Isolation of Saccharomycopsis species from plant material.

Saccharomycopsis species are natural organic sulphur auxotrophs. Their genomes do not encode genes for the uptake and assimilation of sulphate and thus these species cannot grow on media lacking e.g. methionine. Due to the similarity between sulphate and selenate, uptake and assimilation of selenate occurs through the same pathway starting from sulphate transporters encoded by the homologs of the SUL1 and SUL2 genes in S. cerevisiae. Lack of these transporters renders Saccharomycopsis species resistant to selenate levels that are toxic to other microorganisms. We used this feature to enrich environmental samples for Saccharomycopsis species. This led to the isolation of S. schoenii, S. lassenensis and a hitherto undescribed Saccharomycopsis species with limited by-catch of other yeasts, mainly belonging to Metschnikowia and Hanseniaspora. We performed growth and predation assays to characterize the potential of these new isolates as predacious yeasts. Most Saccharomycopsis species are temperature sensitive and cannot grow at 37°C; with the exception of S. lassenensis strains. Predation assays with S. schoenii and S. cerevisiae as prey indicated that predation was enhanced at 20°C compared to 30°C. We crossed an American isolate of S. schoenii with our German isolate using marker directed breeding. Viable progeny indicated that both strains are interfertile and belong to the same biological species. S. lassenensis is heterothallic, while S. schoenii and the new Saccharomycopsis isolate, for which we suggest the name S. geisenheimensis sp. nov., are homothallic.

Integrated genome-transcriptome analysis unveiled the mechanism of Debaryomyces hansenii-mediated arsenic stress amelioration in rice.

Globally, rice is becoming more vulnerable to arsenic (As) pollution, posing a serious threat to public food safety. Previously Debaryomyces hansenii was found to reduce grain As content of rice. To better understand the underlying mechanism, we performed a genome analysis to identify the key genes in D. hansenii responsible for As tolerance and plant growth promotion. Notably, genes related to As resistance (ARR, Ycf1, and Yap) were observed in the genome of D. hansenii. The presence of auxin pathway and glutathione metabolism-related genes may explain the plant growth-promoting potential and As tolerance mechanism of this novel yeast strain. The genome annotation of D. hansenii indicated that it contains a repertoire of genes encoding antioxidants, well corroborated with the in vitro studies of GST, GR, and glutathione content. In addition, the effect of D. hansenii on gene expression profiling of rice plants under As stress was also examined. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed 307 genes, annotated in D. hansenii-treated rice, related to metabolic pathways (184), photosynthesis (12), glutathione (10), tryptophan (4), and biosynthesis of secondary metabolite (117). Higher expression of regulatory elements like AUX/IAA and WRKY transcription factors (TFs), and defense-responsive genes dismutases, catalases, peroxiredoxin, and glutaredoxins during D. hansenii+As exposure was also observed. Combined analysis revealed that D. hansenii genes are contributing to stress mitigation in rice by supporting plant growth and As-tolerance. The study lays the foundation to develop yeast as a beneficial biofertilizer for As-prone areas.

Efficient PCR-based gene targeting in isolates of the nonconventional yeast Debaryomyces hansenii.

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates. It uses a simple polymerase chain reaction (PCR)-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology.

Insights into the identification and evolutionary conservation of key genes in the transcriptional circuits of meiosis initiation and commitment in budding yeast.

Initiation of meiosis in budding yeast does not commit the cells for meiosis. Thus, two distinct signaling cascades may differentially regulate meiosis initiation and commitment in budding yeast. To distinguish between the role of these signaling cascades, we reconstructed protein-protein interaction networks and gene regulatory networks with upregulated genes in meiosis initiation and commitment. Analyzing the integrated networks, we identified four master regulators (MRs) [Ume6p, Msn2p, Met31p, Ino2p], three transcription factors (TFs), and 279 target genes (TGs) unique for meiosis initiation, and three MRs [Ndt80p, Aro80p, Rds2p], 11 TFs, and 948 TGs unique for meiosis commitment. Functional enrichment analysis of these distinct members from the transcriptional cascades for meiosis initiation and commitment revealed that nutritional cues rewire gene expression for initiating meiosis and chromosomal recombination commits cells to meiosis. As meiotic chromosomal recombination is highly conserved in eukaryotes, we compared the evolutionary rate of unique members in the transcriptional cascade of two meiotic phases of Saccharomyces cerevisiae with members of the phylum Ascomycota, revealing that the transcriptional cascade governing chromosomal recombination during meiosis commitment has experienced greater purifying selection pressure (P value = 0.0013, 0.0382, 0.0448, 0.0369, 0.02967, 0.04937, 0.03046, 0.03357 and < 0.00001 for Ashbya gossypii, Yarrowia lipolytica, Debaryomyces hansenii, Aspergillus fumigatus, Neurospora crassa, Kluyveromyces lactis, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, and Schizosaccharomyces octosporus, respectively). This study demarcates crucial players driving meiosis initiation and commitment and demonstrates their differential rate of evolution in budding yeast.

Pantothenate Auxotrophy in a Naturally Occurring Biocontrol Yeast.

The genus Hanseniaspora is characterized by some of the smallest genomes among budding yeasts. These fungi are primarily found on plant surfaces and in fermented products and represent promising biocontrol agents against notorious fungal plant pathogens. In this work, we identify pantothenate auxotrophy of a Hanseniaspora meyeri isolate that shows strong antagonism against the plant pathogen Fusarium oxysporum. Furthermore, strong biocontrol activity in vitro required both pantothenate and biotin in the growth medium. We show that the H. meyeri isolate APC 12.1 can obtain the vitamin from plants and other fungi. The underlying reason for the auxotrophy is the lack of two key pantothenate biosynthesis genes, but six genes encoding putative pantothenate transporters are present in the genome. By constructing and using a Saccharomyces cerevisiae reporter strain, we identified one Hanseniaspora transporter that conferred pantothenate uptake activity to S. cerevisiae. Pantothenate auxotrophy is rare and has been described in only a few bacteria and in S. cerevisiae strains that were isolated from sake. Such auxotrophic strains may seem an unexpected and unlikely choice as potential biocontrol agents, but they may be particularly competitive in their ecological niche and their specific growth requirements are an inherent biocontainment strategy preventing uncontrolled growth in the environment. Auxotrophic strains, such as the H. meyeri isolate APC 12.1, may thus represent a promising strategy for developing biocontrol agents that will be easier to register than prototrophic strains, which are normally used for such applications. IMPORTANCE As a precursor of the essential coenzyme A (CoA), pantothenate is present in all organisms. Plants, bacteria, and fungi are known to synthesize this vitamin, while animals must obtain it through their diet. Pantothenate auxotrophy has not been described in naturally occurring, environmental fungi and is an unexpected property for an antagonistic yeast. Here, we report that yeasts from the genus Hanseniaspora lack key enzymes for pantothenate biosynthesis and identify a transporter responsible for the acquisition of pantothenate from the environment. Hanseniaspora isolates are strong antagonists of fungal plant pathogens. Their pantothenate auxotrophy is a natural biocontainment feature that could make such isolates interesting candidates for new biocontrol approaches and allow easier registration as plant protection agents than prototrophic strains.

PCR-based gene targeting in Hanseniaspora uvarum.

Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H. uvarum. Addition of short flanking-homology regions of 56-80 bp to these selection markers via PCR was sufficient to promote gene targeting. We report here the deletion of the H. uvarum LEU2 and LYS2 genes with these marker genes via two rounds of consecutive transformations, each resulting in the generation of auxotrophic strains (leu2/leu2; lys2/lys2). The hereby constructed leucine auxotrophic leu2/leu2 strain was subsequently complemented in a targeted manner, thereby further validating this approach. PCR-based gene targeting in H. uvarum was less efficient than in Saccharomyces cerevisiae. However, this approach, combined with the availability of two marker genes, provides essential tools for directed gene manipulations in H. uvarum.

Yeast diversity during the spontaneous fermentation of wine with only the microbiota on grapes cultivated in Japan.

Making wine via spontaneous fermentation without sulfur dioxide and commercial yeast (spontaneous winemaking) is increasing in recent year, but there is scant research regarding microbial communities present in Japan during spontaneous winemaking using culture-independent molecular methods. We analyzed fungal communities and populations during laboratory-scale spontaneous winemaking using sterilized labware to avoid winery-resident microbes. In the spontaneous fermentation of four grape varieties (Pinot Noir, Riesling, Koshu, and Koshusanjaku) grown in the same Japanese vineyard, our analysis of yeast and other fungal species by next-generation sequencing based on the ITS1 region demonstrated that Saccharomyces cerevisiae was eventually dominant in seven of 12 fermentation batches (three replications for each grape variety), whereas non-Saccharomyces species (e.g., Schizosaccharomyces japonicus, Lachancea dasiensis, and Hanseniaspora valbyensis) became dominant in four batches at the end of fermentation. In another batch, lactic acid bacteria (LAB) became dominant and the fermentation remained incomplete. Diverse microbes were involved in the spontaneous fermentation (particularly in Koshusanjaku), indicating that residual sugar remained and lactic and acetic acid largely increased. Compared to the control wine made with SO2 and commercial yeast, the concentration of lactic acid was 47-fold higher in the must dominated by L. dasiensis, and the concentrations of acetic acid and lactic acid were 10-fold and 20-fold higher in the must dominated by LAB, respectively. Even when indigenous S. cerevisiae became dominant, the finished wines obtained high sensory-analysis scores for complexity but low scores for varietal typicality, indicating the risk of fermentation with unselected wild yeast on the grapes grown in Japan.

A new hypothesis for the origin of the lager yeast Saccharomyces pastorianus.

Saccharomyces pastorianus, which is responsible for the production of bottom-fermented lager beer, is a hybrid species that arose from the mating of the top-fermenting ale yeast Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus around the start of the 17th century. Based on detailed analysis of Central European brewing records, we propose that the critical event for the hybridization was the introduction of top-fermenting S. cerevisiae into an environment where S. eubayanus was present, rather than the other way around. Bottom fermentation in parts of Bavaria preceded the proposed hybridization date by a couple of hundred years and we suggest that this was carried out by mixtures of yeasts, which may have included S. eubayanus. A plausible case can be made that the S. cerevisiae parent came either from the Schwarzach wheat brewery or the city of Einbeck, and the formation of S. pastorianus happened in the Munich Hofbräuhaus between 1602 and 1615 when both wheat beer and lager were brewed contemporaneously. We also describe how the distribution of strains from the Munich Spaten brewery, and the development by Hansen and Linder of methods for producing pure starter cultures, facilitated the global spread of the Bavarian S. pastorianus lineages.

Insights into the transcriptional regulation of poorly characterized alcohol acetyltransferase-encoding genes (HgAATs) shed light into the production of acetate esters in the wine yeast Hanseniaspora guilliermondii.

Hanseniaspora guilliermondii is a well-recognized producer of acetate esters associated with fruity and floral aromas. The molecular mechanisms underneath this production or the environmental factors modulating it remain unknown. Herein, we found that, unlike Saccharomyces cerevisiae, H. guilliermondii over-produces acetate esters and higher alcohols at low carbon-to-assimilable nitrogen (C:N) ratios, with the highest titers being obtained in the amino acid-enriched medium YPD. The evidences gathered support a model in which the strict preference of H. guilliermondii for amino acids as nitrogen sources results in a channeling of keto-acids obtained after transamination to higher alcohols and acetate esters. This higher production was accompanied by higher expression of the four HgAATs, genes, recently proposed to encode alcohol acetyl transferases. In silico analyses of these HgAat's reveal that they harbor conserved AATs motifs, albeit radical substitutions were identified that might result in different kinetic properties. Close homologues of HgAat2, HgAat3, and HgAat4 were only found in members of Hanseniaspora genus and phylogenetic reconstruction shows that these constitute a distinct family of Aat's. These results advance the exploration of H. guilliermondii as a bio-flavoring agent providing important insights to guide future strategies for strain engineering and media manipulation that can enhance production of aromatic volatiles.

A Versatile Toolset for Genetic Manipulation of the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is an ascomycetous yeast that frequently dominates the population in the first two days of wine fermentations. It contributes to the production of many beneficial as well as detrimental aroma compounds. While the genome sequence of the diploid type strain DSM 2768 has been largely elucidated, transformation by electroporation was only recently achieved. We here provide an elaborate toolset for the genetic manipulation of this yeast. A chromosomal replication origin was isolated and used for the construction of episomal, self-replicating cloning vectors. Moreover, homozygous auxotrophic deletion markers (Huura3, Huhis3, Huleu2, Huade2) have been obtained in the diploid genome as future recipients and a proof of principle for the application of PCR-based one-step gene deletion strategies. Besides a hygromycin resistance cassette, a kanamycin resistance gene was established as a dominant marker for selection on G418. Recyclable deletion cassettes flanked by loxP-sites and the corresponding Cre-recombinase expression vectors were tailored. Moreover, we report on a chemical transformation procedure with the use of freeze-competent cells. Together, these techniques and constructs pave the way for efficient and targeted manipulations of H. uvarum.

The effect of vine variety and vintage on wine yeast community structure of grapes and ferments.

AIMS: The yeast community structure associated with grapes is an essential part of the wine-growing chain with a significant effect on wine quality. The aim of the present study was to evaluate the effect of the varietal factor on the yeast community assembly on grapes and during must fermentation. METHODS AND RESULTS: We analysed the wine yeast populations associated with four different grape varieties from the Greek national collection vineyard of Lykovryssi. The vintage effect was also considered by sampling the grapes for two consecutive years. Fourteen yeast species were recovered and genotyped to distinct subpopulations. A relatively stable yeast community structure was detected across vintages, with Hanseniaspora guilliermondii being the core species of the vineyard under study. The detected species subpopulations shared a relatively high genetic similarity with several genotypes persisting across vintages. CONCLUSIONS: It was shown that different grape cultivars were associated with distinct yeast communities, pointing to their possible implication on wine chemical diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Present findings show that the varietal factor is an important sharpener of the vineyard-associated wine yeast community, which may interfere with the organoleptic profile of the resulting wines.

Charting Shifts in Saccharomyces cerevisiae Gene Expression across Asynchronous Time Trajectories with Diffusion Maps.

During fermentation, Saccharomyces cerevisiae metabolizes sugars and other nutrients to obtain energy for growth and survival, while also modulating these activities in response to cell-environment interactions. Here, differences in S. cerevisiae gene expression were explored over a time course of fermentation and used to differentiate fermentations, using Pinot noir grapes from 15 unique sites. Data analysis was complicated by the fact that the fermentations proceeded at different rates, making a direct comparison of time series gene expression data difficult with conventional differential expression tools. This led to the development of a novel approach combining diffusion mapping with continuous differential expression analysis (termed DMap-DE). Using this method, site-specific deviations in gene expression were identified, including changes in gene expression correlated with the non-Saccharomyces yeast Hanseniaspora uvarum, as well as initial nitrogen concentrations in grape musts. These results highlight novel relationships between site-specific variables and Saccharomyces cerevisiae gene expression that are linked to repeated fermentation outcomes. It was also demonstrated that DMap-DE can extract biologically relevant gene expression patterns from other contexts (e.g., hypoxic response of Saccharomyces cerevisiae) and offers advantages over other data dimensionality reduction approaches, indicating that DMap-DE offers a robust method for investigating asynchronous time series gene expression data. IMPORTANCE In this work, Saccharomyces cerevisiae gene expression was used as a biosensor to capture differences across and between fermentations of Pinot noir grapes from 15 unique sites representing eight American Viticultural Areas. This required development of a novel analysis method, DMap-DE, for investigation of asynchronous gene expression data. It was demonstrated that DMap-DE reveals biologically relevant shifts in gene expression related to cell-environment interactions in the context of hypoxia and fermentation. Using these data, it was discovered that gene expression by non-Saccharomyces yeasts and initial nitrogen content in grape musts are correlated with differences in gene expression among fermentations. These findings highlight important relationships between site-specific variables and gene expression that may be used to understand why foods and beverages, including wine, possess sensory characteristics associated with or derived from their place of origin.

Development of Genetic Modification Tools for Hanseniasporauvarum.

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

CRISPR/Cas9-Mediated Genome Engineering Reveals the Contribution of the 26S Proteasome to the Extremophilic Nature of the Yeast Debaryomyces hansenii.

The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.

Metschnikowia pulcherrima represses aerobic respiration in Saccharomyces cerevisiae suggesting a direct response to co-cultivation.

The use of non-Saccharomyces species as starter cultures together with Saccharomyces cerevisiae is becoming a common practice in the oenological industry to produce wines that respond to new market demands. In this context, microbial interactions with these non-Saccharomyces species must be considered for a rational design of yeast starter combinations. Previously, transcriptional responses of S. cerevisiae to short-term co-cultivation with Torulaspora delbrueckii, Candida sake, or Hanseniaspora uvarum was compared. An activation of sugar consumption and glycolysis, membrane and cell wall biogenesis, and nitrogen utilization was observed, suggesting a metabolic boost of S. cerevisiae in response to competing yeasts. In the present study, the transcription profile of S. cerevisiae was analyzed after 3 h of cell contact with Metschnikowia pulcherrima. Results show an over-expression of the gluco-fermentative pathway much stronger than with the other species. Moreover, a great repression of the respiration pathway has been found in response to Metschnikowia. Our hypothesis is that there is a direct interaction stress response (DISR) between S. cerevisiae and the other yeast species that, under excess sugar conditions, induces transcription of the hexose transporters, triggering glucose flow to fermentation and inhibiting respiration, leading to an increase in both, metabolic flow and population dynamics.

Tolerance to Oxidative Stress in Budding Yeast by Heterologous Expression of Catalases A and T from Debaryomyces hansenii.

The function of catalases A and T from the budding yeast Saccharomyces cerevisiae (ScCta1 and ScCtt1) is to decompose hydrogen peroxide (H2O2) to mitigate oxidative stress. Catalase orthologs are widely found in yeast, suggesting that scavenging H2O2 is crucial to avoid the oxidative damage caused by reactive oxygen species (ROS). However, the function of catalase orthologs has not yet been experimentally characterized in vivo. Here, we heterologously expressed Debaryomyces hansenii DhCTA1 and DhCTT1 genes, encoding ScCta1 and ScCtt1 orthologs, respectively, in a S. cerevisiae acatalasemic strain (cta1Δ ctt1Δ). We performed a physiological analysis evaluating growth, catalase activity, and H2O2 tolerance of the strains grown with glucose or ethanol as carbon source, as well as under NaCl stress. We found that both genes complement the catalase function in S. cerevisiae. Particularly, the strain harboring DhCTT1 showed improved growth when ethanol was used as carbon source both in the absence or presence of salt stress. This phenotype is attributed to the high catalase activity of DhCtt1 detected at the exponential growth phase, which prevents intracellular ROS accumulation and confers oxidative stress resistance.

Genetic and transcriptomic evidences suggest ARO10 genes are involved in benzenoid biosynthesis by yeast.

Benzenoids are compounds associated with floral and fruity flavours in flowers, fruits and leaves and present a role in hormonal signalling in plants. These molecules are produced by the phenyl ammonia lyase pathway. However, some yeasts can also synthesize them from aromatic amino acids using an alternative pathway that remains unknown. Hanseniaspora vineae can produce benzenoids at levels up to two orders of magnitude higher than Saccharomyces species, so it is a model microorganism for studying benzenoid biosynthesis pathways in yeast. According to their genomes, several enzymes have been proposed to be involved in a mandelate pathway similar to that described for some prokaryotic cells. Among them, the ARO10 gene product could present benzoylformate decarboxylase activity. This enzyme catalyses the decarboxylation of benzoylformate into benzaldehyde at the end of the mandelate pathway in benzyl alcohol formation. Two homologous genes of ARO10 were found in the two sequenced H. vineae strains. In this study, nine other H. vineae strains were analysed to detect the presence and per cent homology of ARO10 sequences by PCR using specific primers designed for this species. Also, the copy number of the genes was estimated by quantitative PCR. To verify the relation of ARO10 with the production of benzyl alcohol during fermentation, a deletion mutant in the ARO10 gene of Saccharomyces cerevisiae was used. The two HvARO10 paralogues were analysed and compared with other α-ketoacid decarboxylases at the sequence and structural level.

Contribution of the mitogen-activated protein kinase Hog1 to the halotolerance of the marine yeast Debaryomyces hansenii.

Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1 M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.

Yeast population dynamics during spontaneous fermentation of icewine and selection of indigenous Saccharomyces cerevisiae strains for the winemaking in Qilian, China.

BACKGROUND: Icewine produced in China is becoming popular, but there is only limited knowledge available on the yeast population that occurs during fermentation and also on the selection of indigenous Saccharomyces cerevisiae strains for its production. In this work, we first investigated yeast species and the evolution of yeast population in spontaneous fermentations of icewine produced in the Qilian region of China and then analyzed the biodiversity and important enological properties of S. cerevisiae isolates. RESULTS: Seven species of five genera including S. cerevisiae, S. uvarum, Torulaspora delbrueckii, Hanseniaspora uvarum, Lachancea thermotolerans, Metschnikowia aff. fructicola and H. osmophila were identified by the colony morphology on Wallerstein Laboratory Nutrient medium and sequence analysis of the 26S rRNA gene D1/D2 domain. Saccharomyces cerevisiae, H. uvarum and L. thermotolerans were the dominant species, representing almost 87% of the total yeast isolates. Microvinification with seven preselected S. cerevisiae strains were performed on Vidal. All selected strains could complete fermentations, and the enochemical parameters were within the acceptable ranges of the wine industry. W5B3 produced higher amounts of ethyl hexanoate and ethyl octanoate than other strains. R3A10 was a low volatile acid producer and the corresponding icewine presented the highest values on some odorants including ß-damascenone, 1-octen-3-ol, ethyl 2-methylbutyrate, and isoamyl alcohol. Vidal icewines fermented with R3A10, R3A16 and W5B3 were well accepted by the judges because of superior sensory quality. CONCLUSION: Three indigenous strains (R3A10, R3A16 and W5B3) could be used as starters and could potentially improve the regional character of the icewine in Qilian. © 2020 Society of Chemical Industry.

Melatonin and glycolytic protein interactions are related to yeast fermentative capacity.

Melatonin is an indole amine that interacts with some proteins in mammals, such as calreticulin, calmodulin or sirtuins. In yeast, melatonin is synthetized and interacts with glycolytic proteins during alcoholic fermentation in Saccharomyces cerevisiae. Due to its importance as an antioxidant molecule in both Saccharomyces and non-Saccharomyces yeasts, the aim of this study was to determine the intracellular and extracellular synthesis profiles of melatonin in four non-Saccharomyces strains (Torulaspora delbrueckii, Hanseniaspora uvarum, Starmeralla bacillaris and Metschnikowia pulcherrima) and to confirm whether glycolytic enzymes can also interact with this molecule in non-conventional yeast cells. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry, and proteins bound to melatonin were immunopurified by melatonin-IgG-Dynabeads. Melatonin was produced in a similar pattern in all non-Saccharomyces yeast, with M. pulcherrima and S. bacillaris being the highest producers. However, melatonin only bound to proteins in two non-conventional yeasts, S. bacillaris and T. delbrueckii, which specifically had higher fermentative capacities. Sequence analysis showed that most proteins shared high levels of homology with glycolytic enzymes, but an RNA-binding protein, the elongation alpha factor, which is related to mitochondria, was also identified. This study reports for the first time the interaction of melatonin with proteins inside non-Saccharomyces yeast cells. These results reinforce the possible role of melatonin as a signal molecule, likely related to fermentation metabolism and provide a new perspective for understanding its role in yeast.