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Title: Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri

Abstract

We present a genome-scale metabolic reconstruction for the archaeal methanogen Methanosarcina barkeri. This reconstruction represents the first large-scale, predictive model of a methanogen and an archael species. We characterize this reconstruction and compare it to those from the prokaryotic, eukaryotic, and archael domains. We further apply constraint-based methods to stimulate the metabolic fluxes and resulting phenotypes under different environmental and genetic conditions. These results are validated by comparison to experimental growth measurements and phenotypes of M. barkeri on different substrates. The predicted growth phenotypes for mutants of the methanogenic pathway were found to have a high level of agreement with experimental findings. The active reactions and pathways under selected growth conditions are presented and characterized. We also examined the efficiency of the energy-conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase reaction. This work demonstrates that a reconstructed metabolic network can serve as an in silico analysis platform to predict cellular phenotypes, characterize methanogenic growth, improve the genome annotation, and further uncover the metabolic characteristics of methanogenesis.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
896062
Report Number(s):
PNNL-SA-52250
TRN: US200703%%488
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Molecular Systems Biology, 2
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; EFFICIENCY; GENETICS; HYDROGENASES; MUTANTS; SIMULATION; SUBSTRATES; archael metabolism; metabolic modeling; methanogenesis; Methanosarcina barkeri; network reconstruction

Citation Formats

Feist, Adam, Scholten, Johannes C., Palsson, Bernard O., Brockman, Fred J., and Ideker, Trey. Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri. United States: N. p., 2006. Web. doi:10.1038/msb4100046.
Feist, Adam, Scholten, Johannes C., Palsson, Bernard O., Brockman, Fred J., & Ideker, Trey. Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri. United States. doi:10.1038/msb4100046.
Feist, Adam, Scholten, Johannes C., Palsson, Bernard O., Brockman, Fred J., and Ideker, Trey. Tue . "Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri". United States. doi:10.1038/msb4100046.
@article{osti_896062,
title = {Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri},
author = {Feist, Adam and Scholten, Johannes C. and Palsson, Bernard O. and Brockman, Fred J. and Ideker, Trey},
abstractNote = {We present a genome-scale metabolic reconstruction for the archaeal methanogen Methanosarcina barkeri. This reconstruction represents the first large-scale, predictive model of a methanogen and an archael species. We characterize this reconstruction and compare it to those from the prokaryotic, eukaryotic, and archael domains. We further apply constraint-based methods to stimulate the metabolic fluxes and resulting phenotypes under different environmental and genetic conditions. These results are validated by comparison to experimental growth measurements and phenotypes of M. barkeri on different substrates. The predicted growth phenotypes for mutants of the methanogenic pathway were found to have a high level of agreement with experimental findings. The active reactions and pathways under selected growth conditions are presented and characterized. We also examined the efficiency of the energy-conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase reaction. This work demonstrates that a reconstructed metabolic network can serve as an in silico analysis platform to predict cellular phenotypes, characterize methanogenic growth, improve the genome annotation, and further uncover the metabolic characteristics of methanogenesis.},
doi = {10.1038/msb4100046},
journal = {Molecular Systems Biology, 2},
number = ,
volume = ,
place = {United States},
year = {Tue Jan 31 00:00:00 EST 2006},
month = {Tue Jan 31 00:00:00 EST 2006}
}
  • Hydrogenotrophic methanogenesis occurs in multiple environments, ranging from the intestinal tracts of animals to anaerobic sediments and hot springs. Energy conservation in hydrogenotrophic methanogens was long a mystery; only within the last decade was it reported that net energy conservation for growth depends on electron bifurcation. In this work, we focus onMethanococcus maripaludis, a well-studied hydrogenotrophic marine methanogen. To better understand hydrogenotrophic methanogenesis and compare it with methylotrophic methanogenesis that utilizes oxidative phosphorylation rather than electron bifurcation, we have built iMR539, a genome scale metabolic reconstruction that accounts for 539 of the 1,722 protein-coding genes ofM. maripaludisstrain S2. Our reconstructedmore » metabolic network uses recent literature to not only represent the central electron bifurcation reaction but also incorporate vital biosynthesis and assimilation pathways, including unique cofactor and coenzyme syntheses. We show that our model accurately predicts experimental growth and gene knockout data, with 93% accuracy and a Matthews correlation coefficient of 0.78. Furthermore, we use our metabolic network reconstruction to probe the implications of electron bifurcation by showing its essentiality, as well as investigating the infeasibility of aceticlastic methanogenesis in the network. Additionally, we demonstrate a method of applying thermodynamic constraints to a metabolic model to quickly estimate overall free-energy changes between what comes in and out of the cell. Finally, we describe a novel reconstruction-specific computational toolbox we created to improve usability. Together, our results provide a computational network for exploring hydrogenotrophic methanogenesis and confirm the importance of electron bifurcation in this process. Understanding and applying hydrogenotrophic methanogenesis is a promising avenue for developing new bioenergy technologies around methane gas. Although a significant portion of biological methane is generated through this environmentally ubiquitous pathway, existing methanogen models portray the more traditional energy conservation mechanisms that are found in other methanogens. In conclusion, we have constructed a genome scale metabolic network ofMethanococcus maripaludisthat explicitly accounts for all major reactions involved in hydrogenotrophic methanogenesis. Our reconstruction demonstrates the importance of electron bifurcation in central metabolism, providing both a window into hydrogenotrophic methanogenesis and a hypothesis-generating platform to fuel metabolic engineering efforts.« less
  • A sulfate-reducing vibrio was isolated from a methanogenic enrichment with choline as the sole added organic substrate. This oganism was identified as a member of the genus Desulfovibrio and was designated Desulfovibrio strain G1. In a defined medium devoid of sulfate, a pure culture of Desulfovibrio strain G1 fermented choline to trimethylamine, acetate, and ethanol. In the presence of sulfate, more acetate and less ethanol were formed from choline than in the absence of sulfate. When grown in a medium containing sulfate, a coculture of Desulfovibrio strain G1 and Methanosarcina barkeri strain Fusaro degraded chloline almost completely to methane, ammonia,more » and hydrogen sulfide and presumably to carbon dioxide. Methanogenesis occurred in two distinct phases separated by a lag of about 6 days. During the first phase of methanogenesis choline was completely converted to trimethylamine, acetate, hydrogen sulfide, and traces of ethanol by the desulfovibrio.M. barkeri fermented trimethylamine to methane, ammonia, and presumably carbon dioxide via dimethyl- and methylamine as intermediates. Simultaneously, about 60% of the acetate expected was metabolized. In the second phase of methanogenesis, the residual acetate was almost completely catabolized.« less
  • Production of methane by Methanosarcina barkeri from H/sub 2/-CO/sub 2/ was studied in fed-batch culture under phosphate-limiting conditions. A transition in the kinetics of methanogenesis from an exponentially increasing rate to a constant rate was due to depletion of phosphate from the medium. The period of exponentially increasing rate of methanogenesis was extended by increasing the initial concentration of phosphate in the medium. Addition of phosphate during the constant period changed the kinetics to an exponentially increasing rate of methanogenesis, indicating the reversibility of phosphate depletion. The relation between methanogenesis and growth of M. barkeri was investigated by measuring themore » incorporation of phosphorus, supplied as KH/sub 2//sup 32/PO/sub 4/, in the medium. At a low (1 ..mu..M) initial concentration of phosphate in the medium and during the constant period of methanogenesis, there was no net cell growth. At a higher (10 ..mu..M) initial concentration of phosphate, cell growth proceeded linearly with time after phosphate had been removed from the medium by uptake into cells.« less
  • A bacterial consortium capable of sucrose degradation primarily to CH/sub 4/ and CO/sub 2/ was constructed, with acetate as the key methanogenic precursor. In addition, the effect of agar immobilization on the activity of the consortium was determined. The primary fermentative organism, Escherichia coli, produced acetate, formate, H/sub 2/, and CO/sub 2/ (known substrates for methanogens), as well as ethanol and lactate, compounds that are not substrates for methanogens. Oxidation of the nonmethanogenic substrates, lactate and ethanol, to acetate was mediated by the addition of Acetobacterium woodii and Desulfovibrio vulgaris. The methanogenic stage was accomplished by the addition of themore » acetophilic methanogen Methanosarcina barkeri and the hydrogenophilic methanogen Methanobacterium formicicum. Results of studies with low substrate concentrations (0.05 to 0.2% (wt/vol)), a growth-limiting medium, and the five-component consortium indicated efficient conversion (40%) of sucrose carbon to CH/sub 4/. Significant decreases in yields of CH/sub 4/ and rates of CH/sub 4/ production were observed if any component of the consortium was omitted. Approximately 70% of the CH/sub 4/ generated occurred via acetate. Agar-immobilized cells of the consortium exhibited yields of CH/sub 4/ and rates of CH/sub 4/ production from sucrose similar to those of nonimmobilized cells. The rate of CH/sub 4/ production decreased by 25% when cysteine was omitted from reaction conditions and by 40% when the immobilized consortium was stored for 1 week at 4/sup 0/C.« less