Escherichia Coli Dh10b And Stenotrophomonas Maltophilia R551-3 Total Metabolism Of Imidacloprid And Its Nitro Reduction Mechanism

This research mainly investigated the cometabolism of neonicotinoid insecticide imidacloprid (IMI) by Escherichia coli DH10B and the optimal IMI degradation conditions, then the corresponding IMI nitroreductase genes were knockouted by using red-mediated recombination system; at last, the plasmids containing the IMI nitroreductase genes were constructed and overexpressed.It was found that E.coli DH10B could degrade IMI, in the presence of sodium succinate as co-substrate, while not with sucrose as co-substrate. Mass spectrum indicated that E.coli DH10B converted IMI (=N-NO2) into the metabolite urea IMI (=0). Interestingly, as compared with S. maltophpilia strain R551-3 in the degradation of IMI, E.coli DHIOB did not degrade IMI under acid pH condition. After optimization of transformation conditions, the formation of urea IMI increased 92% and the degradation IMI increased 86%.The putative genes concerning IMI nitro reduction were further knockouted. First, menadione inhibition indicated that IMI degradation and nitroreduction to form urea IMI was inhibited, which implying that molybdoprotein are involved in the IMI degradation and nitro reduction. According to bioinformatics analysis, three genes YagR,XdhA and XdhD were selected for deletion by the Red/ET recombination system. It has been found that both the generation of urea IMI and the degradation of IMI have been dramaticially deduced when the YagR gene was knockouted. It can be explained that the YagR protein in E. coli DH10B may be the IMI nitroreductase. While the E. coli DH10B△XdhA did not reduce the IMI degradation and urea IMI formation. It indicates that XdhA protein is not the IMI nitroreuctase.S. maltophilia is the bacterium for studying on IMI metabolism. S. maltophilia R551-3 was determined the genome. In the presence of succinate as cosubstrate, S. maltophilia R551-3 metabolized IMI to urea, olefin metabolites; while with sucrose as cosubstrate, the IMI nitroreduction was inhibited. The cytoplasm of S. maltophilia R551-3 can transform IMI to urea IMI with succinate as cosubstrate, indicating that IMI nitroredutase is soluble protein. Therefore referenced the literatures and the genome of S. maltophilia R551-3, NQO, carbonyl reducates, or aldehyde oxidase(AOX) was selected for cloning and expression. We constructed successfully the expression plasmids of smal₂429,2638,3642,1569.0358,0139 and 1991 genes.