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.

分子氢（H ₂）被认为是未来替代化石燃料的理想能源载体。由含氧光合作用驱动的生物技术 H ₂生产看起来很有前景，因为生物催化剂和 H ₂合成主要依赖光、水和 CO ₂而不是稀有金属。该生物过程需要将光合水氧化装置与产生 H _{2 的}氢化酶偶联。然而，该策略受到氧（O ₂）的同时释放的阻碍，氧是大多数氢化酶的强抑制剂。在这里，我们通过引入 O ₂耐受氢化酶转化为光养细菌，即蓝藻模型菌株集胞藻。PCC 6803。为此，编码来自真养产碱菌( Re SH)的可溶性、O ₂耐受性和 NAD(H) 依赖性氢化酶的基因簇被功能性转移到具有敲除天然 O ₂敏感性的集胞藻菌株。氢化酶。有趣的是，光合活性细胞产生了 O ₂耐受性Re SH，其活性在体外和体内得到证实。此外，Re SH 启用了构建的应变Syn_ReSH ⁺ 利用H ₂作为唯一的电子源来固定CO ₂。SYN _重新SH ⁺ 也能生成H ₂暗发酵的条件下，以及在光的存在下，培养细胞内NADH过量的条件下。这些发现强调了Re SH 和蓝藻氧化还原代谢之间的高度联系。该研究为进一步工程奠定了基础，例如通过 NADPH 或铁氧还蛋白将电子转移到Re SH，最终实现光合作用驱动的 H ₂生产。