Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis

Biocatalysis is an attractive route for the synthesis of complex organic molecules, such as pharmaceuticals, due to the properties of enzymes (high specificity and high catalytic rate). Ideally, we would be able to use enzymes in continuous-flow reactors to benefit from the advantages of continuous-flow chemistry (flexibility, control, product stream purity, low capital cost and improved yields for some reactions). However, continuous-flow applications for biocatalysis face substantial technical obstacles, particularly for enzymes that require cofactors. In the work presented here we tackle two of these obstacles: the provision of cofactor and cofactor recycling in flow, and enzyme immobilization without loss of activity. This is achieved through the production of modular biocatalysts that retain and recycle their cofactors, and that allow orthogonal, site-specific covalent conjugation to a surface. This generalizable engineering approach allowed us to build a complex, multistep flow reactor that outperforms previously published systems for cofactor-dependent continuous-flow biocatalysis. Tackling the loss of expensive cofactors is a key challenge in continuous-flow biocatalysis. This work reports immobilized enzymes with a tethered cofactor that is channelled between a recycling and a catalysis module facilitating total turnover numbers of NAD+ and ATP exceeding 10,000 in biocatalytic flow systems.