邻苯二酚生物感应分子的研制及其功能验证

It is important that low and unstable degradation ability and low tolerance of microorganisms is true in the bioremediation field of aromatic pollutants (AP). The directed evolution plays an essential role in the environmental bioremediation. However, due to the lack of a high-throughput screening method, the enzyme catalytic activity in the biodegradation of AP isn't improved significantly. Catechol is one of key intermediates in AP metabolism. Therefore, AP bioavailability could be monitored by catechol concentration. In this project, the regulatory protein OrfS of the catechol metabolic pathways will be mutated and combined with the promoter PtadD2 and green fluorescent protein gene. The positive correlation occurred between fluorescence intensity and catechol concentration in cell; OrfS mutants and PtadD2-fluorescence system will be functionally expressed in Pseudomonas putida within a vector, establishing a rapid detection method to monitor catechol concentration. The fluorescence intensity can indicate the catalytic efficiency of aniline dioxygenase quickly, which will be applied into the high-throughput screening method for the directed evolution of aniline dioxygenase. This study will offer that the bioavailability of AP in the environment could be quickly detected, and a high-throughput screening method could be used in directed evolution of key enzymes in the AP metabolic pathways; meanwhile, catechol, an important chemical intermediates, can also be produced, which is benefit to the resource recycling of environmental pollutants

芳香族污染物（AP）的生物降解存在代谢速率低、菌株耐受性差等重要问题。虽然定向进化在生物修复领域发挥着重要作用，由于缺乏高通量筛选方法，AP代谢酶的催化活性尚没有得到大幅度提高。邻苯二酚是AP的关键代谢产物，通过邻苯二酚可以检测AP的生物利用度。本项目中，调控蛋白OrfS将被改造成能被邻苯二酚特异结合并能诱导启动子PtadD2表达下游荧光蛋白的生物感应分子，通过细胞荧光强度可以反映邻苯二酚浓度的变化；在Pseudomonas putida中同时表达OrfS突变体和PtadD2-荧光系统，建立邻苯二酚浓度变化的快速检测方法；该方法将用作高通量筛选工具对苯胺双加氧酶进行定向进化改造，通过荧光强度能快速揭示该酶降解苯胺生成邻苯二酚的催化效率。本项目不仅能对环境中AP的生物利用度进行快速检测，也为A P代谢途径关键酶的定向改造提供高通量筛选方法，还可得到重要化工原料邻苯二酚，有利于环境污染物资源化

定向进化在生物修复领域发挥着重要作用，由于缺乏高通量筛选方法，芳香族污染物代谢酶的催化活性尚未得到大幅提高。邻苯二酚是芳香族污染物的关键代谢产物，通过邻苯二酚可以检测芳香族污染物的生物利用度。对比分析调控蛋白xylE、OrfS、DntR生物活性，选取DntR为改造对象，通过3轮随机突变DntR配体结合域，用流式细胞仪筛选得到一个能结合邻苯二酚并能诱导启动子PdntR表达下游荧光蛋白GFP的突变体。在这个邻苯二酚生物感应系统中，邻苯二酚的浓度与诱导的转录激活作用存在剂量关系，对邻苯二酚可检出浓度为5 μM，半数效应浓度（EC50）为10.2 μM；将该系统将用作高通量筛选工具对苯胺双加氧酶的TdnA1亚基进行进化改造，经过2轮突变筛选得到一个比野生型比酶活提高3.37倍的突变体，表明该方法为芳香族污染物代谢途径关键酶的定向改造提供了的高通量筛选方法，有利于环境污染物的资源化。