About This Project

Soluble methane monooxygenase (sMMO) is an enzyme capable of fixing atmospheric methane and when applied at scale could help mitigate climate change, yet it has several characteristics hindering its industrial application. This project sets out to engineer and evolve sMMO to increased catalytic rates, by utilizing breakthroughs in heterologous methane metabolism and continuous accelerated directed evolution in E. coli, while producing the world’s first true synthetic methanotroph.

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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 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 [3].

Methane monooxygenase (MMO) enzymes naturally catalyze oxidation of CH₄ to methanol in a one-step reaction at ambient conditions [4]. Oxidation of dilute CH₄ at scale may be possible in engineered systems using methanotrophs or cell-free MMO, for example via flow-through reactors [5][6], or expression in plants [7]. Achieving cost-effective, scalable low-CH4 oxidation requires a 10-fold efficiency boost to reach $100/t CO2e [6].

Specific Bottleneck

Engineering MMO or screening many 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) [4][8]. However, sMMO engineering thus far has focused on manufacturing applications with high CH₄ concentrations [9][10][11][12].

Specific affinity (a˚_S), defined as V_max/k_M, is a metric for enzyme activity at very low substrate concentration that should be maximized[5]. pMMO has been favored over sMMO in proposed technologies owing to a lower apparent k_M [5][6]; however, few sMMO variants have been characterized [13], and other natural or engineered variants might achieve lower k_M and/or higher a˚_S. In heterologous expression or cell-free deployment, suppressing non-specific oxidation of methanol to toxic formaldehyde will also be crucial [14][15].

Actionable Goals

While design constraints for an application scenario are needed to establish clear performance targets for sMMO engineering, work should begin now to engineer sMMO. Goals are higher a˚_S of the MMOH hydroxylase component of sMMO, as well as suppression of non-specific oxidation of methanol to toxic formaldehyde. A k_M < 50 nM would be comparable to the lowest whole-cell k_M value measured for a methanotroph [16]. This work should involve protein engineering and screening a large number of sMMO variants.