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Title: Redirection of metabolism for hydrogen production

Abstract

This project is to develop and apply techniques in metabolic engineering to improve the biocatalytic potential of the bacterium Rhodopseudomonas palustris for nitrogenase-catalyzed hydrogen gas production. R. palustris, is an ideal platform to develop as a biocatalyst for hydrogen gas production because it is an extremely versatile microbe that produces copious amounts of hydrogen by drawing on abundant natural resources of sunlight and biomass. Anoxygenic photosynthetic bacteria, such as R. palustris, generate hydrogen and ammonia during a process known as biological nitrogen fixation. This reaction is catalyzed by the enzyme nitrogenase and normally consumes nitrogen gas, ATP and electrons. The applied use of nitrogenase for hydrogen production is attractive because hydrogen is an obligatory product of this enzyme and is formed as the only product when nitrogen gas is not supplied. Our challenge is to understand the systems biology of R. palustris sufficiently well to be able to engineer cells to produce hydrogen continuously, as fast as possible and with as high a conversion efficiency as possible of light and electron donating substrates. For many experiments we started with a strain of R. palustris that produces hydrogen constitutively under all growth conditions. We then identified metabolic pathways and enzymes importantmore » for removal of electrons from electron-donating organic compounds and for their delivery to nitrogenase in whole R. palustris cells. For this we developed and applied improved techniques in 13C metabolic flux analysis. We identified reactions that are important for generating electrons for nitrogenase and that are yield-limiting for hydrogen production. We then increased hydrogen production by blocking alternative electron-utilizing metabolic pathways by mutagenesis. In addition we found that use of non-growing cells as biocatalysts for hydrogen gas production is an attractive option, because cells divert all resources away from growth and to hydrogen. Also R. palustris cells remain viable in a non-growing state for long periods of time.« less

Authors:
Publication Date:
Research Org.:
The University of Washington
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1029943
Report Number(s):
DOE/07ER64482/4
DOE Contract Number:
FG02-07ER64482
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Hydrogen gas, nitrogenase, bacteria, photosynthesis, Calvin cycle

Citation Formats

Harwood, Caroline S. Redirection of metabolism for hydrogen production. United States: N. p., 2011. Web. doi:10.2172/1029943.
Harwood, Caroline S. Redirection of metabolism for hydrogen production. United States. doi:10.2172/1029943.
Harwood, Caroline S. Mon . "Redirection of metabolism for hydrogen production". United States. doi:10.2172/1029943. https://www.osti.gov/servlets/purl/1029943.
@article{osti_1029943,
title = {Redirection of metabolism for hydrogen production},
author = {Harwood, Caroline S.},
abstractNote = {This project is to develop and apply techniques in metabolic engineering to improve the biocatalytic potential of the bacterium Rhodopseudomonas palustris for nitrogenase-catalyzed hydrogen gas production. R. palustris, is an ideal platform to develop as a biocatalyst for hydrogen gas production because it is an extremely versatile microbe that produces copious amounts of hydrogen by drawing on abundant natural resources of sunlight and biomass. Anoxygenic photosynthetic bacteria, such as R. palustris, generate hydrogen and ammonia during a process known as biological nitrogen fixation. This reaction is catalyzed by the enzyme nitrogenase and normally consumes nitrogen gas, ATP and electrons. The applied use of nitrogenase for hydrogen production is attractive because hydrogen is an obligatory product of this enzyme and is formed as the only product when nitrogen gas is not supplied. Our challenge is to understand the systems biology of R. palustris sufficiently well to be able to engineer cells to produce hydrogen continuously, as fast as possible and with as high a conversion efficiency as possible of light and electron donating substrates. For many experiments we started with a strain of R. palustris that produces hydrogen constitutively under all growth conditions. We then identified metabolic pathways and enzymes important for removal of electrons from electron-donating organic compounds and for their delivery to nitrogenase in whole R. palustris cells. For this we developed and applied improved techniques in 13C metabolic flux analysis. We identified reactions that are important for generating electrons for nitrogenase and that are yield-limiting for hydrogen production. We then increased hydrogen production by blocking alternative electron-utilizing metabolic pathways by mutagenesis. In addition we found that use of non-growing cells as biocatalysts for hydrogen gas production is an attractive option, because cells divert all resources away from growth and to hydrogen. Also R. palustris cells remain viable in a non-growing state for long periods of time.},
doi = {10.2172/1029943},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Mon Nov 28 00:00:00 EST 2011},
month = {Mon Nov 28 00:00:00 EST 2011}
}

Technical Report:

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  • The work proposed to be accomplished in the previous funding period was to develop a procedure for genetic exchange based on conjugation mediated by broad host-range plasmids. Such a system has recently been identified that employs IncQ group plasmids and a Desulfovibrio desulfuricans G100A derivative as recipient. During the search for conjugation, we also identified a defective bacteriophage capable of generalized transduction of fragments of chromosomal DNA between mutants of Desulfovibrio desulfuricans. Some of the factors influencing the production and transduction by this defective phage have been investigated. A curious observation was made concerning the response of colonies of thesemore » sulfate-reducing bacteria upon exposure to air. All the cells of a colony do not die. Some survive, most likely by producing sulfide at a rate sufficient to provide an anaerobic environment. Dramatic colony morphological changes occur and these have been documented by scanning and transmission electron microscopy. Finally a small endogenous plasmid has been isolated from Desulfovibrio desulfuricans G100A. It has been stably subcloned into a sequencing vector, and nested deletions of this plasmid are being prepared. This plasmid may be useful for the development of a shuttle cloning vector that could be used in more diverse Desulfovibrio. Many of the techniques now to be used in the mutant analysis of hydrogenase genes in the sulfate-reducing bacteria have been successfully applied in an analysis of hydrogenase functions of Rhodobacter capsulatus. 8 figs., 2 tabs.« less