NRPS ENGINEERING OF EPOXYKETONE PROTEASOME INHIBITORS

Abstract

Bacterial natural products arising from non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), or hybrids of both are of immense pharmaceutical interest. Epoxomicin, an NRPS/PKS hybrid product biosynthesised by the actinomycete G. coeruleoviolacea ATCC53904, represents an important class of epoxyketone-containing proteasome inhibitors with potential as an anti-cancer therapeutic. In recent years, several strategies have been developed for the rational engineering of NRPS systems to generate synthetic NRPSs capable of producing the desired peptide. In this study, we utilise these strategies to engineer the epoxomicin NRPS with the goal of producing novel epoxyketone-containing compounds with improved bioactivity. Our efforts to use Streptomyces as a host for NRPS engineering because of its tolerance of high-GC foreign DNA were unsuccessful due to failed conjugation from E. coli. Through NRPS engineering in E. coli, using codon-optimised epoxomicin BGC, we successfully produced twenty-seven novel NRPS/PKS hybrid compounds. However, we were unsuccessful in producing epoxyketone-containing compounds, possibly due to high specificity of the epoxomicin PKS and Acyl-Coenzyme A Dehydrogenase-like (ACAD) enzyme. Taken together, this study demonstrates the engineering of NRPS/PKS hybrids and proposes future experiments to improve the success rate of producing epoxyketone-containing compounds.