Peptide-based natural products known as non-ribosomal peptides (NRPs) are one of the most promising resources for drug discovery and development owing to a broad range of biologically important properties, including antimicrobial, anticancer, and immunosuppressive activities. They are biosynthesized by multimodular enzymes known as non-ribosomal peptide synthetases (NRPSs). Adenylation (A) domains act as the gatekeepers of NRPSs ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. The A-domain catalyst serves as an entry point for an amino acid into NRPS machinery. Therefore, the A-domain makes them attractive targets for the reprogramming of NRPS machinery for the production of non-native NRPs. Aryl acid building blocks are most commonly observed in iron-chelating siderophores, but are not limited to them. The non-ribosomal codes toward aryl acid substrates are poorly understood. Very little is known about the reprogramming of aryl acid A-domains. Here we show that a single asparagine-to-glycine mutation in an aryl acid A-domain creates novel enzyme specificities toward a wide range of non-native aryl acids. The reprogrammed catalyst is capable of activating the non-native aryl acids functionalized with nitro, cyano, bromo, and iodo, albeit with no enzymatic activity of wild-type enzyme toward these substrates (Figure 1). Co-crystal structures with non-hydrolysable aryl-AMP analogues revealed the origins of substrate promiscuity expansion, highlighting an enlarged substrate binding pocket of the enzyme. Our finding may be exploited to produce diversified aryl acid-containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis.

Reprogramming aryl acid adenylation domains for non-native building blocks

2019 Fall National Meeting

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