Molecular Mechanism Invovled In The Biodegradation Of Fenoxaprop-p-ethyl And 2-methyl-6-ethylaniline

With the development of modern agriculture,excessive use of herbicide led to environmental pollution has increasingly attracted attention of the society.Many means and methods are proposed and updated to solve pesticide residues problem.Microorganisms as widespread disintegrator in soil,its ability to degrade herbicides has become a study hotspot in recent years.Aryloxyphenoxypropionate and chloroacetanilide herbicides are the most used herbicides in China except glyphosate.In the two kinds of herbicides,fenoxaprop-p-ethyl(FE)and acetochlor were the largest variety of production volume and sales volume.In this study,FE was used as the main research target to reveal its microbial metabolic pathway and degradation mechanism.The degradation mechanism of acetochlor also was further improved,elucidating the related genes and enzymes involving in the degradation of MEA.The results will provide the theoretical basis and microbial resources for bioremediation and research of herbicide resistant transgenic crops.An efficient FE-degrading consortium W1 was enriched from the long-term pesticide-contaminated soil in the study.Four intermediate metabolites during the FE degradation,fenoxaprop acid(FA),6-chloro-2,3-dihydrobenzoxazol-2-one(CDHB),2-amino-5-chlorophenol(2A5CP),2-(4-hydroxy-phenoxy)-propionic acid(HPP),were detected and identified using HPLC-MS.The upstream metabolism pathway of FE was proposed by consortium W1 based on these intermediate metabolites.Bacterial diversity analysis showed that the consortium W1 still had a rich diversity and the majority of the microorganisms were uncultured.An efficient FE-transforming strain DL-2 was isolated from the consortium W1 and identified as Acinetobacter sp.The intermediate metabolite FA was identified in the FE degradation by strain DL-2 using HPLC-MS.Strain DL-2 could not further degrade FA.The FE hydrolase gene afeH was cloned from Acinetobacter sp.DL-2 genome by shotgun library.The FE hydrolysis esterase AfeH contained a catalytic triad Ser-Glu-His(Ser159,Asp253 and His283)and the common G-X-S-X-G esterase motif in the a/p-hydrolase family protein.AfeH was identified as a new member of the esterase VII family.The afeH could be expressed efficiently in E.coli BL21(DE3).The pure recombinant enzyme rAfeH was obtained by Ni-NAT and the enzymatic characteristics of rAfeH were further studied.rAfeH had a broad substrate spectrum,which not only had activity on the hydrolysis of AOPPs,the esterase substrate p-nitrophenyl esters and carboxylic acidgeneral glycerol esters also could be hydrolyzed.An efficient CDHB-degrading strain DL-8,isolated from the consortium W1,was identified as Pigmentiphaga sp.and its genome was sequenced for the first time.The intermediate metabolite of CDHB degradation,2A5CP,was identified using HPLC-MS and it could spontaneously polymerize to formation of 9-chloro-2-amino-3H-phenoxazin-3-one(CAPO)by GC-MS.Therefore,the whole metabolism process of CDHB was proposed by strain DL-8.A highly active CDHB hydrolase(CbaA)was purified 20-fold with 4%recovery from strain DL-8 using ammonium sulfate precipitation,DEAE anion exchange and hydrophobic interaction chromatography and enzymatic characteristics of pure CbaA was further studied.Peptide fragments of CbaA was consistent with the orf5248 in scaffold 112 of strain DL-8 genome.The amino acid sequence of CbaA contained the cyclase and aryl carboxamide hydrolase domains.CbaA homology sequence alignment in PDB database found that CbaA contained a catalytic triad His-His-Glu(His137,His288 and Glu301),however,CbaA lost conserved metal binding region of HXGTHXDXPXH.Therefore,CbaA may be a new member of the amide hydrolase family.The rest cells of recombinant strain E.coli BL21(DE3-pET-cbaA),were transformation of CDHB to 2A5CP and BOA to o-aminophenol(2AP)using HPLC and HPLC-MS,respectively.The mutant strain DL-8,which cbaA was deleted using the single crossover homologous recombination,lost the ability to degrade CDHB.The 2A5CP metabolic gene cluster was found and validate by genome researching improving the microbial metabolic pathway and mechanism of CDHB.MEA is an important intermediate product of the photolysis,hydrolysis and biodegradation of chloroacetanilide herbicides.The mutant strain MEA3-1Mut from strain Sphingobium sp.MEA3-1 was obtained by continuous subculture,which could only convert MEA to a undenfitied product.Wild type strain can mineralize most alkyl substituted aniline or phenol compounds and 4-hydroxy-2,6-dimethylaniline(4-OH-DMA),however,the mutant strain could only transform these compounds,and not degradation of 4-OH-DMA.However,strain MEA3-1 could not degrade aniline,which suggested that the metabolic of MEA was different from the traditional metabolism of aniline compounds.Metabolic intermediates of MEA were identified by strains MEA3-1 and MEA3-1Mut using HPLC-MS.Combine with the substrate spectrum of strain MEA3-1,the biodegradation pathway of MEA was proposed for the first time.The initial reaction of MEA metabolism was formation of 4-OH-MEA in para hydroxylation;then spontaneous oxidation to formation of 2-methyl-6-ethyl-benzoquinone imine(MEBQI);MEBQI could spontaneous hydrolysis of deammonia to form MEHQ in aqueous solution,and MEHQ was hydroxylated to form 3-hydroxy-2-methyl-6-ethyl-benzoquinone(3-OH-MEHQ)which could be opened the benzene ring in the dioxygenase action.The whole genome of strain MEA3-1 was sequenced by 454 sequencing technology and 7 completely circular plasmids sequences were spliced using bioinformatics analysis and PCR verification,which the largest plasmid length up to 300 kb and the smallest only 6 kb.Comparison of plasmids profiles of wild type and mutant type showed that two fragments was lost on the plasmid pMEA02 of wild type strain MEA3-1.Therefore,we speculated that a key gene(cluster)involved in MEA degradation was located in the pMEA02.The proteomes of the wild type and mutant strains were blasted using 2-D electrophoresis.The results found that mass spectrometry data of 3 distinct protein spots were located in the congtigs 10 and 20 of the pMEA02 and the MEHQ monooxygenase meaA was found on the contig 20 by bioinformatics analysis.MeaA shared 25%similarity containing the monooxygenase conserved Trp86,His94 and His367 amino acid residues with the oxygenase part of two-component flavin C2-hpah,suggesting that MeaA may be a new member of the flavin monooxygenase family.A suspected reductase gene meaB was found in the upstream of meaA gene,which had only less 10%similarity with C1-hpah,but it had 40%similarity with cytochrome C with 30%coverage.Promoter analysis showed that meaA and meaB shared the same promoter,and the two genes are constitutively expressed according to reverse transcription PCR results.The strain MEA3-1Mut with monooxygenase genes meaBA could mineralize MEA completely and meaBA into Psedomonas putida KT2440 also could detect the 4-OH-MEA degradation ability.The E.coli with meaBA could not detect the ability on the degradation of MEHQ regardless of its promoter or T7 promoter.The cytochrome P450 specific inhibitor metyrapone had an obvious influence on the biodegradation of MEA.Therefore,the metabolism reaction of MEA into the 4-OH-MEA was probably completed by the cytochrome P450 system.The process of MEBQI spontaneous hydrolysis of deammonia to form MEBQ in aqueous solution was simulated by using Nessler's reagent colorimetric method.