Перегляд за Автор "Sibirny, Andriy"

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Deficiency in frataxin homolog ue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation
(2009) Pynyaha, Yuriy; Boretsky, Yuriy; Fedorovych, Daria; Fayura, Lubov; Levkiv, Andriy; Ubiyvovk, Vira; Protchenko, Olha; Philpott, Caroline; Sibirny, Andriy; Борецький, Юрій
Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B2) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Dyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-L-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Dyfh1 mutant was *3–3.5 times higher as compared to the parental strain. It produced 50–70 times more riboflavin in iron sufficient synthetic media relative to the parental wildtype strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Dyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.
Development of a transformation system for gene knock - out in the flavinogenic yeast Pichia guilliermondii
(2007) Boretsky, Yuriy; Pynyaha, Yuriy; Boretsky, Volodymyr; Kutsyaba, Vasyl; Protchenko, Olga; Philpott, Caroline; Sibirny, Andriy; Борецький, Юрій
Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5′-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib− Ura+ phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3–12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3′ end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8–0.9 kb sequences homologous to the target gene.
Identification of the genes a¡ecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis
(2011) Boretsky, Yuriy; Pynyaha, Yuriy; Boretsky, Volodymyr; Fedorovych, Dariya; Fayura, Lyubov; Protchenko, Olha; Philpott, Caroline; Sibirny, Andriy
Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondii Dvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondii Dfra1-45 mutant accumulated 1.8–2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Dvma1-17 and Dfes1-77 knockout strains could not grow at 37 1C in contrast to the wild-type strain and the Dfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 1C. Although the Dfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Dvma1-17 and Dfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.
Improved method for expression and isolation of the Mycoplasma hominis arginine deiminase from the recombinant strain of Escherichia coli
(2013) Fayura, Lyubov; Boretsky, Yuriy; Pynyaha, Yuriy; Wheatley, Denys; Sibirny, Andriy; Борецький, Юрій
Arginine deiminase is a promising anticancer drug active against melanoma, hepatocarcinoma and other tumors. Recombinant strains of Escherichia coli that express arginine deiminase from pathogenic bacteria Mycoplasma have been developed. However, production costs of heterologous arginine deiminase are high due to use of an expensive inducer and extraction buffer, as well as using diluted culture for enzyme induction. We report on a new advanced protocol for Mycoplasma hominis arginine deiminase expression, extraction and renaturation. The main improvements include manipulation with dense suspensions of E. coli, use of lactose instead of isopropyl -d-1-thiogalactopyranoside as an inducer and a cheaper but not less efficient buffer for solubilization of arginine deiminase inclusion bodies. In addition, supplementation of the storage culture medium with glucose and substrate (arginine) significantly stabilized the recombinant arginine deiminase producer. Homogenous preparations of recombinant arginine deiminase were obtained using anion-exchange and hydrophobic chromatography. The purified enzyme retained a specific activity of 30–34 U/mg for 12 months when stored at 4 ◦C in 20 mM sodium phosphate buffer pH 7.2 containing 1 M NaCl.
Improving the efficiency of plasmid transformation in Shewanella oneidensis MR-1 by removing ClaI restriction site
(2014) Rachkevych, Nazarii; Sybirna, Kateryna; Boyko, Solomiya; Boretsky, Yuriy; Sibirny, Andriy; Борецький, Юрій
Here we demonstrate that elimination of ClaI restriction site from the sequence of a plasmid DNA increases the efficiency of transformation of Shewanella oneidensis MR-1 significantly. To achieve reliable transformation of S. oneidensisMR-1 plasmids either lacking ClaI site or isolated from primary transformants of S. oneidensis should be used.
Mutations and environmental factors affecting regulation of riboflavin synthesis and iron assimilation also cause oxidative stress in the yeast Pichia guilliermondii
(2007) Boretsky, Yuriy; Protchenko, Olga; Prokopiv, Tetiana; Mukalov, Igor; Fedorovych, Daria; Sibirny, Andriy
Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. However, the mechanisms of such regulation are not known. We found that mutations causing riboflavin overproduction and iron hyperaccumulation (rib80, rib81 and hit1), as well as cobalt excess or iron deficiency all provoke oxidative stress in the Pichia guilliermondii yeast. Iron content in the cells, production both of riboflavin and malondialdehyde by P. guilliermondii wild type and hit1 mutant strains depend on a type of carbon source used in cultivation media. The data suggest that the regulation of riboflavin biosynthesis and iron assimilation in P. guilliermondii are linked with cellular oxidative state.
Mutations and environmental factors affecting regulation of riboflavin synthesis and iron assimilation also cause oxidative stress in the yeast Pichia guilliermondii
(2007) Boretsky, Yuriy; Protchenko, Olga; Prokopiv, Tetiana; Mukalov, Igor; Fedorovych, Daria; Sibirny, Andriy; Борецький, Юрій
Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. However, the mechanisms of such regulation are not known. We found that mutations causing riboflavin overproduction and iron hyperaccumulation (rib80, rib81 and hit1), as well as cobalt excess or iron deficiency all provoke oxidative stress in the Pichia guilliermondii yeast. Iron content in the cells, production both of riboflavin and malondialdehyde by P. guilliermondii wild type and hit1 mutant strains depend on a type of carbon source used in cultivation media. The data suggest that the regulation of riboflavin biosynthesis and iron assimilation in P. guilliermondii are linked with cellular oxidative state.
Novel arginine deiminase-based method to assay L-arginine in beverages
(2016) Stasyuk, N.; Gayda, G.; Fayura, Lyubov; Boretskyy, Yuriy; Gonchar, Mykhailo; Sibirny, Andriy; Борецький, Юрій
A highly selective and sensitive enzymatic method for the quantitative determination of L-arginine (Arg) has been developed. The method is based on the use of recombinant bacterial arginine deiminase (ADI) isolated from the cells of a recombinant strain Escherichia coli and o-phthalaldehyde (OPA) as a chemical reagent. Ammonia, the product of the enzymatic digestion of Arg by ADI, reacts with OPA and forms in the presence of sulfite a product, which can be detected by spectrophotometry (S) and fluorometry (F). The linear concentration range for Arg assay in the final reaction mixture varies for ADI-OPA-F variant of the method from 0.35 lM to 24lM with the detection limit of 0.25 lM. For ADI-OPA-S variant of the assay, the linearity varies from 0.7 lM to 50 lM with the detection limit of 0.55 lM. The new method was tested on real samples of wines and juices. A high correlation (R = 0.978) was shown for the results obtained with the proposed and the reference enzymatic method.
Oversynthesis of Riboflavin in the Yeast Pichia guilliermondii is Accompanied by Reduced Catalase and Superoxide Dismutases Activities
(2013) Prokopiv, Tetyana; Fedorovych, Dariya; Boretsky, Yuriy; Sibirny, Andriy; Борецький, Юрій
Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. Under iron deprivation conditions, Pichia guilliermondii cells increase production of riboflavin and malondialdehyde and the formation of protein carbonyl groups, which reflect increased intracellular content of reactive oxygen species. In this study, we found that P. guilliermondii iron deprived cells showed dramatically decreased catalase and superoxide dismutase activities. Previously reported mutations rib80, rib81, and hit1, which affect repression of riboflavin synthesis and iron accumulation by iron ions, caused similar drops in activities of the mentioned enzymes. These findings could explain the previously described development of oxidative stress in iron deprived or mutated P. guilliermondii cells that overproduce riboflavin. Similar decrease in superoxide dismutase activities was observed in iron deprived cells in the nonflavinogenic yeast Saccharomyces cerevisiae.
Pichia guilliermondii
(2009) Sibirny, Andriy; Boretsky, Yuriy; Борецький, Юрій
Pichia guilliermondii (asporogenous strains of this species are designated as Candida guilliermondii ) is the model organism of a group so named “ flavinogenic yeasts ” capable of riboflavin oversynthesis during starvation for iron. Besides, some strains of this species efficiently convert xylose to xylitol, an anti-caries sweetener. However, there are also pathogenic C. guilliermondii strains. This species has been used for studying enzymology of riboflavin synthesis due to overproduction of participating enzymes and intermediates under iron-limiting conditions as well as for identification of genes of negative and positive action involved in such a regulation. Besides, P. guilliermondii was used for identification and studying the properties of the systems for active transport of riboflavin in the cell (riboflavin permease) and out of the cell (riboflavin “ excretase ” ). The genetic line of P. guilliermondii with high fertility has been selected and the methods of classic genetics (hybridization and analysis of meiotic segregation) have been developed. More recently, tools for molecular genetic studies of P. guilliermondii have been developed which include collection of host strains, vectors with recessive and dominant markers, several transformation protocols including that for gene knock out. Recently, the genome of this yeast species was sequenced and become publicly available ( http://www.broad.mit.edu ).
Positive selection of mutants defective in transcriptional repression of riboflavin synthesis by iron in the flavinogenic yeast Pichia guilliermondii
(2005) Boretsky, Yuriy; Kapustyak, Kostyantyn; Fayura, Lyubov; Stasyk, Oleh; Stenchuk, Mykola; Bobak, Yaroslav; Drobot, Lyudmyla; Sibirny, Andriy; Борецький, Юрій
It is known for many years that iron represses synthesis of riboflavin (RF) and most of RF-synthesizing enzymes in several yeast species, known as flavinogenic yeasts. However, the mechanism of such repression is not known. We have found that iron represses transcription of RIB1 and RIB7 genes coding for the first and the last enzymes of RF biosynthesis in the model flavinogenic organism Pichia guilliermondii. To decipher molecular mechanisms of iron-dependent repression, isolation and study of the regulatory mutants defective in corresponding regulation is desirable. However, no suitable methods for isolation of such mutants were previously available. We have produced a single-point transition mutation in the RIB1 gene. The corresponding rib1-86 mutant exhibits leaky phenotype and is unable to grow in iron-sufficient minimal medium without exogenous RF. However, it can grow in minimal iron-deficient medium without RF, or in iron-sufficient medium upon introduction of the previously-isolated regulatory mutation rib81, which leads to increase in RF production. Using the rib1-86 mutant as parental strain, a collection of mutants able to grow in iron-sufficient medium without exogenous RF has been isolated. The mutants appeared to be defective in regulation of RF biosynthesis and iron homeostasis and were divided into six new complementation groups. Study of one corresponding mutant, red6, showed derepression of RIB1 mRNA synthesis in iron-sufficient medium.
Recombinant arginine-degrading enzymes in metabolic anticancer therapy and bioanalytics
(2015) Stasyk, Oleh; Boretsky, Yuriy; Gonchar, Mykhailo; Sibirny, Andriy
Tumor cells often exhibit specific metabolic defects due to the aberrations in oncogene-dependent regulatory and/or signaling pathways that distinguish them from normal cells. Among others, many malignant cells are deficient in biosynthesis of certain amino acids and concomitantly exhibit elevated sensitivity to deprivation of these amino acids. Although the underlying causes of such metabolic changes are still not fully understood, this feature of malignant cells is exploited in metabolic enzymotherapies based on single amino acid, e.g., arginine, deprivation. To achieve efficient arginine depletion in vivo, two recombinant enzymes, bacterial arginine deiminase and human arginase I have been evaluated and are undergoing further development. This review is aimed to summarize the current knowledge on the application of arginine-degrading enzymes as anticancer agents and as bioanalytical tools for arginine assays. The problems that have to be solved to optimize this therapy for clinical application are discussed.
The Pleiotropic Nature of rib80, hit1 , and red6 Mutations Affecting Riboflavin Biosynthesis in the Yeast Pichia guilliermondii
(2007) Fayura, Lyubov; Fedorovych, Daria; Prokopiv, Tetiana; Boretsky, Yuriy; Sibirny, Andriy
The yeast Pichia guilliermondii is capable of riboflavin overproduction under iron deficiency. The rib80, hit1 , and red6 mutants of this species, which exhibit impaired riboflavin regulation, are also distinguished by increased iron concentrations in the cells and mitochondria, morphological changes in the mitochondria, as well as decreased growth rates (except for red6 ) and respiratory activity. With sufficient iron supply, the rib80 and red6 mutations cause a 1.5–1.8-fold decrease in the activity of such Fe–S cluster proteins as aconitase and flavocytochrome b2, whereas the hit1 mutation causes a six-fold decrease. Under iron deficiency, the activity of these enzymes was equally low in all of the studied strains.