【作者】 李娜；

【导师】 何健；

【作者基本信息】 南京农业大学 ， 微生物学， 2018， 博士

【摘要】 3,6-二氯水杨酸(3,6-dichlorosalicylate,3,6-DCSA)是除草剂麦草畏微生物降解的脱甲基产物。麦草畏(3,6-二氯-2-甲氧基苯甲酸)是一种广谱高效的激素型除草剂,广泛应用于玉米、高粱和小麦等农田中杂草的防治,特别是在美国孟山都生物技术公司构建的抗麦草畏的转基因作物大规模推广种植后,麦草畏年使用量从1.5万吨急剧增加到6.0万吨以上。因此有必要深入研究麦草畏及其降解中间产物在环境中的迁移、转化和微生物降解机制,为评估麦草畏规模化应用后的环境行为和生态安全性提供理论依据。已有研究表明,微生物代谢是环境中麦草畏降解的主要途径。目前国内外已分离到的菌株降解麦草畏的起始步骤均为脱甲基,生成无除草活性的产物3,6-DCSA。3,6-DCSA为有毒难降解氯代芳香化合物,在土壤和农产品中累积对生态环境和人类健康具有潜在的危害,然而目前3,6-DCSA的降解过程以及参与降解的关键基因和酶还未见报道,因此从基因和酶水平阐明3,6-DCSA的微生物降解代谢机制具有非常重要的理论和实际应用价值。本论文以麦草畏降解菌株Rhizorhabdus dicambivorans Ndbn-20为研究材料,筛选得到一株不能降解3,6-DCSA的突变菌株,通过对野生株和突变株基因组比较分析、降解酶功能验证、降解基因敲除、转录研究以及代谢产物鉴定,我们揭示了 3,6-DCSA微生物降解过程及参与的基因和酶。本论文为深入研究麦草畏的降解代谢机制及环境行为提供了理论依据。取得的主要研究结果如下:1、突变株筛选与3,6-DCSA降解基因簇的初步鉴定菌株Ndbn-20在1/5LB(不添加麦草畏和3,6-DCSA)培养基中连续传30代,获得一株失去3,6-DCSA降解能力的突变株Ndbn-20m。基因组比对分析结果表明突变株Ndbn-20m的染色体DNA上丢失了一个约64.1Kb的大片段。该片段上有一个基因簇dsmR1DABCEFGR2,其中dsmABC编码一个三组分的细胞色素P450单加氧酶,dsmD,dsmE,dsmF和dsmG分别编码龙胆酸双加氧酶、富马酰丙酮酸水解酶、转运蛋白和马来酰乙酸还原酶,dsmR1和dsmR2分别编码LuxR家族和IcIR家族调控蛋白。转录研究结果表明dsmR1DABCEFG共8个基因在一个转录单元上,且3,6-DCSA显著诱导基因簇dsm中各基因的转录。而且在菌株Ndbn-20的基因组中还发现另外一个受到3,6-DCSA诱导的基因簇,包括一个龙胆酸双加氧酶基因gtdA副和一个谷胱甘肽依赖型脱氯酶基因dsmH2。2、三组分细胞色素P450单加氧酶DsmABC负责3,6-DCSA的羟基化DsmA与一些催化抗生素羟基化的细胞色素P450单加氧酶具有30%～36%相似性,在细胞色素P450系统进化树中,DsmA形成一个独立的分支。DsmB和DsmC分别为铁氧还蛋白和铁氧还蛋白还原酶,组成电子传递链。将dsmA和dsmABC分别连接到广宿主载体pBBR1MCS-2并导入不能降解3,6-DCSA的菌株Sphingobium quisquiliarum DC-2 中,重组菌株 DC-2(pBBR-dsmABC)获得 了降解 3,6-DCSA 的能力,并生成一个羟基化产物;而只表达氧化酶组分DsmA的重组菌株DC-2(pBBR-dsmA)不能降解3,6-DCSA。采用同源重组单交换技术获得dsmA插入突变的突变株Ndbn-20ΔdsmA,该突变株失去了对3,6-DCSA的降解能力,而回补dsmA后该突变株恢复了降解3,6-DCSA的能力。采用UHPLC/MS和X射线单晶衍射的方法鉴定了 3,6-DCSA的羟基化产物是3,6-二氯龙胆酸(3,6-DCGA),表明羟基化位置为苯环上5-碳原子,DsmABC 为 3,6-DCSA5-羟化酶。3、谷胱甘肽依赖型脱氯酶DsmH2负责3,6-DCGA的脱氯菌株Ndbn-20的粗酶只有在添加谷胱甘肽(GSH)时具有3,6-DCGA脱氯酶活,推测3,6-DCGA的降解是在谷胱甘肽S-转移酶(GST)催化下脱氯。通过基因组比对分析发现在菌株Ndbn-20基因组中有两个GST基因dsmH1和dsmH2。酶学研究结果表明,DsmH1和DsmH2都能够催化3,6-DCGA脱氯,且DsmH2的3,6-DCGA脱氯酶活远高于DsmH1。基因转录和敲除结果表明在菌株Ndbn-20中,负责3,6-DCGA脱氯的是DsmH2,而不是DsmH1。核磁氢谱分析结果表明3,6-DCGA脱氯产物为3-氯龙胆酸。另外,通过系统发育和功能分析,将这四个细菌来源的脱氯酶PcpC,LinD,DsmH1和DsmH2划分为一个新的GST类群,命名为‘eta’。4、龙胆酸1,2-双加氧酶DsmD主要负责3-氯龙胆酸开环在菌株Ndbn-20中有两个龙胆酸1,2-双加氧酶基因dsmD和gtdA。酶学结果表明,DsmD和GtdA均能降解龙胆酸和3-氯龙胆酸。DsmD对3-氯龙胆酸的降解效率远高于GtdA降解3-氯龙胆酸的速率,且高于其降解龙胆酸的速率;GtdA降解龙胆酸的速率高于降解3-氯龙胆酸的速率,表明DsmD的最适底物为3-氯龙胆酸,而GtdA的最适底物为龙胆酸。基因转录和敲除结果表明在菌株Ndbn-20中,DsmD主要负责3,6-DCGA脱氯产物3-氯龙胆酸的开环降解,而GtdA主要负责龙胆酸的开环降解。5、DsmG催化2-氯马来酰丙酮酸还原脱氯dsmG与马来酰乙酸还原酶TfdF(P27137.1)的氨基酸序列具有最高的相似度(39%)。TfdF能够催化2-氯马来酰乙酸还原脱氯,而3-氯龙胆酸的开环产物2-氯马来酰丙酮酸的结构与2-氯马来酰乙酸相似,因此推测DsmG具有2-氯马来酰丙酮酸脱氯活性。酶活实验表明外源表达纯化的DsmG能够以NADH为还原力,将2-氯马来酰丙酮酸还原脱氯生成马来酰丙酮酸。以上研究从基因和酶水平上阐明了菌株Ndbn-20中3,6-DCSA的降解过程:3,6-DCSA在单加氧酶DsmABC催化下5号位羟化生成3,6-DCGA,3,6-DCGA在脱氯酶DsmH2催化下生成3-氯龙胆酸,3-氯龙胆酸在双加氧酶DsmD催化下开环生成2-氯马来酰丙酮酸,然后在脱氯酶DsmG作用下生成马来酰丙酮酸。马来酰丙酮酸的降解还有待于进一步研究。相关的降解基因位于两个基因簇上。更多还原

【Abstract】 3,6-Dichlorosalicylate(3,6-DCSA)is the demethylation product of herbicide dicamba.Dicamba(3,6-dichloro-2-methoxybenzoic acid)is a low mammalian toxicity,broad-spectrum and high-efficiency hormone herbicide,and widely used to control a variety of broadleaf weeds in corn,sorghum and wheat fields.Especially after the large-scale planting of GM dicamba-resistant crops constructed by Monsanto Biotechnology Company,the annual use of dicamba significantly increased from 15,000 tons to more than 60,000 tons.Thus,the catabolism and ecological effects of dicamba needs to be studied.In the environment,dicamba is mainly degraded through microbial metabolism.In reported dicamba-utilizing bacteria,dicamba is initially demethylated to generate 3,6-dichlorosalicylate(3,6-DCSA).3,6-DCSA is a toxic and persistent chlorinated aromatic compound that has potential hazards to the ecological environment and human health as it accumulates in soil and agricultural products.However,the catabolic pathway and the genes and enzymes involved have remained unknown.Therefore,it is of great theoretical and practical value to elucidate the microbial catabolic pathway of 3,6-DCSA and the enzymes and genes involved.In this study,a dicamba-degrading’ strain Rhizorhabdus dicambivorans Ndbn-20 was used as the research material,we revealed the microbial catabolism of 3,6-DCSA and the genes and enzymes involved.The main results obtained are as follows:1.Screen of the 3,6-DCSA-degradation-deficient mutant and the preliminary identification of the degrading gene clustersA 3,6-DCSA-degradation-deficient mutant,Ndbn-20m,was acquired by continuous transfers of the strain on 1/5 Luria-Bertani(LB)agar without addition of 3,6-DCSA.Pairwise comparison of the mutant genome with that of the wild-type strain revealed that a 64.1-kb DNA fragment was absent in mutant Ndbn-20m.ORF analysis showed that there was a gene cluster,designated dsmR1DABCEFGR2(3,6-DCSA monooxygenase),existed in the 64.1-kbfragment.dsmABC encoded a three-component cytochrome P450,dsmD,dsmE,dsmF and dsmG encoded gentisate dioxygenase,fumarylpyruvate hydrolase,transporter and maleylacetate reductase,respectively.Genes dsmR1 and dsmR2 encoded LuxR family transcriptional regulator and IclR family transcriptional regulator,respectively.RT-PCR results showed that dsmD,dsmA,dsmB,dsmC,dsmE,dsmF,dsmG and dsmR1 were organized in an operon.RT-qPCR results showed that all the genes in cluster dsm were significantly induced by 3,6-DCSA.In addition,we found that another gene cluster,which contained a gentisate dioxygenase gene gtdA and a glutathione-dependent dehalogenase gene dsmH2,was also obviously induced by 3,6-DCSA.2.The three-component cytochrome P450 monooxygenase DsmABC was responsible for the 5-hydroxylation of 3,6-DCSADsmA shared 30%-36%identities with some cytochrome P450 monooxygenases that catalyze the hydroxylation of antibiotics.In the phylogenetic tree constructed by neighbor-joining(NJ)algorithm based on related cytochrome P450 monooxygenases,DsmA was located within this NJ tree,but formed a monophyletic branch.DsmB and DsmC were ferredoxin and ferredoxin reductase,respectively,and constituted an electron transfer chain.Fragments containing dsmA or dsmABC were ligated into the broad-host-range plasmid pBBR1MCS-2,and then introduced into Sphingobium quisquiliarum DC-2,which could not degrade 3,6-DCSA and salicylate.Recombinant DC-2(pBBR-dsmABC)acquired the ability to transform 3,6-DCSA,and correspondingly,a hydroxylation product was produced.Recombinant DC-2(pBBR-dsmA)could not transform 3,6-DCSA.Mutant Ndbn-20ΔdsmA could not degrade 3,6-DCSA.Re-introduction of dsmA into Ndbn-20ΔdsmA restored the.ability to degrade 3,6-DCSA.Single-crystal X-ray diffraction analysis showed that the DsmABC-catalyzed hydroxylation occurred at the C-5 of 3,6-DCSA,forming 3,6-dichlorogentisate(3,6-DCGA).The above results indicated that DsmABC was a 3,6-DCSA 5-hydroxylase.3.The glutathione-depende-nt dehalogenase DsmH2 was responsible for the 6-dechlorination of 3,6-DCGAThe cell lysate of strain Ndbn-20 could transform 3,6-DCGA only in the presence of GSH,indicating that 3,6-DCGA was dechlorinated by a glutathione S-transferase(GST)in strain Ndbn-20.By genome comparison and blast,two GSTs,dsmH1and dsmH2,were found in the genome of strain Ndbn-20.Enzymatic results showed that both DsmHland DsmH2 displayed 3,6-DCGA dehalogenase activity.DsmH2 had significantly higher catalytic efficiency toward 3,6-DCGA than DsmH1.Transcription and disruption analysis revealed that DsmH2,but not DsmHl,was responsible for the 6-dechlorination of 3,6-DCGA in strain Ndbn-20 in vivo.1H NMR analysis demonstrated that the 6-chlorine but not the 3-chlorine of 3,6-DCGA was replaced by a hydrogen,generating 3-chlorogentisate.Furthermore,we propose a novel ’eta’ class of GSTs to accommodate the four bacterial dehalogenases PcpC,LinD,DsmH1,and DsmH2.4.The gentisate 1,2-dioxygenase DsmD was mainly responsible the cleavage of 3-chlorogentisateThere were two gentisate 1,2-dioxygenase genes,dsmD and gtdA,existed in the genome of strain Ndbn-20,Enzymatic study showed that both DsmD and GtdA could catalyzed the cleavage of gentisate and 3-chlorogentisate.DsmD had significantly higher catalytic efficiency toward 3-chlorogentisate than gentisate,and than that of GtdA;and GtdA had significantly higher catalytic efficiency toward gentisate than 3-chlorogentisate.These results indicated that the optimal substrate for DsmD is 3-chlorogentisate,while the optimal substrate for GtdA is gentisate.Transcription and disruption analysis revealed that DsmD,but not GtdA,was mainly responsible for the cleavage of 3-chlorogentisate,and GtdA was mainly responsible for the cleavage of gentisate in strain Ndbn-20 in vivo.5.The reductive dehalogenase DsmG catalyzed the reductive dechlorination of 2-chloromaleylpyruvatedsmG shared the highest identity with maleylacetate reductase TfdF(39%).TfdF could catalyze the reductive dechlorination of 2-chloromaleylacetate,and 2-chloromaleylpyruvate,the cleavage product of 3-chlorogentisate,was structurally similar to 2-chloromaleylacetate.Enzymatic study showed that the exogenously expressed and purified DsmG could catalyzed the reductive dechlorination of 2-chloromaleylpyruvate to maleylpyruvate in the presence of NADH.Base on the above results,we basically elucidated the catabolic pathway of 3,6-DCSAin strain Ndbn-20.Two gene clusters are involved in this pathway.3,6-DCSA is 5-hydroxylated to 3,6-DCGA by monooxygenase DsmABC,3,6-DCGA is subsequently 6-dechlorinated to 3-chlorogentisate by dehalogenase DsmH2,3-chlorogentisate is further cleaved to 2-chloromaleylpyruvate by gentisate 1,2-dioxygenase DsmD.2-chloromaleylpyruvate was reductively dechlorinated to maleylpyruvate by a reductive dehalogenase DsmG.Further study is needed to elucidate the catabolism of maleylpyruvate in strain Ndbn-20.更多还原