Genome-Scale Metabolic Reconstruction and Hypothesis Testing in the Methanogenic Archaeon Methanosarcina acetivorans C2A

Abstract

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...

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