BIOSYNTHESIS OF ANTHRACYCLINE ANTIBIOTICS

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This proposal requests continuation of our study of the biochemistry and genetics of anthracycline antibiotic biosynthesis for a five year period. During this time we will (i) isolate and study the properties of the socalled " aromatic polyketide synthases" and their structural genes, (ii) define the genetic and biochemical organization of anthracycline antibiotic biosynthetic pathways with special emphasis on their regulation at DNA, RNA and protein levels, and (iii) explore the possibilities for expressing Streptomyces genes in other actinomycetes and creating new anthracycline antibiotics by genetic engineering. The major project during this period will be a study of tetracenomycin C, and antitumor antibiotic produced by Streptomyces glaucescens. We will complete the cloning of the tcm genes then dissect the tcm gene cluster by standard techniques individual tcm genes. Their structures will be studied by nucleotide sequencing, S1 mapping, and other methods to locate the regulatory regions and transcriptional/translational start/stop sites. This information and the results of Northern hybridizations will be used to identify the transcriptional units within the tcm gene cluster. The enzymes catalyzing the formation of the aromatic polyketide intermediates of TcmC biosythesis will be overproduced by inserting their genes in suitable expression vectors to facilitate the study of their physical and biochemical properties. In this way we will establish information about the regulation of TcmC biosynthesis at the DNA, RNA and protein levels. Subsidiary projects will involve cloning the genes for the production of elloramycin, an analog of TcmC; using the tcm genes as probes for genes governing related biochemical pathways in Streptomyces; expressing the tcm genes in other actinomycetes; and exploring the possibilities for the construction of hybrid anthracyclines by genetic engineering.