About This Project

Methane (CH₄) emissions drive ~30% of global warming, yet existing oxidation technologies are ineffective at low CH₄ concentrations (<1000 ppm). Soluble methane monooxygenases (sMMO) offer a promising biocatalytic solution, but its engineering faces expression and efficiency challenges. A recently developed mini-sMMO shows enhanced activity and scalability. This project aims to optimize mini-sMMO for solubility, stability and high-yield CH₄ oxidation into methanol at low concentrations.

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Motivating Factor

CH4 emissions have contributed ~30% of global warming to date [1], and natural sources may increase via feedback to warming [2]. Technologies for oxidizing atmospheric CH4, area CH4 emissions, and unavoidable point sources could mitigate climate change. While CH4 >44,000 ppm can be flared, CH4 atmospheric pollution (2 ppm) and area emissions (<1000 ppm) are too dilute to be oxidized at scale using existing technologies [3].

Methane monooxygenases (MMOs) from methanotrophic bacteria oxidize CH4 into methanol in a one-step reaction at ambient conditions [4]. Oxidation of dilute CH4 at scale may be possible in engineered systems using whole-cell or cell-free MMOs, e.g. via flow-through reactors [5][6]. Enhancing oxidation rate at low (2-1000 ppm) CH4 concentrations is required for feasible cost and scalability of these applications. Estimates indicate efficiency improvements must enable up to 10-fold cost reduction for oxidation of 500 ppm CH4 to reach $100/t CO2e in a reactor [6].

Specific Bottleneck

Engineering MMOs may provide a path to efficient CH4 oxidation. Of the two MMO families, soluble MMO (sMMO), a three-part enzymatic system, is a stronger candidate for engineering, since it faces fewer challenges to heterologous expression, handling and study compared to particulate MMO (pMMO) [4].

However, sMMO engineering has several caveats: (a) its engineering has been focused on applications under high CH4 conditions, i.e., pure CH4 [7][8]; (b) its expression has been mainly successful when using methanotrophs for either homologous (M. trichosporium OB3b) or heterologous expression (M. album BG8, M. parvus OBBP) [9] ; (c) when using more popular bacterial systems for protein production and engineering like E. coli, cloning of all three components plus the GroESL chaperone is required for successful expression [7][10]; (d) improving the activity of sMMOs requires the engineering of all three components, which typically are studied one by one devoid of the overall context [8].

Actionable Goals

Recently, a two-component version of Methylococcus capsulatus (Bath) sMMO (mini-sMMO) was engineered. It comprises trimmed versions of its hydrolase (MMOH) and reductase (MMOR) components in a single chain, and the N-terminal MMOH-binding region of the regulatory component (MMOB) in another chain, scaffolded by human apoferritin (huHF) [9]. Strikingly, mini-sMMO exhibits an in vitro methanol turnover of 0.32 s−1 and higher yield (3.0 g/L) and productivity (0.11 g/L/h) using recombinant E. coli in comparison to methanotrophs in whole-cell experiments on a fed-batch bioreactor, under CH4 and air mixtures at 3:7 v/v.

Due to its smaller size, this mini-sMMO is a perfect model system for further computational and experimental engineering, aiming to eliminate the need of a nanoparticle scaffold for its soluble production and function, improve its methanol turnover in vitro beyond 0.32 s-1 and obtaining high CH4 to methanol conversion yields even at lower CH4 air mixtures (<1000 ppm).