In the model yeast Saccharomyces cerevisiae, Trk1 is the main K+ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K+ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins' abilities to support the growth of cells under various cultivation conditions such as low K+ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb+ . Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K+ and high salt and that the overproduction of S. cerevisiae's own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li+ ions.
- MeSH
- biologický transport MeSH
- draslík metabolismus MeSH
- fylogeneze MeSH
- proteiny přenášející kationty * genetika MeSH
- Saccharomyces cerevisiae - proteiny * metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- Yarrowia * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of the study was to screen Yarrowia lipolytica strains for keto acid production and determine optimal conditions for pyruvic acid biosynthesis from glycerol by the best producer. The analyzed parameters were thiamine concentration, medium pH, stirring speed, and substrate concentration. The screening was performed in flask cultures, whereas pyruvic acid production was carried out in 5-L stirred-tank reactor with 2 L of working volume. In total, 24 Y. lipolytica strains were compared for their abilities to produce pyruvic and α-ketoglutaric acids. The total concentration of both acids ranged from 0.1 to 15.03 g/L. Ten strains were selected for keto acid biosynthesis in bioreactor. The Y. lipolytica SKO 6 strain was identified as the best producer of pyruvic acid. In the selected conditions (thiamine concentration 1.5 μg/L, pH 4.0, stirring speed 800 rpm, 150 g/L of glycerol), the strain Y. lipolytica SKO 6 produced 99.3 g/L of pyruvic acid, with process yield of 0.63 g/g and volumetric production rate of 1.18 g/L/h. Higher titer of pyruvic acid was obtained during fed-batch culture with 200 g/L of glycerol, reaching 125.8 g/L from pure glycerol (yield 0.68 g/g) and 124.4 g/L from crude glycerol (yield 0.62 g/g). Results obtained for the strain Y. lipolytica SKO 6 proved the suitability of microbial production of pyruvic acid at industrial scale.
- MeSH
- bioreaktory MeSH
- glycerol analýza metabolismus MeSH
- kultivační média chemie MeSH
- kyselina pyrohroznová analýza metabolismus MeSH
- kyseliny ketoglutarové analýza metabolismus MeSH
- techniky vsádkové kultivace MeSH
- thiamin analýza MeSH
- Yarrowia růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The objective of the study was to compare the efficiency of two species of yeast, Yarrowia lipolytica (YL) and Saccharomyces cerevisiae (SC), with or without a probiotic supplement, added to feed for piglets, on the basis of haematological blood indices and the gut microbiota. A total of 360 piglets (the average 27-d-old) were allotted to dietary treatments: 1) the basal control(C) diet, 2) the C diet + probiotic(P) (a mixture of Bacillus licheniformis and Bacillus subtilis), 3) the C diet +3%YL(Y), 4) the C diet +3%YL + probiotic(YP), 5) the C diet +3%SC(S) and 6) the C diet +3%SC + probiotic(SP). The study showed that YL yeast can be used in compound feeds for piglets interchangeably with SC yeast. The effect of YL on haematological blood parameters and the microbes colonizing the gut proved to be more beneficial than the effect of SC yeast. The combined application of YL or SC with a probiotic had a more favourable effect on the gut microbiota than the use of yeast alone. It should be noted, however, that supplementation of the compound feed with YL in combination with a probiotic reduced the multiplication of coliform bacteria and Escherichia coli in the intestinal contents, while the feed containing SC together with a probiotic did not. The dietary study confirmed that YL in combination with a probiotic is highly suitable for feeding piglets.
- MeSH
- dieta MeSH
- krmivo pro zvířata MeSH
- potravní doplňky MeSH
- prasata krev mikrobiologie MeSH
- probiotika * MeSH
- Saccharomyces cerevisiae MeSH
- střevní mikroflóra * MeSH
- Yarrowia MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts. Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives. The comparison of innovative CRISPR-Cas expression strategies in yeasts presented here may also serve as guideline to implement and refine CRISPR-Cas systems for highly efficient genome editing in other eukaryotic organisms.
- MeSH
- bodová mutace MeSH
- chromozomy hub MeSH
- CRISPR-Cas systémy * MeSH
- editace genu metody MeSH
- geneticky modifikované mikroorganismy MeSH
- guide RNA, Kinetoplastida MeSH
- klonování DNA MeSH
- kvasinky genetika MeSH
- metabolické inženýrství MeSH
- Pichia genetika MeSH
- regulace genové exprese u hub MeSH
- Saccharomyces cerevisiae genetika MeSH
- technologie gene drive MeSH
- Yarrowia genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Fungi are commonly involved in dairy product spoilage and the use of bioprotective cultures can be a complementary approach to reduce food waste and economic losses. In this study, the antifungal activity of 89 Lactobacillus and 23 Pediococcus spp. isolates against three spoilage species, e.g., Yarrowia lipolytica, Rhodotorula mucilaginosa and Penicillium brevicompactum, was first evaluated in milk agar. None of the tested pediococci showed antifungal activity while 3, 23 and 43 lactobacilli isolates showed strong antifungal activity or total inhibition against Y. lipolytica, R. mucilaginosa and P. brevicompactum, respectively. Then, the three most promising strains, Lactobacillus paracasei SYR90, Lactobacillus plantarum OVI9 and Lactobacillus rhamnosus BIOIII28 at initial concentrations of 105and 107CFU/ml were tested as bioprotective cultures against the same fungal targets in a yogurt model during a 5-week storage period at 10 °C. While limited effects were observed at 105CFU/ml inoculum level, L. paracasei SYR90 and L. rhamnosus BIOIII28 at 107CFU/ml respectively retarded the growth of R. mucilaginosa and P. brevicompactum as compared to a control without selected cultures. In contrast, growth of Y. lipolytica was only slightly affected. In conclusion, these selected strains may be good candidates for bioprotection of fermented dairy products.
- MeSH
- antifungální látky farmakologie MeSH
- Lactobacillus fyziologie MeSH
- mléčné výrobky mikrobiologie MeSH
- Pediococcus fyziologie MeSH
- Penicillium růst a vývoj MeSH
- potravinářská mikrobiologie MeSH
- probiotika MeSH
- Rhodotorula růst a vývoj MeSH
- Yarrowia růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species.
- MeSH
- Candida genetika metabolismus MeSH
- mitochondriální proteiny genetika metabolismus MeSH
- molekulární evoluce * MeSH
- proteiny s vysokou pohyblivostí genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Yarrowia genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytokinin oxidase/dehydrogenase (CKO; EC 1.5.99.12) irreversibly degrades the plant hormones cytokinins. A recombinant maize isoenzyme 1 (ZmCKO1) produced in the yeast Yarrowia lipolytica was subjected to enzymatic deglycosylation by endoglycosidase H. Spectrophotometric assays showed that both activity and thermostability of the enzyme decreased after the treatment at non-denaturing conditions indicating the biological importance of ZmCKO1 glycosylation. The released N-glycans were purified with graphitized carbon sorbent and analyzed by MALDI-TOF MS. The structure of the measured high-mannose type N-glycans was confirmed by tandem mass spectrometry (MS/MS) on a Q-TOF instrument with electrospray ionization. Further experiments were focused on direct analysis of sugar binding. Peptides and glycopeptides purified from tryptic digests of recombinant ZmCKO1 were separated by reversed-phase chromatography using a manual microgradient device; the latter were then subjected to offline-coupled analysis on a MALDI-TOF/TOF instrument. Glycopeptide sequencing by MALDI-TOF/TOF MS/MS demonstrated N-glycosylation at Asn52, 63, 134, 294, 323 and 338. The bound glycans contained 3-14 mannose residues. Interestingly, Asn134 was found only partially glycosylated. Asn338 was the sole site to carry large glycan chains exceeding 25 mannose residues. This observation demonstrates that contrary to a previous belief, the heterologous expression in Y. lipolytica may lead to locally hyperglycosylated proteins.
- MeSH
- glykosylace MeSH
- klonování DNA MeSH
- kukuřice setá enzymologie MeSH
- molekulární sekvence - údaje MeSH
- oxidoreduktasy metabolismus MeSH
- polysacharidy analýza MeSH
- rekombinantní proteiny izolace a purifikace MeSH
- sekvence aminokyselin MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- stabilita enzymů MeSH
- tandemová hmotnostní spektrometrie MeSH
- Yarrowia enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Yarrowia lipolytica plasma-membrane Na+/H+ antiporter, encoded by the YlNHA2 gene, is a very efficient exporter of surplus sodium from the cytosol. Its heterologous expression in Saccharomyces cerevisiae wild-type laboratory strains increased their sodium tolerance more efficiently than the expression of ZrSod2-22 antiporter from the osmotolerant yeast Zygosaccharomvces rouxii.
- MeSH
- antiportéry MeSH
- chlorid sodný chemie MeSH
- financování organizované využití MeSH
- prahové limitní hodnoty MeSH
- Saccharomyces cerevisiae cytologie chemie metabolismus MeSH
- sodík chemie metabolismus škodlivé účinky MeSH
- viabilita buněk MeSH
- Yarrowia cytologie chemie metabolismus MeSH
- Zygosaccharomyces cytologie chemie metabolismus MeSH
The family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.
- MeSH
- antiportéry metabolismus MeSH
- buněčná membrána metabolismus MeSH
- časové faktory MeSH
- draslík metabolismus MeSH
- exprese genu MeSH
- financování organizované MeSH
- fungální proteiny metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- Saccharomyces cerevisiae cytologie MeSH
- sekvenční analýza proteinů MeSH
- sodík metabolismus MeSH
- soli farmakologie MeSH
- transport proteinů MeSH
- Yarrowia fyziologie metabolismus účinky léků MeSH
