Metabolic engineering of Escherichia coli for the utilization of methylsuccinate, the product of methane activation via fumarate addition.

Methylsuccinate is a branched-chain, 5-carbon (C5) dicarboxylate that can be generated from the O-independent activation of methane via fumarate addition. However, no established metabolic pathway enables growth and product synthesis from methylsuccinate. Here, we report a synthetic pathway that converts methylsuccinate into two precursor metabolites: pyruvate and acetyl-CoA. The pathway was constructed through rational design and validated both in vitro and in vivo using E. coli as the host. Subsequently, growth on methylsuccinate as the sole carbon source was achieved using two parallel strategies: adaptive laboratory evolution and enzyme mining. Through the latter approach, we identified a heterologous electron transfer pathway mediated by previously uncharacterized enzymes and integrated into E. coli enabling the conversion of methylsuccinyl-CoA to mesaconyl-C4-CoA. The engineered strain demonstrated efficient growth on various C5 dicarboxylates including methylsuccinate, mesaconate, and itaconate, with a specific growth rate of 0.11 h on methylsuccinate. This study represents an important step toward achieving synthetic methanotrophy, as the engineered strain can serve as a platform for screening potential methane activation enzymes and ultimately as a production chassis for the bioconversion of methane into various value-added products.