Study Of Cytochrome P450 And N-Methylnicotinamide Hydroxylase Involved In The Degradation Of Nicotine By Aspergillus Oryzae 112822

As the main alkaloid in Nicotiana tabacum,nicotine which is carcinogenic and addictive can easily induce various diseases by metabolizing N-nitrosamines in human body while stimulating nicotinic acetylcholine receptors promoting to release massive neurotransmitters by central nervous system.Naturally,toxic residues from cigarettes,nicotine-containing pesticides and insecticides are discharged indiscriminately which are enriched seriously polluting the environment and destroying the ecological balance because of difficulties in degradation.Therefore,developing new efficient and harmless methods for realizing complete degradation of nicotine and its metabolites have both ecological and social benefits.Nicotine-degrading microorganisms which grow with nicotine as the only source of carbon and nitrogen can directly reduce nicotine content through biodegradation under different metabolic mechanisms after long-term natural evolution.In particular,bacterial nicotine degradation pathways has been studied thoroughly which can be classified into the pyridine pathway represented by Arthrobacter nicotinovorans,the pyrrolidine pathway represented by Pseudomonas putida and variant pathway of pyridine pathway and pyrrolidine pathway,VPP pathway,represented by Ochrobactrum thizosphaerae.While nicotine N-demethyl pathway in fungi is studied rarely which has only been reported that the first step of nicotine N-demethylation to generate nornicotine except for Microsporum gypseum,Cunninghamella echinulata,Pellicularia filamentosa and Aspergilus oryzae.And key enzymes involved in the complete nicotine degradation pathway still remain unknown.In 2010,our laboratory proposed a novel fungal nicotine N-demethyl pathway based determination of intermediates by A.oryzae 112822 isolated from the surface of tobacco leaves.A.oryzae 112822 converts nicotine to nornicotine firstly during this pathway.After dehydrogenation,the pyrrolidine ring can be spontaneously opened removing a molecule of acetic acid to produce N-methylnicotinamide(NMN).The ?-position of NMN pyridine ring is hydroxylated to 2-hydroxy-N-methylnicotinamide(2HNMN).2HNMN is subsequently hydrolyzed to produce 2,3-dihydroxypyridine(2,3DHP)and succinic acid.Eventually this new pathway ends with the entrance into TCA cycle.But studies on related enzymes and their genes in this pathway have not been reported at present.Cytochrome P450 monooxygenases(P450s,CYPs)superfamily participates in heterotoxin metabolism across organisms widely within diverse communities.CYP82E4 in tobacco leaf cells and CYP2A6 and CYP2B6 in human liver cells mediate nicotine-demethylation.So we hypothesized that A.oryzae 112822 has the similar way to degradate nicotine.Previously,we had proved that the NND was induced expression with resting cells by A.oryzae 112822 in culture conditions with or without nicotine induction.Combining peptide fingerprinting analysis with transcriptome analysis,we had screened 10 NND candidate genes from P450 database of A,oryzae following cyp577a4,cyp613d1,cyp584e5,cyp53a13,cyp548d3,cyp55a5v2,cyp5113a1,cyp531c3,cyp620h1 and cyp620h2.In this thesis,we investigated for the key enzyme firstly which catalyzed nicotine to nornicotine by A.oryzae 112822.After sequence analysis,candidate nicotine N-demethylase(NND),cyp55a5v2,cyp620h1 and cyp620h2.and cytochrome P450 reductase(CPR)gene Ao.cpr.The cyp55a5v2 gene is a 1227 bp DNA fragment with a predicted 20 amino-acid signal peptide and 3 predicted N-glycosylation sites.The cyp620h1 gene is a 1578 bp DNA fragment with a predicted 23 amino-acid signal peptide.The cyp620h2 gene is a 1587 bp DNA fragment with a predicted 20 amino-acid signal peptide and 3 predicted N-glycosylation sites and the Ao.cpr gene is a 2088 bp DNA fragment with a predicted 26 amino-acid signal peptide and 3 predicted N-glycosylation sites.Primers were designed according to A.oryzae RIB40 genome(GenBank:GCA₀00184455.3)inserted into Kozak sequence(GCCACC)before the start codons ATG to improve transcription and translation efficiency.Using genome of A.oryzae 112822 as the template,DNA fragments of cyp55a5v2(1167 bp),cyp620h1nt(1509 bp),cyp620h2nt(1527 bp)and Ao.cpr(2010 bp)were amplified by PCR removing predicted N-signal peptides.The recombinant plasmids pYES2-cyp55a5v2.pYES2-cyp620h1nt and pYES2-cyp620h2nt were constructed for soluble intracellular expression in Saccharomyces cerevisiae W303.Western-blotting detected molecular masses of recombinant CYP55A5v2.CYP620H1nt and CYP620H2nt were?45 kDa.?58 kDa and?59 kDa which were in accord with predicted molecular masses of 44.3 kDa.57.8 kDa and 58.5 kDa.The recombinant plasmid pPICzaA-Ao.cpr was constructed for soluble intracellular expression in Pichia pastoris KM71.Western-blotting detected the molecular mass of the recombinant Ao.CPR was?75 kDa which was in accord with predicted molecular mass of 73.9 kDa.In the presence of 200 mM NADPH as the electron donor and 0.5 mM cytochrome C of horse heart(10 mM KPB pH 7.4)as the electron acceptor.recombinant Ao.CPR produced?1.5 nmol reduction product per minute per pg with absorption at 550 nm.In the presence of 2 mM NADPH,300 ?g recombinant Ao.CPR and sufficient O2 with 0.2 g/mL nicotine(50 mM KPB,pH 7.5)as substrates,TLC analysis showed that microsome of recombinant CYP620Hlnt degraded nicotine and HPLC showed that the retention time of catalytic compound was identical to the retention tumes of nornicotine at 28? for 12 h.ESI-MS showed a molecular ion peak at m/z 149.09[M+H]+ in this reaction solution which was in accord with predicted molecular mass of nornicotine(148.10).Therefore.we demonstrated the NND activity of recombinant CYP620H1nt with the assistance of recombinant Ao.CPR.The CRISPR/Cas9 gene editing technology has been successfully applied in fungi realizing targeted transformation and direcyed modification.Our lab confirmed the correct function of the CRISPR/Cas9 system constructed in A.oryzae 112822 by knocking-out the polyketide synthase gene wA(GenBank:XM₀01822648.1)successfully which regulates pigment synthesis.In this thesis,we aimed for knocking-out the target-DNA cyp620h1 in A.oryzae 112822.Using pUC57 as the template,linear sgRNA expression plasmids pUC57-?cyp620h1-sgRNA1/2/3/4/5 were amplified by PCR inserted into 5 different 20-bp fragments of sgRNA.To construct sgRNA-Cas9 co-targeting vectors BPCF-?cyp620h1-sgRNA1/2/3/4/5,reading frames?cyp620h1-sgRNA1/2/3/4/5 obtained by enzymatic cleavage and linear BPCF were connected by T4 DNA ligase.Using the pyrithiamine(PT)resistance gene(ptrA)as the screening marker,the homology-directed repair template HDRT-ptrA for target-DNA consisted a 997-bp fragment upstream of cyp620h1,the 2028-bp gene ptrA(GenBank:AF217503.1)and a 1025-bp fragment downstream of cyp620h1.The connected 4300-bp fragment of HDRT-ptrA obtained by PCR amplification were co-transformed to A.oryzae 112822 with linear sgRNA-Cas9 co-targeting vectors mediated by PEG with 1 ?g/mL PT.Transformants with good growth were selected for multiple passages with 2 ?g/mL PT until genome was stable.Designed a pair of primers for ptrA gene and target-DNA homologous region,we successfully obtained an anti-PT(PTR)strain in which the target-DNA was targeted knockout by phenotype screening and PCR verification with genome as template.This will be followed by verifying the NND catalytic activity comparing the difference in nicotine degradation ability by before and after targeting knockout the candidate NND gene cyp620h1 in this PTR strain.As one of nicotine degrading intermediates by A.oryzae 112822,NMN is a typical six-membered heterocyclic compound whose pyridine ring was catalyzed by N-methylnicotinamide hydroxylase with ?-hydroxylation converting NMN to 2HNMN.It was reported that xanthine dehydrogenases(XDHs)could specifically bind to the ?-position with ?-hydroxylation ingoring any side chains.Futhermore,there was only one predicted xdh gene in A.oryzae RIB40 genome.Therefore,it's hypothesized that the XDH encoded by xdh has same catalytic activity as N-methylnicotinamide hydroxylase.In this thesis,crude enzyme was obtained by 40?70%ammonium sulfate graded precipitation of cell-free axtracts by A.oryzae 112822 under 5 mM NMN-induced culture conditions.In the presence of 5 mM NAD+and sufficient O2 with 2 mM NMN(50 mM KPB,pH 6.5)as the substrate.TLC and HPLC showed that the crude enzyme degraded NMN at 28? for 12 h.ESI-MS result showed a molecular ion peak at m/z 174.99[M+Na]+in this reaction solution which was in accord with predicted molecular mass of 2HNMN(152.00).In the presence of 1 mM MTT and 0.1 mM PMS with 3 mM hypoxanthine(50 mM Tris-HCl,pH 8.0)as the substrate,the native gradient PAGE gel which had XDH activity detected by reactive dyeing under light-proof conditions detected molecular mass of?270 kDa.The peptide fingerprinting analysis was performed after purification of this strip which showed the highest match of 62.41%with XDH from A.oryzae RIB40.Primers were designed according to the predicted xdh gene from A.oryzae RIB40 inserted into the Kozak fragment before the start codons ATG.The xdh DNA fragment was obtained by PCR amplification with the genome of A.oryzae 112822 as the template.Nucleotide sequence aligments indicated homology with the predicted xdh gene.This 4080 bp fragment encoding 1359 amino acids has 7 predicted N-glycosylation sites.The recombinant plasmid pPICzaA-xdh was constructed for soluble intracellular expression in Pichia pastoris KM71.Western-blotting detected molecular mass of the recombinant XDH was?175 kDa which was larger than the predicted molecular mass(148.3 kDa).After glycans fully cleaved by N-glycoside endonuclease PNGaseF.SDS-PAGE showed that the molecular mass of the single subunit was?150 kDa which was in accord with the predicted molecular mass.Native gradient PAGE showed that the molecular weight of the recombinant XDH was?342 kDa which indicated its active state was a bisubunit dimeric protein.In the presence of 5 mM NAD+and sufficient O2 with 1 mM NMN(50 mM KPB,pH 6.5)as the substrate,TLC showed that the NMN degradation activity of the recombinant XDH at 28? for 48 h.ESI-MS showed a molecular ion peak m/z at 174.99[M+Na]+ in this reaction solution which was in accord with the predicted molecular mass of 2HNMN(152.00).Thus,the xdh gene derived from A.oryzae 112822 was identified as XDH which possesses N-methylnicotinamide hydroxylase activity.Combining the determination of NMN degradation activity and XDH activity,we identified and heterologous expressed a new metabolism-related enzyme in this pathway.Focusing on metabolism-related enzymes and their genes in nicotine-degrading strain A.oryzae 112822,we demonstrated the NND catalytic activity of cytochrome P450 monooxygenase CYP620Hlnt and the N-methylnicotinamide hydroxylase activity of xanthine dehydrogenase XDH providing an significant basis for elucidating the molecular mechanisms of key enzymes and their genes in fungal nicotine degradation metabolism pathway.