowledge about their occurrence in the food and/or feed chains and Burkholderia ubonensis also due to its ability to generate biologically active compounds with antimicrobial activity; Klebsiella pneumoniae, Serratia marcescens and Pseudomonas putida due to safety concerns. K. pneumoniae is excluded from future QPS evaluations. Chlamydomonas reinhardtii is recommended for QPS status with the qualification 'for production purposes only'; Clostridium tyrobutyricum is recommended for QPS status with the qualification 'absence of genetic determinants for toxigenic activity'; Candida oleophila has been added as a synonym of Yarrowia lipolytica. The Panel clarifies the extension of the QPS status for genetically modified strains.

Divergence in the Saccharomyces Species' Heat Shock Response Is Indicative of Their Thermal Tolerance.

The Saccharomyces species have diverged in their thermal growth profile. Both Saccharomyces cerevisiae and Saccharomyces paradoxus grow at temperatures well above the maximum growth temperature of Saccharomyces kudriavzevii and Saccharomyces uvarum but grow more poorly at lower temperatures. In response to thermal shifts, organisms activate a stress response that includes heat shock proteins involved in protein homeostasis and acquisition of thermal tolerance. To determine whether Saccharomyces species have diverged in their response to temperature, we measured changes in gene expression in response to a 12 °C increase or decrease in temperature for four Saccharomyces species and their six pairwise hybrids. To ensure coverage of subtelomeric gene families, we sequenced, assembled, and annotated a complete S. uvarum genome. In response to heat, the cryophilic species showed a stronger stress response than the thermophilic species, and the hybrids showed a mixture of parental responses that depended on the time point. After an initial strong response indicative of high thermal stress, hybrids with a thermophilic parent resolved their heat shock response to become similar to their thermophilic parent. Within the hybrids, only a small number of temperature-responsive genes showed consistent differences between alleles from the thermophilic and cryophilic species. Our results show that divergence in the heat shock response is mainly a consequence of a strain's thermal tolerance, suggesting that cellular factors that signal heat stress or resolve heat-induced changes are relevant to thermal divergence in the Saccharomyces species.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 18: Suitability of taxonomic units notified to EFSA until March 2023.

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms, intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 38 microorganisms notified to EFSA between October 2022 and March 2023 (inclusive) (28 as feed additives, 5 as food enzymes, food additives and flavourings, 5 as novel foods), 34 were not evaluated because: 8 were filamentous fungi, 4 were Enterococcus faecium and 2 were Escherichia coli (taxonomic units that are excluded from the QPS evaluation) and 20 were taxonomic units (TUs) that already have a QPS status. Three of the other four TUs notified within this period were evaluated for the first time for a possible QPS status: Anaerobutyricum soehngenii, Stutzerimonas stutzeri (former Pseudomonas stutzeri) and Nannochloropsis oculata. Microorganism strain DSM 11798 has also been notified in 2015 and as its taxonomic unit is notified as a strain not a species, it is not suitable for the QPS approach. A. soehngenii and N. oculata are not recommended for the QPS status due to a limited body of knowledge of its use in the food and feed chains. S. stutzeri is not recommended for inclusion in the QPS list based on safety concerns and limited information about the exposure of animals and humans through the food and feed chains.

Divergence in the Saccharomyces species' heat shock response is indicative of their thermal tolerance.

The Saccharomyces species have diverged in their thermal growth profile. Both S. cerevisiae and S. paradoxus grow at temperatures well above the maximum growth temperature of S. kudriavzevii and S. uvarum, but grow more poorly at lower temperatures. In response to thermal shifts, organisms activate a stress response that includes heat shock proteins involved in protein homeostasis and acquisition of thermal tolerance. To determine whether Saccharomyces species have diverged in their response to temperature we measured changes in gene expression in response to a 12°C increase or decrease in temperature for four Saccharomyces species and their six pairwise hybrids. To ensure coverage of subtelomeric gene families we sequenced, assembled and annotated a complete S. uvarum genome. All the strains exhibited a stronger response to heat than cold treatment. In response to heat, the cryophilic species showed a stronger response than the thermophilic species. The hybrids showed a mixture of parental stress responses depending on the time point. After the initial response, hybrids with a thermophilic parent were more similar to S. cerevisiae and S. paradoxus, and the S. cerevisiae × S. paradoxus hybrid showed the weakest heat shock response. Within the hybrids a small subset of temperature responsive genes showed species specific responses but most were also hybrid specific. Our results show that divergence in the heat shock response is indicative of a strain's thermal tolerance, suggesting that cellular factors that signal heat stress or resolve heat induced changes are relevant to thermal divergence in the Saccharomyces species.

Understanding the role of GRE3 in the erythritol biosynthesis pathway in Saccharomyces uvarum and its implication in osmoregulation and redox homeostasis.

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.

Comparative genomics of infective Saccharomyces cerevisiae strains reveals their food origin.

Fungal infections are less studied than viral or bacterial infections and often more difficult to treat. Saccharomyces cerevisiae is usually identified as an innocuous human-friendly yeast; however, this yeast can be responsible for infections mainly in immunosuppressed individuals. S. cerevisiae is a relevant organism widely used in the food industry. Therefore, the study of food yeasts as the source of clinical infection is becoming a pivotal question for food safety. In this study, we demonstrate that S. cerevisiae strains cause infections to spread mostly from food environments. Phylogenetic analysis, genome structure analysis, and phenotypic characterization showed that the key sources of the infective strains are food products, such as bread and probiotic supplements. We observed that the adaptation to host infection can drive important phenotypic and genomic changes in these strains that could be good markers to determine the source of infection. These conclusions add pivotal evidence to reinforce the need for surveillance of food-related S. cerevisiae strains as potential opportunistic pathogens.

Saccharomyces cerevisiae wine strains show a wide range of competitive abilities and differential nutrient uptake behavior in co-culture with S. kudriavzevii.

The wine industry has implemented complex starters with multiple yeast species as an efficient method to improve certain wine properties. Strains' competitive fitness becomes essential for its use in such cases. In the present work, we studied this trait in 60 S. cerevisiae strains from different origins, co-inoculated with a S. kudriavzevii strain, and confirmed it to be associated with the strains' origin. To gather deeper knowledge about the characteristics of strains with highly competitive ability versus the rest, microfermentations using representative strains from each group were performed and the carbon and nitrogen sources uptake was analysed. Our results demonstrate that despite wine strains being the subclade with the highest competitive ability, they present a wide range of behaviors as well as nutrient uptake dynamics, which points to a heterogeneous nature of domestication processes. An interesting strategy was observed in the highly competitive strains (GRE and QA23), the nitrogen sources uptake in the competition was accelerated and the sugar fermentation was slowing despite the fermentation finish at the same time. Therefore, this competition study, using particular combinations of strains, expands the knowledge in the field of the usage of mixed starters in wine manufactured products.

A novel aminotransferase gene and its regulator acquired in Saccharomyces by a horizontal gene transfer event.

BACKGROUND: Horizontal gene transfer (HGT) is an evolutionary mechanism of adaptive importance, which has been deeply studied in wine S. cerevisiae strains, where those acquired genes conferred improved traits related to both transport and metabolism of the nutrients present in the grape must. However, little is known about HGT events that occurred in wild Saccharomyces yeasts and how they determine their phenotypes. RESULTS: Through a comparative genomic approach among Saccharomyces species, we detected a subtelomeric segment present in the S. uvarum, S. kudriavzevii, and S. eubayanus species, belonging to the first species to diverge in the Saccharomyces genus, but absent in the other Saccharomyces species. The segment contains three genes, two of which were characterized, named DGD1 and DGD2. DGD1 encodes dialkylglicine decarboxylase, whose specific substrate is the non-proteinogenic amino acid 2-aminoisobutyric acid (AIB), a rare amino acid present in some antimicrobial peptides of fungal origin. DGD2 encodes putative zinc finger transcription factor, which is essential to induce the AIB-dependent expression of DGD1. Phylogenetic analysis showed that DGD1 and DGD2 are closely related to two adjacent genes present in Zygosaccharomyces. CONCLUSIONS: The presented results show evidence of an early HGT event conferring new traits to the ancestor of the Saccharomyces genus that could be lost in the evolutionary more recent Saccharomyces species, perhaps due to loss of function during the colonization of new habitats.

A Dynamic Genome-Scale Model Identifies Metabolic Pathways Associated with Cold Tolerance in Saccharomyces kudriavzevii.

Saccharomyces kudriavzevii is a cold-tolerant species identified as a good alternative for industrial winemaking. Although S. kudriavzevii has never been found in winemaking, its co-occurrence with Saccharomyces cerevisiae in Mediterranean oaks is well documented. This sympatric association is believed to be possible due to the different growth temperatures of the two yeast species. However, the mechanisms behind the cold tolerance of S. kudriavzevii are not well understood. In this work, we propose the use of a dynamic genome-scale model to compare the metabolic routes used by S. kudriavzevii at two temperatures, 25°C and 12°C, to decipher pathways relevant to cold tolerance. The model successfully recovered the dynamics of biomass and external metabolites and allowed us to link the observed phenotype with exact intracellular pathways. The model predicted fluxes that are consistent with previous findings, but it also led to novel results which we further confirmed with intracellular metabolomics and transcriptomic data. The proposed model (along with the corresponding code) provides a comprehensive picture of the mechanisms of cold tolerance that occur within S. kudriavzevii. The proposed strategy offers a systematic approach to explore microbial diversity from extracellular fermentation data at low temperatures. IMPORTANCE Nonconventional yeasts promise to provide new metabolic pathways for producing industrially relevant compounds and tolerating specific stressors such as cold temperatures. The mechanisms behind the cold tolerance of S. kudriavzevii or its sympatric relationship with S. cerevisiae in Mediterranean oaks are not well understood. This study proposes a dynamic genome-scale model to investigate metabolic pathways relevant to cold tolerance. The predictions of the model would indicate the ability of S. kudriavzevii to produce assimilable nitrogen sources from extracellular proteins present in its natural niche. These predictions were further confirmed with metabolomics and transcriptomic data. This finding suggests that not only the different growth temperature preferences but also this proteolytic activity may contribute to the sympatric association with S. cerevisiae. Further exploration of these natural adaptations could lead to novel engineering targets for the biotechnological industry.

Identification and assessment of non-conventional yeasts in mixed fermentations for brewing bioflavored beer.

The increasing demand for more flavored and complex beers encourages the investigation of novel and non-conventional yeasts with the ability to provide a combination of bioflavoring and low ethanol yields. The present study identified 22 yeasts isolated from different brewing sources, including the fermentation by-products known as yeast sludges, and characterized a selection of strains to find the more suitable for the aforementioned aims. HPLC and GC-FID analysis of its brewing products were performed. The most promising results were obtained with the non-conventional yeasts Pichia kudriavzevii MBELGA61 and Meyerozyma guilliermondii MUS122. The former, isolated from a Belgian wheat beer sludge, was capable of growing in wort (17.0°Bx., 20 °C) with very low ethanol yields (1.19 % v/v). Besides, upon mixed fermentations with Saccharomyces cerevisiae, was suitable to produce volatile compounds such as ethyl acetate, 2-phenyl ethanol and isoamyl alcohol, with characteristic fruity notes. M. guilliermondii MUS122, isolated from a golden ale beer sludge, partially attenuated the wort with low production of ethanol and biomass. In addition, provided some fruity and floral nuances to the aroma profile of mixed fermentations with brewer's yeast. The results suggest that these strains favor the development of more fruity-flowery aroma profiles in beers. Furthermore, they are suitable for use in mixed fermentations with Saccharomyces brewer's strains, although the ethanol level did not decrease significantly.

Characterization of kefir yeasts with antifungal capacity against Aspergillus species / Caracterización de levaduras de kéfir con capacidad antifúngica frente a especies de Aspergillus

Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp., and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing, and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against Aspergillus flavus and Aspergillus parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them—belonging to S. cerevisiae and P. kudriavzevii species—generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence-related traits, growth at high temperatures, enzymatic activities, and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.(AU)

Modelling the physiological status of yeast during wine fermentation enables the prediction of secondary metabolism.

Saccharomyces non-cerevisiae yeasts are gaining momentum in wine fermentation due to their potential to reduce ethanol content and achieve attractive aroma profiles. However, the design of the fermentation process for new species requires intensive experimentation. The use of mechanistic models could automate process design, yet to date, most fermentation models have focused on primary metabolism. Therefore, these models do not provide insight into the production of secondary metabolites essential for wine quality, such as aromas. In this work, we formulate a continuous model that accounts for the physiological status of yeast, that is, exponential growth, growth under nitrogen starvation and transition to stationary or decay phases. To do so, we assumed that nitrogen starvation is associated with carbohydrate accumulation and the induction of a set of transcriptional changes associated with the stationary phase. The model accurately described the dynamics of time series data for biomass and primary and secondary metabolites obtained for various yeast species in single culture fermentations. We also used the proposed model to explore different process designs, showing how the addition of nitrogen could affect the aromatic profile of wine. This study underlines the potential of incorporating yeast physiology into batch fermentation modelling and provides a new means of automating process design.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 17: suitability of taxonomic units notified to EFSA until September 2022.

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms, intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, new information was found leading to the withdrawal of the qualification 'absence of aminoglycoside production ability' for Bacillus velezensis. The qualification for Bacillus paralicheniformis was changed to 'absence of bacitracin production ability'. For the other TUs, no new information was found that would change the status of previously recommended QPS TUs. Of 52 microorganisms notified to EFSA between April and September 2022 (inclusive), 48 were not evaluated because: 7 were filamentous fungi, 3 were Enterococcus faecium, 2 were Escherichia coli, 1 was Streptomyces spp., and 35 were taxonomic units (TUs) that already have a QPS status. The other four TUs notified within this period, and one notified previously as a different species, which was recently reclassified, were evaluated for the first time for a possible QPS status: Xanthobacter spp. could not be assessed because it was not identified to the species level; Geobacillus thermodenitrificans is recommended for QPS status with the qualification 'absence of toxigenic activity'. Streptoccus oralis is not recommended for QPS status. Ogataea polymorpha is proposed for QPS status with the qualification 'for production purposes only'. Lactiplantibacillus argentoratensis (new species) is included in the QPS list.

Characterization of kefir yeasts with antifungal capacity against Aspergillus species.

Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp., and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing, and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against Aspergillus flavus and Aspergillus parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them-belonging to S. cerevisiae and P. kudriavzevii species-generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence-related traits, growth at high temperatures, enzymatic activities, and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.

Adaptive evolution in the Saccharomyces kudriavzevii Aro4p promoted a reduced production of higher alcohols.

The use of unconventional yeast species in human-driven fermentations has attracted a lot of attention in the last few years. This tool allows the alcoholic beverage industries to solve problems related to climate change or the consumer demand for newer high-quality products. In this sense, one of the most attractive species is Saccharomyces kudriavzevii, which shows interesting fermentative traits such as the increased and diverse aroma compound production in wines. Specifically, it has been observed that different isolates of this species can produce higher amounts of higher alcohols such as phenylethanol compared with Saccharomyces cerevisiae. In this work, we have shed light on this feature relating it to the S. kudriavzevii aromatic amino acid anabolic pathway in which the enzyme Aro4p plays an essential role. Unexpectedly, we observed that the presence of the S. kudriavzevii ARO4 variant reduces phenylethanol production compared with the S. cerevisiae ARO4 allele. Our experiments suggest that this can be explained by increased feedback inhibition, which might be a consequence of the changes detected in the Aro4p amino end such as L26 Q24 that have been under positive selection in the S. kudriavzevii specie.

Update of the list of QPS-recommended microbiological agents intentionally added to food or feed as notified to EFSA 16: suitability of taxonomic units notified to EFSA until March 2022.

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms, intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge, safety concerns and occurrence of antimicrobial resistance. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. Of the 50 microorganisms notified to EFSA in October 2021 to March 2022 (inclusive), 41 were not evaluated: 10 filamentous fungi, 1 Enterococcus faecium, 1 Clostridium butyricum, 3 Escherichia coli and 1 Streptomyces spp. because are excluded from QPS evaluation, and 25 TUs that have already a QPS status. Nine notifications, corresponding to seven TUs were evaluated: four of these, Streptococcus salivarius, Companilactobacillus formosensis, Pseudonocardia autotrophica and Papiliotrema terrestris, being evaluated for the first time. The other three, Microbacterium foliorum, Pseudomonas fluorescens and Ensifer adhaerens were re-assessed. None of these TUs were recommended for QPS status: Ensifer adhaerens, Microbacterium foliorum, Companilactobacillus formosensis and Papiliotrema terrestris due to a limited body of knowledge, Streptococcus salivarius due to its ability to cause bacteraemia and systemic infection that results in a variety of morbidities, Pseudonocardia autotrophica due to lack of body of knowledge and uncertainty on the safety of biologically active compounds which can be produced, and Pseudomonas fluorescens due to possible safety concerns.

Functional divergence in the proteins encoded by ARO80 from S. uvarum, S. kudriavzevii and S. cerevisiae explain differences in the aroma production during wine fermentation.

Phenylethanol (PE) and phenylethyl acetate (PEA) are commonly desired compounds in wine because of their rose-like aroma. The yeast S. cerevisiae produces the PE either through de novo biosynthesis by shikimate pathway followed by the Ehrlich pathway or the direct phenylalanine catabolism via Ehrlich pathway, and then converted into PEA. Previous work demonstrated that, compared to S. cerevisiae, other Saccharomyces species, such as S. kudriavzevii and S. uvarum, produce higher concentrations of PE and PEA from the precursor phenylalanine, which indicates differential activities of the biosynthetic-involved enzymes. A previous in-silico analysis suggested that the transcriptional activator Aro80p is one of the best candidates to explain these differences. An improved functional analysis identified significant radical amino acid changes in the S. uvarum and S. kudriavzevii Aro80p that could impact the expression of the catabolic genes ARO9 and ARO10, and hence, the production of PE from phenylalanine. Indeed, wine S. cerevisiae strains carrying the S. uvarum and S. kudriavzevii ARO80 alleles increased the production of both compounds in the presence of phenylalanine by increasing the expression of ARO9 and ARO10. This study provides novel insights of the unidentified Aro80p regulatory region and the potential usage of alternatives ARO80 alleles to enhance the PE and PEA concentration in wine.

Genome-wide effect of non-optimal temperatures under anaerobic conditions on gene expression in Saccharomyces cerevisiae.

Understanding of thermal adaptation mechanisms in yeast is crucial to develop better-adapted strains to industrial processes, providing more economical and sustainable products. We have analyzed the transcriptomic responses of three Saccharomyces cerevisiae strains, a commercial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and a commercial bioethanol strain, Ethanol Red, grown at non-optimal temperatures under anaerobic chemostat conditions. Transcriptomic analysis of the three strains revealed a huge complexity of cellular mechanisms and responses. Overall, cold exerted a stronger transcriptional response in the three strains comparing with heat conditions, with a higher number of down-regulating genes than of up-regulating genes regardless the strain analyzed. The comparison of the transcriptome at both sub- and supra-optimal temperatures showed the presence of common genes up- or down-regulated in both conditions, but also the presence of common genes up- or down-regulated in the three studied strains. More specifically, we have identified and validated three up-regulated genes at sub-optimal temperature in the three strains, OPI3, EFM6 and YOL014W. Finally, the comparison of the transcriptomic data with a previous proteomic study with the same strains revealed a good correlation between gene activity and protein abundance, mainly at low temperature. Our work provides a global insight into the specific mechanisms involved in temperature adaptation regarding both transcriptome and proteome, which can be a step forward in the comprehension and improvement of yeast thermotolerance.

Generation of intra- and interspecific Saccharomyces hybrids with improved oenological and aromatic properties.

Non-wine yeasts could enhance the aroma and organoleptic profile of wines. However, compared to wine strains, they have specific intolerances to winemaking conditions. To solve this problem, we generated intra- and interspecific hybrids using a non-GMO technique (rare-mating) in which non-wine strains of S. uvarum, S. kudriavzevii and S. cerevisiae species were crossed with a wine S. cerevisiae yeast. The hybrid that inherited the wine yeast mitochondrial showed better fermentation capacities, whereas hybrids carrying the non-wine strain mitotype reduced ethanol levels and increased glycerol, 2,3-butanediol and organic acid production. Moreover, all the hybrids produced several fruity and floral aromas compared to the wine yeast: ß-phenylethyl acetate, isobutyl acetate, γ-octalactone, ethyl cinnamate in both varietal wines. Sc × Sk crosses produced three- to sixfold higher polyfunctional mercaptans, 4-mercapto-4-methylpentan-2-one (4MMP) and 3-mercaptohexanol (3MH). We proposed that the exceptional 3MH release observed in an S. cerevisiae × S. kudriavzevii hybrid was due to the cleavage of the non-volatile glutathione precursor (Glt-3MH) to detoxify the cell from the presence of methylglyoxal, a compound related to the high glycerol yield reached by this hybrid. In conclusion, hybrid generation allows us to obtain aromatically improved yeasts concerning their wine parent. In addition, they reduced ethanol and increased organic acids yields, which counteracts climate change effect on grapes.