异丙甲草胺脱烷基酶基因的克隆及脱烷基酶特性和功能的研究

Chloroacetanilide herbicides are a class of highly efficient preemergence herbicides that are widely used for the control of annual grass and broadleaf weeds. Presently, it is the second largest herbicide in the world, and acetochlor and metolachlor were the main chloroacetanilide herbicides. Due to that acetochlor was suspected to be carcinogenic and chronic toxic, it was forbidden in European Union and USA since 2013, thus the usage of metolachlor, which was relatively low toxic and non- carcinogenic, are increasing significantly. To date, the molecular bases for the microbial degradation of metolachlor are still unknown. In structure, metolachlor has a chiral carbon and a chiral axis and the N- side chain was methoxyaethyl, whereas acetochlor has only a chiral axis and the N- side chain was ethoxymethyl. The difference in molecular structure led to that the reported acetochlor dealkylases CndABC and EthB could not catalyze the dealkylation of metolachlor. Strain Paracoccus sp. FLY-8 could completely degrade and utilize metolachlor，and the degradation of metolachlor in this strain was initiated by dealkylation under the catalysis of an unknown dealkylase. This project will try to clone the metolachlor dealkylase by using genomics analysis and transposon insertion mutation technology, study the enzymatic characteristics, function and kinetics, and chiral selectivity of the enzyme to the isomers of metolachlor. This project will elucidate the molecular mechanism of metolachlor dealkylation in strain FLY-8, and provide scientific basis for the study of the environmental behavior of metolachlor, and the enzymatic mechanism for the effect of N- side chain structure on the biodegradability of chloroacetanilide herbicides.

氯乙酰胺除草剂是一类高效、使用量世界第二的除草剂，主要品种有乙草胺和异丙甲草胺。因乙草胺有致癌性和毒性，欧美已于2012年禁用，因此低毒无致癌性的异丙甲草胺用量显著增加。然而对异丙甲草胺微生物降解的生理和遗传机制研究还未见报道。异丙甲草胺的N-侧链是甲氧乙基，含有1个手性碳和一个手性轴（而乙草胺为N-乙氧甲基，一个手性轴），结构差异导致已报道的乙草胺脱烷基酶不能降解异丙甲草胺。异丙甲草胺降解菌Paracoccus sp. FLY-8通过一个未知的脱烷基酶催化异丙甲草胺脱烷基。本项目采用插入突变和基因组学等技术从FLY-8中克隆异丙甲草胺脱烷基酶基因，研究脱烷基酶特性、动力学和功能，及脱烷基酶降解异丙甲草胺各异构体的手性选择性。本项目将揭示菌株FLY-8中异丙甲草胺脱烷基的分子机制，为深入研究氯乙酰胺除草剂N-侧链结构影响其生物可降解性的酶学机制，及异丙甲草胺的环境行为提供材料和理论依据。

氯乙酰胺类除草剂是一类分子结构有氯乙酰胺基团的高效除草剂，使用量居世界第二位，主要品种包括乙草胺、丁草胺、甲草胺、异丙甲草胺（商品化产品都尔主要成分S-型，金都尔主要成分R-型）和异丙草胺等，上述除草剂的大量使用在消除杂草危害、提高劳动生产率的同时，其残留也对耕地土壤造成严重的污染，研究表明微生物在消除环境中除草剂其中非常重要的作用。本项目围绕副球菌FLY-8及其他降解菌株，通过基因组学、转座子随机插入突变和基因回补技术，揭示细菌降解异丙甲草胺分子机制。测定了菌株FLY-8全基因组，克隆到异丙甲草胺脱烷基酶基因cndA2, CndA2为三组分非血红素氧化酶系，在辅因子NADH存在下通过CndA2-Red-Fdx组成酶系将S-型异丙甲草胺转化成CMEPA，体外酶学特性发现该酶系只能降解S-型异丙甲草胺缺不能R-型异并甲草胺；测定了硝酸盐还原菌ZZ-1，克隆异丙甲草胺脱卤酶bphA2；通过比较基因组学在鞘酯菌DE-13中克隆到异丙甲草胺代谢关键酶基因meaXY，MeaXY为2-甲基-6-乙基苯胺羟化酶基因；通过转基因技术将cndA基因转入拟南芥叶绿体中，获得能够降解甲草胺、乙草胺和丁草胺的拟南芥植株。本项目揭示了微生物降解异丙甲草胺的分子机制，为该类物质的土壤污染微生物修复提供了材料。