Elucidation of the coumarin degradation by Pseudomonas sp. strain NyZ480

As a phytotoxin and synthetic chemical, coumarin (COU) is known for its hepatotoxicity and carcinogenicity. However, no thorough characterization of its microbial degradation has been reported. Here, Pseudomonas sp. strain NyZ480 was isolated for its capability of utilizing COU as the sole carbon source. Studies on its growth and degradation efficiency of COU under various conditions suggested that strain NyZ480 performed the optimum degradation at 30 ℃, pH 7, and 0.5 mM COU was completely removed within 4 h with 1% inoculum. HPLC and LC-MS analyses indicated that dihydrocoumarin (DHC), melilotic acid (MA) and 3-(2,3-dihydroxyphenyl)propionate (DHPP) were the upstream biotransformation intermediates of COU. Enzyme assay established that the initial reaction transforming COU to DHC required an NAD(P)H-dependent reductase, followed by the hydrolysis of DHC to generate MA, and the third reaction catalyzing the monooxygenation of MA to DHPP utilized a strict NADH-dependent hydroxylase. Combining genomics and transcriptomics, we proposed that the COU downstream degradation (from DHPP) was catalyzed by enzymes encoded by a gene cluster homologous to the mhp cluster for 3(3-hydroxyphenyl)propionate degradation via DHPP in E. coli. This study thoroughly identified the intermediates from the COU catabolism, providing essential insights into the molecular evidences of its biodegradation pathway.