Study On The Molecular Mechanism Of Carbendazim Degradation By Rhodococcus Sp.L1

Carbendazim is a highly effective broad-spectrum benzimidazole fungicide,it can control a variety of fungal diseases.With the widespread application of carbendazim,the residues of carbendazim can be detected in water,soil and agricultural products,which poses a threat to the environment as well as human health.Microbial degradation is the main way to eliminate carbendazim residues in the environment.Although many carbendazim-degrading strains have been isolated and their degradation characteristics have been studied,the research related to their degradation gene mainly focuses on the carbendazim hydrolase gene which responsible for the hydrolysis of carbendazim to 2-aminobenzimidazole,and the complete degradation mechanism of carbendazim has not been elucidated yet.The purpose of this study is to isolate the strain which can completely degrade carbendazim,to study the related genes degrading carbendazim,and to analyze the microbial degradation mechanism of carbendazim.The main results are as follows:1.Isolation and identification of carbendazim-degrading bacteria and its degradation characteristics.By the method of enrichment culturing,a carbendazim-degrading strain was isolated,it was preliminarily identified as Rhodococcus sp.and named strain L1according to the morphological characteristics,physiological and biochemical characteristics and the analysis of its 16S r RNA sequence.The results of degradation characteristics of carbendazim showed that the strain L1 could use carbendazim as the sole carbon and nitrogen source for growth,and it could degrade up to 95%of 0.52 m M carbendazim within 36 hours.The optimum temperature and p H for the strain to degrade carbendazim were 30℃and 7.0,respectively.The degradation pathway of carbendazim in strain L1 was investigated by HPLC and MS/MS,on this basis,the pathway was preliminarily proposed as:strain L1first hydrolyzed carbendazim to 2-aminobenzimidazole,which was further converted to 2-hydroxybenzimidazole and then subjected to ring cleavage,yielding the carbon source for the growth of strain L1.2.Cloning and expression of carbendazim hydrolase gene cbm A and the study of its evolutionary relationship.The carbendazim hydrolase gene cbm A was cloned from strain L1 by PCR.It is1431 bp in length and encodes a hydrolase of 476 amino acids.The analysis of its amino acid sequence showed that Cbm A belongs to AS signature amidase.The cbm A gene was heterologously expressed in E.coli,and Cbm A was purified by Strep Tactin Sepharose FF.Cbm A could hydrolyze carbendazim to 2-aminobenzimidazole.The optimum temperature and p H for the hydrolysis of carbendazim by Cbm A were 25℃and 7,respectively,and the K_mof Cbm A was 6.63±0.66μM.At the concentration of1 m M,Li⁺,Mg²⁺,Ca²⁺have no significant effect on the activity of Cbm A;Fe²⁺,Fe³⁺,Mn²⁺,and Ni²⁺slightly inhibit the activity of Cbm A;Zn²⁺,Cu²⁺and Hg²⁺inhibit the activity of Cbm A,especially Hg²⁺,which completely inhibits the activity of Cbm A;while Co²⁺slightly promotes the activity of Cbm A.Cbm A has a relatively narrow substrate spectrum.In addition to carbendazim,it could only hydrolyze propanil and 4-nitroacetanilide,but the corresponding activity is higher than that of carbendazim.By the method of overlap extension PCR,the putative conserved catalytic triad Ser157-Ser181-Lys82 in Cbm A was mutated to alanine（Ala）.After heterologous expression and purification,the hydrolysis activity of the mutants against carbendazim was determined,and the result showed that all the mutants had no hydrolysis activity against carbendazim,which proved that Cbm A is an amidase signature enzyme containing the highly conserved catalytic triad Ser-Ser-Lys.After aligning the amino acid sequence of Cbm A with proteins in NR database,the result showed that proteins with a similarity of more than 80%to Cbm A were from Rhodococcus sp..Therefore,the gene cbm A was speculated to be conserved in Rhodococcus sp.,and it could also be cloned from other strains of Rhodococcus sp.which were stocked previously in our lab such as Rhodococcus qingshengii djl-6、Rhodococcus qingshengii YL-1、Rhodococcus erythropolis PR4 and Rhodococcus sp.WH99.The gene cbm A in these strains were heterologously expressed in E.coli and purified by Strep Tactin Sepharose FF.It was showed that only Cbm A in Rhodococcus qingshengii djl-6、Rhodococcus qingshengii YL-1 and Rhodococcus erythropolis PR4could hydrolyze carbendazim to 2-aminobenzimidazole,and Cbm A in these strains have more than 90%similarity to that of strain L1,the K_m of Cbm A in Rhodococcus qingshengii djl-6,Rhodococcus qingshengii YL-1 and Rhodococcus erythropolis PR4were 3.10±0.38μM,3.71±0.71μM and 3.47±0.47μM,respectively.But the Cbm A in Rhodococcus sp.WH99 could not hydrolyze carbendazim to 2-AB,as its amino acid sequence is only 77.87%similar to that in strain L1.Therefore,although the gene cbm A is conserved in Rhodococcus sp.,it loses the hydrolysis activity of carbendazim when its protein has low similarity with that of strain L1.The phylogenetic trees were constructed with the 16S r RNA sequence of these strains and the amino acid sequence of Cbm A,it was showed that the two trees had the same clustering relationship,indicating that Cbm A is conserved in Rhodococcus sp.and its amino acid sequence presents the same evolutionary trend with its 16S r RNA sequence.3.Transcriptome analysis of Rhodococcus sp.L1 and cloning of 2-aminobenzimidazole degradation genes.Strain L1 was pre-cultured with 0.38 m M 2-aminobenzimidazole and 0.38 m M glucose,respectively.It was found that the cells of pre-cultured with glucose had a lag period of about 5 h in degrading 2-aminobenzimidazole,while there is no such phenomenon in the strain pre-cultured with 2-aminobenzimidazole and it could completely degrade 0.38 m M 2-aminobenzimidazole in 6 h,indicating that the degradation of 2-aminobenzimidazole in strain L1 was induced by the substrate.The transcriptomes of strain L1 under induced and uninduced conditions were measured,and the differentially expressed genes were analyzed.Among the 2121 up-regulated genes,a gene cluster with an up-regulation fold greater than 16 was found.These genes were cloned into p RESQ and then transformed into strain WH99 that does not have 2-aminobenzimidazole degradation activity to construct recombinant strains,but none of them showed degradation activity against 2-aminobenzimidazole.However,after constructing the recombinant strain with gene cluster orf387-orf390 by the same way,it could degrade 2-aminobenzimidazole to 2-hydroxybenzimidazole.Based on this,it is speculated that gene cluster of orf387-orf390 is responsible for the degradation of 2-aminobenzimidazole to 2-hydroxybenzimidazole,but whether one or more genes are specifically responsible for this function has not yet been determined,and further research is needed.