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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, Calvin Henard2, Mary Lidstrom3, Jeremy Semrau4, Jessica Swanson5, Wenyu Gu6, Lisa Stein7, 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 using methanotrophs or cell-free MMO, for example via enhanced natural CH4 sinks, flow-through reactors [5][6], or expression in plants [7].

Specific Constraint

The particulate MMO (pMMO) family is dominant and essential in most methanotrophs, and may have a deployment advantage over soluble MMO (sMMO) due to its occurrence within a membrane, where CH₄ is more soluble. To enable CH4 oxidation technologies using pMMO, it will be valuable to identify pMMO variants with optimal activity and/or to engineer pMMO. However, it has proved difficult to isolate functional pMMO or discover its mechanism, let alone engineer or screen variants [4]. 

Expression of pMMO variants in methanotrophs is complicated by the toxicity of the PmoC subunit in E. coli, which prevents the use of E. coli to produce plasmids encoding the pmo operon for transfer into methanotrophs for homologous expression or genomic recombination [8][9]. Further, methanotroph electroporation efficiency is too low for electroporation of in vitro-constructed plasmids [10]. CRISPR-based editing is limited by low efficiency in methanotrophs [11][12] and the presence of two genomic pmo loci in many methanotrophs13. Methods for expressing pMMO variants in methanotrophs should be developed in order to enable analysis and engineering of pMMO. Such tools would also be broadly enabling across methanotroph research.

Actionable Goals

Methods for expressing pMMO variants, such as point mutants or epitope-tagged pMMO, must be developed to enable study and engineering of pMMO. Examples of enabling methods include:

• discovery or development of a host that can tolerate the pMMO gene sequence on a plasmid and enable conjugation or electroporation of the plasmid into methanotrophs

• evelopment of a high-efficiency genome editing systems for MMO editing in situ

• discovery or development of a heterologous host in which pMMO variants can be functionally expressed.

Additional information

Open Questions

The Motivating Factor could be better supported by:

• quantitative analyses of flow-through reactor designs based on MMO or methanotrophs and potential revenue-generating co-products, indicating their feasibility and the degree to which oxidation rates would need to be improved for a reactor to be economical

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 pMMO

Related problem statements 

• Discover or engineer efficient soluble methane monooxygenase

• Methods to express and screen 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 [13][14]. 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/yr14 [15]; 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 [13]. The goals of this problem statement also apply to methanol production.