[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.

[Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81]. / Geneticheskii kontrol' biosinteza riboflavina u drozhzhei Pichia guilliermondii. Obnaruzhenie novogo reguliatornogo gena RIN81.

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 riboflavin analogs on Pichia guilliermondii growth]. / Vliianie analogov riboflavina na rost Pichia guilliermondii.

The work was aimed at studying the biological activity of 32 structural riboflavin (RF) analogs substituted at positions 7, 8 and 10 as well as with a modified structure of the isoalloxazine cycle and the side D-ribityl chain. An RF-dependent mutant of the yeast Pichia guilliermondii MS1 was used as a test organism. The strain could grow in a medium without the vitamin in the presence of 2-thio-RF and analogs with esterified hydroxyls in the side chain, viz. tetraacetate and tetrabutyrate of RF. The antagonistic properties were distinctly displayed only by D-ribityl derivatives of vitamin B2 with a substitution of CF3, Cl, H, NH2 and N(CH3)2 for one or the both methyl groups. The inhibition indices for such analogs varied from 0.52 to 10.8. The action of the antivitamins on the yeast growth was competitively eliminated by adding RF to the growth medium. The antivitamin activity of an analog abruptly decreased or disappeared if (i) a bulky substituent such as N-piperidyl or hydroxyethylamine was incorporated at position 8 of an antimetabolite molecule, (ii) the D-ribityl side chain was substituted by a D-galactyl, D-sorbityl, L-ramnityl, 2-hydroxyethyl or methyl group and (iii) the structure of isoalloxazine cycle was modified. The analogs which were phosphorylated by RF kinase (ATP: RF-5'-phosphotransferase, EC 2.7.1.26) from P. guilliermondii were shown to be effective antivitamins. Therefore, the antagonistic effect of vitamin B2 analogs in yeast cells is realized at the level of coenzyme forms.

[Substrate and inhibitory specificity of riboflavin kinase from Pichia guilliermondii yeast]. / Issledovanie substratnoi i ingibitornoi spetsifichnosti riboflavinkinazy iz drozhzhei Pichia guilliermondii.

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.