Synthesis Of An Artificial Consortium For Phenanthrene Biodegradation And Analysis Of Its Crucial Metabolic Mechanism

With the extensive exploitation and extensive application of coal and petroleum,polycyclic aromatic hydrocarbons(PAHs)produced in the process of refining and combustion are accumulated in large amounts.Phenanthrene is one of the main components of organic pollutants of PAHs and is a model compounds,microbial remediation is one of the main means to remove phenanthrene in the environment.In this study,first of all,based on a large number of literature and database investigations,17 genes related to the degradation pathway of phenanthrene were found and artificially synthesized,and assembled into 3 functional modules according to their functions and matches,constructed with E.coli BL21 as the host,E.coli BL21-M1,E.coli BL21-M2 and E.coli BL21-M3-1 were obtained,in which E.coli BL21-M1 had an aromatic ring cleavage module and E.coli BL21-M2 had salicylic acid synthesis module,and catechol metabolism module in E.coli BL21-M3-1.The mixed culture of the three engineering bacteria constitutes an artificial mixed bacteria system,and the best degradation conditions of the mixed culture of the three engineering bacteria are obtained through single factor experiment and response surface analysis: the inoculation time interval is 6 h,and the inoculation ratio is 1:1:1,under the condition of IPTG concentration of 2 m M,the 7-day degradation rate of 100mg/L phenanthrene by the artificial mixed bacteria system was 72.67%.The above results indicate that the synthetic E.coli flora in this study has a good phenanthrene degradation capacity.The degradation ability of the artificial mixed bacteria system consisting of E.coli BL21-M1,E.coli BL21-M2 and E.coli BL21-M3-1 at different concentrations of phenanthrene from 10 mg/L to 500 mg/L was investigated respectively.It shows that the system has good degradation ability to different concentrations of phenanthrene.Through enzyme activity experiments combined with GC-MS analysis of intracellular and extracellular metabolites,it was clarified that the key metabolic mechanism of phenanthrene degradation in artificial flora was analyzed: E.coli BL21 i has dioxygenase activity,which works during the phenanthrene ring-opening process.Therefore,all three engineering bacteria can convert phenanthrene to 1,2-dihydroxy phenanthrene or 9,10-dihydroxy phenanthrene,and can secrete it into the extracellular environment;E.coli BL21-M2 can cleave phenanthrene by its own dioxygenase,and can also convert phenanthrene cleaved products into trans-cinnamic acid,2,4-di-tert-butylphenol and cateol in the cell through other routes of salicylic acid synthesis,and secrete 2,4-di-tert-butylphenol and catechol to the outside of the cell;E.coli BL21-M3-1 can continuously used the phenanthrene in the culture environment and the 2,4-di-tert-butylphenol and catechol in the system under the effect of its own dioxygenase and catechol 1,2-dioxygenase.The three engineering bacteria showed a synergistic effect in the degradation of phenanthrene.Through the analysis of crucial metabolic mechanism,considering the use of batch supplementary bacterial degradation to make the metabolic flow as simple as possible,so as to exert the best degradation performance of each engineering strain.The degradation results of phenanthrene under the this method showed that the three engineering strains showed further excellent degradation performance to phenanthrene,and the degradation rate of phenanthrene in 21 days could reach 90.66%,indicating that the degradation of phenanthrene by batch supplementation method more completely.The results show that the construction and mechanism analysis of phenanthrene-degrading artificial flora provides a new idea and method for the degradation of phenanthrene.