The red mutations impair the regulation of flavinogenesis and metal homeostas in yeast Pichia guilliermondii

A method of positive selection of mutants with impaired regulation of flavinogenesis and metal homeostasis in yeast Pichia guilliermondii was developed. This positive selection system was based on the isolation of pseudo-wild-type revertants (the Rib+ phenotype) in riboflavin-dependent rib1-86 mutant (the Rib- phenotype) of yeast P. guilliermondii. Mutation rib1-86 blocks activity of the GTP cyclohydrolase II catalyzing the first step in riboflavin (RF) biosynthesis. Study of a collection of spontaneous Rib+ revertants allowed the identification of a considerably large number of genetic loci responsible for the suppression of rib1-86, which include both previously identified three loci (rib80, rib81, and hit1) and six new loci designated red1-red6 (reduction). A comparative analysis of the wild-type strain and red mutants revealed that these mutants had higher activity levels of GTP cyclohydrolase and RF-synthase, elevated levels of RF biosynthesis, enhanced Fe/Cu reductase activity and higher total iron content in cells and that they are characterized by enhanced sensitivity to transition metals (Fe(III), Cu(II), Cd(II), Co(II), Zn(II), Ag(I), and to H2O2. The metal hypersensitivity of mutant cells can be prevented by an increased amount of extracellular iron ions. Mutations red1 and red6 synergistically interact with the locus rib81 in the course of RF biosynthesis. Obviously, each RED gene plays an important role in the regulation of both flavinogenesis and metal homeostasis in P. guilliermondii cells.     

Effect of rib83 mutation on riboflavin biosynthesis and iron assimilation in Pichia guilliermondii]

The monogenic rib83 mutation blocked riboflavin oversynthesis in the yeast Pichia guilliermondii and lowered iron acquisition by cells, their ferric reductase activity, and the growth rate in iron-deficient media. Mutants with the combined mutations of rib83 with rib80 and rib81 (the last two mutations impair the negative control of riboflavin synthesis and thus cause its oversynthesis) were unable to depress the enzymes of flavinogenesis (GTP cyclohydrolase and riboflavin synthase) and to overproduce riboflavin in both iron-deficient and iron-sufficient media. This suggests that the rib83 mutation is epistatic with respect to the rib80 and rib81 mutations. The RIB83 gene may positively control both riboflavin synthesis and iron acquisition in the yeast P. guilliermondii.     

The red Mutations Impair the Regulation of Flavinogenesis and Metal Homeostasis in Yeast Pichia guilliermondii

A method of positive selection of mutants with impaired regulation of flavinogenesis and metal homeostasis in yeast Pichia guilliermondii was developed. This positive selection system was based on the isolation of pseudo-wild-type revertants (the Rib⁺ phenotype) in riboflavin-dependent rib1-86 mutant (the Rib^– phenotype) of yeast P. guilliermondii. Mutation rib1-86 blocks activity of the GTP cyclohydrolase II catalyzing the first step in riboflavin (RF) biosynthesis. Study of a collection of spontaneous Rib⁺ revertants allowed the identification of a considerably large number of genetic loci responsible for the suppression of rib1-86, which include both previously identified three loci (rib80, rib81, andhit1) and six new loci designated red1–red6 (reduction). A comparative analysis of the wild-type strain and red mutants revealed that these mutants had higher activity levels of GTP cyclohydrolase and RF-synthase, elevated levels of RF biosynthesis, enhanced Fe/Cu reductase activity and higher total iron content in cells and that they are characterized by enhanced sensitivity to transition metals (Fe(III), Cu(II), Cd(II), Co(II), Zn(II), Ag(I)) and to H₂O₂. The metal hypersensitivity of mutant cells can be prevented by an increased amount of extracellular iron ions. Mutations red1 and red6 synergistically interact with the locus rib81 in the course of RF biosynthesis. Obviously, each RED gene plays an important role in the regulation of both flavinogenesis and metal homeostasis in P. guilliermondii cells.     

Riboflavin overproduction in 4-aminopyrazole[3,4-d]pyrimidine-treated yeast Pichia guilliermondii

More than 90 mutants resistant to the adenine analogue 4-aminopyrazole[3,4-d]pyrimidine (4-APP), were isolated from a wild-type strain of yeast Pichia guilliermondii. Some of Appr mutants accumulated noticeable amounts of products absorbing at 260 nm in the culture medium, probably nucleotides and their derivatives. In comparison to the parent strain, the mutant Appr-27 synthesized greater amounts of xanthine and uracil suggesting the presence of defects in the regulation of de novo biosynthesis of purines and pyrimidines. The regulatory mutations rib80 and rib81 are known to cause riboflavin (RF) overproduction and derepression of RF-producing enzyme synthesis in P. guilliermondii. The mutant Appr-27 was crossed to the rib81 strain. The yield of RF biosynthesis in some meiotic segregants was significantly higher than that in segregants from the diploid rib81/RIB81. Apparently, rib81 and appr mutations were combined in single genome on the favorable genetic background. An increase in RF production was also found in strains with appr mutations induced directly in the genome of the RF oversynthesizing strain rib80 rib81. These results indicate that introduction of appr mutations into genome of P. guilliermondii can intensify their RF overproduction.     

Riboflavin Overproduction in 4-Aminopyrazolo[3,4-d]pyrimidine-Resistant Mutants of Yeast Pichia guilliermondii

More than 90 mutants resistant to the adenine analogue 4-amino-pyrazolo[3,4-d]pyrimidine (4-APP), were isolated from a wild-type strain of yeast Pichia guilliermondii. Some of the App ^rmutants accumulated noticeable amounts of products absorbing at 260 nm in the culture medium, probably nucleotides and their derivatives. In comparison to the parent strain, the mutant App ^r-27 synthesized greater amounts of xanthine and uracil suggesting the presence of defects in the regulation of de novo biosynthesis of purines and pyrimidines. The regulatory mutations rib80 and rib81 are known to cause riboflavin (RF) overproduction and derepression of synthesis of corresponding enzymes in P. guilliermondii. The mutant App ^r-27 was crossed to the rib81 strain. The yield of RF biosynthesis in some meiotic segregants was significantly higher than that in segregants from the diploid rib81/RIB81. Apparently,rib81 and app ^r mutations were combined in a single genome on the favorable genetic background. An increase in RF production was also found in strains with app ^r mutations induced directly in the genome of the RF oversynthesizing strain rib80 rib81. These results indicate that introduction of app ^r mutations into the genome of P. guilliermondii can intensify their RF overproduction.     

Effect of the rib83Mutation on Riboflavin Synthesis and Iron Acquisition in the Yeast Pichia guilliermondii

Abstract–Monogenicrib83mutation blocked riboflavin oversynthesis in the yeast Pichia guilliermondiiand lowered iron acquisition by cells, their ferric reductase activity, and the growth rate in iron-deficient media. Mutants with the combined mutations of rib83with rib80and rib81(the last two mutations impair the negative control of riboflavin synthesis and thus cause its oversynthesis) were unable to depress the enzymes of flavinogenesis (GTP cyclohydrolase and riboflavin synthase) or overproduce riboflavin in both iron-deficient and iron-sufficient media. This suggests that rib83mutation is epistatic with respect to rib80and rib81mutations. The RIB83gene may positively control both riboflavin synthesis and iron acquisition in the yeast P. guilliermondii.