Engineered Methylobacterium extorquens grows well on methoxylated aromatics due to its formaldehyde metabolism and stress response

Abstract Lignin is a vast yet underutilized source of renewable energy. The microbial valorization of lignin is challenging due to the toxicity of its degradation intermediates, particularly formaldehyde. In this study, we engineered Methylobacterium extorquens to metabolize lignin-derived methoxylated aromatics, vanillate (VA) and protocatechuate (PCA), by introducing the van and pca gene clusters. Compared to Pseudomonas putida, M. extorquens exhibited better formaldehyde detoxification, enabling robust growth on VA without accumulation of formaldehyde. Genetic analyses confirmed that formaldehyde oxidation and stress response systems, rather than C1 assimilation, were important for VA metabolism. Additionally, VA and PCA were found to disrupt membrane potential, contributing to their inherent toxicity. Our findings establish M. extorquens as a promising chassis for lignin valorization and provide a framework for engineering formaldehyde-resistant microbial platforms.