In Vivo Assembly of Photosystem I‐Hydrogenase Chimera for In Vitro PhotoH₂ Production

Abstract Photosynthetic hydrogen (photoH 2 ) production is an elegant approach to storing solar energy. The most efficient strategy is to couple the hydrogen‐producing enzyme, the hydrogenase (H 2 ase), directly to photosystem I (PSI), which is a light‐driven nanomachine found in photosynthetic organisms. PSI–H 2 ase fusions have been tested in vivo and in vitro. Both approaches have each their specific advantages and drawbacks. Here, a system to combine both approaches by assembling PSI–H 2 ase fusions in vivo for in vitro photoH 2 production is established. For this, cyanobacterial PSI–H 2 ase fusion mutants are generated and characterized concerning photoH 2 production in vivo. The chimeric protein is purified and embedded in a redox polymer on an electrode where it successfully produces photoH 2 in vitro. The combination of in vivo and in vitro processes comes along with reciprocal benefits. The in vivo assembly ensures that the chimeric protein is fully functional and suited for the fabrication of bioelectrodes in vitro. At the same time, the photoelectrochemical in vitro characterization now permits to analyze the assemblies in detail. This will open avenues to optimize in vivo and in vitro approaches for photoH 2 production in a target‐oriented manner in the future.