Genome Sequencing Of Chlorpyrifos Degrading Paracoccus Sp. Strain TRP,Cloning And Functional Verification Of Cpd Gene

A feasible way to remediate chlorpyrifos (CP) contaminated water and soil environments is toscreen chlorpyrifos degrading microorganisms because of its important significance in thebiodegradation of pesticide residues. Paracoccus sp. TRP is a strain capable of efficiently degradingchlorpyrifos. Paracoccus sp. TRP was selected as the research object in this study. The basiccharacteristics of the strain, the cloning of a new chlorpyrifos degrading gene, and the relationshipsbetween the structure and function of the chlorpyrifos degrading enzyme were investigated in this study.The contents are as follows:Strain TRP isolated in our lab was renovated.The strain can rapidly degrade chlorpyrifos from the2ndday to the4thday, and the degradation rate of50mg/L CP reached to83.3%at the7thday. The strainis resistant to5~100μg/mL Amp and not resistant to5~100μg/mL Tc and Gm. The strain can't producesugar lipid biosurfactants.The chlorpyrifos degrading enzyme of the strain TRP was constitutive.Solexa sequencing technology was used to determine the genome sequence of Paracoccus sp. TRP(GenBank accession number AEPN00000000). Genome size is about3.9Mb with GC content of62.89%. The number of predicted CDS is3961.155metabolic maps were produced, of which25xenobiotic related metabolic maps were found, including metabolic maps for herbicide atrazine anddenitrification.96tandem repeats,200transposons,147IS,45tRNA and5sets of rRNA were alsofound in the genome sequence. Paracoccus sp. TRP and Paracoccus denitrificans Pd1222are closelyrelated in evolutionary distance. Most of the genes are highly homologous. All the four genes of redoxenzymes for denitrification similar to those of P. denitrificans Pd1222were identified in Paracoccus sp.TRP. The analysis of the genome of strain TRP can help us identify more different degrading genes andfurther clarify microbial degradation pathways for chlorpyrifos. This is so far the first report about thegenome sequencing of Paracoccus sp. capable of biodegrading chlorpyrifos.Chlorpyrifos degrading gene cpd was cloned from Paracoccus sp.TRP. Gene size is927bp withGC content of60.73%. The homology between cpd gene and other organophosphorus degrading genesalready published is low, indicating that it is a new organophosphorus degrading gene. CPD proteinconsists of308amino acid residues. Molecular weight is33.8kDa. Charge is-7.0. Isoelectric point is4.8305. CPD protein, a member of the esterase_lipase superfamily, not only has Ser155-Asp251-His281catalytic triad and motif GDSAG but also has the conserved substrate pocket ILYIHGGGWSFCSA.Thesize of CPD fusion protein expressed in the E.coli BL21(DE3) is about53.9KDa.CPD protein hasesterase activity. CPD protein esterase activity needs no metal ions. CPD protein esterase activity wassignificantly inhibited by1mmol/L PMSF,1mmol/L DEPC and0.025%SDS. CPD protein esteraseactivity was significantly enhanced by0.025%Tween-20. CPD esterase activity needs His and Ser.Within15min, the degradation rate of50mg/L CP by CPD enzyme is63.5%±2.14. The enzymeactivity is59.62U/mg. Strain TRP△cpd, in which cpd gene was knocked out, was successfullyconstructed by inserting tetracycline box into the cpd gene of strain TRP. Within5days, the degradationrate of50mg/L CP by strain TRP△cpd and strain TRP were27.3%and78.5%, respectively.The degradation rate of CP by strain TRP△cpd significantly reduced, indicating that cpd gene plays animportant role in chlorpyrifos degradation of strain TRP.The3D structure of CPD protein is a tightly folded globular structure consisting of the alphahelixes, beta strands and coils. The active site Ser155, Asp251and His281in catalytic triad located atthe flexible loops between β strand and α helix. Molecular docking between CPD and CP showed thatspace distance between O atom from hydroxyl group of active site Ser155and P atom from CP is near,which facilitates to produce nucleophilic attack to degrade CP to form TCP and DETP. The mutation ofactive site Ser155to Ala was performed by PCR mediated site-directed mutagenesis technology, namelyS155A. Within15min, the degradation rate of50mg/L CP by CPD-S155A enzyme is5.15%±1.03,enzyme activity is7.85U/mg. Ser155mutation for Ala of the CPD protein reduced the degradation rateof CP by more than10times, indicating that Ser155plays a key role in the degradation of CP by CPDenzyme.