A new tetracycline antibiotic from a Dactylosporangium species. Fermentation, isolation and structrure elucidation.

Abstract

An actinomycete identified as a Dactylosporangium sp. produces a new tetracycline, 4a-hydroxy-8-methoxychlortetracycline (Sch 34164). The addition of magnesium ions to complex fermentation media increased the antibiotic titers. Sch 34164 was isolated by solvent extraction and Sephadex G-25 column chromatography. The novel structure was proposed based on spectroscopic analysis. The shift of C-4a (35 to 77 ppm) and C-8 (140 to 163 ppm) in the 13C NMRas compared to chlortetracycline was indicative of the novel hydroxyl and methoxysubstituents, respectively.An actinomycete, designated SCC1695, isolated from a sample collected in the Kasie Valley of Zambiawas found to produce in fermentation a newtetracycline antibiotic. The antibiotic was isolated from fermentation broth by solvent extraction and purified by Sephadex G-25 column chromatography. The producing strain was characterized and found to have the macroscopic, microscopic and whole-cell hydrolysis properties of the genus Dactylosporangium.This paper describes the production, isolation, physico-chemical properties and structure elucidation of Sch 341641}. Taxonomy and biological properties2) will be reported elsewhere. FermentationThe fermentation studies were carried out in shake flasks and 14-liter fermentors. An aliquot of the frozen whole broth was used to inoculate a germination medium composed of Cerelose 0.1 %, potato starch 2.4%, beef extract 0.3%, yeast extract 0.5% Tryptone 0.5% and CaCO3 0.2%. The pH of the mediumwas adjusted to 7.0 prior to sterilization. The seed culture was incubated at 30°Con a rotary shaker at 300 rpm for 48 hours. Twenty five ml of the resulting seed culture were transferred to a 2-liter Erlenmeyer flask containing 500 ml of the above mediumand incubated for an additional 48 hours at 30°C. The entire content was used to inoculate a 14-liter fermentor containing 10 liters of the production medium consisting of Cerelose 1.0 %>soluble starch 2 %, yeast extract 0.5 %, NZ-Amine 0.5%, CaCO3 0.4% and 1 mMCoCl2 1 ml. The fermentation was carried out at 30°C, 300rpm and aeration of 3.5 liters per minute for 96 hours.Antibiotic production in shake flasks and tanks was monitored at 72 and 96 hours by whole broth disc-agar diffusion assay against Staphylococcus aureus and Escherichia coli. Since the initial titers were extremely low, experiments in shake flasks focusing on the production mediumwere initiated to increase antibiotic titers. The fermentation mediumdiscussed previously was supplemented with various ions at the concentration shown in Table 1. All ionic solutions were added post steriliza-