Systems-Level Dissection of Methanol Adaptation in Methylorubrum extorquens

Abstract

The transition to a sustainable bioeconomy demands robust microbial platforms capable of converting single-carbon (C1) feedstocks like methanol into value-added chemicals. Methylorubrum extorquens represents a key candidate for this role. However, utilizing its metabolic potential is critically hindered by two fundamental barriers: (1) the ‘strain selection problem’, the need to navigate the high genomic diversity of the species (e.g., the complex, plasmid-heavy AM1) to identify a stable and efficient strain, and (2) the ‘adaptation problem’, the need to elucidate the mechanisms of methanol tolerance to overcome its inherent cytotoxicity. This dissertation addresses these two barriers sequentially through an integrated systems biology approach. First, the ‘strain selection problem’ was addressed through a comprehensive pan-genome analysis of all available, completely sequenced M. extorquens strains. This study quantitatively validated that M. extorquens PA1, while eliminating non-essential genetic burdens like plasmids, fully preserves the core C1 metabolic network. These findings identified PA1 as a superior, streamlined strain and provided the necessary controlled system for subsequent adaptation studies. Next, to elucidate the molecular mechanisms of methanol tolerance, the model strain M. extorquens AM1 was subjected to adaptive laboratory evolution (ALE) and analyzed through integrated genomic and transcriptomic approaches. The evolved strains exhibited convergent mutations in the metY gene, involved in methionine biosynthesis, and the kefB gene, associated with cellular energy conservation. This demonstrates at a systems level that M. extorquens adapts to methanol stress by fine-tuning metabolic pathways to mitigate byproduct toxicity and by efficiently managing cellular energy. Finally, the interplay between rational design and evolution was investigated by tracking the adaptation of an engineered strain (PA1 Δfdh234) with a simplified formate oxidation step. Unexpectedly, evolution bypassed the engineered catabolic target and converged on a single point mutation in metY-1, demonstrating that, under reduced catabolic redundancy, ensuring substrate specificity through toxicity mitigation is a higher evolutionary priority than enhancing catabolic flux. This reveals a fundamental trade-off in which microbes prioritize metabolic stability and fidelity over mere rate enhancement. Therefore, this dissertation presents two critical outcomes for C1-biomanufacturing: (1) a new design principle (substrate specificity > catabolic flux) that provides a new perspective on engineering robust methylotrophic strains, and (2) validated, high-priority engineering targets (metY, kefB) for the development of next-generation robust methylotrophic platforms.