Sphingomonas sp. DC-6降解乙草胺的代谢途径及分子机制研究

Chloroacetamide herbicides are among the most important class of herbicides used in corn, cotton, soybean and many other crops for control of annual grass and broadleaf weeds. The most commonly used chloroacetamide herbicides in the world is acetochlor. However, chloroacetamide herbicides persist for a long time in soil, and the residues are highly toxic to some aquatic organisms and are carcinogenic to human, and consistently injure subsequent rotation crops, especially in sandy soils with low organic matter. Biodegradation is the most important factor in the fate of chloroacetamide herbicides in the environment. However, the microbial degradation pathway of chloroacetamide herbicide has not been elucidated, and the genes and enzymes involved in this pathway have not been studied. Sphingomonas sp. DC-6 was able to completely degrade many chloroacetamide herbicides, such as acetochlor, butachlor, alachlor and propisochlor, and use these substrates as the sole carbon source for growth. The target of this program is to elucidate the microbial degradation pathway of acetochlor by the methods of metabolites identification and substrates utilization, to clone the genes involved in this pathway, and to study the enzymatic characteristics of the key enzyme N-dealkylase, which catalyze the N-dealkylation of acetochlor. The results of this program will provide the biochemical and genetic foundation of microbial degradation pathway of acetochlor, and benefit both the bioremediation of chloroacetamide herbicide-contaminated environments and the genetic engineering of chloroacetamide herbicide-resistant crops.

氯代乙酰胺类除草剂在全世界广泛应用，但其残留破坏生态环境、危害人体健康且对作物有药害。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，但目前对该类除草剂微生物降解的生物学机制研究还不够深入，完整的代谢途径及参与的酶和基因尚不清楚。Sphingomonas sp. DC-6完全降解多种氯代乙酰胺除草剂（乙草胺、甲草胺、丁草胺和异丙草胺），本项目以该类除草剂中使用量最大的乙草胺为靶标，通过底物利用和产物质谱鉴定等技术研究该菌降解乙草胺过程中的各种中间产物，揭示乙草胺降解的完整途径；从该菌中克隆乙草胺降解代谢过程的各个降解酶基因，并验证其功能；研究乙草胺降解关键酶N-脱烷基酶的特性，通过突变发现影响N-脱烷基酶降解活性的氨基酸位点。本项目从生化和分子生物学角度揭示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构建提供理论和技术依据。

氯代乙酰胺类除草剂在全世界广泛应用，但其残留破坏生态环境、危害人体健康且对作物有药害。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，但目前对该类除草剂微生物降解的生物学机制研究还不够深入，完整的代谢途径及参与的酶和基因尚不清楚。Sphingomonas sp. DC-6完全降解多种氯代乙酰胺除草剂（乙草胺、甲草胺、丁草胺和异丙草胺），本项目以该类除草剂中使用最普遍的乙草胺为靶标，通过底物利用和产物质谱鉴定等技术研究该菌降解乙草胺过程中的各种中间产物。乙草胺首先在脱烷基酶Cnd的催化下脱烷基生成CMEPA，CMEPA 在酰胺酶CmeH催化下脱氯乙酰基转化为MEA，MEA在两组分黄素单加氧酶MeaXY的催化下在对位进行羟基化，生成的4-OH-MEA会自发氧化和脱氨基，产物MEHQ在另外一个二组分黄素单加氧酶MeaAB的催化下生成3-OH-MEHQ，3-OH-MEHQ可进一步开环，最后进入TCA循环代谢。克隆了3个乙草胺降解新基因，分别为乙草胺脱烷基酶基因cndABC（负责乙草胺降解第一步脱烷基）, CMEPA酰胺酶基因cmeH（负责乙草胺降解产物CMEPA脱氯乙酰基）和MEA单加氧酶基因meaXY（负责乙草胺降解下游产物MEA羟基化）。研究乙草胺降解关键酶N-脱烷基酶Cnd和酰胺酶CmeH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。项目执行期间发表相关论文16篇（标注了项目号），其中SCI论文15篇，包括在微生物学权威刊物Appl. Environ. Microbiol.上发表论文3篇。中文核心期刊论文1篇。获得3项国家发明专利（包括1项2014年江苏省百件优秀专利），申请了1项国际专利。