Rhodococcus sp.CD-9降解丙草胺代谢途径及N-脱烷基酶基因克隆的研究

Chloroacetamide herbicides are the most important class of preemergence herbicides which are widely used in the world. The commonly used chloroacetamide herbicides are alachlor, acetochlor, pretilachlor and butachlor. To date, CndABC and EthBAD are the only two N-dealkylases which could catalyze the cleavage of the C-N bond of alachlor, acetochlor and butachlor. However, neither of them could degrade pretilachlor. Strain Rhodococcus sp. CD-9 was isolated by our lab, and it could completely degrade and utilize pretilachlor as the sole carbon source for growth. The degradation of pretilachlor in this strain was initiated by cleavage of the C-N bond under the catalysis of an unknown N-dealkylase. This project is to identify the degradation pathway of pretilachlor by the method of substrates utilization and metabolites identification, to clone and express the N-dealkylase gene of Rhodococcus sp. CD-9 through genomic analysis combined with general molecular manipulation, to study the enzymatic characteristics, function and kinetics of N-dealkylase, and the effect of the oxygen atom position of alkyl linkage to nitrogen atom on the catalytic efficiency of the N-dealkylase. The expected results of this study will elucidate the pretilachlor degradation pathway by strain CD-9, and provide theoretical foundation for the further study of the structure and function of N-dealkylase and the bioremediation of pretilachlor-contaminated environments.

氯代酰胺类除草剂是世界上广泛使用的一类芽前除草剂，其代表品种包括甲草胺、乙草胺、丙草胺和丁草胺等。已报道催化甲草胺、乙草胺和丁草胺C-N键断裂的N-脱烷基酶CndABC和EthBAD都不能降解丙草胺。Rhodococcus sp. CD-9是本课题组分离的一株能降解并利用丙草胺为唯一碳源生长的菌株，该菌株通过未知N-脱烷基酶催化丙草胺C-N键断裂作为起始降解步骤。本项目拟通过底物利用和产物鉴定技术解析菌株CD-9降解丙草胺的代谢途径；运用基因组学和常规分子生物学操作相结合的手段克隆和表达其N-脱烷基酶基因，并研究其酶学特性、动力学参数及N上连接烷基中O的相对位置对N-脱烷基酶催化效率的影响。预期结果将解析菌株CD-9降解丙草胺的途径，并为深入研究N-脱烷基酶结构与功能及丙草胺污染环境的生物修复提供理论依据。

氯代酰胺类除草剂是世界上广泛使用的一类芽前除草剂，但其残留破坏生态环境、危害人体健康且对作物有药害。其代表品种包括甲草胺、乙草胺、丙草胺和丁草胺等。微生物降解是环境中氯代乙酰胺类除草剂残留消除的最主要因素，已报道催化甲草胺、乙草胺和丁草胺C-N键断裂的N-脱烷基酶CndABC和EthBAD都不能降解丙草胺，目前对微生物降解丙草胺的机理研究还很不深入，完整的降解途径及参与的基因还不清楚。Rhodococcus sp. B2 (原为CD-9)能高效降解多种氯代乙酰胺除草剂（甲草胺、乙草胺、甲草胺、丙草胺、丁草胺和异丙草胺）的菌株。本项目以该菌株为材料，解析丙草胺降解相关基因，此外还继续分离丙草胺及其下游产物降解菌株，为研究丙草胺降解菌的多样性提供材料。通过底物利用和产物质谱鉴定等技术研究该菌降解丙草胺的产物，丙草胺在脱烷基酶EthBADB2的催化下O-脱烷基生成为N-羟乙基-2-氯-N-(2,6 -二乙基苯基)-乙酰胺。克隆了2个丙草胺降解新基因，分别为丙草胺脱烷基酶基因EthBADB2（负责丙草胺降解第一步脱烷基）, CDEPA酰胺酶基因mmH（负责丙草胺降解产物CDEPA脱氯乙酰基）。研究丙草胺降解关键酶脱烷基酶EthBADB2和酰胺酶MmH的特性。本项目从生化和分子生物学角度揭示了微生物降解氯代乙酰胺类除草剂的代谢机制，为该类除草剂残留污染的微生物降解修复及抗该类除草剂转基因作物的构建提供理论和技术依据。