Mer overexpression in Methanosarcina acetivorans affects growth and methanogenesis during substrate adaptation

Abstract

Evidence suggests that multienzyme complexes are involved in biological methane production (methanogenesis), although the composition of the Wolfe Cycle methanogenesis complexes may vary between diverse methanoarchaeal taxa. Methylenetetrahydromethanopterin reductase (Mer) is the first committed step in C1 oxidation to CO₂ during methylotrophic methanogenesis. However, Mer is downregulated when cells use acetate as a substrate. We hypothesized that Mer overexpression during methylotrophic methanogenesis would be beneficial, while overexpression during acetoclastic methanogenesis would be detrimental for energy conservation. To test this hypothesis, we overexpressed Mer and characterized strain physiology on methanol, acetate, and when switching substrates. We found that Mer overexpression results in faster growth on methanol, with less C fixation into biomass, and no effect on methanogenesis. Growth on acetate was not affected by Mer overexpression, but switching between substrates was affected. The native Mer overexpressing strain was slower to adjust from methanol to acetate and vice-versa. These data suggest that tight regulation of Mer expression is necessary to regulate C flux through methylotrophic versus acetoclastic methanogenesis pathways in Methanosarcina.IMPORTANCEMethanoarchaea thrive near the "thermodynamic limit of life" and have likely evolved efficient mechanisms to control flux of substrates to conserve energy. Methylenetetrahydromethanopterin reductase (Mer) is a highly conserved, key enzyme in the Wood-Ljungdahl and Wolfe Cycle methanogenesis pathways. Our study sheds light on how Mer enzyme stoichiometry affects methanogenesis and suggests avenues for engineering the organism to promote renewable fuel or bioproduct synthesis.

Keywords: Methanosarcina; Wood-Ljungdahl; archaea; methane; methanogenesis; one-carbon.

Fig 1

Construction of pNB746, pMW1, and strain validation. (a) Plasmid map of pNB746 for att:mer⁺ strain construction and (b) plasmid map of pMW1 for obtaining the att:mer^his-strep+ strain. Following transformation into M. acetivorans, isolates for att:mer⁺ (NB210 [c]) and att:mer^his-strep+ (NB249 [d]) strains were screened by diagnostic PCR. Black arrows show bands indicating expected sizes for plasmid integration. (e) SDS-PAGE of 3 µg of crude extracts from parent (NB34), att:mer⁺ (NB210), att:mer^+his-strep (NB249), and 1 µg of His-NTA purified Mer from NB249 analyzed on a 4% stacking/12.5% separating gel and stained with Coomassie Blue. (f) Immunoblot of the gel from panel a incubated with custom anti-MA3733 rabbit antibody and anti-rabbit HRP conjugated antibody for qualitative analysis of Mer overexpression (att:mer⁺). Red arrows indicate position of native and his-tagged Mer.

Fig 2

Growth curves of parent versus Mer overexpression strains. (a) Methanol-adapted growth curve. (b) Acetate-adapted growth curve. (c) Growth curves when methanol-adapted cells are inoculated into acetate. (d) Growth curves when acetate-adapted cells are inoculated into methanol. Each curve represents an average of 10 biological replicates. Error bars are omitted for clarity.

Fig 3

Effect of Mer overexpression on biomass production and rate of methanogenesis. (a–d) Biomass produced by parent and Mer overexpression strains when adapted to grow on methanol (a), on acetate (b), or when switched from methanol to acetate (c) or from acetate to methanol (d). Each measurement was obtained from 10 biological replicates. (e–h) Rate of methane produced by parent and Mer overexpression strains when adapted to grow on methanol (e), on acetate (f), or when switched from methanol to acetate (g) or from acetate to methanol (h). Each measurement was obtained from two biological and four to five technical replicates (n = 9). Error bars represent standard deviation.

Fig 4

Metabolic efficiency of Mer overexpression strains versus parent strain. (a) Relative metabolic efficiency of parent and overexpression strains when adapted to methanol or acetate and when switched from methanol to acetate or acetate to methanol. (b) Two-dimensional hysteresis representation of relative metabolic efficiency indicating Mer overexpression primes metabolism for methylotrophic methanogenesis.

Fig 5

Effect of Mer overexpression when switching substrates. Inferred flux through methanogenesis pathways in the Mer overexpression strain when switching from methanol to acetate (a) and when switching from acetate to methanol (b). Arrow sizes are according to percent change in flux through pathway. Green ovals represent energy-conserving steps. Red ovals represent energy-consuming steps. Ac-CoA, acetyl-coenzyme A; ACDS, acetyl-CoA decarbonylase/synthase; Fmd, formyl-methanofuran dehydrogenase; Fpo, proton-pumping F₄₂₀-methanophenazine oxidoreductase; Ftr, formyl-methanofuran:H₄MPT formyl transferase; Hdr, heterodsulfide reductase; Mch, methenyl-H₄MPT cyclohydrolase; Mcr, methyl-coenzyme M reductase; Mer, methylenetetrahydromethanopterin; Mtd, F₄₂₀-dependent methylene-H₄MPT dehydrogenase; Mtr, methyl-coenzyme M methyltransferase; Rnf, Rhodobacter nitrogen fixation.