The Preliminary Study On Molecular Catabolic Mechanism Of Nicotine By Shinella Sp.HZN7

Because of the harmful effects,nicotine has been treated as Toxic Release Inventory.Therefore,it is important to remove nicotine from tobacco waste,or even convert it to valuable compounds.Microbial treatment is one of the recommended technologies for the rapid decomposition of tobacco waste.Research on mechanisms of nicotine degradation can reveal chemical biology mechanisms and provide further theoretical foundation.In this study,we report the identification and characterization of the novel nicotine-degrading strain Shinella sp.HZN7.Strain HZN7 has a high degradation rate at 30?35 0C and pH values of 6.5?8.0.This strain can degrade 500 mg/L nicotine completely within 3 h at 30 ? and pH 7.0.The biodegradation of nicotine by Shinella sp.HZN7 involves fiveintermediate metabolites:6-hydroxy-nicotine(6HN),6-hydroxy-N-methylmyosmine,6-hydroxypseudooxynicotine(6HPON),6-hydroxy-3-succinoyl-pyridine(HSP),and 2,5-dihydroxypyridine(2,5-DHP),as detected by ultraviolet spectrophotometry,HPLC,and LC-MS.We generated three mutants,N7-W18,N7-X5 and N7-M17 by transposon mutagenesis,in which the nicotine-degrading pathway terminated at 6HN,6HPON,and HSP,respectively.The production of the five intermediate metabolites and their order in the degradation pathway were confirmed in the three mutants,indicating that strain HZN7 degrades nicotine via a variant of the pyridine and pyrrolidine pathways.The mutant gene from strain N7-X5,orf2,was cloned by self-formed adaptor PCR,but the nucleotide and amino acid sequence showed no similarity to any gene or gene product with defined function.However,orf2 disruption and complementation suggested that the orf2 gene is essential for the conversion of 6HPON to HSP in strain HZN7.This is the first study to provide genetic evidence for this variant nicotine degradation pathway.