Conservation, origin, and remodeling of the phenyl acetate degradation pathway

Aromatic hydrocarbons are widespread environmental compounds whose microbial degradation is essential for detoxification, yet the genomic distribution and evolutionary stability of many catabolic pathways remain poorly understood. The phenylacetic acid (PAA) hybrid pathway integrates aerobic and anaerobic features and represents a central route for aromatic compound degradation; however, its prevalence, evolutionary conservation and regulatory organization across prokaryotes have not been comprehensively reassessed.Here, we investigated the global distribution, structural conservation and evolutionary dynamics of the PAA pathway across prokaryotic genomes. We performed a large-scale comparative genomic analysis of 6,536 bacterial and archaeal genomes, leveraging functionally annotated genomes to enable consistent identification of pathway components, integrating protein architecture profiling, genomic context organization, phylogenetics, motif and structural analyses of phenylacetate-CoA ligase (PAL) and cophylogenetic inference. Using stringent criteria, we identified 853 high-confidence PAL predictions across 825 genomes (≈12%), revealing substantial previously unrecognized PAA metabolic potential. Structural analyses confirmed the conservation of key catalytic features, supporting functional annotation of newly identified PALs. Genomic context analyses revealed extensive pathway remodelling across lineages, yet a conserved core of local regulatory elements mediated by PaaX, PaaR and PaaY was maintained. Notably, PaaX and PaaR were, for the first time, predicted to co-occur within the same genomic context organization in several groups. In contrast, archaeal genomes predominantly encode incomplete PAA configurations, suggesting functional divergence and/or pathway fragmentation.Together, these results demonstrate that the PAA pathway is more widespread and evolutionarily dynamic than previously appreciated, combining deep enzymatic conservation with flexible genomic organization. Finally, the conservation of the core epoxidase subunits PaaA and PaaC across PAL-encoding genomes supports their use as robust genomic markers of PAA pathway potential.