The alpha,beta-epoxyketone proteasome inhibitor TMC-86A was discovered as a previously unreported metabolite of Streptomyces chromofuscus ATCC49982, and the gene cluster responsible for its biosynthesis was identified via genome sequencing. Incorporation experiments with [C-13-methyl]L-methionine implicated an alpha-dirriethyl-beta-keto acid intermediate in the biosynthesis of TMC-86A. Incubation of the chemically synthesized alpha-dimethyl-beta-keto acid with a purified recombinant flavin-dependent enzyme that is conserved in all known pathways for epoxyketone biosynthesis resulted in formation of the corresponding alpha-methyl-alpha,beta-epoxyketone. This transformation appears to proceed via an unprecedented decarboxylation dehydrogenation mono-oxygenation cascade. The biosynthesis of the TMC-86A warhead is completed by cytochrome P450-mediated hydroxylation of the alpha-methyl-alpha,beta-epoxyketone.