[Functional characteristic of the CefT transporter of the MFS family involved in the transportation of beta-lactam antibiotics in Acremonium chrysogenum and Saccharomyces cerevisiae].

Vectors for the expression of the CefT transporter of the MFS family in Acremonium chrysogenum--a producer of beta-lactam antibiotic cephalosporin C--and in Saccharomyces cerevisiae as a fusion with the cyan fluorescent protein (CFP) have been created. The subcellular localization of the CefT-CFP hybrid protein in yeast cells has been investigated. It was shown that the CefT-CFP hybrid protein is capable of complementation of the qdr3, tpo 1, and tpo3 genes encoding for orthologous MFS transporters of Saccharomycetes, making the corresponding strains resistant to spermidine, ethidium bromide, and hygromycin B. High-yield strain VKM F-4081D of A. chrysogenum, expressing the cefT-cfp fusion, was obtained by an agrobacteria conjugated transfer. It was also shown that the constitutive expression of cefT in A. chrysogenum VKM F-4081D led to a change in the biosynthetic profiles of cephalosporin C and its precursors. This resulted in a 25-35% decrease in the finite product accumulated in the cultural liquid with a simultaneous increase in the concentration of its intermediators.

[Chromosomal polymorphism of Acremonium chrysogenum strains producing cephalosporin C].

Using pulse electrophoresis in controlled homogenous electric field we conducted molecular karyotyping of highly-productive and laboratory strains of Acremonium chrysogenum generating antibiotic cephalosporin C (cefC). Differences in size of several chromosomes of highly active strain CB26/8 compared to the wild-type strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the highly active strain was not associated with alteration of localization and copy number ofcephalosporin C biosynthesis and transport genes. A cluster of "early" cefC biosynthesis genes is located on chromosome VI (4.4 Mb); a cluster of the "late genes", on chromosome II (2.3 Mb). Both clusters are presented as a single copy perA. chrysogenum genome in the wild-type and in CB26/8 producer strains. Based on comparative analysis of laboratory and industrial cefC producers, a karyotype scheme for A. chrysogenum strains of various origins was designed.

[The concentration dynamics of inorganic polyphosphates during the cephalosporin C synthesis by Acremonium chrysogenum].

The contents of five fractions of energy-rich inorganic polyphosphates (polyPs), ATP, and H(+)-ATPase activity in the plasma membrane were determined in a low-activity cephalosporin C (cephC) producer Acremonium chrysogenum ATCC 11550 and selected highly efficient producer strain 26/8 grown on glucose or a synthetic medium providing for active synthesis of this antibiotic. It was shown that strain 26/8 on the synthetic medium produced 26-fold higher amount of cephC as compared with strain ATCC 11550. This was accompanied by a drastic decrease in the cell contents of ATP and the high-molecular-weight fractions polyP2, polyP3, and polyPS with a concurrent increase in the low-molecular-weight fraction polyP1. These data suggest that polyPs are involved in the cephC synthesis as a source of energy. H(+)-ATPase activity insignificantly changed at both low and high levels of cephC production. This confirms the assumption that A. chrysogenum has other alternative antibiotic transporters in addition to cefT. The obtained results can be used for optimizing commercial-scale cephC biosynthesis.

[Structure peculiarities of cell walls of Acremonium chrysogenum--an autotroph of cephalosporin C].

Alterations of cell walls of Acremonium chrysogenum occurring at intensive synthesis of cephalosporin C has been studied. It is shown, using electron microscopy, that the cell wall of the cells ofATCC 11550 strain ("wild" type) became looser and thicker during growth. The cell wall of the cells of strain 26/8 (hyperautotroph of cephalosporin C) considerably degraded by the end of the stationary phase. Biochemical analysis has shown that these alterations entailed decrease of the proteins' content covalently or noncovalently linked with the polysaccharides of cell walls of both strains. An increase of sensitivity of cell walls of the strain-superproducer to an activity of lytic enzymes of chitinase, laminarinase, proteinase K, and lyticase preparation has been observed during the growth, but this increase has not been found in the case of "wild" type strain. The obtained results evidence to the structure failure of the cell wall of A. chrysogenum entailing the intensive creation of antibiotic.

[Genetic transformation of the mycelium fungi Acremonium chrysogenum].

The system of transformation of heterologous genes under the method of agrobacterial transfer into Acremonium chrysogenum ATCC 11550 wild-type strains, natural producents of beta-lactam antibiotic cephalosporin C, and strains highly producing cephalosporin C 26/8 revealed by the multistage selection on its basis were developed. Vectors for agrobacterial transformation of A. chrysogenum containing expression cassettes of genes encoding resistance to geneticin (G418) and bleomicin (Zeocin) antibiotics under control of Ashbya gossypii and Saccharomyces cerevisiae TEF1 promoters were constructed. A comparable assessment of agrotransformation methods while co-cultivating fungi and agrobacterial cells on filters and in deep culture was conducted. Transformants, selected by resistance to geneticin and bleomicin, were characterized by PCR and Southern blot analyses.

[A study of steroid hydroxylation activity of Curvularia lunata mycelium].

It has been demonstrated that the mycelium of Curvularia lunata at the end of the logarithmic growth phase displays a maximal 11-hydroxylase activity towards cortexolone (4-6 g/l) used for transformation as a microcrystalline suspension in phosphate buffer. The mycelium at a later stage of fungal growth displays an elevated 14-hydroxylase activity, necessary for generation of 14-hydroxyandrostenedione. The effects of different forms of substrate added to the reaction mixture, age and concentration of mycelium, and fungal clones tolerant to salts of heavy metals (0.35-0.5%) were studied to remove the side 14-hydroxylation, accompanying the main cortexolone transformation. Mycelia of the fungal clones tolerant to Co2+ and Cu2+ displayed a weak hydroxylase activity or its complete absence and an elevated content of melanin, the biosynthesis of which is intensified under adverse conditions. The results obtained suggest that the transformation of steroids by the studied C. lunata strain is a detoxication of foreign compounds.

[Induced variability in Acremonium chrysogenum, a producer of neutral-alkaline proteases and peptidases]. / Indutsirovannaia izmenchivost' Acremonium chrysogenum produtsenta neitral'no-shchelochnykh proteaz i peptidaz.

Lethal and mutagenic effects of N-nitrosomethyl biuret on the organism producing a complex of proteolytic enzymes i. e. Acremonium chrysogenum were studied. It was shown that methionine inhibited production of the protease complex in the induced prototrophic mutants. The selected mutants were classified according to the level of enzyme biosynthesis induction by methionine.

[Ratio of the mutation spectrum to the physiological state of the Penicillium chrysogenum cell. Auxotrophic mutations induced by UV rays at different stages of DNA replication]. / Zavisimost' spektra mutatsii of fiziologicheskogo sostoianiia kletki Penicillium chrysogenum. Auksotrofnye mutatsii, indutsirovannye UF-luchami v ravlichnye stadii replikatsii DNK.

The spectrum of the mutations obtained with exposure of nonactivated spores of Pen. chrysogenum to N-nitrozo-N-methylbiuret (NMB) was studied and it was shown that the mutations associated with blocking of amino acid synthesis predominated. When activated spores of Pen. chrysogenum were treated with the mutagen, correlation was observed between the spectrum of the induced mutations, the physiological state of the cells and the exposure time. It is supposed that the mutagen molecule affects the replicating part of the DNA molecule. Schemes for predominating liberation of the mutations according to the markers in time were developed. This may give an idea of the order of the markers in genome.

[Penicillin formation by biochemical mutants of Penicillium chrysogenum and their spontaneous revertants]. / Obazovanie penitsillina biokhimicheskimi mutantami Penicillium chrysogenum i ikh spontannymi revertantami.

The capacity for the antibiotic production in the auxotrophs of Penicillium chrysogenum with various deficiency and their revertants was studied. It was found that the capacity for penicillin synthesis was impaired to various degrees in the majority of the auxotrophs. Variants with the penicillin production levels by 13--20 per cent higher than those in the initial prototrophic strain were isolated for the first time in selection of the eukaryotes with the method of obtaining highly active revertants from auxotrophs according to the scheme "prototroph-auxotroph-prototroph".

[Ratio of the mutation spectrum to the physiological state of the Penicillium chrysogenum cell. Auxotrophic mutations induced by N-nitroso-N-methylbiuret in exposure at different stages of DNA replication]. / Zavisimost' spektra mutatsii ot fiziologicheskogo sostoianiia kletki Penicillium chrysogenum. Auksotrofnye mutatsii, indutsirovannye N-nitrozo-N-metilbiuretom pri vozdeistvii na raznye stadii replikatsii DNK.

The effect of UV light on activated conidia of Penicillium chrysogenum, strain 39 was studied. It was found that the spectrum of the auxotrophic mutations induced by UV light during replication of DNA changed with the dose of the mutagen and was specific to every dose. The schemes of predominating mutation induction during DNA replication under the effect of 2 doses of UV light were developed.

[Lethal and mutagenic action of nitrosomethylbiuret on a culture of the mycophilic fungus, Didymocladium ternatum]. / Letal'noe i mutagennoe deistvie nitrozometilbiureta na kul'turu mikofil'nogo griba Didymocladium ternatum

The effect of N-nitrozo-N-methylbiuret (NMB) on various features of Didymocladium ternatum, an organism producing termatin was studied. It was found that the lethal effect of NMB depended on the exposure time, prolongation of which resulted in increased numbers of the plus variants, morphological mutations and reverses into the populations. Variants possessing 8 times higher activity as compared to the initial culture were obtained as a result of selection of active variants.

[Mutagenic action of N-nitrosodimethylurea on Actinomyces rimosus and Penicillium chrysogenum]. / Mutagennoe deistvie N-nitrozodimetilmocheviny na Actinomyces rimosus i Penicillium chrysogenum

High mutagenic activity of N-nitrozodimethylurea (NDMU), an agent of the group of the nitrozo compounds not studied in detail was shown with respect to prototrophic and auxotrophic strains of Actinomyces rimosus, an organism producing oxytetracycline and Penicillium chrysogenum, an organism producing penicillin. The rate of direct and back mutations in the auxotrophic strain of Act. rimosus under the effect of NDMU was many times higher than that of spontaneous mutations. NDMU was used at one of the selection stages at which a more active variant of Act. rimosus was obtained. This is evident of a possible use of the mutagen for induction of variation with respect to the quantitative feature of oxytetracycline production. A great number of morphologically changed forms and biochemical mutants of Pen. chrysogenum formed under the effect of this substance. NDMU induced a mutant of Pen. chrysogenum capable of selective synthesis of 6-aminopenicillinic acid without addition of the precursor.