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Context

CH₄ emissions have contributed ~30% of global warming to date (IEA, 2022), and natural sources may increase via feedback to warming (Zhang, 2023). Technologies for oxidizing atmospheric CH₄, area CH₄ emissions, and unavoidable point sources could substantially mitigate climate change. While CH₄ above ~44,000 ppm can be flared, ~75% of CH₄ pollution is atmospheric (2 ppm) or area emissions below 1000 ppm that are too dilute to be oxidized at scale using existing technologies (Abernathy, 2023).

Methane monooxygenase (MMO) enzymes, found in methanotrophic bacteria, naturally catalyze oxidation of CH₄to methanol in a one-step reaction at ambient conditions (Tucci, 2024). Oxidation of dilute CH₄ at scale may be possible in engineered systems using methanotrophs or cell-free MMO, for example via flow-through reactors (Lidstrom, 2023; Yoon, 2009), or expression in plants (Spatola, 2023). Enhancing oxidation rate at low (2-1000 ppm) CH₄ concentrations is necessary to achieve feasible cost and scalability for these applications. For example, 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 (Yoon, 2009).

Significance

Engineering MMO or screening many natural variants 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 study, handling and heterologous expression compared to particulate MMO (pMMO) (Tucci, 2024; Reginato, 2024). Nonetheless, sMMO presents barriers to engineering. The MMOH hydroxylase component of sMMO is challenging to express in E. coli. One study reported MMOH expression in E. coli using chaperones and solubility-enhancing mutations, but stopped short of engineering enhanced activity (Bennett, 2021). Protein engineering platforms in methanotrophs have been developed, but studies using these have been limited to only a few variants owing to the difficulty of handling methanotrophs (Lock, 2017). A system for robustly expressing and screening diverse natural and engineered sMMO variants would enable progress on engineering or discovering efficient sMMO.

Goals

Methods should be developed for robust expression and screening of diverse natural and engineered sMMO variants with active MMOH. Such methods should handle >10⁵ variants, and should either enable active natural sMMO variants to be expressed without modification, or should enable rapid design of solubility-enhanced versions of natural variants without reducing their activity. Approaches might include developing a platform in E. coli, perhaps building on the work of Bennett et al. (Bennett, 2021); developing a platform in a non-model organism that can more easily express active MMOH; or developing a platform in methanotrophs that is amenable to high-throughput approaches. We note that the company Industrial Microbes has been pursuing sMMO engineering, but their work aside from (Bennett, 2021) has not been shared publicly.

Open Questions

The significance could be made more clear by:

• Providing a clearer description of the target deployment scenario for engineered sMMO to ground the motivation and guide engineering.

Assumptions

This problem statement assumes:

• Technologies for oxidation of atmospheric CH4 or area emissions using flow-through reactors are not economically prohibited by CapEx or the cost of moving air
• The methane oxidation rate that would enable economical flow-through reactors is within a feasible range for engineered sMMO

Other information

Biological CH₄ oxidation technologies could also substantially improve the sustainability of methanol production and economically drive mitigation of point-source CH4 emissions through methanol manufacturing (Haynes, 2014; Shivananda, 2016). A high-specificity methane-to-methanol oxidation catalyst that operates at ambient temperatures could: 1) obviate high temperatures and pressures (200-300 ˚C, 50-100 atm) of the current industrial process, thereby reducing process emissions by up to ~0.25 Gt CO₂/yr (Haynes, 2014); and 2) enable one-step manufacturing process that has lower CapEx than the current two-step process, thereby enabling economical use of dilute or low-flux CH₄ sources that are currently leaked to the atmosphere (Haynes, 2014). The goals of this problem statement also apply to methanol production.