[Characteristics of obtaining protoplasts from 2 strains of Tolypocladium inflatum subsp. Blastosporum]. / Osobennosti obrazovaniia protoplastov u dvukh shtammov Tolypocladium inflatum subsp. Blastosporum.

The optimal conditions for preparing protoplasts with high yields by using the cells of two (low and high potent) isogenic cyclosporine-producing Tolypocladium strains were developed. A specific medium containing 0.5 per cent yeast autolysate (by dry weight) and 3 per cent glucose was used. When grown on this medium the cells of the highly potent strain 847 acquired a yeast-like shape. High yields of protoplasts prepared from the low potent strain 43 mycelium were obtained via prior incubation with 0.01 M dithiothreitol followed by treatment with a complex of enzymes from Helix pomatia for 1.5 to 2 hours was used. For preparation of the protoplasts with employing the highly potent strain 847 cells the prior incubation with dithiothreitol was not required, but it was necessary to employ a mixture of the enzyme complex (Helix pomatia), drizilase (Irpex lacteus) and chitinase (Streptomyces griseus) for 18 hours. The electron microscopic data on the two isogenic strains and their protoplasts are presented. The protoplasts proved to be a suitable initial material for investigating bioenergetic processes at the subcellular level and further genetic improvement of the strains.

[Activity of glycolysis enzymes in cyclosporine-producing Tolypocladium sp]. / Aktivnost' fermentov glikoliza u Tolycocladium sp.-produtsenta tsiklosporina.

Enzymes of various glycolysis stages, i.e. hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase, were detected in cyclosporine-producing organisms belonging to Tolypocladium. The initial activity of the enzymes in the highly active strain was much higher than that in the starting low active strain. During the fermentation the activity of the glycolysis enzymes per 1 mg of mycelium protein in the both strains increased. This was accompanied by a decrease in respiration activity. Therefore, there was a direct correlation between cyclosporine biosynthesis and glycolytic activity of the mycelium in Tolypocladium sp.

[High-molecular polyphosphates and pyrophosphate in cyclosporin- producing Tolypocladium sp. and their role in the processes of growth and antibiotic synthesis]. / Vysokomolekuliarnye polifosfaty i pirofosfat u produtsenta tsiklo- sporina Tolypocladium sp. i ikh rol' v protsessakh rosta i antibiotikoobrazovaniia.

The contents of high-energy phosphorous compounds, i.e. three fractions of polyphosphates, pyrophosphate, and ATP were determined in isogenic strains of Tolypocladium sp. differing in cyclosporine production levels. The content of polyphosphates was 1 to 2 orders of magnitude greater than that of pyrophosphate or ATP and did not depend on the strain productivity. During the period of the mycelial intensive growth and at the beginning of antibiotic synthesis, the level of polyphosphates lowered 2-3-fold and the content of pyrophosphate markedly decreased as well. The activities of polyphosphatase and pyrophosphatase during the culture growth and cyclosporine biosynthesis was higher in the highly productive strain.

[Cyclosporin biosynthesis and dynamics of bioenergetic processes in cyclosporin-producing strain]. / Biosintez tsiklosporina i dinamika bioénergeticheskikh protsessov u produtsenta tsiklosporina.

An attempt was made to show a correlation between definite bioenergetic parameters of the cells of the cyclosporine-producing culture and biosynthesis of cyclosporine. It was found that the three strains producing cyclosporine used in the study had an alternative cyanide-resistant pathway along with the classical cytochrome chain. In the strain forming only traces of the cyclopeptide during fermentation of the cyanide-resistant respiration constituted 60 to 80%. In the isogenic highly productive strains the cyanide-resistant respiration appeared to be markedly decreased beginning from the 1st day of fermentation and during the maximum biosynthesis of cyclosporine (on day 4 or 5 of fermentation) it reached zero. The ATP content in the cells of the highly productive strain, despite its decrease by the antibiotic biosynthesis peak, remained at a much higher level than that in the strain producing only traces of cyclosporine. A procedure for isolating functionally active mitochondria from the protoplasts was developed and a bioenergetic characterization of the mitochondria isolated from the strains with different antibiotic productions is presented.

[Antibiotic production by variants of Penicillium chrysogenum isolated after protoplast formation and exposure to mutagens at the protoplast stage]. / Antibiotikoobrazovanie u variantov Penicillium chrysogenum, poluchennykh posle protoplastirovaniia i obrabotki mutagenami na stadii protoplastov.

A procedure for protoplast formation in the penicillin-producing organism Penicillium chrysogenum was developed. The yield of the protoplasts was high, the protoplasts were stable and capable of regeneration. Two types of the protoplast regeneration were revealed. The spores and protoplasts were treated with UV light and N-nitroso-N'-methyl biuret and their effect on production of the antibiotic by the isolated variants was studied. It was shown that the protoplasts of P. chrysogenum were more liable to the mutagenic effect of UV light and nitroso methyl biuret than the fungus conidia. It is possible to use this specific feature in intensification of selection aimed at isolation of highly productive strains of P. chrysogenum.