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

Funding Information: P.W., A.F., and J.A. contributed equally to this work. The authors are grateful to the Bundesministerium für Bildung und Forschung (BMBF) in the framework of the project CyFun (03SF0652A). The authors also thank Prof. Wolfgang Lubitz (Max Planck Institute for Chemical Energy Conversion, Mülheim an der Ruhr) for providing the DvMF[NiFe]-H2ase used for the fabrication of the H2 microsensor. Part of the project was funded by the research training group GRK2341 “Microbial Substrate Conversion (MiCon)” of the German research council (DFG) and the Dietmar Hopp Stiftung. P.W. is grateful for the financial support provided by the China Scholarship Council (CSC). F.C. is grateful to the support provided by FCT–Fundação para a Ciência e a Tecnologia, I.P. through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020). Open access funding enabled and organized by Projekt DEAL. Funding Information: P.W., A.F., and J.A. contributed equally to this work. The authors are grateful to the Bundesministerium für Bildung und Forschung (BMBF) in the framework of the project CyFun (03SF0652A). The authors also thank Prof. Wolfgang Lubitz (Max Planck Institute for Chemical Energy Conversion, Mülheim an der Ruhr) for providing the DvMF[NiFe]‐Hase used for the fabrication of the H microsensor. Part of the project was funded by the research training group GRK2341 “Microbial Substrate Conversion (MiCon)” of the German research council (DFG) and the Dietmar Hopp Stiftung. P.W. is grateful for the financial support provided by the China Scholarship Council (CSC). F.C. is grateful to the support provided by FCT–Fundação para a Ciência e a Tecnologia, I.P. through MOSTMICRO‐ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020). 2 2 Publisher Copyright: © 2023 The Authors. Advanced Energy Materials published by Wiley-VCH GmbH.Photosynthetic hydrogen (photoH2) production is an elegant approach to storing solar energy. The most efficient strategy is to couple the hydrogen-producing enzyme, the hydrogenase (H2ase), directly to photosystem I (PSI), which is a light-driven nanomachine found in photosynthetic organisms. PSI–H2ase 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–H2ase fusions in vivo for in vitro photoH2 production is established. For this, cyanobacterial PSI–H2ase fusion mutants are generated and characterized concerning photoH2 production in vivo. The chimeric protein is purified and embedded in a redox polymer on an electrode where it successfully produces photoH2 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 photoH2 production in a target-oriented manner in the future.publishersversionpublishe