Screening,Cloning,Expression And Analysis Of Nicotine Demethylase From Aspergillus Oryzae

As a major alkaloid in tobacco plants,nicotine is widely found in tobacco products.It is well know that smoking is harmful to human beings,a large number of people death occur due to smoking.By the year 2020,it is also expected to exceed 9 million deaths annually.Nicotine is a sympathomimetic drug that releases catecholamines,increases heart rate and cardiac contractility,constricts cutaneous and coronary blood vessels,transiently increases blood pressure,reduces sensitivity to insulin,may aggravate or precipitate diabetes,and may contribute to endothelial dysfunction.With the increase in the use of tobacco products,the tobacco industry produces a large number of solid and liquid tobacco waste containing high concentrations of nicotine.Nicotine is easily dissolved in water and causes groundwater pollution,and its water pollution disturbs the ecological balance of the soil.Therefore,it is significant to remove nicotine from tobacco waste to prevent contamination of soil and water.It has been reported that nicotine degradation widely exists in human beings,plants and microorganisms.In the human body,nicotine is metabolized by the liver into various metabolites,of which the six major metabolites are cotinine,1'-(R)-2'-(S)-cis nicotine-N-oxide/1'-(S)-2'-(S)-trans,nicotine-N-oxide,N-methylnicotinium ion,nicotine glucuronide,2'-hydroxynicotine,and nornicotine.Nicotine N-demethylation is frequently an important pathway in the metabolism of xenobiotics,but this route is,in most species,a minor pathway in the metabolism of nicotine.Currently,the enzymes involved in the demethylation of nicotine are CYP2A6,CYP2B6,and CYP2D6.Nicotine is also catalyzed by nicotine demethylase CYP82E4 to produce demethylnicotine in tobacco plants.Microorganisms have a strong ability to degrade nicotine,and the development and use of nicotine-degrading microbial resources have a major impact on maintaining ecological balance.Therefore,many nicotine metabolisms have been studied.There are three major pathways for nicotine degradation in microorganisms:the pyridine pathway,the pyrrole pathway,and the demethylation pathway.Arthrobacter follows the pyrrole pathway and Pseudomonas follows the pyridine pathway.The fungus follows the demethylation pathway.The studies of nicotine degradation in fungus are quite rare,and only discovered the initial step of nicotine demethylation.Our group filled the gaps in nicotine degradation stud by first proposed a nicotine degradation path way in Aspergillus oryzae 112822 and identified of some important metabolites.But the enzymes and molecular mechanism incolving in the nicotine degradation pathway are still unknown.It has been determined that nicotine demethylation is catalyzed by P450 monooxygenases,which are mostly membrane proteins.The overexpression of membrane proteins is a major bottleneck in the study of their function and structure.A variety of heterologous expression systems have been developed to express membrane proteins.For example,E.coli expression systems,yeast expression systems,baculovirus/insect cell expression systems,mammalian expression systems,and cell-free expression systems.There are advantages and disadvantages for the expression of membrane proteins,so it is very important to choose the appropriate expression system.E.coli is the most commonly used system for expressing recombinant proteins.Some researchers have developed some experimental strategies to optimize the expression of P450 in E.coli,such as N-terminal modification,co-expression of chaperone proteins,vector selection,and codon-optimized gene synthesis.These strategies are worth learning for the expression of P450.In this paper,the demethylation of nicotine in the first step of degradation of nicotine by A.oryzae was studied.The first step reaction requires the participation of CYP and CPR.Our previous work has screened nine candidate proteins by enzyme separation,peptide mapping,and transcriptome sequencing.Genes of CYP1,CYP2,CYP3,CYP4,CYP5,CYP7,CYP8,CYP9,and CYP10 were cloned,and NADPH-cytochrome P450 reductase(CPR)genes of A.oryzae and Saccharomyces cerevisiae were also cloned.We extracted RNA from A.oryzae and S.cerevisiae and reverse transcribed it into cDNA.We successfully cloned the corresponding CYP and CPR genes using cDNA as a template.Subsequently,in order to carry out work on the heterologous expression and function of these 9 CYPs and 2 CPRs,we first tried to express CYP and CPR in E.coli.A.oryzae CPR and Saccharomyces cerevisiae CPR successfully obtained soluble expression and had electron transport activity.CYP 1,CYP2,CYP5,CYP8,and CYP 10 were also expressed,but with the exception of CYP8,all of them formed inclusion bodies and could not detect the activity of the enzyme protein.Only CYP8 was able to combine with CO to form a characteristic absorption peak at 450 nm,and CYP8 was tested with Ao.CPR and Se.CPR for nicotine degradation activity.Nicotine reduction and nornicotine were not detected.It has been reported that CYP8 is a nitric oxide reductase that can directly reduce NO to N2O without the participation of CPR in the presence of NAD(P)H.To verify the substrate activity of CYP8,we determined the catalytic activity of CYP8 by measuring the 340 nm decrease in the characteristic absorption peak of NADH.It was found that in the presence of NADH,CYP8 can reduce the absorption peak at 340 nm,indicating that CYP8 has the ability to catalyze NO.According to peptide mapping and transcriptome analysis,the peptide matching rate of CYP1 was 10%,and transcription was the highest,suggesting that CYP1 is most likely a nicotine demethylase.Therefore,in order to increase the soluble expression of CYP1 in E.coli,several expression vectors were constructed by truncation of the N-terminal transmembrane domain and modification of the N-terminus by the addition of a hydrophilic amino acid sequence.Five N-terminal modifications of CYP1 were performed,1.Native(MA-);2.CYP1?(3-28);3.CYP1(17a);4.CYP1(2C3);5.CYP1(2E1).Four different expression vectors,including pCWori,pET21b,pET22b,and pET28b,and four different E.coli expression systems,including DH5a,BL21,Rosetta gamiB,and JM109,were used to express CYP1.To explore the optimal N-terminal modified sequence of CYP1 and the combination of the best expression vector and expression host.Five N-terminally modified CYP1 clones were successfully cloned and the corresponding four expression vectors were successfully constructed.Finally,56 strains of expression strains were screened out,but only 15 of them could express the protein of interest and were all expressed as inclusion bodies.This paper successfully cloned 9 CYP of A.oryzae and CPR of A.oryzae and Saccharomyces cerevisiae,verified the substrate catalytic activity of CYP8 and explored the expression of N-terminal sequence modification of CYP1.Finally,we compared the amino acid sequences of the known nicotine demethylases in humans and plants in the A.oryzae genome and found that the CYP620 subfamily has higher amino acid sequence similarities.Combined with peptide mapping and transcriptome analysis,it was found that CYP620H1 and CYP620H2 are most likely nicotine demethylases in A.oryzae.There are still many problems and deficiencies in the research.The research and exploration in this paper provide some reference and experience for the follow-up research.