Discover or engineer efficient soluble methane monooxygenase

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Origin: This problem statement was created as part of a collaborative effort between Spark Climate Solutions and Homeworld Collective to identify and share priority problems at the intersection of biotech and atmospheric methane removal. The Context for this problem statement was originally shared by Wenyu Gu at a Homeworld workshop on protein engineering and climate tech.

Contributors:  Paul Reginato1, Lisa Stein2, Mary Lidstrom3, Jeremy Semrau4, Jessica Swanson5, Wenyu Gu6, Noah Helman7, James Weltz8, Mark Hansen9, Paige Brocidiacono10, Ariana Caiati11, Erin Wilson12

Lead contact: Paul Reginato ([email protected])

Problem Statement

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, found in methanotrophic bacteria, naturally catalyze oxidation of CH₄to 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]. 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 [6]. 

Specific Constraint

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) [4] [8]. However, sMMO engineering thus far has focused on manufacturing applications with high CH₄ concentrations [9][10][11][12]. sMMO activity should be engineered using high-throughput methods in an expression platform where the enzyme can fold well [9][10]. 

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 screening of natural sMMO variants from uncultured microbes derived from metagenomic sequence data, in addition to protein engineering.

Additional information

Open Questions

The Motivating Factor could be made more clear by:

• Statement of the current estimated cost impact of catalyst inefficiencies on methane oxidation technologies, relative to other components of the technology. 

The Specific Constraint 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.

The Actionable Goals could be made more granular by:

• Providing a target for V_max of sMMO

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

Related problem statements 

• Genetic tools for manipulation of pMMO

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 [17][18]. 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/yr13 [19]; 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 [17]. Engineering sMMO is also relevant to production of methanol and other chemicals.