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 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.