Structural Analysis And Functional Studies Of PAHs Degradation Gene Cluster In Rhodococcus Ruber OA1

Among the severe environmental pollutants, polycyclic aromatic hydrocarbons (PAHs)represent a group of the priority pollutants because of their toxicity and relative persistencyin the environment due to the high thermodynamic stability of the benzene moiety. It hasbecome a growing concern of the world to investigate the eliminating ways of thesepollutants. Microbial degrading and transforming of PAHs has become a hot topic ofresearch since its high efficiency. During the previous work, Rhodococcus ruber strainOA1(G+bacteria) was isolated which had high performance in both PAHs and benzoatecatabolizing. This study illustrated the molecular mechanisms and gene ragulation of PAHscatabolization in Rhodococcus ruber OA1preliminary by investigating the organization,structure, founctions, which provided scientific basis for the detection, prevention, controland bioremediation of PAHs pollution in the environment.R. ruber OA1was used to construct a Fosmid genomic library. The genomic DNA ofOA1was extracted by phenol after the cells were lysed by lysozyme. The DNA wasrandomly sheared and end-repaired by T4DNA Polymerase and T4Polynucleotide Kinase.The insert fragments about40kb were recovered by electrophoresis and ligated toCopyControl pCC2FOS vector with the Fast-LinkTMDNA Ligase, then transfected the hostbacterium E.coli EPI300-T1Rafter being packaged into phage with MaxPlaxTMLambdaPackaging Extracts in vitro. As a result, the Rhodococcus ruber OA1Fosmid genomiclibrary was constructed successfully, which titer was1x104CFU/ml.Based on the ring hydroxylating dioxygenase genes in PAHs biodegradation (RHD)which had been reported in NCBI, a pair of primer was designed for coloning the coresequence of RHD in R. ruber OA1, and the core fragment of RHD gene (about300bp) wasamplified by using the primer pair. The RHD fragment was labeled by DIG High PrimeDNA Labeling and Detection Starter Kit I as a target gene to screen the positive clone withPAHs degrading gene cluster from the genomic library by the method of colony in situhybridization. Two positive clones A#and B#which harboured PAHs catabolic geneclusters were obtained from the hybridization plate.By sequencing and the function analysis of the positive plasmid DNA sequences, aprotocatechuate catabolic gene cluster related to PAHs degradation was found, whose keygene was protocatechuate3,4-dioxygenase (P34D) gene. The P34D gene was amplified with the designed specific primer, and the cloned gene was connected with pET-30a(+)vector carring T7promotor then transformed to E. coli BL21(DE3), and the high levelexpression of recombinants were obtained. SDS-PAGE showed that the expressed proteinwere secreted to the supernant after the optimization of expression condition in E. coliBL21, Enzymatic assay revealed that the heterologous expressed P34D had the ability tocatalyze cracking protocatechuate. The results showed that the gene cluster found in thestudy was a protocatechuate degradation related gene cluster, and R. ruber OA1had thegenetic basis to degrade aromatic compounds via the protocatechuate metabolic pathway.