| Polycyclic aromatic hydrocarbons are persistent organic pollutions widespread in environment. They are teratogenetic, carcinogenic and mutagenic and harmful to human health and environment. A bacterium Sphingobium sp. FB3that could degrade a mixture of PAHs including phenanthrene, anthracene, fluoranthene, pyrene and benzo[a]pyrene had been isolated in the previous work. It was efficient on degradation of a four-ring fluoranthene. It could grow up with fluoranthen as sole carbon and energy. There were few studies about genes involved in fluoranthene degradation. The critical genes involved in degradation of fluoranthene in Sphingobium sp. FB3had been cloned and the enzyme encoding by the genes had been analyzed in this study.A random insertional mutagenesis library had been established by inserting Mariner randomly into genomic DNA of Sphingobium sp. FB3. Mutants were inoculated in liquid MSM with fluoranthen as sole carbon and energy. Whether there was color generated or different color compared with inoculated with Sphingobium sp. FB3in liquid MSM were used as selection markers. Two mutants with number5-41and12-45that could not make the liquid MSM generate color and three ones with number1-45,5-49and6-91that generated color rapidly were found. Amplification and sequencing of flanking sequences of these five mutants were performed. The insertion sites of Mariner in mutant5-41and12-45were genes that encoded a subunit of terminal oxygenase and ferredoxin of ring-hydroxylating dioxygenase, respectively. It was considered that these genes may be associated with fluoranthene degradation by strain FB3. The insertion sites of Mariner in mutant1-45,5-49and6-91were genes that encoded tartrate dehydrogenase, transposase and integrase according to the results from flank sequences of Meriner and genome sequence of FB3.Genome sequence of Sphingobium sp. FB3had been sequenced. It consisted of4.6MB and included155scaffolds. The percentage of G+C was63.47%. The gene be inserted by Mariner in mutant5-41named flnAlf was located on scaffold91where there was gene named flnA2f downstream flnAlf. The gene be inserted by Mariner in mutant12-45named flnA3was located on scaffold120. There was a gene named flnA4which was18.3kb downstream from flnA3. These two scaffolds included a number of genes involved in PAHs degradation.FlnA1f, flnA2f, flnA3and flnA4that encoded ring-hydroxylating dioxygenase FlnA had been co-expressed in E coli. BL21(DE3). E coli. BL21FlnA could degrade43%of fluoranthene parent compound in12h and generate monohydroxyfluoranthene which could be detected by GC-MS. These proved that FlnA was a crucial enzyme and the four genes above were involved in fluoranthene degradation by Sphingobium sp. FB3. No corresponding metabolite could be generated when used E coli. BL21FlnA12that expressed flnAlf and flnA2f only for fluoranthene degradation. It shows that FlnA3and FlnA4were necessary for FlnA. The study of substrate range of FlnA showed that it could also convert phenanthrene, anthracene and pyrene into corresponding metabolites. The3D structure of α subunit of terminal oxygenase of ring-hydroxylating dioxygenase in Sphingobium sp. FB3had been predicted using the corresponding α subunit in Sphingobium yanoikuyae B1as template. The active site of α subunit had also been predicted. Docking between active site of α subunit and benzo[a]pyrene had been studied. There were four orientations for benzo[a]pyrene to fit in active site. Degradation efficiency of benzo[a]pyrene parent compound could be improved when the residue PHE223of α subunit was substituted with LEU223and it had the same situation to phenanthrene, anthracene, fluoranthene and pyrene. It showed that the number223residue of FlnA1f was crucial in PAHs degradation with ring-hydroxylating dioxygenase. |
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