P130: Next generation sequencing allows first insights into the biosynthetic pathways of epoxyketone proteasome inhibitors

Microbes are a prolific source of anti-cancer molecules, and one such class of compounds is the proteasome inhibitors (PIs). These molecules inhibit the normal functioning of the proteasome (an essential protein degradation complex), by covalent attachment via a reactive warhead resulting in cell death. Epoxomicin and eponemycin are two naturally occurring PIs that were isolated from marine actinomycete species. They are particularly interesting as they possess an unusual epoxyketone warhead and analogues of each compound (Bortezomib and Carfilzomib respectively) have been approved for the treatment of multiple myeloma. While the mechanism by which the epoxyketone binds to and arrests the function of the proteasome has been described, the biosynthesis of the natural products is unknown. Of particular interest to us is the assembly of the unique epoxyketone moiety. Unveiling the biosynthetic pathways to assemble these molecules could enable production of large quantities of starting material for synthetic derivatives and precursors. We have sequenced the genomes of both producing organisms using next-generation technologies, a challenging task due to their high GC content. Using a bioinformatic approach we identified the gene clusters responsible for both epoxomicin and eponemycin biosynthesis and have confirmed this assignment by heterologous expression studies. We are now in a position to investigate the fine details of these pathways and to probe the origin of the unique warhead functionality. This research represents the identification of the first biosynthetic gene clusters for expoxyketone PIs, and we suggest unprecedented biotransformations for the construction of the unusual epoxyketone warhead.