Biodegradation Of Biphenyl By Dyella Ginsengisoli LA-4 And Cloning, Expression Of Bph Genes

The purpose of this dissertation is to investigate the characteristics and molecular genetics research on catabolic genes of a newly isolated bacterium Dyella with the ability to degrade biphenyl.The physio- biochemical characterization,Biolog analysis and 16S rDNA molecular identification were performed.The characteristics of strain LA-4 on growth and biphenyl degradation were also investigated and the proposed metabolic pathway of biphenyl degradation by strain LA-4 was presumed.Meanwhile,bioaugmentation of biphenyl degradation with strain LA-4 as augmented inoculums was studied,and the PCR-DGGE fingerprints analysis was used to reveal the structural changes of microbial community. Finally,a complete bph gene cluster was amplified from strain LA-4 by genome walking method.The bphA1A2,bphA3,bphA4,bphB,bphC and mfphA genes were cloned and heterologously expressed in Escherichia coli.Strain LA-4 is a novel biphenyl-degrading bacterium.It was identified as genus Dyella ginsengisoli belonging to Gammaproteobacteria according to the morphological and physio-biochemical characteristics,Biolog analysis and 16S rDNA sequence analysis.Strain LA-4 has been deposited as a patent bacterium in China General Microorganism Culture Center with the accession number CGMCC 2723.The 16S rDNA sequence of strain LA-4 showed the highest similarity with that of Dyella ginsengisoli Gsoil 3046^T(=DSM 18387^T) (98.22%identity).The 16S rDNA sequence of strain LA-4 has been deposited in the GenBank database under accession number EF191354.Strain LA-4 was resistant to kanamycin and streptomycin,and sensitive to other antibiotics.The optimal conditions for both growth and degradation were as follows: temperature 30℃,pH6.0 and shaking speed 150 r/min.The surfactant,Tween 80,could accelerate the biodegradation process when the concentration was less than 40 mg/L.And strain LA-4 was able to grow on and degrade biphenyl in the medium containing 3%NaCl.It could tolerate to Zn²⁺ in the biphenyl degradation.The crude cell extracts of strain LA-4 exhibited the 2,3-dihydroxybiphenyl dioxygenase activity,and it was proved that the enzyme was a constitutive extradiol dioxygenase.A yellow intermediate was observed during the biphenyl degradation by strain LA-4,and it was suggested that meta-cleavage occured.The major metabolites formed from biphenyl,such as 2-hydroxy-6-oxo-6- phenylhexa-2,4-dienoic acid(HOPDA) and benzoic acid,were identified by LC-MS. The suspended cells of strain LA-4 were inoculated into biphenyl treatment system,and the results showed that the cells of strain LA-4 can shorten the start-up time of the system.Different inoculums had the significant impacts on bioaugmentation,and the results demonstrated that the system with 3.96%inoculums had the strongest removal efficiency,which had the relatively stable function when the biphenyl concentration of influent was 500-1000 mg/L.The PCR-DGGE was used to reveal the structural changes of microbial community in the treatment system.The results showed that the microbial diversity was decreased in the activated sludge system and 1.98%inoculums system.As the biphenyl concentration was increasing,strain LA-4 was disappeared in 1.98%inoculums system.However,the introduced strain LA-4 was persistent in the augmented systems inoculated 3.96%strain.A complete 12,186 bp bph gene cluster was amplified by genome walking method,and the sequence of this cluster has been deposited in the GenBank database under accession number EU258607.Based on the sequence analysis,the gene organization of bph gene cluster from strain LA-4 was shown as bphA1A2-ORF1-bphA3A4BCXOX1-ORF2-bphX2X3D.12 ORFs in 13 ORFs obtained in this study showed homology with the previously characterized bph genes.Insterestingly,a novel ORF(ORF2) was firstly cloned from bph gene cluster, which was located between bphX1 and bphX2.The bph gene cluster from strain LA-4 belonged to LB400-type gene cluster.Meanwhile,it was determined that the ORF2 gene encoded a meta-fission hydrolase designated as MfphA,because the amino acid sequence of ORF2 exhibited 75%similarity with the sequence of 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas putida UCC22(BAB62053).The sequence of all ORFs exhibited lower similarity(about 63-89%identities) with previously characterized bph genes.The bphA1A2,bphA3,bphA4,bphB,and bphC genes were cloned and heterologously expressed in Escherichia coli BL21(DE3).The recombinant biphenyl dioxygenase(BPDO_LA-4) could biotransform biphenyl and various polychlorinated biphenyls(PCBs).BPDO_LA-4 showed the best activity to transform biphenyl with the specific activity 134.9 nmol NADH/min(mg protein).It was able to transform the biphenyl/PCBs in the following order: biphenyl＞4-CB＞2,5-CB＞2,2'-CB＞2,5,2'-CB＞3,3'-CB＞2,5,3'-CB＞4,4'-CB＞2,5,4'-CB.But the BPDO_LA-4 had no activity on 2,2',5,5'- tetrachlorobiphenyl.Meanwhile, BPDO_LA-4 could also transform dibenzofuran and flavanone.It was detected that BPDO_LA-4 could introduce two hydroxyl into the biphenyl or PCB congeners at ortho and meta carbons, and 3,4-dihydroxylation was not observed by GC-MS and UV-Vis.According to the amino acid sequences analysis of BphC_LA-4,it was a two-domain typeâ… Fe(â…¡)-dependent extradiol dioxygenase.The expression product of BphC_LA-4 in E. coli BL21 was purified using an(?)KTA explorer.The specific activity was 118.3 U/mg for 2,3-dihydroxybiphenyl,which was the best substrate for BphC_LA-4.The optimal pH for BphC_LA-4 was 8.0,and the enzyme showed its maximal activity at 40℃.According to the specificity constant(K_cat/K_m),BphC LA-4 was able to cleave dihydroxylated substrates in the following order of specificity:2,3-dihydroxybiphenyl＞3-methylcatechol＞catechol＞4-chlorocatechol＞4-methylcatechol.According to the amino acid sequences analysis and the three-dimensional structure of MfphA,it was indicated that MfphA encoded by the novel gene located in bph gene cluster belonged to theα/βhydrolase family,which could hydrolyze the meta-fission products(MFPs) during the biodegradation process of monocyclic compounds.The mfphA gene was heterologously expressed in E.coli,and the expression product was purified using an AKTA explorer.The purified MfphA could transform MFPs,and the highest specific activity was 2.0 U/mg for HODA among these substrates.MfphA showed the maximum activity at pH7.0,and the optimal temperature was 70℃.Meanwhile,MfphA was more stable for storage at low temperature.It was determined that MfphA was able to hydrolyze the substrates in the following specificity order:6-methyl-HODA＞HODA＞5-methyl-HODA＞6-phenyl-HODA＞5-chloro-HODA.