Synergistic effect of rib80 and hit mutations on riboflavin biosynthesis and iron transport in the yeast Pichia guilliermondii

Mutant strains of the yeast Pichia guilliermondii, carrying both rib80 and hit mutations in a haploid genome, were derived from previously obtained strains with defective rib80 or hit genes, exerting negative control of the riboflavin biosynthesis and iron transport in Pichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of the rib80 and hit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of 55Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

The pleiotropic nature of <Emphasis Type="Italic">rib80, hit1</Emphasis>, and <Emphasis Type="Italic">red6</Emphasis> mutations affecting riboflavin biosynthesis in the yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis>

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 b ₂, 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.     

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.     

Biochemical functions of RIB5 and RIB6 gene products participating in riboflavin biosynthesis in the yeast Pichia guilliermondii

The biosynthesis of riboflavin precursor 6,7-dimethyl-8-ribityllumazine was studied in extracts of Pichia guilliermondii yeast mutants of rib5 and rib6 genotypes with impaired synthesis of proteins P1 and P2, respectively. It was shown that synthesis of 6,7-dimethyl-8-ribityllumazine took place in extracts of rib5 mutant (active P1 protein) in the presence of 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and the compound formed from ribose-5-phosphate by extracts of rib6 mutant (active P2 protein). No lumazine was formed in extracts of rib6 mutant from pyrimidine substrate and ribose-5-phosphate preincubated with extracts of rib5 mutant. Hence, P1 protein (the product of RIB5 gene) participates in the biosynthesis of 6,7-dimethyl-8-ribityllumazine from 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and aliphatic intermediate which is formed from ribose-5-phosphate, under the action of P2 protein (the product of RIB6 gene).     

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.     

Purification and properties of the riboflavin kinase of the yeast Pichia guilliermondii]

Riboflavin kinase (E.C.2.7.1.26) was isolated from the cells of the yeast Pichia guilliermondii. The enzyme was 680-fold purified uzing ammonium sulphate fractionation, chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50 and gel-filtration through Sephadex G-75. Purified enzyme preparation was free from phosphatases and FAD-synthetase. The pH optimum was 8,7, the temperature optimum-45 degrees C. The enzyme was activated by Zn2+, Mg2+ and Co2+ ions. Km for riboflavin was 1,0x10(-5) M, for ATP -- 6,7X10(-6) M. Riboflavin kinase catalyzed the phosphorylation of riboflavin analogues with the substitution of methyl groups at positions 7 and 8. UTP, GTP, ADP and CTP, besides ATP, were phosphate donors. AMP inhibited the enzyme activity. Molecular weight of the enzyme was 28000, as estimated by gel-filtration through Sephadex G-150. Purified riboflavin kinase was stable under storage.     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) 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. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 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 Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 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 Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

Oversynthesis of riboflavin by yeast Pichia guilliermondii in response to oxidative stress

The effect of oxidative stress on riboflavin (vitamin B2) biosynthesis and iron accumulation in flavinogenic yeast P. guilliermondii was investigated. Treatment of P. guilliermondii cells with superoxidgenerating agent methylviologen leads to elevated production of malondialdyhyd (MDA) which reflects the overall cellular oxidation state. Increased iron content in the cells and enhanced productivity of flavinogenesis under these conditions has been shown too. Significant increasing of MDA and riboflavin production by yeast cells under iron deficiency was observed. Riboflavin overproducing P. guilliermondii mutant strains rib80, rib81 and hit, possess high iron transport and synthesize increased quantity of MDA. The role of riboflavin overproduction and activation of iron assimilation in the P. guilliermondii antioxidant defence is discussed.     

Substrate and inhibitory specificity of riboflavin kinase from Pichia guilliermondii yeast]

The interaction of purified riboflavin kinase (EC 2.7.1.26) from Pichia guilliermondii with 44 structural vitamin B2 analogues is studied. The presence of D-ribityl lateral chain in an analogue structure is found to be necessary for the substrate activity. The substitution of CH3 groups in the 7 and 8 positions of isoalloxazine ring in the riboflavin molecule for CF3, Cl, H, NH2 and N(CH3)2 resulted in the decrease of the analogue affinity to riboflavin kinase as compared with the natural substrate, vitamin B2. The most efficient enzyme inhibitors of analogues without substrate properties turned to be trifluoromethylisoalloxazines, containing 2'-hydroxyethyl group at N10. The elongation of D-ribityl lateral chain, the elimination of change of CH3-groups in the 7 and 8 positions for CF3- Cl-, COOH-substitutors resulted in the decrease of the inhibitory effect of flavines. Modifications in the structure of isoalloxazine ring, etherification of OH-groups in the lateral D-ribityl chain, and the introduction of volume substitutors (N-piperidyl, D-ribitylamine, hydroxyethylamine) prevented the interaction of the analogue with riboflavin kinase. Flavin nucleotides (FMN and FAD) did not affect the rate of vitamin B2 phosphorylation.     

Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation

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 Δyfh1 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 Δyfh1 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 wild-type 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 Δyfh1 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.     

Regulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis by riboflavin and iron in riboflavin-deficient mutants of Pichia guilliermondii yeast.

The effect of riboflavin and iron on 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate was investigated in the cultures of the yeast Pichia guilliermondii (rib2 mutants) with the blocked second reaction to flavinogenesis. It was shown that riboflavin inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich and iron-deficient cells of mutants with low riboflavin requirements. Cycloheximide did not prevent the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis caused by riboflavin starvation. 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis, while 7-methyl-8-trifluoro-methyl-10-(beta-hydroxyethyl)izoalloxazine and galactoflavin exerted only a slight effect on this process. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells was significantly higher than in iron-rich cells. The 2,2'-dipyridyl treatment of iron-rich cells caused the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide abolished this effect. The results suggest that the activity of the first enzyme of flavinogenesis (guanylic cyclohydrolase) is under the control of feedback inhibition by flavins and the biosynthesis of this enzyme is regulated by iron.     

Regulation of the lysine biosynthesis in Pichia guilliermondii

The regulatory properties of four enzymes (homocitrate synthase, -aminoadipate reductase, saccharopine reductase, saccharopine dehydrogenase) involved in the lysine biosynthesis of Pichia guilliermondii were investigated and compared with the regulatory patterns found in other yeast species. The first enzyme of the pathway, homocitrate synthase, is feedback-inhibited by L-lysine. Some other amino acids (-aminoadipate, glutamate, tryptophan, leucine) and lysine analogues are also inhibitors of one or more enzymes. It is shown that only the synthesis of homocitrate synthase is weakly repressed by L-lysine.     

Xylitol and riboflavin accumulation in xylose-grown cultures of Pichia guilliermondii

Seven strains of Pichia guilliermondii (Candida guilliermondii, asexual state) from diverse isolation sources were examined for the production of xylitol and riboflavin in xylose-grown cultures. Under the conditions tested, all strains produced xylitol from xylose; conversion efficiencies varied, on a strain-specific basis, from 7% to 36% of the initial substrate. Four of seven strains metabolized xylitol immediately as xylose levels became depleted. The remaining three strains metabolized xylitol slowly and incompletely. Surprisingly, utilization of xylitol showed an apparent relationship with riboflavin production. Strains that readily metabolized xylitol produced at least threefold greater levels of riboflavin than did strains that used xylitol slowly. Moreover, riboflavin accumulation took place during xylitol consumption. P. guilliermondii strains that produced the highest levels of riboflavin on xylose produced significantly less riboflavin when grown on glucose or directly on xylitol. Received: 24 April 1996 / Received revision: 29 July 1996 / Accepted: 24 August 1996     

Synergistic effect ofrib80 andhit Mutations on riboflavin biosynthesis and iron transport in the yeastPichia guilliermondii

Mutant strains of the yeastPichia guilliermondii, carrying bothrib80 andhit mutations in a haploid genome, were derived from previously obtained strains with defectiverib80 orhit genes, exerting negative control of the riboflavin biosynthesis and iron transport inPichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of therib80 andhit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of⁵⁵Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81

The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied. The mutants were isolated from a genetic line of Pichia guilliermondii. Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium. An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium. The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class. The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency. Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81. Thus, riboflavin biosynthesis in P. guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81. The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes. The hybrids of rib81 mutants with natural nonflavinogenic strain P. guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium. Apparently, the strain NF1453-1 contains an unaltered gene RIB81.     

Effect of glucose and its derivatives on systems of riboflavin uptake and excretion in the yeast Pichia guilliermondii]

Riboflavin uptake by washed cells of riboflavin deficient mutant MS1-3 of Pichia guilliermondii yeast was strongly depressed by D-glucose, L-sorbose, alpha-methyl-D-glucoside, sucrose, trehalose, maltose and salicin but not by D-mannose, D-galactose, D-fructose or ribitol. Glucose decreased also the initial uptake rate of riboflavin analogue, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine; the inhibition having a competitive character (Ki==5,7 mM). Apparently riboflavin permease is able to accept not only riboflavin and its analogues but also glucose and some of glucose derivates. Cells preloaded with riboflavin and transferred into riboflavin-free medium excreted vitamin B2 into the medium. This excretion was strongly stimulated by D-glucose, D-fructose, D-mannose but not by citrate or succinate. In contrast to riboflavin, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine was not excreted into the medium even in the presence of glucose. The rate of riboflavin excretion depended on temperature and pH of incubation medium (pH optimum approximately 7.0) and was decreased in the presence of different inhibitors of energy metabolism. It seems that the exit of riboflavin from the cells is accomplished by energy-dependent specific system of excretion (excretase) which in some properties is different from that of riboflavin permease.     

Origin of the ribityl side-chain of riboflavin from the ribose moiety of guanosine triphosphate in Pichia guilliermondii yeast.

In wild-type cells and some riboflavin-deficient mutants of P. guilliermondii GTP is transformed to the ribitylated intermediates 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and 5-amino-2,6-dihydroxy-4-ribitylaminopyrimidine of the riboflavin biosynthetic path. We were able to show that these compounds were formed in vitro as well as in permeabilized cells by reactions including a reductive conversion of the product of GTP cyclohydrolase II action upon GTP. In order to analyse the pyrimidine derivates, 6,7-dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine were synthesized by reaction of pyrimidines with diacetyl. The formation of ribitylated pyrimidines was shown to be strictly dependent on the presence of NADPH2. The data obtained indicate that the reductive step is catalyzed by a 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-reductase. 6,7-Dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine isolated from the incubation mixtures have been identified by chromatography and by their ultraviolet and fluorescence spectra.