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Zygosaccharomyces rouxii is an osmotolerant and halotolerant yeast that can participate in fermentation. To understand the mechanisms of salt and sugar tolerance, the transcription levels of Z. rouxii M 2013310 under 180 g/L NaCl stress and 600 g/L glucose stress were measured. The transcriptome analysis showed that 2227 differentially expressed genes (DEGs) were identified under 180 g/L NaCl stress, 1530 DEGs were identified under 600 g/L glucose stress, and 1278 DEGs were identified under both stress conditions. Then, KEGG enrichment analyses of these genes indicated that 53.3% of the upregulated genes were involved in the ergosterol synthesis pathway. Subsequently, quantitative PCR was used to verify the results, which showed that the genes of the ergosterol synthesis pathway were significantly upregulated under 180 g/L NaCl stress. Finally, further quantitative testing of ergosterol and spotting assays revealed that Z. rouxii M 2013310 increased the amount of ergosterol in response to high salt stress. These results highlighted the functional differences in ergosterol under sugar stress and salt stress, which contributes to our understanding of the tolerance mechanisms of salt and sugar in Z. rouxii.

Zygosaccharomyces rouxii produces high levels of 4-hydroxy-2,5-dimethyl-3(2H)-furanone in YPD medium supplemented with 120 g/L D-fructose and 180 g/L NaCl after 5 d. D-fructose has a stress effect on Z. rouxii, and GSH-Px is a main enzyme involved in the defense of Z. rouxii against oxygen stress according to our previous report. In order to further explore the molecular mechanism of the glutathione metabolism pathway in Z. rouxii in response to D-fructose stress, changes in the expression of genes and proteins involved in the synthesis of glutathione precursor amino acids and enzymes were observed. In addition, changes in the intermediates related to glutathione synthesis in Z. rouxii were reported. The results indicated that some gene-encoding enzymes involved in the glutamate, cysteine and glycine biosynthesis pathways and key genes involved in glutathione synthesis were upregulated. The expression levels of other genes, except SHMT, were consistent with the qRT-PCR results. The contents of γ-glutamylcysteine and glutathione amide in the D-fructose group were higher than those in the control group. In the D-fructose stress groups, the metabolic flux towards glutathione synthesis was increased. These results might provide more in-depth and detailed theoretical support for the oxidative stress defense mechanism of Z. rouxii under D-fructose stress.

Interspecies hybridization is an important evolutionary mechanism in yeasts. The genus Zygosaccharomyces in particular contains numerous hybrid strains and/or species. Here, we investigated the genome of Zygosaccharomyces strain MT15, an isolate from Maotai-flavor Chinese liquor fermentation. We found that it is an interspecies hybrid and identified it as Zygosaccharomyces pseudobailii. The Z. bailii species complex consists of three species: Z. bailii, which is not a hybrid and whose 10 Mb genome is designated ‘A’, and two hybrid species Z. parabailii (‘AB’ genome, 20 Mb) and Z. pseudobailii (‘AC’ genome, 20 Mb). The A, B and C subgenomes are all approximately 7%–10% different from one another in nucleotide sequence, and are derived from three different parental species. Despite being hybrids, Z. pseudobailii and Z. parabailii are capable of mating and sporulating. We previously showed that Z. parabailii regained fertility when one copy of its MAT locus became broken into two parts, causing the allodiploid hybrid to behave as a haploid gamete. In Z. pseudobailii, we find that a very similar process occurred after hybridization, when a deletion of 1.5 kb inactivated one of the two copies of its MAT locus. The half-sibling species Z. parabailii and Z. pseudobailii therefore went through remarkably parallel but independent steps to regain fertility after they were formed by separate interspecies hybridizations., Several natural interspecies hybrids in Zygosaccharomyces overcame sterility by inactivating a MAT locus and undergoing whole-genome duplication.

The brewing industry is experiencing a period of change and experimentation largely driven by customer demand for product diversity. This has coincided with a greater appreciation of the role of yeast in determining the character of beer and the widespread availability of powerful tools for yeast research. Genome analysis in particular has helped clarify the processes leading to domestication of brewing yeast and has identified domestication signatures that may be exploited for further yeast development. The functional properties of non-conventional yeast (both Saccharomyces and non- Saccharomyces) are being assessed with a view to creating beers with new flavours as well as producing flavoursome non-alcoholic beers. The discovery of the psychrotolerant S. eubayanus has stimulated research on de novo S. cerevisiae × S. eubayanus hybrids for low-temperature lager brewing and has led to renewed interest in the functional importance of hybrid organisms and the mechanisms that determine hybrid genome function and stability. The greater diversity of yeast that can be applied in brewing, along with an improved understanding of yeasts' evolutionary history and biology, is expected to have a significant and direct impact on the brewing industry, with potential for improved brewing efficiency, product diversity and, above all, customer satisfaction. Fil: Gibson, B.. Vtt Technical Research Centre Of Finland; Finlandia Fil: Geertman, J. M. A.. Heineken Supply Chain; Países Bajos Fil: Hittinger, C. T.. University of Wisconsin; Estados Unidos Fil: Krogerus, K.. Vtt Technical Research Centre Of Finland; Finlandia. Aalto University; Finlandia Fil: Libkind Frati, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales.; Argentina Fil: Louis, E. J.. University of Leicester; Reino Unido Fil: Magalhães, F.. Vtt Technical Research Centre Of Finland; Finlandia. Aalto University; Finlandia Fil: Sampaio, J.P.. Universidade Nova de Lisboa; Portugal

The aim of this pioneering study was to determine the biodiversity of non- Saccharomyces yeasts in ancient distilleries located in the La Mancha region, which is the principal area for the production of bioethanol and grape-based distillates in Spain. In this study, the yeast populations that were present during the process of extraction of alcohol and residual sugars from the byproducts of vinification, such as piquettes, pomace and grape skins, were studied. Non- Saccharomyces yeasts were identified by PCR-RFLP analysis of the 5.8S rRNA genes and, when necessary, by sequencing the D1/D2 domain of the 26S and/or 5.8S rRNA genes. Further, fermentation and the assimilation of carbon compounds were studied, to identify potential industrial applications. Phylogenetic trees and heat-maps were constructed for the genetic and phenotypic traits, respectively. Twenty yeast species belonging to eight genera were identified ( Torulaspora , Candida , Zygosaccharomyces , Pichia , Hanseniaspora , Kluyveromyces , Ogataea and Saccharomycodes ). Pichia galeiformis , Candida lactis-condensi , Hanseniaspora osmophila and Torulaspora delbrueckii were the most abundant species and were found principally in sweet and fermented piquettes.

Zygosaccharomyces rouxii complex comprises three yeasts clusters sourced from sugar- and salt-rich environments: haploid Zygosaccharomyces rouxii, diploid Zygosaccharomyces sapae and allodiploid/aneuploid strains of uncertain taxo- nomic affiliations. These yeasts have been characterized with respect to gene copy number variation, karyotype variability and change in ploidy, but func- tional diversity in stress responses has not been explored yet. Here, we quanti- tatively analysed the stress response variation in seven strains of the Z. rouxii complex by modelling growth variables via model and model-free fitting meth- ods. Based on the spline fit as most reliable modelling method, we resolved dif- ferent interstrain responses to 15 environmental perturbations. Compared with Z. rouxii CBS 732 T and Z. sapae strains ABT301 T and ABT601, allodiploid strain ATCC 42981 and aneuploid strains CBS 4837 and CBS 4838 displayed higher multistress resistance and better performance in glycerol respiration even in the presence of copper. l-based logarithmic phenotypic index high- lighted that ABT601 is a slow-growing strain insensitive to stress, whereas ABT301 T grows fast on rich medium and is sensitive to suboptimal conditions. Overall, the differences in stress response could imply different adaptation mechanisms to sugar- and salt-rich niches. The obtained phenotypic profiling contributes to provide quantitative insights for elucidating the adaptive mecha- nisms to stress in halo- and osmo-tolerant Zygosaccharomyces yeasts.

Zygosaccharomyces rouxii and the related species Zygosaccharomyces sapae (hereafter referred to as Z. rouxii complex) are protoploid hemiascomycete yeasts relevant in the elaboration and spoilage of foodstuff. Divergence of Z. rouxii complex before whole genome duplication, leading to the genus Saccharomyces, makes these yeasts very attractive for genome evolution study. Relatively little is known, however, about the diversity in this branch at the genetic and physiological levels. In this work, we investigated Z. rouxii complex, encompassing strains that in other works have been studied separately and comparing them in a comprehensive way. We showed that the majority of strains are unusually heterogeneous in their ribosomal DNA, a signal of relaxation of concerted evolution. Further analysis showed that they have hypervariable karyotypes, different levels of ploidy, and that housekeeping markers vary both in copy number and sequence. Overall, the results provide compelling evidence that the strains considered in this study are a complex of haploid, aneuploid and diploid mosaic lineages. The reproductive mode and life cycle of Zygosaccharomyces could lead to this unsuspected diversity.

The ability of Zygosaccharomyces bailii to grow at low pH and in the presence of considerable amounts of weak organic acids, at lethal condition for Saccharomyces cerevisiae , increased the interest in the biotechnological potential of the yeast. To understand the mechanism of tolerance and growth effect of weak acids on Z. bailii , we evaluated the physiological and macromolecular changes of the yeast exposed to sub lethal concentrations of lactic acid. Lactic acid represents one of the important commodity chemical which can be produced by microbial fermentation. We assessed physiological effect of lactic acid by bioreactor fermentation using synthetic media at low pH in the presence of lactic acid. Samples collected from bioreactors were stained with propidium iodide (PI) which revealed that, despite lactic acid negatively influence the growth rate, the number of PI positive cells is similar to that of the control. Moreover, we have performed Fourier Transform Infra-Red (FTIR) microspectroscopy analysis on intact cells of the same samples. This technique has been never applied before to study Z. bailii under this condition. The analyses revealed lactic acid induced macromolecular changes in the overall cellular protein secondary structures, and alterations of cell wall and membrane physico-chemical properties.

Species-specific primer pairs that produce a single band of known product size have been developed for members of the Zygosaccharomyces clade including Zygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces kombuchaensis, Zygosaccharomyces lentus, Zygosaccharomyces machadoi, Zygosaccharomyces mellis and Zygosaccharomyces rouxii. An existing primer pair for the provisional new species Zygosaccharomyces pseudorouxii has been confirmed as specific. The HIS3 gene, encoding imidazole-glycerolphosphate dehydratase, was used as the target gene. This housekeeping gene evolves slowly and is thus well conserved among different isolates, but shows a significant number of base pair changes between even closely related species, sufficient for species-specific primer design. The primers were tested on type and wild strains of the genus Zygosaccharomyces and on members of the Saccharomycetaceae. Sequencing of the D1/D2 region of rDNA was used to confirm the identification of all nonculture collection isolates. This approach used extracted genomic DNA, but in practice, it can be used efficiently with a rapid colony PCR protocol. The method also successfully detected known and new hybrid strains of Z. rouxii and Z. pseudorouxii. The method is rapid, robust and inexpensive. It requires little expertise by the user and is thus useful for preliminary, large-scale screens.

The nonconventional yeast Zygosaccharomyces bailii has been proposed as a new host for biotechnological processes due to convenient properties such as its resistance to high sugar concentrations, relatively high temperatures and especially to acidic environments. We describe a series of new expression vectors specific for Z. bailii and the resulting improvements in production levels. By exploiting the sequences of the endogenous plasmid pSB2, 2microm-like multicopy vectors were obtained, giving a fivefold increase in production. A specific integrative vector was developed which led to 100% stability in the absence of selective pressure; a multiple-integration vector was constructed, based on an rRNA gene unit portion cloned and sequenced for this purpose, driving the insertion of up to 80 copies of the foreign construct. Moreover, we show the construction of the first stable auxotrophic mutant of Z. bailii, obtained by targeted gene deletion applied to ZbLEU2. The development of molecular tools for the Z. bailii manipulation has now reached a level that may be compatible with its industrial exploitation; the production of organic acids is a prominent field of application.

The yeast Zygosaccharomyces bailii, known to have peculiar resistance to several environmental constraints, is very little known with respect to its genetics and life cycle. In addition to molecular and biochemical studies, cytofluorimetric and morphological analyses can also add information necessary to shed light on its interesting features. In the present study, the DNA and protein content as well as the cellular morphology of Z. bailii populations growing in minimal medium supplemented with different carbon sources and with the addition of different organic acids were investigated. The results show the occurrence of a multibudded phenotype and of a low, but significant percentage of binucleate cells occurring in the early-stationary phase. These traits appear to be different in comparison with the better-known laboratory yeast Saccharomyces cerevisiae. Experiments and speculations about these features and possible implications with Z. bailii main characteristics are discussed.

Zygosaccharomyces rouxii is an osmotolerant yeast growing in the presence of high concentrations of salts and/or sugars. The maintenance of intracellular potassium homeostasis is essential for osmostress adaptation. Zygosaccharomyces rouxii is endowed with only one typical potassium transporter (ZrTrk1). We characterized ZrTrk1 activity and its contribution to various physiological parameters in detail. Our results show that ZrTrk1 is a high-affinity K(+) transporting system efficiently discriminating between K(+) and Li(+) and indicate the presence of another, currently unknown K(+) importing system with a low affinity in Z. rouxii cells. Upon ZrTrk1 heterologous expression in Saccharomyces cerevisiae, it confers cells with a remarkably high lithium tolerance (even to wild-type strains) due to preventing Li(+) influx into cells, and is able to complement a plasma-membrane hyperpolarization and cell sensitivity to cationic compounds caused by the lack of endogenous K(+) transporters. Intracellular pH measurements with pHluorin, whose coding sequence was integrated into the genome, showed that the expression of ZrTrk1 also complements a decrease in intracellular pH in S. cerevisiae trk1Δ trk2Δ cells. Our data corroborate a tight connection between potassium and proton transporters in yeasts and provide new insights into Z. rouxii cation homeostasis and the basis of its high osmotolerance.

Due to increased occurrence of infections and food spoilage caused by yeast, there is an unmet need for new antifungal agents. The arginine-β-(2,5,7-tri-tert-butylindol-3-yl) alanine-arginine (R-Tbt-R) motif was previously proved useful in the design of an antifungal tripeptide. Here, an array of peptidomimetics based on this motif was investigated for antifungal and hemolytic activity. The five most promising modified tetrapeptide analogues ( 6: and 9-12: contain an additional C-terminal hydrophobic residue, and these were found to exhibit antifungal activity against Saccharomyces cerevisiae (MIC 6 and 12 μg mL(-1)) and Zygosaccharomyces bailii (MIC 6-25 μg mL(-1)). Four compounds ( 6: and 9-11: , had limited hemolytic activity (

The genetic diversity of the types or authentic strains of 20 facultative synonyms of Pichia membranifaciens (E.C. Hansen) E.C. Hansen was revealed on the basis of large-subunit (26S) rDNA D1/D2 domain and internal transcribed spacer region sequencing and electrophoretic karyotyping. At least five separate species were recognized among the strains studied. Fourteen strains were confirmed to belong to P. membranifaciens. Strain CBS 241, an authentic strain of Zygosaccharomyces chevalieri Guilliermond var. fermentati Saito, should be assigned to Pichia manshurica Santa Maria. Strain CBS 243, an authentic strain of Zygopichia chiantigiana Castelli, is conspecific with CBS 2287, the type strain of Pichia fluxuum (Phaff & Knapp) Kreger-van Rij. Strain CBS 1367, the type of Zygosaccharomyces bisporus Anderson, belongs to Pichia kluyveri Bedford var. kluyveri. Strain CBS 989, the type of Cryptococcus californicus Anderson & Skinner, represents a distinct species, for which a new combination, Candida californica comb. nov., is proposed. The taxonomic status of strains CBS 189, the type of Pichia calliphorae Klocker, and CBS 214, the type of Pichia derossii Castelli, remain to be studied further. Their D1/D2 sequences and chromosomal DNA banding patterns were similar to those of P. membranifaciens, but their internal transcribed spacer sequences differed significantly.

We have evaluated the effect of grape maturity and cold maceration prior to fermentation on the yeast ecology during wine fermentation. Non-Saccharomyces strains were selectively isolated and identified using two rapid PCR techniques, namely enterobacterial repetitve intergenic consensus-PCR and PCR-intron splice sites, in various wine fermentation conditions. These identifications were further complemented and confirmed by restriction fragment length poymorphism and sequencing analysis of the 5.8S-ITS and D1/D2 ribosomal regions, respectively. Eleven species belonging to five genera were identified. Candida stellata, Hanseniaspora uvarum and Hanseniaspora osmophila were the dominant species, representing almost 90% of the isolates. Minor strains presented different species of the genera Candida, Issatchenkia, Zygoascus and Zygosaccharomyces. Selective isolation made it possible to isolate some species that were hardly related to the wine-making process, such as Issatchenkia hanoiensis, a new species that has only been described recently.

26S rDNA D1/D2 sequencing was used to characterise a number of food-associated Zygosaccharomyces rouxii strains held at the National Collection of Yeast Cultures. In the course of this study, four strains (NCYC 1682, NCYC 3042, NCYC 3060 and NCYC 3061) were identified which appeared, based on their D1/D2 sequences, to belong to a novel Zygosaccharomyces species. However, subsequent sequence analysis showed that NCYC 1682, NCYC 3060 and NCYC 3061 possess two highly divergent copies of the nuclear-encoded ADE2, HIS3 and SOD2 genes, indicating these three strains are in fact hybrids. NCYC 3042, however, does appear to represent a novel species which may be hypothesized to have crossed with Z. rouxii and given rise to hybrid strains. Additional approaches to define precise taxonomic status and mechanisms of hybrid genome formation amongst yeast species are discussed.

We cloned two genes from the salt-tolerant yeast Zygosaccharomyces rouxii: ZrSOD2 for the cell membrane Na+/H+-antiporter and ZrPMA1 for the cell membrane H+-ATPase. The products of these genes play cooperative roles in the salt-tolerance of Z. rouxii, and the function of the ZrPMA1 product is regulated at the transcription level. We constructed a yeast expression vector that is able to co-express the ZrSOD2 and ZrPMA1 genes. Single expression of ZrSOD2 was effective in conferring salt-tolerance, and although a slight synergic effect was observed with co-expression of ZrSOD2 and ZrPMA1, the usefulness of this co-expression is likely to be minimal with regard to salt-tolerance.

Glycerol is a key compound in the regulation of several metabolic pathways in Saccharomyces cerevisiae. From this yeast most of the genes involved in glycerol consumption, production and transport are now available. Some of the mechanisms involving glycerol metabolism and transport are common to other yeasts. This work presents a search for GPD1/2, GUT1, GUP1/2 and FPS1 orthologues in a series of hemiascomycetous yeasts. All the genes cloned were able to complement S. cerevisiae mutant phenotypes and presented a high degree of similarity to the corresponding genes in this yeast. A phylogenetic analysis is presented. The allocation of GUP genes in the membrane bound O-acyl transferases (MBOAT) family is suggested as more consistent than their inclusion in the TC-DB/glycerol uptake family.

We examined the effects of heterologous expression of the open reading frames (ORF) of two genes on salt tolerance and glycerol production in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Δgpd2Δ). When the ORF of the Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) was expressed under the control of the GAL10 promoter, salt tolerance and glycerol production increased; when the ORF of the glycerol dehydrogenase gene (ZrGCY1) was expressed under the control of the GAL1 promoter, no such changes were observed. Zrgcy1p had a weak effect on glycerol production. These results suggest that Zrgpd1p is the primary enzyme involved in Z. rouxii glycerol production, following a mechanism similar to that of S. cerevisiae (Gpd1p). When the ORFs of the S. cerevisiae glycerol 3-phosphatase gene (GPP2) and ZrGPD1 were simultaneously expressed, glycerol production increased, compared with that in yeast expressing only ZrGPD1.

Molecular tools for the production of heterologous proteins and metabolic engineering applications of the non-conventional yeast Zygosaccharomyces bailii were developed. The combination of Z. bailii's resistance to relatively high temperature, osmotic pressure and low pH values, with a high specific growth rate renders this yeast potentially interesting for exploitation for biotechnological purposes as well as for the understanding of the biological phenomena and mechanisms underlying the respective resistances. Looking forward to these potential applications, here we present the tools required for the production and the secretion of different heterologous proteins, and one example of a metabolic engineering application of this non-conventional yeast, employing the newly developed molecular tools.

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.

Genera currently assigned to the Saccharomycetaceae have been defined from phenotype, but this classification does not fully correspond with species groupings determined from phylogenetic analysis of gene sequences. The multigene sequence analysis of Kurtzman and Robnett [FEMS Yeast Res. 3 (2003) 417-432] resolved the family Saccharomycetaceae into 11 well-supported clades. In the present study, the taxonomy of the Saccharomyctaceae is evaluated from the perspective of the multigene sequence analysis, which has resulted in reassignment of some species among currently accepted genera, and the proposal of the following five new genera: Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Zygotorulaspora.

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.

Zygosaccharomyces rouxii, a salt-tolerant yeast isolated from the soy sauce process, produces fusel alcohols (isoamyl alcohol, active amyl alcohol and isobutyl alcohol) from branched-chain amino acids (leucine, isoleucine and valine, respectively) via the Ehrlich pathway. Using a high-throughput screening approach in microtiter plates, we have studied the effects of pH, temperature and salt concentration on growth of Z. rouxii and formation of fusel alcohols from branched-chain amino acids. Application of minor variations in pH (range 3-7) and NaCl concentrations (range 0-20%) per microtiter plate well allowed a rapid and detailed evaluation of fermentation conditions for optimal growth and metabolite production. Conditions yielding the highest cell densities were not optimal for fusel alcohol production. Maximal fusel alcohol production occurred at low pH (3.0-4.0) and low NaCl concentrations (0-4%) at 25 degrees C. At pH 4.0-6.0 and 0-18% NaCl, considerable amounts of alpha-keto acids, the deaminated products from the branched-chain amino acids, accumulated extracellularly. The highest cell densities were obtained in plates incubated at 30 degrees C. The results obtained under various incubation conditions with (deep-well) microtiter plates were validated in Erlenmeyer shake-flask cultures.

Zygocin, a protein toxin produced and secreted by a killer virus-infected strain of the osmotolerant yeast Zygosaccharomyces bailii, kills a great variety of human and phytopathogenic yeasts and filamentous fungi. Toxicity of the viral toxin is envisaged in a two-step receptor-mediated process in which the toxin interacts with cell surface receptors at the level of the cell wall and the plasma membrane. Zygocin receptors were isolated and partially purified from the yeast cell wall mannoprotein fraction and could be successfully used as biospecific ligand for efficient one-step purification of the 10-kDa protein toxin by receptor-mediated affinity chromatography. Evidence is presented that zygocin-treated yeast cells are rapidly killed by the toxin, and intensive propidium iodide staining of zygocin-treated cells indicated that the toxin is affecting cytoplasmic membrane function, most probably by lethal ion channel formation. The presented findings suggest that zygocin has potential as a novel antimycotic in combating fungal infections.

Here we show that 320–800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.

Zygosaccharomyces kombuchaensis was recently discovered in the 'tea fungus' used to make fermented tea. Z. kombuchaensis was shown by ribosomal DNA sequencing to be a novel species, and a close relative of Zygosaccharomyces lentus, from which it could not be distinguished by conventional physiological tests. Z. lentus was originally established as a new taxon by growth at 4 degrees C, sensitivity for heat and oxidative stress, and lack of growth in aerobic shaken culture at temperatures above 25 degrees C. Subsequent analysis of Z. kombuchaensis reveals that this species shares these unusual characteristics, confirming its close genealogical relationship to Z. lentus. Detailed physiological data from a number of Z. kombuchaensis and Z. lentus strains clearly demonstrate that these two species can in fact be distinguished from one another based on their differing resistance/sensitivity to the food preservatives benzoic acid and sorbic acid. The spoilage yeasts Zygosaccharomyces bailii and Z. lentus are resistant to both acetic acid and sorbic acid, whereas Z. kombuchaensis is resistant to acetic acid but sensitive to sorbic acid. This would indicate that Z. kombuchaensis strains lack the mechanism for resistance to sorbic acid, but possess the means of resistance to acetic acid. This observation would therefore suggest that these two resistance mechanisms are different, and that in all probability acetic and sorbic acids inhibit yeast growth by different modes of action. Z. kombuchaensis strains were also sensitive to benzoic acid, again suggesting inhibition dissimilar from that to acetic acid.

A new ascosporogenous yeast, Zygosaccharomyces kombuchaensis sp. n. (type strain NRRL YB-4811, CBS 8849), is described; it was isolated from Kombucha tea, a popular fermented tea-based beverage. The four known strains of the new species have identical nucleotide sequences in domain D1/D2 of 26S rDNA. Phylogenetic analysis of D1/D2 and 18S rDNA sequences places Z. kombuchaensis near Zygosaccharomyces lentus. The two species are indistinguishable on standard physiological tests used for yeast identification, but can be recognized from differences in restriction fragment length polymorphism patterns obtained by digestion of 18S-ITS1 amplicons with the restriction enzymes DdeI and MboI.

The nonconventional yeast Zygosaccharomyces bailii has been proposed as a new host for biotechnological processes due to convenient properties such as its resistance to high sugar concentrations, relatively high temperatures and especially to acidic environments. We describe a series of new expression vectors specific for Z. bailii and the resulting improvements in production levels. By exploiting the sequences of the endogenous plasmid pSB2, 2μm-like multicopy vectors were obtained, giving a fivefold increase in production. A specific integrative vector was developed which led to 100% stability in the absence of selective pressure; a multiple-integration vector was constructed, based on an rRNA gene unit portion cloned and sequenced for this purpose, driving the insertion of up to 80 copies of the foreign construct. Moreover, we show the construction of the first stable auxotrophic mutant of Z. bailii, obtained by targeted gene deletion applied to ZbLEU2. The development of molecular tools for the Z. bailii manipulation has now reached a level that may be compatible with its industrial exploitation; the production of organic acids is a prominent field of application. © 2010 Federation of European Microbiological Societies.

Saccharomyces cerevisiae acquires a resistance to high, toxic levels of acetic acid by destabilizing Fps1p, the plasma membrane aquaglyceroporin through which this acid - in its undissociated state - enters the cell. In this study, Fps1p loss was shown to confer resistances to acetic acid, acrolein and allyl alcohol, not just in S. cerevisiae but also in the osmotolerant spoilage yeast Zygosaccharomyces rouxii. However, in Z. rouxii, the loss of Fps1p severely compromised the use of acetamide and several other small amides as sources of nitrogen, an indication that these amides enter the cells of this yeast by passive diffusion through the Fps1p pore. Saccharomyces cerevisiae cannot grow on acetamide, but was conferred with an ability to use this and other small amides as nitrogen sources by heterologous expression of a Z. rouxii ORF (ZrAMD1) with protein sequence identity to the amdS-encoded amidase of Aspergillus nidulans. This capacity of ZrAMD1-expressing S. cerevisiae to assimilate amide nitrogen was severely compromised by the loss of Fps1p. ZrAMD1 appears to encode the major amidase of Z. rouxii as a Zramd1Delta deletant mutant had, like the Zrfps1Delta deletant, lost the ability to assimilate small amides as sources of nitrogen.

Saccharomyces cerevisiae acquires a resistance to high, toxic levels of acetic acid by destabilizing Fps1p, the plasma membrane aquaglyceroporin through which this acid – in its undissociated state – enters the cell. In this study, Fps1p loss was shown to confer resistances to acetic acid, acrolein and allyl alcohol, not just in S. cerevisiae but also in the osmotolerant spoilage yeast Zygosaccharomyces rouxii. However, in Z. rouxii, the loss of Fps1p severely compromised the use of acetamide and several other small amides as sources of nitrogen, an indication that these amides enter the cells of this yeast by passive diffusion through the Fps1p pore. Saccharomyces cerevisiae cannot grow on acetamide, but was conferred with an ability to use this and other small amides as nitrogen sources by heterologous expression of a Z. rouxii ORF (ZrAMD1) with protein sequence identity to the amdS-encoded amidase of Aspergillus nidulans. This capacity of ZrAMD1-expressing S. cerevisiae to assimilate amide nitrogen was severely compromised by the loss of Fps1p. ZrAMD1 appears to encode the major amidase of Z. rouxii as a Zramd1Δ deletant mutant had, like the Zrfps1Δ deletant, lost the ability to assimilate small amides as sources of nitrogen.

Saccharomyces cerevisiae has been used for millennia in the production of food and beverages and is by far the most studied yeast species. Currently, it is also the most used microorganism in the production of first-generation bioethanol from sugar or starch crops. Second-generation bioethanol, on the other hand, is produced from lignocellulosic feedstocks that are pretreated and hydrolyzed to obtain monomeric sugars, mainly D-glucose, D-xylose and L-arabinose. Recently, S. cerevisiae recombinant strains capable of fermenting pentose sugars have been generated. However, the pretreatment of the biomass results in hydrolysates with high osmolarity and high concentrations of inhibitors. These compounds negatively influence the fermentation process. Therefore, robust strains with high stress tolerance are required. Up to now, more than 2000 yeast species have been described and some of these could provide a solution to these limitations because of their high tolerance to the most predominant stress conditions present in a second-generation bioethanol reactor. In this review, we will summarize what is known about the non-conventional yeast species showing unusual tolerance to these stresses, namely Zygosaccharomyces rouxii (osmotolerance), Kluyveromyces marxianus and Ogataea (Hansenula) polymorpha (thermotolerance), Dekkera bruxellensis (ethanol tolerance), Pichia kudriavzevii (furan derivatives tolerance) and Z. bailii (acetic acid tolerance). ispartof: Fems Yeast Research vol:15 issue:6 ispartof: location:England status: published

We cloned two genes from the salt-tolerant yeast Zygosaccharomyces rouxii: ZrSOD2 for the cell membrane Na(+)/H(+)-antiporter and ZrPMA1 for the cell membrane H(+)-ATPase. The products of these genes play cooperative roles in the salt-tolerance of Z. rouxii, and the function of the ZrPMA1 product is regulated at the transcription level. We constructed a yeast expression vector that is able to co-express the ZrSOD2 and ZrPMA1 genes. Single expression of ZrSOD2 was effective in conferring salt-tolerance, and although a slight synergic effect was observed with co-expression of ZrSOD2 and ZrPMA1, the usefulness of this co-expression is likely to be minimal with regard to salt-tolerance.

26S rDNA D1/D2 sequencing was used to characterise a number of food-associated Zygosaccharomyces rouxii strains held at the National Collection of Yeast Cultures. In the course of this study, four strains (NCYC 1682, NCYC 3042, NCYC 3060 and NCYC 3061) were identified which appeared, based on their D1/D2 sequences, to belong to a novel Zygosaccharomyces species. However, subsequent sequence analysis showed that NCYC 1682, NCYC 3060 and NCYC 3061 possess two highly divergent copies of the nuclear-encoded ADE2, HIS3 and SOD2 genes, indicating these three strains are in fact hybrids. NCYC 3042, however, does appear to represent a novel species which may be hypothesized to have crossed with Z. rouxii and given rise to hybrid strains. Additional approaches to define precise taxonomic status and mechanisms of hybrid genome formation amongst yeast species are discussed.

We examined the effects of heterologous expression of the open reading frames (ORF) of two genes on salt tolerance and glycerol production in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Deltagpd2Delta). When the ORF of the Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) was expressed under the control of the GAL10 promoter, salt tolerance and glycerol production increased; when the ORF of the glycerol dehydrogenase gene (ZrGCY1) was expressed under the control of the GAL1 promoter, no such changes were observed. Zrgcy1p had a weak effect on glycerol production. These results suggest that Zrgpd1p is the primary enzyme involved in Z. rouxii glycerol production, following a mechanism similar to that of S. cerevisiae (Gpd1p). When the ORFs of the S. cerevisiae glycerol 3-phosphatase gene (GPP2) and ZrGPD1 were simultaneously expressed, glycerol production increased, compared with that in yeast expressing only ZrGPD1.

Molecular tools for the production of heterologous proteins and metabolic engineering applications of the non-conventional yeast Zygosaccharomyces bailii were developed. The combination of Z. bailii's resistance to relatively high temperature, osmotic pressure and low pH values, with a high specific growth rate renders this yeast potentially interesting for exploitation for biotechnological purposes as well as for the understanding of the biological phenomena and mechanisms underlying the respective resistances. Looking forward to these potential applications, here we present the tools required for the production and the secretion of different heterologous proteins, and one example of a metabolic engineering application of this non-conventional yeast, employing the newly developed molecular tools. © 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.

Zygocin, a protein toxin produced and secreted by a killer virus-infected strain of the osmotolerant yeast Zygosaccharomyces bailii, kills a great variety of human and phytopathogenic yeasts and filamentous fungi. Toxicity of the viral toxin is envisaged in a two-step receptor-mediated process in which the toxin interacts with cell surface receptors at the level of the cell wall and the plasma membrane. Zygocin receptors were isolated and partially purified from the yeast cell wall mannoprotein fraction and could be successfully used as biospecific ligand for efficient one-step purification of the 10-kDa protein toxin by receptor-mediated affinity chromatography. Evidence is presented that zygocin-treated yeast cells are rapidly killed by the toxin, and intensive propidium iodide staining of zygocin-treated cells indicated that the toxin is affecting cytoplasmic membrane function, most probably by lethal ion channel formation. The presented findings suggest that zygocin has potential as a novel antimycotic in combating fungal infections.

Glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in yeast are connected to the beta-1,3-glucan network via a beta-1,6-glucan moiety. Addition of gentiobiose or beta-1,6-glucan oligomers to growing cells affected the construction of a normal layer of GPI-dependent cell wall proteins at the outer rim of the Saccharomyces cerevisiae cell wall. Treated S. cerevisiae cells secreted significant amounts of cell wall protein 2, were much more sensitive to the lytic action of zymolyase 20T and displayed a marked increase in sensitivity to the small amphipathic antimicrobial peptide MB-21. Similar results in terms of sensitization of yeast cells to the antimicrobial peptide were obtained with the notorious food spoilage yeast Zygosaccharomyces bailii. Our results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.

Yeast GeneFilters® containing all Saccharomyces cerevisiae open reading frame (ORF) sequences were used to elucidate gene activity in the osmotolerant yeast Zygosaccharomyces rouxii. Labelled cDNA derived from Z. rouxii was targeted to spotted S. cerevisiae ORFs. Approximately 90–100% homology of Z. rouxii genes with those of S. cerevisiae was required for definitive identification of the cDNAs hybridised to GeneFilter®. Hybridised labelled cDNAs were visualised as small spots on the microarray, providing simultaneous information on homologous genes present in Z. rouxii and on their level of gene activity. Cross-hybridisation of the GeneFilters® displayed 155 as yet unidentified genes of Z. rouxii hybridising to S. cerevisiae ORFs. From those 155 genes, the activity of 86 genes was influenced as a result of NaCl stress. In comparison with S. cerevisiae 24% of Z. rouxii genes revealed a different transcription behaviour following NaCl stress. All of these genes had no previously defined function in osmotic-stress response in Z. rouxii. Therefore, cross-hybridisation of GeneFilters® proves to be an appropriate and straightforward method for screening transcripts in Z. rouxii, which provides an extension of the knowledge of genes present in a yeast genus other than S. cerevisiae.