Study On The Pathway And Mechanism Of Imidacloprid Metabolism By Pseudoxanthomonas CGMCC 6648 And Escherichia Coli DH10B

A large number of long-term applications of imidacloprid(IMI)have produced the pesticide residue in the environments and therefore caused the potential threat to human health.Microbial metabolism is one of the important pathways of IMI degradation in environmnets and so far,two pathways involving hydroxylation and nitroreduction have been found.In hydroxylation pathway,the main metabolites are 5-hydroxy IMI and olefin IMI.The insecticidal activity of 5-hydroxy IMI is lower than IMI and the insecticide activity of olefin IMI is about 19 times higher than IMI.In nitroreduction pathway,the metabolite guanidine IMI is harmful to mammals as compare with IMI.Hence,it is indispensable for us to explore the microbial metabolism of IMI,which is conducive to repair the pollution of IMI in environment as well as decrease the amount of the application amount of IMI and reduce environmental toxicity.The author's laboratory has screened a bacterium Pseudoxanthomonas indica CGMCC 6648 which have a high IMI degradation ability.In present study,the author continued to explore the metabolic pathway of IMI in P.indica CGMCC 6648.P.putida CGMCC 6648 had different IMI degradation ability and metabolic pathway when using glucose and sodium pyruvate as co-metabolic substrate respectively.Using glucose as co-metabolic substrate and the pre-cultivated time was 18 h,the main metabolites of transformation of IMI by the resting cells of CGMCC 6648 were 5-hydroxy IMI and olefin IMI and the metabolite nitroso IMI was not detected,and the amount of 5-hydroxy IMI and olefin IMI were 260mg/L,7.78mg/L respectively.When Use sodium pyruvate as co-metabolic substrate and the pre-cultivated time was 18 h,the main metabolites were 5-hydroxy IMI?olefin IMI and nitroso IMI,and the production of 5-hydroxy IMI?olefin IMI and nitroso IMI were 152.93 mg/L,42.9mg/L and 0.48 mg/L respectively.The maximum amount of nitroso IMI was 2.22 mg/L when the pre-cultivate time was 24 h.In the genomic DNA of P.indica CGMCC 6648,there exists 13 monooxygenase genes,they are as follows:0479(aspartyl-asparaginyl beta-hydroxylase)?0642(2-octaprenyl-3-methyl-6-methoxy-1?4-benzoquinol hydroxylase)?0826(alkanal monooxygenase)?0864(UbiH/UbiF/VisC/COQ6 family ubiquinone biosynthesis hydroxylase)?1219(Putative monooxygenase2-polyprenyl-6-methoxyphenol hydroxylase)?1812(molybdopterin-binding aldehyde oxidase and xanthine dehydrogenase)?2337(vanillate monooxygenase)?2450(phenylalanine 4-monooxygenase)?2490(phenol hydroxylase)?3100(2-octaprenyl-3-methyl-6-methoxy-1?4-benzoquinol hydroxylase)?3337(2-octaprenyl-6-methoxyphenyl hydroxylase)?3675(antibiotic biosynthesis monooxygenase)?3825(nitronate monooxygenase)?kynurenine-3-monooxygenase(this enzyme has been previously cloned and over-expressed by the former graduate student Zhai shan).The present studies carried out the cloning and heterologous expression of these oxygenase genes in Escherichia coli Rosetta.These recombinant strains were prepared for the resting cells and sodium pyruvate was used as co-substrate,the transformation was conducted for 4 d,in 37? and at speed of 220 rpm in rotary shaker.HPLC analysis indicated that the recombinant E.coli over-expressing 0864?2450?and 0479 had 1.27?1.49?and 1.75-fold higher IMI hydroxy activity than the control of host without harboring heterogenous gene,respectively.The protein 2450 is phenylalanine 4-monooxygenase and it was mainlythe soluble fraction by SDS-PAGE analysis.The over-expressed protein 2450 with histag was purified by by Ni2+ chelating His-Tag resin.The purified protein was primarily examined its monooyxgenase activity by using phenylalanine as substrate and HPLC analysis proved its monooxygenase activity.The transformation of IMI by.the cell free extract of E.coli expressing the protein 2450 was 1.8 times higher than the control without 2450 expression.The purified protein of 2450 was checked its IMI hydroxylation,5-hydroxy IMI was examined,whereas the control without enzyme addition.This was due to the phenylalanine hydroxylase 2450 needs reduced neopterin and iron.The Fe2+alone also can hydroxylate IMI to 5-hydroxy IMI.In the previous study,we found that the bacterial hydroxylation of IMI needs NADH or NADPH as co-factor,and NADPH has two folds higher effeicacy of IMI hydroxylation than NADH.Except the glucose-6-phosphate dehydrogenase of HMP pathway,the two enzymes MaeA and MaeB in malic acid and pyruvic acid conversion pathway also regenerate NADPH.In present study,the homologous recombination system of Red/ET was applied to knock out maeA?maeB and the double knockout of maeA and maeB.Double gene knockouted strains of maeA and maeB has a lower IMI reduction than the control without gene knockout as well as the non-trace knockout strain.Amount of IMI degradation of double knockout strains by 24.2 mg/L in the control group decreased to 11.9 mg/L(p<0.05).In conclusion,the current study focused on the metabolism of the largest pesticide IMI by P.inidca CGMCC 6648 and found that this bacterium has the IMI nitroreduction pathway;and searched 13 morioxygenase genes and an aldehyde oxidase gene in the genomic DNA of CGMCC 6648.The twelve monooxygenase coding genes were cloned and over-expressed into the hosting strain E.coli Rosetta and examined the IMI hydroxylation of the recombinant strains.The three mutants with the knockouted maeA and maeB were constructed and examined its effects on IMI metabolism.The present studies will help to understand the microbial metabolism of IMI.