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The integration of enzyme-catalyzed reactions in flow systems has been boosted during the last few years. Flow chemistry has been proposed in modern synthetic chemistry as a technology for process...

Expanding the toolbox of enzymatic reactions accessible to organic chemists is one of the major goals in biocatalysis. Here, we describe the development of an acyltransferase variant from Mycobacterium smegmatis (MsAcT) where a strategic Ser/Cys exchange in the catalytic triad dramatically expanded its synthetic capability, yielding a biocatalyst able to efficiently catalyse the formation of thioesters and tertiary amides in water. Preparative scale (250 mM) biotransformations were performed starting from different thiols and secondary amines with excellent yields and reactions times, using vinyl esters as acylating agents. The high substrate-tocatalyst ratio and the cofactor independence make this process a sustainable and cost-effective procedure that was successfully applied to the synthesis of acetyl-CoA as well as structurally simpler analogues. Computational studies provided insights into enzymatic selectivity and substrate recognition.The enzyme toolbox available for synthetic chemistry dramatically expanded in the last few years thanks to new high-throughput screening techniques and protein engineering. 1,2 Enzymatic reactions can be nowadays used in preparative chemistry, enabling new easy, mild, and high yielding methods. Nevertheless, a few key-reactions are noticeably missing from this toolbox especially in terms of productivity and biocatalyst availability, such as thioester and tertiary amide preparation; proof of concepts have been reported, but no suitable preparative biocatalytic

As an alternative to the traditional chemical synthesis or in vivo production of l-pipecolic acid, we have developed two ex vivo strategies using purified and immobilised enzymes for the production of this key building block.