Redundant and scattered genetic determinants for coumarin biodegradation in Pseudomonas sp. strain NyZ480.

Coumarin (COU) is both a naturally derived phytotoxin and a synthetic pollutant which causes hepatotoxicity in susceptible humans. Microbes have potentials in COU biodegradation; however, its underlying genetic determinants remain unknown. Pseudomonas sp. strain NyZ480, a robust COU degrader, has been isolated and proven to grow on COU as its sole carbon source. In this study, five homologs of xenobiotic reductase A scattered throughout the chromosome of strain NyZ480 were identified, which catalyzed the conversion of COU to dihydrocoumarin (DHC) in vitro . Phylogenetic analysis indicated that these COU reductases belong to different subgroups of the old yellow enzyme family. Moreover, two hydrolases (CouB1 and CouB2) homologous to the 3,4-dihydrocoumarin hydrolase in the fluorene degradation were found to accelerate the generation of melilotic acid (MA) from DHC. CouC, a new member from the group A flavin monooxygenase, was heterologously expressed and purified, catalyzing the hydroxylation of MA to produce 3-(2,3-dihydroxyphenyl)propionate (DHPP). Gene deletion and complementation of couC indicated that couC played an essential role in the COU catabolism in strain NyZ480, considering that the genes involved in the downstream catabolism of DHPP have been characterized (Y. Xu and N. Y. Zhou, Appl Environ Microbiol 86:e02385-19, 2020) and homologous catabolic cluster exists in strain NyZ480. This study elucidated the genetic determinants for complete degradation of COU by Pseudomonas sp. strain NyZ480.IMPORTANCECoumarin (COU) is a phytochemical widely distributed in the plant kingdom and also artificially produced as an ingredient for personal care products. Hence, the environmental occurrence of COU has been reported in different places. Toxicologically, COU was proven hepatotoxic to individuals with mutations in the CYP2A6 gene and listed as a group 3 carcinogen by the International Agency for Research on Cancer and thus has raised increasing concerns. Until now, different physicochemical methods have been developed for the removal of COU, whereas their practical applications were hampered due to high cost and the risk of secondary contamination. In this study, genetic evidence and biochemical characterization of the COU degradation by Pseudomonas sp. strain NyZ480 are presented. With the gene and strain resources provided here, better managements of the hazards that humans face from COU could be achieved, and the possible microbiota-plant interaction mediated by the COU-utilizing rhizobacteria could also be investigated.