共代谢调控睾丸酮丛毛单胞菌降解喹啉的分子机制研究

Comamonas testosteroni exists ubiquitously in the aerobic processes in wastewater treatment. It can effectively degrade polycyclic aromatic hydrocarbons (PAHs), nitrogen-containing heterocyclic aromatic hydrocarbons, steroids, and other refractory organic compounds by coordinating a variety of different enzymes and plays an important role in the petrochemical wastewater treatment. Investigation of mechanisms of the cometabolism of two or more refractory compounds can improve our understanding of utilization of these compounds as sole carbon and energy source for Comamonas testosteroni in treatment of complex petrochemical wastewater and provide instruments to enhance the treatment performance. In the project, the quinoline-degrading Comamonas testosteroni strain isolated by our lab will be used as model organism to investigate cometabolism of the selected refractory compounds. The specific genes for key enzymes in degrading these compounds, such as dehydrogenase/carbonyl reductase will be located in the bacterial genome. The transcription of the genes and the enzyme activities in the presence of quinoline and/or the related compounds will be investigated. By analyzing the regulatory regions of the quinoline-degrading gene operon, the promoter, operator and other regulatory protein binding sites will be identified and the interaction of the purified regulatory proteins with the regulatory region will be investigated in the presence of quinoline and/or the related compounds. After the cometabolism model is established, the expression of 3α-hydroxy-steroid dehydrogenase (3α-HSD) will be analyzed, through which the prevalence of Comamonas testosteroni in wastewater is determined. The mechanisms of regulation of the specific enzymes in cometabolism will guide the development of bioaugmentation and improve treatment efficiency of the refractory compounds in wastewater.

睾丸酮丛毛单胞菌是废水处理中普遍存在的一类好氧细菌，其可以通过共代谢调节多种酶的合成来分解水中甾体、含氮杂环和多环芳烃等化合物，在废水处理中具有十分重要的地位。研究睾丸酮丛毛单胞菌共代谢的分子机制，有助于解释细菌在复杂污染条件下的营养摄取行为，进而通过调控专性细菌生殖和底物利用范围来强化废水处理效率。本项目以专性降解喹啉的睾丸酮丛毛单胞菌为研究对象，从TCA循环过程和关键酶的表达入手，逐步分析关键降解酶（羟化酶和加氧酶）的特异表达和特异基因的编码属性，确定喹啉降解基因簇的启动子与操纵子位置，探索复杂基质条件下共代谢喹啉过程与基因编码的关系；进一步构建实验室模型，通过分析睾丸酮丛毛单胞菌3α-HSD基因表达，定量评价废水处理中睾丸酮丛毛单胞菌的丰度，深入研究共代谢条件下特异酶的生成与抑制机制，获得在转录水平调控细菌共代谢特性和快速生殖的方法来强化废水处理效率，推动生物强化技术的应用与发展。

针对喹啉降解基因催化机制和调控机制不明确的问题，以专性降解喹啉的睾丸酮丛毛单胞菌为研究对象，该项目利用PCR及转录组测序技术，分析了睾丸酮丛毛单胞菌降解喹啉过程中菌株的降解特性及特异基因表达，揭示了睾丸酮丛毛单胞菌降解喹啉的作用机制及废水中外源物质存在对降解喹啉的影响，提出了相分离法负载菌株高效处理喹啉事故废水的技术，为推动生物强化技术的应用与发展提供理论支持，主要研究成果如下：.（1）通过PCR技术，采用喹啉降解基因引物，在喹啉降解菌睾丸酮丛毛单胞菌基因组DNA上发现了控制2-羟基喹啉转变为2,8-二羟基喹啉基因。SDS-PAGE电泳表明，喹啉培养基中条带数量有所增加，其中多出的大小为43.0-66.2 kDa和22.0-31.0 kDa的2个条带很有可能是控制喹啉降解的关键性蛋白酶。.（2）利用转录组测序技术对菌株进行测序分析，研究了睾丸酮丛毛单胞菌对喹啉的降解途径和相关基因。参与喹啉降解的bphB基因在两组实验组中均显著上调，ligABC基因在喹啉浓度为100 mg/L降解实验组中显著上调。此外差异基因主要富集在KEGG数据库中ABC转运系统与能量代谢中，这与底物的识别转运、信号转导、基因表达调控及能量提供有关。.（3）系统的研究了外源有机物、生长因子以及金属离子对喹啉降解过程的影响。外源物质对喹啉降解情况的影响主要包括促进喹啉的羟基化；促进喹啉羟基化生产的2-羟基喹啉的进一步代谢；对Comamonas testosteroni QYY产生毒害作用，抑制细菌的生长。不同的外源有机物及生长因子往往只具有三种影响中的其中一种，而金属离子由于浓度的不同则会产生不同影响，在特定浓度范围内以其中一种影响为主。.（4）合成了通道良好的多孔聚醚砜小球，并将其用于Comamonas testosteroni QYY细胞的好氧反应器，以去除生活污水中意外释放的染料废水中的喹啉、苯酚和其他难降解化合物。负载Comamonas testosteroni QYY的聚醚砜小球能抵抗有机负荷冲击，提高总有机碳去除率，并且达到喹啉100%去除，经过聚醚砜小球负载细菌处理后可以有效去除废水中有毒化合物，包括氨基甲酸苯酯、2-硝基甲苯和邻苯二甲酸二辛酯。