Expression And Functional Confirmation Of Genes Involved In Dibenzofuran Metabolism By Rhodococcus Sp. Strain P52

Dioxins are persistent organic pollutants that are deleterious to human health and environments because of their high toxicity and carcinogenicity,including polychlorinated derivatives of dibenzo-p-dioxin(PCDD)and dibenzofuran(PCDF).These chemicals are produced as by-products from processes such as incineration,pulp bleaching,and chemical synthesis and are stable in the environment.Bioremediation is a promising technology for cleanup of these pollutants due to its lower-cost performance and no secondary pollution when compared with physical and chemical approaches.In this thesis,Rhodococcus sp.strain p52 which harbors two plasmids involved in degrading dibenzofuran was used as an target bacterium of study.We examined on the expression and functional identification of genes encoding extradiol dioxygenase and hydrolase which were involved in dibenzofuran metabolism.Complete genome sequencing of Rhodococcus sp.strain p52 was performed to gain insights into the genetic elements involved in the degradation of dibenzofuran.The genome of p52 contains two gene clusters,located in two different degradative plasmids,respectively,involved in dibenzofuran degradation.The gene clusters include dfdA and dbfA genes coding angular dioxygenase,dfdB and flnD genes coding extradiol dioxygenase,and dfdC and flnE genes coding hydrolase.In this study,the genes responsible for extradiol dioxygenase and hydrolase of dibenzofuran metabolism were obtained by degenerate polymerse chain reaction(PCR).Meanwhile,we performed the heterologous expression of the genes in Ecoli BL21(DE3)and Rosetta gamiTM 2(DE3)plysS,using the pET-32a(+)as expressing vector.SDS-PAGE experiments showed that the target genes were expressed and the molecular weight of the corresponding expression products is consistent with the theoretical value,comparing with the host strain contains empty plasmid vector induced simultaneously with IPTG.The expression products of genes dfdB,dfdC and flnE were mainly expressed in the form of soluble protein in host strain E.coli BL21(DE3),however,the extradiol dioxygenase gene flnD was expressed mainly as a inclusion bodies in Rosetta gamiTM 2(DE3)plysS.Based on the above results,the biological function of extradid dioxygenase DfdB and hydrolase DfdC in recombinant strain E.coli BL21(DE3)was also preliminarily explored in this thesis.Meanwhile,we studied the effect of temperature,pH,and metal ions on the enzymatic activity of extradiol dioxygenase DfdB.Regarding to gene flnD,we renatured the inclusion bodies in order to obtain bioactive protein.The results showed that the recombinant of gene dfdB encoding extradiol dioxygenase is involved in the transformation of 2,3-dihydroxybiphenyl(DHB)to HPDA,then the product of gene dfdC encoding hydrolase attacks HPDA.When the temperature is 30℃,pH is 8.0,extradiol dioxygenase DfdB has the highest biological activities,while different metal ions has inhibition effect distinctly on the enzyme activity.The extradiol dioxygenase gene flnD were expressed in the form of inclusion bodies and turned to be soluble after renaturation,however,its biological function needs to be further verified.In general,the present study revealed the molecular genetics mechanism of dioxins-degradation.It provides a theoretical foundation for the development and utilization of microorganism to remove dioxins and other pollutants,therefore,facilitating bioremediation of dioxin pollutions in the environment.