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Extending Cofactor-Tethered Nanomachines to Complex Multienzyme Redox Cascades in Continuous Flow
Zinnia Dsouza1, Jan-Simon Jeshua Friedrichs1, Okke Melse1
1Chair of Chemistry of Biogenic Resources, Campus Straubing for Biotechnology and Sustainability, Technical University of Munich, Straubing, Germany.
Chemsuschem
|July 25, 2026
Summary
Researchers developed self-sufficient biocatalytic nanomachines that retain and recycle essential cofactors, enabling efficient cofactor-autonomous multienzyme cascades for chemical synthesis. This innovation streamlines redox transformations in continuous flow systems.
Area of Science:
- Biocatalysis and synthetic biology
- Chemical engineering
- Biotechnology
Background:
- Multienzyme cascades offer sustainable alternatives to fermentation for producing chemicals and fuels from biogenic feedstocks.
- Cofactor loss and complex regeneration are significant challenges in continuous-flow redox biocatalysis.
- Self-sufficient nanomachines with tethered cofactors present a solution for intramolecular cofactor retention and recycling.
Purpose of the Study:
- To demonstrate the integration of self-sufficient biocatalytic nanomachines into a complete, cofactor-autonomous multienzyme redox cascade.
- To establish a proof-of-concept for a 10-step glucose-to-isobutanol pathway using fused enzymes and tethered nicotinamide cofactors.
- To define design principles for future cell-free synthesis systems.
Main Methods:
- Enzymes were fused into redox pairs using peptide linkers with a single cysteine for attaching polyethylene glycol (PEG)-modified nicotinamide cofactors.
- Monomeric enzymes were rationally selected for structural compatibility and soluble expression.
- The nanomachines were tested in both batch and continuous-flow operations for isobutanol production.
Main Results:
- The engineered nanomachines successfully produced isobutanol in a 10-step cascade without requiring free nicotinamide adenine dinucleotide (NAD+).
- A high total turnover number of approximately 1.4 × 10^4 was achieved for the tethered PEG-NAD+ cofactor.
- The use of an ultrastable analogue, PEG-cNAD+, demonstrated the modularity and robustness of the platform.
Conclusions:
- Cofactor-autonomous multienzyme redox cascades can be effectively implemented in continuous flow systems using self-sufficient nanomachines.
- This approach overcomes cofactor instability and loss issues, paving the way for efficient cell-free synthesis.
- The developed platform provides a foundation for designing next-generation biocatalytic systems for sustainable chemical production.
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