title: Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2production.
abstract: The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2production, 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 H2and self-reports its own H2production through fluorescence. This model system represents a unique method of developing hydrogenase-based H2production 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.
authors.affiliation: GeneBiologics, LLC, Boulder, Colorado.
authors.forename: Matt S A
authors.initials: MS
authors.lastname: Wecker
authors.affiliation: United States Air Force Academy, Department of Chemistry, Colorado Springs, Colorado.
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Beaton
authors.affiliation: United States Air Force Academy, Department of Chemistry, Colorado Springs, Colorado.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Chado
authors.affiliation: National Renewable Energy Laboratory, MS 3313, 15013 Denver West Parkway, Golden, Colorado, 80401.
authors.forename: Maria L
authors.initials: ML
authors.lastname: Ghirardi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D046969:Clostridium acetobutylicum / Q000201:enzymology / Q000235:genetics; D057166:High-Throughput Screening Assays; D006859:Hydrogen / Q000032:analysis / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism; D008027:Light; D060847:Metabolic Engineering; D058433:Photobioreactors / Q000382:microbiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006859:Hydrogen; D006864:Hydrogenase
keywords: H2 production; R. capsulatus H2 sensor; high-throughput screening; nitrogenase; photobiohydrogen