Molecular Mechanism Research Of Nicotine Degradation In Agrobacterium Tumefaciens S33

Nicotine is a main toxic compound in tobacco and tobacco wastes.Since nicotine is soluble in water,the improper treatment of tobacco waste can therefore seriously pollute the environment and threaten the health of human being.Biological methods of employing microbes to treate nicotine are more promising than physical and chemical degradation processes because they are highly efficient,cost-competitive and environmentally friendly.Previously,Agrobacterium tumefaciens S33 which was isolated from tobacco rhizosphere based on its strong ability to degrade nicotine.And the strain was found to employs a novel pathway,which is a variant of pyridine pathways of nicotine degradation in Arthrobacter and pyrrolidine pathway in Pseudomonas.In this work,the biochemical and molecular mechanism involved in the nicotine degradation by Agrobacterium tumefaciens S3 3 was studied.We carried out genome sequencing of strain S33 by using high-throughput Illumina sequencing techniques,in order to perform a deep study of nicotine degradation pathway.According to genome sequencing result,the primers were designed for amplifying the DNA fragment of the gap regions among the Scaffold 16.Then these gap regions were amplified by using PCR and sequenced.The genome of strain S33 was annotated on the Rast wed sever.According to the result of gene annotation,there are some functional genes for mobile element protein,transcription regulator,conjugative transfer protein,chromosome partitioning protein and plasmid replication protein,then speculated that Scaffold 16 was transferred into strain A.tumefaciens S33 by conjugation from other bacteria.Sequence analysis showed high similarity between the genes related to nicotine degradation in strain S3 3 and those from other strains.Then the predicted genes involved in nicotine degradation in A.taumefaciens S33 according to the annotation were analyzed at the relative transcription level.Firstly,their relative transcription level in nicotine medium or glucose and ammonium mediun were tested by RT-PCR analysis.Further the transcriptome of A.tumefaciens S33 was sequenced,the result confirmed that all target genes appeared to be up-regulated in nicotine-induced A.tumefaciens S33,the same results were obtained by RT-PCR analysis.In order to confirm the genes of nicotine dehydrogenase(Ndh),the enriched enzyme was digested with trypsin and then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF/MS).The results were aligned with the fully annotated genome to identify their encoding genes,which showed that the Ndh sample from A.tumefaciens S33 contained three proteins of Mr 80 Da(NdhA),73 Da(Pno)and 16 Da(NdhB).Then conserved sequences of three proteins were analyzed,and the result showed that NdhA contains the conserved molybdopterin-binding domain,Pno has conserved flavin binding motif,and NdhB has conserved[2Fe-2S]binding motif.There are 4 bp overlap between ndhA and ndhB,while pno not only keep away from ndhA but also is bigger than the middle-sized subunit of Ndh and ketone dehydrogenase from A.nicotinovorans.In order to further confirm the encoding genes of Ndh,experiments were carried out to independently knockout the genes ndhA,pno and ndhB.The results demonstrated that the knockout strains could not grow with nicotine,but were able to grow with 6-hydroxy-3-succinylpyridine(HSP),except tthe knockout strains of pno could not grow with 6-hydroxynicotine.Complementing the three genes in the mutant strains could recover the growth of strains on nicotine,respectively.The intermediates of nicotine degradation by resting cells of strains S33-?pno were analyzed with LC-MS.The result shows that nicotine was catalyzed by Ndh and Hno into 6-hydroxynicotine,6-hydroxy-N-methylmyosmine and 6-hydroxypseudooxynicotine,however HSP and 2,5-DHP were not detected.Considering that there is a gene encoding aldehyde dehydrogenase in nicotine degradation gene cluster of the genome,we speculate that Ndh is composed of NdhA and NdhB,while Pno catalyzes 6-hydroxypseudooxynicotine into 6-hydroxy-3-succinylsemialdehyde-pyridine.6-Hydroxynicotine oxidase(Hno)is the second key enzyme in the nicotine degradation of strain S33.The enzyme was purified from A.tumefaciens S33,the purified enzyme was digested with trypsin and then analyzed by MALDI-TOF/MS.Compared the result with the genome of strain S33,the gene(1,314 bp)encoding Hno was determined.The hno was expressed successfully in E.coli BL21(DE3),and purified His-tag Hno was obtained.The catalytic products of Hno were determined by LC-MS,the results indicated that 6-hydroxynicotine was oxidized by Hno into 6-hydroxy-N-methylmyosmine,which was then hydrolyzed spontaneously into 6-hydroxy-pseudooxynicotine.The HSP hydroxylase(Hsh)was purified from A.tumefaciens S33,and its properties were analyzed.The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine(2,5-DHP)and succinic acid.Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium.It encodes a protein composed of 391 amino acids with 62%identity to HSP hydroxylase(HspB)from Pseudomonas putida S16,which degrades nicotine via the pyrrolidine pathway.Considering the application potential of 2,5-DHP in agriculture and medicine,an enzymatic route was developed to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and NADH-regenerating system,via which around 0.53±0.03 mM 2,5-DHP was produced from 0.76±0.01mM HSP with molar conversion as 69.7%.In conclusion,the molecular mechanism of nicotine degradation in A.tumefaciens S33 was studied by genome sequencing,transcriptome sequencing and enzymes purification and biochemical characterization,.The results indicated that Ndh and Hno from A.tumefaciens S33 share highest identity in amino acid sequences with Ndh and Hno from Arthrobacter,while Hsh has highest identity in amino acid sequences with HspB from Pseudomonas.Moreover,Hpo,Nfo,Ami and Iso from A.tumefaciens S33 have high identity with the same enzyme from P.putida S16.All results comfirmed that A.tumefaciens S33 has a variant of pyridine and pyrrolidine pathways to degrade nicotine as found previously by identification of nicotine biotransformation intermediates in this lab.There are still some important questions required to answer in the project although great efforts have been input in this work.For example,the enzymes and genes involved in the step of transformation of 6-hydroxy-3-succinylsemialdehyde-pyridine into HSP need further analysis and verification.Meanwhile,the physiological electron acceptor of Ndh is not clear.