Engineering O 2 -Tolerant Chimeric Hydrogenases Optimized for Ferredoxin Coupling in Synechocystis sp. PCC 6803

ACS Synthetic Biology, vol. 14, pp. 4478–4495

Abstract

The development of hydrogenases capable of operating under oxygenic photosynthetic conditions remains a key challenge for sustainable biohydrogen production. In this study, we developed a series of chimeric NAD + -reducing [NiFe]-hydrogenases (SH) combining structural elements from the O 2 -tolerant SH of Cupriavidus necator ( Cn SH) and the ferredoxin-interacting SH of Synechocystis sp. PCC6803 ( Syn SH). By engineering chimeric HoxU and HoxF subunits, we developed constructs─MixSH, Ch-HoxEF Syn +U Cn, and Ch-HoxU swapCTD ─that successfully couple the Cn HoxYH hydrogenase module to the Syn HoxEFU reductase module while retaining O 2 tolerance and enhancing interaction with reduced ferredoxin. The lithoautotrophic growth of C. necator confirmed the tolerance of these variants to O 2, while activity assays in Synechocystis demonstrated partial hydrogenase function, including H 2 consumption and fermentative H 2 production. Notably, Ch-HoxEF Syn +U Cn retained ferredoxin interaction despite lacking the [4Fe4S] U4 cluster, showing [2Fe2S] F2 in HoxF as a functional ferredoxin-binding site. Moreover, we achieved the artificial integration of a [2Fe2S] cluster into Cn HoxF and identified the Cn HoxF N-terminal domain as structurally and functionally analogous to Syn HoxE. Although electron transfer efficiency and activity in Synechocystis remained limited, this work validates the modular engineering of [NiFe]-hydrogenases, uniting O 2 -tolerance with ferredoxin interaction and offering a foundational step toward photosynthesis-coupled H 2 production.

Authors 11

1. Elisabeth Lettau Aachen iAMB-Institute of Applied Microbiology

   Technische Universität Berlin · RWTH Aachen University

   Affiliation as printed

   RWTH Aachen University, iAMB−Institute of Applied Microbiology, Worringerweg 1, 52074 Aachen, Germany

   Technische Universität Berlin, Institute for Chemistry, Straße des 17. Juni 135, 10623 Berlin, Germany

2. Petra Till

   Helmholtz Centre for Environmental Research · TU Wien

   Affiliation as printed

   Helmholtz Centre for Environmental Research−UFZ-Leipzig, Department of Microbial Biotechnology, Permoserstraße 15, 04318 Leipzig, Germany

   TU Wien, Institute of Chemical, Environmental and Bioscience Engineering−ICEBE, Gumpendorfer Straße 1a, 1060 Wien, Austria

3. Jörg Toepel

   Helmholtz Centre for Environmental Research

   Affiliation as printed

   Helmholtz Centre for Environmental Research−UFZ-Leipzig, Department of Microbial Biotechnology, Permoserstraße 15, 04318 Leipzig, Germany

4. Jens Appel

   University of Kassel

   Affiliation as printed

   Universität Kassel, Institut für Biologie, Heinrich-Plett-Straße 40, 34132 Kassel, Germany

5. Marko Boehm

   University of Kassel

   Affiliation as printed

   Universität Kassel, Institut für Biologie, Heinrich-Plett-Straße 40, 34132 Kassel, Germany

6. Christian Lorent

   Technische Universität Berlin

   Affiliation as printed

   Technische Universität Berlin, Institute for Chemistry, Straße des 17. Juni 135, 10623 Berlin, Germany

7. Christian Teutloff

   Freie Universität Berlin

   Affiliation as printed

   Freie Universität Berlin, Institute für Physik, Arnimallee 14, 14195 Berlin, Germany

8. Robert Ludwig Mach

   TU Wien

   Affiliation as printed

   TU Wien, Institute of Chemical, Environmental and Bioscience Engineering−ICEBE, Gumpendorfer Straße 1a, 1060 Wien, Austria

9. Kirstin Gutekunst

   University of Kassel

   Affiliation as printed

   Universität Kassel, Institut für Biologie, Heinrich-Plett-Straße 40, 34132 Kassel, Germany

10. Bruno Bühler

    Helmholtz Centre for Environmental Research

    Affiliation as printed

    Helmholtz Centre for Environmental Research−UFZ-Leipzig, Department of Microbial Biotechnology, Permoserstraße 15, 04318 Leipzig, Germany

11. Lars Lauterbach corresponding Aachen iAMB-Institute of Applied Microbiology

    Fraunhofer Institute for Molecular Biology and Applied Ecology · RWTH Aachen University

    Affiliation as printed

    Fraunhofer-Institut für Molekularbiologie und Angewandte Ökologie IME, Forckenbeckstr. 6, 52074 Aachen, Germany

    RWTH Aachen University, iAMB−Institute of Applied Microbiology, Worringerweg 1, 52074 Aachen, Germany