Molecular Biology Of The Key Enzymes And Genes Associated With Nicotine Degradation By Aspergillus Oryzae 112822 And Study Of The Molecular Mechanism Of Levansucrase From Bacillus Licheniformis 8-37-0-1 For Product Specificity

This thesis consists of two parts:molecular biology of the key enzymes and genes associated with nicotine degradation by Aspergillus oryzae 112822 and study of the molecular mechanism of levansucrase from Bacillus licheniformis 8-37-0-1 for product specificity.Part ?:molecular biology of the key enzymes and genes associated with nicotine degradation by Aspergillus oryzae 112822Nicotine is the most abundant tobacco alkaloid,as a kind of hypertoxic N-heterocyclic compound,nicotine is the leading cause of smoking addiction.Annually,large quantities of tobacco wastes containing high concentration of nicotine are accumulated from tobacco industry.Due to the high water-solubility,nicotine can easily enter groundwater system and disturb the ecological balance of soil,displaying potential ecotoxicological risk.Many microorganisms growing in tobacco leaves and soil for tobacco cultivation can utilize nicotine as the carbon and nitrogen sources for their growth.These efficient nicotine-degrading microorganisms have attracted much attention due to their potential for detoxication of tobacco wastes.Nicotine-degrading microorganisms have different pathways for nicotine degradation,thus far,a variety of nicotine-degrading bacteria have been reported successively,such as Arthrobacter nicotinovorans,Pseudomonas putida S16,and Ochrobactrum sp.Strain SJY1,etc.Three nicotine degradation pathways have been fully elucidated in bacteria:nicotine degradation in Arthrobacter sp.begins with hydroxylation of the pyridine-ring to form 6-hydroxy-L-nicotine(6-HN),called the pyridine pathway;nicotine degradation in Pseudomonas sp.starts from the dehydrogenation at the pyrrolidine-ring to form N-methylmyosmine(NMM),called the pyrrolidine pathway;other bacteria degrade nicotine through the VPP pathway,in which nicotine is initially transformed into 6-hydroxypseudooxynicotine(6-HPON)through the pyridine pathway,and then the 6-HPON is further converted into 6-hydroxy-3-succinoyl-pyridine(HSP)which turns into the pyrrolidine pathway.In bacteria,the enzymes involved in the first two pathways have been elucidated in detail,and several isoenzymes involved in the VPP pathway have also been reported.Compared with nicotine-degrading bacteria,few fungal strains,including Microsporum gypseum ATCC11395,Pellicularia filamentosa JTS-208,Cunninghamella echinulata IFO-4444,and Aspergillus oryzae 112822 in our laboratory have been reported to have nicotine-degrading ability.Nicotine degradation in fungi begins with N-demethylation of nicotine to form nornicotine.So far,the description of nicotine degradation in the first three fungi has been limited to the first step,and the nicotine demethylases have not been reported.In our previous work,based on analysis of the metabolites,a detailed nicotine degradation pathway of fungi was firstly proposed in the world,namely the demethylation pathway of A.oryzae 112822.However,related genes and enzymes involved in this demethylation pathway have not been revealed,and the underlying molecular mechanisms remain unknown.Aimed at the nicotine demethylation pathway of A.oryzae 112822,we performed a comparative transcriptome analysis to explore the molecular mechanisms of nicotine degradation in fungi at the overall level.Complex metabolic adjustment,involving detoxification,transport,and oxidative stress responses accompanied by increased energy investment,were developed for nicotine tolerance and degradation,the molecular models for nicotine degradation by A.oryzae 112822 were proposed based on in-depth information analysis.Moreover,according to the analysis of differentially expressed genes(DEG),characteristics of reactions,multiple sequence alignment and phylogenetic relations,15 cyp genes that potentially participate in N-demethylation of nicotine were screened out,namely cyp577A4,cyp613D1,cyp584E5,cyp53A13,cyp548D3,cyp684A2,cyp52G4,cyp55A5v2,cyp5113A1,cyp531C3,cyp620H2,cyp573A5,cyp58K1,cyp5095A1 and cyp5080B2.It is speculated that an 8.78-fold up-regulated gene(A0090023000011)encode amine oxidase catalyzing the dehydrogenation at the pyrrolidine-ring of nornicotine.The molybdopterin hydroxylase with[2Fe-2S]cluster,FAD and molybdenum cofactor(Moco)binding domain encoded by xdh gene(A0090003001099),was preliminarily speculated to catalyze the hydroxylation of sp2-Ccarbon at ?-position of pyridine-ring of NMN.The N-heterocyclic aromatic hydroxylase containing three conserved fingerprint motifs[GXGXXG,(D/E)XXIGADG,and GDA(A/C)H]was encoded by a 4.29-fold up-regulated gene(AO090102000087),which was proposed to be responsible for the pyridine-ring ?-hydroxylation of 2-HNMN.These candidate genes that potentially participate in nicotine demethylation pathway could be major targets for nicotine degradation study in A.oryzae 112822,laying the foundation for further revealing the molecular mechanisms of nicotine degradation in filamentous fungi.Nicotine degradation by A.oryzae 112822 is initiated by nicotine N-demethylation that leads to nornicotine formation,which is consistent with the initial reactions catalyzed by CYP82E4 and CYP82E5v2 in Nicotiana sp.as well as the nicotine N-demethylation catalyzed by CYP2A6 and CYP2B6 in human hepatocyte.Cytochrome P450 monooxygenases(CYP)constitute a large superfamily of heme-containing monooxygenases which distribute in a variety of organisms,they are generally involved in detoxification of drugs and xenobiotics.Moreover,the initial reaction of nicotine degradation by A.oryzae 112822 requires O2 and NADPH,which are characteristics of the CYP-catalyzed reaction.Accordingly,it was reasonably speculated that nicotine N-demethylation in A.oryzae 112822 might also be catalyzed by a specific CYP.A total of 142 CYPs are annotated in the reference genome of A.oryzae RIB40.Sequence alignment was performed against database of A.oryzae RIB40 using tobacco nicotine demethylase CYP82E4 and the highest identity is merely 27%,CYP620H1,CYP620H3,and CYP620H2 occupy the top three.In order to lock the nicotine demethylase of A.oryzae 112822,the native enzyme catalyzing the N-demethylation of nicotine was partially purified and analyzed using peptide fingerprint mapping,and then mapped to the CYP database of A.oryzae RIB40.Nine CYPs have the match ratio more than 10%,namely CYP5113A1,CYP6001C12,CYP51F1,CYP6001A1,CYP620H1,CYP577A4,CYP5109A1,CYP531C3 and CYP55A5v2.Among them,CYP577A4,CYP55A5v2,CYP5113A1,and CYP531C3 also appeared in the results of transcriptome analysis.CYP620H1 and CYP620H2 shared the identity of 69.4%and appeared in the results of peptide fingerprint analysis and transcriptome analysis respectively.Therefore,we focused on the expression and activity determination of cyp577A4,cyp55A5v2,cyp5113A1,cyp531C3,cyp620H1,and cyp620H2,together with the up-regulated cyp613D1,cyp584E5,cyp53A13,and cyp548D3 in the transcriptome analysis.These 10 cyp candidate genes were cloned and proved to be successfully expressed in Pichia pastoris KM71 and the resting cells of CYP620H1/KM71 displayed the ability of nicotine degradation.Moreover,the NADPH-cytochrome P450 reductases(CPR)from A.oryzae 112822 and Saccharomyces cerevisiae for electron transport were also successfully expressed and purified from E.coli BL21,the enzymatic activity assay was performed using equine heart cytochrome c(Ecyto C)at 550 nm.The microsome of CYP620H1-harbored P.pastoris KM71 was prepared and the nicotine N-demethylation activity was feebly detected by addition of exogenous CPR.It was preliminarily speculated that the electron transport system between CYP anchored on the microsomal membrane system and the exogenous CPR might be imperfect for lack of the effective organization by living cells,and the content of endogenous CPR of P.pastoris KM71 was insufficient.Therefore,the coexpression plasmids for cyp and cpr were constructed to verify the nicotine N-demethylation activity and perform the biochemical characterization of CYP,and the research is ongoing.Aimed at the hydroxylation of sp2-carbon at ?-position of pyridine-ring of NMN in the nicotine demethylation pathway of A.oryzae 112822,we found that this kind of reactions are catalyzed by molybdopterin hydroxylases with[2Fe-2S]cluster,FAD and Moco binding domain,regardless of the length and type of side chain of pyridine-ring.Thus far,quite a few molybdopterin hydroxylases have been identified to catalyze the ?-hydroxylation of pyridine-ring in the nicotine and nicotinic acid degradation pathways of bacteria,playing critical roles in the biodegradation of N-heterocycle compounds.Therefore,we have tried to express A.oryzae 112822 xdh gene encoding molybdopterin hydroxylase in P.pastoris for the sake of Moco assembly.Detailly,expression of total xdh gene and separate xdhl[2Fe-2S],xdh2[FAD],and xdh3[Moco]genes was performed,they were all proved to be successfully expressed via western blot analysis,this is the first exploration of heterogenous expression of fungal XDH.Through the acitivity assay for resting cells of XDH/KM71,only to find that the control KM71 itself displayed high metabolic ability for NMN,so it needs to further purify XDH for verification of NMN?-hydroxylation activity.Meanwhile,we have constructed CRISPR-Cas9 gene-editing system in A.oryzae 112822 to knock out the candidate genes for verification of their functions in vivo.The cas9 gene from Streptococcus pyogenes was codon-optimized and fused with SV40-NLS,which was inserted into the filamentous fungi expression vector pBARGPE1 and pBARGPE1-eGFP,and the bar gene on pBARGPE1 was replaced with the pyrithiamine(PT)resistance gene ptrA.The recombinant plasmids were linearized and transformed into protoplast of A.oryzae 112822 using PEG-CaCl2 method,and Cas9 nuclease was proved to be successfully expressed under the control of glyceraldehyde triphosphate promoter.Moreover,the analysis of fusion protein Cas9-eGFP via fluorescence microscopy found that Cas9 nuclease was accurately located into the nucleus under the guidance of SV40-NLS.Subsequently,the wA gene(AO090102000545)involved in pigment formation and its mutation will lead to color change of conidia from green to white,was selected as the target for activity verification of CRISPR-Cas9 system in A.oryzae 112822.Three target sites were selected according to the sequence analysis of wA,the transcription of sgRNA in vivo was designed to be controlled by U6 promoter and terminator,and the transcription box was constructed into the cas9 expression plasmid.After transformation,the mutant strains were screened out through PT resistance and white spore phenotype,and positive strains appeared under the targeted action of sgRNA,suggesting that CRISPR-Cas9 system was successfully constructed in A.oryzae 112822.Compared with the traditional method for gene knock-out based on spontaneous homologous recombination,the efficiency of CRISPR-Cas9 system has been greatly improved,which laid the foundation for further research of key enzymes and its genes involved in the nicotine demethylation pathway of A.oryzae 112822.Part ?:study of the molecular mechanism of levansucrase from Bacillus licheniformis 8-37-0-1 for product specificityLevansucrases(EC 2.4.1.10)belong to the glycoside hydrolase 68 family,synthesize fructan with ?(2-6)-linkages in the main chain using sucrose as substrate,also referred to as fructosyl-transferases.Levansucrases from Gram-positive bacteria catalyze the formation of high molecular weight levan,whereas most levansucrases from Gram-negative bacteria mainly produce fructo-oligosaccharides(FOS).Levansucrases from the two sources share similar overall structures and highly conserved essential motifs,however,the substrate-entering channels of levansucrases from gram-positive bacteria become narrow due to the protruding structure formed by amino acids on the surface.Therefore,it was speculated that the product specificity was related to surface structure of substrate-entering channel.We reported a levansucrase(BI_SacB)from Bacillus licheniformis 8-37-0-1 that leads to high yield(47.5 g/L)of polymerized levan with molecular weight of up to 2.8X 104.Accordingly,residues Tyr246,Asn251,Lys372,and Arg369 of Bl_SacB predicted to localize on the surface of the substrate-entering channel were selected for mutagenesis.Our results indicated that mutations could significantly affect the degree of polymerization(DP)and product linkage specificity.Thus far,no structural information is available for levansucrase in complex with FOS-type acceptor substrate.Given the structural requirement for the fructosyl donor and acceptor docking and the geometry of the active cavity,the residues located on the surface of the substrate-entering channel can participate in the acceptor-enzyme interaction.Thus,the substitution at residues Tyr246,Asn251,Lys372,and Arg369 may weaken the interaction between the enzyme and the acceptor molecule,the mutants may lose the coordination and orientation for acceptor,and the acceptor was attacked by the nucleophilic donor at alternative hydroxyl oxygen to synthesize saccharides with altered linkage types.Mutations also made it easier for dissociation of products,and the mechanism of transglycosylation reaction shifted from processive to non-processive which results in accumulation of FOS with different DP.This work revealed the molecular mechanism of levansucrase for product specificity.