Epoxyketone proteasome inhibitors have attracted much interest due to their potential as anti-cancer drugs. not yet been fully elucidated. Nonetheless bioinformatics analysis has shown four genes to be conserved across the epoxomicin and eponemycin gene clusters and these genes encode a non-ribosomal peptide synthetase (NRPS) i.e. EpnG/EpxD; a polyketide synthase (PKS) i.e. EpnH/EpxE; an acyl-CoA dehydrogenase (ACAD) homolog i.e. EpnF/EpxF; and a cytochrome P450 monooxygenase i.e. EpnI/EpxC. Based on this analysis a biosynthetic pathway in which a hybrid NRPS-PKS assembly line generates a carboxylic acid that is subsequently modified by the ACAD and cytochrome P450 to form the terminal epoxyketone moiety was previously proposed. Notably however the recently identified gene cluster for macyranone biosynthesis lacks a gene encoding a cytochrome P450 which led to an alternative acyl-carrier-bound proposed mechanism for epoxyketone formation involving the ACAD homolog MynC and possibly the type II thioesterase MynH. Determine 1 Biosynthesis of a peptidyl epoxyketone in 357.2384 [M+H]+ 4 as a means to quickly and systematically identify the enzymes necessary for the generation of the terminal epoxyketone moiety. Due to its genetic tractability fast growth rate and well-characterized metabolism is a particularly attractive host for studying the biosynthesis of natural products.[16-19] Furthermore the reconstitution of NRPS-PKS activity in provides a superior platform for the overproduction and diversification of their products.[20-21] To determine the enzymes required for the biosynthesis of the terminal epoxyketone pharmacophore we co-expressed the four genes from the eponemycin gene cluster (on three plasmids. The plasmids were co-transformed into BAP1 which contains a chromosomal copy of the phosphopantetheinyl transferase encoding gene (Physique 1A). Consequently cultures of JL6 were supplemented with 1 mM hexanoic acid or octanoic acid at the time of induction to promote the incorporation of these alternative fatty acyl groups into the assembly line. The culture extracts of JL6 and the control strain were analyzed by liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based comparative metabolomics  and two new compounds (3 and 4) with masses consistent with molecular formulae of C18H32N2O5 and C20H36N2O5 respectively were detected in high large quantity in the extracts of JL6 but were absent from your extracts of the control strain (Figures 1B and S1-2). Further HRMS/MS analysis confirmed 3 and 4 to be consistent with a C6 or C8 fatty acyl group condensed with serine leucine and malonate but it did not give insight VCL into the structure of the terminal moiety resulting from modifications (-)-Epicatechin gallate around the malonate (Figures S1 and S2). To uncover the exact molecular structure of new compounds we then scaled up the production cultures and purified ~2 mg of the major (-)-Epicatechin gallate product 4 for NMR spectroscopic analysis. Compound 4 was purified by following the fractions using LC-MS and the yield of 4 was ~0.2 mg/L. Analysis of the 1D (1H 13 and 2D (HSQC COSY HMBC) NMR spectra of purified 4 (-)-Epicatechin gallate confirmed the presence of an acylated peptide composed of an eight-carbon fatty acyl chain serine and leucine connected to a methyl-substituted epoxyketone (Figures (-)-Epicatechin gallate 1 S3-7 and Table S3). Notably the proton and carbon shifts of the methyl-substituted epoxyketone and leucine residue are in strong agreement with those reported for a standard of 2 which has these same functionalities. Moreover the methyl-substituted quaternary epoxide ring was supported by HMBC correlations from your C3 methyl protons (δH 1.52) to both the quaternary carbon C2 (δC 59.5) and the oxygenated methylene carbon C1 (δC 52.7) and by HMBC correlations from your C1 protons (δH 2.91 and 3.28) to C2 and C3 (δC 16.9) (Figures 1 S7 and Table S3). HMBC correlations from your C1 and C3 protons to the ketone carbon C4 (δC 208.8) also confirmed the connectivity of the epoxide (Figures 1 S7 and Table S3). Thus based on our HRMS and NMR analyses we decided 4 to be a new terminal epoxyketone compound. To probe the.