Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H sub(2) production

The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H sub(2) as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H sub(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 sub(2). The resulting strain photoproduces H sub(2) and self-reports its own H sub(2) production through fluorescence. This model system represents a unique method of developing hydrogenase-based H sub(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 sub(2). Biotechnol. Bioeng. 2017; 114: 291-297. The authors have created a self-reporting bacterial system for the detection and selection of improved H sub(2)-production characteristics. They describe photosynthetic, hydrogenase-based H sub(2) production in Rhodobacter capsulatus in the absence nitrogenase activity. The system has potential use in the directed evolution of hydrogenases, hydrogenase assembly proteins, and hydrogenase fusion proteins; in the selection and modeling of increased H sub(2) metabolism in bacteria; and in optimizing photosynthetic production of H sub(2).