E. Coli DH10B Co-metabolizes Imidacloprid And Separation And Purification Of 5-hydroxy Imidacloprid Dehydratase From Stenotrophomonas Maltophilia CGMCC 1.1788

Neonicotinoid insecticides imidacloprid?IMI?can be transformed by Stenotrophomonas maltophilia CGMCC 1.1788 via hydroxylation pathway.In the heterologous expression of the genes from Stenotrophomonas maltophilia CGMCC 1.1788,we found that Escherichia coli DH10B can also co-metabolically transform IMI.In this paper,we primarily explored the biotransformation pathway of IMI by Escherichia coli DH10B by HPLC and LC-MS analysis.Then the monooxygenase coding genes from DH10B genomic DNA were knockouted by Red/ET recombination technology.The conditional lethal monooxygenase coding genes were cloned and over-expressed.The above constructed strains were evaluated its biotransformation of IMI and compared with the wild DH10B.The 5-hydroxy IMI dehydratases from E.coli DH10B and S.maltophilia CGMCC 1.1788 were separated by salting out using ammonium sulfate precipitation and then the precipitated proteins were further purified by gel chromatography.HPLC and LC/MS analysis showed that the products of cometabolism of IMI by E.coli DH10B are 5-hydroxy IMI and olefin IMI respectively,which indicating that E.coli DH10B has the same IMI metabolic pathway with S.maltophilia.Time course of transformation of IMI by E.coli DH10B indicated that DH10B degraded 1.16 mmol/L of IMI with degradation rate of 67%,and formed 0.23 mmol/L of 5-hydroxy IMI and 0.17 mmol/L of olefin IMI in 6 d and in the presence of 100 mmol/L succinate.There are 13 of monooxygenase and hydroxylase coding genes in the genomic DNA of E.coli DH10B?genbank database accession number:NC₀10473?.The monooxygenase coding gene were knockouted by Red/ET recombination technology,of which replaced the target gene with sacB and neo from the plasmid PSNR mediated by the plasmid pSC101-BAD-gbaA.The hydroxylation of IMI by the mutated strains was compared with the wild DH10B by HPLC analysis and the results indicated that the DH10B mutated with rutA,ssuD,mhpA,visC,ybiX,ygiN,yqeB,yqeC,ycfD and ydhR respectively did not reduce the amount of formed 5-hydroxy IMI.The conditional lethal ubiB,ubiH and ubiF were cloned and over-expressed in E.coli Rosetta respectively.However,the successful over-expressed ubiB,ubiH and ubiF did not increase the amount of formed 5-hydroxy IMI.The above results indicated that the selected monooxygenases have no function of IMI hydroxylation.The author further conducted the separation and purification of 5-hydroxy IMI dehydratase.The cell of E.coli DH10B was broken by French press,and the cell-free extracts were salted out using ammonium sulfate precipitation to sediment proteins.The proteins were examined its activity of 5-hydroxy IMI dehydratase by using HPLC to examine the conversion of 5-hydroxy IMI to olefin IMI.All the collected proteins from E.coli DH10B had no 5-hydroxy IMI dehydratase activities.However,it is interesting that the precipitated proteins salted out by 40%ammonium sulfate can convert 5-hydroxy IMI to two new products peaked with retention time of 3.1 and 5.6 min respectively.LC-MS analysis indicated that the two metabolites were the product cleavaged the methylene brige of IMI and its adduct with succinic acid.The CGMCC 1.1788 proteins sediments collected at the concentration of 20%and 40%of ammonium sulfate can transform 5-hydroxy IMI into olefin IMI.The proteins collected at the concentration of 20%ammonium sulfate were further separated by gel chromatography and 7 protein peaks were observed.HPLC analysis of the olefin IMI formed by the peaked proteins indicated that the proteins at peak 3 had the 5-hydroxy IMI dehydratase activity.The 5-hydroxy IMI dehydratase in the peak 3 is now purified by SDS-PAGE and identified by MALDI-TOF/TOF mass analysis.