恶臭假单胞菌SJTE-1降解17α-乙炔基雌二醇的分子机制研究

17α-ethinylestradiol is one typical pollutant of environmental endocrine disrupting chemicals and pharmaceuticals and personal care products, with high biological activity, stable structure, strong toxicity and low remove efficiency. Biodegradation is the efficient method to eliminate this chemical. But as the unclear genetic background of the degradation strains, the wide existence of gene redundancy, and the lack of gene modification system, the biodegrading mechanism and metabolic pathways of 17α-ethinylestradiol in bacteria are still not clear. Pseudomonas putida SJTE-1 isolated by applicants has been confirmed with the capability of using 17α-ethinylestradiol as sole carbon source and biodegrading it into chemicals with no estrogenic activity. Its whole genome sequence and comparative proteomic analysis results showed that its genome contains multiple potential degradation genes. In this project, taking strain SJTE-1 as the research object, we plan to identify the degradation metabolites of 17α-ethinylestradiol and render its metabolic pathway. With the existing efficient continuous and scarless gene modification system, we will delete the potential 17α-ethinylestradiol-degrading genes one by one, construct the single- and multiple-knockout mutants, analyze their degradation properties, and determine the key degradation genes. Then, we will express the key genes in vitro, study the enzymatic properties and catalytic mechanisms of the recombinant proteins, and finally reveal the molecular degradation mechanism of 17α-ethinylestradiol in Pseudomonas. This research can promote the biodegradation mechanism study of 17α-ethinylestradiol, and provide an important theoretical basis for its environmental bioremediation.

17α-乙炔基雌二醇为口服避孕药与激素补充药的主要有效成分，是一种重要的环境污染物，结构稳定，难去除，毒性强。生物降解是其有效去除手段；但因降解菌株遗传背景不明、基因冗余存在和基因改造系统缺乏，17α-乙炔基雌二醇的生物降解机制与代谢途径仍不明确。申请人已鉴定的恶臭假单胞菌SJTE-1可直接以17α-乙炔基雌二醇为唯一碳源并将其高效降解；全基因组与蛋白组学结果显示其含有多个潜在降解基因。本项目拟以该菌株为对象，鉴定其代谢17α-乙炔基雌二醇的产物，绘制其代谢通路；利用已有的假单胞菌无痕连续基因改造系统，对预测的17α-乙炔基雌二醇降解基因进行单敲除和多基因组合敲除，鉴定突变体的降解性质，确定降解关键基因；异源表达关键基因，研究重组蛋白的酶学性质与催化机制，最终阐明假单胞菌降解17α-乙炔基雌二醇的分子机理。本研究可推动微生物降解17α-乙炔基雌二醇的机制研究，为其环境修复应用提供理论依据。

17α-乙炔基雌二醇为口服避孕药与激素补充药的主要有效成分，是一种重要的环境污染物。生物降解是其有效去除手段；但其机制与代谢途径仍不明确。本项目的主要研究内容是以假单胞菌菌株SJTE-1为对象，鉴定并明确其代谢17α-乙炔基雌二醇的中间产物、降解基因与关键酶等，阐明分子机理。主要进展包括：1. 鉴定了恶臭假单胞菌SJTE-1对不同浓度17α-乙炔基雌二醇的代谢性质。确定其可将17α-乙炔基雌二醇代谢转化为雌酮后，E1进一步在C9位发生了羟化与酮化开环，再进行后续降解。2.解析了假单胞菌降解雌激素的整体代谢网络与相关通路，证实了细菌可全面特异提高相关代谢通路蛋白的表达水平和重要的能量代谢水平来适应胁迫环境。3.确定了假单胞菌SJTE-1胞内存在多个酶参与雌激素的代谢，多个同工酶催化了第一步关键反应；两个短链脱氢酶和两P450是该菌株转化17α-乙炔基雌二醇的关键酶。4. 鉴定了多个可调控雌激素降解酶活性的转录因子，明确多个不同家族的转录因子同时调控单个酶的多重调控模式。5.分离鉴定了多株环境适应性强的雌激素高效降解菌株，解析了其降解性质与降解酶等。本项目达到了预期研究目标，可推动微生物降解17α-乙炔基雌二醇的机制研究，为其环境修复应用提供理论依据。