Cloning And Functional Analysis Of The Genes Encode Two-Component Monooxygenase MeaXY In Sphingomonads

Due to extensive use of chloroacetanilide herbicides over the past sixty years,bacteria have evolved catabolic pathways to mineralize these compounds.In the upstream pathway of microbial metabolism of amide herbicides,chloroacetanilide herbicides are transformed into the two common metabolites 2-methyl-6-ethylaniline(MEA)and 2,6-diethylaniline(DEA)through N-dealkylation and amide hydrolysis.The pathway downstream of MEA is initiated by the hydroxylation of aromatic rings followed by its conversion to a substrate for ring-cleavage after several steps.Most of the upstream genes have been identified.However,The gene responsible for the initial hydroxylation and opening of MEA are still unknown.We have carried out the following studys:1.Isolation of the MEA degradation-deficient mutant strain and analysis of its metabolic pathway.Sphingobium baderi DE-13 could completely degrade MEA and use it as a sole carbon source for growth.Through continuous transfer on LB medium,two MEA degradation-deficient mutant strains DE-13-E9 and DE-13-D2 were isolated.The DE-13-E9 mutant showed no degradation activity toward MEA,and another mutant strain DE-13-D2 can transform MEA to accumulate intermediate substance,losing the ability to completely mineralized MEA.HPLC-MS/MS identified the intermediates accumulated in MEA of mutant strain DE-13-D2:4-hydroxyl-2-methyl-6-ethylaniline(4-OH-MEA),2-methyl-6-ethyl-benzoquinone imine(MEBQI)and 2-methyl-6-ethyl-hydroquinone(MEHQ).Therefore,it is speculated that the metabolic initiation reaction of MEA is the formation of 4-OH-MEA.2.Prediction of MEA-catabolic genes based on comparative genomic analysis.To find the genes lost in those MEA degradation-deficient mutants,the complete genome of strain DE-13 and the draft genome of the mutant DE-13-E9 and DE-13-D2 were sequenced.The complete genome(4.58 Mb)of strain DE-13 revealed nine replicons,consisting of one circular chromosome and eight circular plasmids.The draft genome(4.48 Mb)of the DE-13-E9 mutant contained 159 contigs.The draft genome(4.42 Mb)of the DE-13-D2 mutant contained 84 contigs.Pairwise comparison results showed that compared to the wild type,the DE-13-E9 mutant lost a 14-kb fragment,which was named F-E9,the DE-13-D2 mutant lost a 44.6-kb fragment,which was named F-D2.The candidate genes involved in the degradation of MEA were then predicted in fragment F-E9.This fragment identified 13 open reading frames(ORFs>500 bp),one designated meaX was homologous to the genes encoding the oxygenase component of the two-component flavoprotein monooxygenase system.MeaX consists of 406 amino acid residues and shares 27%and 24.8%identities with HsaA from Rhodococcus sp.strain RHA1 and MeaA from Sphingobium sp.strain MEA3-1,respectively.3.Genetic complementation,heterologous expression of MeaXY and enzyme assays.The fragments containing meaX,orf4 and meaXorf4 genes were cloned into the vector pBBR1MCS2 and were introduced into S.baderi DE-13-E9 by triparental conjugative.S.baderi DE-13-E9 which containing plasmid pBmeaX and pBmeaXorf4 have the ability to transform MEA.Recombinant MeaX,MeaY and ORF4 were individually overexpressed in E.coli BL21.In vitro recombination experiments showed that MeaXY was a two component flavin dependent monooxygenase that catalyzes the hydroxylation of MEA and DEA by NADH and FMN cofactor.The Km values of MeaXY for MEA/DEA were 0.43±0.053 ?M,0.53±0.024 ?M,respectively.The corresponding kcat/Km values were 1.72±0.084 s-1·?M-1,1.49±0.12s-1·?M-1.MeaXY-hydroxylated DEA and MEA were purified using silica gel column chromatography and subjected to nuclear magnetic resonance(NMR)analysis.4.A preliminary study on the downstream ring cleavage gene of aromatic ring.The structure of 3-OH-MEHQ and 1,2,4-Benzenetriol are similar,it is speculated that the two open-loop approach are also similar,which was 1,2,4-benzenetriol 1,2-double-oxygen pathway.Analysis of the double oxygenase gene in the complete map of wild strain DE-13,we cloned a gene encoding a double-oxygenase of 1,2,4-Benzenetriol named meaC.After E.coli BL21 heterologous expression and purification of cobalt column,SDS-PAGE protein electrophoresis showed that the size of MeaC was about 34 kDa.In vitro recombinant MeaC with 1,2,4-Benzenetriol-1,2-double oxygenase activity and catechol-1,2-monooxygenase activity.Insertion mutation dioxygenase gene meaC in strain DE-13 by triparental conjugative.It was found that mutant strain DE-13(pJQ-TYmeaC)could't degrade 1,2,4-Benzenetriol as wild strain DE-13.The role of meaC will be validated in the future work.