Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part I. The synthesis of echinomycin.

Washed suspensions of Streptomyces echinatus, and protoplasts derived from them, have been shown to synthesise echinomycin in the absence of growth. Protoplast suspensions free from significant contamination with unlysed mycelia are obtained by incubation with lysozyme followed by filtration through layers of tightly packed glass wool. Although physiologically young cells produce a better yield of protoplasts, optimal antibiotic biosynthesis is achieved with protoplasts prepared from mycelia about to enter the stationary phase of growth i.e., approximately 24 h after inoculation into a nutrient broth--salts seed medium. As judged by the incorporation of label from L-[methyl-14C]methionine, echinomycin synthesis proceeds for about 1 h after preparation of washed suspensions, but the kinetics of incorporation by intact cells and protoplasts are different. Uptake of labelled methionine by protoplasts is critically dependent upon the presence of sucrose as osmotic stabiliser and is drastically reduced if galactose, calcium, or magnesium is omitted from the suspending buffer. Uptake by intact, washed cells is essentially independent of nutrients in the medium. Small quantities of 11 materials other than echinomycin are detectable in chloroform extracts after labelling with L-[methyl-14C]methionine; some of these may represent precursors in the biosynthesis of the antibiotic. All amino acid constituents of echinomycin as well as tryptophan, a putative precursor of the quinoxaline chromophores, are actively incorporated into echinomycin by protoplasts and resting cells, but not with equal efficiency.