Calicheamicin gamma1I (1) from Micromonospora echinospora spp. calichensis is the most prominent of the enediyne family and its unprecedented molecular architecture in conjunction with its superb biological activity and therapeutic value brand 1 an excellent target for the study of natural product biosynthesis and self-resistance. While the synthetic achievements toward 1 have been monumental, the total synthesis of 1 is secondary to the isolation of 1 from large fermentations of M. echinospora and thus, methods to produce mass amounts of 1 and potentially useful variants are still desperately needed. Furthermore, the notably unusual architecture of 1 implies the participation of a remarkably novel biosynthetic pathway. Through a multi- disciplinary approach, the fundamental goal of this proposal is to present rational strategies from which to build a foundation of knowledge regarding 1 biosynthesis and self-resistance. Specifically, we will i) clone the gene cluster encoding for 1 biosynthesis and self-resistance in Micromonospora; ii) elucidate the nucleotide sequence of this unparalleled gene cluster; iii) develop a transformation system for Micromonospora echinospora; iv) provide functional assignments for the putative genes involved in aryltetrasaccharide assembly; v) localize the specific 1 self-resistance genes within this cluster; and vi) elucidate the mechanism by which the corresponding encoded 1 self-resistance proteins function. Achieving these aims will not only provide pioneering discoveries in mechanistic enzymology (formation of aglycone diynenes, hydroxylamine glycosides, thioester sugars, and phenolic-rhamnosides as well as unprecedented modes of resistance to exceptionally reactive natural products), but may also provide access to the rational biosynthetic modification of enediyne structure for new drug leads, biosynthetic methods to introduce 1-targeting or tagging ligands, the potential to construct enediyne overproducing strains and possibly even an enediyne combinatorial biosynthesis program. Should the self-resistance mechanism(s) be applicable, these studies may also lead to gene therapy approaches (via introduction of 1 drug resistance genes into bone marrow cells) for increasing tolerable chemotherapeutic dose levels of 1.
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