BIOSYNTHESIS OF MAYTANSINOIDS AND ANALOGS

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Maytansine (1) is a very potent antitumor agent originally isolated by Kupchan and coworkers from the plant, Maytenus serrata (formerly M. ovatus), where it occurs in extremely low concentration (0.00002 percent of dry weight). Its isolation was only possible by virtue of its extraordinary biological potency, using a rigorous bioassay-guided fractionation. 1 and a number of congeners, mostly with variations in the ester side chain, were also isolated from other plants of the family Celastraceae, the highest concentration (12 mg/kg dry weight) being found in Putterlickia verrucosa. Notably, members of the maytansinoid family, the ansamitocins, were subsequently isolated from cultures of Nocardia species as well as some other Actinomycetes, raising the question whether the compounds isolated from the plant materials were produced by the plants or by symbiotic or contaminating microorganisms. The latter possibility was further reinforced by the isolation of maytansinoids from several mosses. Despite its extraordinary potency as an antitumor agent, clinical trials of 1 as an anticancer drug gave disappointing results due to dose limiting toxicity. Structure-activity studies, limited to the naturally occurring compounds and those accessible by semisynthesis from them, did not result in significant improvements of the therapeutic ratio. However, these structural variations were of necessity limited primarily to the C-3 ester side chain and the region around its attachment site; backbone modifications and functional group modifications in other regions of the molecule have remained largely unexplored due to inaccessibility of the compounds. This provides the ultimate rationale for the proposed project. Under this project, the PI will delineate, at the biochemical and at the genetic level, the biosynthesis of the microbial maytansinoids, the ansamitocins. The complete sequence analysis of the ansamitocins biosynthetic gene cluster will then lay the foundation for the engineering of mutant organisms harboring and expressing altered clusters in which specific biosynthetic genes have been deleted, added or replaced by functional equivalents from other biosynthetic pathways. These will be analyzed for the production of analogs of the natural maytansinoid structures. Secondly, the microbial maytansinoid biosynthesis genes and their sequences will be used to analyze the DNA of 1-producing plants and of microorganisms isolated form them for the presence of homologous genes in order to identify the true biosynthetic origin of the maytansinoid structures isolated from these plants.