A novel MMO that is active and readily expressible across hosts would enable methane mitigation technologies.
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Contributors: Eli Hornstein1, Arjun Khakar2, Verena Kriechbaumer3, Amy Rosenzweig4, Jonas Wilhelm5, Rachel Strickman6, Paul Reginato7
Author contributions: This problem statement was outlined at a Workshop on Biological Methane Removal hosted by Homeworld Collective and Spark Climate Solutions in November, 2024. Paul Reginato and Rachel Strickman developed the outline into a final draft. All other authors outlined the problem statement at the workshop and gave feedback on drafts.
Lead contact: Paul Reginato ([email protected])
Motivating Factor
Novel technology for methane removal (MR) at 1-100 MtCH4 scale is needed, particularly for oxidizing atmospheric concentrations (2 ppm) (NASEM, 2024, Abernethy, 2024) and emissions at 2-1000 ppm that are too dilute to be scalably oxidized with existing technology (Abernethy, 2023).
Several MR strategies have been proposed that would leverage biological CH4 oxidation in engineered contexts. One example is methane oxidation bioreactors (Lidstrom, 2024); another is engineered crops or managed trees expressing methane monooxygenase (MMO) in their leaves or roots, which could oxidize CH4 in soil or ambient air (Strand et al. 2022, Spatola Rossi et al. 2023).
Development of MMO-bearing plants or optimizing CH4 oxidation in reactors will require engineering, heterologous expression, and/or or context-specific characterization of MMO. However, significant challenges limit such manipulation of MMO (Tucci, 2024; Rosenzweig, 2025; Reginato, 2024), preventing progress on technologies leveraging MMO.
Specific Bottleneck
Expression of functional MMO in heterologous hosts is challenging for a variety of reasons. The particulate MMO (pMMO) is a membrane-bound multi-subunit complex. The biophysical conditions and electron delivery pathways needed for high MMO activity are undetermined, and are thought to be strongly coupled to host biology (Tucci and Rosenzweig 2024). Heterologously-expressed pMMO has had low or no activity (, Spatola Rossi, 2023, Gou, 2006). Further, the PmoC subunit of pMMO is toxic to cloning hosts (Stolyar 1999), preventing generation of mutant libraries and substantially hindering efforts to engineer and study pMMO.
The soluble MMO (sMMO) is also a multi-subunit complex, for which functional heterologous expression is hindered by the need for chaperone proteins (Zill, 2022; Bennett, 2021). sMMO activity comparable to lower activities observed in its native context has been achieved in an optimized E. coli host (Bennett, 2021), but those results are challenging to generalize due to host-specific biology.
An alternative pathway to MMO engineering and research would be to pursue de novo engineering of an MMO that is readily heterologously expressible with high activity. Such an engineered MMO would be beneficial even if new research achieves a complete understanding of native MMO function, since it could help circumvent the challenges associated with heterologously expressing a functional multi-protein complex with dependencies on host biology.
Actionable Goals
De novo MMO proteins should be developed that achieve high methane oxidation rates (on par with native MMOs) and can function across diverse heterologous hosts. De novo MMOs would ideally be non-toxic single-domain monomeric proteins that rely minimally on host-specific biology for function, including folding and electron delivery.
The mechanism by which the de novo MMO will obtain the reducing equivalents required for methane oxidation is critically important. The de novo MMO could be fused to a reductant-producing protein or localized to a cellular component with a supply of an appropriate reductant that is readily available across hosts. Characterization of alternative reductants from potential heterologous MMO hosts, in vitro or in natural methanotrophs, might be used to inform other aspects of protein design.
Consideration should also be given to maximizing access of de novo MMO to methane, which is non-polar and is more soluble in a lipid bilayer than the aqueous cytoplasm. Improved access could be achieved by engineering a de novo MMO as a membrane protein. Complementary host-engineering work to optimize methane accessibility could involve establishing lipid-rich environments at the localization region of the MMO.