Study Of Cytochrome P450 Monooxygenases Associated With The Degradation Of Nicotine In Aspergillus Oryzae 112822

Nicotine is a toxic N-heterocyclic compound found in tobacco.Waste from tobacco processing contains high levels of nicotine,and nicotine accumulation is a serious threat to humans and the environment.Unlike physical and chemical methods,nicotine-degrading microorganisms can grow and reproduce with nicotine as the sole carbon and nitrogen source,enabling green and environmentally friendly nicotine degradation with promising applications.In our laboratory,previous graduate students have screened and obtained a strain of Aspergillus oryzae 112822 from tobacco leaves and confirmed that the first step of the nicotine degradation in this strain is the demethylation nicotine to produce nornicotine,and its nicotine N-demethylase(NND)was inducible.By joint analysis of peptide fingerprinting of partially purified NNDs and nicotine-induced transcriptome of A.oryzae 112822,10 cytochrome P450 monooxygenases(CYPs)were chosen as the candidate NNDs(Ao.CYPs).In this work,6 candidate NND genes cyp613DI(cyp2),cyp548D3(cyp5),cyp55A5v2(cyp8),cyp5113A1(cyp9),cyp620H1 and cyp620H2 of A.oryzae 112822 and a single cytochrome P450 reductase(CPR)gene in the genome of the strain 112822 were investigated.The main results were as follows:1.Heterologous expression of the genes of candidate Ao.CYPs and Ao.CPR and determination of their recombinant protein activityAn Escherichia coli expression system was constructed to express the candidate genes Nd-cyp620H1,Nd-cyp620H2 and Nd-Ao.cpr,with N-terminal membrane anchoring deletion(labled Nd-),SDS-PAGE revealed that both Nd-CYP620H1 and Nd-CYP620H2 existed as inclusion bodies in the cell,whereas Nd-Ao.CPR was solubilised and expressed in the cytoplasm,and 1 μg of recombinant Nd-Ao.CPR reduced 1.75 nM equine heart cytochrome C per minute.Meanwhile,the Pichia pastoris expression system was constructed to express cyp620H1,cyp620H2 and NdAo.cpr.Western blot demonstrated the expression of the 3 recombinant proteins in P.pastoris.The recombinant Nd-Ao.CPR showed electron transfer activity,but neither recombinant CYP620H1 and CYP620H2 showed CO binding properties or nicotine Ndemethylation,and CYP620H1 did not show ECOD(Ethoxycoumarin O-deethylase)activity previously reported.In addition,the Ao.CYPs-Nd-Ao.CPR fusion protein expression system was constructed in P.pastoris and the expression and activity of 6 candidate Ao.CYPs fused respectively to Nd-Ao.CPR were examined.All the 6 fusion proteins expressed in the cytoplasm showed electron transfer activity of Nd-Ao.CPR,but no CYP-related activity was detected.2.Overexpression of the candidate Ao.CYPs genes in A.oryzae 1 12822The overexpression plasmid pBARGPE1-ptrA-A o.cyps was constructed using the pyridine thiamine resistance gene(ptrA)as a screening marker.After protoplast transformation of the strain 112822.6 strains overexpressing strains of 6 candidate Ao.CYPs genes were successfu lly constructed.The transcription levels of the candidate genes in 6 overexpression strains were determined by qPCR.The relative expression levels of cyp2,cyp5,cyp8,cyp9,cyp620H1 and cyp620H2 were 4.8,3.3,17.6,15.2,5.4 and 11.5 times higher than those in original strain,respectively.The results of nicotine degradation activity showed that CYP620H2 overexpression strain had a higher nicotine degradation efficiency,which was 400%higher than that of original strain 1 12822 at the time of the nicotine degradation reactions for 24 h.3.Knockout of the candidate Ao.CYPs genes in A.oryzae 112822Based on CR1SPR-Cas9 technology,knockout plasmid pBARGPE1-sgRNA(Acyps)-Cas9 and homologous directed repair template HDRT-ptrA with ptrA as knockin gene were constructed.After protoplast transformation of strain 112822,5 knockout strains with the ptrA replacement candidate Ao.cyps,named Δcyp2,Δcyp5,Δcyp8,Δcyp9 and Δcyp620H2.were successfully screened.The results of detection of nicotine degradation activities of 5 knockout strains showed that the nicotine degradation activity of the Δcyp620H2 almost disappeared,and its nicotine degradation efficiency was 91.3%lower than that of the original strain at the time of the nicotine degradation reaction for 48 h.In this work,the heterologous expression studies of the genes of Ao.CYPs and Ao.CPR showed that deletion of the N-terminal membrane anchoring domain did not affect the function of Ao.CPR.The recombinant Nd-Ao.CPR in both E.coli and P.pastoris expression systems showed electron transfer activity,whereas the recombinant Nd-Ao.CYPs and Ao.CYPs expressed in the same system did not show activity.Further research on their heterologous expression and catalytic systems should be carried out.The nicotine degradation activities of Ao.CYPs genes overexpression strains.or genes knockout strains were respectively different from that of the original strain,confirmed the critical role of cyp620H2 in nicotine degradation in A.oryzae 112822.which provides an important reference for the discovery of fungal nicotine degrading enzymes and the elucidation of their molecular mechanisms.