Engineering E. coli for H₂ -driven hydroxylation reactions through the heterologous production of hydrogenases and methane monooxygenases

Introduction: The conversion of the potent greenhouse gas methane to methanol presents a promising method for reducing emissions while producing valuable chemicals. Unlike standard industrial catalysts, soluble methane monooxygenases (sMMO) offer an environmentally friendly alternative by converting methane to methanol under mild conditions^1,2. In vivo systems maintain enzyme stability and activity in a controlled cellular environment, thereby increasing the overall efficiency of the reaction. E. coli offers faster growth rates and easier genetic manipulation than methanotrophic organisms, making it a more practical host for bioengineering applications targeting methanol production.

Goals: This study explores using an engineered E. coli system for methane-to-methanol conversion by introducing synthetic metabolic pathways alongside a soluble NAD⁺-reducing hydrogenase (SH) for cofactor recycling to increase process sustainability^1,3. A connected zero-gap electrolyzer system with selective permeability decouples mass and electron transfer, allowing precise redox control and facilitate proton exchange without direct contact between the cathode and anode⁴.

Materials & Methods: Our engineered E. coli features two plasmid integrations encoding 1) SH for H₂-driven NAD⁺-reduction and 2) sMMO for methane oxidation. In addition, the adaptable plasmid design allows rapid sMMO mutagenesis to expand the substrate range.

Results: We successfully achieved robust heterologous expression of active SH in E. coli under challenging aerobic conditions. We also demonstrated sMMO production in E. coli, opening new ways for methane-to-methanol conversion in a highly versatile host system¹.

Summary: We develop a sustainable method to convert methane to methanol in engineered E. coli, reducing emissions and creating valuable chemicals. Enhancing sMMO activity and NADH recycling via SH with a zero-gap cell offers a low-energy alternative to traditional methanol production and enables precise redox control for broader electrobiochemical applications.

1] D. Zill, E. Lettau, C. Lorent, F. Seifert, P. K. Singh, L. Lauterbach, ChemBioChem 2022, 23, e202200195.

2] F. J. Tucci, A. C. Rosenzweig, Chem. Rev. 2024, 124, 1288–1320.

3] A. Al-Shameri, N. Borlinghaus, L. Weinmann, P. N. Scheller, B. M. Nestl, L. Lauterbach, Green Chem. 2019, 21, 1396–1400.

4] R. Rad, T. Gehring, K. Pellumbi, D. Siegmund, E. Nettmann, M. Wichern, U.-P. Apfel, Cell Reports Physical Science 2023, 4, 101526.