Rerouting Cellular Electron Flux To Increase the Rate of Biological Methane Production

Abstract

Methanogens are anaerobic archaea that grow by producing methane, a gas that is both an efficient renewable fuel and a potent greenhouse gas. We observed that overexpression of the cytoplasmic heterodisulfide reductase enzyme HdrABC increased the rate of methane production from methanol by 30% without affecting the growth rate relative to the parent strain. Hdr enzymes are essential in all known methane-producing archaea. They function as the terminal oxidases in the methanogen electron transport system by reducing the coenzyme M (2-mercaptoethane sulfonate) and coenzyme B (7-mercaptoheptanoylthreonine sulfonate) heterodisulfide, CoM-S-S-CoB, to regenerate the thiol-coenzymes for reuse. In Methanosarcina acetivorans, HdrABC expression caused an increased rate of methanogenesis and a decrease in metabolic efficiency on methylotrophic substrates. When acetate was the sole carbon and energy source, neither deletion nor overexpression of HdrABC had an effect on growth or methane production rates. These results suggest that in cells grown on methylated substrates, the cell compensates for energy losses due to expression of HdrABC with an increased rate of substrate turnover and that HdrABC lacks the appropriate electron donor in acetate-grown cells.

FIG 1

Putative models for the role of HdrABC during methylotrophic growth. (A) In an electron confurcation mechanism, HdrABC uses electrons from both FdxH₂ and F₄₂₀H₂ in a 1:1 ratio to reduce two molecules of CoM-S-S-CoB and bypasses Rnf, Fpo, and HdrED energy-conserving enzyme complexes. (B) In a direct-reduction mechanism, HdrABC bypasses either Rnf or Fpo ion translocation steps by using FdxH₂ or F₄₂₀H₂ as an electron donor. (C) HdrABC uses a bifurcation mechanism to reduce CoM-S-S-CoB while interchanging electrons between FdxH₂ and F₄₂₀H₂ electron carriers. The model in panel B most closely matches the experimental data. k_a is the rate of HdrABC enzyme activity; k_CH4 is the rate of methane production; t_g is the generation time.

FIG 2

Construction of plasmid pJC1 and strain verification. (A) Map of plasmid pJC1 containing the hdrABC* operon under the control of the Ptet promoter. The asterisks indicate the locations of point mutations in the hdrABC operon. (B) Agarose gel showing PCR identification of each strain.

FIG 3

Phenotypes of parent and hdrABC mutant strains. (A) Growth curve of methanol-adapted strains. Growth data were collected from triplicate biological replicates. (B and C) The rates of methane production by cell suspensions were measured from cells grown on methanol (B) and acetate (C). Methane production was measured in triplicate biological and triplicate technical replicates. *, P < 0.0001. (D) Heterodisulfide reductase activity in methanol-grown cell extracts. Assays were performed in triplicate. The error bars indicate the standard deviations and may not be visible behind the symbols.

FIG 4

HdrABC uncouples methanogenesis from cell growth. (A) In cell suspension assays, the rate of methane production is dependent on the amount of HdrABC enzyme activity in the cell. (B) The growth rate and methane production rate are correlated by a second-order relationship. (C) Metabolic efficiency of each strain. The values are normalized to the ΔhdrABC mutant strain. (D) HdrABC enzyme activity has a strong negative correlation with metabolic efficiency and fits a direct-reduction mechanism model (R² = 0.9981). The error bars indicate the standard deviations and may not be visible behind the symbols.

FIG 5

Methylotrophic methanogenesis pathway in M. acetivorans. The green ovals indicate energy-conserving steps. The red oval indicates an energy-consuming step. Yellow indicates an [Na⁺]-dependent energy conservation step. Ac-CoA, acetyl-coenzyme A; Fpo, proton-pumping F₄₂₀-methanophenazine oxidoreductase; Mtr, methyl-coenzyme M methyltransferase.