Novosphingobium sp. FND-3降解呋喃丹的分子机制研究

Carbofuran is a broad-spectrum N-methylcarbamate pesticide, which has ever been widely used to control insects, nematodes of crops. Although China has limited the usage of carbofuran due to its high toxicity and prohibited its use in vegetables, fruits, tea and Chinese medical herbs, it is still used in other crops. The residue of carbofuran is harmful to ecosystem and poses a threat to the health of humans. Microbial metabolism is an effective and enviromental friendly approach to eliminate the pollution of carbofuran in the environment, and the increasing attention has been put on the this approach. However, the molecular mechanism of degradation of carbofuran has not been elucidated. Novosphingobium sp. FND-3 was isolated by our lab; it is able to use carbofuran as the sole carbon source for growth. Strain FND-3 could degrade carbofuran through the typical pathway, which was initiated by the hydrolysis of its N-methylcarbamate linkage. Additionally, strain FND-3 has other two steps for initiating the degradation of carbofuran (1, hydrolysis of the ether bond of its furan ring; 2, hydroxylation of its phenyl ring). We have cloned the genes which is responsible for the first two steps in the typical degradation pathway of carbofuran in strain FND-3. They are carbofuran hydrolase gene cehA1 and carbofuran phenol monooxgenase genes cfdC1C2. The target of this project is to clone the other genes involved in the typical degradation pathway of carbofuran as well as the genes responsible for the other two initial dergradation steps. The results of the present study will elucidate the degradation mechanism of carbofuran by strain FND-3 from the gene level and provide the theoretical basis for the bioremediation of the carbofuran-contaminated environments.

呋喃丹是一种曾经广泛使用的高毒氨基甲酸酯类杀虫剂。虽然呋喃丹现在是我国的限制使用农药品种，但是它的残留仍然破坏生态环境和威胁人体健康。微生物降解是消除环境中呋喃丹污染的有效手段，但是目前对微生物降解呋喃丹的分子机制还未阐明。Novosphingobium sp. FND-3是本课题组分离的能以呋喃丹为唯一碳源生长的菌株。该菌株除了具有以水解呋喃丹氨基甲酸酯键为起始步骤的典型呋喃丹降解途径之外，还具有另外两种起始降解步骤（1、水解其呋喃环的醚键；2、对其苯环进行羟化）。我们已经克隆了菌株FND-3典型呋喃丹降解途径中负责前两步的基因（呋喃丹水解酶基因cehA1和呋喃酚单加氧酶基因cfdC1C2）。本项目将继续克隆其典型降解途径中的其它基因以及负责另外两种起始降解步骤的基因。预期结果将从基因水平上解析菌株FND-3降解呋喃丹的机制，为呋喃丹污染环境的生物修复提供理论支撑。

呋喃丹是一种曾经广泛使用的高毒氨基甲酸酯类杀虫剂，它的残留破坏生态环境和威胁人体健康。微生物降解是消除环境中呋喃丹污染的有效手段，但目前对微生物降解呋喃丹的机理研究还很不充分，完整的降解途径及参与的基因还不清楚。本项目拟以呋喃丹降解菌株FND-3为材料，研究其降解呋喃丹的分子机制。取得以下结果：1、完成了菌株FND-3完整基因组的测定及解析，但由于该菌株的降解性状不稳定，后续没有进一步研究；2、新分离到一株性状稳定的呋喃丹降解菌株Sphingobium sp.CFD-1并研究了其降解特性；3、克隆了菌株CFD-1的呋喃丹水解酶基因cehACFD-1，对其进行了异源表达和纯化，鉴定了其决定催化活性的关键氨基酸位点；4、研究了来自不同菌株的呋喃丹水解酶CehA的底物偏好性及其相关的的氨基酸位点，并以此为基础建立了cehA基因的环境行为模型；5、克隆了菌株CFD-1呋喃酚羟化酶基因cfdCR，对其进行了异源表达和纯化，并研究了其酶学特性；6、克隆了菌株CFD-1环氧水解酶基因cfdF和双加氧酶基因cfdE；7、综合基因功能及相关中间代谢产物的鉴定结果提出了菌株CFD-1降解呋喃丹的途径及相关基因，解析了该菌株降解呋喃丹的分子机制。这些结果将为呋喃丹污染环境的生物修复提供理论支撑。. 总体来说，本项目完成了预期研究目标。项目主持人以通讯作者发表标注项目号SCI录用文章9篇(第1标注7篇),中文文章1篇；其中包括Appl. Environ. Microbiol 3篇。培养研究生5名，其中博士2名，硕士3名。