[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.

[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.

[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.

[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 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.

[Genotoxic and mutagenic activity of antineoplastic anthracyclines and their aglycones: study in two test-systems].

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.

[Effect of surface-active substances on the dynamics of intracellular pH in 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].

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 development of a rapid semiautomatic enzymatic analyzer of beta-lactam antibiotics].

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.

[Physiological characteristics of the strains of Cryptococcus deffluens--producers of penicillin acylase].

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.

[Selection and physiological study of culture of Bacillus circulans-- producer of butirosin].

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.

[Study of possible biological transformation of kanamycin to amikacin].

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.

[Use of analogs of primary metabolites in the selection of the producer of polymyxin 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.

[Isolation of protoplasts of Xanthomonas rubrilineans and their use in the study of localization of aminopeptidases].

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.

[Isolation of nisin from native solution and its purification on cationites].

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.

[Effect of penicillin precursors on antibiotic biosynthesis in various strains].

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.

[Effect of low molecular weight regulators on the biosynthesis of rifamycin B by Amycolatopsis mediterranei strains].

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.

[Elaboration of a semiautomated enzymatic analyzer of glucose for the control of biosynthesis of antibiotics].

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.

[Phosphate regulation of the processes of growth and biosynthesis of gentamicin in Micromonospora purpurea var. violaceae].

The influence of orthophosphate on isogenic strains of the gentamicin-producing organism with various levels of the antibiotic production was studied. An increase in the content of inorganic phosphate in the medium led to inhibition of the growth and gentamicin biosynthesis in all the strains: the low-active strains were more stable to the effect of the phosphate as compared to the highly active strains. Heterogeneity of the population of the strains was shown in respect of decreasing the growth rate and the level of the antibiotic biosynthesis inhibition in the presence of orthophosphate.

[Ways of developing the first and second generation biocatalysts for antibiotic production].

Methods for development of bioengineering systems of different types useful in synthesis and transformation of antibiotics are discussed. It was shown that in development of monoenzymatic biocatalysts on the basis of immobilized cells and in preparation of immobilized cultures producing secondary metabolites with enzymological engineering directed action on the cells could be provided which made it possible to establish highly efficient bioengineering systems. Various means for providing the directed action and method for estimation of the carrier-culture interation are proposed.

[Morpho-functional analysis of isogenic strains of Escherichia coli-- producers of penicillinacylases].

A population of Escherichia coli strains producing penicillinacylase (PA) and differing in the level of their enzyme activity was studied. Their structural-functional analysis showed that selection according to the property of PA production yielded strains with aberrations in the processes of cell division. The population of microbial cells producing PA had a correlation between its morphological composition and enzyme activity, and the two characteristics depended on the conditions under which the strains were cultivated.

[Lipids of initial and mutant cultures of Penicillium chrysogenum, producers of penicillin].

Comparative investigation of lipogenesis in 2 initial and 4 mutant strains of Penicillium chrysogenum showed that there were no noticeable differences in the composition of the lipid fatty acids in these strains. Certain shifts in the ratio of definite lipid fatty acids in the mutant strain deficient by synthesis of lysine and isoleucine (increased contents of oleic acid) were revealed. A marked influence of the physiological conditions on lipogenesis in the mutant with multiple deficiency by amino acids, vitamins and nucleotides was observed.