Molecular hydrogen (H₂) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H₂ production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H₂ syntheses rely mainly on light, water, and CO₂ and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H₂-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O₂) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O₂-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O₂-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O₂ sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O₂ tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH⁺ to utilize H₂ as sole electron source to fix CO₂. Syn_ReSH⁺ also was able to produce H₂ under dark fermentative conditions as well as in presence of light, under conditions fostering intracellular NADH excess. These findings highlight a high level of interconnection between ReSH and cyanobacterial redox metabolism. This study lays a foundation for further engineering, e.g., of electron transfer to ReSH via NADPH or ferredoxin, to finally enable photosynthesis-driven H₂ production.

Sara Lupacchini

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