Pathway And Molecular Mechanism Of Phthalic Acid Esters Biodegradation By Microbe

Phthalic acid esters(PAEs)are widely used as plasticizer in production and daily necessities,but they posed a great threat to mankind for their extensive use and hazards of hepatotoxicity,carcinogenicity and endocrine interference.Biodegradation has been proved to be an efficient technology in the removal of PAEs.At present,the research on PAEs biodegradation by microbe mainly focused on the screening of pure strains,bodegradation characteristics and metabolites identification,while research on the degradable enzymes,degradation genes and biodegrading consortium were few,especially the molecular mechanism of PAEs biodegradation has not been elucidated.In this paper,based on two PAEs degrading bacteria isolated,a high efficient consortium USTB-YA was constructed.Through genome sequencing and annotation,cloning and expression of crucial degradation genes,activity verification of recombinant enzyme,the pathway and molecular mechanism of DBP biodegradation by consortium USTB-YA were clarified.The main work and research progress of this paper are as follows:(1)Two bacterial strains were successfully isolated from activated sludge and characterized as Microbacterium sp.USTB-Y and Comamonas sp.USTBZA1 based on 16S rDNA sequence analysis.The two strains had the strongest biodegradability under conditions of temperature 30℃,initial pH 7.5 and inoculum volume ratio of 6%.DBP of 50 mg/L could be biodegraded with 91.4%by strain USTB-Y within 12 h which conformed to the first-order reaction,and diethyl phthalate(DEP)of 50 mg/L could be completely biodegraded by strain USTBZA1 within 24 h which conformed to the Gompertz model.(2)Based on the analysis of HPLC,GC-MS and LC-MS,phthalic acid(PA)was identified as the end-product of DBP biodegradation by USTB-Y and USTBZA1 could completely biodegraded DEP through de-esterification metabolites of monoethyl phthalate(MEP)and PA.The consortium USTB-YA composed of Microbacterium sp.USTB-Y and Comamonas sp.USTBZA1 was constructed for the first time,wich could biodegrade 98%of 50 mg/L DBP within 12 h,all of the product PA within 21 h and 96.8%of DBP in soil within 48 h cooperating with indigenous microorganisms.(3)The genome of Microbacterium sp.USTB-Y was sequenced and annotation firstly,which contained a total genome length of 4109641 bp,3922 protein coding sequences(CDS),and 46 genes involved in 15 xenobiotics metabolic pathways.The genome of strain USTBZA1 contained 5684228 bp,4273 CDS and 151 genes involved in 19 xenobiotics metabolic pathways,which had rich degradation genes for organic pollutant.(4)The genome of strain USTB-Y contained 7 esterase genes and 47 hydrolase genes,but no genes responsible for PA biodegradation.Two hydrolase genes mices01 and mices02 were screened out based on homology alignment.Through cloning and expression of the two genes,activity verification of recombinant enzyme,it was proved that mices02 and mices01 were critical hydrolase genes for the hydrolysis of DBP to MBP and PA.There were 3 esterase genes and 41 hydrolase genes in the genome of strain USTBZA1,and the whole gene clusters for PA(pht)and protocatechuic acid(PCA)(lig)biodegradation were found.(5)Based on functional gene analysis and biodegradation experiments,the pathway and molecular mechanism of DBP biodegradation by consortium USTB-YA were clarified.DBP was first biodegraded into MBP and PA sequentially by hydrolases of MiCEs02 and MiCEs01 in USTB-Y;Then PA was biodegraded into PCA by hydroxylation,dehydrogenation and decarboxylation under the catalysis of dioxygenase,dehydrogenase and decarboxylase encoded by pht gene cluster in USTBZA1,and PCA was biodegraded into oxaloacetic acid and pyruvic acid under the catalysis of dioxygenase,dehydrogenase,hydrolase,hydratase and aldolase encoded by lig gene cluster in USTBZA1,and finally entered into TCA cycle for complete degradation.