LAUSR

The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by‐product of its nitrogenase‐catalyzed nitrogen‐fixing activity. Such H2 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 H2. The resulting strain photoproduces H2 and self‐reports its own H2 production through fluorescence. This model system represents a unique method of developing hydrogenase‐based H2 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 H2. Biotechnol. Bioeng. 2017;114: 291–297. © 2016 Wiley Periodicals, Inc.