A Chimera between Particulate and Soluble Methane Monooxygenases Produces Methanol Using Endogenous NADH in <i>Escherichia coli</i>

Methane monooxygenases (MMOs), found in methanotrophs as particulate (pMMO) or soluble (sMMO) forms, catalyze methane oxidation through overcoming high activation energy of the stable C–H bond under ambient conditions and are thus promising biocatalysts for environmentally and economically benign utilization of methane; however, their mass production and industrial application remain unsettled due to membrane-associated, unclarified electron transfer to the catalytic site of pMMO and multicomponent, complex structure and function of sMMO. Here, we developed a chimeric MMO (cMMO) through reassembling the catalytic dicopper center of pMMO and the endogenous NADH-dependent FAD domain of sMMO on a robust scaffold, which is favorable for molecular editing. The cMMO was produced as a catalytically active enzyme (turnover frequency, ∼0.1 s–1) in a large quantity from Escherichia coli. Notably, cMMO-expressing E. coli successfully converted methane to methanol without a requirement of exogenous reductants, indicating that this innovative approach offers an attractive platform for biocatalytic methane utilization.