[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.

[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.

[Genotoxic and mutagenic activity of antineoplastic anthracyclines and their aglycones: study in two test-systems]. / Genotoksicheskaia i mutagennaia aktivnost' protivoopukholevykh antratsiklinov i ikh aglikonov: izuchenie v test-sistemakh.

Mutagenic (Ames tests) and genotoxic (SOS chromotest) activities of highly-efficient natural anthracycline monosaccharides possessing antitumor activity-daunorubicin (also known as daunomycin or rubomycin), doxorubicin (adriamycin), and carminomycin-were studied. At the same time, the hypothesis was tested that intercalation of the antibiotic moiety into the helix of cell DNA, which was mediated by the saccharide amino group, played a crucial role in genotoxicity of these anthracyclines. The hydrolysis products of these antibiotics (the corresponding aglycones) and aclacynomycin A (an anthracycline trisaccharide), as well as aclavinone (its derivative aglycone), were studied. All these compounds lacked the saccharide amino group necessary for intercalation. It was found that all anthracycline monosaccharides studied had a strong mutagenic effect on strain TA98 and a moderate effect on strain TA100 of Salmonella typhimurium. Aclacynomycin A was found to have no mutagenic effect on any strain. Lack of the glycoside amino group did not necessarily result in loss of mutagenic activity in the derivative aglycones of anthracycline monosaccharides: they exhibited moderate mutagenic activity in strain TA98 and low but significant activity in strain TA100. The S9 microsomal fraction did not alter the mutagenic activity of either anthracycline monosaccharides or their aglycones; however, it dramatically increased the mutagenic activity of aclavinone: correspondence between positive responses in Ames tests and the SOS chromotest was found. Apparently, the mutagenic activity of the substances studied in bacterial cells was mediated by inducing the SOS-repair process. If the compound contained the amino glycoside moiety, functional and structural precursors of the SOS response were formed via intercalation of the reagents into the DNA duplex; if the substance did not contain this moiety, the precursors were formed via ionic interaction.

[Effect of surface-active substances on the dynamics of intracellular pH in Fusidium coccineum]. / Vliianie poverkhnostno-aktivnykh veshchestv na dinamiku vnutrikletochnogo pH u Fusidium coccineum.

Correlation between the dynamics of intracellular pH and antibiotic synthesis in Fusidium coccineum strains with different biosynthetic capacity was studied. At the beginning of the intensive antibiotic synthesis the intracellular pH in the low-active and highly-active strains was minimum and maximum respectively. In the highly active strain an increase in the intracellular pH of the cytoplasm after the addition of Tween-80 and Factor-d2 analogs to the growth medium was observed.

[Role of biocatalysis in the creation and improvement of production of beta-lactam antibiotics in Russia]. / Rol' biokataliza v sozdanii i sovershenstvovanii proizvodstva betalaktamnykh antibiotikov v Rossii.

The paper presents the results of the studies carried out by the authors within 20 years on development of processes for production of beta-lactam antibiotics with using biocatalysis. The proposed general principles for the development of efficient biocatalytic technologies are discussed in regard to production of the key compounds and synthesis of beta-lactams. The paper includes 4 parts concerned with comparison of the biocatalytic and chemical processes for production of beta-lactam antibiotics, requirements to the quality of the biocatalysts used and criteria for estimation of the efficiency of the stage of the technological biocatalyst production. The criteria provided determination of the optimal biocatalyst for production of the key compounds in the synthesis of beta-lactams. A retrospective analysis of the biocatalytic processes for production of 6-amino-penicillanic acid is presented and the impact of the activity and the form of the biocatalyst on the process efficiency is substantiated. Various schemes of the enzymatic synthesis of beta-lactam antibiotics and the approaches to the improvement of the technological processes including those with the use of immobilized microbial cultures at the stage of the production of the initial biosynthetic antibiotics are described. The prospects for the improvement of the processes for production of drugs with the use of biocatalysis are indicated and the main trends of the research required for the large scale use of the immobilized enzymes, microbial cells, oligo-enzymatic and poly-enzymatic systems in transformation and synthesis of organic compounds are defined.

[The development of a rapid semiautomatic enzymatic analyzer of beta-lactam antibiotics]. / Razrabotka fermentativnogo poluavtomaticheskogo ékspress-analizatora betalaktamnykh antibiotikov.

A new method and a device for enzymatic express assay of beta-lactam antibiotics are described. The principle of the device operation is based on the use of native enzymes specific to the antibiotics assayed. The method makes it possible to exclude the influence of the buffer capacity of the samples on the results of the assay. The data on the testing of the method and device with model solutions of benzylpenicillin are presented. The relative error of a single measurement does not exceed 2 per cent. The time of an assay is not more than 3 minutes. Recommendations on the use of the device in research studies and manufacture are presented.

[Study of possible biological transformation of kanamycin to amikacin]. / Issledovanie vozmozhnosti biologicheskoi transformatsii kanamitsina v amikatsin.

For transformation of kanamycin A (Km) to amikacin (Ak) with acylating enzymes from B. circulans, a culture producing butirosin (Btn), cellular and acellular conversion systems and methods for chemical and biological identification of Km, Ak and Btn were developed. The level of conversion of Km to Ak in vivo and in vitro did not exceed 2 per cent.

[Selection and physiological study of culture of Bacillus circulans-- producer of butirosin]. / Selektsionnoe i fiziologicheskoe issledovanie kul'tury Bacillus circulans--produtsenta butirozina.

For isolating a highly active variant of the butirosin-producing culture, a strain forming trace amounts of the antibiotic substance was used. Exposure to nitrosomethylbiuret and nitrosoguanidine and the use of selective media containing streptomycin and butirosin resulted in a 30-fold increase in the strain productivity. Thin layer chromatography of the produced antibiotic substance in the solvent system developed by the authors, mass spectrometry and assay of the antimicrobial spectrum in regard to ++gram-positive and ++gram-negative bacteria by using the known aminoglycosidine-inactivating enzymes revealed that the substance was identical to butirosin. Along with the major product, the fermentation broth contained up to 5 per cent of ribostamycin.

[Physiological characteristics of the strains of Cryptococcus deffluens--producers of penicillin acylase]. / Izuchenie fiziologicheskikh osobennostei shtammov Cryptococcus diffluens--produtsentov penitsillin-V-atsilaz.

A fermentation medium balanced by the main components was developed for Cryptococcus diffluens strains producing penicillin-V-acylases (PA). It was shown that the culture needed for production of the enzyme was inductor, which was phenoxyacetic acid (POAA). Additional introduction of ethanol to the medium provided an increase in production of PA by 36 per cent and the culture growth by 25 per cent. Introduction of one of the following substances to the medium with POAA and ethanol i.e. (CN3COO)2Ca, FeSO4, proline or asparagine provided an additional increase in the production level of PA by 24 to 94 per cent. The use of the medium varieties will permit one to isolate highly productive cells of the culture.

[Isolation of protoplasts of Xanthomonas rubrilineans and their use in the study of localization of aminopeptidases]. / Poluchenie protoplastov Xanthomonas rubrilineans i ikh ispol'zovanie dlia izucheniia lokalizatsii aminopeptidaz.

The culture of Xanthomonas rubrilineans was able to synthesize a number of intracellular aminopeptidases. To study localization of the enzymes in the cells, a protoplasting procedure was developed providing the yield of 99.7 per cent. The following subcellular fractions were isolated: periplasmic, cytoplasmic and membranous. It was shown that alanine aminopeptidase was a cytoplasmic enzyme and glutamate peptidase was a membrane-bound enzyme.

[Use of analogs of primary metabolites in the selection of the producer of polymyxin B]. / Ispol'zovanie analogov pervichnykh metabolitov v selektsii produtsenta polimiksina B.

A number of amino acids were found to have effects on the growth of the polymyxin B-producing culture and biosynthesis of the antibiotic by it. Of special importance was the stimulating effect by methionine. Four selection stages were carried out with using structural analogs of purines and amino acids as selective factors. There were no stable variants with increased antibiotic productivity among the mutants resistant to the analogs of purines and leucine. The levels of polymyxin B accumulation by the variants resistant to 4-fluorophenylalanine were 30 to 50 per cent higher than those in the controls and the variants were characterized by low morphological and antibiotic production variation in the subcultures. The mechanisms of the methionine physiological effect and the prospects of using analogs of the primary metabolites in improvement of the culture producing polymyxin B are discussed.

[Isolation of nisin from native solution and its purification on cationites]. / Vydelenie nizina iz nativnogo rastvora i ego ochistka na kationitakh.

It was found possible to use organic sorbents and in particular carboxylic cationites for isolation of nisin from the fermentation broth filtrate and its purification. Nisin is known as a polypeptide antibiotic applied as a preservative. The sorbents were shown to have high exchange capacities by the isolated substance and mechanical strength and resistance. They also proved to be highly stable.

[Effect of penicillin precursors on antibiotic biosynthesis in various strains]. / Vliianie predshestvennikov penitsillinov na biosintez antibiotika u razlichnykh shtammov.

The regularities of biosynthesis of 6-aminopenicillanic acid (6-APA), benzylpenicillin (BP) and phenoxymethylpenicillin (PMP) by the strains under the investigation did not significantly differ. In the absence of the precursor both the strains mainly synthesized 6-APA. Phenylacetic acid (PAA) and phenoxyacetic acid (POAA) provided directed biosynthesis: the fungus synthesized BP or PMP depending on the precursor nature. When the amount of the precursors was not sufficient, 6-APA was synthesized along with the penicillins. PAA proved to be a more active precursor than POAA. When both precursors were present in the fermentation broth, only BR was synthesized. An important distinction of strain 316A was its increased sensitivity to PAA especially in the initial period. After an increase in the PAA concentration the growth rate of strain 316A lowered to a greater extent than that of strain 284A. This was likely to determine the higher levels of penicillin production by strain 316A in the presence of POAA, a nontoxic precursor. A procedure for supplying the precursors was developed. Under the laboratory conditions it provided high levels of the penicillin production.

[Effect of low molecular weight regulators on the biosynthesis of rifamycin B by Amycolatopsis mediterranei strains]. / Vliianie nizkomolekuliarnykh reguliatorov na biosintez rifamitsina B shtammami Amycolatopsis mediterranei.

Formation of differentiation regulators of the A-factor group in representatives of Nocardia and Nocardia-like actinomyces: N. asteroides, N. brasiliensis, Amycolatopsis mediterranei and "Streptomyces listeri" was observed. The effect of the regulators of different nature (barbital, A-factor and B-factor) on biosynthesis of rifamycin B by A. mediterranei strains was studied. It was shown that the A-factor stimulated rifamycin B production in the adifferentiated low active variant isolated from a natural population of the active strain VNIIA 1713 of the rifamycin B-producing culture. B-Factor insignificantly inhibited biosynthesis of rifamycin B in the studied strains of A. mediterranei.

[Elaboration of a semiautomated enzymatic analyzer of glucose for the control of biosynthesis of antibiotics]. / Razrabotkapoluavtomaticheskogo fermentnogo analizatora gliukozy dlia kontrolia protsessov biosinteza antibiotikov.

The results of developing a semiautomatic apparatus with oxygen detection for enzymatic control of glucose concentration are presented. The design of a glucose sensitive electrode is based on an oxygen probe and a membrane with immobilized glucose oxidase. Materials for the probe were chosen and the operating conditions for measuring temperature, pH, linear agitation velocity and other parameters were optimized. The semiautomatic analyzer was constructed and its main characteristics were studied. The results of the apparatus testing during biosynthesis of various antibiotics are presented. It was shown that the required glucose concentration in the cultivation medium was provided for any specific circumstances in relation to the carbohydrate source.