Isolation Of 2,6-dichloro-4-nitrophenol Degrading Strain,Identification Of The Metabolic Pathway And Cloning Of The Dechlorinase Gene

Chlorinated nitrophenol compounds(CNPs)are widely used in the chemical production in industrial and agricultural areas,such as the production of fungicides,pesticides,dyes,etc.As a result of the substitution of the strong electron-withdrawing groups of nitro and chloride groups,CNPs have long half-lives in the environment and are recalcitrant to be degraded.CNAs are highly toxic to animals as well as human beings and some CNPs are even teratogenic and carcinogenic.The pollution of CNPs mainly results from the irregular emissions of wastes from industry,leading to the serious pollution of water,soil and air and even serious damage to human health and ecosystem safety.Microorganisms play key roles in degradation of pollutants in environments,and the use of microorganisms to clean up the contamination of CNPs is a kind of economic,safe and effective in situ bioremediation method,which has the great potential in the applications.Currently,very few studied on the microbial degradation of 2,6-dichloro-4-nitro phenol(DCNP)have been reported and the biodegradation pathway and the degradation mechanism especially the dehalogenation mechanism remain unclear.In this study,DCNP was used as the target compound,isolation of high efficient degradation strains and investigation of the degradation mechanism,cloning the degrading gene(cluster)and the functional identification of the degrading genes were carried out.The illumination of the complete metabolic pathway and molecular mechanisms involved in DCNP degradation will provide theoretical guidance for the microbial degradation of CNPs and provide technical support for the bioremediation of CNPs-contaminated sites.In this study,soil samples were collected from the First Pesticide Plant in Suzhou and Kunshan Green Pesticide Plant.After continuous enrichment cultures,a high efficiency degradation bacterium named 22-1 was isolated from the DCNP enrichment.Based on the 16s rRNA gene sequence analysis,the strain 22-1 was identified as Ensifer sp.Degradation test research showed that strain could use DCNP as the sole carbon,nitrogen and energy sources for growth,and could mineralize DCNP completely.The optimal temperature for degradation was 25? and the optimal degradation initial pH value was 8.0.It was found that 1 mM Fe3+,Mn2+ and Ca2+ could improve the degradation rate of DCNP,while,other icons,such as 1 mM Cu2+ and Co2+ had strong inhibit on the rate of degradation.Using the second generation sequencing technology Illumina Miseq to sequence the whole genome of strain 22-1 and using bioinformatics methods through GenBank database and microbial metabolism(KEGG,UM-BBD)comparison and analysis,the DCNP degradation gene(cluster)was found:cnpA,2,4,6-trichlorophenol monooxygenase;cnpB,NAD(FAD)-dependent dehydrogenase;cnpC,putative NAD(P)H-dependent FMN reductase;cnpD,catechol 1,2-dioxygenase;cnpE,FMN adenylyltransferase;cnpR,LysR family transcriptional regulator;orfl,maleylacetate reductase.In this series of genes,predicting cnpA is the key enzyme genes of strain 22-1 degradation DCNP,so by knock out cnpA gene to verify the prediction of the function of the gene cluster.Found that when the cnpA gene has been successfully knock out,mutant strain lost the degradation function of DCNP,thus we can verify the predict gene cluster is involved in the DCNP degradation.The wild type strain 22-1 and mutant strains 22-AcnpA with different substrate processing,were extracted of total RNA,then reversing transcription generated cDNA.We designed 8 pairs of primers in this experiment,the theory length of each pair of primers amplification is between 500-1000 bp,overlapping between these fragments,such fragments can cover athe whole cnpBADCE gene clusters.PCR results showed that,RNA as a template directly,8 pairs of primers to amplify nothing.Instead,cDNA as the template,8 pairs of primers could amplify the corresponding fragment,the purpose of this suggests that these five genes transcription in the same unit(cnpB,cnpA,cnpD,cnpC and cnpE).they are a total of transcription.By using the method of PCR to clone oxidoreductase CnpAB gene segments(cnpA and cnpB)from strain 22-1,they were heterologously expressed in E.coli BL21(DE3)and purified by nickel column affinity chromatography.Found that no degradation activity was found for the single component,and the combinations of CnpA/CnpB showed degradation activity for DCNP with adding the cofactor NADPH.So,CnpA needs CnpB supplied reducing power hydrogen and electronics.The enzymology characteristics study of CnpAB found that,the optimal reaction temperature 30?,the optimum pH is 8.0.It was found that 1 mM of Ni+2,Zn2+ and Fe3+ had a strong inhibitory effect to the enzyme activity,and 1 mM Ca2+,Cue+,K+,Al3+,Mn2+,Co2+ also to have certain inhibitory effect to enzyme activity.The supernatant of component CnpA and CnpB reaction system was extracted,and the extracts be used to derivatize experiment.By gas chromatogram mass spectrometery,the metabolited during the degradation were identified,and the metabolic pathway of DCNP in strain 22-1 was proposed:DCNP was transformed into 2,6-dichloquinone,2,6-dichloquinone was further hydrolyzed to generate 6-chlorohydroxyhydroquinone,and then 6-chlorohydroxyhydroquinol was produced,finally 6-chlorohydroxyhydroquinol was mineralized through the TCA cycle.