Differential analysis of ergosterol function in response to high salt and sugar stress in Zygosaccharomyces rouxii.

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.

The revenge of Zygosaccharomyces yeasts in food biotechnology and applied microbiology.

Non-conventional yeasts refer to a huge and still poorly explored group of species alternative to the well-known model organism Saccharomyces cerevisiae. Among them, Zygosaccharomyces rouxii and the sister species Zygosaccharomyces bailii are infamous for spoiling food and beverages even in presence of several food preservatives. On the other hand, their capability to cope with a wide range of process conditions makes these yeasts very attractive factories (the so-called "ZygoFactories") for bio-converting substrates poorly permissive for the growth of other species. In balsamic vinegar Z. rouxii is the main yeast responsible for converting highly concentrated sugars into ethanol, with a preference for fructose over glucose (a trait called fructophily). Z. rouxii has also attracted much attention for the ability to release important flavor compounds, such as fusel alcohols and the derivatives of 4-hydroxyfuranone, which markedly contribute to fragrant and smoky aroma in soy sauce. While Z. rouxii was successfully proposed in brewing for producing low ethanol beer, Z. bailii is promising for lactic acid and bioethanol production. Recently, several research efforts exploited omics tools to pinpoint the genetic bases of distinctive traits in "ZygoFactories", like fructophily, tolerance to high concentrations of sugars, lactic acid and salt. Here, I provided an overview of Zygosaccharomyces industrially relevant phenotypes and summarized the most recent findings in disclosing their genetic bases. I suggest that the increasing number of genomes available for Z. rouxii and other Zygosaccharomyces relatives, combined with recently developed genetic engineering toolkits, will boost the applications of these yeasts in biotechnology and applied microbiology.

Fruit host-dependent fungal communities in the microbiome of wild Queensland fruit fly larvae.

Bactrocera tryoni (Froggatt), the Queensland fruit fly (Qfly), is a highly polyphagous tephritid fly that is widespread in Eastern Australia. Qfly physiology is closely linked with its fungal associates, with particular relationship between Qfly nutrition and yeast or yeast-like fungi. Despite animal-associated fungi typically occurring in multi-species communities, Qfly studies have predominately involved the culture and characterisation of single fungal isolates. Further, only two studies have investigated the fungal communities associated with Qfly, and both have used culture-dependant techniques that overlook non-culturable fungi and hence under-represent, and provide a biased interpretation of, the overall fungal community. In order to explore a potentially hidden fungal diversity and complexity within the Qfly mycobiome, we used culture-independent, high-throughput Illumina sequencing techniques to comprehensively, and holistically characterized the fungal community of Qfly larvae and overcome the culture bias. We collected larvae from a range of fruit hosts along the east coast of Australia, and all had a mycobiome dominated by ascomycetes. The most abundant fungal taxa belonged to the genera Pichia (43%), Candida (20%), Hanseniaspora (10%), Zygosaccharomyces (11%) and Penicillium (7%). We also characterized the fungal communities of fruit hosts, and found a strong degree of overlap between larvae and fruit host communities, suggesting that these communities are intimately inter-connected. Our data suggests that larval fungal communities are acquired from surrounding fruit flesh. It is likely that the physiological benefits of Qfly exposure to fungal communities is primarily due to consumption of these fungi, not through syntrophy/symbiosis between fungi and insect 'host'.

Improving RNA content of salt-tolerant Zygosaccharomyces rouxii by atmospheric and room temperature plasma (ARTP) mutagenesis and its application in soy sauce brewing.

Derived from RNA, 5'-ribonucleotides, especially Inosine-5'-monophosphate (IMP) and guanosine-5'-monophosphate (GMP), can enhance the umami taste of soy sauce. In this study, the RNA content of three different salt-tolerant yeasts was examined. The most valuable strain was subjected to atmospheric and room-temperature plasma (ARTP) mutagenesis, which improved its RNA content by 160.54%. Regular fermentation with RNA-enhanced strain failed to increase the amount of 5'-ribonucleotides in the soy sauce due to hydrolysis by phosphatase. A two-stage fermentation strategy was then carried out. Aroma compounds were mainly synthesized in the first stage, and RNA-enriched biomass was massively produced in the second stage followed by heat treatment to inactivate phosphatase. After the proposed strategy was applied, IMP and GMP in the soy sauce reached 68.54 and 89.37 mg/L, respectively. Moreover, the amounts of key aroma compounds and organic acids significantly increased. Results may provide new insights for improving the quality of soy sauce through microorganism breeding and fermentation control.

Effect of co-culture with Tetragenococcus halophilus on the physiological characterization and transcription profiling of Zygosaccharomyces rouxii.

Zygosaccharomyces rouxii and Tetragenococcus halophilus are widely existed and play vital roles during the manufacture of fermented foods such as soy sauce. The aim of this study was to elucidate the effect of T. halophilus CGMCC 3792 on the physiological characterizations and transcription profiling of Z. rouxii CGMCC 3791. Salt tolerance analysis revealed that co-culture with T. halophilus enhanced the salt tolerance of Z. rouxii during salt stress. Analysis of the volatile compounds revealed that co-culture reduced the level of 1-butanol, improved the level of octanoic acid which all were produced by T. halophilus and reduced the level of phenylethyl alcohol produced by Z. rouxii. The presence of Z. rouxii decreased the contents of 3,4-dimethylbenzaldehyde and acetic acid produced by T. halophilus. In addition, co-culture improved the content of benzyl alcohol significantly. Analysis of membrane fatty acid showed that co-culture improved the content of palmitic (C16:0) and stearic (C18:0) acids in cells of Z. rouxii, and reduced the contents of myristic (C14:0), palmitoleic acid (C16:1) and oleic acid (C18:1). In order to further explore the interactions between the two strains, RNA-seq technology was used to investigate the effect of co-culture with T. halophilus on the transcription profiling of Z. rouxii. By comparing cells incubated in co-culture group with cells incubated in single-culture group, a total of 967 genes were considered as differentially expressed genes (DEGs). Among the DEGs, 72 genes were up-regulated, while 895 genes were down-regulated. These DEGs took party in various activities in cells of Z. rouxii, and the result showed co-culture with T. halophilus had a positive effect on proteolysis, the attachment of a cell to another cell, extracellular protein accumulation, energy metabolism, and a negative effect on oxidative phosphorylation, small molecular substances metabolism, DNA replication and repair, and transcription in cells of Z. rouxii. Results presented in this study may contribute to further understand the interactions between Zygosaccharomyces rouxii and Tetragenococcus halophilus.

Zygosaccharomyces pseudobailii, another yeast interspecies hybrid that regained fertility by damaging one of its MAT loci.

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.

Pathogenicity of Zygosaccharomyces bailii and Other Yeast Species to Mexican Fruit Fly (Diptera: Tephritidae) and Mass Rearing Implications.

Yeasts from all immature life stages of Mexican fruit fly Anastrepha ludens (Loew) (Diptera: Tephritidae) from diet, insectary air, and rearing materials were isolated, identified and evaluated for pathogenicity. Fifteen species of yeasts with one to genus level were identified from 72 yeast cultures obtained. Zygosaccharomyces bailii was the only yeast found to be highly pathogenic to Mexican fruit fly. Seventy-two hours post inoculation, the diet in bioassay cups with Z. bailii consistently showed signs of fermentation with gas bubbling causing the migration of larvae to the walls and lids of bioassay cups. The spent diet from Z. balii-infested cups was crusty, cracked and had a pasty layer. Many larvae were small, moribund, and discolored, appearing caramel or blackish. Insect yield loss with Z. bailii in comparison to that of control ranged from 10 to 44% for larvae and 14 to 47% for pupae. Additionally, Z. bailii caused a reduction in mean pupal weight. The weakly pathogenic yeasts produced significantly less yield of larvae and pupae than the nonpathogenic ones included Trichosporon montevideense, Clavispora lucitaniae, Candida sp., C. rugosa, and Rhodotorula mucilaginosa. Yield loss of larvae caused by this group ranged from 12 (C. lusitaniae) to 18% (R. mucilaginosa). Yield losses for pupa were similar to that of larvae. The mean pupa weight for these species was above the minimum acceptable (16.50 mg) for the SIT program. The nonpathogenic yeast produced yields of larvae and pupae similar to the control included Cryptococcus diffluens, Pichia kudriavzevii, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Trichosporon asahii, Debaryomyces hansenii, Candida tropicalis, Cryptococcus sp., Candida parapsilosis, and Hanseniaspora opuntiae. In conclusion, the identification and management of insect pathogenic yeasts, such as Z. balii in mass rearing systems of Mexican fruit fly must be considered to avoid their potential negative effects.

Effects of dielectric barrier discharge plasma on the inactivation of Zygosaccharomyces rouxii and quality of apple juice.

This work aimed to evaluate the effects of dielectric barrier discharge (DBD) plasma on inactivation of spoilage yeast Zygosaccharomyces rouxii (Z. rouxii), in apple juice. Results showed that DBD plasma treatment at 90 W for 140 s resulted in about 5-log reduction of Z. rouxii in apple juice. The levels of extracellular nucleic acids and proteins as well as contents of H2O2 and NO2- in yeast extract-peptone-dextrose (YPD) medium increased significantly after DBD plasma treatment at 90 W for 40-200 s. The increases in membrane permeability and generation of reactive species would likely contribute to DBD plasma-mediated inactivation of Z. rouxii. DBD plasma caused significant changes in pH, titratable acidity, and certain color parameters of apple juice, but had no effect on the contents of total soluble solids, reducing sugar, and total phenolics. This study provides key implications for the application of DBD plasma in fruit juice processing.

Transcriptional Response to Lactic Acid Stress in the Hybrid Yeast Zygosaccharomyces parabailii.

Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae Furthermore, formate dehydrogenase (FDH) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production.IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC 60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains.

Can Interspecies Hybrid Zygosaccharomyces rouxii Produce an Allohaploid Gamete?

In soy sauce manufacturing, Candida versatilis plays a role in the production of volatile flavor compounds, such as volatile phenols, but limited accessible information on its genome has prevented further investigation regarding aroma production and breeding. Although the draft genome sequence data of two strains of C. versatilis have recently been reported, these strains are not similar to each other. Here, we reassess the draft genome sequence data for strain t-1, which was originally reported to be C. versatilis, and conclude that strain t-1 is most probably not C. versatilis but a gamete of hybrid Zygosaccharomyces rouxii Phylogenetic analysis of the D1/D2 region of the 26S ribosomal DNA (rDNA) sequence indicated that strain t-1 is more similar to the genus Zygosaccharomyces than to C. versatilis Moreover, we found that the genome of strain t-1 is composed of haploid genome content and divided into two regions that show approximately 100% identity with the T or P subgenome derived from the natural hybrid Zygosaccharomyces rouxii, such as NBRC110957 and NBRC1876. We also found a chromosome crossing-over signature in the scaffolds of strain t-1. These results suggest that strain t-1 is a gamete of the hybrid Z. rouxii, generated by either meiosis or chromosome loss following reciprocal translocation between the T and P subgenomes. Although it is unclear why strain t-1 was misidentified as C. versatilis, the genome of strain t-1 has broad implications for considering the evolutionary fate of an allodiploid.IMPORTANCE In yeast, crossing between different species sometimes leads to interspecies hybrids. The hybrid generally cannot produce viable spores because dissimilarity of parental genomes prevents normal chromosome segregation during meiotic division, leading to a dead end. Thus, only a few natural cases of homoploid hybrid speciation, which requires mating between 1n gametes of hybrids, have been described. However, a recent study provided strong evidence that homoploid hybrid speciation is initiated in natural populations of the budding yeast, suggesting the potential presence of viable 1n gametes of hybrids. The significance of our study is finding that the strain t-1, which had been misidentified as Candida versatilis, is a viable 1n gamete derived from hybrid Zygosaccharomyces rouxii.

Mechanism for Restoration of Fertility in Hybrid Zygosaccharomyces rouxii Generated by Interspecies Hybridization.

The mechanism of whole-genome duplication (WGD) in yeast has been intensively studied because it has a large impact on yeast evolution. WGD has shaped the genomic architecture of modern Saccharomyces cerevisiae; however, the mechanism for restoring fertility after interspecies hybridization, which would be involved in the process of WGD, has not been thoroughly elucidated. In this study, we obtained a draft genome sequence of the salt-tolerant yeast Zygosaccharomyces rouxii NBRC110957 and revealed that it is a hybrid lineage of Z. rouxii (allodiploid) with two subgenomes equivalent to NBRC1876. Because this allodiploid yeast can mate with other allodiploid strains and form spores, it can be a good model of restoring fertility after interspecies hybridization. We observed that NBRC110957 and NBRC1876 contain six mating-type-like (MTL) loci. There are no large deletions or deleterious mutations in MTL loci, except for several-base-pair deletions in the X region in certain MTL loci. We also assigned only one mating-type (MAT) locus that exclusively determines mating types from six MTL loci. These results suggest that it is possible to recover mating competence regardless of whether cells lose one MAT locus through random gene loss by mitotically dividing after interspecies hybridization. Moreover, we propose that perturbation of gene expression and substantial breakdown of MAT heterozygosity caused by chromosomal rearrangement at MTL loci play roles in restoring the mating competence of allodiploids. This scenario can provide a mechanism for restoring fertility after interspecies hybridization that is compatible with random gene loss models and suggests genomic plasticity during WGD in yeast.IMPORTANCE A whole-genome duplication occurred in an ancestor of the baker's yeast Saccharomyces cerevisiae The origins of this complex and multifaceted process, which requires intra- or interspecies hybridization followed by dysfunction of one mating-type (MAT) locus to regain mating competence, has not been thoroughly elucidated. In this study, we provide a mechanism for regaining fertility in an interspecies hybrid, Zygosaccharomyces rouxii The draft genome sequence analysis and mating test showed that the Z. rouxii strain used in this study is an intact interspecies hybrid, suggesting that it is possible to recover fertility regardless of whether cells lose one MAT locus.

Selection of Zygosaccharomyces rouxii strains resistant to cadmium with improved removal abilities through ultraviolet-diethyl sulfate cooperative mutagenesis.

Cd2+ resistance and bioaccumulation capacity were selected from parental Zygosaccharomyces rouxii (CRZ-0) while maintaining NaCl tolerance using protoplast mutagenesis technology. Ultraviolet-diethyl sulfate (UV-DES) cooperative mutagenesis, followed by preliminary screening and rescreening, was used to select the mutant strain CRZ-9. CRZ-9 grew better than CRZ-0 in YPD medium with 20 or 50 mg L-1 of Cd2+. Scanning electron microscopy observations and flow cytometry tests indicated that CRZ-9 was more effective at eliminating reactive oxygen species (ROS) generated by Cd2+, which led to less cellular structural damage and lower lethality. Furthermore, compared with CRZ-0, CRZ-9 exhibited increased potential for application with higher Cd2+ removal ratio, wider working pH range, and lower biomass dosage in Cd2+ bioaccumulation. The mutant strain CRZ-9 possessed improved Cd2+ resistance and bioaccumulation capacity and therefore is a promising strain to remove Cd2+ from wastewater.

Incidence of osmophilic yeasts and Zygosaccharomyces rouxii during the production of concentrate grape juices.

Zygosaccharomyces rouxii is the main spoilage yeast of grape juice concentrates. Detection and identification of Z. rouxii during the production of grape juice concentrate is critical to prevent spoilage in the final product. In this work, three grape juice concentrate processing plants were assessed by identifying osmophilic yeasts in juices and surfaces during different stages of a complete production line. Subsequently, molecular typing of Z. rouxii isolates was done to determine the strain distribution of this spoilage yeast. Osmotolerant yeast species, other than Z. rouxii, were mainly recovered from processing plant environments. Z. rouxii was only isolated from surface samples with grape juice remains. Z. rouxii was largely isolated from grape juice samples with some degree of concentration. Storage of grape juice pre-concentrate and concentrate allowed an increase in the Z. rouxii population. A widely distributed dominant molecular Z. rouxii pattern was found in samples from all three processing plants, suggesting resident microbes inside the plant.

Influence of yeast and lactic acid bacterium on the constituent profile of soy sauce during fermentation.

Soy sauce is a Japanese traditional seasoning composed of various constituents that are produced by various microbes during a long-term fermentation process. Due to the complexity of the process, the investigation of the constituent profile during fermentation is difficult. Metabolomics, the comprehensive study of low molecular weight compounds in biological samples, is thought to be a promising strategy for deep understanding of the constituent contribution to food flavor characteristics. Therefore, metabolomics is suitable for the analysis of soy sauce fermentation. Unfortunately, only few and unrefined studies of soy sauce fermentation using metabolomics approach have been reported. Therefore, we investigated changes in low molecular weight hydrophilic and volatile compounds of soy sauce using gas chromatography/mass spectrometry (GC/MS)-based non-targeted metabolic profiling. The data were analyzed by statistical analysis to evaluate influences of yeast and lactic acid bacterium on the constituent profile. Consequently, our results suggested a novel finding that lactic acid bacterium affected the production of several constituents such as cyclotene, furfural, furfuryl alcohol and methional in the soy sauce fermentation process.

Differential hypersaline stress response in Zygosaccharomyces rouxii complex yeasts: a physiological and transcriptional study.

The Zygosaccharomyces rouxii complex comprises three distinct lineages of halotolerant yeasts relevant in food processing and spoilage, such as Z. sapae, Z. rouxii and a mosaic group of allodiploid strains. They manifest plastic genome architecture (variation in karyotype, ploidy level and Na(+)/H(+) antiporter-encoding gene copy number), and exhibit diverse tolerances to salt concentrations. Here, we investigated accumulation of compatible osmolytes and transcriptional regulation of Na(+)/H(+) antiporter-encoding ZrSOD genes during salt exposure in strains representative for the lineages, namely Z. sapae ABT301(T) (low salt tolerant), Z. rouxii CBS 732(T) (middle salt tolerant) and allodiploid strain ATCC 42981 (high salt tolerant). Growth curve modelling in 2 M NaCl-containing media supplemented with or without yeast extract as nitrogen source indicates that moderate salt tolerance of CBS 732(T) mainly depends on nitrogen availability rather than intrinsic inhibitory effects of salt. All the strains produce glycerol and not mannitol under salt stress and use two different glycerol balance strategies. ATCC 42981 produces comparatively more glycerol than Z. sapae and Z. rouxii under standard growth conditions and better retains it intracellularly under salt injuries. Conversely, Z. sapae and Z. rouxii enhance glycerol production under salt stress and intracellularly retain glycerol less efficiently than ATCC 42981. Expression analysis shows that, in diploid Z. sapae and allodiploid ATCC 42981, transcription of gene variants ZrSOD2-22/ZrSOD2 and ZrSOD22 is constitutive and salt unresponsive.

Assessing physio-macromolecular effects of lactic acid on Zygosaccharomyces bailii cells during microaerobic fermentation.

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.

Two glycerol uptake systems contribute to the high osmotolerance of Zygosaccharomyces rouxii.

The accumulation of glycerol is essential for yeast viability upon hyperosmotic stress. Here we show that the osmotolerant yeast Zygosaccharomyces rouxii has two genes, ZrSTL1 and ZrSTL2, encoding transporters mediating the active uptake of glycerol in symport with protons, contributing to cell osmotolerance and intracellular pH homeostasis. The growth of mutants lacking one or both transporters is affected depending on the growth medium, carbon source, strain auxotrophies, osmotic conditions and the presence of external glycerol. These transporters are localised in the plasma membrane, they transport glycerol with similar kinetic parameters and besides their expected involvement in the cell survival of hyperosmotic stress, they surprisingly both contribute to an efficient survival of hypoosmotic shock and to the maintenance of intracellular pH homeostasis under non-stressed conditions. Unlike STL1 in Sa. cerevisiae, the two Z. rouxii STL genes are not repressed by glucose, but their expression and activity are downregulated by fructose and upregulated by non-fermentable carbon sources, with ZrSTL1 being more influenced than ZrSTL2. In summary, both transporters are highly important, though Z. rouxii CBS 732(T) cells do not use external glycerol as a source of carbon.

Influence of sanitizers on the lipopolysaccharide toxicity of Escherichia coli strains cultivated in the presence of Zygosaccharomyces bailii.

The influence of sublethal concentrations of two sanitizers, liquid iodophor and liquid hypochlorite (LH), on the growth rates and toxicity of food-borne pathogenic Escherichia coli strains grown in the presence of spoilage yeast Zygosaccharomyces bailii was assessed. When grown in combination with Z. bailii both E. coli O113 and E. coli O26 exhibited slower growth rates, except when E. coli O113 was grown in combination with Z. bailii at 0.2% LH. The growth rate of Z. bailii was not impacted by the addition of the sanitizers or by communal growth with E. coli strains. LAL and IL-6 results indicated a decrease in toxicity of pure E. coli cultures with comparable profiles for control and sanitizer exposed samples, although the LAL assay proved to be more sensitive. Interestingly, pure cultures of Z. bailii showed increased toxicity measured by LAL and decreased toxicity measured by IL-6. LAL analysis showed a decrease in toxicity of both E. coli strains grown in combination with Z. bailii, while IL-6 analysis of the mixed cultures showed an increase in toxicity. The use of LAL for toxicity determination in a mixed culture overlooks the contribution made by spoilage yeast, thus demonstrating the importance of using the appropriate method for toxicity testing in mixed microbe environments.

Prenylated flavonoids from the stems and leaves of Desmodium caudatum and evaluation of their inhibitory activity against the film-forming growth of Zygosaccharomyces rouxii F51.

In order to provide scientific evidence for the relationship between the traditional usage, stems and leaves of Desmodium caudatum being used for protecting miso from spoilage, and its Japanese name (miso-naoshi), phytochemical study on the stems and leaves of this plant was carried out. Seven new prenylated flavonoids (1-3, 15-18), together with 19 known compounds (4-14, 19-26), were isolated, and the structures of new compounds were elucidated by extensive spectroscopic analyses. The minimum inhibitory concentrations (MICs) of 28 flavonoids, including 17 compounds (1, 2, 4, 5, 7-14, 20-22, 24, 25) isolated in this study and 11 flavonoids (27-37) previously isolated from the roots of this plant, against the film-forming yeast of Zygosaccharomyces rouxii F51 were determined. Fifteen compounds (2, 4, 5, 11, 12, 14, 21, 22, 25, 27, 28, 32-35) inhibited the film-forming growth of Z. rouxii F51 (MIC values, 7.8-62.5 µg/mL), among which 2",2"-dimethylpyran-(5",6":7,8)-5,2'-dihydroxy-4'-methoxy-(2R,3R)-dihydroflavonol (11) demonstrated potent inhibitory activity with an MIC value of 7.8 µg/mL.

Molecular tools and protocols for engineering the acid-tolerant yeast Zygosaccharomyces bailii as a potential cell factory.

Microorganisms offer a tremendous potential as cell factories, and they are indeed used by humans for centuries for biotransformations. Among them, yeasts combine the advantage of unicellular state with a eukaryotic organization, and, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycetales budding yeast, is widely known for its peculiar tolerance to various stresses, among which are organic acids. Despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here, we describe in detail protocols for transformation, for target gene disruption or gene integration, and for designing episomal expression plasmids helpful for developing and further studying the yeast Z. bailii.