Isolation,Identification Of 2-phenoxybenzoate (2-PBA) Degrading Strain And The Metabolic Mechanism Of 2-PBA And Diphenyl Ether

Diphenyl ether and its derivatives,such as phenoxybenzoate?PBA?,diphenyl ether herbicides and halogenated diphenyl ether,are widely used in agriculture and industries.And they are also an important pollutants which attract widely public concern.Previous studies show that diphenyl ether compounds are harmful to the ecological environment and health of human and other organisms.Thus,the study of microbial degradation of diphenyl ether compounds has great theoretical and practical importance.Microbial metabolism is the most important factor in the degradation of diphenyl ether compounds in the environment.A variety of bacterial strains able to degrade diphenyl ether compounds,such as diphenyl ether,3-PBA and 4-PBA,have been isolated and characterized.Two degradation pathways of diphenyl ether have been reported:the lateral deoxygenation and the angular dioxygenation.However,isolate capable of degrading 2-PBA,an important diphenyl ether compound,has not been reported.In this study,two strains,SC₃ and SC₄,capable of degrading 2-PBA were isolated by enrichment method and identified as two novel species in the genus Sphingbium by polyphasic taxonomic analysis.The metabolic productions of diphenyl ether and 2-PBA degradation by strain SC₃ were studied by high-performance liquid chromatography?HPLC?and liquid chromatograph-mass spectrometer?LC-MS?and a novel angular dioxygenase gene dpe,which is responsible for the angular deoxygenation of 2-PBA and diphenyl ether,was cloned.The major results of the current study are as follows:1.Isolation and polyphasic taxonomic studies of 2-PBA-degrading strains.Two 2-PBA-degrading strains,SC₃ and SC₄ were isolated from pesticide contaminated soil sediment using 2-PBA as the sole source of carbon and energy.The degradation experiment results showed that both of the strains were able to completely degrade 2-PBA and diphenyl ether and use them as sole carbon source for growth.Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SC₃T formed a monophyletic lineage in the genus Sphingobium,and showed highest similarity to S.abikonense KCTC 2864T?97.0%?,followed by S.lactosutens?96.8%?and S.cloacae ?96.7%?.The DNA-DNA relatedness between strain SC₃T and its closest phylogenetic neighbor S.abikonense KCTC 2864T was 36.8±4.7%.The genomic DNA G+C content of strain SC₃T was 62.9 mol%.On the basis of phenotypic,chemotaxonomic,phylogenetic as well as genotypic data,strain SC₃T represents a novel species within the genus Sphingobium,for which the name Sphingobium phenoxybenzoativorans sp.nov.is proposed.Strain SC 4 showed the highest 16S rRNA gene homology with Sphingobium phenoxybenzoativorans SC₃T?97.08%?.The DNA-DNA hybridization with strain S.phenoxybenzoativorans SC₃T was 44.2±1.52%.Based on the polyphasic analysis results,strain SC 4 was identified as a new species belonging to the genus Sphingobium,for which the name Sphingobium diphenylethervorans sp.nov.is proposed.2.Identification of the metabolic productis of 2-PBA and diphenyl ether degradation by strain SC₃.Strain SC₃ was able to degrade 2-PBA,diphenyl ether,2,3-dihydroxy benzoic acid,benzoic acid,phenol and catechol.However,it was unable to degrade 3-PBA and 4-PBA,which were the isomers of 2-PBA.The optimum degradation condition for the degradation of 2-PBA by strain SC₃ was as follows:temperature 30?and pH 7.0.Two metabolites phenyl-2,4-dienoate and phenol were identified through HPLC and LC-MS analysis during the degradation of 2-PBA or diphenyl ether by strain SC 3.Based on these results,a novel aromatic ring cleavage mechanism of 2-PBA or diphenyl ether through angular dioxygenation was proposed in strain SC₃:carbon?C1?which bonded to the oxygen atom and the adjacent carbon?C2?in the aromatic ring are both oxidized and produces the chemically unstable hemiacetal-like intermediates,which are spontaneously converted to phenyl-2,4-dienoate.Phenyl-2,4-dienoate was then hydrolyzed to phenol and muconic acid semialdehyde.3.The cloning and characterization of the 2-PBA angular dioxygenase gene dpeA1A2.a)Cloning and verification of 2-PBA angular dioxygenase gene dpeA1A2.The draft genome of strain SC 3 was sequenced using the Illumina Hiseq2000 techniques.The total length of the genome was 5,044,557 bp,the predicted ORF was 4906 and the G+C%content was 62.2%.The strategy to clone the 2-PBA angular dioxygenase gene was based on the assumption that since 2-PBA showed similarity with these automatic compounds,the 2-PBA angular dioxygcnase this gene should share homology with some reported angular dioxygenses,which were responsible for the angular dioxygenation of dibenzofuran,carbazole,dibenzothiophene and dibenzo-p-dioxin.The dot plot result of 11 reported angular dioxygenses with the genome of strain SC₃ retrieved 9 putative angular dioxygense genes;and among the 9 genes,a gene dpeA1A2,which located in contig 0053,was confirmed to encode the 2-PBA angular dioxygenase by the complement analysis.Phylogenetic analysis revealed that DpeA1A2 belonged to the Type V RHO?Rieske non-heme iron aromatic ring-hydroxylating oxygenase?.DpeAl had the conserved[2Fe-2S]sites?CXH17CX2H?of RHO,which contained two His and two Cys residues.In addition,DpeAl also had a conserved Fe???sites[EX4DX2HX4H],which contained the conserved residues Asp?235?,Glu?230?and His?238,243?.b)The disruption,complement and induction of dpeA1A2The dpeA1A2 gene was successfully disrupted by a single cross over event,and the dpeA1A2-disrupted mutant lost the ability to degrade 2-PBA and diphenyl ether,suggesting that gene dpeA1A2 was the sole angular dioxygenase gene responsible for degradation of 2-PBA and diphenyl ether in strain SC 3.Real-time PCR results indicated that the transcription of the dpeA1A2 was induced by 2-PBA and diphenyl ether.4.The cloning of the ferredoxin gene dpeB and reductase gene dpeC and the reconstruction of the 2-PBA angular dioxygenase Dpe in vitro.The strategy to identify the ferredoxin and reductase components was based on the assumption that since Dpe belonged to the Type V RHO family,its ferredoxin and reductase components should share homology with the ferredoxin and reductase components of some reported Type V RHOs.Based on this strategy,a[2Fe-2S]ferredoxin gene dpeB and a GR reductase gene dpeC were retrieved from the genome of strain SC₃.dpeB encoded 105 amino acids and dpeC encoded 409 amino acids.The four components of Dpe,DpeAl,DpeA2,DpeB and DpeC were expressed in E. coli BL21?DE3?using pET29a?+?expression system and purified by Ni-affinity chromatography.The purified DpeAl,DpeA2,DpeB and DpeC were mixed and constituted in vitro.The enzymatic assay showed that the whole enzyme DpeAlA2BC showed the 2-PBA angular dioxygenase activity.The HPLC and LC-MS analysis results of the products showed that DpeA1A2BC transferred 2-PBA or diphenyl ether to 6-pheny-2,4-dienoate.Thus we confirmed that DpeB and DpeC were able to transfer electrons for DpeA1A2.