An Atypical Acyl‐CoA Synthetase Enables Efficient Biosynthesis of Extender Units for Engineering a Polyketide Carbon Scaffold

Abstract

Acyl-CoAs are key precursors of primary and secondary metabolism. Their efficient biosynthesis is often impeded by the limited substrate specificity and low in vivo activity of acyl-CoA synthetases (ACSs) due to regulatory acylation of the catalytically important lysine residue in motif A10 (Lys-A10). In this study, we identified an unusual ACS (UkaQ) from the UK-2A biosynthetic pathway that naturally lacks the Lys-A10 residue and exhibits extraordinarily broad substrate specificity. Protein engineering significantly improved its stability and catalytic activity, enabling it to synthesize a large variety of acyl-CoAs with highly robust activity. By combining it with permissive carboxylases, we produced a large array of polyketide extender units and obtained six novel halobenzyl-containing antimycin analogues through an engineered biosynthetic pathway. This study significantly expands the catalytic mode of ACSs and provides a potent tool for the biosynthesis of acyl-CoA-derived natural products.

Current State‐of‐the‐Art Toward Chemoenzymatic Synthesis of Polyketide Natural Products

“…Commonly, malonates are chemically appended with the side chain of choice (if not commercially available) before enzymatic ligation with a malonyl‐CoA ligase, such as MatB, [37] which has demonstrated success in both in vitro and in vivo settings [38–42] . This approach also lends itself to a broad array of monocarboxylic acids, which can be ligated to CoA using one of many available promiscuous acyl‐CoA ligases, such as AcsA, FACL, or UkaQ, before serving as a precursor in polyketide biosynthesis [43–45] . With this, an effective understanding of how various thioester building blocks are interconverted has enabled the construction of a chemoenzymatic roadmap between them.…”

“…[38][39][40][41][42] This approach also lends itself to a broad array of monocarboxylic acids, which can be ligated to CoA using one of many available promiscuous acyl-CoA ligases, such as AcsA, FACL, or UkaQ, before serving as a precursor in polyketide biosynthesis. [43][44][45] With this, an effective understanding of how various thioester building blocks are interconverted has enabled the construction of a chemoenzymatic roadmap between them. Leveraging synthetic coupling or acylation methods in tandem with acyl-CoA dehydrogenases (AcDHs), enoyl-CoA carboxylase/reductases (ECRs), and MatB, recent efforts have identified and coupled enzymes with high substrate tolerance to access a broad array of thioester substrates.…”