Biomimetic synthesis and structural refinement of the macrocyclic dimer aminoglycoside 66-40C--the remarkably selective self-condensation of a putative aldehyde intermediate in the submerged culture medium producing sisomicin.

Aminoglycoside 66-40C, an unprecedented 16-membered bis-azadiene macrocyclic natural product isolated from the Micromonospora producer of the antibiotic sisomicin, was synthesized following a biomimetic strategy which definitively established its origin as arising from a remarkably selective non-enzymatic macro-dimerization.

Molecular cloning and sequence analysis of the sisomicin biosynthetic gene cluster from Micromonospora inyoensis.

Micromonospora inyoensis produces sisomicin (Sm), an aminoglycoside antibiotic. The gene cluster of sisomicin biosynthesis spanning ca. 47 kb consists of 37 ORFs encoding various proteins for sisomicin biosynthesis, regulation, resistance and transport. The comparative genetic studies on the biosynthetic genes of sisomicin and gentamicin (Gm) reveal a similar biosynthetic route and provide a framework for the future biosynthetic studies.

[Study of the conditions for reproducing the processes of antibiotic biosynthesis. Calculating the process of sisomycin biosynthesis]. / Issledovanie uslovii vosproizvodimosti protsessov biosinteza antibiotikov. Masshtabirovanie protsessa biosinteza sizomitsina.

Conditions were experimentally studied for reproducing sisomicin biosynthesis by up-to-down scaling. It was shown that respiration intensity as a parameter of antibiotic biosynthesis scaling-down had certain limitations connected with multistage oxygen mass transfer. The parameter could be used only when the limiting stages of oxygen mass transfer coincided. It was confirmed experimentally that it was possible to apply the previously proposed theoretical method to estimation of the limiting stage of oxygen mass transfer.

[Formation of A-factor-type regulators by representatives of the genus Micromonospora]. / Obrazovanie reguliatorov tipa A-faktora predstaviteliami roda Micromonospora.

It is known that substances similar in their chemical structure and biological action to A-factor, a regulator of Streptomyces griseus differentiation are widely observed in streptomycetes. The present paper is concerned with studies on capacity for producing A-factor-like regulators in 13 strains belonging to 11 species of genus Micromonospora. It was shown that 7 strains had an A-factor-deficient test mutant effect analogous to the effect of A-factor. Relation of A-factor-like substance production to biosynthesis of sisomicin and sporogenesis was investigated in M. fusca var. sisomicini, strain RIA 1572. A-Factor-like substances were isolated from submersion culture of strain 1572 on the 6th-8th day of the culture growth. By the time it corresponded to the highest number of the spores in the medium. The highest levels of sisomicin were detected on the 3rd day of the fermentation process. After that period its concentration markedly lowered. Exogenic addition of A-factor had no effect on sisomicin biosynthesis but resulted in increased numbers of the spores.

[Inhibitor of aminoglycoside phosphotransferases from Micromonospora sp.--a producer of sisomicin]. / Ingibitor aminoglikozid-fosfotransferaz iz Micromonospora sp.--produtsenta sizomitsina.

A substance inhibiting the activity of aminoglycoside-3-'-phosphotransferases of both the actinomycetous and the bacterial origin was detected in the filtrates of the culture fluid and mycelium of Micromonospora sp. producing sisomicin. The activity of the substance against the aminoglycoside inactivating enzymes of different types was studied. It was shown that the quantity of the inhibitor in the culture fluid of Micromonospora sp. correlated with intensity of sisomocin production. Under conditions not providing production of the antibiotic the inhibitory activity was lacking. The inhibitor was purified by ion exchange chromatography on Amberlite CG-50 and KM-cellulose (NH+4 form), gel filtration through Sefadex G-50 and preparative paper chromatography. Stability of the inhibitor at different pH values and different temperatures, its sensitivity to certain enzymes and behaviour in high voltage electrophoresis were studied. The results of ultrafiltration showed that the molecular weight of the inhibitor was relatively low: less than 1000 D.

[Experimental study of vibration agitation in antibiotic biosynthetic processes in laboratory apparatus]. / Eksperimental'noe izuchenie vibratsionnogo peremeshivaniia v protsessakh biosinteza antibiotikov v usloviiakh laboratornykh apparatov.

The use of vibroagitation in biosynthesis of antibiotics was shown to be in principle possible. The results of the experiments in 3-liter apparatus equipped with devices for vibroagitation and turbine mixers are presented.

[Significance of pH for the growth of the sisomicin producer and biosynthesis of the antibiotic]. / Znachenie pH dlia rosta produtsenta sizomitsina i biosinteza antibiotika.

Dependence of the mycelium growth and sisomycin production on the pH values of the fermentation broth was studied in cultures of various ages for 6 hours with the method of pH control within the preset levels. It was shown that the values of pH within the range of 7.25 - 7.6 were optimal. The pH values of 7.5 - 8.0 and 7.0 - 8.0 were optimal for the growth of the 24-hour culture and biosynthesis of the antibiotic by it respectively. pH 7.5 was optimal for the growth of the 48-hour mycelium and biosynthesis of the antibiotic by it.

The structure of aminoglycoside antibiotic 66-40G produced by Micromonospora inyoensis.

Aminoglycoside antibiotic 66-40G is a minor component produced in the fermentation of Micromonospora inyoensis. Its structure has been established as 3''-de-N-methyl-sisomicin (4) by spectroscopic means and by direct comparison with an authentic sample obtained from photochemical oxidative de-N-methylation of sisomicin (1).

Micromonospora-produced sisomicin components.

A sisomicin fermentation carried out in the presence of (methyl-14C)-L-methionine resulted in a crude mixture, composed of methyl-14C-labeled sisomicin as a major component; and two 4''-C-desmethylsisomicin (66-40B and 66-40D) isomer-like components, an unidentified component and a gentamicin A-like antibiotic as minor components. When (methyl-14C)-L-methionine was added in an early stage of the fermentation (24 hours), incorporation of methyl-14C-label into polar components (e.g., gentamicin A-like antibiotic) preceded that into sisomicin. Chromatographic evidence for the bioconversion of (methyl-14C)-gentamicin A to a radioactive sisomicin-like product (possibly (3''-N-methyl-14C)-sisomicin) was seen, when a Micromonospora blocked mutant was incubated in the presence of the former antibiotic.

A new broad spectrum aminoglycoside antibiotic, G-52, produced by Micromonospora zionensis.

G-52 is a new broad spectrum aminoglycoside produced by a species of the genus Micromonospora, Micromonospora zionensis. It has been differentiated from other known related antibiotics by a variety of chemical and biological methods. Its in vitro and in vivo spectrum of activity appears to be quite similar to that of verdamicin and gentamicin but is differentiated from them by its increased activity against 6'-N-acetylating strains.

Biotransformation, a new approach to aminoglycoside biosynthesis. I. Sisomicin.

Utilizing a mutant of Micromonospora inyoensis which requires the addition of 2-deoxystreptamine for sisomicin production, the bioconversion of 2-deoxstreptamine containing pseudodisaccharides and pseudotrisaccharides into sisomicin was demonstrated. The trisaccharides tested were structurally related minor components found in the sisomicin or gentamicin fermentations. Based upon the specificity of the structural configuration of those compounds which were converted to sisomicin versus those which were not, a pathway for the biosynthesis of sisomicin is proposed.