Biosynthesis and bioengineering of epoxyketone proteasome inhibitors

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"Epoxyketones are an important class of bacterial nonribosomal peptides that target the proteolytic -subunit of the proteasome. They inspired the development of carfilzomib, an epoxyketone approved in 2012 for the treatment of multiple myeloma. Oprozimib, a second-generation orally available anticancer epoxyketone, and KZR-616, an epoxyketone that selectively targets the immune proteasome, are currently in phase II clinical trials. All three are manufactured via chemical synthesis, which is costly and unsustainable. We aim to develop cheaper and more sustainable biocatalytic approaches for epoxyketone production.We have investigated the biosynthesis of TMC-86A and eponemycin, two closely related epoxyketones produced by Actinobacteria. The biosynthetic gene clusters for these metabolites have been cloned using transformation-associated recombination (TAR) in yeast and expressed in a heterologous host. TAR-based methods have been used to create in-frame deletions in each of the biosynthetic genes and characterisation of the metabolites accumulated in each of the mutants has provided extensive insights into the nature and order of each of the biosynthetic steps. We have also shown using in vitro biochemical methods that the epoxyketone pharmacophore of these metabolites is assembled from the -dimethyl--keto acid product of a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) by an unusual trifunctional flavin-dependent decarboxylase-dehydrogenase-monooxygenase (epoxyketone synthase). This enzyme has been shown to accept several analogues of the natural substrate.In this project, we aim to determine the X-ray crystal structure of the epoxyketone synthase, providing a basis for rational engineering to further broaden its substrate tolerance, and develop a mutasynthesis approach for the production of epoxyketones that are structurally related to the compounds currently undergoing clinical trials."