About This Project

A low-cost, rapid, and highly sensitive assay is needed to measure methane gas oxidation rates by methane monooxygenase (MMO) enzymes. In this project, we will develop a simple, yet innovative, electrochemical method that can quantify short- and long-term MMO activity. This method will unlock the door for existing and new MMO researchers to more quickly develop and screen the activity of native and engineered MMOs.

Ask the Scientists

Motivating Factor

A novel assay for CH₄ oxidation is needed to enable engineering and heterologous expression of MMO variants for CH₄ mitigation technologies. New technology for CH₄ 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 for existing technology (Abernethy, 2023). Several proposed MR strategies leverage biological CH₄ oxidation in engineered contexts. One is CH₄ oxidizing bioreactors (Lidstrom, 2024); another is engineered crops or managed trees expressing MMOs 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 MMOs. However, significant challenges limit manipulation of MMOs (Tucci, 2024; Rosenzweig, 2025; Reginato, 2024), preventing progress on such technologies.

Specific Bottleneck

A key challenge limiting progress on manipulating and characterizing MMOs is a lack of a high-sensitivity and high throughput assay for facile measurement of CH₄ oxidation activity in cell culture or isolated MMO samples. Current protocols involve monitoring production of ¹³C-labeled methanol by gas chromatography-mass spectrometry (GC-MS). This method, which requires serial delivery of ¹³CH₄ to individual samples, is time-consuming, laborious, and relatively low-sensitivity (~10 µM lower limit of detection) (Ro, 2018). With these limitations, it is difficult to (1) distinguish MMO variants with different activities; (2) measure oxidation rates at very low CH₄ concentrations; and (3) detect incremental progress towards CH₄ oxidation in variants with very low activity. Our abilities to characterize MMO variants, develop heterologous MMO expression systems, and engineer de novo MMOs are thus curtailed significantly.

Actionable Goals

An assay should be developed that enables rapid evaluation of MMO activity at high sensitivity. The assay should be lower cost compared to current methods so that adoption is improved. Analytical instrumentation to quantify trace levels of methanol can be upwards of $200,000 or more (GC-MS). Background methanol levels from labs can contaminate samples, requiring use of expensive ¹³C-labeled compounds. Quantifying methanol is direct evidence of MMO activity, but if a verified indirect method that overcomes the cost, time, limited sensitivity, and errors associated with traditional quantification methods can be developed, more rapid evaluation of MMOs may be possible. The assay should have high sensitivity, such that trace levels of MMO activity can be detected and small differences across MMOs can be observed. Rapid results on the order of seconds to minutes are needed to increase the ability to process dozens of samples per day.