分枝杆菌YC-RL4高效降解酞酸酯的遗传调控网络解析

Phthalate is widely applied as plasticizer and additive of other chemical products. Phthalate is universally distributed in the environment, and is extremely difficult to degrade under natural conditions, which has done damage to environment safety and health of human. Microbial metabolism is the main approach for elimination of phthalate. Some strains including Mycobacterium sp. YC-RL4 and Gordonia alkanivorans YC-RL2 isolated previously by the applicant were capable of degrading phthalate efficiently. Based on the accomplished degradation properties, cloning and identification of esterase genes and complete genome sequencing of strain YC-RL4, the following experiments will be performed： (1) demonstrating the efficient catalytic mechanism of the esterases DPH1, DPH2 and MPH1 by analyzing the enzymatic characteristics and 3D structures; (2)screening genes involved in phthalate metabolism by analysis of the transcriptome and proteome of strain YC-RL4 under phthalate induction; (3)verifying the candidate genes by RT-qPCR and gene knockout using CRISPR/Cas9, and analyzing the interaction of regulatory proteins and DNA. In conclusion, the model of phthalate metabolism and regulation network will be built, and the molecular mechanism of phthalate degradation will be proposed. The results will be theoretically and practically significant for the application and sustainable development of resources of phthalate degradation.

酞酸酯被广泛用作塑化剂及化工产品的添加剂，在环境中分布广泛，自然降解极慢，严重危害环境安全和人类健康，微生物代谢是酞酸酯降解的主要途径。申请者已分离到酞酸酯高效降解菌Mycobacterium sp.YC-RL4、Gordonia alkanivorans YC-RL2，并完成了YC-RL4对酞酸酯的降解特性、酯酶基因克隆及鉴定、全基因组测序等工作。本项目拟在此基础上进行以下研究：（1）进行酞酸酯酶DPH1、DPH2和MPH1酶学特征与酶三维结构分析以解析其催化的分子机制；（2）通过酞酸酯诱导下转录组和蛋白组的研究鉴定酞酸酯代谢相关基因；（3）用RT-qPCR、CRISPR/Cas9基因敲除验证候选基因，BIAcore分析调控蛋白与DNA的相互作用，最终构建酞酸酯降解的完整遗传调控网络，全面深入揭示酞酸酯的降解机理，研究结果将对酞酸酯降解资源的开发与可持续应用具有重要的理论和实践意义。

酞酸酯被广泛用作塑化剂及化工产品的添加剂，在环境中分布广泛，自然降解极慢，严重危害环境安全和人类健康，微生物代谢是酞酸酯降解的主要途径。以酞酸酯高效降解菌Mycobacterium sp. YC-RL4为研究对象，通过大容量基因组文库文库构建，从中鉴定了5个二酯酶基因（dphM1、dphM2、dphM3、dphM4和dphM5）；选取对邻苯二甲酸丁酯（DBP）水解活性较高的dphM1和dphM3，进行酶学性质及催化机制的系统研究。在大肠杆菌中对DphM1和DphM3进行异源表达，借助组氨酸标签获得高纯度重组蛋白，并探究了温度、pH、金属离子及其它酶抑制剂对酶活性的影响；以常见的13种酞酸酯为底物，进行酶促反应动力学分析，发现DphM3的最适底物为中等长度侧链的DBP，DphM1的最适底物为短侧链的邻苯二甲酸甲酯（DMP）。通过X射线衍射解析蛋白质晶体结构，发现DphM3含有10个α螺旋和8个β片层，构成帽子结构域和催化结构域，通过DphM3与DBP的分子对接及定点突变，鉴定了催化三联体（Ser156-His281-Asp251）、氧离子洞（Ala157和Gly85）及参与DBP结合的多种氨基酸（L204、V209、F39、Y183、F195、W188、S213、W86、Y94、I203和D155）；同源建模获得DphM1的三维结构，含有帽子结构域和催化结构域。通过DphM1与DBP的分子对接及定点突变，鉴定了催化三联体（Ser210-His336-Asp306）、氧离子洞（Gly138、Gly139和Ala211）及参与DBP结合的氨基酸（W240、L259、Y238、I264、L308、D209、F140、F267和W340）。通过三代测序技术获得了菌株YC-RL4全基因序列，从中筛选到一个PAEs单酯酶基因MphMB1并进行了功能验证，鉴定到参与邻苯二甲酸代谢的1个pht基因簇和2个pca基因簇。综合以上结果，推测了菌株YC-RL4对酞酸酯的完整代谢通路。在酞酸酯胁迫下，分离到多株酞酸酯降解菌，包括两株耐碱的戈登氏菌、一株耐盐的红球菌和一株耐酸的肠杆菌。本研究为酞酸酯的生物降解提供了丰富的微生物资源，对系统阐述菌株的高效降解能力及菌株的实际应用提供了理论参考与指导。