Thioester-mediated biocatalytic amide bond synthesis with in situ thiol recycling

Activation of carboxylic acids to thioesters plays an important role in biology. However, biochemical studies and biotechnological applications are hampered by a general lack of access to thioesters, especially those based on HS-Coenzyme A. Here we show that a generic thioester recycling enzyme by exploiting a promiscuous activity of carboxylic acid reductase (CARsr-A). The adenylation domain of CARsr (CARsr-A) catalyses the conversion of a wide range of carboxylic acids to acyl-S-Coenzyme A and other thioesters in good yields. CARsr-A was used in situ as part of a recycling system to regenerate thioesters for acyl-S-Coenzyme A dependent enzymes in one-pot reactions. This concept of thioester recycling was demonstrated with a range of acyltransferases allowing formation of diverse amides and non-native acylation of lysine sidechains in a histone-derived peptide using the epigenetic writer, lysine acetyltransferase HATp300. Overall, these results establish a generic platform for thioester formation towards amide formation and beyond.