Polymer-Based Module for NAD+ Regeneration with Visible Light

The regeneration of enzymatic cofactors by cell-free synthetic modules is a key step towards producing a purely synthetic cell. Herein, we demonstrate the regeneration of the enzyme cofactor NAD⁺ by photo-oxidation of NADH under visible-light irradiation by using metal-free conjugated polymer nanoparticles. Encapsulation of the light-active nanoparticles in the lumen of polymeric vesicles produced a fully organic module able to regenerate NAD⁺ in an enzyme-free system. The polymer compartment conferred physical and chemical autonomy to the module, allowing the regeneration of NAD⁺ to occur efficiently, even in harsh chemical environments. Moreover, we show that regeneration of NAD⁺ by the photocatalyst nanoparticles can oxidize a model substrate, in conjunction with the enzyme glycerol dehydrogenase. To ensure the longevity of the enzyme, we immobilized it within a protective silica matrix; this yielded enzymatic silica nanoparticles with enhanced long-term performance and compatibility with the NAD⁺-regeneration system.