Development of a Rhodobacter capsulatus self‐reporting model system for optimizing light‐dependent, [FeFe]‐hydrogenase‐driven H₂ production

The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H 2 as a by‐product of its nitrogenase‐catalyzed nitrogen‐fixing activity. Such H 2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase‐based system (Ghirardi et al., 2009 Photobiological hydrogen‐producing systems. Chem Soc Rev 38(1):52–61). Here, we report the insertion of a Clostridium acetobutylicum [FeFe]‐hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H 2 . The resulting strain photoproduces H 2 and self‐reports its own H 2 production through fluorescence. This model system represents a unique method of developing hydrogenase‐based H 2 production in R. capsulatus , may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase‐associated genes, and provides a means of screening for increased metabolic production of H 2 . Biotechnol. Bioeng. 2017;114: 291–297.