Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts

Abstract

Here, we demonstrate bioconversion of methane to muconic acid, a dicarboxylic acid that can be upgraded to an array of platform chemicals, by three gammaproteobacterial methanotrophs. All engineered methanotrophs expressing a heterologous dihydroxyshikimate dehydratase, protocatechuate decarboxylase, and catechol dioxygenase produced muconic acid from methane, with the highest titer (12.4 mg MA per L), yield (2.8 mg MA per g CH₄), and specific productivity (1.2 mg MA per g dcw, 48 hr) synthesized by Methylotuvimicrobium buryatense, Methylococcus capsulatus, and Methylotuvimicrobium alcaliphilium, respectively. Methylotuvimicrobium alcaliphilum genome-scale model-guided strain engineering predicted that disruption of the pyruvate dehydrogenase or shikimate dehydrogenase would significantly enhance flux to the heterologous muconic acid pathway in this organism. However, knock-out of these targets caused a growth defect, and coupled with similar muconic acid titers (∼1 mg L⁻¹), resulted in minimal flux enhancement to muconic acid in these genetically-modified strains. The shikimate dehydrogenase mutant's ability to grow without aromatic amino acid supplementation revealed that M. alcaliphilum likely encodes an unidentified enzyme or pathway with shikimate biosynthetic capacity, which prevents maximal flux through the synthetic muconic acid pathway. This study expands the suite of products that can be generated from methane using methanotrophic biocatalysts, lays the foundation for green production of muconic acid-derived polymers from methane, and highlights the need for further analysis of methanotroph biosynthetic potential to guide refinement of metabolic models and strain engineering.