About This Project

Genetic tools that enable the transversal of methanotroph pMMO expression plasmids through E. coli are needed to realize CH4 mitigation biotechnologies. This project will develop genetic tools that enable cloning and mutagenesis of the pMMO genes in E. coli and subsequent expression of wild-type and mutant pMMO variants in native methanotrophs. This project will increase our understanding of pMMO and guide the development of biotechnologies for atmospheric CH4 capture.

Ask the Scientists

Motivating Factor

Methane (CH₄) has caused 30% of global warming to date. If all known CH₄ emissions mitigation strategies are implemented, meeting 2°C targets will require removal of 25-120 Mt CH₄/year from the atmosphere (currently 2 ppm and rising) and dilute area sources (<1000 ppm)2,3. However, no technologies exist for economical CH₄ oxidation below 1000 ppm; thus, major breakthroughs are needed.

Several CH₄ removal approaches under consideration rely on enzymatic CH₄ oxidation by CH₄ monooxygenase (MMO) from methanotrophs, and development of biotechnologies using these unique microbes requires strain engineering or context-specific characterization of MMO4^-6

Specific Bottleneck

Particulate MMO (pMMO) is considered to have the greatest potential for methane oxidation due to its high activity and dominance in nature4. However, methodological challenges to rapidly generate pMMO variants render it practically impossible to engineer and characterize pMMO at a rate needed for effective technology development across several CH4 removal approaches. Specific challenges are directly related to pMMO expression toxicity (lethality) in cloning hosts7–10.

Actionable Goals

Methods for cloning pMMO in heterologous hosts should be developed to enable structure-function analyses and engineering of pMMO. Such tools would be broadly enabling for pMMO expression in native and heterologous hosts. Examples of enabling methods include:

• Development of genetic tools that limit pMMO expression/toxicity in heterologous hosts but allow native expression levels in methanotrophs.
• Discovery or development of a heterologous or native host in which pMMO variants can be functionally expressed.