Rewiring cyanobacterial photosynthesis by the implementation of an oxygen-tolerant hydrogenase.

Molecular hydrogen (H) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H syntheses rely mainly on light, water, and CO and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH to utilize H as sole electron source to fix CO. Syn_ReSH also was able to produce H 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 H production.