Methanosarcina acetivorans strain C2A is a marine methanogenic archaeon notable for its substrate utilization, genetic tractability, and novel energy conservation mechanisms. To help probe the phenotypic implications of this organism's unique metabolism, we have constructed and manually curated a genome-scale metabolic model of M. acetivorans, iMB745, which accounts for 745 of the 4,540 predicted protein-coding genes (16%) in the M. acetivorans genome. The reconstruction effort has identified key knowledge gaps and differences in peripheral and central metabolism between methanogenic species. Using flux balance analysis, the model quantitatively predicts wild-type phenotypes and is 96% accurate in knockout lethality predictions compared to currently available experimental data. The model was used to probe the mechanisms and energetics of by-product formation and growth on carbon monoxide, as well as the nature of the reaction catalyzed by the soluble heterodisulfide reductase HdrABC in M. acetivorans. The genome-scale model provides quantitative and qualitative hypotheses that can be used to help iteratively guide additional experiments to further the state of knowledge about methanogenesis. Methanogenic archaea are unique in their ability to grow on low-energy substrates, such as acetic acid, by converting them into methane and other by-products. Methanogens are a critical part of the global carbon cycle, consuming by-products of other natural bioprocesses that would otherwise be recalcitrant in sulfate-poor, anaerobic environments (12). They also play an important role in global warming, since methane is a greenhouse gas 20 times as potent as carbon dioxide (42) and methanogenesis is the primary mechanism for the emission of methane into the atmosphere (2).Methanosarcina is the only known genus of methanogens with members that can utilize all of the known methanogenic pathways (acetoclastic, methylotrophic, hydrogenotrophic, and methyl reducing) (71). This metabolic diversity makes these species more permissive to metabolic and genetic manipulations than other methanogens. To capitalize on this characteristic, the genomes of three Methanosarcina species have been sequenced (15,22,38). In addition, genetic tools have been developed for several of these species, including methods for directed mutagenesis and regulated expression of specific genes (3,34,73,74).The constraint-based reconstruction and analysis (COBRA) strategy is a powerful paradigm for consolidating large amounts of metabolic knowledge and synthesizing that knowledge into quantitative phenotypic predictions (45, 51). For the performance of constraint-based analysis on an individual organism, its metabolic network is reconstructed from the bottom up, beginning with a sequenced and annotated genome and ending with a network of reactions and reaction-gene associations that directly link genotype and phenotype (68). Many metabolic reconstructions have been curated by hand and have been used to make useful predictions, such as the identification of put...

Methanosarcina barkeri is an Archaeon that produces methane anaerobically as the primary byproduct of its metabolism. M. barkeri can utilize several substrates for ATP and biomass production including methanol, acetate, methyl amines, and a combination of hydrogen and carbon dioxide. In 2006, a metabolic reconstruction of M. barkeri, iAF692, was generated based on a draft genome annotation. The iAF692 reconstruction enabled the first genome-Scale simulations for Archaea. Since the publication of the first metabolic reconstruction of M. barkeri, additional genomic, biochemical, and phenotypic data have clarified several metabolic pathways. We have used this newly available data to improve the M. barkeri metabolic reconstruction. Modeling simulations using the updated model, iMG746, have led to increased accuracy in predicting gene knockout phenotypes and simulations of batch growth behavior. We used the model to examine knockout lethality data and make predictions about metabolic regulation under different growth conditions. Thus, the updated metabolic reconstruction of M. barkeri metabolism is a useful tool for predicting cellular behavior, studying the methanogenic lifestyle, guiding experimental studies, and making predictions relevant to metabolic engineering applications.