Salinosporamide A is a potent irreversible proteasome inhibitor presently in phase Ib human clinical trials for the treatment of multiple myeloma and other cancers. This marine bacterial natural product has a distinctive mechanism of action based on its ?-lactam-?-lactone pharmacophore that differs from the only FDA-approved proteasome inhibitor, the peptide boronate bortezomib. During the last period of support, we established the biosynthetic foundation of salinosporamide assembly and discovered a number of novel enzymatic reactions in halogenation, prephenate biochemistry, and polyketide precursor supply. Translation of this basic knowledge allowed us to rationally design through genetic engineering new salinosporamide analogues for biological evaluation. This work helped determine the structure-activity relationships within the salinosporamide family of anticancer agents. Despite our significant progress to date, we still only have a cursory understanding of how salinosporamide is biosynthesized due to its unprecedented assembly from novel molecular building blocks. Numerous questions remain, while new opportunities have surfaced in response to discoveries made in this ongoing research program. Opportunities in enzyme discovery, synthetic biology, chemoenzymatic synthesis, genome mining, and proteasome biochemistry are uniquely suited for this natural product biosynthetic program. To accomplish the broad goals outlined in this application, we propose a multidisciplinary project involving five specific aims. First, we plan to functionally and structurally characterize the SalC ketosynthase and its key biosynthetic role in the formation of the ?-lactam-?-lactone core of salinosporamide. Second, we will apply the function of SalC to develop a streamlined chemoenzymatic synthesis of salinosporamide derivatives based on a focused library of ?-lactam-?-lactones from synthetic acylamino acid thioesters with recombinant salinosporamide biosynthetic enzymes. Third, we aim to functionally characterize the biosynthetic enzymes responsible for the synthesis of salinosporamide's novel amino acid residue, cyclohexenylalanine, which is paramount to its potent proteasome binding affinity. Fourth, we will functionally characterize the dedicated proteasome ?-subunit SalI and its hypothesized role in S. tropica self-resistance against salinosporamide. And fifth, we plan to develop new crotonyl-CoA reductase-based expression systems for the engineered biosynthesis of new polyketide synthase extender units with halogenated (fluorine and chlorine) and branched side chains for the design of new polyketide molecules. PUBLIC HEALTH RELEVANCE: The recent FDA approval of the peptide boronate bortezomib (Velcade) as a first-in-class inhibitor of the 20S proteasome to treat multiple myeloma and other cancers has fueled the discovery and development of new drugs to address the long-term utility of bortezomib due to dose-limiting toxicities and the development of resistance. One of the most promising drug candidates currently in human clinical trials is salinosporamide A (USAN name marizomib), a natural product possessing a complex, densely functionalized ?-lactam-?-lactone pharmacophore that is chemically distinct from bortezomib and other peptide-based proteasome inhibitors. Because of the clinical promise and mechanistic novelty of salinosporamide A, we aim to characterize the fundamental mechanism of salinosporamide biosynthesis in order to develop bioengineering approaches to enhance the productivity of the natural product and rationally design analogs for biological evaluation.