Identification Of Carbofuran Hydrolase Gene And Monooxygenase Monooxygenase Gene In Sphingomonas Sp. CDS-1 Genome

Carbofuran,2,3-Dihydro-2,2-dimethyl-7-benzofuranol N-methylcarbamate,is acarbamate pesticide which has been used widely in agricultural production.It is not only an inhibitor of acetylcholinesterase,an important enzyme in signal transduction in mammalian central nervous system,but also an endocrine disruptor.Although the use of carbofuran is restricted now in China,its residues are still destroying the ec ological environment and threatening the human health.Microbial degradation is an effective measure to eliminate the environment pollution of carbofuran,but the mole cular mechanism of microbial degradation of carbofuran has not been clarified.Sphingomonas sp.CDS-1 is a carbofuran-degrading strain preserved in our laboratory and it can use carbofuran as the sole carbon source for growth.Previous experiments showed that the bacteria first hydrolyzed carbofuran to furanol under the action of a hydrolase,and then converted the furan phenol to a red metabolite under the action of an oxidase.However,the key genes in the degradation pathway of phenol have not been reported yet.In the present study,Sphingomonas sp.CDS-1 was used as the research material to analyze its genome sequence,identify key genes of carbofuran and furanol degradation,and to elucidate the degradation mechanism at the gene level.At the same time,the related genes cloned were recombined,expressed and proteins were purified and characterized.The main results are as follows:(1)Through analysed genome of strain CDS-1,we found that it had a similarity of 99%between the sequences of carbofuran hydrolase gene cehA in strains CDS-1 and carbaryl hydrolase gene cehA' in AC 100,and only one amino acid coded was different.However,Hashimoto M did not detect the degradation activity of carbaryl hydrolase CehA on carbofuran in strain AC 100.We obtained the carbaryl hydrolase gene cehA' by point mutation of carbofuran hydrolase gene cehA gene of strain CDS-1,carried out heterologous expression in Ecoli BL21 and detected the enzyme activity on carbofuran.We found that the carbaryl hydrolase CehA' also obtained after mutation still had a strong degradation activity.(2)We removed the 87 bases of carbofuran hydrolase gene cehA and the carbaryl hydrolase gene cehA ',which encoded the signal peptide,carried out heterologous expression and tested the degradation activity against carbofuran.Through the HPLC detection,it was found that thecarbofuran hydrolase CehA and the carbaryl hydrolase CehA',which removed the N-terminal signal peptide,still had strong degradation activity to carbofuran(3)Through compared genome of strain CDS-1 with published genome sketch of strain KN65.2,we found a furanol single monooxygenase gene cfdC with the highest similarity of 48%(NCBI).Through the knockout and complement of the cfdC gene of bacteria CDS-1,we found that the mutant CDQC,which was knocked out of the cfdC gene,lost the ability to degrade furanol,and the strain CDQC-C,which was restored cfdC gene,was able to degrade furanol.These results suggest that cfdC is the key gene responsible for the degradation of furanol in strain CDS-1.However,the gene cfdC could be expressed in E.coli,but its activity of degrading furanol was not detected.Thus,it was speculated that CfdC might require specific reductase component.(4)Through the analysis of the CDS-1 genome,we found that five possible orf,which encoded reductases paired with furanol monooxygenase CfdC,orf,l489,orf1586,orf3663,orf2902,orf1721 respectively.We obtained five recombinant expression plasmid by linking these five possible reductase genes to pET-29a carrier.The cfdC gene and T7 promoter were attached to the pBBR1MCS-5 vector to obtain the recombinant expression plasmid pBB-T7C containing cfdC gene and the T7 promoter.Then the five expression plasmids containing possible reductase gene were expressed respectively in E.coli BL21(DE3)with the pBB-T7C expression plasmid,then the whole cell catalysis for carbofuran phenol was carried out.Finally,two effective reductase genes,orf3663 and orf1721,were obtained.It was also shown that the single oxygenase CfdC indeed require the involvement of a reductase to degrade carbofuran phenol.(5)The furanol monooxygenase gene cfdC and the reductase gene orf3663 were expressed in E.coli BL21(DE3)respectively,and the expressed products were puri fied.An enzymatic reaction system was established to study their enzymatic properti es.In addition to monooxygenase and reductase,addition of cofactor FMN,NADH,was required in the reaction system.Through the identification of degradation prod ucts by LC-MS it was concluded that the product furan catalyzed by monooxygenas e CfdC in strain CDS-1 was 2,3-dihydro-2,2-dimethy1-4,7-dihydroxybenzofuran.