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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.
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.
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.
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.