BIOSYNTHESIS OF ANTHRACYCLINE ANTIBIOTICS

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This proposal requests support of a study of the molecular genetics and biochemistry of anthracycline antibiotic formation by streptomycetes. The biosynthesis of a new anthracycline metabolite with potent antitumor activity in vivo, tetracenomycin C (7), by Streptomyces glaucescens ETH 22794 will be studied as a model of the formation of all anthracycline antibiotics. The experimental approach will first involve construction of a set of bacterial mutants having point blocks in the tetracenomycin biosynthetic pathway by treatment of S. glaucescens with physical and chemical mutagens. These mutants will then be used as hosts for transformation with DNA segments from the parent organism in shotgun gene cloning experiments, using Streptomyces cloning vectors known to function in S. glaucescens. Genetic and biochemical complementation of the blocked mutants' ability to produce 7 after transformation will be assessed by bioassay for the restoration of antibiotic formation; chemical and physical methods will be used to affirm this and the presence of the associated enzymes. Gene amplification with high copy number vectors, or the use of high level expression vectors will be explored as a means for increasing the production of the enzymes and intermediates of tetracenomycin C biosynthesis. Subsidiary work will be done to probe the cellular location of the antibiotic production genes, and the possibility for expression of these genes in other bacterial species. The results of the research program will exemplify a new approach to research on the biosynthesis of antibiotics, and will establish general parameters necessary for increasing the production of valuable antibiotics.