The pre-whole genome duplication (WGD) Zygosaccharomyces clade comprises several allodiploid strain/species with industrially interesting traits. The salt-tolerant yeast ATCC42981 is a sterile and allodiploid strain which contains two subgenomes, one of them resembling the haploid parental species Z. rouxii. Recently, different mating-type-like (MTL) loci repertoires were reported for ATCC42981 and the Japanese strain JCM22060, which are considered two stocks of the same strain. MTL reconstruction by direct sequencing approach is challenging due to gene redundancy, structure complexities, and allodiploid nature of ATCC42981. Here, DBG2OLC and MaSuRCA hybrid de novo assemblies of ONT and Illumina reads were combined with in vitro long PCR to definitively solve these incongruences. ATCC42981 exhibits several chimeric MTL loci resulting from reciprocal translocation between parental haplotypes and retains two MATa/MATα expression loci, in contrast to MATα in JCM22060. Consistently to these reconstructions, JCM22060, but not ATCC42981, undergoes mating and meiosis. To ascertain whether the damage of one allele at the MAT locus regains the complete sexual cycle in ATCC42981, we removed the MATα expressed locus by gene deletion. The resulting MATa/- hemizygous mutants did not show any evidence of sporulation, as well as of self- and out-crossing fertility, probably because incomplete silencing at the chimeric HMLα cassette masks the loss of heterozygosity at the MAT locus. We also found that MATα deletion switched off a2 transcription, an activator of a-specific genes in pre-WGD species. These findings suggest that regulatory scheme of cell identity needs to be further investigated in Z. rouxii protoploid yeast.

• Klíčová slova
• MinION, Zygosaccharomyces, chimeric loci, interspecies hybridization, mating-type, sexual cycle, yeast,
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• biologický transport genetika   MeSH
• fermentace genetika   MeSH
• fruktosa metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• genový knockdown MeSH
• glukosa metabolismus   MeSH
• proliferace buněk genetika   MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Zygosaccharomyces genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fruktosa MeSH
• fungální proteiny MeSH
• glukosa MeSH

The osmotolerant and cell wall properties of the two most studied wild-type Zygosaccharomyces rouxii strains (CBS 732 and ATCC 42981) were examined. Differences in their (1) tolerance to high salt content in the medium, (2) resistance to the lysing enzymes Lyticase and Zymolyase, (3) cell-wall polymer content and (4) cell wall micromorphology suggested that the less osmotolerant CBS 732 strain possesses a more rigid cell wall than the more osmotolerant ATCC 42981, whose cell wall seems to be more flexible and elastic.

• MeSH
• antifungální látky farmakologie   MeSH
• buněčná stěna fyziologie   ultrastruktura   MeSH
• endo-1,3-beta-glukanasa farmakologie   MeSH
• fyziologická adaptace MeSH
• hypertonický solný roztok metabolismus   MeSH
• multienzymové komplexy farmakologie   MeSH
• osmolární koncentrace MeSH
• proteasy farmakologie   MeSH
• Saccharomyces cerevisiae fyziologie   MeSH
• Zygosaccharomyces cytologie   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antifungální látky MeSH
• endo-1,3-beta-glukanasa MeSH
• hypertonický solný roztok MeSH
• lyticase MeSH Prohlížeč
• multienzymové komplexy MeSH
• proteasy MeSH

Zygosaccharomyces rouxii is a hemiascomycetous yeast known for its high osmotolerance, the basis of which still remains unknown. By exploring the Génolevures I database, four Z. rouxii fragments homologous to Saccharomyces cerevisiae centromeres were identified. Two of them were subjected to further analysis. Their function as centromeres in Z. rouxii was proved, and they were localized to Z. rouxii chromosomes II and VII, respectively. The species-specificity of centromeres was observed; plasmids with a Z. rouxii centromere were not recognized as centromeric in S. cerevisiae, and a S. cerevisiae centromere did not function as a centromere in Z. rouxii. Constructed plasmids bearing Z. rouxii centromeres serve as the first specific centromeric plasmids, and thus contribute to the so-far limited set of genetic tools needed to study the Z. rouxii specific features.

• MeSH
• centromera genetika   MeSH
• DNA fungální genetika   MeSH
• DNA primery genetika   MeSH
• druhová specificita MeSH
• genetické vektory MeSH
• plazmidy genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence nukleotidů MeSH
• transformace genetická MeSH
• Zygosaccharomyces genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA fungální MeSH
• DNA primery MeSH

The osmotolerant yeast Zygosaccharomyces rouxii is sensitive to the toxic L-proline analogue, L-azetidine-2-carboxylate (AZC). The possibility of use of the Saccharomyces cerevisiae MPR1 gene (ScMPR1) encoding the AZC-detoxifying enzyme as a dominant selection marker in Z. rouxii was examined. The heterologous expression of ScMPR1 in two Z. rouxii strains resulted in AZC-resistant colonies, but that of ScMPR1 as a dominant marker gene in vectors was affected by a high frequency of spontaneously resistant colonies. The same was found for an AZC-sensitive S. cerevisiae strain in which the ScMPR1 was expressed. In both yeasts, ScMPR1 can be used only as an auxiliary marker gene.

• MeSH
• acetyltransferasy genetika   metabolismus   MeSH
• antibiotická rezistence genetika   MeSH
• bakteriální transformace MeSH
• genetické inženýrství MeSH
• genetické markery MeSH
• kultivační média chemie   MeSH
• kyselina azetidinkarboxylová antagonisté a inhibitory   farmakologie   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   fyziologie   MeSH
• selekce (genetika) MeSH
• technika přenosu genů MeSH
• Zygosaccharomyces genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetyltransferasy MeSH
• genetické markery MeSH
• kultivační média MeSH
• kyselina azetidinkarboxylová MeSH
• Mpr1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH