Gene Cloning And Characterization Of The Tetrahydrofolate-dependent Methyltransferase Catalyzing The O-demethylation Of Dicamba In Sphingomonas Sp.Ndbn-20

Dicamba(3,6-dichloro-2-methoxybenzoic acid)is a kind of synthetic benzoic acid herbicide,which has been widely used in a variety of crops to control broadleaf weeds.In addition,due to its low cost,high efficiency,broad weed control spectrum,environmental friendliness and low mammalian toxicity,dicamba is considered to be a suitable target herbicide for engineering of herbicide-resistant transgenic crops.Recently,the biotechnology company Monsanto has successfully developed dicamba-resistant soybean and cotton by introducing a dicamba monooxygenase(DMO)gene.These genetically modified(GM)crops have received regulatory approval and were planned to enter commercial use in 2015.The increasing usage of dicamba has raised concerns regarding its ecotoxicity,dissipation and biodegradation in the environment.Furthermore,the dicamba degradation or detoxification gene has potential applications in the construction of herbicide-resistant transgenic crops.Dicamba is chemically stable,but it does not persist in soil and water,suggesting that its dissipation in the environment is biologically mediated.To date,several microorganisms capable of degrading dicamba have been described.The degradation of dicamba in bacteria is initiated by O-demethylation to generate 3,6-dichlorosalicylic acid(DCSA),a compound without herbicidal activity.There were two kinds of O-demethylases have been reported:DMO in aerobic bacterium Stenotrophomonas maltophilia DI-6 and a tetrahydrofolate(THF)-dependent demethylase system(Mtv)in anaerobic bacterium Moorella thermoacetica.In our previous study,aerobic dicamba-degrading bacterium Sphingomonas sp.Ndbn-20 was isolated from compost sample.The strain degrades dicamba initially via O-demethylation,which catalyzed by an unknown THF-dependent demethylase.In this study,a novel THF-dependent dicamba O-demethylation mechnism was identified in the strain.The unpublished data obtained by our partner Beijing DBN Technology Group Co.,Ltd showed that Arabidopsis thaliana introduced with the THF-dependent dicamba O-demethylase genes dmt50 or dmt66 acquired obvious resistance to dicamba,suggesting that genes dmt50 and dmt66 have potential applications in the engineering of transgenic herbicide-resistant crops.Furthermore,our studies also revealed that Mthfr66 is a multifunctional enzyme,which has dicamba demethylase activity and 5-methyl-THF reductase activity.The main results were showed as following:1.Gene cloning of THF-dependent dicamba demethylase and identification of its functionThe draft genome of Sphingomonas sp.Ndbn-20 was sequenced by Illumina Hiseq 2000 technology.The genome was resolved into 127 contigs consisting of 5.35Mb,and 4911 genes were predicted.To identify the putative THF-dependent dicamba demethylase gene,the amino acid sequences of the reported four kinds of O-demethylases such as cytochrome P450(e.g.CYP51A1),rieske non heme iron oxidase(e.g.DMO),tetrahydrofolate-dependent demethylase(e.g.DesA)and(e.g.MtvB)were used for BLASTP searches of the genome.This screen resulted in the identification of three putative THF-dependent methyltransferase gene clusters,which were located in scaffold 02 scaffold 50 and scaffold 66,respectively.The methyltransferase in scaffold 02 showed the highest homology to LigM(43%identity)and DesA(42%identity),respectively;whereas the methyltransferase in scaffold 66 shared 46%and 45%identities to LigM and DesA,respectively.ORF analysis revealed that three genes exhibiting similarities with mthfr(encoding 5,10-methylene-THF reductase),dhc(encoding a bifunctional enzyme 5,10-methenyl-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase)and mthc(formyl-THF deformylase)were existed downstream of each methyltransferase gene.The three genes were reported to involve in THF metabolism.The three putative methyltransferase genes or gene clusters were ligated into the broad-host-range vector pBBR1MCS-2,and introduced into Sphingonomas sp.DC-6,which lacks dicamba-degrading ability,respectively.Whole-cell transformation experiments showed that the recombinants DC-6pBBRdmt02c(containing methyltransferase gene cluster from scaffold 02)and DC-6pBBRdmt02(only containing methyltransferase gene from scaffold 02)could not degrade dicamba,whereas DC-6pBBRdmt50c(containing methyltransferase gene cluster from scaffold 50)and DC-6pBBRdmt66c(containing methyltransferase gene cluster from scaffold 66)acquired the abilities to convert dicamba,suggesting that methyltransferase gene cluster in scaffold 50 and scaffold 66 was involved in the O-demethylation of dicamba;In addition,transformant DC-6pBBRdmt50(only containing the methyltransferase gene from scaffold 50)and DC-6pBBRdmt66(only containing the methyltransferase gene from scaffold 66)could also convert dicamba,but was much slower than DC-6pBBRdmt50c and DC-6pBBRdmt66c,indicating that genes mthfr,dhc and mthc,which involved in THF metabolism,play important role in dicamba O-demethylation in vivo.Real-time quantitative PCR analysis showed that the transcriptions of dmt,mthfr,mthc and dhc in scaffold 50 and scaffold 66 were detected in conditions with or without dicamba induction,and there were no obvious increases in the transcription levels of these genes under inducing dicamba condition,indicating that the transcription of the dmt operon was constitutive.2.Expression,purification and characterization of Dmt50 and Dmt66The three methyltransferase genes in scaffold 02,scaffold 50 and scaffold 66 were cloned into pET 29a(+)and expressed in E.coli BL21(DE3),respectively.The recombinant proteins were purified by Ni-affinity chromatography.Enzymatic assays showed that the methyltransferases from scaffold 50 and scaffold 66,but not scaffold 02,could convert dicamba to DCSA in the presence of THF;simultaneously,5-methyl-THF was identified as another product by HPLC and MS/MS analysis.Thus,the methyltransferase gene in scaffold 50 and scaffold 66 were designated as dmt50(dicamba methyl transferase)and dmt66 in this study.Dmt66 was severely inhibited by the heavy metal ions Ni2+,Hg2+,Pb2+,Cr2+ and Ag+ but not obviously affected by the monovalent cations K+,Na+,and Li+ or the divalent cations Fe2+ and Mg2+.The divalent cation-chelating agent EDTA did not inhibit the demethylase activity.Demethylase activity was detected ranging from 4 to 45? and at pH values ranging from 5.0 to 10.0,with the highest activity being detected at 30? and pH 8.0.The purified Dmt50 and Dmt66 had a specific activities of 72±5 nmol-1 min-1mg and 63±7 nmol-1 min-1mg towards dicamba,respectively.Dmt66 was feedback inhibited by product 5-methyl-THF,but not DCSA.3.Expression,purification and characterization of 5,10-methylene-THF reductase Mthfr66The 5,10-methylene-THF reductase gene in scaffold 66 was expressed using pET 29a(+)system,and the recombinant proteins were purified by Ni-affinity chromatography.Enzymatic assays showed that Mthfr66 was a multifunctional enzyme and showed the 5-methyl-THF reductase activity.Whereas other four Mthfr from Sphingobium quisquiliarum DC-2,Sphingobium jiangsuense BA-3 and Escherichia coli K-12 had no dicamba O-demethylase activity,and only showed 5-methyl-THF reductase activities in the presence of menadione.The activity of Mthfr66 was severely inhibited by the heavy metal ions Zn2+,Ag+,Pb2+,Hg2+,Cr2+ and Ni2+,but not ob-viously affected by the monovalent cations K+,Na+,and Li+ or the divalent cations Fe2+ and Mg2+.The highest activity of Mthfr66 being detected at 30? and pH 8.0.