Genetic and functional characterization of a novel meta-pathway for degradation of naringenin in Herbaspirillum seropedicae SmR1

Summary In this study, a random mutant library of Herbaspirillum seropedicae SmR1 was constructed by Tn5 insertion and a mutant incapable of utilizing naringenin as a carbon source was isolated. The Tn5 transposon was found to be inserted in the fdeE gene (Hsero_1007), which encodes a monooxygenase. Two other mutant strains in fdeC (Hsero_1005) and fdeG (Hsero_1009) genes coding for a dioxygenase and a putative cyclase, respectively, were obtained by site‐directed mutagenesis and then characterized. Liquid Chromatography coupled to mass spectrometry (LC‐MS)/MS analyses of culture supernatant from the fdeE mutant strain revealed that naringenin remained unaltered, suggesting that the FdeE protein is involved in the initial step of naringenin degradation. LC‐MS/MS analyses of culture supernatants from the wild‐type (SmR1) and FdeC deficient mutant suggested that in H. seropedicae SmR1 naringenin is first mono‐oxygenated by the FdeE protein, to produce 5,7,8‐trihydroxy‐2‐(4‐hydroxyphenyl)−2,3‐dihydro‐4H‐chromen‐4‐one, that is subsequently dioxygenated and cleaved at the A‐ring by the FdeC dioxygenase, since the latter compound accumulated in the fdeC strain. After meta‐cleavage of the A‐ring, the subsequent metabolic steps generate oxaloacetic acid that is metabolized via the tricarboxylic acid cycle. This bacterium can also modify naringenin by attaching a glycosyl group to the B‐ring or a methoxy group to the A‐ring, leading to the generation of dead‐end products.