About This Project

Methane removal at 1-100 MtCH₄ scale requires novel oxidation technologies, especially for atmospheric and dilute emissions. Methane monooxygenases (MMOs) enable biological CH₄ oxidation but face expression and activity challenges in heterologous hosts. Inspired by recently engineered miniaturized MMOs, we propose the computational design of de novo MMOs (dnMMOs), aiming for heterologously expressed, soluble, highly active enzymes that overcome current limitations and enhance MR efficiency.

Ask the Scientists

Motivating Factor

Novel technology for methane removal (MR) at 1-100 MtCH4 scale is needed, particularly for oxidizing atmospheric concentrations (2 ppm) [1][2] and emissions at 2-1000 ppm that are too dilute to be scalably oxidized with existing technology [3].

Several MR strategies have been proposed that would leverage biological CH4 oxidation in engineered contexts. One of such strategies is methane oxidation bioreactors based on using aerobic methanotrophs [4]. Such development would require heterologous expression, engineering, and/or context-specific characterization of methane monooxygenases (MMOs).

These MMOs exist in two forms: soluble (sMMOs) and particulate (pMMOs), with the former being better described at the structural and functional level [5]. Still, both types of MMOs impose significant challenges, particularly regarding heterologous expression in conventional host organisms, limiting their characterization and engineering [5] and preventing progress on technologies leveraging MMOs.

Specific Bottleneck

Expression of functional MMO in heterologous hosts faces many challenges. pMMO is a membrane-bound multi-subunit complex. The biophysical conditions and electron transfer pathways for high pMMO activity are undetermined, but thought to be strongly coupled to host biology [5]. Heterologously-expressed pMMO have shown low or no activity [6][7], and its PmoC subunit is toxic to cloning hosts [8], thus hindering efforts to engineer and study pMMO.

The sMMO is also a multi-subunit complex, for which functional heterologous expression is hindered by the need for chaperone proteins [9][10]. sMMO activity comparable to lower activities observed in its native context has been achieved in an optimized E. coli host [10], but those results are challenging to generalize due to host-specific biology.

An alternative pathway would be to pursue de novo engineering of an MMO that is readily heterologously expressible with high activity, thus circumventing the challenges associated with natural MMOs.

Actionable Goals

Recently, two engineered miniaturized soluble variants of sMMO (mini-sMMO) and pMMO (pMMO-mimic) from M. capsulatus (Bath) have been developed [11] [12]. They are built only from domains essential for or enhancing catalytic activity, scaffolded by human apoferritin (huHF). Strikingly, mini-sMMO exhibits an in vitro methanol turnover of 0.32 s−1 and leads to higher yield (3.0 g/L) and productivity (0.11 g/L/h) using recombinant E. coli in comparison to methanotrophs, under CH4 and air mixtures at 3:7 v/v [11]. While the turnover rate of pMMO-mimic is modest (0.084 s−1), at par with wt pMMO, it can be integrated into a hydrogel, greatly increasing their overall stability and cumulative production of methanol when compared to the non-scaffolded counterpart [12].

Building on this work, we propose to computational design de novo MMOs (dnMMOs), aiming to eliminate the need of a nanoparticle scaffold for soluble production and function and achieve higher methanol turnover in vitro.