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

Abstract

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.

Keywords: H2 production; R. capsulatus H2 sensor; high-throughput screening; nitrogenase; photobiohydrogen.