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.

Fig 1

Properties of the Methanosarcina acetivorans model and comparison to other existing Methanosarcina models. (A) After curation, the metabolic model contained reactions related to the synthesis of essential biomass components, cell wall components, and methanogenesis, among others. (B to D) Comparison of the electron transport chains in the three available Methanosarcina models. Reactions indicated by red arrows are reversible in that model. Note that the curated M. acetivorans model includes the Rnf complex and the soluble heterodisulfide reductase (HdrABC) and excludes Frh and Vht, two hydrogenases known to be inactive in M. acetivorans but present and active in M. barkeri. mphen, oxidized methanophenazine; mphenh2, reduced methanophenazine; hsfd, heterodisulfide; com, coenzyme M; cob, coenzyme B.

Fig 2

Analysis of the ion-pumping stoichiometry of Rnf and Mrp during growth on acetate (A) and carbon monoxide (B). Circles represent simulated combinations of ion-pumping stoichiometries for the two pumps. Each colored line is a contour representing a constant predicted growth yield. The experimental growth yields are 2.4 g (dry weight)/mmol during growth on acetate and 2.5 g (dry weight)/mol during growth on CO. Only the combination of a 3 Na/2 e⁻ stoichiometry for Rnf and a 1 Na⁺/1 H⁺ stoichiometry for Mrp was consistent with experimental data. See the supplemental material for the analogous simulation of growth on methanol.

Fig 3

Analysis of growth of M. acetivorans on carbon monoxide. (A) Regeneration of coenzyme F₄₂₀ during growth on carbon monoxide for M. barkeri (left) and proposed pathway for M. acetivorans (right). co2, carbon dioxide; na1, Na⁺. (B) Theoretical ATP yields during growth on CO differed depending on the by-product produced and whether sodium-pumping Mtr or its nonpumping bypass reaction (Mtr_b) was active. Red, formate generation; black, acetogenesis; blue, methanogenesis (with Mtr); green, methyl sulfide production (with Mtr); gold, methyl sulfide production (with Mtr_b); dark red, methanogenesis (with Mtr_b). An ATP/CO ratio of at least 0.22 is required to overcome the non-growth-associated maintenance requirement, and an additional 65 mmol ATP/g (dry weight) is required for growth. m5hbc, co-methyl-co-5-hydroxybenzimidazolylcobamide. (C) FBA predicts that limitation of CO dehydrogenase activity leads to formate production.

Fig 4

Studies on the soluble heterodisulfide reductase HdrABC during growth on methanol. (A) Hypothesized bifurcation mechanism of the soluble heterodisulfide reductase HdrABC during growth on methylotrophic substrates in M. acetivorans. The alternative hypothesis is that the reaction does not involve F₄₂₀ (dashed lines). formmfr(b), formylmethanofuran(b). (B) Flux balance analysis predicts that during growth on methylotrophic substrates (methanol shown), HdrABC is not used when Rnf is available, but a Δrnf mutant is predicted to carry flux through HdrABC. The growth rate for the Δrnf mutant is predicted to be about 20% less than that for the wild type with bifurcation and 35% less without.