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 × 10⁴ 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.