Cloning And Expression Of Key Genes Involved In The Degradation Of Carbofuran By Sphingomonas Sp.CDS-1

Carbofuran is a kind of carbamate pesticide which has been used widely.It is an inhibitor of cholinesterase,which is an important enzyme in the signal transmission process of central nervous system of mammals.So the carbofuran in the environment will pose a potential danger to the health of human beings.Microbial degradation is thought to be the main factor to eliminate carbofuran residues in the environment,but the current research on the degradation mechanism of carbofuran is inadequate,and the complete degradation pathway and the key genes is still unclear.Sphingomonas sp.CDS-1 is a carbofuran-degrading bacterium preserved in our laboratory and it can use carbofuran as the sole carbon source for growth.The previous research showed that carbofuran was initially hydrolyzed to carbofuran phenol by CDS-1,and then carbofuran phenol was converted to a red metabolite 3-(2-hydroxy-2-methylpropyl)cyclohexa-3,5-diene-1,2-dione.However,the genes involved in the degradation pathway have not been reported.In the present study,Sphingomonas sp.CDS-1 was used as the research material to clone the key genes of carbofuran degradation,and to elucidate the degradation mechanism at the gene level.The main results are as follows:(1)The mutant library of CDS-1 was constructed by the method of transposon Tn5 insertion mutagenesis and five mutants,named as CSM1,CSM2,CSM3,CSM4 and CSM5 were obtained.Among these mutants,CSM1,CSM2 and CSM3 could transform carbofuran to carbofuran phenol,but couldn't degrade carbofuran phenol,CSM4 and CSM5 were able to degrade carbofuran phenol,but not carbofuran.(2)By means of SEFA-PCR,the fragments on the upstream and downstream of the Tn5 inserted site in each mutant strain were amplified,which were sequenced and spliced.Then a 5463 bp fragment was combined from SEFA-PCR products of CSM1,CSM2 and CSM3 and a 8365 bp fragment was combined from the SEFA-PCR products of CSM4 and CSM5.(3)The analysis of the ORFs in the 5463 bp fragment showed that the Tn5 insertion sites in CSM2 and CSM3 were located in a putative monooxygenase gene(orfC)(the highest similarity is 48%),while the insertion site in CSM1 was located in a putative TetR family regulator gene(orfA)(the highest similarity is 42%)on the upstream of orfC.In addition,an unknown function gene(orfB)was situated between orfA and orfC.Referring to the degradation pathway of carbofuran,monooxygenase gene orfC was probably the key gene responsible for the degradation of carbofuran phenol.Similarly,the ORFs in the 8365 bp fragment were analyzed,and the Tn5 insertion sites in CSM4 and CSM5 were found to be located in a gene with 99%similarity to a reported esterase gene cehA,.Therefore cehA might be in charge of the conversion of carbofuran to carbofuran phenol.However,the specific function of orfC and cehA still required further research.(4)The cehA gene knockout strain CDKA of CDS-1 was constructed by the means of single crossover homologous recombination.Recombination strain CDKA showed the same characteristicas as mutant strains CSM4 and CSM5 and also lost the ability to convert carbofuran to carbofuran phenol.The cehA with its native promoter gene was cloned into a wide host vector pBBR1MCS-5 to construct the complementary plasmid pBB-cehA,which was introduced into CDKA,resulted in the functional complementation strain CDKA-A,it was able to degrade both carbofuran and carbofuran phenol,which was the same as strain CDS-1.Then the cehA gene was cloned into the expression vector pET-24b to construct the recombinant plasmid pET-AE,which was transformed into E.coli BL21 for inducible expression,the expression product was able to convert carbofuran to carbofuran phenol,the result demonstrated that cehA gene was responsible for the hydrolysis of carbofuran to carbofuran phenol.Thus far,the cehA gene was confirmed to be in charge of the conversion of carbofuran to carbofuran phenol in CDS-1.(5)Strain CDKC was constructed by the knockout of orfC gene in CDS-1,it showed the same characteristic as mutant strains CSM1,CSM2 and CSM3 and also lost the ability to degrade carbofuran phenol,but they could convert carbofuran to carbofuran phenol.The orfABC gene cluster containing its 283 bp upstream region as the possible promoter P1 was cloned into pBBR1MCS-5 to construct the complementary plasmid plasmid pBB-ABC,which was transformed into E.coli DH5a to construct recombinant strain E.coli(pBB-ABC),then pBB-ABC was introduced into CDKC by triparental conjugation,resulted in strain CDKC-ABC,it restored the ability of degrading carbofuran phenol.Similarly,orfBC containing P1 and orfC containing PI were ligated to pBBR1MCS-5,respectively,to construct recombinant plasmids pBB-BC and pBB-C,and recombinant strains E.coli(pBB-BC)and E.coli(pBB-C),then the recombinant plasmids pBB-BC and pBB-C were introduced into CDKC respectively by the same method as that of pBB-ABC,and the recombinant strains CDKC-BC and CDKC-C were obtained,they both regained the ability of degrading carbofuran phenol,indicating that orfC gene was the gene responsible for the degradation of carbofuran phenol,and the TetR family regulator gene orfA and the unknown function gene orfB were not required for the expression of orfC gene.The recombinant plasmid pBB-C was also introduced into Sphingomonas sp.RW1,Sphingobium sp.BHC-A and Pseudomonas putida KT2440 respectively,to construct recombinant strains.KT-C and E.coli(pBB-C)failed to get the ability of degrading carbofuran phenol,but strains RW-C and BHC-C obtained the ability of degrading carbofuran phenol and converted it to the red metabolite 3-(2-hydroxy-2-methylpropyl)cyclohexa-3,5-diene-1,2-dione.On the basis of these experimental evidences,we could determine that monooxygenase gene orfC was the gene responsible for the degradation of carbofuran phenol in CDS-1,but why the E.coli(pBB-ABC)and KTABC could not degrade carbofuran phenol,we speculated that they were both lack of a flavin dependent oxidoreductase for providing the monooxygenase orfC with FADH2,but Sphingomonas sp.RW1 and Sphingobium sp.BHC-A had a close genetic relationship with Sphingomonas sp.CDS-1,so they had the flavin dependent oxidoreductase in their cells and could provide FADH2 for the monooxygenase orfC,and the recombinant strains RW-C and BHC-C could convert carbofuran phenol into the red metabolite 3-(2-hydroxy-2-methylpropyl)cyclohexa-3,5-diene-1,2-dione.However,this flavin dependent oxidoreductase gene in CDS-1 still requires further research.