The objective of the proposed work is the biosynthesis of analogs of rapamycin in specifically constructed mutants of Streptomyces hygroscopicus ATCC 29253, the rapamycin producer. These novel compounds will be tested for their biological activity as immunosuppressive agents and will be valuable starting materials for chemical modification. This objective will be accomplished through (1) characterizing the (rap) genes involved in rapamycin biosynthesis, (2) introducing mutations into specific sites in rap DNA and replacing the wild type gene with its mutant counterpart, (3) fermenting the mutants, isolating the rapamycin analogs and determining their structure and (4) evaluating the novel rapamycin analogs for their biological activity. Steps 1 and 2 involve transposon mutagenesis, gene cloning and disruption, nucleotide sequencing and gene replacement which will be done in the Leonard Katz (P.I.) and Richard Hutchinson (Co-P.I.) laboratories at Abbott Laboratories and the University of Wisconsin, respectively. Step 3 will be performed at Abbott under the direction of James McAlpine (Co-P.l.). Novel rapamycin analogs will turned over to K. Mollison and B. Lane at Abbott Laboratories for evaluation of their biological properties. This proposal is based on our recently acquired understanding of the organization of the genes (eryA) involved in the biosynthesis of 6-deoxyerythronolide B (6-dEB), the 14-membered macrocyclic lactone precursor of the macrolide antibiotic erythromycin; on our ability to introduce mutations into eryA which resulted in the formation of structural analogs of 6-dEB of predicted structure; and on our belief that the genes for rapamycin biosynthesis will be organized similarly and amenable to analogous manipulations. Thus it will be possible to pre-select the structure of the compounds desired and construct a family of genetically engineered S. hygroscopicus strains each producing a rapamycin analog carrying a change at a unique functional site of the molecule. These compounds, or their chemically modified derivatives, can be used in binding studies with rapamycin binding proteins, in functional tests for immunosuppressive activity, and in bioavailability studies. The proposed research presents a novel method to make structural analogs of a natural compound that would be difficult to make by conventional chemical approaches. Our work will provide molecules that will shed light on the structure/activity relationships of macrocyclic immunosuppressants and could potentially lead to the discovery of improved forms of such therapeutic agents. It will also expand knowledge about microbial (Streptomyces) genetics and the structure of complex polyketide synthases, thus setting the stage for definitive studies of this unusual enzymology.
|Lomovskaya, N; Fonstein, L; Ruan, X et al. (1997) Gene disruption and replacement in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253. Microbiology 143 ( Pt 3):875-83|
|Lomovskaya, N; Hong, S K; Kim, S U et al. (1996) The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production. J Bacteriol 178:3238-45|