[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.

[Riboflavin transport in mutant of Pichia guilliermondii yeast with damaged glucose uptake]. / Transport riboflavina u mutanta drozhzhei Pichia guilliermondii s narushennym pogloshcheniem glukozy.

The mutant of P. guilliermondii yeast MS-50 which had lost the ability to grow in a glucose-containing medium but not in the presence of sucrose or maltose was selected. The mutant has a damaged glucose uptake from the medium. The riboflavin permease activity of the MS1-50 mutant is not impaired; glucose inhibits the riboflavin uptake by this strain. Thus, riboflavin permease does not promote glucose transport into yeast cells.

[Overproduction of riboflavin in mutants of Pichia guilliermondii yeasts resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine]. / Sverkhsintez riboflavina u mutantov drozhzhei Pichia gluilliermondii, rezistentnykh k 7-metil-8-triftometil-10-(1'-D-ribitil)izoalloksazinu.

Mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine were selected from the yeast Pichia guilliermondii, strain MS14-A10, possessing a multiple sensitivity to antibiotics and antimetabolites. A lot of such mutants displayed an elevated flavinogenic activity. The investigation of the properties of three mutants with the highest flavinogenic activity, viz. RZ4, RZ7 and RZ11, has shown that their capability for riboflavin overproduction does not stem from disordered regulation of the de novo purine biosynthesis, from the damaged transport of iron ions into the cell, or from changes in the allosteric properties of GTP cyclohydrolase. A twofold increase in the specific activities of GTP cyclohydrolase and riboflavin synthase was observed in the two mutants, RZ4 and RZ11. These data suggest that the mechanism for repression of the synthesis of flavinogenic enzymes, in which iron ions are involved, is impaired in these mutants. The reason for riboflavin overproduction in the mutant RZ7 remains obscure.

[Coordinate regulation of riboflavin permease and alpha-glucosidase synthesis in the yeast Pichia guilliermondii]. / Koordinirovannaia reguliatsiia sinteza piboflavinpermeazy i alf'a-gliukozidazy u drozhzhei Pichia guilliermondii.

Riboflavin permease of the yeast Pichia guilliermondii appear to be inducible transport system. Its synthesis is induced by sucrose, maltose, alpha-methyl-D-glocoside, melizitose and raffinose, but not by D-glucose, trehalose or cellobiose. The synthesis of riboflavin permease in the presence of sucrose of maltose is depressed by cycloheximide, actinomycin D and 8-hydroxyquinoline. These results suggest that the synthesis of riboflavin permease is regulated on the transcription level. The inducers of riboflavin permease are also able to induce the synthesis of alpha-glucosidase. The mutants have been selected in which the synthesis of riboflavin permease occurs constitutively; the synthesis of alpha-glucosidase in the mutants is also constitutive. Growing of the yeast in a medium with high content of glucose results in a parallel decrease of the riboflavin permease and alpha -- glucosidase activities. These data are indicative of corrdinate regulation of riboflavin permease and alpha -- glucosidase in P. guilliermondii. Suboptimal or excessive content of vitamin B2 in the medium does not affect the level of riboflavin permease in this yeast species.

[Effect of glucose and its derivatives on systems of riboflavin uptake and excretion in the yeast Pichia guilliermondii]. / Vliianie gliukozy i ee proizvodnykh na sistemy pogloshcheniia i ekskretsii riboflavina udrozhzhei 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.

[Active transport of riboflavin in the yeast Pichia guilliermondii. Detection and some properties of the cryptic riboflavin permease]. / Aktivnyi transport riboflavina u drozhzhei Pichia guilliermondii. Obnaruzhenie i nekotorye svoistva kripticheskoi riboflavinpermeazy.

In contrast to cells and protoplasts of the wild strain intact cells and protoplasts of riboflavin (RF)-deficient mutants of Pichia guilliermondii yeast possessing multiple sensitivity to antibiotics and antimetabolites were found capable to accomplish active transport of RF. The accumulation of RF against concentration gradient was energized by endogenous energy sources and was strongly depressed by uncouplers of oxidative phosphorylation and by inhibitors of respiration. RF transport was also blocked by the agents which destroy the permeability barrier and by sucrose. Cyclohemide did not inhibit the transport of vitamin B2. Uptake of RF depends on temperature and pH of the incubation mixture. RF permease possessed rather stringent substrate specificity and did not catalyze transport of FMN and FAD. RF uptake in two strains tested, MS1-3 and MS1, followed saturation kinetics (Km = 0.17 mM and 2.0 mM, respectively) and was not connected with RF auxotrophy. The ability for exogenous RF uptake was controlled by recessive allele. Restoration of normal resistance to antibiotics and antimetabolites was accompanied by a decrease in the permease affinity for substrate.

[Riboflavin transport in cells of riboflavin-dependent yeast mutants]. / O transporte riboflavina v kletki riboflavinzevisimykh mutantov drozhzhei.

Riboflavin was transported at a high rate into yeast cells of Pichia guilliermondii and Schwanniomyces occidentalis mutants capable of growth in a medium containing low concentrations of riboflavin, and having multiple susceptibility to some antibiotics and antimetabolites. Sucrose and sodium azide inhibited transport of riboflavin. Other riboflavin dependent mutants of Pichia guilliermondii, Pichia ohmeri, Torulopsis candida, and Saccharomyces cerevisiae, also growing in media containing low concentrations of riboflavin, were not capable of its active transport.

[Variability in the flavinogenic activity of Pichia guilliermondi yeasts]. / Izmenchivnost'flavinogennoi aktivnosti drozhzhei Pichia guilliermondi

The natural and induced variability of the flavinogenic activity was studied in the strain of Pichia guilliermondii ATCC 9058. The flavinogenic activity of the collection strain showed normal distribution; the amount of riboflavin(RF) accumulated in the medium differed several times in the extreme variants. In the clones with the maximum and minimum accumulation of RF, the distribution of the variants was asymmetric, due to the appearance of the cells with an average flavinogenic activity. The clones have acquired almost the same flavinogenic activity after being transferred eight times on a fresh medium. The asymmetric distribution of the variants according to their flavinogenic activity was found also in the case of the clones obtained from the UV-irradiated cells. The mutants have been isolated, which synthesized 3-30 times more RF than the parent strain in the presence of iron doses optimal for the growth. Five mutants that were most active in producing RF differed in the sensitivity of their flavinogenesis to high concentrations of iron, yeast autolysate, and carbon sources.

[Nature of riboflavin precursors in Pichia guilliermondi yeasts]. / Priroda predshestvennikov riboflavina u drozhzhei Pichia guilliermondii

Thirty-nine riboflavin-deficient mutants have been isolated from three yeast strains of Pichia guilliermondii (ATSS 9058, VKM Y-1256, VKM Y-1257) and F5-121 mutant which is capable of production of large amounts of riboflavin in the presence of iron in the medium. All mutants were divided into five groups according to the nature of precursors accumulated in the medium and growth reaction in media with 6,7-dimethyl-8-ribityllumasine and diacetyl. The mutants of the first group did not accumulate specific precursors of riboflavin either in the cells or in the medium. The mutants of the second, third and fourth groups accumulated, after the incubation with diacetyl, 2-amino-4-hydroxy-6,7-dimethylpteridine, 2-amino-4-hydroxy-6,7-dimethyl-8-ribitylpteridine and 6,7-dimethyl-8-ribityllumasine; therefore, they synthesized the following precursors of riboflavin: 2,4,5-triamino-6-hydroxy-pyrimidine, 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine. The mutants of the fifth group accumulated 6,7-dimethyl-8-ribityllumasine in the medium and lacked riboflavin synthetase activity, as was confirmed by enzymatic studies.