Pseudomonas sp.LUNF2高效降解PAEs的酶促催化及转录调控机制

邻苯二甲酸酯(Phthalic Acid Esters，PAEs)是环境雌激素类的普遍环境污染物，严重危害生态环境安全，微生物代谢是消除其污染的主要途径，而代谢机理研究的不足严重限制了生物修复技术的突破。本项目在假单胞菌LUNF2高效降解 PAEs的特征鉴定、基因组分析的基础上，拟重点研究：（1）由转录组学和蛋白组学技术克隆PAEs水解酶基因dphPM1和mphPM1，敲除基因验证其功能；（2）鉴定PAEs水解酶的酶学特征，由结构生物学方法和BIAcore技术研究水解酶高效催化PAEs的分子机制；（3）结合遗传学方法、BIAcore鉴定dphPM1基因的启动子Pdph及转录调控蛋白DphR，由EMSA、DNase I足迹法探究DphR与启动子的互作，解析dphPM1基因的转录调控机制。研究成果将深入揭示菌株高效代谢PAEs的分子机理，促进PAEs高效降解工程菌或工程酶的开发及环境修复应用。

Phthalic acid esters (PAEs) are common environmental pollutants with the property of environmental estrogens，which have done serious harm to ecological environment. Microbial metabolism is the main solution of eliminating PAEs, but the lack of degradation mechanism greatly limits the breakthrough of remediation technology. Based on the accomplished characterization of Pseudomonas sp. LUNF2 efficiently degrading PAEs and its genomic analysis, this project will focus on: (1) cloning the PAEs hydrolase genes dphPM1 and mphPM1 by analysis of transcriptome and proteome of strain LUNF2, and verifying the function of the hydrolases by gene knockout; (2) identifying the enzymatic characteristics of PAEs hydrolases，revealing the molecular mechanism of hydrolases efficiently catalyzing PAEs by technology of structural biology and Biacore； (3) identifying the promoter Pdph of dphPM1 and transcriptional factor DphR by genetics method and Biacore, analyzing the interaction between DphR and the promoter by EMSA and DNase I footprinting，and discovering the transcriptional regulation mechanism of the gene dphPM1. Taken together, the molecular mechanism of strain LUNF2 efficiently metabolizing PAEs will be elucidated in depth. The results will contribute to the development of engineered bacteria or enzymes capable of efficiently degrading PAEs and their application to the remediation of environment.