As a model of secondary metabolism, I propose to continue a study of the biochemistry and genetics of the production of erythromycin A (erythro'A), a metabolite of Saccharopolyspora erythraea that is classified chemically as a macrolide and has broad spectrum antiinfective activity. Information about the biosynthesis of macrolides, like erythro'A, can be used to increase the production of known drugs and to make new drugs by genetic engineering. This use is an attractive prospect for macrolides because many of them have medically valuable antibiotic activity. Since the principal portion of macrolides is made by the polyketide pathway, which represents hundreds of different structural types, it is likely that numerous biochemical perturbations are possible. This chemical diversity could make the search for new drugs by genetic engineering especially fruitful. Antibiotic production is a characteristic of actinomycetes that has attracted considerable attention. Genetic studies are likely to uncover significant 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 possibility of using the macrolide PKS genes and genes that regulate macrolide antibiotic production to construct strains that overproduce secondary metabolites is very promising; thus, information about the regulatory mechanisms should have wide-spread utility in biotechnology. Our goals for the next four years, listed in the priority in which they will be pursued, are as follows. 1) Complete characterization of the ery structural and regulatory genes. This goal will be reached through: (a) Cloning and analysis of other genes required for the action of 6- deoxyerythronolide B and erythromycin D hydroxylases. (b) Cloning and analysis of the TDP-4-keto-6-deoxy-D-glucose epimerase and reductase genes involved in TDP-L-mycarose formation. (c) Characterization of other ery genes in the region upstream of eryA1 by sequence analysis and gene disruption and replacement expts, in collaboration with researchers at Abbott Laboratories. (d) Identification of the genes controlled by eryC1 or other regulatory genes found in the ery cluster and exploration of the molecular mechanisms involved. 2. Investigation of metabolic sources of propionate, the main erythronolide building block. This goal will be reached through: (a) Cloning and analysis of valine dehydrogenase (vdh) and threonine dehydratase (tdt) genes. (b) Analysis of the regulation of vdh and tdt expression in relation to their role in amino acid utilization. (c) Determination of the effect of inactivation of the vdh and tdt genes on erythromycin production.
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