M. Blumauerová

How many genes are required for the synthesis of chlortetracycline

When contemplating the applicability of the theory of polygenic heredity to the development of views on the genetic regulation of the biosynthesis of secondary metabolites one encounters two extreme concepts. The first assumes that with increasing knowledge of the molecular basis of the biosynthesis of these compounds the views of polygenic heredity (formulated at the beginning of this century) cannot fundamentally contribute to the interpretation of new experimental data and to the formulation of a new working hypothesis in accord with the present state of our knowledge. The second concept proceeds from the simple fact that there is no unified theory of genetics at the present time to account satisfactorily for the heredity of complicated quantitative characters, such as the number of eggs laid, milk production, body weight, the production of various pigments etc. Most geneticists studying the improvement of industrial strains producing antibiotics are of the opinion that an increase in the production of these substances represents a very complicated matter and, basically, possesses all the features of polygenic heredity.

Regulation of biosynthesis of secondary metabolites. IX. The biosynthetic activity of blocked mutants of Streptomyces aureofaciens.

The biosynthetic activity of mutants with altered morphological and biochemical characteristics, obtained from two strains ofStreptomyces aureofaciens by means of physical and chemical mutagens, was studied. The majority of mutants formed pigments different from pigments of the parent strains, which did not usually give fluorescence in UV-light and, in addition, differed as to the solubility in organic solvents. The production of further secondary metabolites was investigated chromatographically in the extracts from mycelium and in the fermentation fluid of submerged cultures. According to the results of chromatographical analysis, the obtained mutants can be divided into 12 metabolic types. In most of them the production of substances was found which are different both mutually and from the metabolites of parent strains in physical and chemical properties. A direct correlation was observed between the character of colonies and biosynthetic properties of mutants.

Macrotetrolide antibiotics produced byStreptomyces globisporus

Macrotetrolides isolated from a new producer,Streptomyces globisporus, were identified as nonactin, monactin, dinactin and trinactin. Spectroscopic characterization of these compounds was extended by13NMR spectra. Chemical ionization with ammonia as reactive gas was proposed for mass-spectroscopic characterization of their mixtures. Their biological activity was confirmed by using larvae of the Colorado potato beetle (Leptinotarsa decemlineata) as a new test model.

Biological activity of hydroxyanthraquinones and their glucosides toward microorganisms.

Five mono- and dihydroxyanthraquinones as well as 12 of their glucosides (both free and acetylated) were tested with six different microbial species using the plate-diffusion method. None of the tested substances was active againstEscherichia coli, 15 of the 17 substances displayed an activity towardBacillus subtilis, Bacillus cereus, Candida albicans, Saccharomyces cerevisiae andStreptomyces aurecfaciens. Relationships between the substance type and biological activity are discussed.

Studies on the production of daunomycinonederived glycosides and related metabolites inStreptomyces coeruleorubidus andStreptomyces peucetius

Strains ofStreptomyces coeruleorubiduě ISP 5145,JA 10092 and 39–146, differing mutually in antibiotic activity, were found to produce identical spectrum of metabolites (at least nine antibiotically active glycosides, 13-dihydrodaunomycinone, ε-rhodomycinone and a larger number of unidentified compounds); only trace quantities of daunomycin and daunomycinone could be detected. A fraction of glycosides with a higher Rf (0.4–0.7), isolated from strain 39–146, could be transformed to daunomycin by mild hydrolysis and to daunomycinone by total hydrolysis.Streptomyces peucetius IMI 101 335 differed fromStreptomyces coeruleorubidus in an increased production of ε-rhodomycinone and a lower content of glycosides; the zone of daunomycin was most pronounced among the glycoside spots.Streptomyces coeruleorubidus 39-146 exhibited the highest activity in a medium containing 3.5% soluble starch, 3.0% soybean meal, 0.3% NaCl and 0.3% CaCo3; glucose was a more useful carbon source for the remaining strains The activity ofStreptomyces coeruleorubidus was inhibited by 1-propanol, Na-propionate,5,5-diethylbarbiturate and bacitracin. Ferrous sulphate stimulated the production of glycosides only in strain JA 10092, decreasing simultaneously the production of aglycones.

Biotransformations of anthracyclinones inStreptomyces coeruleorubidus andStreptomyces galilaeus

The ability to transform biologically exogenous daunomycinone, 13-dihydrodaunomycinone, aklavinone, 7-deoxyaklavinone, ε-rhodomycinone, ε-isorhodomycinone and ε-pyrromycinone was studied in submerged cultures of the following strains: wildStreptomyces coeruleorubidus JA 10092 (W1) and its improved variants 39–146 and 84–17 (type P1) producing glycosides of daunomycinone and of 13-dihydro-daunomycinone, together with ε-rhodomycinone, 13-dihydrodaunomycinone and 7-deoxy-13-dihydro-daunomycinone; in five mutant types ofS. coeruleorubidus (A, B, C, D, E) blocked in the biosynthesis of glycosides and differing in the production of free anthracyclinones; in the wildStreptomyces galilaeus JA 3043 (W2) and its improved variant G-167 (P2) producing glycosides of ε-pyrromycinone and of aklavinone together with 7-deoxy and bisanhydro derivatives of both aglycones; in two mutant typesS. galilaeus (F and G) blocked in biosynthesis of glycosides and differing in the occurrence of anthracyclinones. The following bioconversions were observed: daunomycinone → 13-dihydrodaunomycinone and 7-deoxy-13-di-hydrodaunomycinone (all strains); 13-dihydrodaunomycinone → 7-deoxy-13-dihydrodaunomycinone (all strains); daunomyeinone or 13-dihydrodaunomycinone → glycosides of daunomyeinone and of 13-dihydrodaunomycinone, identical with metabolites W1 and P1 (type A), or only a single glycoside of daunomyeinone (type E); aklavinone → ε-rhodomycinone (types A and B); aklavinone → 7-deoxyaklavinone and bisan-hydroaklavinone (type C); ε-rhodomycinone → ζ-rhodomycinone (types C, E); ε-rhodomycinone → glycosides of ε-rhodomycinone (types W2, P2); ε-isorhodomycinone → glycosides of ε-isorhodomycinone (types W2, P2); ε-pyrromycinone → a glycoside of ε-pyrromycinone (types W1, P1). 7-Deoxyaklavinone remained intact in all tests. Exogenous daunomyeinone suppressed the biosynthesis of its own glycosidea in W1 and P1; it simultaneously increased the production of ε-rhodomyeinone in P1.

The study of variability and strain selection inStreptomyces atroolivaceus

A seven-step selection procedure (repeated UV irradiation, single-step application of nitrous acid, and natural selection, following each mutagenic treatment) made it possible to increase production of mithramycin byStreptomyces atroolivaceus from 40–50 μg/ml to 680–830 μg/ml,i.e. roughly by 15 to 19-fold. The UV radiation was more effective when applying lower doses, yielding about 5% survival, 2.3% survival was obtained after the treatment with nitrous acid.N-Methyl-N′-nitro-N-nitrosoguanidine applied in a buffer pH 9.0) at doses yielding more than 99% killing was less effective than the two former mutagens.

Intra- and interspecific cosynthetic activity of mutants ofStreptomyces coeruleorubidus andStreptomyces galilaeus impaired in the biosynthesis of anthracyclines

Cosynthesis of anthracycline compounds was followed in five phenotypic groups of mutants ofStreptomyces coeruleorubidus (A - E), blocked in the biosynthesis of the daunomycine complex, and in two mutant types ofStreptomyces galilaeus (F, G) blooked in the biosynthesis of glycosides of ε-pyrromy-cinone and aklavinone. Glycosides of daunomycinone and 13-dihydrodaunomycinone were produced in combinations A + B, A + C, A + D, A + E and A + F, ε-rhodomycinone was synthesized in combinations A + E, A + F, B + E and B + F. During the cultivation of types B - E with type G or F non-anthra-cycline compounds, typical ofS. galilaeus, were cosynthesized. No cosynthesis could be observed in other combinations of the mutant types. Negative results were also obtained with combinations of mutants of the same group and during cultivation of all mutant types with streptomycetes not producing anthracyclines. A scheme illustrating metabolic pathways leading to the biosynthesis of daunomycinone, aklavinone, ε-pyrromycinone and ε-rhodomycinone inS. coeruleorubidus andS. galilaeus was constructed.

Production of quinomycin A inStreptomyces lasaliensis

In addition to lasalocid, an oligoether coccidiostatic compound, other compounds are synthesized byStreptomyces lasaliensis. Mutants producing either of two antibiotics, lasalocid A or quinomycin A (an antibiotic of quinoxaline character), were obtained by natural selection and by mutagenesis. Methods of isolation, purification and estimation of both compounds were established.

Developmental mutants ofStreptomyces coeruleorubidus, a producer of anthracyclines: Isolation and preliminary characterization

AbstbactThe wild strainStreptomyces coeruleorubidus JA 10092 was found to segregate into two spontaneous morphological variants (spo-1 andbld-1) with a different ability to form aerial mycelium in media with glucose as the main carbon source. Six new types of developmental mutants were obtained from the bald variantbld-1 after treatment with mutagens (UV light, γ radiation, nitrous acid) and after natural selection Formation of the aerial mycelium was fully suppressed in thebld-2 type growing on media both with glucose and with starch. The other types were bald only on starch media, forming the aerial mycelium on media with glucose; typesspo-2, spo-3, spo-4 andspo-5 differed in size, shape and surface structure of spores, the typewhi formed asporogenous aerial hyphae.