Molecular Mechanism Of Nicotine Degradation In Strain Pseudomonas Geniculata N1

Nicotine,the main toxic alkaloid in tobacco,is an environmental hazard of N-heterocyclic compound,which seriously threatens the ecological environment and human health.Strain Pseudomonas geniculata N1 is capable of degrading nicotine efficiently through a pathway variant from pyridine and pyrrole pathway(VPP pathway).VPP pathway,reported in recent years,comprises of the upstream of pyridine pathway and downstream of pyrrole pathway.To date,compared with the research of pyridine pathway and pyrrole pathway,the molecular mechanism of VPP pathway has not been illustrated deeply.Therefore,the research of strain Pseudomonas geniculata N1 may help to provide insights into the molecular mechanism of VPP pathway and understand the evolvement of the nicotine-degrading bacterium.Pseudomonas geniculata N1,isolated from tobacco leaves,utilizes nicotine as a sole carbon and nitrogen source.By detecting the intermediate metabolites,strain N1 has been identified as a VPP pathway microorganism to degrade nicotine.The whole genome sequencing provides the information of the nicotine degradation related genes.After analysis of genome,we predict three genes related to nicotine degradation and use RT-qPCR to detect the change of transcriptional level in strain N1.The genes increase several folds in the transcriptional level induced by nicotine,indicating that the genes are related to nicotine degradation.After expression in vitro,the function of two crucial nicotine-degrading genes(hisD and vD)were identified.The gene hisD is the key gene in VPP pathway by connecting upstream of pyridine pathway and downstream of pyrrole pathway.HisD converts 6-Hydroxypseudooxynicotine(6HPON)to 6-Hydroxy-3-succinoyl-pyridine(HSP).Due to some cofactors,HisD cannot function in E.coli but Pseudomonas KT2440.The gene vD can catalyze HSP into 2,5-DHP with the existence of NADH,which is the first reaction of the downstream of pyrrole pathway.The protein VD,expressed in E.coli,has high capabilities to react after the research of biochemical properties.