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Cyanobacterial in vivo solar hydrogen production using a photosystem I–hydrogenase (PsaD-HoxYH) fusion complex

Abstract

Photosynthetically produced hydrogen is an attractive, sustainable fuel. Semiartificial in vitro techniques have been successfully implemented in which hydrogenases were attached to isolated photosystems for hydrogen production. However, in vitro systems are in general short lived as metabolic processes that support self-repair and maintenance are missing. So far, photosystem–hydrogenase fusions have been tested in vitro only. Here, we report photosynthetic hydrogen production using a photosystem I–hydrogenase fusion in vivo. The NiFe-hydrogenase HoxYH of the cyanobacterium Synechocstis sp. PCC 6803 was fused to its photosystem I subunit PsaD in close proximity to the 4Fe4S cluster FB, which ordinarily donates electrons to ferredoxin. The resultant psaD-hoxYH mutant grows photoautotrophically, achieves a high concentration of photosynthetically produced hydrogen of 500 μM under anaerobic conditions in the light and does not take up the generated hydrogen. Our data indicate that photosynthetic hydrogen production in psaD-hoxYH is most likely based on both oxygenic and anoxygenic photosynthesis.

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Fig. 1: Hydrogen production in WT and Δhox/hoxYH related to the optical density at 750 nm (OD750) of the cultures.
Fig. 2: Strategy for the in vivo fusion of the Hox hydrogenase (HoxYH) of Synechocystis to its PSI by fusing the hydrogenase moiety hoxY to a truncated version of the PSI subunit psaD.
Fig. 3: Growth, hydrogenase activity, localization of the hydrogenase in WT and psaD-hoxYH (which is a ΔhoxΔpsaD/psaD-hoxYH mutant) and immunoblot analyses.
Fig. 4: Transitory in vivo photoH2 and O2 production in WT and psaD-hoxYH in darkness and upon illumination under anaerobic conditions in the absence and presence of DCMU.
Fig. 5: Assumed photosynthetic electron flow to the hydrogenase (HoxYH) in the psaD-hoxYH mutant.
Fig. 6: Lasting in vivo photoH2 production in WT and psaD-hoxYH under anaerobic conditions in continuous light in the absence and presence of DCMU.
Fig. 7: Light dependence of lasting photoH2 production in WT and psaD-hoxYH under anaerobic conditions in continuous light.
Fig. 8: Glucose dependence of lasting photoH2 production in WT and psaD-hoxYH in the presence of DCMU.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and the supplementary file. Source data for Figs. 1, 3a–c, 4a–f, 6a–f, 7a–d and 8a–d and for Supplementary Fig. 5 are provided with the paper.

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Acknowledgements

We thank R. Schulz for giving us a scientific home. HoxH and HoxY antibodies were provided by P. Nixon. This study was financed by grants from the Bundesministerium für Bildung und Forschung (BMBF, FP3 09) and the Deutsche Forschungsgemeinschaft (GU1522/2-1).

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J.A. and K.G. conceived the research. J.A. and V.H. constructed and characterized the mutants. M.B. performed the immunoblot analysis. J.A., V.H., M.B. and K.G. analysed data. K.G. wrote the manuscript and supervised the work.

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Correspondence to Kirstin Gutekunst.

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Appel, J., Hueren, V., Boehm, M. et al. Cyanobacterial in vivo solar hydrogen production using a photosystem I–hydrogenase (PsaD-HoxYH) fusion complex. Nat Energy 5, 458–467 (2020). https://doi.org/10.1038/s41560-020-0609-6

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