紫色酸性磷酸酶介导下小球藻参与工业废水中有机磷生物降解及其作用机制研究

Organophosphorus pollutants from industrial sewage are not easily degraded in the aquatic environment and cause irreversible pollution to aquatic environment due to its chemical stability. Chlorella pyrenoidosa is a typical microalga for sewage treatment, but the degraded and removal effect on organophosphorus are not satisfied. Therefore, to clarify the regulation mechanism of organophosphorus biodegradation by C. pyrenoidosa and to modify microalgal strain to achieve the ability of high-efficient organophosphorus biodegradation are the urgent problems which need to be solved. Previously, we reported engineered C. pyrenoidosa overexpressed with purple acid phosphatase from marine diatom Phaeodactylum tricornutum could effectively biodegrade two typical organophosphorus pesticides. Based on these, this project intends to use this engineered strain as object to study its response and biodegradation effect of different typical aquatic organophosphorus pollutants; to analyze the physiological and biochemical characteristics of this engineered strain under different typical aquatic organophosphorus pollutants conditions; to identify the subcellular localization of purple acid phosphatase and its metabolic transport pathway under different typical aquatic organophosphorus pollutants conditions; to analyze the expression change of metabolites and key nodes in microalgae introduced with purple acid phosphatase by metabolomics and transcriptomics. This will reveal the correlation mechanism between phenotype and molecular regulation and systematically explain the molecular mechanism and regulation network of organophosphorus biodegradation by engineered strain. This project will provide theoretical support and new idea for the improvement of microalgal germplasm for aquatic organophosphorus biodegradation.

工业废水中的有机磷污染物由于自身的化学稳定性，流入水环境不易降解，会对水环境造成不可逆污染。蛋白核小球藻虽然作为污水处理典型藻，但是其针对有机磷的降解和去除效果仍不够理想。因此，厘清蛋白核小球藻生物降解有机磷的调控机制以及改良藻株实现高效降解有机磷污染物是目前亟需解决的难题。我们前期工作发现，将海洋硅藻三角褐指藻的紫色酸性磷酸酶基因导入蛋白核小球藻中表达可以有效降解两种典型有机磷农药。本项目拟基于该工程藻株进一步研究其对不同水体典型有机磷污染物的响应和降解；分析工程藻株在不同有机磷下的生理、生化特征；鉴定紫色酸性磷酸酶的亚细胞定位及其对不同有机磷的代谢转运途径；通过代谢组学和转录组学解析紫色酸性磷酸酶作用下微藻体内代谢物和重要基因的表达变化，揭示其表型与分子调控间的关联机制，系统全面地阐释微藻降解有机磷的分子机制和调控网络，为微藻种质改良用于水体有机磷污染处理提供理论支撑和新的思路。

水环境污染问题是当今热点的生态问题，严重威胁着水环境安全。由于工业生产和使用等环节中的大量释放，有机污染物在工业废水中的含量急剧增加，大多数有机污染物（如有机磷等）化学稳定性强，在水环境中不易降解，因此对水环境极易造成不可逆的影响。微藻作为“碳达峰、碳中和”目标进程下最具前景的生物系统，是理想的污染物低碳处理系统。能否利用微藻这类低碳生物系统对有机污染物进行绿色降解呢？如何改进传统藻株对有机污染物的处理效果是目前研究的热点。研究以紫色酸性磷酸酶介导微藻参与工业废水中有机磷污染物降解为目标，明确了紫色酸性磷酸酶基因导入微藻中可以有效改良基因工程藻株实现高效降解有机磷污染物，明确紫色酸性磷酸酶对有机磷的偏好性，即紫色酸性磷酸酶可以介导微藻优先利用有机磷进行细胞代谢，通过具体解析有机磷污染物降解过程，确定具有金属核心活性位点的紫色酸性磷酸酶作为非特异性磷酸单酯酶以生物降解有机磷污染物，随后为细胞代谢提供足够的无机磷用于基因工程藻株生物能源的生产。此外，我们发现基于微藻共代谢途径的抗生素污染物高效降解体系，明确雨生红球藻对氟喹诺酮类抗生素具有降解作用，确定甘油作为碳源添加后可以促进抗生素的高效降解，通过具体解析抗生素降解过程，发现细胞色素P450蛋白参与微藻共代谢途径的抗生素降解过程中，为雨生红球藻生物降解抗生素并提升虾青素含量提供了潜在的分子机制解释。基于此，我们提出了基于微藻体系的低碳生物降解污染物策略，以同时实现污染物降解和生物质资源化高效利用的双重效益。上述研究工作为微藻种质改良用于水体有机污染物处理提供理论支撑和新的思路。

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