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.