Cloning and characterization the nicotine degradation enzymes 6-hydroxypseudooxynicotine amine oxidase and 6-hydroxy-3-succinoylpyridine hydroxylase in Pseudomonas geniculata N1

Microbial catabolism plays a crucial role in the removal of toxic alkaloids including nicotine from tobacco waste. Pseudomonas geniculata N1, an effective nicotine-degrader, possesses a variant of the pyridine and pyrrolidine (VPP) nicotine catabolic pathway. In this study, a 20-kbp gene cluster was found to contain eight nicotine-degrading genes. In comparison to Sphingomonas melonis TY, Ochrobactrum sp. SJY1, Shinella sp. HZN7, and Agrobacterium tumefaciens S33, these genes are tightly clustered in the P. geniculata N1 genome. The gene hisD encoding a 6-hydroxypseudooxynicotine (6-HPON) amine oxidase was heterologously expressed in Pseudomonas putida KT2440 and the recombinant strain acquired the ability to transform 6-HPON to 6-hydroxy-3-succinoylpyridine (HSP). The vD gene encodes a flavin-containing NADH-dependent monooxygenase, which converted HSP to 2,5-dihydroxypyridine was cloned in E. coli and characterized. Both genes, hisD and vD, were significantly upregulated in response to nicotine. This study expands our knowledge of the VPP nicotine catabolic pathway in bacteria.