海洋微生物Gallaecimonas xiamenensis RL01对邻苯二甲酸酯的降解机理及关键酯酶研究

Environmental pollution caused by extensive use of plastics has become more and more serious and the pollution of plastics to the marine environment has drawn much attention in recent years. The threats of plasticizers (an important component of plastics) to human health and environment have been extensively investigated. This study focused on the problems of marine plastic pollution. Phthalic acid esters (PAEs, one kind of typical plasticizer) were selected as substrate for biodegradation and one marine bacterial strain Gallaecimonas xiamenensis RL01, which could efficiently degrade various PAEs, was selected as the target strain for investigation. In this project, the complete metabolic pathway of PAEs in strain RL01 will be identified by substrate utilization and intermediates identification. Genes contributing to the hydrolysis of ester bonds in PAEs will be obtained via genomic library construction and genome sequencing and analysis. Functional verification, real-time quantitative PCR analysis and gene knockout of target genes will be performed to confirm the function of the genes in the metabolism of PAEs. Enzymatic analysis of the key esterases will be done to reveal their enzymatic and catalytic kinetic characteristics. Finally, the metabolic mechanisms of PAEs in strain RL01 will be systematically elucidated from the aspects of physiology, biochemistry and molecular mechanism. This study will provide theoretical basis for further study on the mechanism of PAEs transformation in marine ecosystem and the bioremediation of plastic pollution.

塑料大量使用所带来的环境污染问题日益突出，特别是其对海洋环境的污染在近几年被大量报道。塑化剂作为塑料的重要组分，其对人体健康及生态环境的危害已得到广泛且深入的研究。本研究针对海洋塑料污染问题，选择典型塑化剂邻苯二甲酸酯为研究对象，以可降解邻苯二甲酸酯的海洋细菌Gallaecimonas xiamenensis RL01为研究材料，拟通过底物利用和中间代谢产物鉴定解析菌株RL01降解邻苯二甲酸酯的完整代谢途径；利用基因组文库构建及基因组测序与分析获得参与邻苯二甲酸酯酯键水解的相关酶基因；通过功能验证、实时荧光定量PCR分析及基因敲除确定相关酶在邻苯二甲酸酯降解过程中的功能；基于酶学分析揭示关键酶的酶学及催化动力学特征。最终，从生理生化及分子机制水平系统阐明菌株RL01对邻苯二甲酸酯的降解机理，为深入研究海洋生态系统中邻苯二甲酸酯的转化机制及塑料污染的生物修复提供理论依据。

塑料大量使用所带来的环境污染问题日益突出，特别是其对海洋环境的污染在近几年被大量报道。塑化剂作为塑料的重要组分，其对人体健康及生态环境的危害已得到广泛且深入的研究。本研究针对海洋塑料污染问题，选择典型塑化剂邻苯二甲酸酯（phthalic acid esters, PAEs）为研究对象，广泛的开展了海洋微生物对邻苯二甲酸酯类塑化剂的降解研究，以邻苯二甲酸二（2-乙基己基）酯(di-(2-ethylhexyl) phthalate, DEHP)为底物开展了降解菌的分离，获得了降解菌6株（其中海洋细菌5株）。其中，（1）从红壤中分离获得的DEHP降解菌Gordonia terrae RL-JC02，对其环境适应性、代谢途径、降解动力学及生物修复潜能开展了系统的研究，相关研究结果已发表在期刊Science of the Total Environment（2020, 733, 139138），申请并已获授权发明专利1项；（2）对分离至海洋潮间带的DEHP降解菌Mycolicibacterium phocaicum RL-HY01开展了降解特性、代谢机理及模拟原位修复研究，相关研究结果已发表在期刊Science of the Total Environment（2021, 791, 148303），并已申请发明专利1项（目前已进入实质审查阶段）；（3）对分离自海洋沉积物的DEHP降解菌Gallaecimonas xiamenensis RL01开展了系统的研究，基于LC-MS/MS的分析，确定了菌株RL01首先通过β-氧化逐步将DEHP转化为邻苯二甲酸二乙酯（diethyl phthalate, DEP），进一步通过酯键水解将DEP转化邻苯二甲酸（phthalic acid, PA），同时，通过基因组测序与分析，获得了参与第二个酯键水解的酯酶MehpH并完成了功能验证，筛选到参与第一个酯键水解的酯酶DehpH，目前已完成克隆、表达、蛋白纯化及功能验证，正在进行野生型菌株体内基因敲除与DehpH蛋白催化位点核心氨基酸的点突变；（4）对于其他已分离的海洋降解菌，目前已完成了初步的菌株鉴定与降解特性研究，接下来将有序开展菌株对PAEs的降解机理研究。本项目的开展，为阐明海洋微生物对邻苯二甲酸酯类塑化剂的降解机理及相关环境污染的生物修复提供了重要的理论依据。