Semisynthetic [FeFe]-hydrogenase with stable expression and H2 production capacity in a photosynthetic microbe

Hydrogen (H2) is a promising future chemical energy carrier and feedstock with several renewable production options, including electrolyzers and biological/bioinspired systems. The top H2 producers in nature are [FeFe]-hydrogenases, high turnover metalloenzymes with a complex maturation process that can be circumvented by artificial synthetic activation. Here, we report the expression and activation of group A and D [FeFe]-hydrogenases in a photosynthetic host organism, the unicellular cyanobacterium Synechocystis PCC 6803. The hydrogenase from Solobacterium moorei (group A) facilitates high in vivo H2 production from purely photoautotrophically generated substrates and unmistakably links to the metabolism of the photosynthetic host. Cells harboring the non-native, semisynthetic enzyme retain their H2 production capacity for several days after synthetic activation. This work expands both the number and the diversity of [FeFe]-hydrogenases examined in a photosynthetic background and provides important insights for future investigations into the development and understanding of biological and biohybrid H2 production systems.