Building carbon-carbon bonds using a biocatalytic methanol condensation cycle

Abstract

Methanol is an important intermediate in the utilization of natural gas for synthesizing other feedstock chemicals. Typically, chemical approaches for building C-C bonds from methanol require high temperature and pressure. Biological conversion of methanol to longer carbon chain compounds is feasible; however, the natural biological pathways for methanol utilization involve carbon dioxide loss or ATP expenditure. Here we demonstrated a biocatalytic pathway, termed the methanol condensation cycle (MCC), by combining the nonoxidative glycolysis with the ribulose monophosphate pathway to convert methanol to higher-chain alcohols or other acetyl-CoA derivatives using enzymatic reactions in a carbon-conserved and ATP-independent system. We investigated the robustness of MCC and identified operational regions. We confirmed that the pathway forms a catalytic cycle through (13)C-carbon labeling. With a cell-free system, we demonstrated the conversion of methanol to ethanol or n-butanol. The high carbon efficiency and low operating temperature are attractive for transforming natural gas-derived methanol to longer-chain liquid fuels and other chemical derivatives.

Keywords: bio-butanol; bio-ethanol; cell-free synthesis; metabolic engineering; methanol metabolism.

Fig. 1.

Conversion of methanol to higher n-alcohols. (A) The MCC is the combination of RuMP with NOG that bypasses ATP dependency. See Table S1 for details. (B) The major MCC mode uses the more active X5P-phosphoketolase (Xpk). (C) The minor MCC mode can achieve the same result with the less active F6P-phosphoketolase (Fpk).

Fig. 2.

Simulation and in vitro demonstration of a kinetic trap. (A) Ensemble model robustness analysis (EMRA) of the core MCC pathway (19). Y_R,M is the fraction of robust models constrained to a steady state. The variation of phosphoketolase is shown in red and variation of transaldolase in blue. (B) Simulated distribution of acetic acid isotopes using ¹³C-formaldehyde as the substrate (C) In vitro production of acetic acid and GC-MS analysis using ¹³C-formaldehyde as substrate. Seven enzymes (Hps, Phi, Tkt, Tal, Rpe, Rpi, and F/Xpk) were used to produce acetyl-phosphate from ¹³C-formaldehyde. For GC analysis, the AcP was then converted to acetate by acetate kinase using ATP recycling by glucokinase. An acetate standard curve was established with R² = 0.998 up to 5 mM to ensure reliable quantitation. Assays were independently run in triplicate (n = 3) with error bars representing SD.

Fig. 3.

¹³C Tracing from ¹³C-formaldehyde and formate to ethanol. (A) The mass spectra of all four ethanol isotope standards including unlabeled ethanol, [1-¹³C]-ethanol, [2-¹³C]-ethanol, and double-labeled [1,2-¹³C]-ethanol. All spectra were normalized to the most abundant internal peak. Only double-labeled ethanol has a significant 48 ion (red asterisks). (B) Mass spectrum of ethanol experimentally produced from ¹³C-formaldehyde, unlabeled formate, and unlabeled R5P using the full MCC pathway with formate dehydrogenase. The assays were analyzed after 2 h at room temperature. Formate was oxidized to CO₂ by formate dehydrogenase to provide the necessary NADH to reduce acetyl-CoA to ethanol. (C) The “No Tal” control contained the same conditions as the full pathway except for omitting transaldolase. No 48 ion was detected for the control. m/z, mass to charge ratio; R.A., relative abundance.

Fig. 4.

Ethanol and n-butanol production from formaldehyde or methanol using MCC. (A) Steady-state production of ethanol from formaldehyde with MCC using formic acid as electron source; 6 mM formaldehyde, 10 mM formate, and 0.5 mM R5P were added at the 0-, 1-, and 2-h points. (B) Sugar phosphate measurement over time using HPIC-PAD for the batch conversion of formaldehyde to acetyl-phosphate. Most of the carbon rearrangement occurred within the first minute. F6P and R5P standards overlap so the combine area is provided in the full assay. (C) Conversion of methanol to ethanol and (D) to n-butanol over 24 h. The productivity drops after five hours, likely due to instability of intermediates. The alcohol production assays were independently run in triplicate. Methanol consumption (for full assay only) is shown in red circles, whereas ethanol and n-butanol production is shown with diamonds. Blue colors indicates the full MCC pathway, whereas green illustrates the “No Tal” control. Assays were independently run in triplicate (n = 3), with error bars representing SD.