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

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 PA1 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 PA1 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 C 1 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 PA1 as a promising chassis for lignin valorization and provide a framework for engineering formaldehyde-resistant microbial platforms. IMPORTANCE In developing biotechnological solutions for a circular economy, it is critical to valorize all parts of renewable resources, such as lignocellulose from vegetative components of agricultural crops and from bioenergy feedstocks. After chemical breakdown, the aromatics arising from lignin present significant challenges for use due to their toxicity. Here, we address one component of this challenge—the methoxy groups that get released as formaldehyde—and show that existing biotechnological platform organisms with strong formaldehyde metabolism, such as Methylobacterium extorquens , can be transformed into highly capable utilizers of methoxylated aromatics.