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bioRxiv · 10.1101/2025.10.01.679765

Genus-specific remodeling of carbon and energy metabolism facilitates acetoclastic methanogenesis in Methanosarcina spp. and Methanothrix spp.

Downing, B.·Gupta, D.·Shalvarjian, K. E.·Nayak, D. D.
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Abstract

Methanogenic archaea (methanogens) are microorganisms that obligately produce methane as a byproduct of their energy metabolism. While most methanogens grow on CO2+H2, isolates of the Genus Methanosarcina and Methanothrix can use acetate as the sole substrate for methanogenesis. Methanogenic growth on acetate, i.e., acetoclastic methanogenesis, is hypothesized to require two distinct genetic modules: one for the activation of acetate to acetyl-CoA and the other for producing a chemiosmotic gradient using electrons derived from ferredoxin. In Methanosarcina spp., the activation of acetate to acetyl-CoA is mediated by acetate kinase (Ack) and phosphotransacetylase (Pta) whereas Methanothrix spp. encode AMP-forming acetyl-CoA synthetases (Acs). The Rhodobacter nitrogen fixation complex (Rnf) or Energy converting hydrogenase (Ech) are critical for energy conservation in Methanosarcina spp. during growth on acetate, and a F420:phenazine oxidoreductase-like complex (Fpo) likely plays an analogous role in Methanothrix spp. Here, we tested the proposed modularity of these pathways to facilitate acetoclastic methanogenesis. First, we surveyed over a hundred genomes within the Class Methanosarcinia to show that the genomic potential for acetoclastic methanogenesis using distinct combinations of modules is widespread. We then used the genetically tractable strain, Methanosarcina acetivorans, to build all modular combinations for acetoclastic methanogenesis. Our results indicate that Acs, while functional, cannot replace Ack+Pta to rescue acetate growth in M. acetivorans. Similarly, the Fpo bioenergetic complex cannot replace Rnf. As such, our work suggests that, in addition to horizontal gene transfer of core catabolic modules, acetoclastic metabolism in methanogens requires changes in energetic modules too. IMPORTANCEA large fraction of biogenic methane is derived from acetate, yet acetoclastic methanogens, i.e., methanogens that grow on acetate, remain poorly characterized due to their slow growth. Two groups of methanogens, Methanosarcina spp. and Methanothrix spp., perform acetoclastic methanogenesis using distinct sets of genes for acetate activation and energy conservation. It is widely hypothesized that these genetic modules from Methanosarcina spp. and Methanothrix spp. are functionally analogous and would thus be interchangeable. To test this hypothesis, we engineered different combinations of modules for acetoclastic growth in Methanosarcina acetivorans. Our results challenge this hypothesized paradigm of modularity, and we posit that other changes to the carbon and electron transfer pathways were crucial for the emergence of acetoclastic methanogenesis.

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Downing, B., Gupta, D., Shalvarjian, K. E., Nayak, D. D.. 2025-10-01. Genus-specific remodeling of carbon and energy metabolism facilitates acetoclastic methanogenesis in Methanosarcina spp. and Methanothrix spp.. https://doi.org/10.1101/2025.10.01.679765

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