A novel assay for methane oxidation is needed to enable engineering and heterologous expression of MMO variants for methane mitigation technologies.
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Contributors: Amy Rosenzweig1, Eli Hornstein2, Arjun Khakar3, Verena Kriechbaumer4, 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, 2022; Spatola Rossi 2023).
Development of MMO-bearing plants or optimizing CH4 oxidation in reactors will require engineering and/or heterologous expression of MMO. However, significant challenges limit such manipulation of MMO (Tucci, 2024; Rosenzweig, 2025; Reginato, 2024), preventing progress on such technologies.
Specific Bottleneck
A key challenge limiting progress on manipulating and characterizing MMO is a lack of a high-sensitivity and high throughput assay for facile measurement of methane oxidation to methanol at low methanol concentrations in cell culture or isolated MMO samples. Current protocols involve monitoring production of isotopically-labeled methanol by gas chromatography-mass spectrometry (GC-MS). This method, which requires serial delivery of the gaseous methane substrate to individual samples, is time-consuming, laborious, and relatively low-sensitivity (~10 µM lower limit of detection) (Ro, 2018). The low throughput of this assay (100 samples per week, including controls and replicates) precludes efforts to engineer and screen new variants.
With these limitations on throughput and sensitivity, it is difficult to 1) distinguish MMO variants with different activities; 2) measure oxidation rates at very low methane concentrations (e.g. near atmospheric concentration); and 3) detect incremental progress towards methane oxidation in engineered or heterologous variants with very low activity. Our abilities to characterize MMO variants, build a sequence-function framework for MMO catalysis, develop heterologous MMO expression systems, and engineer de novo MMOs are thus curtailed significantly.
Actionable Goals
An assay should be developed to assay CH4 oxidation to methanol, with sensitivity to detect nM or sub-nM methanol and throughput to test at least 1000 samples per week (i.e., ~150 distinct test conditions, accounting for controls and replicates), or ideally 10000 samples per week. A higher-throughput assay enabling up to millions of measurements per week, perhaps using fluorescence-activated cell sorting (FACS) to read out methanol concentrations, is also desirable.