利福霉素SV生物合成基因簇转录调控网络的解析

Rifamycin produced by Amycolatopsis mediterranei is an ansamycin antibiotic. It and its derivatives have been the first-line drugs effective in the treatment of infection caused by pathogenic mycobacteria i.e., Mycobacterium tuberculosis and Mycobacterium leprae. Due to its clinical importance, research in rifamycin biosynthesis has been continuously active over the years. However, the regulation of rifamycin biosynthetic gene cluster (rif) has not been reported while the coordinated regulation between the secondary and primary metabolisms in A. mediterranei are unclear. Recently the AMED_0655, RifQ and GlnR proteins are identified as candidates for regulating the transcription of rif cluster, probably in a coordinated network. This project is aimed to explore the regulatory mechanism of these proteins via a series of in vivo and in vitro experiments. Then a mechanistic model for the proposed regulatory network may be formulated and novel production strains may be constructed accordingly to improve the productivity of rifamycin.

利福霉素是由地中海拟无枝酸菌产生的萘环类安沙抗生素，是治疗结核病和麻风病的一线药物。由于临床上的重要价值，利福霉素的生物合成研究一直倍受关注。迄今为止，对该生物合成基因簇（rif）转录调控的研究未见报道；同时，地中海拟无枝酸菌中次级代谢与初级代谢之间的协同调控机制也不清楚。本项目将首先分别解析新发现的三个可能与rif转录调控相关的蛋白（AMED_0655，RifQ和GlnR）调控利福霉素生物合成基因簇转录的机制，通过一系列的体内体外实验，总结出利福霉素生物合成的调控网络及作用机制；然后在此理论基础上对利福霉素的生产菌株进行改造，以期提高其利福霉素的产量。本项目研究同时具有重要的基础生物学意义和广泛的应用前景。

在本项目的资助下，我们充分解析了地中海拟无枝酸菌中rif cluster的转录受RifZ、GlnR以及RifQ三重调控的网络模型：RifZ是rif cluster的途径特异性调控因子，它可以直接激活整个rif cluster的转录；GlnR既可以通过结合rifK和rifZ的启动子来直接控制这两个基因的转录，又可以通过正调控rifZ的表达来间接地控制整个rif cluster的转录；RifQ可以直接负调控基因rifP（编码利福霉素泵出蛋白）的转录，而利福霉素B则可以使RifQ失去结合rifP启动子的能力，从而间接地实现了对rifP的转录调控。