The so-called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T . By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase-mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX-loxP module in Z. rouxii ATCC 42981 G418-resistant mutants previously constructed by replacing the MATαP expression locus with the loxP-kanMX-loxP cassette.
- MeSH
- antibakteriální látky farmakologie MeSH
- centromera genetika MeSH
- fungální léková rezistence účinky léků genetika MeSH
- genetické inženýrství MeSH
- genetické markery MeSH
- integrasy genetika MeSH
- plazmidy genetika MeSH
- Zygosaccharomyces účinky léků genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Na(+)/H(+) antiporters may recognize all alkali-metal cations as substrates but may transport them selectively. Plasma-membrane Zygosaccharomyces rouxii Sod2-22 antiporter exports Na(+) and Li(+), but not K(+). The molecular basis of this selectivity is unknown. We combined protein structure modeling, site-directed mutagenesis, phenotype analysis and cation efflux measurements to localize and characterize the cation selectivity region. A three-dimensional model of the ZrSod2-22 transmembrane domain was generated based on the X-ray structure of the Escherichia coli NhaA antiporter and primary sequence alignments with homologous yeast antiporters. The model suggested a close proximity of Thr141, Ala179 and Val375 from transmembrane segments 4, 5 and 11, respectively, forming a hydrophobic hole in the putative cation pathway's core. A series of mutagenesis experiments verified the model and showed that structural modifications of the hole resulted in altered cation selectivity and transport activity. The triple ZrSod2-22 mutant T141S-A179T-V375I gained K(+) transport capacity. The point mutation A179T restricted the antiporter substrate specificity to Li(+) and reduced its transport activity, while serine at this position preserved the native cation selectivity. The negative effect of the A179T mutation can be eliminated by introducing a second mutation, T141S or T141A, in the preceding transmembrane domain. Our experimental results confirm that the three residues found through modeling play a central role in the determination of cation selectivity and transport activity in Z. rouxii Na(+)/H(+) antiporter and that the cation selectivity can be modulated by repositioning a single local methyl group.
- MeSH
- bodová mutace MeSH
- draslík metabolismus MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- hydrofobní a hydrofilní interakce MeSH
- kationty metabolismus MeSH
- konformace proteinů MeSH
- lithium metabolismus MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- Na(+)-H(+) antiport chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sodík metabolismus MeSH
- substrátová specifita MeSH
- Zygosaccharomyces chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- biologický transport MeSH
- delece genu MeSH
- fyziologický stres MeSH
- glycerol metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- kultivační média chemie MeSH
- mikrobiální viabilita MeSH
- organické látky metabolismus MeSH
- osmoregulace * MeSH
- osmotický tlak MeSH
- regulace genové exprese u hub účinky léků MeSH
- symportéry genetika metabolismus MeSH
- Zygosaccharomyces genetika růst a vývoj metabolismus fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
Three different transport systems exist to accumulate a sufficient amount of potassium cations in yeasts. The most common of these are Trk-type transporters, which are used by all yeast species. Though most yeast species employ two different types of transporters, we only identified one gene encoding a potassium uptake system (Trk-type) in the genome of the highly osmotolerant yeast Zygosaccharomyces rouxii, and our results showed that ZrTrk1 is its major (and probably only) specific potassium uptake system. When expressed in Saccharomyces cerevisiae, the product of the ZrTRK1 gene is localized to the plasma membrane and its presence efficiently complements the phenotypes of S. cerevisiae trk1∆ trk2∆ cells. Deletion of the ZrTRK1 gene resulted in Z. rouxii cells being almost incapable of growth at low K(+) concentrations and it changed some cell physiological parameters in a way that differs from S. cerevisiae. In contrast to S. cerevisiae, Z. rouxii cells without the TRK1 gene contained less potassium than the control cells and their plasma membrane was significantly hyperpolarized compared with those of the parental strain when grown in the presence of 100 mM KCl. On the other hand, subsequent potassium starvation led to a substantial depolarization which is again different from S. cerevisiae. Plasma-membrane hyperpolarization did not prevent the efflux of potassium from Z. rouxii trk1Δ cells during potassium starvation, and the activity of ZrPma1 is less affected by the absence of ZrTRK1 than in S. cerevisiae. The use of a newly constructed Z. rouxii-specific plasmid for the expression of pHluorin showed that the intracellular pH of the Z. rouxii wild type and the trk1∆ mutant is not significantly different. Together with the fact that Z. rouxii cells contain a significantly lower amount of intracellular potassium than identically grown S. cerevisiae cells, our results suggest that this highly osmotolerant yeast species maintain its intracellular pH and potassium homeostasis in way(s) partially distinct from S. cerevisiae.
- MeSH
- biologická adaptace MeSH
- biologický transport MeSH
- buněčná membrána fyziologie MeSH
- delece genu MeSH
- DNA fungální genetika metabolismus MeSH
- draslík metabolismus MeSH
- geny hub * MeSH
- homeostáza MeSH
- homologní rekombinace MeSH
- koncentrace vodíkových iontů MeSH
- membránové potenciály MeSH
- proteiny přenášející kationty genetika metabolismus MeSH
- regulace genové exprese u hub * MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika fyziologie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- sekvenční seřazení MeSH
- Zygosaccharomyces genetika fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- financování organizované MeSH
- genetické vektory MeSH
- plazmidy genetika MeSH
- Saccharomyces cerevisiae genetika MeSH
- sekvence nukleotidů MeSH
- transformace genetická MeSH
- Zygosaccharomyces genetika MeSH
- Publikační typ
- srovnávací studie MeSH
- MeSH
- acetyltransferasy genetika metabolismus MeSH
- antibiotická rezistence MeSH
- bakteriální transformace MeSH
- finanční podpora výzkumu jako téma 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 fyziologie MeSH
- technika přenosu genů MeSH
- Zygosaccharomyces genetika metabolismus MeSH
