R. G. Condon

Micromonospora-Produced Gentamicin Components

After the chromatographic separation of [methyl-14C]gentamicin major (C) components from a large-scale radioactive fermentation (Lee et al., 1974), [methyl-14C]gentamicin minor (polar) components (A, B, B1, X2, and G-418) were isolated from subsequent chromatography of the remaining antibiotic mixture. When l-[methyl-14C]methionine was added at the onset of biosynthesis of the gentamicin components, incorporation of label into the minor components preceded incorporation into the major components. Degradation occurred when [methyl-14C]gentamicin major components (C1, C2 and C1a) were added respectively to the gentamicin-producing culture medium and shaken. Images

Biotransformation of Sisomicin to Gentamicin C2b

Sisomicin was transformed to gentamicin C2b by Micromonospora rhodorangea NRRL 5326. The mechanisms involved in the biotransformation are the 6′-N-methylation and the (4′-5′)-reduction. The progression of the methylation was followed by the isotope technique, but the reduction reaction was not monitored.

Antibiotic Biosynthesis by Cofermentation of Blocked Mutants of Two Micromonospora Species

Two aminocyclitol-negative Micromonospora mutants representing two different species, M. purpurea and M. inyoensis, and blocked at different steps in the biosynthetic pathway were paired and cofermented for the synthesis of antibiotics. The two blocked mutants were incapable of producing antibiotics alone except when 2-deoxystreptamine was added. When combined they produced gentamicins A, X2, C1a, and C2b, which all have an amino group at the 2′ position, and gentamicin B, which has a hydroxyl group at this position instead. Images

Biosynthetic pathway leading to gentamicin C2b.

Incubation of Micromonospora purpurea SC 1210 (NRRL 5467) with L-[methyl-14C]methionine yielded [methyl-14C]gentamicin A and methyl-14C-labeled antibiotic JI-20A in a molar radioactivity ratio of 9:2. We did not isolate methyl-14C-labeled gentamicin X2, which was expected as an intermediate based on the biosynthetic pathways proposed by others. Addition of methyl-14C-labeled antibiotic JI-20A to M. purpurea SC 1124 (NRRL 8102) yielded [methyl-14C]-gentamicin C1a and [methyl-14C]gentamicin C2b in variable molar radioactivity ratios. These data do not support the biosynthetic pathway leading to gentamicin C2b proposed by Testa and Tilley.

Method for Qualitative Determination of 2-Deoxystreptamine

We have developed a qualitative method to assay for the presence of 2-deoxystreptamine in hydrolysates of crude aminoglycoside preparations using a 2-deoxystreptamine-requiring idiotrophic mutant. The assay involves (i) incubation of a 2-deoxystreptamine-requiring mutant with 1 mg of a hydrolyzed preparation from a crude unknown antibiotic mixture per ml, and (ii) examination of the resultant incubation mixture for production of antibiotic(s) by disk assay.

Formation of Methylated and Phosphorylated Metabolites During the Fermentation Process of Verdamicin

In an attempt to understand the biosynthetic processes leading to the formation of verdamicin (end product), we have examined the patterns of the formation of methylated and phosphorylated metabolites, which resulted from either the addition of l-[methyl-14C]methionine or [32P]KH2PO4 to the fermentation. Incorporation of label from l-[methyl-14C]methionine into the bioactive sisomicin, verdamicin, and the chromatographically polar components increased with the progression of time. Two methylated bioinactive metabolites were found in the culture broth after removal of the methylated bioactive metabolites. In contrast to the bioactive metabolites, incorporation of the methyl-14C label into the two methylated bioinactive metabolites decreased with the progression of time. A phosphorylated bioinactive metabolite (nonmethylated) was also found in the culture broth, fermented in the presence of [32P]KH2PO4. The role of the phosphorylated metabolite in the biosynthesis of the bioactive metabolites cannot yet be explained. Images