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

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.