A distinct 2,2′-diphenic acid-mediated pyrene degradation pathway in a novel PAH degrader, <i>Glutamicibacter soli</i> ENR6

Bacteria play an important role in the biodegradation of polycyclic aromatic hydrocarbons (PAHs), especially the more recalcitrant high-molecular-weight compounds like pyrene. In this study, we isolated and characterized a novel pyrene-degrading bacterium, <i>Glutamicibacter soli</i> ENR6, from petroleum-contaminated soil. Strain ENR6 was able to utilize various PAHs as sole carbon sources and degrade 88.8% of 50 mg L^-1 pyrene and 76.8% of 100 mg L^-1 pyrene within 15 days. During pyrene degradation, eight metabolites were detected, with 2,2'-diphenic acid emerging as the predominant intermediate. By day 7, 40.6% of the degraded pyrene had accumulated in the form of 2,2'-diphenic acid, which was subsequently metabolized via the phthalate pathway. To our knowledge, this is the first report identifying 2,2'-diphenic acid as an intermediate in pyrene degradation. Integrated genomic and transcriptomic analyses revealed four gene clusters (clusters I-IV) involved in pyrene degradation in strain ENR6. Reverse transcription-quantitative PCR (RT-qPCR) analysis demonstrated that these clusters were specifically induced by pyrene and its intermediates, with genes <i>chr_3400-chr_3404</i> in cluster I strongly associated with 2,2'-diphenic acid degradation. Based on these findings, we propose a distinct 2,2'-diphenic acid-centered pathway for pyrene degradation in strain ENR6. This study not only broadens the bacterial repertoire available for PAH bioremediation but also provides new insights into the metabolic diversity of PAH-degrading microorganisms.IMPORTANCEPolycyclic aromatic hydrocarbons (PAHs) are widespread, highly toxic, and persistent environmental pollutants. Microbial biodegradation offers a sustainable approach for PAH remediation, yet the metabolic mechanisms remain incompletely understood. In this study, we isolated and characterized a novel pyrene-degrading bacterium, <i>Glutamicibacter soli</i> ENR6, thereby expanding the bacterial repertoire available for PAH bioremediation. The identification of 2,2'-diphenic acid as a key intermediate reveals a previously unrecognized pathway of pyrene degradation, in which the subsequent catabolism of 2,2'-diphenic acid is mediated by a newly identified ring-hydroxylating dioxygenase. This pathway provides an alternative metabolic entry point into the broadly conserved phthalate pathway when the classical 1-hydroxy-2-naphthoic acid-centered pathway is inhibited. The distinct 2,2'-diphenic acid-mediated pyrene degradation pathway elucidated in strain ENR6 highlights metabolic diversity among PAH-degrading microorganisms and provides a mechanistic foundation for applying this strain in the bioremediation of PAH-contaminated sites.