[The microbial synthesis of flavin nucleotides: a review].

Recent data on the synthesis and hydrolysis of flavin nucleotides in yeast and bacteria and the regulation of this process are summarized. Specific examples are provided and the prospects of the use of genetically modified microorganisms for the industrial manufacturing of flavin mononucleotide and flavin dinucleotide are considered.

[The pleiotropic nature of rib80, hit1, and red6 mutations affecting riboflavin biosynthesis in the yeast Pichia guilliermondii].

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 hitl mutation causes a six-fold decrease. Under iron deficiency, the activity of these enzymes was equally low in all of the studied strains.

[Selection and properties of mutant yeast Pichia guilliermondii strains resistant to chromium (VI)].

Yeast Pichia guilliermondii strains L3 and L2, exposed to UV mutagenesis, produced over 80 mutants capable of growing on media containing 1.5 mM bichromate (Cr(VI)). The mutations making the strains resistant to Cr(VI) were dominant or semidominant. The mutants varied in Cr(VI) resistance, the degree of chromium accumulation in the cells (from 0.1 to 11.6 mg/g dry cells), and the degree of Cr(VI) reduction (from 50% to complete disappearance of bichromate from the culture liquid). Chromium accumulation in mutant cells depended on medium composition, Cr(VI) concentration, and the time of exposure to Cr(VI). The resistance to bichromate can be caused by various reasons: decrease in chromium absorption, altered ability to reduce Nr(VI), or damage of sulfate transport mechanisms.

[The reversion of Pichia guilliermondii transformants to the wild-type phenotype]. / Zakonomernosti vosstanovleniia fenotipa dikogo tipa u transformantov drozhzhei Pichia guilliermondii.

Pichia guilliermondii strain with blocked GTP cyclohydrolase II was transformed using replicative plasmids and their fragments containing the structural gene RIB7 of this enzyme. Experiments showed that the presence of an ARS element and the promoter region of the gene in the genome of transformants reduces the probability of their reversion to the wild-type phenotype. Different types of recombination in the yeast P. guilliermondii are discussed.

[Riboflavin overproduction in 4-aminopyrazole[3,4-d]pyrimidine-treated yeast Pichia guilliermondii]. / Sverkhsintez riboflavina u drozhzhei Pichia guilliermondii pri deistvii 4-aminopirazol[3,4-d]pirimidina.

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.

[Effect of rib83 mutation on riboflavin biosynthesis and iron assimilation in Pichia guilliermondii]. / Vliianie mutatsii rib83 na biosintez riboflavina i assimiliatsiiu zheleza u drozhzhei 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.

[Synergistic effect of rib80 and hit mutations on riboflavin biosynthesis and iron transport in the yeast Pichia guilliermondii]. / Sovmestnoe vliianie mutatsii rib80 and hit na biosintez riboflavina i transport zheleza u drozhzhei 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.

Iron uptake by the yeast Pichia guilliermondii. Flavinogenesis and reductive iron assimilation are co-regulated processes.

Pichia guilliermondii cells overproduce riboflavin (vitamin B2) in responce to iron deprivation. The increase in ferrireductase activity in iron-starved P. guilliermondii cells correlated with the increase in flavin excretion. As in Saccharomyces cerevisiae, a typical b-type cytochrome spectrum was associated with the plasma membrane fraction of P. guilliermondii and the cell ferrireductase activity was strongly inhibited by diphenylene-iodonium, an inhibitor of flavoproteins, in both yeasts. Mutants of P. guilliermondii with increased ferrireductase activity were selected for further investigation of the relationship between iron reduction/uptake and flavin production. The obtained mutation has been called hit (high iron transport). A hit mutant with a single recessive mutation showed the following phenotype: high ferrireductase activity, increased rate of iron uptake and elevated flavinogenic activity. Cu(II) (50 microns) strongly inhibited the growth of the hit mutant compared to the wild-type. The mutant cells grown in copper-supplemented medium (5-25 microns) showed an increase of the ferrireductase activity (up to 2-3 fold). The copper content of the mutant cells grown under these conditions was also higher (1.5-2 fold) than that of the wild-type. The role of the HIT gene of P. guillermondii in the regulation of iron, copper and flavin metabolisms is discussed.

[Selection and mutant properties of Pichia guilliermondii yeasts with derepressed GTP cyclohydrolase, the enzyme of the 1st step in flavinogenesis]. / Selektsiia i svoistva mutantov drozhzhei Pichia guilliermondii s derepressirovannym fermentom pervogo etapa flavinogeneza--GTF-tsiklogidrolazoi.

A positive method is proposed for selecting Pichia guilliermondii mutants with derepressed GTP cyclohydrolase. Mutants with the incompletely blocked gene RIB2 were used as parent strains; these can grow in a medium without riboflavin (RF) only if the enzyme is derepressed as the result of iron deficiency in cells. Strains growing in a medium without RF at the optimal supply of cells with iron were selected as regulatory mutants. The mutants accumulated 6,7-dimethylpterin in high concentrations and a small amount of RF in the medium and in the cells. The activity of GTP cyclohydrolase rather than that of RF synthase increased in the mutants; the activity of RF kinase and FAD pyrophosphorylase was not elevated. Hybrids produced by crossing the regulatory mutants with wild type strains did not accumulate 6,7-dimethylpterin in the medium and the activity of the GTP cyclohydrolase did not increase; this is indicative of the negative regulation for the expression of the structural gene for GTP cyclohydrolase. The authors propose a model for the regulation of GTP cyclohydrolase and RF synthase at the gene level involving iron ions as a corepressor.

[Activity of the enzyme of the 2d step of flavinogenesis, 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate reductase, in Pichia guilliermondii yeasts]. / Ob aktivnosti fermenta vtorogo étapa flavinogeneza 2,5-diamino-6-oksi-4-ribozilaminopirimidin-5'-fosfatreduktazy u drozhzhei Pichia guilliermondii.

The activity of 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate reductase, an enzyme of the second step of flavinogenesis, was studied in the yeast Pichia guilliermondii using a new fluorimetric technique. The activity of the enzyme was found in extracts of the wild-type strain and the genotype rib mutants but not in extracts of the FM 1-4 (rib 2) mutant. Interallelic complementation was revealed in the gene rib 2. The activity of the enzyme was identical in extracts of the cells grown in the medium with either a high or a low iron content which synthesized riboflavin at a low or a high rate, respectively. Therefore, the synthesis of this enzyme in P. guilliermondii is not regulated with iron.

[Respiratory system of Pichia guilliermondii yeasts with different levels of flavinogenesis]. / Dykhatel'naia sistema drozhzhei Pichia guilliermondii pri razlichnykh urovniakh flavinogeneza.

The yeast Pichia guilliermondii was grown on media with different content of iron and its respiration system was studied. When the yeast was cultivated on a complete medium, its respiratory chain operated at the maximum rate in the exponential growth phase, i. e. all the three points of phosphorylation were involved; cytochrome oxidase was the only terminal oxidase. When the growth was decelerated and at the stationary phase, the alternative autooxidable cyanide-resistant pathway inhibited with salicyl hydroxamate partly functioned. Iron deficiency in the medium resulted in a two-three-fold decrease in the content of total and non-hemin iron in the cells, changes in the character and rate of growth, a decrease in the biomass yield, a high rate of flavinogenesis, a slight decrease in the respiration activity, though no drastic changes in the respiration system occurred. This system is represented, as in the case of cells grown on a complete medium, by a typical cytochrome system and an alternative autooxidable pathway. The absence of principal differences in the respiration systems of normal and iron-deficient cells, as well as the operation of the first point of coupling in flavinogenic cells, makes it doubtful that Fenh-proteins of the first segment of the respiratory chain are involved in the regulation of flavinogenesis.

[The activity of enzymes involved in synthesis and hydrolysis of flavin adenine dinucleotide is Pichia guilliermondii studies at different levels of flavinogenesis]. / Issledovanie aktivnosti fermentov, uchastvuiushchikh v sinteze i gidrolize flavinadenin-dinkleotida u drozhzhei Pichia guilliermondii pri razlichnykh urovniakh flavinogeneza.

The activity of FAD-pyrophosphorylase and FAD-hydrolase (nucleotidepyrophosphatase) was studied in extracts of Pichia guilliermondii ATCC 9058 capable of riboflavin over-production. The specific activity of the enzymes was highest at the logarithmic growth phase (2.6 and 3.8 mcmoles of FAD per 1 min per 1 mg of protein X10(-5), respectively), and did not increase upon the induction of riboflavin overproduction. A decrease in the content of hemin compounds and a low content of flavins in the cells of Pichia guilliermondii mutants had no considerable effect on the activity of the two enzymes. When the yeast was cultivated on a medium containing hexadecane, an increase in the content of FAD in the cells was not accompanied with a rise in the activity of FAD-pyrophosphorylase. The activity of the enzyme did not change when succinate and lactate, the substrates of FAD-containing enzymes, were used as the source of carbon. The activity of FAD-pyrophosphorylase increased only when iron-deficient cells of the yeast were grown or incubated on a medium containing glycine; this stimulation was inhibited by cycloheximide.

[A flavinogenic mutant of the yeast Pichia guilliermondii with impaired iron transport]. / Flavinogennyi mutant drozhzhei Pichia guilliermondii s povrezhdennym transportom zheleza

A mutant of the yeast Pichia guilliermondii was produced by means of UV; the mutant was capable of riboflavin overproduction in the presence of high concentrations of iron in the medium. The content of total and non-hemin iron and cytochrome c, and the activity of catalase, were lower in the cells of the mutant than in the parent cells, while the activity of riboflavin synthetase was higher. The content of iron in the cells increased when the mutant was cultivated on media with citric acid, siderochromes of Klebsiella aerogenes, Neurospora crassa, Rhodotorula glutinis, cultural broth of Pichia ohmeri, and autolysate of brewer's yeast, whereas the flavinogenous activity of the cells decreased. Rotenone inhibited respiration of the intact cells of the mutant producing elevated amounts of riboflavin; therefore, flavinogenesis was not regulated by non-hemin iron on the first segment of the respiratory chain. Overproduction of riboflavin in the mutant of Pichia guilliermondii was proved to be a recessive property.