The missing enzymatic link in syntrophic methane formation from fatty acids

Abstract

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H₂, CO₂, or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO₂ to methane by electrons derived from β-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop-driven reduction of CO₂ by acyl-coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO₂ and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of β-oxidation in (methyl)menaquinone-containing organisms.

Keywords: diheme oxidoreductase; methylmenaquinone; microbial methane formation; redox loop; syntrophy.

Fig. 1.

Syntrophic degradation of organic matter to methane. (A) Major metabolic processes involving primary fermenters, secondary fermenters, and methanogenic archaea (for simpler presentation, acetogenic conversion of monomers is not depicted here). (B) Model for syntrophic β-oxidation of butyrate to two acetates coupled to the reduction of protons or CO₂. The enzyme mediating electron transfer from reduced ETF to FDH has not been studied before and was assigned to noncharacterized DUF224 based on omics-based predictions.

Fig. 2.

Analysis of the quinone and EMO from S. aciditrophicus. (A) Ultra-performance liquid chromatography analysis of quinones with identical UV/vis spectra as 8-MKK (Inset). (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis of enrichment of EMO (the minor band below is most probably an EMO proteolysis product). M, mass standard; SE, size exclusion chromatography. (C) UV/vis spectra of solubilized EMO in the oxidized (as isolated) and dithionite-reduced form. (D) Redox titration of the heme b cofactors in the presence of redox dyes. Data points are fitted to two Nernst curves with n = 1 and midpoint potentials as indicated. (E) Reduction of solubilized EMO by CHCoA in the presence of ETF and CHCoA DH. Excess CHCoA reduced approximately one heme b equivalent. Light brown circles represent data points of differences in absorption 413 nm minus 427 nm and khaki diamonds of difference absorption 560 nm minus 580 nm.

Fig. 3.

Model of syntrophic scFA oxidation coupled to CO₂ reduction in S. aciditrophicus. The model scFA butyrate is oxidized to two acetates coupled to the reduction of two CO₂ to formate. Alternatively, the NADH formed by 3-hydroxybutyryl-CoA DH is regenerated by a soluble hydrogenase, and the H₂ formed diffuses across the membrane without the involvement of a transporter. Protons that contribute to proton motive force (pmf) generation are shown in green; those that diminish pmf are shown in red. Note that transport of protons during uptake and excretion is based on assumptions and has not been studied in this work.

Fig. 4.

Phylogenetic analysis of emo genes in prokaryotes. EMO from S. aciditrophicus served as query sequence during BLASTp analysis. The presence of conserved His ligands of heme b and the genomic context with regard to etf and β-oxidation genes are indicated. Syntrophic bacteria are highlighted by bold typeface. The numbers refer to bootstrap values; the scale bar indicates the number of substitution per site. Red crosses mark selected pathogenic bacteria.