Isolation & Characterization Of 3-Phenoxybenzoic Acid-Degrading Strain BA3 (Sphingobium Sp.) And Construction Of Genetically Engineered Microorganism

A strain of bacteria capable of utilizing 3-phenoxybenzoic acid as sole carbon source for growth was isolated from petroleum-contaminated soil. This bacteria, designated as BA3 (GenBank Accession No:GQ176409), was identified as Sphingobium sp. according to its physiological & biochemical characteristic and the similarity analysis of its 16S rRNA sequence.The investigation of the biological characteristics of strain B A3 showed that, the optimal pH value for the growth of strain BA3was 5.0-8.0, the optimal pH value for the growth of strain BA3was 7.0, the optimal temperature was 30℃. The aeration was related positively to strain growth. The optimal osmotic pressure for the strain BA3 was 5-15mg·L-1 NaCl. The optimal medium for the growth of strain BA3 was glucose as carbon source and organic nitrogen as nitrogen source. BA3 has resistance to streptomycin.Strain BA3 was able to degrade 99% of 100mg L-1 3-phenoxybenzoic acid in 60h. The optimal pH and temperature for the degradation were 7.0 and 30℃, respectively. The degradation efficiency was related positively to initial inoculum size.Strain BA3 utilized catechol, protocatechuic acid, resorcinol andhydroquinol but not phenol, p-nitrophenol and 2,4-dinitrophenolas sole carbon source.The reported catechol 1,2-dioxygenase genes from Sphingobium were aligned and degenerated primers were designed according to the conserved sequence.The catechol 1,2-dioxygenase gene sequence was amplified from BA3 total genomic DNA. According to the analysis of online-software and GenBank database, this sequence had high similarity to Sphingobium sp.YBL3(U825703) and Sphingobium sp.YBL2(U825702).The pyrethroid hydrolase gene e (pytH) gene was cloned by the method of shotgun cloning, and the sequences were determined and analyzed. PytH gene was amplified from the genomic DNA of Sphingobium sp. JZ-2 by PCR. Recombinant plasmids pPYTH was constructed by ligating pytH gene into broad host vector pBBRMCS-5.With the help of plasmid RK600, PYTH was transferred into Sphingobium sp. BA3 to constructBA3-pytH. BA3-pytH can degrade the selected genetically engineered microorganisms (GEM) can degrade Permethrin and 3-PBA simultaneously. BA3-pytH.showed higher degrading ability than the original strain JZ-2. All these results indicate that BA3-pytH. was a promising GEM in bioremediation of pyrethroid contaminated environment.