Catalysis

Thioester Mediated Biocatalytic Amide Bond Synthesis with In Situ Thiol Recycling

Authors

Abstract

The conversion of carboxylic acids to thioesters is a key step in the biosynthesis of natural products, resulting in activation of the acyl groups for a broad range of subsequent biochemical reactions, such as acylations and alkylations. In particular, the thioesters of Coenzyme A (CoA-SH) play disproportionate roles in many metabolic pathways, from fatty acids, polyketides and peptides to epigenetic post-translational modifications, such as N-, O- and S-acylations of proteins. However, lack of access to a broad range of structurally diverse thioesters and the structural complexity of CoA-SH have limited the wider exploitation of thioesters in biochemistry, cell biology and biotechnology. Here we report an in-situ recycling system of thioesters from free carboxylic acids that aims to address these challenges. We show that the adenylation domain of the carboxylic acid reductase from Segniliparus rugosus (CARsr-A) can function as an efficient and robust acyl-S-CoA synthetase, accepting a wide range of carboxylic acids as substrates. In addition, CARsr-A was able to generate thioesters from structurally simpler thiols such as pantetheine. The resulting thioesters were shown to be substrates for acyltransferases leading to diverse amides, including more challenging targets such as pharmaceutically relevant aryl amides. Importantly, CoA-SH is regenerated in situ and can be used in sub-stoichiometric quantities. In a further application, the histone-derived peptide H4-20 was successfully acylated via thioester activation with a range of natural and unnatural ‘clickable’ carboxylic acids, by combination with the epigenetic ‘writer’ lysine acetyltransferase HATp300. Overall, this broad-spectrum biocatalyst for thioester synthesis, allowing in-situ CoA-SH recycling in combination with a range of thioester-dependent enzymes, provides a generic platform for thioester-dependent cell-free biosynthesis, with potential to open up applications beyond amide bond formation.

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