Extending Cofactor‐Tethered Nanomachines to Complex Multienzyme Redox Cascades in Continuous Flow

Multienzyme cascades are emerging as alternatives to fermentation for converting biogenic feedstocks into value‐added chemicals and fuels. A major bottleneck in redox transformations is the loss of costly and unstable cofactors in continuous‐flow systems, typically necessitating co‐immobilization and complex regeneration schemes. Self‐sufficient biocatalytic nanomachines, created by fusing enzymes with tethered cofactors, offer a streamlined solution by enabling intramolecular cofactor retention and recycling. Here, we demonstrate that such nanomachines can be integrated into a complete redox‐dependent cascade, exemplified by a 10‐step glucose‐to‐isobutanol pathway. Oxidoreductases were fused into redox pairs using peptide linkers containing a single cysteine for covalent attachment of polyethylene glycol (PEG)‐modified nicotinamide cofactors, with rational selection of monomeric enzymes ensuring structural compatibility and soluble expression. The resulting nanomachines remained catalytically competent and produced isobutanol in both batch and continuous‐flow operation without the addition of free NAD + . A total turnover number of ∼1.4 × 10 4 was achieved for tethered PEG–NAD + , among the highest reported for immobilized nicotinamide cofactors. Use of the ultrastable analogue PEG–cNAD + further demonstrates the modularity of the platform. Together, these results establish a proof‐of‐concept for cofactor‐autonomous multienzyme redox cascades in continuous flow and define design principles for future cell‐free synthesis systems.