BIOSYNTHESIS OF ANTHRACYCLINE ANTIBIOTICS

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I propose to continue a study of the biochemistry and genetics of the production of tetracenomycin C (Tcm C), a metabolite of Streptomyces glaucescens that is classified chemically as an anthracycline and has moderate antitumor properties, as a model of secondary metabolism. Information about the biosynthesis of polyketides, like Tcm C, can be used to increase the production of known drugs. In attempts to make new drugs by genetic engineering, which is an attractive prospect for anthracyclines because some of them have clinically valuable antitumor and notable anti- HIV activity, the knowledge that the polyketides represent hundreds of different structural types suggests that numerous perturbations of this biochemistry are possible. This could make the search for new drugs by genetic engineering especially fruitful. Antibiotic production is a characteristic of Streptomyces that has attracted considerable attention. Probing its genetics is likely to uncover significantly new information because secondary metabolism, a unique characteristic of the slow growth (stationary) phase of laboratory cultures, is quite unlike the primary metabolism of rapidly growing cells that has been the focal point of most studies of prokaryotic genetics. Moreover, the value of using the PKS genes and genes that regulate antibiotic production to construct strains that overproduce secondary metabolites has been demonstrated; thus, information about the regulatory mechanisms should have wide-spread utility in biotechnology. Our goals for the next five years, listed in the priority in which they will be pursued, are as follows. 1. Enzymology of the tcm polyketide synthase (PKS). We will study the enzymology of polyketide metabolism by (i) determining the properties of the enzymes produced by the tcm PKS genes, (ii) using these enzymes to develop a system for the production of Tcm F2 nd Tcm F1 (or D3) in vitro, and then (iii) investigating the effect of substituting the components of other PKS's for the different enzymes of the tcm PKS. New metabolites may be produced in this work. 2. Regulation of the expression of the tcm genes. We will investigate the genetics of Tcm C production by (i) elucidating the transcriptional organization of the tcmIII/VI/Ia/II, tcmVII, and tcmAR genes, (ii) determining whether expression of the tcm PKS, tcmVII and tcmA genes is controlled by the tcmR gene or vice-versa, and (iii) searching for genes or physiological factors that control expression of the tcmIII/VI/Ia/II and tcmAR genes. This will provide insight into the regulation of secondary metabolism. 3. Properties of the fatty acid synthase (FAS) genes. We will determine the effect of deleting the Orf1 gene of the S. glaucescens ACP-II region on cell growth and development, and the production of the Tcm C metabolites. This work will reveal possible interactions between polyketide and fatty acid metabolism and whether the Orf1 gene is part of the FAS.