Functional Identification Of Key Genes Involved In The Triclocarban Catabolism By Sphingomonas Sp.YL-JM2C

Triclocarban is a type of polychlorinated biphenyl aromatic compound,which is widely utilized as an antibacterial agent in daily life.Triclocarban is regarded as one of pollutants and attracts much attention because of its strong stability,difficult degradability and substantially accumulation in the environment.Currently,there are few reports about microbial degradation of triclocarban.Sphingomonas sp.YL-JM2 C is the first one capable of degrading triclocarban.However,the degradation pathway of triclocarban by strain YL-JM2 C is still only speculated at chemical level,and there is no relevant research at molecular biological and biochemical level.In this study,through Bioscreen C plate-reader system,the growth characteristics of triclocarban degrading by strain YL-JM2 C was explored: the optimal triclocarban concentration was 4 mg/L-8 mg/L,and the optimal pH value was pH=7-7.5 for its growth.It was found out that,as a carbon source,triclocarban was better utilized by strain YL-JM2 C in the optimized R2 A minimal medium which contains 0.01% yeast extract.Through biotransformation experiment with strain YL-JM2 C,it was proved the strain YL-JM2 C could degrade triclocarban with 68.77% decrease after 10 hours.During the study of downstream pathway of triclocarban,three putative catechol 1,2-dioxygenase genes stcA1,stcA2,stcA3 and three putative catechol 2,3-dioxygenase genes stcE1,stcE2,stcE3 were found out through bioinformatics analysis.All six genes were heterologous expressed in E.coli and the extract of E.coli containing stcA1,stcA2,stcA3,stcE1,stcE2 exhibited enzymatic activity to catechol as well as its derivative chlorocatechol.Kinetic parameter was evaluated through protein concentration examination and enzyme activity.The transcriptional response of the above five catechol dioxygenase genes was analyzed by Real-time PCR.The result showed that all five catechol dioxygenase genes were constitutive expressed.In addition,basing on bioinformatics analysis,five putative amidase genes,which may be involved in the upstream degradation pathway of triclocarban,were found out.According to color reaction,these five putative amidase maybe catalyze hydrolysis reaction of triclocarban.In this study,as many as five functional catechol dioxygenases from a single strain were revealed,which has not been reported previously.StcA1,StcA2 and StcA3 belong to type II catechol 1,2-dioxygenase,while StcE1 and StcE2 are treated as a novel type of catechol 2,3-dioxygenase.And five putative amidase genes may be involved in the hydrolysis reaction of triclocarban,although further clarification is required.This study provides a theoretical basis for the further study of triclocarban metabolic mechanism and the bioremediation of triclocarban-contaminated environment.