海洋拟无枝菌酸菌的基因组挖掘和天然产物沉默基因簇的靶向特异激活

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis. Development of new anti-TB with potential new molecular mechanism is facing an urgent situation according to the severe drug resistance and TB-HIV co-infection. Marine microbes play a crucial role for drug leads discovery. In addition, the development and application of genome sequencing technology provide an efficient tool for people to study microbial natural products. This proposal describes a genome mining- and integrated cryptic gene clusters activation-based anti-TB natural products investigation strategy. Based on pool sequencing genomics data of 3000 strains of marine microbes, a systematic bioinformatics analysis has been performed and highlighted serial strains of the genus Amycolatopsis. The novel secondary metabolites biosynthetic gene clusters will be identified and subsequently the double Reporter-Guided Mutant Selection (dRGMS), heterologous expression, and inducible expression will be applied for activating the expression of targeted cryptic gene clusters. A nanoDESI and MS/MS molecular networking method has been established for quick de-replication and identification of natural products. The bioassay-guided isolation and structure elucidation will be performed for purification of targeted compounds. This systematic anti-TB drug leads investigation platform will provide an efficient approach to solve the shortage of anti-TB drugs in clinic.

结核病(Tuberculosis, TB)为结核分枝杆菌引起的慢性传染病，耐药结核的卷土重来以及TB与HIV共感染急需具有新颖结构与作用机制的抗结核药物的研制。海洋微生物是药物开发的一个重要资源，基因组测序技术的发展和应用为人类研究微生物天然产物药物的研究提供了更高效率的工具。本项目拟对本实验室已经获得3000多株海洋放线菌混合测序的基因组进行生物信息学分析，对选择出海洋拟无枝菌酸菌潜力菌株进行基因组三代测序，组装和注释，鉴定新的合成基因簇。进一步以自主开发的双报告基因引导的基因簇激活系统(dRGMS)为主，同时整合异源表达，可控性诱导表达等合成生物学手段，实现对目标隐性基因簇的激活；基于已经构建的NanoDESI和二级质谱小分子网络技术的天然产物快速排重方法，快速发现有效表达的新颖化合物，通过活性追踪分离、鉴定新颖化合物，这为抗TB药物先导化合物的研究具有重要的意义。

海洋微生物是微生物药物研究的重要来源。临床上多耐药病原菌尤其是革兰氏阴性菌的严峻形势，迫切需要具有新的药物作用机制的药物候选物。本项目结合基因组挖掘、突变生物合成以及秀丽隐杆线虫-铜绿假单胞菌的感染模型活性筛选相结合的策略，研究海洋拟无枝菌酸菌Amycolatopsis methanolica 239T的活性化合物。通过对Amycolatopsis methanolica 239T的合成基因簇进行分析，结合对其发酵液提取物活性追踪、二级质谱小分子网络策略进行化合物的快速鉴定，优选出能够产生amychelin类铁载体化合物对应的基因簇进行深入研究。结合突变生物合成，我们重点对合成基因簇中的水杨酸生物合成旁路进行研究，饲喂多种含氟和含氯的前体物质，成功分离和鉴定一系列新颖amychelin类铁载体化合物。其中，化合物fluoroamychelin I能延长铜绿假单胞菌感染的救秀丽线虫的寿命，EC50为1.4 M，效果优于阳性对照抗生素ceftazidime和meropenem。此外，化合物fluoroamychelin I在体外实验中，不能直接抑制铜绿假单胞菌的生长，因此，该化合物是通过毒力因子阻断作用机制而起作用的。本项目的研究成果可进一步开发成药物先导化合物。