Expression of a Clostridial [FeFe]-Hydrogenase in Chlamydomonas reinhardtii Prolongs Photo-Production of Hydrogen from Water Splitting

The high oxygen (O₂) sensitivity of green algal [FeFe]-hydrogenases is a significant limitation for the sustained production of hydrogen gas (H₂) from photosynthetic water splitting. To address this limitation we replaced the native [FeFe]-hydrogenases with a more O₂-tolerant clostridial [FeFe]-hydrogenase CaI in Chlamydomonas reinhardtii strain D66ΔHYD (hydA1⁻ hydA2⁻) that contains insertionally inactivated [FeFe]-hydrogenases genes. Expression and translocation of CaI in D66ΔHYD led to the recovery of H₂ photoproduction at ~ 20% of the rates of the wild-type parent strain D66. We show for the first time that a bacterial [FeFe]-hydrogenase can be expressed, localized and matured to a catalytically active form that couples to photosynthetic electron transport in the green alga C. reinhardtii. The lower rates of O₂ inactivation of CaI led to more sustained H₂ photoproduction when cultures were challenged with O₂ or kept under prolonged illumination at solar intensities. These results provide new insights into the requisites for attaining photobiological H₂ production from water splitting using a more O₂-tolerant hydrogenase.