Identifying the Minimal Enzymes Required for Biosynthesis of Epoxyketone Proteasome Inhibitors

Abstract

Epoxyketone proteasome inhibitors have attracted much interest due to their potential as anti-cancer drugs. While the biosynthetic gene clusters for several peptidyl epoxyketone natural products have recently been identified, the enzymatic logic involved in the formation of the terminal epoxyketone pharmacophore has been relatively unexplored. Here, we report the identification of the minimal set of enzymes from the eponemycin gene cluster necessary for the biosynthesis of novel metabolites containing a terminal epoxyketone pharmacophore in Escherichia coli, a versatile and fast-growing heterologous host. This set of enzymes includes a non-ribosomal peptide synthetase (NRPS), a polyketide synthase (PKS), and an acyl-CoA dehydrogenase (ACAD) homolog. In addition to the in vivo functional reconstitution of these enzymes in E. coli, in vitro studies of the eponemycin NRPS and 13C-labeled precursor feeding experiments were performed to advance the mechanistic understanding of terminal epoxyketone formation.

Epoxomicin and Eponemycin Biosynthesis Involves gem‐Dimethylation and an Acyl‐CoA Dehydrogenase‐Like Enzyme

“…During the preparation of our manuscript, Liu et al. published a study on the identification of the minimal gene cluster for the biosynthesis of epoxyketone proteasome inhibitors and came to the same conclusions . They showed that in an E. coli heterologous system, only the genes for the NRPS/PKS machinery ( epnG / epnH ), the acyl CoA‐dehydrogenase ( epnF ), and the external addition of fatty acids are necessary to produce an epoxyketone compound.…”

“…During the preparation of our manuscript,L iu et al published as tudy on the identification of the minimal gene cluster for the biosynthesis of epoxyketonep roteasome inhibitors and came to the same conclusions. [8] They showedt hat in an E. coli heterologouss ystem,o nly the genes for the NRPS/PKS machinery (epnG/epnH), the acyl CoA-dehydrogenase (epnF), and the externala ddition of fatty acids are necessary to produce an epoxyketone compound. Feeding of isotope-enriched precursors in the same setupa nd MS analysis indicated that two methylg roups are incorporateda nd one carbon from acetate is lost by decarboxylation duringe poxyketoneb iosynthesis.…”

“…A recent study on eponemycin biosynthesis in Escherichia coli by Liu et al. will be discussed in detail below …”

“…Indeed, analysis of both the Δ epnF and Δ epxF knockout strains strongly indicates that the conserved ACADs are essentially involved in the formation of the epoxyketone warhead of proteasome inhibitors. This is in agreement with the results from Liu et al., who showed that in E. coli , only EpnF and the NRPS/PKS machinery are needed to form an epoxyketone . Curiously, the deletion of both CYP genes epnI and epnK resulted in the abolishment of eponemycin production, whereas epoxomicin was still accumulated in the Δ epxC CYP mutant.…”

“…[7] Ar ecent study on eponemycin biosynthesis in Escherichia coli by Liu et al will be discussed in detail below. [8] Both the epoxomicin (epx)a nd eponemycin (epn)g ene clusters encodealinear non-ribosomal peptides ynthetase/polyketide synthetase (NRPS/PKS) assembly line that mostl ikely catalyzes the construction of an acylated peptide and the extension of the terminal leucine residue. Comparison of the pathways indicates the involvement of an acyl-CoA dehydrogenase( ACAD) and ac ytochrome P450 (CYP) enzyme in the formation of the a',b'-epoxyketone, as the respective genes are highlyc onserved.…”