Cell-Free Noncanonical Redox Cofactor Systems

Noncanonical redox cofactor systems utilize nicotinamide adenine dinucleotide (phosphate), NAD(P)H, mimics to perform biotransformation reactions. Compared to systems utilizing native NAD(P)H, these noncanonical redox cofactors can offer decreased cost of cofactor supply, improved system activities, and can even supply reducing power directly to targeted reactions in complex biological environments. When these systems are operated in cell-free settings, the high level of user control afforded by direct access to the reaction system enables specific tuning of cofactor parameters, enzyme activity, and reaction progression to maximize system productivity. In this chapter, we will describe methods for constructing these cell-free noncanonical redox cofactor systems. Specifically, methods, design concepts, and system adaptation will be discussed for applying noncanonical redox cofactors to both purified protein-based and crude lysate-based biotransformation systems.