(629a) In silico Analysis of Anaerobic Methane Oxidation By Methanosarcina Acetivorans

There is a renewed industrial interest in capturing methane, major constituent of greenhouse and shale gas. Anaerobic oxidation of methane (AOM) has been evaluated to be more efficient in terms of carbon conversion of methane to valuable products. However, there has been limited success in isolation and characterization of anaerobic methanotrophs due to the fact that they are often found in consortia of organisms and, thus, are not easily cultured in lab. Interestingly, trace methane oxidation has been demonstrated in the methanogenic archaeon Methanosarcina acetivorans. While the pathways describing the metabolism of native substrates of this organism have been well established, relevant pathways for methane oxidation remain unexplained. This talk is aiming at addressing thermodynamically feasible routes for anaerobic methane oxidation by M. acetivorans leading to proposal of optimized pathways for methane (co)utilization and candidate biofuel production. To this end, an updated genome-scale metabolic model of M. acetivorans is introduced (iMAC868 containing 868 genes, 840 reactions, and 712 metabolites) by integrating information from two previously reconstructed metabolic models (i.e., iVS941 and iMB745), modifying 17 reactions, adding 19 new reactions, and revising 64 gene-protein-reaction (GPR) associations based on newly available sources. The new model establishes improved predictions of growth yields on native substrates and is capable of correctly predicting the dispensability for 27 out of 28 gene deletion mutants. The iMAC868 model enables prediction of thermodynamically feasible pathway for co-utilization of methane and bicarbonate in the presence of ferric, nitrate, sulfate, and manganese dioxide as external electron acceptors. We find that the reversal of the acetate to methane pathway is the prominent route for AOM by M. acetivorans, which produces acetyl-CoA, the precursor for the production of ethanol, 1-butanol, and iso-butanol. We also find that the methylotrophic pathway must carry a minimum mandatory flux to ensure thermodynamic feasibility. The implications of acetate and CO₂ production, and the interplay between externally supplied electron acceptors and various by-products are discussed. This effort paves the way in informing the search for thermodynamically feasible ways of (co)utilizing novel carbon substrates in archaea, in general, and in M. acetivorans, specifically, since the archaeon possess a diverse carbon utilization capability and its genetic tools are well studied among archaea.