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

Abstract

The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H 2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H 2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system. 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 2. The resulting strain photoproduces H 2 and self-reports its own H 2 production through fluorescence. Furthermore, this model system represents a unique method of developing hydrogenase-based H 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 2.

Authors:
 [1];  [2];  [2];  [3]
  1. GeneBiologics, LLC, Boulder, CO (United States)
  2. United States Air Force Academy, Colorado Springs, CO (United States)
  3. National Renewable Energy Lab. (NREL), Golden, CO (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1339249
Alternate Identifier(s):
OSTI ID: 1400579
Report Number(s):
NREL/JA-2700-66611
Journal ID: ISSN 0006-3592
Grant/Contract Number:
AC36-08GO28308; AC36-08GC28308z
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Biotechnology and Bioengineering
Additional Journal Information:
Journal Volume: 114; Journal Issue: 2; Journal ID: ISSN 0006-3592
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; R. capsulatus H2 sensor; high-throughput screening; photobiohydrogen; nitrogenase; H2 production

Citation Formats

Wecker, Matt S. A., Beaton, Stephen E., Chado, Robert A., and Ghirardi, Maria L. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production. United States: N. p., 2016. Web. doi:10.1002/bit.26076.
Wecker, Matt S. A., Beaton, Stephen E., Chado, Robert A., & Ghirardi, Maria L. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production. United States. doi:10.1002/bit.26076.
Wecker, Matt S. A., Beaton, Stephen E., Chado, Robert A., and Ghirardi, Maria L. Wed . "Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production". United States. doi:10.1002/bit.26076. https://www.osti.gov/servlets/purl/1339249.
@article{osti_1339249,
title = {Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production},
author = {Wecker, Matt S. A. and Beaton, Stephen E. and Chado, Robert A. and Ghirardi, Maria L.},
abstractNote = {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. 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. Furthermore, 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.},
doi = {10.1002/bit.26076},
journal = {Biotechnology and Bioengineering},
number = 2,
volume = 114,
place = {United States},
year = {Wed Aug 17 00:00:00 EDT 2016},
month = {Wed Aug 17 00:00:00 EDT 2016}
}

Journal Article:
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  • Mutants of Rhodobacter capsulatus unable to grow photoautotrophically with H{sub 2} and CO{sub 2} were isolated. Those lacking uptake hydrogenase activity as measured by H{sub 2}-dependent methylene blue reduction were analyzed genetically and used in complementation studies for the isolation of the wild-type genes. Results of further subcloning and transposon Tn5 mutagenesis suggest the involvement of a minimum of five genes. Hybridization to the 2.2-kilobase-pair SstI fragment that lies within the coding region for the large and small subunits of Bradyrhizobium japonicum uptake hydrogenase showed one region of strong homology among the R. capsulatus fragments isolated, which we interpret tomore » mean that one or both structural genes were among the genes isolated.« less
  • Comparisons of hydrogen-deuterium solvent exchange rate constants for the NH protons of wild-type Pro {yields} Ala-35 (P35A) and Tyr {yields} Phe-75 (Y75F) Rhodobacter capsulatus ferrocytochromes c{sub 2} were made by {sup 1}H-{sup 15}N heteronuclear multiple-quantum correlation spectroscopy. Exchange rate constants increased for the NH protons of residues 45-46, 54, 57-58, 60-61, 82-87, 98, and 100 with Y75F and 16-18, 20, 34, 37, 43, 45-46, and 58 with P35A. The increases in exchange rate constants are consistent with changes in unfolding equilibria and protein dynamics. In Y75F the exchange rate constants of the observable NH protons of the helix spanning Pro-79-Asp-89,more » namely Phe-82-Leu-87, increase to a similar degree, suggesting that this helix is a single cooperative unfolding unit compatible with the local unfolding model. As the oxidation-reduction potential of Y75F is 59 mV lower than wild-type cytochrome c{sub 2} (367 mV), the dynamic changes in this mutant, compared to wild-type, are proposed to be important determinants of the oxidation-reduction potential. Several differences between wild-type and Y75F are in common with P35A, a mutation which does not affect the oxidation-reduction potential, implying that not all observed dynamic changes are functionally important.« less
  • The peptide resonances of the {sup 1}H and {sup 15}N nuclear magnetic resonance spectra of ferrocytochrome c{sub 2} from Rhodobacter capsulatus are sequentially assigned by a combination of 2D {sup 1}H-{sup 1}H and {sup 1}H-{sup 15}N spectroscopy, the latter performed on {sup 15}N-enriched protein. Short-range nuclear Overhauser effect (NOE) data show {alpha}-helices from residues 3-17, 55-65, 69-88, and 103-115. Within the latter two {alpha}-helices, there are three single 3{sub 10} turns, 70-72, 76-78, and 107-109. In addition {alpha}H-NH{sub i+1} and {alpha}H-NH{sub i+2} NOEs indicate that the N-terminal helix (3-17) is distorted. Compared to horse or tuna cytochrome c and cytochromemore » c{sub 2} of Rhodospirillium rubrum, there is a 6-residue insertion at residues 23-29 in R. capsulatus cytochrome c{sub 2}. The NOE data show that this insertion forms a loop, probably an {Omega} loop. {sup 1}H-{sup 15}N heteronuclear multiple quantum correlation experiments are used to follow NH exchange over a period of 40 h. As the 2D spectra are acquired in short time periods (30 min), rates for intermediate exchanging protons can be measured. Comparison of the NH exchange data for the N-terminal helix of cytochrome c{sub 2} of R. capsulatus with the highly homologous horse heart cytochrome c shows that this helix is less stable in cytochrome c{sub 2}.« less