Molecular Mechanism Of Insecticide Carbofuran Degradation By Sphingobium Sp.strain CFD-1

Carbofuran is a highly toxic carbamate insecticide and an inhibitor of acetylcholinesterase,which is an important enzyme in the signal transmission process of the central nervous system.Therefore,carbofuran residues in the environment pose a potential threat to non-target organisms and humans.The study of the microbial degradation of carbofuran not only has important theoretical significance,but also has application prospects in the field of bioremediation.Some carbofuran-degrading microorganisms have been reported and their degradation characteristics have been studied.However,the research on degradation related genes mainly the carbamate hydrolase gene,which is responsible for the hydrolysis of carbofuran to carbofuran phenol,while the successive degradation pathway of carbofuran phenol and the involved genes are still unclear.The purpose of this study is to isolate the bacteria that can completely degrade carbofuran,to study the metabolic pathways and related genes and enzymes,and to elucidate the molecular mechanism of carbofuran degradation.The main results are as follows:1.Isolation and identification of carbofuran-degrading bacteria and its growth and degradation characteristicsA bacterial strain CFD-1 that can grow with carbofuran as the sole carbon source was isolated through the enrichment culturing method.And the strain was preliminary identified as Sphingobium sp.according to its morphological,physiological and biochemical characteristics and 16S r RNA gene polygenetic analysis.The optimum temperature and Ph for the degradation of carbofuran by strain CFD-1 were 30°C and 7.Under these conditions,strain CFD-1 can completely degrade 1.0 m M carbofuran within 14 h.The investigation on the degradation pathway showed that it first converts carbofuran into carbofuran phenol through the action of a hydrolase,then converts carbofuran phenol into4-hydroxycarbofuran phenol,then 4-hydroxycarbofuran phenol is subjected to the catalysis of a series of enzymes,yielding the carbon source material that can be used by strain CFD-1 for the growth.2.Cloning,expression and enzymatic characteristics of carbamate hydrolase gene cehACFD-1The carbamate hydrolase gene cehA_CFD-1 in strain CFD-1 was cloned by PCR,which is responsible for hydrolyzing carbofuran to carbofuran phenol.The cehA_CFD-1 was heterologously expressed,purified and its enzymatic properties were studied.CehA_CFD-1displayed maximal enzymatic activity at 40℃and p H 7.And the V_max,K_m and k_cat of CehA_CFD-1 for carbofuran were 0.017μM·s^-1·mg^-1,133.22±5.70μM,and 9.48±0.89 s^-1,respectively.His351,His353,His453 and His459 are the key amino acid sites of CehA_CFD-1,any mutation of them will cause it to lose the catalytic activity.CehA_CFD-1 can hydrolyze a variety of carbamate insecticides,and the catalytic activity is in the order of carbaryl>carbofuran>oxamyl>aldicarb>propoxur>isoprocarb,but it does not hydrolyze methomyl.Gene knockout and complementation experiments showed that cehA_CFD-1 is the only gene responsible for the hydrolysis of carbofuran in strain CFD-1.3.Study on the substrate preference of carbamate hydrolase CehAWith cehA_CFD-1 as the template,the carbamate hydrolase genes cehA_AC100,cehA_OXA20,cehA_KN65.2,cehA_CDS-1 from different strains are obtained by overlap PCR,and they were collectively referred to as cehAS（cehA stands for any one of cehAS）.The nucleotide sequence similarity of these cehAS is as high as 99%.They were heterologously expressed and purified to obtain pure CehAS enzymes.The catalytic activity of CehAS against bicyclic carbamate insecticides（carbofuran and carbaryl）,monocyclic carbamate insecticides（isoprocarb and propoxur）and linear carbamate insecticides（oxamyl and aldicarb）were compared,and the results showed that when amino acid at position 152amino acid residue is Phe,CehA shows a preference for carbaryl,whereas when it is Leu,the enzyme shows preference for carbofuran.Only residue 494 of CehA is a fatty amino acid（Ala/Ile）,while residue 570 is Thr,a hydroxyl amino acid.Alternatively,residue 494of CehA is Thr,while residue 570 is a fatty amino acid（Ala/Ile）.These enzymes show good catalytic activity towards monocyclic and linear carbamates.If the amino acid residues at both positions 494 and 570 of CehA are the hydroxy amino acid Thr,their activity against monocyclic and linear carbamates is very poor（difference in catalytic activity is about10-20 times）.Combining the analysis of the upstream and downstream IS sequences of cehA in different host strains,the environment in which the host strains were isolated from,the catalytic activity on different carbamate insecticides and the natural carbamate insecticide physostigmine.it is proposed that when comparing the catalytic activity of different CehAS enzymes against a given substrate,the best performing CehA often comes from a bacterial strain living in an environment that contains this substrate.4.Cloning,expression and enzymatic characteristics of carbofuran phenol monooxygenase gene cfdCBy sequencing and analyzing the genome of strain CFD-1,the gene cfdC encoding the oxidase component of carbofuran phenol monooxygenase was speculated.The results of gene knockout and complementation experiments show that cfdC was the only gene encoding carbofuran phenol hydroxylase oxidase component in strain CFD-1.Genomic analysis predicted the gene cfd R that encodes the reductase component that can cooperate with cfdC.Then they were heterologously expressed and purified.Studies on the enzymatic properties of CfdCR showed that it can use reducing power NADH but not NADPH,and prefers FMN as the cofactor in comparison with FAD.CfdCR displayed maximal enzymatic activity at 25℃and p H 5.CfdCR can hydroxylate carbofuran phenol to produce4-hydroxycarbofuran phenol,and can also hydroxylate 1-naphthol,2-naphthol and5-amino-1-naphthol.5.Cloning and functional identification of epoxide hydrolase gene cfd F and dioxygenase gene cfd EThrough the analysis of the upstream and downstream sequences of cfdC,a gene cluster named cfd,that may be involved in the degradation of carbofuran phenol was speculated.It includes the epoxide hydrolase gene cfd F and the cleavage dioxygenase gene cfd E,which were involved in the metabolism of 4-hydroxycarbofuran phenol.Through the knockout and complementation of genes cfd F and cfd E and identifying the metabolites produced during the degradation of carbofuran phenol by the knockout strains,it is determined that the enzyme encoded by gene cfd F is responsible for catalyzing the furan ring hydrolysis of 4-hydroxycarbofuran phenol,and the enzyme encoded by gene cfd E is responsible catalyzes the cleavage the benzene ring of the furan ring hydrolysis product.Although the epoxide hydrolase gene cfd F and the cleavage dioxygenase gene cfd E were heterologously expressed and purified,the substrates were not commercially available and unstable and difficult to prepare,and the enzyme function could not be verified in vitro.In summary,this study isolated a strain CFD-1 that can grow with carbofuran as the sole carbon source,and elucidated the degradation process of carbofuran in strain CFD-1from the level of genes and enzymes:Carbofuran was catalyzed by the enzyme CehA_CFD-1to hydrolyze and break the ester bond to generate carbofuran phenol,which was converted to 4-hydroxycarbofuran phenol under the hydroxylation of the two-component monooxygenase CfdCR;then the furan ring of 4-hydroxycarbofuran phenol was hydrolyzed under the action of epoxide hydrolase Cfd F,and the benzene ring of the hydrolysate is cleaved under the action of cleavage dioxygenase Cfd E,yielding the carbon source material that can be used by strain CFD-1 for the growth.Through the study of CehA substrate preference,an environmental behavior model revealing the horizontal transfer and evolution of the cehA gene in the environment is speculated.This study elucidated the molecular mechanism of carbofuran degradation by microorganisms for the first time,and provided a theoretical basis for microbial remediation of carbofuran pollution.