Evolutionary Integration of a Foreign Aromatic Catabolic Pathway Drives Metabolic Trade-offs in Methylobacterium extorquens PA1

Abstract Bacteria often acquire novel metabolic functions through horizontal gene transfer, allowing them to utilize new carbon sources. However, to benefit from these new pathways, they must be integrated with the host’s native metabolism. In nature, this process is fine-tuned via selection, enabling bacteria to exploit new niches. Alternate routes for pathway integration might yield distinct patterns of trade-offs, leading to differentiation within the adapting population. Understanding the metabolic mechanisms underlying these trade-offs provides insight into what maintains metabolic diversity in the environment. Lignin, a complex aromatic biopolymer, serves as an ideal substrate for exploring these questions, as monomers require complex metabolic pathways to break aromatic rings and be fed directly into central metabolism. In this study, we used the phyllosphere bacterium Methylobacterium extorquens PA1 to examine how a previously engineered catabolic gene cluster enabling lignin monomer utilization integrates with central metabolism. To this end, we experimentally evolved strains on lignin monomers vanillate (VA) and protocatechuate (PCA). Whole-genome sequencing revealed substrate-specific mutations that collectively reprogram stress responses and carbon storage regulation. These mutations resulted in myriad metabolic trade-offs: VA adapted strains showed diminished growth on PCA, PCA adapted strains largely cross-adapted to VA, and both VA and PCA evolution decreased growth rate on non-aromatic native substrates like succinate. These findings illuminate how evolution optimizes catabolic flux and resource allocation to efficiently integrate a foreign pathway following horizontal gene transfer, and the pleiotropic effects of such optimization. Importance Microbial use of aromatics derived from lignin, whether in natural ecosystems or in biomass conversion, has been a major research focus. While aromatic pathways are well-known to have broad substrate specificity, pulling in aromatic molecules at various levels of conversion, it is unclear how much evolutionary constraint there is upon adaptation to each of these steps. By experimentally evolving Methylobacterium extorquens carrying a foreign lignin-derived aromatic catabolic pathway, we demonstrate that adaptation involves not just increased pathway flux but also regulatory rewiring and reallocation of resources, especially through PHB metabolism and the TCA cycle. The results reveal substrate-specific trade-offs and cross-adaptation, illustrating how new functions reshape fitness landscapes. These insights on metabolic innovation, pleiotropy, and diversification following horizontal gene transfer have broad implications for microbial evolution and metabolic engineering. Footnotes ↵* Co-first authors