Deconstructing Methanosarcina acetivorans into an acetogenic archaeon

Abstract

The reductive acetyl-coenzyme A (acetyl-CoA) pathway, whereby carbon dioxide is sequentially reduced to acetyl-CoA via coenzyme-bound C1 intermediates, is the only autotrophic pathway that can at the same time be the means for energy conservation. A conceptually similar metabolism and a key process in the global carbon cycle is methanogenesis, the biogenic formation of methane. All known methanogenic archaea depend on methanogenesis to sustain growth and use the reductive acetyl-CoA pathway for autotrophic carbon fixation. Here, we converted a methanogen into an acetogen and show that Methanosarcina acetivorans can dispense with methanogenesis for energy conservation completely. By targeted disruption of the methanogenic pathway, followed by adaptive evolution, a strain was created that sustained growth via carbon monoxide-dependent acetogenesis. A minute flux (less than 0.2% of the carbon monoxide consumed) through the methane-liberating reaction remained essential, indicating that currently living methanogens utilize metabolites of this reaction also for anabolic purposes. These results suggest that the metabolic flexibility of methanogenic archaea might be much greater than currently known. Also, our ability to deconstruct a methanogen into an acetogen by merely removing cellular functions provides experimental support for the notion that methanogenesis could have evolved from the reductive acetyl-coenzyme A pathway.

Keywords: Methanosarcina; acetogenic; acetyl-CoA pathway; methanogenic.

Fig. 1.

The acetyl CoA pathway and methanogenesis. Whether H₄F or H₄MPT is the pterin cofactor (-N) in the methyl branch of the acetyl-CoA pathway (black) is not indicated; activated acetyl group is either used anabolically (green) or catabolically (red); methanogenesis (orange), reduction of methyl-N to methane involves Mtr (N⁵-methyl-H₄MPT:HS-CoM methyltransferase), Mcr (methyl-S-CoM reductase), and Hdr (heterodisulfide reductase); for simplicity, thiol-containing cofactors coenzyme A, B, and M are abbreviated, and the input of electrons is not shown.

Fig. 2.

Converting a methanogen into an acetogen. Biomass production (blue, OD₅₇₈), CO consumption (black, values have to be multiplied by 5 to get the actual amounts), and formation of acetate (green), formate (purple), and methane (red) in M42 (A, wild type) and MKOmtrSF (B, suppressor of mtr deletion, SI Appendix, Table S1) during carboxidotrophic growth. (B, Inset) Methane formation in a differently scaled y-axis; shown are mean values and their SDs (error bars) of 6 (M42) or 5 (MKOmtrSF) independent cultures (serum bottles), respectively; the result was reproduced twice.

Fig. 3.

Methyl reduction versus methyl group dependence in M. acetivorans. MKOmtr3 (lacking Mtr, SI Appendix, Table S1) could be grown on a combination of acetate (40 mM) and MeOH (A, Balch tubes) in which acetate oxidation drives methyl reduction to methane for energy conservation or of CO (150 kPa) and MeOH (B, serum bottles) in which CO-dependent acetogenesis serves energy conservation, while MeOH is required for anabolism. (Insets) Correlation of biomass yield (ΔOD, OD₅₇₈ at the end of experiment minus OD₅₇₈ at the beginning of experiment) and MeOH concentration supplemented; for each MeOH concentration, mean values and their SDs (error bars) of five independent cultures are shown.

Fig. 4.

Hdr and Mcr in M. acetivorans. Specific activity of heterodisulfide reductase (A) and methyl-S-CoM reductase (B), determined as given in Materials and Methods, in M42 grown on MeOH (gray), M42 grown on CO (blue), MKOmtr3 grown on CO+MeOH (1 mM) (green), MKOmtrSF grown on CO (red), and MKOmtrhdr grown on CO (yellow). (A) The membranous fraction is indicated by a darker shade of color, the cytoplasmic by a lighter shade of color; data shown correspond to mean values and their SD (error bars) resulting from three biological replicates, except for Hdr activity in M42 grown on MeOH, where only a single (cytoplasmic) or technical replicate (membrane) measurement is shown.