Identification of indigenous yeast flora isolated from the five winegrape varieties harvested in Xiangning,China

Inoculated fermentation by selected indigenous yeast strains from a specific location could provide the wine with unique regional sensory characteristics. The identification and differentiation of local yeasts are the first step to understand the function of yeasts and develop a better strain-selection program for winemaking. The indigenous yeasts in five grape varieties, Chardonnay, Cabernet Franc, Cabernet Sauvignon, Marselan, and Merlot cultivated in Xiangning, Shanxi, China were investigated. Eight species of seven genera including Aureobasidium pullulans, Candida zemplinina, Hanseniaspora uvarum, Hanseniaspora occidentalis, Issatchenkia terricola, Metschnikowia pulcherrima, Pichia kluyveri, and Saccharomyces cerevisiae were identified using Wallerstein Laboratory Nutrient medium with sequencing of the 26S rDNA D1/D2 domain. H. uvarum and S. cerevisiae were the predominant species, while most non-Saccharomyces species were present in the whole fermentation process at different levels among the grape varieties. The genotypes of S. cerevisiae from each microvinification were determined by using interdelta sequence analysis. The 102 isolates showed eight different genotypes, and genotype III was the predominant genotype found. The distribution of S. cerevisiae strains during the fermentation of Marselan was also studied. Six genotypes were observed among the 92 strains with different genotypes of competitiveness at different sampling stages. Genotype V demonstrated the potential for organizing starter strains and avoiding inefficient fermentation. In general, this study explored the yeast species in the grapes grown in Xiangning County and provided important information of relationship of local yeast diversity and its regional wine sensory characteristics.     

Inter‐kingdom signaling — symbiotic yeasts produce semiochemicals that attract their yellowjacket hosts

In recent studies, the yeast species Hanseniaspora uvarum and Lachancea thermotolerans were isolated from the digestive tract of four North American yellowjacket species (Hymenoptera: Vespidae), and attraction of yellowjackets to brewer's yeast, Saccharomyces cerevisiae (all Saccharomycetaceae), growing on fruit powder was demonstrated. We tested the hypothesis that Vespula spp. are attracted to cultures of H. uvarum and L. thermotolerans and their respective volatiles. In field experiments, we found that H. uvarum and L. thermotolerans are attractive to three species of yellowjacket, but only when grown on grape juice‐infused yeast peptone dextrose (YPD) agar. Using gas chromatography‐mass spectrometry, we analyzed the headspace volatiles produced by these yeasts, and field tested an 18‐component yeast synthetic semiochemical blend. This synthetic blend attracted western yellowjackets, Vespula pensylvanica (Saussure), but no other yellowjacket species. Acetic acid or ethanol added to the synthetic blend at biologically relevant doses either had no effect or significantly lowered trap captures. Our results demonstrate that yeast symbionts isolated from the digestive tract of yellowjackets are attractive to their hosts. Further research is needed to identify the volatiles mediating attraction of species other than V. pensylvanica to the yeast cultures.     

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

Erythritol is produced in yeasts via the reduction of erythrose into erythritol by erythrose reductases (ERs). However, the genes codifying for the ERs involved in this reaction have not been described in any Saccharomyces species yet. In our laboratory, we recently showed that, during alcoholic fermentation, erythritol is differentially produced by Saccharomyces cerevisiae and S. uvarum species, the latter being the largest producer. In this study, by using BLAST analysis and phylogenetic approaches the genes GRE3, GCY1, YPR1, ARA1 and YJR096W were identified as putative ERs in Saccharomyces cerevisiae Then, these genes were knocked out in our S. uvarum strain (BMV58) with higher erythritol biosynthesis compared to control S. cerevisiae wine strain, to evaluate their impact on erythritol synthesis and global metabolism. Among the mutants, the single deletion of GRE3 markedly impacts erythritol production, although ΔYPR1ΔGCY1ΔGRE3 was the combination that most decreased erythritol synthesis. Consistent with the increased production of fermentative by-products involved in redox balance in the Saccharomyces uvarum strain BMV58, erythritol synthesis increases at higher sugar concentrations, hinting it might be a response to osmotic stress. However, the expression of GRE3 in the S. uvarum strain was found to peak just before the start of the stationary phase, being consistent with the observation that erythritol increases at the start of the stationary phase, when there is low sugar in the medium and nitrogen sources are depleted. This suggests that GRE3 plays its primary function to help the yeast cells to maintain the redox balance during the last phases of fermentation.     

Mitochondrial DNA polymorphism of the yeast <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> var. <Emphasis Type="Italic">uvarum</Emphasis>

Genetic relationships among forty-one strains of Saccharomyces bayanus var. uvarum isolated in different wine regions of Europe and four wild isolates were investigated by restriction analysis (RPLP) of mitochondrial DNA (mtDNA) with four restriction endonucleases, AluI, DdeI, HinfI and RsaI. No clear correlation between origin and source of isolation of S. bayanus var. uvarum strains and their mtDNA restriction profiles was found. On the whole, the mtDNA of S. bayanus var. uvarum is much less polymorphic than that of S. cerevisiae. This observation is in good agreement with results obtained by electrophoretic karyotyping. Unlike wine S. cerevisiae, strains of S. bayanus var. uvarum display a low level of chromosome length polymorphism.     

Proteomic characterization of extracellular vesicles produced by several wine yeast species

In winemaking, the use of alternative yeast starters is becoming increasingly popular. They contribute to the diversity and complexity of wine sensory features and are typically used in combination with Saccharomyces cerevisiae, to ensure complete fermentation. This practice has drawn the interest on interactions between different oenological yeasts, which are also relevant in spontaneous and conventional fermentations, or in the vineyard. Although several interactions have been described and some mechanisms have been suggested, the possible involvement of extracellular vesicles (EVs) has not yet been considered. This work describes the production of EVs by six wine yeast species (S. cerevisiae, Torulaspora delbrueckii, Lachancea thermotolerans, Hanseniaspora uvarum, Candida sake and Metschnikowia pulcherrima) in synthetic grape must. Proteomic analysis of EV-enriched fractions from S. cerevisiae and T. delbrueckii showed enrichment in glycolytic enzymes and cell-wall-related proteins. The most abundant protein found in S. cerevisiae, T. delbrueckii and L. thermotolerans EV-enriched fractions was the enzyme exo-1,3-β-glucanase. However, this protein was not involved in the here-observed negative impact of T. delbrueckii extracellular fractions on the growth of other yeast species. These findings suggest that EVs may play a role in fungal interactions during wine fermentation and other aspects of wine yeast biology.     

Convergent adaptation of Saccharomyces uvarum to sulfite,an antimicrobial preservative widely used in human-driven fermentations

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 VII^XVI and XI^XVI 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.     

Yeast species associated with spontaneous wine fermentation of Cabernet Sauvignon from Ningxia,China

The eastern base of the Helan Mountains in Ningxia is a fast developing wine production area in China. Of urgent necessity to the Ningxia wine industry is to be able to produce wines with typical regional characteristics. It is well known that autochthonous yeast species and strains play an important role in introducing local character or terroir into the winemaking practice. The aim of this study was to investigate indigenous yeast species diversity and preselect desirable S. cerevisiae strains in the Ningxia region. Four hundred wine-related yeast colonies were isolated from Cabernet Sauvignon musts in three vineyards at the beginning, middle and final stages of spontaneous fermentations. Yeast species were first classified according to colony morphologies on Wallerstein Laboratory Nutrient Agar (WL) and confirmed by sequencing of the D1/D2 domain of the 26S rRNA gene. Nine unique colony morphology types were profiled on WL agar and three new types were found. After sequence analysis of representative colonies from each WL group, nine yeast species were identified, namely H. uvarum, H. occidentalis, M. pulcherrima, C. zemplinina, H. vineae, I. orientalis, Z. bailii, P. kluyveri and S. cerevisiae. The non-Saccharomyces yeasts appeared mainly in the early stage of the fermentation while H. uvarum, I. orientalis and P. kluyveri were able to remain in the final stage. Fourty-six S. cerevisiae isolates were preselected according to physiological characteristics and twelve strains with valuable fermentation properties were obtained.     

Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> subspecies

There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomyces cerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleotide based on a conserved tRNA region, specific for ascomycetes. Tandem repeats are randomly dispersed in all eukaryotic genomes and tRNA genes are conserved and present in several copies in different chromosomes. As an example, the method was applied to discriminate native M. pulcherrima strains but it could be extended to differentiate strains from other non-Saccharomyces species. The biodiversity of species and strains found in the grape ecosystem is a potential source of new enzymes, fungicides and/or novel sustainable methods for biological control of phytopathogens.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

Genetic relationship and biological status of the industrially important yeast <Emphasis Type="Italic">Saccharomyces eubayanus</Emphasis> Sampaio et al.

The genomes of the recently discovered yeast Saccharomyces eubayanus and traditional S. cerevisiae are known to be found in the yeast S. pastorianus (syn. S. carlsbergensis), which are essential for brewing. The cryotolerant yeast S. bayanus var. uvarum is of great importance for production of some wines. Based on ascospore viability and meiotic recombination of the control parental markers in hybrids, we have shown that there is no complete interspecies post-zygotic isolation between the yeasts S. eubayanus, S. bayanus var. bayanus and S. bayanus var. uvarum. The genetic data presented indicate that all of the three taxa belong to the same species.     

Yeast Diversity Isolated from Grape Musts During Spontaneous Fermentation from a Brazilian Winery

Saccharomyces and non-Saccharomyces yeast species from a winery located in Brazil were identified by ribosomal gene-sequencing analysis. A total of 130 yeast strains were isolated from grape surfaces and musts during alcoholic fermentation from Isabel, Bordeaux, and Cabernet Sauvignon varieties. Samples were submitted to PCR–RFLP analysis and genomic sequencing. Thirteen species were identified: Candida quercitrusa, Candida stellata, Cryptococcus flavescens, Cryptococcus laurentii, Hanseniaspora uvarum, Issatchenkia occidentalis, Issatchenkia orientalis, Issatchenkia terricola, Pichia kluyveri, Pichia guilliermondii, Pichia sp., Saccharomyces cerevisiae, and Sporidiobolus pararoseus. A sequential substitution of species during the different stages of fermentation, with a dominance of non-Saccharomyces yeasts at the beginning, and a successive replacement of species by S. cerevisiae strains at the final steps were observed. This is the first report about the yeast distribution present throughout the alcoholic fermentation in a Brazilian winery, providing supportive information for future studies on their contribution to wine quality.     

Identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis

Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum. In sourdough A (traditional process with rye flour), C. humilis dominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40°C, respectively, S. cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant. Isolates identified as C. humilis and S. cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S. uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S. cerevisiae. The last four species were also detected in sourdoughs A, B, and C.     

Killer toxin of Hanseniaspora uvarum

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000.Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by -1, 6-d-glucans.     

Genetic reidentification of the pectinolytic yeast strain SCPP as Saccharomyces bayanus var. uvarum

Using genetic hybridization analysis, pulsed-field gel electrophoresis of chromosomal DNA and PCR/RFLP analysis of the MET2 gene, we reidentified 11 Champagne yeast strains. Two of them, SCPP and SC4, were found to belong to Saccharomyces bayanus var. uvarum and the remaining strains to S. cerevisiae. Strain SCPP (CLIB 2025) of S. bayanus var. uvarum is known as a producer of three pectinolytic enzymes. Received: 28 April 2000 / Received revision: 20 July 2000 / Accepted: 25 July 2000     

A Structural and Phylogenetic Study of the HO Gene from Saccharomyces bayanus var. uvarum

A novel HO gene (Uv-HO) was cloned from the Saccharomyces bayanus var. uvarum (abbreviated as S. uvarum in this study) type strain. The coding region of Uv-HO showed relatively high homology (95%) to that of the Sb-HO gene (S. bayanus var. bayanus HO), but not to the HO genes of other Saccharomyces sensu stricto species. However, the 5′ and 3′ non-coding region of Uv-HO showed less similarity (79% and 76% respectively) even to those of the most homologous gene Sb-HO. Motifs of the mating-type control and the cell-cycle control were conserved in the 5′ non-coding region of Uv-HO, but numbers and positions of motifs were different from those of Sb-HO. CHEF-Southern analysis showed that all tested strains of S. bayanus species, including S. uvarum, carried the HO gene on the 1,100-kb chromosome. By HO-typing PCR using mixed primers, which provided a rapid and convenient tool for yeast identification, either the Uv-HO gene or the Sb-HO gene was detected in strains of S. bayanus species, but two strains were found to have both types of HO gene in each genome. These results suggest that S. uvarum has a unique sequence, but might share the same chromosome constitution within S. bayanus species, and that S. bayanus is a heterogeneous species, of which some strains might be natural hybrid.     

Exploration of genetic and phenotypic diversity within Saccharomyces uvarum for driving strain improvement in winemaking

The selection and genetic improvement of wine yeast is an ongoing process, since yeast strains should match new technologies in winemaking to satisfy evolving consumer preferences. A large genetic background is the necessary starting point for any genetic improvement programme. For this reason, we collected and characterized a large number of strains belonging to Saccharomyces uvarum. In particular, 70 strains were isolated from cold-stored must samples: they were identified and compared to S. uvarum strains originating from different collections, regarding fermentation profile, spore viability and stress response. The results demonstrate a large biodiversity among the new isolates, with particular emphasis to fermentation performances, genotypes and high spore viability, making the isolates suitable for further genetic improvement programmes. Furthermore, few of them are competitive with Saccharomyces cerevisiae and per se, suitable for wine fermentation, due to their resistance to stress, short lag phase and fermentation by-products.

     

The [URE3] prion is not conserved among Saccharomyces species

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating inactive form of the nitrogen catabolism regulator Ure2p. To determine whether the [URE3] prion is conserved in S. cerevisiae-related yeast species, we have developed genetic tools allowing the detection of [URE3] in Saccharomyces paradoxus and Saccharomyces uvarum. We found that [URE3] is conserved in S. uvarum. In contrast, [URE3] was not detected in S. paradoxus. The inability of S. paradoxus Ure2p to switch to a prion isoform results from the primary sequence of the protein and not from the lack of cellular cofactors as heterologous Ure2p can propagate [URE3] in this species. Our data therefore demonstrate that [URE3] is conserved only in a subset of Saccharomyces species. Implications of our finding on the physiological and evolutionary meaning of the yeast [URE3] prion are discussed.     

Metabolomic Comparison of Saccharomyces cerevisiae and the Cryotolerant Species S. bayanus var. uvarum and S. kudriavzevii during Wine Fermentation at Low Temperature

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.