The Context for this problem statement was originally shared by Wenyu Gu at a Homeworld Collective workshop on protein engineering and climate tech. This problem statement is also hosted on Homeworld's problem statement repository here.

CH₄ emissions have contributed ~30% of global warming to date (IEA 2022), and natural sources may increase via feedback to warming (Zheng 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 (Abernethy 2023).

Methane monooxygenase (MMO) enzymes, found in methanotrophic bacteria, naturally catalyze oxidation of CH₄ to methanol in a one-step reaction at ambient conditions (Tucci 2024). 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 (Lidstrom 2023, Yoon 2009) or expression in plants (Rossi 2023).

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 (Tucci, 2024). 

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 (Yan et al, 2016). CRISPR-based editing is limited by low efficiency in methanotrophs (Tapscott et al 2019, Nath et al, 2022) and the presence of two genomic pmo loci in many methanotrophs [13]. 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.

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
• development 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.

Open Questions‍

The context 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 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.

The 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

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, Ail 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 ¹ (IRENA 2021); 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). Engineering sMMO is also relevant to production of methanol and other chemicals.

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[1]: 2.3 t CO₂/t methanol for ~98 Mt of methanol manufactured from methane. (IRENA, 2022) specifies emissions of ~0.1 t CO₂/GJ, which converts to CO₂/t by: 0.1 t CO₂/GJ * 17.8 GJ/m³ methanol * 1/0.792 m³ methanol/t = 2.3 CO₂/t methanol (Neutrium, 2023).