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ABSTRACT Hybridization between Saccharomyces cerevisiae and Saccharomyces eubayanus resulted in the emergence of S. pastorianus, a crucial yeast for lager fermentation. However, our understanding of hybridization success and hybrid vigor between these two species remains limited due to the scarcity of S. eubayanus parental strains. Here, we explore hybridization success and the impact of hybridization on fermentation performance and volatile compound profiles in newly formed lager hybrids. By selecting parental candidates spanning a diverse array of lineages from both species, we reveal that the Beer and PB-2 lineages exhibit high rates of hybridization success in S. cerevisiae and S. eubayanus, respectively. Polyploid hybrids were generated through a spontaneous diploid hybridization technique (rare-mating), revealing a prevalence of triploids and diploids over tetraploids. Despite the absence of heterosis in fermentative capacity, hybrids displayed phenotypic variability, notably influenced by maltotriose consumption. Interestingly, ploidy levels did not significantly correlate with fermentative capacity, although triploids exhibited greater phenotypic variability. The S. cerevisiae parental lineages primarily influenced volatile compound profiles, with significant differences in aroma production. Interestingly, hybrids emerging from the Beer S. cerevisiae parental lineages exhibited a volatile compound profile resembling the corresponding S. eubayanus parent. This pattern may result from the dominant inheritance of the S. eubayanus aroma profile, as suggested by the over-expression of genes related to alcohol metabolism and acetate synthesis in hybrids including the Beer S. cerevisiae lineage. Our findings suggest complex interactions between parental lineages and hybridization outcomes, highlighting the potential for creating yeasts with distinct brewing traits through hybridization strategies.IMPORTANCEOur study investigates the principles of lager yeast hybridization between Saccharomyces cerevisiae and Saccharomyces eubayanus. This process gave rise to the lager yeast Saccharomyces pastorianus. By examining how these novel hybrids perform during fermentation and the aromas they produce, we uncover the genetic bases of brewing trait inheritance. We successfully generated polyploid hybrids using diverse strains and lineages from both parent species, predominantly triploids and diploids. Although these hybrids did not show improved fermentation capacity, they exhibited varied traits, especially in utilizing maltotriose, a key sugar in brewing. Remarkably, the aroma profiles of these hybrids were primarily influenced by the S. cerevisiae parent, with Beer lineage hybrids adopting aroma characteristics from their S. eubayanus parent. These insights reveal the complex genetic interactions in hybrid yeasts, opening new possibilities for crafting unique brewing yeasts with desirable traits.

Abstract 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. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. The TUs in the QPS list were updated based on a verification, against their respective authoritative databases, of the correctness of the names and completeness of synonyms. A new procedure has been established to ensure the TUs are kept up to date in relation to recent taxonomical insights. Of 81 microorganisms notified to EFSA between October 2023 and March 2024 (45 as feed additives, 25 as food enzymes or additives, 11 as novel foods), 75 were not evaluated because: 15 were filamentous fungi, 1 was Enterococcus faecium, 10 were Escherichia coli, 1 was a Streptomyces (all excluded from the QPS evaluation) and 46 were TUs that already have a QPS status. Two of the other eight notifications were already evaluated for a possible QPS status in the previous Panel Statement: Heyndrickxia faecalis (previously Weizmannia faecalis) and Serratia marcescens. One was notified at genus level so could not be assessed for QPS status. The other five notifications belonging to five TUs were assessed for possible QPS status. Akkermansia muciniphila and Actinomadura roseirufa were still not recommended for QPS status due to safety concerns. Rhizobium radiobacter can be recommended for QPS status with the qualification for production purposes. Microbacterium arborescens and Burkholderia stagnalis cannot be included in the QPS list due to a lack of body of knowledge for its use in the food and feed chain and for B. stagnalis also due to safety concerns. A. roseirufa and B. stagnalis have been excluded from further QPS assessment.

Abstract This study aimed to investigate how parental genomes contribute to yeast hybrid metabolism using a metabolomic approach. Previous studies have explored central carbon and nitrogen metabolism in Saccharomyces species during wine fermentation, but this study analyses the metabolomes of Saccharomyces hybrids for the first time. We evaluated the oenological performance and intra‐ and extracellular metabolomes, and we compared the strains according to nutrient consumption and production of the main fermentative by‐products. Surprisingly, no common pattern was observed for hybrid genome influence; each strain behaved differently during wine fermentation. However, this study suggests that the genome of the S. cerevisiae species may play a more relevant role in fermentative metabolism. Variations in biomass/nitrogen ratios were also noted, potentially linked to S. kudriavzevii and S. uvarum genome contributions. These results open up possibilities for further research using different “omics” approaches to comprehend better metabolic regulation in hybrid strains with genomes from different species.

Classifying a yeast strain into a recognized species is not always straightforward. Currently, the taxonomic delineation of yeast strains involves multiple approaches covering phenotypic characteristics and molecular methodologies, including genome-based analysis. The aim of this study was to evaluate the suitability of the Average Nucleotide Identity (ANI) calculation through FastANI, a tool created for bacterial species identification, for the assignment of strains to some yeast species. FastANI, the alignment of in silico-extracted D1/D2 sequences of LSU rRNA, and multiple alignments of orthologous genes (MAOG) were employed to analyze 644 assemblies from 12 yeast genera, encompassing various species, and on a dataset of hybrid Saccharomyces species. Overall, the analysis showed high consistency between results obtained with FastANI and MAOG, although, FastANI proved to be more discriminating than the other two methods applied to genomic sequences. In particular, FastANI was effective in distinguishing between strains belonging to different species, defining clear boundaries between them (cutoff: 94–96%). Our results show that FastANI is a reliable method for attributing a known yeast species to a particular strain. Moreover, although hybridization events make species discrimination more complex, it was revealed to be useful in the identification of these cases. We suggest its inclusion as a key component in a comprehensive approach to species delineation. Using this approach with a larger number of yeasts would validate it as a rapid technique to identify yeasts based on whole genome sequences.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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 L26Q24 that have been under positive selection in the S. kudriavzevii specie.

Abstract 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.

Summary 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.

Summary 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: β‐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.

ABSTRACT 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.

Abstract The qualified presumption of safety (QPS) provides a generic pre‐assessment of the safety of microorganisms intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. QPS assessment allows a fast track evaluation of strains belonging to QPS taxonomic units (TUs): species for bacteria, yeast, fungi, protists/microalgae and families for viruses. QPS TUs are assessed for their body of knowledge and safety. Safety concerns related to a QPS TU are reflected, when possible, as ‘qualifications’, which should be tested at strain and/or product level. Based on the possession of potentially harmful traits by some strains, filamentous fungi, bacteriophages, oomycetes, streptomycetes, Enterococcus faecium, Escherichia coli and Clostridium butyricum are excluded from the QPS assessment. Between October 2019 and September 2022, 323 notifications of TUs were received, 217 related to feed additives, 54 to food enzymes, food additives and flavourings, 14 to plant protection products and 38 to novel foods. The list of QPS‐recommended TUs is reviewed every 6 months following an extensive literature search strategy. Only sporadic infections with a few QPS status TUs in immunosuppressed individuals were identified and the assessment did not change the QPS status of these TUs. The QPS list has been updated in relation to the most recent taxonomic insights and the qualifications were revised and streamlined. The qualification ‘absence of aminoglycoside production ability’ was withdrawn for Bacillus velezensis. Six new TUs received the QPS status: Bacillus paralicheniformis with the qualification ‘absence of toxigenic activity’ and ‘absence of bacitracin production ability’; Bacillus circulans with the qualifications for ‘production purposes only’ and ‘absence of cytotoxic activity’; Haematococcus lacustris (synonym Haematococcus pluvialis) and Ogataea polymorpha, both with the qualification ‘for production purposes only’; Lactiplantibacillus argentoratensis; Geobacillus thermodenitrificans with the qualification ‘absence of toxigenic activity’.

Abstract 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.

Abstract 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.

Abstract Elucidation of temperature tolerance mechanisms in yeast is essential for enhancing cellular robustness of strains, providing more economically and sustainable processes. We investigated the differential responses of three distinct Saccharomyces cerevisiae strains, an industrial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and an industrial bioethanol strain, Ethanol Red, grown at sub- and supra-optimal temperatures under chemostat conditions. We employed anaerobic conditions, mimicking the industrial processes. The proteomic profile of these strains in all conditions was performed by sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS), allowing the quantification of 997 proteins, data available via ProteomeXchange (PXD016567). Our analysis demonstrated that temperature responses differ between the strains; however, we also found some common responsive proteins, revealing that the response to temperature involves general stress and specific mechanisms. Overall, sub-optimal temperature conditions involved a higher remodeling of the proteome. The proteomic data evidenced that the cold response involves strong repression of translation-related proteins as well as induction of amino acid metabolism, together with components related to protein folding and degradation while, the high temperature response mainly recruits amino acid metabolism. Our study provides a global and thorough insight into how growth temperature affects the yeast proteome, which can be a step forward in the comprehension and improvement of yeast thermotolerance.

Summary The utilization of S. eubayanus has recently become a topic of interest due to the novel organoleptic properties imparted to beer. However, the utilization of S. eubayanus in brewing requires the comprehension of the mechanisms that underlie fermentative differences generated from its natural genetic variability. Here, we evaluated fermentation performance and volatile compound production in ten genetically distinct S. eubayanus strains in a brewing fermentative context. The evaluated strains showed a broad phenotypic spectrum, some of them exhibiting a high fermentation capacity and high levels of volatile esters and/or higher alcohols. Subsequently, we obtained molecular profiles by generating ‘end‐to‐end’ genome assemblies, as well as metabolome and transcriptome profiling of two Patagonian isolates exhibiting significant differences in beer aroma profiles. These strains showed clear differences in concentrations of intracellular metabolites, including amino acids, such as valine, leucine and isoleucine, likely impacting the production of 2‐methylpropanol and 3‐methylbutanol. These differences in the production of volatile compounds are attributed to gene expression variation, where the most profound differentiation is attributed to genes involved in assimilatory sulfate reduction, which in turn validates phenotypic differences in H2S production. This study lays a solid foundation for future research to improve fermentation performance and select strains for new lager styles based on aroma and metabolic profiles.

Abstract The qualified presumption of safety (QPS) approach was developed to provide a generic pre‐evaluation of the safety of biological agents. 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 are, where possible, confirmed at the species/strain or product level and reflected by ‘qualifications’. The QPS list was updated in relation to the revised taxonomy of the genus Bacillus, to synonyms of yeast species and for the qualifications ‘absence of resistance to antimycotics’ and ‘only for production purposes’. Lactobacillus cellobiosus has been reclassified as Limosilactobacillus fermentum. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS taxonomic units (TU)s. Of the 70 microorganisms notified to EFSA, 64 were not evaluated: 11 filamentous fungi, one oomycete, one Clostridium butyricum, one Enterococcus faecium, five Escherichia coli, one Streptomyces sp., one Bacillus nakamurai and 43 TUs that already had a QPS status. Six notifications, corresponding to six TUs were evaluated: Paenibacillus lentus was reassessed because an update was requested for the current mandate. Enterococcus lactis synonym Enterococcus xinjiangensis, Aurantiochytrium mangrovei synonym Schizochytrium mangrovei, Schizochytrium aggregatum, Chlamydomonas reinhardtii synonym Chlamydomonas smithii and Haematococcus lacustris synonym Haematococcus pluvialis were assessed for the first time. The following TUs were not recommended for QPS status: P. lentus due to a limited body of knowledge, E. lactis synonym E. xinjiangensis due to potential safety concerns, A. mangrovei synonym S. mangrovei, S. aggregatum and C. reinhardtii synonym C. smithii, due to lack of a body of knowledge on its occurrence in the food and feed chain. H. lacustris synonym H. pluvialis is recommended for QPS status with the qualification ‘for production purposes only’.

Problematic fermentations frequently drive economic losses and logistic problems in the winemaking industry. Previous studies have determined thermal conditions leading to problematic fermentations, selecting two contrasting yeast strains for further transcriptomic analysis. Saccharomyces cerevisiae SBB11 showed strong thermosensitivity towards heat shock, while S. cerevisiae PDM was found to be thermotolerant. The aim of this study was to select genes with significantly upregulated expression to be later used as biomarkers for early detection of sluggish fermentation associated with heat shock. Candidate genes were selected from previously obtained RNA-seq data. Alcoholic fermentations were conducted with 4 S. cerevisiae strains SBB11, PDM, M2 and ICV D21. Heat shocks on day 3 of alcoholic fermentation were applied at 36 and 40 °C for 16 h. S. cerevisiae cells were collected at different times after heat shock onset for qPCR analysis of candidate gene expression over time. Three genes showed promising results; SSA1, MGA1 and OPI10 significantly increased expression with respect to the control. The selected genes showed increased expression during the first 9 h post heat shock and are proposed for early detection of sluggish fermentations associated with heat shock.

Different species can find convergent solutions to adapt their genome to the same evolutionary constraints, although functional convergence promoted by chromosomal rearrangements in different species has not previously been found. In this work, we discovered that two domesticated yeast species, Saccharomyces cerevisiae, and Saccharomyces uvarum, acquired chromosomal rearrangements to convergently adapt to the presence of sulfite in fermentation environments. We found two new heterologous chromosomal translocations in fermentative strains of S. uvarum at the SSU1 locus, involved in sulfite resistance, an antimicrobial additive widely used in food production. These are convergent events that share similarities with other SSU1 locus chromosomal translocations previously described in domesticated S. cerevisiae strains. In S. uvarum, the newly described VIIXVI and XIXVI chromosomal translocations generate an overexpression of the SSU1 gene and confer increased sulfite resistance. This study highlights the relevance of chromosomal rearrangements to promote the adaptation of yeast to anthropic environments.

ABSTRACT Yeasts constitute over 1,500 species with great potential for biotechnology. Still, the yeast Saccharomyces cerevisiae dominates industrial applications, and many alternative physiological capabilities of lesser-known yeasts are not being fully exploited. While comparative genomics receives substantial attention, little is known about yeasts’ metabolic specificity in batch cultures. Here, we propose a multiphase multiobjective dynamic genome-scale model of yeast batch cultures that describes the uptake of carbon and nitrogen sources and the production of primary and secondary metabolites. The model integrates a specific metabolic reconstruction, based on the consensus Yeast8, and a kinetic model describing the time-varying culture environment. In addition, we proposed a multiphase multiobjective flux balance analysis to compute the dynamics of intracellular fluxes. We then compared the metabolism of S. cerevisiae and Saccharomyces uvarum strains in a rich medium fermentation. The model successfully explained the experimental data and brought novel insights into how cryotolerant strains achieve redox balance. The proposed model (along with the corresponding code) provides a comprehensive picture of the main steps occurring inside the cell during batch cultures and offers a systematic approach to prospect or metabolically engineering novel yeast cell factories. IMPORTANCE Nonconventional yeast species hold the promise to provide novel metabolic routes to produce industrially relevant compounds and tolerate specific stressors, such as cold temperatures. This work validated the first multiphase multiobjective genome-scale dynamic model to describe carbon and nitrogen metabolism throughout batch fermentation. To test and illustrate its performance, we considered the comparative metabolism of three yeast strains of the Saccharomyces genus in rich medium fermentation. The study revealed that cryotolerant Saccharomyces species might use the γ-aminobutyric acid (GABA) shunt and the production of reducing equivalents as alternative routes to achieve redox balance, a novel biological insight worth being explored further. The proposed model (along with the provided code) can be applied to a wide range of batch processes started with different yeast species and media, offering a systematic and rational approach to prospect nonconventional yeast species metabolism and engineering novel cell factories.

Abstract The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre‐evaluation of the safety of biological agents, intended for addition to food or feed, 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. Schizochytrium limacinum, which is a synonym for Aurantiochytrium limacinum, was added to the QPS list. Of the 78 microorganisms notified to EFSA between October 2020 and March 2021, 71 were excluded; 16 filamentous fungi, 1 Dyella spp., 1 Enterococcus faecium, 7 Escherichia coli, 1 Streptomyces spp., 1 Schizochytrium spp. and 44 TUs that had been previously evaluated. Seven TUs were evaluated: Corynebacterium stationis and Kodamaea ohmeri were re‐assessed because an update was requested for the current mandate. Anoxybacillus caldiproteolyticus, Bacillus paralicheniformis, Enterobacter hormaechei, Eremothecium ashbyi and Lactococcus garvieae were assessed for the first time. The following TUs were not recommended for QPS status: A. caldiproteolyticus due to the lack of a body of knowledge in relation to its use in the food or feed chain, E. hormaechei, L. garvieae and K. ohmeri due to their pathogenic potential, E. ashbyi and C. stationis due to a lack of body of knowledge on their occurrence in the food and feed chain and to their pathogenic potential. B. paralicheniformis was recommended for the QPS status with the qualification ‘absence of toxigenic activity’ and ‘absence of genetic information to synthesize bacitracin’.

Ten different Saccharomyces cerevisiae strains fermented semi-synthetic musts containing a Polyphenolic and Aroma Precursor Fraction (PAF) extracted from Tempranillo grapes. Aroma compounds were studied by Gas Chromatography (GC), GC-Olfactometry and GC-Mass Spectrometry (MS), during fermentation by trapping volatilized aroma, immediately after fermentation and after accelerated aging. Volatiles lost by evaporation during fermentation are mostly fermentative compounds and not grape-related odorants. Isobutanal and some esters are mostly lost during fermentation. In many cases the impact of yeast strain is evident only after aging. Strains could be classified into 3 major clusters with marked differences in fermentative and varietal profiles. Linalool and geraniol were found to have fermentative origin. S. cerevisiae yeast strains can effectively modulate varietal aroma, likely through specific enzymatic activities acting on grape phenolic acids and norisoprenoid aroma precursors and may be specifically used to mitigate some aging-related off odours, such as massoia lactone, guaiacol or TDN.

Abstract The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre‐evaluation of the safety of biological agents, intended for addition to food or feed, to support the work of EFSA's Scientific Panels. It is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of knowledge, safety concerns and antimicrobial resistance. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at 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 36 microorganisms notified to EFSA between April and September 2020, 33 were excluded; seven filamentous fungi (including Aureobasidium pullulans based on recent taxonomic insights), one Clostridium butyricum, one Enterococcus faecium, three Escherichia coli, one Streptomyces spp. and 20 TUs that had been previously evaluated. Three TUs were evaluated; Methylorubrum extorquens and Mycobacterium aurum for the first time and Bacillus circulans was re‐assessed because an update was requested in relation to a new mandate. M. extorquens and M. aurum are not recommended for QPS status due to the lack of a body of knowledge in relation to use in the food or feed chain and M. aurum, due to uncertainty concerning its pathogenicity potential. B. circulans was recommended for QPS status with the qualifications for ‘production purposes only’ and ‘absence of cytotoxic activity’.

Mixed culture wine fermentations combining species within the Saccharomyces genus have the potential to produce new market tailored wines. They may also contribute to alleviating the effects of climate change in winemaking. Species, such as S. kudriavzevii, show good fermentative properties at low temperatures and produce wines with lower alcohol content, higher glycerol amounts and good aroma. However, the design of mixed culture fermentations combining S. cerevisiae and S. kudriavzevii species requires investigating their ecological interactions under cold temperature regimes. Here, we derived the first ecological model to predict individual and mixed yeast dynamics in cold fermentations. The optimal model combines the Gilpin-Ayala modification to the Lotka-Volterra competitive model with saturable competition and secondary models that account for the role of temperature. The nullcline analysis of the proposed model revealed how temperature shapes ecological dynamics in mixed co-inoculated cold fermentations. For this particular medium and species, successful mixed cultures can be achieved only at specific temperature ranges or by sequential inoculation. The proposed ecological model can be calibrated for different species and provide valuable insights into the functioning of alternative mixed wine fermentations.

Abstract The qualified presumption of safety (QPS) was developed to provide a generic safety evaluation for biological agents to support EFSA's Scientific Panels. It is based on an assessment of the taxonomic identity, the body of knowledge, safety concerns and antimicrobial resistance. Safety concerns identified for a taxonomic unit (TU) are where possible to be confirmed at strain or product level, reflected by ‘qualifications’. No new information was found that would change the previously recommended QPS TUs of the 39 microorganisms notified to EFSA between October 2019 and March 2020, 33 were excluded, including five filamentous fungi, five Escherichia coli, two Enterococcus faecium, two Streptomyces spp. and 19 TUs already evaluated. Six TUs were evaluated. Akkermansia muciniphila was not recommended for QPS status due to safety concerns. Clostridium butyricum was not recommended because some strains contain pathogenicity factors. This TU was excluded for further QPS evaluation. Galdieria sulphuraria and Pseudomonas chlororaphis were also rejected due to a lack of body of knowledge. The QPS status of Corynebacterium ammoniagenes (with the qualification ‘for production purposes only’) and of Komagataella pastoris (with the qualification ‘for enzyme production’) was confirmed. In relation to the taxonomic revision of the Lactobacillus genus, previously designated Lactobacillus species will be reassigned to the new species and both the old and new names will be retained in the QPS list.

In European regions of cold climate, S. uvarum can replace S. cerevisiae in wine fermentations performed at low temperatures. S. uvarum is a cryotolerant yeast that produces more glycerol, less acetic acid and exhibits a better aroma profile. However, this species exhibits a poor ethanol tolerance compared with S. cerevisiae. In the present study, we obtained by rare mating (non-GMO strategy), and a subsequent sporulation, an interspecific S. cerevisiae × S. uvarum spore-derivative hybrid that improves or maintains a combination of parental traits of interest for the wine industry, such as good fermentation performance, increased ethanol tolerance, and high glycerol and aroma productions. Genomic sequencing analysis showed that the artificial spore-derivative hybrid is an allotriploid, which is very common among natural hybrids. Its genome contains one genome copy from the S. uvarum parental genome and two heterozygous copies of the S. cerevisiae parental genome, with the exception of a monosomic S. cerevisiae chromosome III, where the sex-determining MAT locus is located. This genome constitution supports that the original hybrid from which the spore was obtained likely originated by a rare-mating event between a mating-competent S. cerevisiae diploid cell and either a diploid or a haploid S. uvarum cell of the opposite mating type. Moreover, a comparative transcriptomic analysis reveals that each spore-derivative hybrid subgenome is regulating different processes during the fermentation, in which each parental species has demonstrated to be more efficient. Therefore, interactions between the two subgenomes in the spore-derivative hybrid improve those differential species-specific adaptations to the wine fermentation environments, already present in the parental species.

Abstract Qualified presumption of safety (QPS) was developed to provide a generic safety evaluation for biological agents to support EFSA's Scientific Panels. The taxonomic identity, body of knowledge, safety concerns and antimicrobial resistance are assessed. Safety concerns identified for a taxonomic unit (TU) are where possible to be confirmed at strain or product level, reflected by ‘qualifications’. No new information was found that would change the previously recommended QPS TUs and their qualifications. The list of microorganisms notified to EFSA was updated with 54 biological agents, received between April and September 2019; 23 already had QPS status, 14 were excluded from the QPS exercise (7 filamentous fungi, 6 Escherichia coli, Sphingomonas paucimobilis which was already evaluated). Seventeen, corresponding to 16 TUs, were evaluated for possible QPS status, fourteen of these for the first time, and Protaminobacter rubrum, evaluated previously, was excluded because it is not a valid species. Eight TUs are recommended for QPS status. Lactobacillus parafarraginis and Zygosaccharomyces rouxii are recommended to be included in the QPS list. Parageobacillus thermoglucosidasius and Paenibacillus illinoisensis can be recommended for the QPS list with the qualification ‘for production purposes only’ and absence of toxigenic potential. Bacillus velezensis can be recommended for the QPS list with the qualification ‘absence of toxigenic potential and the absence of aminoglycoside production ability’. Cupriavidus necator, Aurantiochytrium limacinum and Tetraselmis chuii can be recommended for the QPS list with the qualification ‘production purposes only’. Pantoea ananatis is not recommended for the QPS list due to lack of body of knowledge in relation to its pathogenicity potential for plants. Corynebacterium stationis, Hamamotoa singularis, Rhodococcus aetherivorans and Rhodococcus ruber cannot be recommended for the QPS list due to lack of body of knowledge. Kodamaea ohmeri cannot be recommended for the QPS list due to safety concerns.

Abstract The qualified presumption of safety (QPS) was developed to provide a safety pre‐assessment within EFSA for microorganisms. Strains belonging to QPS taxonomic units (TUs) still require an assessment based on a specific data package, but QPS status facilitates fast track evaluation. QPS TUs are unambiguously defined biological agents assessed for the body of knowledge, their safety and their end use. Safety concerns are, where possible, to be confirmed at strain or product level, and reflected as ‘qualifications’. Qualifications need to be evaluated at strain level by the respective EFSA units. The lowest QPS TU is the species level for bacteria, yeasts and protists/algae, and the family for viruses. The QPS concept is also applicable to genetically modified microorganisms used for production purposes if the recipient strain qualifies for the QPS status, and if the genetic modification does not indicate a concern. Based on the actual body of knowledge and/or an ambiguous taxonomic position, the following TUs were excluded from the QPS assessment: filamentous fungi, oomycetes, streptomycetes, Enterococcus faecium, Escherichia coli and bacteriophages. The list of QPS‐recommended biological agents was reviewed and updated in the current opinion and therefore now becomes the valid list. For this update, reports on the safety of previously assessed microorganisms, including bacteria, yeasts and viruses (the latter only when used for plant protection purposes) were reviewed, following an Extensive Literature Search strategy. All TUs previously recommended for 2016 QPS list had their status reconfirmed as well as their qualifications. The TUs related to the new notifications received since the 2016 QPS opinion was periodically evaluated for QPS status in the Statements of the BIOHAZ Panel, and the QPS list was also periodically updated. In total, 14 new TUs received a QPS status between 2017 and 2019: three yeasts, eight bacteria and three algae/protists.

Abstract The qualified presumption of safety (QPS) procedure was developed to provide a harmonised generic pre‐evaluation to support safety risk assessments of biological agents performed by EFSA's Scientific Panels. The taxonomic identity, body of knowledge, safety concerns and antimicrobial resistance were assessed. Safety concerns identified for a taxonomic unit (TU) are, where possible and reasonable in number, reflected by ‘qualifications’ which should be assessed at the strain level by the EFSA's Scientific Panels. During the current assessment, no new information was found that would change the previously recommended QPS TUs and their qualifications. The list of microorganisms notified to EFSA from applications for market authorisation was updated with 47 biological agents, received between October 2018 and March 2019. Of these, 19 already had QPS status, 20 were excluded from the QPS exercise by the previous QPS mandate (11 filamentous fungi) or from further evaluations within the current mandate (9 notifications of Escherichia coli). Sphingomonas elodea, Gluconobacter frateurii, Corynebacterium ammoniagenes, Corynebacterium casei, Burkholderia ubonensis, Phaeodactylum tricornutum, Microbacterium foliorum and Euglena gracilis were evaluated for the first time. Sphingomonas elodea cannot be assessed for a possible QPS recommendation because it is not a valid species. Corynebacterium ammoniagenes and Euglena gracilis can be recommended for the QPS list with the qualification ‘for production purposes only’. The following TUs cannot be recommended for the QPS list: Burkholderia ubonensis, due to its potential and confirmed ability to generate biologically active compounds and limited of body of knowledge; Corynebacterium casei, Gluconobacter frateurii and Microbacterium foliorum, due to lack of body of knowledge; Phaeodactylum tricornutum, based on the lack of a safe history of use in the food chain and limited knowledge on its potential production of bioactive compounds with possible toxic effects.

Abstract The qualified presumption of safety (QPS) procedure was developed to provide a harmonised generic pre‐evaluation to support safety risk assessments of biological agents performed by EFSA's Scientific Panels. The taxonomic identity, body of knowledge, safety concerns and antimicrobial resistance were assessed. Safety concerns identified for a taxonomic unit are, where possible and reasonable in number, reflected by ‘qualifications’ which should be assessed at the strain level by the EFSA's Scientific Panels. During the current assessment, no new information was found that would change the previously recommended QPS taxonomic units and their qualifications. Between April and September 2018, the QPS notification list was updated with 48 microorganisms from applications for market authorisation. Of these, 30 biological agents already had QPS status, 15 were excluded from the QPS exercise by the previous QPS mandate (five filamentous fungi) or from further evaluations within the current mandate (two notifications of Enterococcus faecium, one of Streptomyces spp. and seven of Escherichia coli). One taxonomic unit was (re)evaluated: Pseudomonas fluorescens had been previously evaluated in 2016, and was now re‐evaluated within this mandate. The revision of the literature supports the previously identified safety concerns (e.g. production of biocompounds with antimicrobial activity and virulence features), preventing the inclusion of P. fluorescens in the QPS list. Mycobacterium setense and Komagataeibacter sucrofermentans were evaluated for the first time. M. setense cannot be considered for the QPS assessment because there are significant safety concerns. K. sucrofermentans (Acetobacter xylinus subsp. sucrofermentans) can be proposed for the QPS list but only for production purposes. The QPS status of Corynebacterium glutamicum is confirmed with the qualification extended to other production purposes.

Fermentations carried out at low temperatures (10–15°C) enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. Notwithstanding, as Saccharomyces cerevisiae is the main species responsible for alcoholic fermentation, other species of the genus Saccharomyces, such as cryophilic species Saccharomyces eubayanus, Saccharomyces kudriavzevii and Saccharomyces uvarum, are better adapted to low-temperature fermentations during winemaking. In this work, a Saccharomyces cerevisiae × S. uvarum hybrid was constructed to improve the enological features of a wine S. cerevisiae strain at low temperature. Fermentations of white grape musts were performed, and the phenotypic differences between parental and hybrid strains under different temperature conditions were examined. This work demonstrates that hybridization constitutes an effective approach to obtain yeast strains with desirable physiological features, like low-temperature fermentation capacity, which genetically depend on the expression of numerous genes (polygenic character). As this interspecific hybridization approach is not considered a GMO, the genetically improved strains can be quickly transferred to the wine industry.

Wineries face unprecedented challenges due to new market demands and climate change effects on wine quality. New yeast starters including non-conventional Saccharomyces species, such as S. kudriavzevii, may contribute to deal with some of these challenges. The design of new fermentations using non-conventional yeasts requires an improved understanding of the physiology and metabolism of these cells. Dynamic modeling brings the potential of exploring the most relevant mechanisms and designing optimal processes more systematically. In this work we explore mechanisms by means of a model selection, reduction and cross-validation pipeline which enables to dissect the most relevant fermentation features for the species under consideration, Saccharomyces cerevisiae T73 and Saccharomyces kudriavzevii CR85. The pipeline involved the comparison of a collection of models which incorporate several alternative mechanisms with emphasis on the inhibitory effects due to temperature and ethanol. We focused on defining a minimal model with the minimum number of parameters, to maximize the identifiability and the quality of cross-validation. The selected model was then used to highlight differences in behavior between species. The analysis of model parameters would indicate that the specific growth rate and the transport of hexoses at initial times are higher for S. cervisiae T73 while S. kudriavzevii CR85 diverts more flux for glycerol production and cellular maintenance. As a result, the fermentations with S. kudriavzevii CR85 are typically slower; produce less ethanol but higher glycerol. Finally, we also explored optimal initial inoculation and process temperature to find the best compromise between final product characteristics and fermentation duration. Results reveal that the production of glycerol is distinctive in S. kudriavzevii CR85, it was not possible to achieve the same production of glycerol with S. cervisiae T73 in any of the conditions tested. This result brings the idea that the optimal design of mixed cultures may have an enormous potential for the improvement of final wine quality.

Abstract The qualified presumption of safety (QPS) concept was developed to provide a harmonised generic pre‐evaluation to support safety risk assessments of biological agents performed by EFSA's scientific Panels. The identity, body of knowledge, safety concerns and antimicrobial resistance of valid taxonomic units were assessed. Safety concerns identified for a taxonomic unit are, where possible and reasonable in number, considered to be ‘qualifications’ which should be assessed at the strain level by the EFSA's scientific Panels. No new information was found that would change the previously recommended QPS taxonomic units and their qualifications. The BIOHAZ Panel confirms that the QPS approach can be extended to a genetically modified production strain if the recipient strain qualifies for the QPS status, and if the genetic modification does not indicate a concern. Between April and September 2017, the QPS notification list was updated with 46 applications for market authorisation. From these, 14 biological agents already had QPS status and 16 were not included as they are filamentous fungi or enterococci. One notification of Streptomyces K‐61 (notified as former S. griseoviridis) and four of Escherichia coli were not considered for the assessment as they belong to taxonomic units that were excluded from further evaluations within the current QPS mandate. Eight notifications of Bacillus thuringiensis and one of an oomycete are pending the reception of the complete application. Two taxonomic units were evaluated: Kitasatospora paracochleata, which had not been evaluated before, and Komagataella phaffii, previously notified as Pichia pastoris included due to a change in the taxonomic identity. Kitasatospora paracochleata cannot be granted QPS status due to lack of information on its biology and to its possible production of toxic secondary metabolites. The species Komagataella phaffii can be recommended for the QPS list when used for enzyme production.

Saccharomyces cerevisiae is the most widespread microorganism responsible for wine alcoholic fermentation. Nevertheless, the wine industry is currently facing new challenges, some of them associate with climate change, which have a negative effect on ethanol content and wine quality. Numerous and varied strategies have been carried out to overcome these concerns. From a biotechnological point of view, the use of alternative non-Saccharomyces yeasts, yielding lower ethanol concentrations and sometimes giving rise to new and interesting aroma, is one of the trendiest approaches. However, S. cerevisiae usually outcompetes other Saccharomyces species due to its better adaptation to the fermentative environment. For this reason, we studied for the first time the use of a Saccharomyces kudriavzevii strain, CR85, for co-inoculations at increasing proportions and sequential inoculations, as well as the effect of aeration, to improve its fermentation performance in order to obtain wines with an ethanol yield reduction. An enhanced competitive performance of S. kudriavzevii CR85 was observed when it represented 90% of the cells present in the inoculum. Furthermore, airflow supply of 20 VVH to the fermentation synergistically improved CR85 endurance and, interestingly, a significant ethanol concentration reduction was achieved.

Background: Although Saccharomyces cerevisiae is the most frequently isolated species in wine fermentation, and the most studied species, other species and interspecific hybrids have greatly attracted the interest of researchers in this field in the last few years, given their potential to solve new winemaking industry challenges. S. cerevisiae x S. kudriavzevii hybrids exhibit good fermentative capabilities at low temperatures, and produce wines with smaller alcohol quantities and larger glycerol quantities, which can be very useful to solve challenges in the winemaking industry such as the necessity to enhance the aroma profile. Methods: In this study, we performed a transcriptomic study of S. cerevisiae x S. kudriavzevii hybrids in low temperature winemaking conditions. Results: The results revealed that the hybrids have acquired both fermentative abilities and cold adaptation abilities, attributed to S. cerevisiae and S. kudriavzevii parental species, respectively, showcasing their industrially relevant characteristics. For several key genes, we also studied the contribution to gene expression of each of the alleles of S. cerevisiae and S. kudriavzevii in the S. cerevisiae x S. kudriavzevii hybrids. From the results, it is not clear how important the differential expression of the specific parental alleles is to the phenotype of the hybrids. Conclusions: This study shows that the fermentative abilities of S. cerevisiae x S. kudriavzevii hybrids at low temperatures do not seem to result from differential expression of specific parental alleles of the key genes involved in this phenotype.

Background: Saccharomyces cerevisiae is generally considered safe, and is involved in the production of many types of foods and dietary supplements. However, some isolates, which are genetically related to strains used in brewing and baking, have shown virulent traits, being able to produce infections in humans, mainly in immunodeficient patients. This can lead to systemic infections in humans. Methods: In this work, we studied S. cerevisiae isolates in an in vitro human endothelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans. Results: The results showed that this food related yeast is able to cross the endothelial barrier in vitro. However, in contrast to C. glabrata and C. albicans, S. cerevisiae showed very low levels of traversal. Conclusions: We conclude that using an in vitro human endothelial barrier model with S. cerevisiae can be useful to evaluate the safety of S. cerevisiae strains isolated from foods.

Abstract The qualified presumption of safety (QPS) concept was developed to provide a harmonised generic pre‐evaluation to support safety risk assessments of biological agents performed by EFSA's scientific Panels. The identity, body of knowledge, safety concerns and antimicrobial resistance of valid taxonomic units were assessed. Safety concerns identified for a taxonomic unit are, where possible and reasonable in number, reflected as ‘qualifications’ which should be assessed at the strain level by the EFSA's scientific Panels. No new information was found that would change the previously recommended QPS taxonomic units and their qualifications. Between the end of September 2016 and March 2017, the QPS notification list was updated with 87 applications for market authorisation. From these, 32 biological agents already had a QPS status, and 37 were not included in the evaluation as they are filamentous fungi or enterococci. Streptomyces species (Streptomyces cinnamonensis, Streptomyces mobaraensis and Streptomyces violaceoruber), Bacillus circulans (three notifications) and Escherichia coli (seven notifications) were re‐confirmed not suitable for QPS. Streptomyces rubiginosus and Streptomyces netropsis, not evaluated within the previous mandate, were also not recommended for QPS. Streptomyces spp. and E. coli will be excluded from further QPS evaluations within the current QPS mandate. Hyphomicrobium denitrificans, which has never been evaluated before, was not recommended for the QPS list and for Pseudomonas amyloderamosa, the QPS assessment was not applicable because it is not a validated species. Lactobacillus animalis was a new taxonomic unit recommended to have the QPS status.

Abstract EFSA was requested to assess the safety of a broad range of biological agents in the context of notifications for market authorisation as sources of food and feed additives, enzymes and plant protection products. The qualified presumption of safety (QPS) assessment was developed to provide a harmonised generic pre‐assessment to support safety risk assessments performed by EFSA's Scientific Panels. The safety of unambiguously defined biological agents (at the highest taxonomic unit appropriate for the purpose for which an application is intended) and the completeness of the body of knowledge were assessed. Safety concerns identified for a taxonomic unit are, where possible and reasonable in number, reflected as ‘qualifications’ in connection with a recommendation for a QPS status. A total of 57 biological agents were notified to EFSA between the end of April 2016 and the beginning of September 2016. From these, 34 biological agents already had a QPS status and did not require further evaluation, and 10 were not included in the evaluation as they are filamentous fungi or enterococci, biological groups which have been excluded from QPS evaluation since 2014. Three notifications for Streptomyces violaceoruber, one for Streptomyces albus, one for Bacillus circulans and four for Escherichia coli were not evaluated for QPS status because these species were recently assessed and considered not suitable for QPS status. Therefore, only four notifications related to three taxonomic units were evaluated for QPS status. Of these, Arthrobacter ramosus and Pseudomonas fluorescens are not recommended for the QPS list. Bacillus smithii is recommended for the QPS status.

Abstract EFSA is requested to assess the safety of a broad range of biological agents in the context of notification for market authorisation as sources of food and feed additives, food enzymes and plant protection products. The qualified presumption of safety (QPS) assessment was developed to provide a harmonised generic pre‐assessment to support safety risk assessments performed by EFSA's scientific Panels. The safety of unambiguously defined biological agents (at the highest taxonomic unit appropriate for the purpose for which an application is intended), and the completeness of the body of knowledge are assessed. Identified safety concerns for a taxonomic unit are, where possible and reasonable in number, reflected as ‘qualifications’ in connection with a recommendation for a QPS status. The list of QPS recommended biological agents was reviewed and updated in the current opinion and therefore becomes the valid list. The 2016 update reviews previously assessed microorganisms including bacteria, yeasts and viruses used for plant protection purposes following an Extensive Literature Search strategy. The taxonomic units related to the new notifications received since the 2013 QPS opinion, were periodically evaluated for a QPS status and the results published as Statements of the BIOHAZ Panel. Carnobacterium divergens, Lactobacillus diolivorans, Microbacterium imperiale, Pasteuria nishizawae, Pediococcus parvulus, Bacillus flexus, Bacillus smithii, Xanthomonas campestris and Candida cylindracea were recommended for the QPS list. All taxonomic units previously recommended for the 2013 QPS list had their status reconfirmed as well their qualifications with the exception of Pasteuria nishizawae for which the qualification was removed. The exclusion of filamentous fungi and enterococci from the QPS evaluations was reconsidered but monitoring will be maintained and the status will be re‐evaluated in the next QPS Opinion update. Evaluation of bacteriophages should remain as a case‐by‐case procedure and should not be considered for QPS status.

In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other 'opportunistic' strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host.

Although the genus Saccharomyces has been thoroughly studied, some species in the genus has not yet been accurately resolved; an example is S. bayanus, a taxon that includes genetically diverse lineages of pure and hybrid strains. This diversity makes the assignation and classification of strains belonging to this species unclear and controversial. They have been subdivided by some authors into two varieties (bayanus and uvarum), which have been raised to the species level by others. In this work, we evaluate the complexity of 46 different strains included in the S. bayanus taxon by means of PCR-RFLP analysis and by sequencing of 34 gene regions and one mitochondrial gene. Using the sequence data, and based on the S. bayanus var. bayanus reference strain NBRC 1948, a hypothetical pure S. bayanus was reconstructed for these genes that showed alleles with similarity values lower than 97% with the S. bayanus var. uvarum strain CBS 7001, and of 99-100% with the non S. cerevisiae portion in S. pastorianus Weihenstephan 34/70 and with the new species S. eubayanus. Among the S. bayanus strains under study, different levels of homozygosity, hybridization and introgression were found; however, no pure S. bayanus var. bayanus strain was identified. These S. bayanus hybrids can be classified into two types: homozygous (type I) and heterozygous hybrids (type II), indicating that they have been originated by different hybridization processes. Therefore, a putative evolutionary scenario involving two different hybridization events between a S. bayanus var. uvarum and unknown European S. eubayanus-like strains can be postulated to explain the genomic diversity observed in our S. bayanus var. bayanus strains.

Saccharomyces cerevisiae plays a beneficial role in health because of its intrinsic nutritional value and bio-functional properties, which is why it is also used as a dietary supplement. However, the perception that S. cerevisiae is harmless has changed due to an increasing number of infections caused by this yeast. Given this scenario, we have tested whether viable strains contained in dietary supplements displayed virulence-associated phenotypic traits that could contribute to virulence in humans. We have also performed an in vivo study of the pathogenic potential of these strains using a murine model of systemic infection by intravenous inoculation. A total of 5 strains were isolated from 22 commercial products and tested. Results highlight one strain (D14) in terms of burden levels in brains and kidneys and ability to cause death, whereas the other two strains (D2 and D4) were considered of low virulence. Our results suggest a strong relationship between some of the virulence-associated phenotypic traits (ability to grow at 39°C and pseudohyphal growth) and the in vivo virulence in a mouse model of intravenous inoculation for isolates under study. The isolate displaying greatest virulence (D14) was evaluated in an experimental murine model of gastrointestinal infection with immunosuppression and disruption of mucosal integrity, which are common risk factors for developing infection in humans, and results were compared with an avirulent strain (D23). We showed that D14 was able to spread to mesenteric nodes and distant organs under these conditions. Given the widespread consumption of dietary supplements, we recommend only safe strains be used.

During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased V max of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD(+)/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter.

The Saccharomyces genus is the main yeast involved in wine fermentations to play a crucial role in the production and release of aromatic compounds. Despite the several studies done into the genome-wide expression analysis using DNA microarray technology in wine S. cerevisiae strains, this is the first to investigate other species of the Saccharomyces genus. This research work investigates the expression of the genes involved in flavor compound production in three different Saccharomyces species (S. cerevisiae, S. bayanus var. uvarum and S. kudriavzevii) under low (12°C) and moderate fermentation temperatures (28°C). The global genes analysis showed that 30% of genes appeared to be differently expressed in the three cryophilic strains if compared to the reference strain (mesophilic S. cerevisiae), suggesting a very close cold adaptation response. Remarkable differences in the gene expression level were observed when comparing the three species, S. cerevisiae, S. bayanus var. uvarum and S. kudriavzevii, which will result in different aroma profiles. Knowledge of these differences in the transcriptome can be a tool to help modulate aroma to create wines with the desired aromatic traits.

Temperature is one of the most important parameters affecting the length and rate of alcoholic fermentation and final wine quality. Wine produced at low temperature is often considered to have improved sensory qualities. However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase, and sluggish or stuck fermentations. To investigate the effects of temperature on commercial wine yeast, we compared its metabolome growing at 12 °C and 28 °C in a synthetic must. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae. This is the case of the cryotolerant yeasts Saccharomyces bayanus var. uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the metabolome of these species growing at 12°C, which we compared with the metabolome of S. cerevisiae (not well adapted at low temperature) at the same temperature. Our results show that the main differences between the metabolic profiling of S. cerevisiae growing at 12 °C and 28 °C were observed in lipid metabolism and redox homeostasis. Moreover, the global metabolic comparison among the three species revealed that the main differences between the two cryotolerant species and S. cerevisiae were in carbohydrate metabolism, mainly fructose metabolism. However, these two species have developed different strategies for cold resistance. S. bayanus var. uvarum presented elevated shikimate pathway activity, while S. kudriavzevii displayed increased NAD(+) synthesis.