Project description:Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732(T)) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1Δ, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.
Project description: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.
Project description:The osmotolerant Zygosaccharomyces rouxii is known for its trait to survive in extreme high sugar environments. This ability determines its role in the fermentation process and leads to yeast spoilage in the food industry. However, our knowledge of the gene expression in response to high sugar stress remains limited. Here, we conducted RNA-sequencing (RNA-seq) under different sugar concentrations of the spoilage yeast, Z. rouxii, which exhibit extremely high tolerance to sugar stress. The obtained differentially expressed genes (DEGs) are significantly different to that of the Saccharomyces cerevisiae, which is sensitive to extreme high sugar stress. Most of the DEGs participated in the "glucan synthesis," "transmembrane transport," "ribosome," etc. In this work, we also demonstrated that the gene ZYRO0B03476g (ZrKAR2) encoding Kar2p can significantly affect the growth of Z. rouxii under high sugar stress. In addition, we combined with a previous study on the genome sequence of Z. rouxii, indicating that several gene families contain significantly more gene copies in the Z. rouxii lineage, which involved in tolerance to sugar stress. Our results provide a gene insight for understanding the high sugar tolerance trait, which may impact food and biotechnological industries and improve the osmotolerance in other organisms.
Project description:Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5' end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding.
Project description:Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H(+) symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H(+) symporter, with Km 0.45 ± 0.07 mM and Vmax 0.57 ± 0.02 mmol h(-1) (gdw)(-1). We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H(+) symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.
Project description: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.
Project description:The highly halotolerant and allopolyploid yeast Zygosaccharomyces rouxii is industrially used for the food production in high concentrations of salt, such as brewing soy sauce and miso paste. Here, we report the draft genome sequence of Z. rouxii NBRC 1876 isolated from miso paste.