The scope and relevance of biocatalysis has expanded over recent years, with cascading enzyme reactions for the conversion of low value renewable feedstocks into high value products represents a keystone of renewable green chemistry . However, one of the main limitations to the application of such enzyme systems to energy-intensive reactions is the cost of providing a continuous supply of diffusible cofactors or cosubstrates [1-2]. Thus there is an emerging requirement to develop successful multi-enzyme cascades using heterogeneous enzyme catalysts coupled with cell-free cofactor regeneration . In particular, whilst using immobilized enzymes in flow offers the advantages of scalability and yield over batch-wise enzymic processes, using cofactor-dependent enzymes in a flow system means that the enzyme, the cofactor recycling enzyme and the cofactor must be immobilized in such a way that the cofactor is free to bind appropriately and participate in reaction with both enzymes .
We have developed a novel and generalizable chemo-genetic enzyme engineering approach that enables the fabrication of multifunctional biocatalysts for modular, multistep, biocatalytic, continuous-flow reactors using cofactor-dependent enzymes . The resultant multifunctional biocatalysts, termed nanomachines, encompass both tethered cofactor recycling and an immobilization protein domain which allows them to be covalently attached to surfaces and used in flow reactor systems (Figure 1). Fit-for-purpose nanomachines to catalyse the synthesis of a variety of chiral pharmaceutical intermediates have been demonstrated, encompassing nanomachines for enzymes ranging from kinases to reductases and utilising tethered cofactors for NAD(H), NADP(H) and ATP-dependent chemistries. Combination of three different nanomachines into a nanofactory yielded up to 100% conversion of glycerol to glycerol-3-phosphate and dihydroxyacetone phosphate (DHAP), with final DHAP yields of up to 60% conversion in batch reactions, and close to 90% yield of 3,4-dihydroxy-2-oxyhexyl phosphate (3S,4R-DHOP; a precursor to the anti-diabetic drug D-fagomine).