A key O -demethylase in the degradation of guaiacol by Rhodococcus opacus PD630.

PD630 is a high oil-producing strain with the ability to convert lignin-derived aromatics to high values, but limited research has been done to elucidate its conversion pathway, especially the upper pathways. In this study, we focused on the upper pathways and demethylation mechanism of lignin-derived aromatics metabolism by PD630. The results of the aromatic carbon resource utilization screening showed that PD630 had a strong degradation capacity to the lignin-derived methoxy-containing aromatics, such as guaiacol, 3,4-veratric acid, anisic acid, isovanillic acid, and vanillic acid. The gene of , which encodes cytochrome P450, showed significant up-regulation when PD630 grew on diverse aromatics. Deletion mutants of and its partner protein resulted in the strain losing the ability to grow on guaiacol, but no significant difference to the other aromatics. Only co-complementation alone of and restored the strain's ability to utilize guaiacol, demonstrating that both genes were equally important in the utilization of guaiacol. assays further revealed that GcoA could convert guaiacol and anisole to catechol and phenol, respectively, with the production of formaldehyde as a by-product. The study provided robust evidence to reveal the molecular mechanism of PD630 on guaiacol metabolism and offered a promising study model for dissecting the demethylation process of lignin-derived aromatics in microbes.IMPORTANCEAryl--demethylation is believed to be the key rate-limiting step in the catabolism of heterogeneous lignin-derived aromatics in both native and engineered microbes. However, the mechanisms of -demethylation in lignin-derived aromatic catabolism remain unclear. Notably, guaiacol, the primary component unit of lignin, lacks demonstration and illustration of the molecular mechanism of guaiacol -demethylation in lignin-degrading bacteria. This is the first study to illustrate the mechanism of guaiacol metabolism by PD630 as well as characterize the purified key -demethylase . This study provided further insight into the lignin metabolic pathway of PD630 and could guide the design of an efficient biocatalytic system for lignin valorization.