Rhizorhabdus dicambivorans Ndbn-20降解除草剂麦草畏的下游代谢途径及分子机制研究

Dicamba is a widely used herbicide with broad herbicide spectrum. With the rapid increase of the genetically modified dicamba-resistant crops，the amount of dicamba will increase dramatically, so it is very important to study the environmental behavior of dicamba. Dicamba is demethylated by soil microbes to generate 3,6-dichlorosalicylic acid (DCSA), but the further metabolic mechanism of DCSA is unclear. Strain Ndbn-20 is capable of mineralizing dicamba. Recently, we screened a mutant of strain Ndbn-20 defective in DCSA degradation. Sequence comparison of the mutant genome with that of the wild type revealed that a DNA fragment including a gene cluster dch28 was missing in the mutant. Gene cluster dch28 contains several putative genes that encode cytochrome P450 monooxygenase, reductive dechlorinase, gentisate 1,2-dioxygenase and fumarylpyruvate hydrolase, respectively. Introduction of the gene cluster into the mutant restored its DCSA degradation ability. This project was designed to study the expression of each gene in the gene cluster and the catalytic activity of the expressed product, identify the structure of intermediates, study the gene transcription and effect of gene deletion on the DCSA degradation. This project will reveal the process and mechanism of DCSA degradation and the enzymes and genes involved in the process, and provide the theoretical basis for in-depth study on the degradation mechanism and ecotoxicology of dicamba in soil.

麦草畏是一种应用广泛、杀草谱广的除草剂，随着抗麦草畏转基因作物的推广，其用量将急剧增加，因此研究麦草畏的降解转化等环境行为具有非常重要的意义。麦草畏在土壤微生物作用下脱甲基生成3,6-二氯水杨酸（DCSA），但DCSA的进一步降解过程和机制还不清楚。菌株Ndbn-20能矿化麦草畏。近期申请人筛选到该菌一个失去DCSA降解能力的突变株，发现其基因组丢失片段包含一个基因簇dch28，该基因簇包含编码疑似细胞色素P450单加氧酶、还原脱氯酶、龙胆酸1,2-双加氧酶和反丁烯二酸单酰丙酮酸水解酶的基因。将基因簇dch28导入突变株能恢复其DCSA降解能力。本项目拟研究该基因簇上各基因的外源表达和表达产物的催化活性，鉴定各降解中间产物的结构，研究各基因转录情况及敲除对菌株降解DCSA的影响，揭示DCSA降解过程及参与的酶和基因，为深入研究麦草畏在土壤中的微生物降解转化机制和生态毒理提供理论依据。

麦草畏是一种广谱高效的除草剂，随着抗麦草畏转基因作物的推广，其用量急剧增加，因此有必要深入研究麦草畏在环境中迁移、转化和降解机制。已有研究表明麦草畏降解的起始步骤均是脱甲基生成无除草活性的3,6-二氯水杨酸（3,6-DCSA）。3,6-DCSA是有毒难降解的氯代芳香化合物，在环境中累积对生态和人类健康具有潜在的危害，然而目前有关3,6-DCSA的降解研究还未见报道。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。.本项目以麦草畏降解菌株Ndbn-20和不能降解3,6-DCSA的突变菌株Ndbn-20M为研究材料，从分子水平基本揭示了3,6-DCSA的完整降解途径。首先，通过异源表达、插入突变、基因回补等方法验证了基因dsmABC的功能，确定其编码的是一个三组分细胞色素P450单加氧酶体系DsmABC，负责催化3,6-DCSA的羟基化，通过X射线单晶衍射确定羟基化位置为苯环上5-C原子，生成3,6-二氯龙胆酸（3,6-DCGA）。然后，基因组比对分析发现在菌株Ndbn-20基因组中有两个编码谷胱甘肽（GSH）依赖型的还原脱氯酶基因dsmH1和dsmH2。酶学研究结果表明，DsmH1和DsmH2都能够催化3,6-DCGA脱氯，核磁氢谱分析结果表明产物为3-氯龙胆酸，且DsmH2的活性远高于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。其次，基因组分析获知在菌株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，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DsmD主要负责3-氯龙胆酸的开环降解，GtdA主要负责龙胆酸的开环降解。最后，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。.完整的降解过程为：3,6-DCSA在细胞色素P450单加氧酶DsmABC催化下生成3,6-DCGA，3,6-DCGA在GSH依赖型脱氯酶DsmH2催化下生成3-氯龙胆酸，3-氯龙胆酸在双加氧酶DsmD