弗氏链霉菌中十字孢碱生物合成的调控机制

Staurosporine, produced by Streptomyces fradiae 4.576, is an excellent compound with antitumor activity (to inhibit protein kinase or DNA topoisomerase). However, the regulatory mechanism of staurosporine biosynthesis has not been reported so far and its production yield is still low, which significantly hindered the development of new derivatives based on staurosporine through chemical semi-synthesis and their clinical application. staR* is an important cluster-situated regulatory gene for staurosporine biosynthesis in S. fradiae. In this project, the regulatory mechanism of staR* will be intensively studied by a series of approaches such as gene disruption, genetic complementation, EMSA, foot printing and transcriptional regulatory analysis. The interactions between regulatory protein and signaling molecules will be also investigated. This project is aimed to reveal regulatory mechanism of staurosporine biosynthesis and to lay foundations for strain optimization and construction of staurosporine high-yield producing strains.

十字孢碱是由弗氏链霉菌4.576产生的一种具有抗肿瘤活性（对蛋白激酶或DNA拓扑异构酶有抑制作用）的化合物。到目前为止有关十字孢碱生物合成的调控机制尚无任何报道，其产量仍处于较低水平，这使得以十字孢碱为前体进行半化学合成获得新衍生物的研究及其在临床药物开发中的应用受到了极大限制。因此本申请在前期工作的基础上，拟通过对弗氏链霉菌4.576中发现的十字孢碱生物合成基因簇中的重要调控基因（staR*）进行基因敲除、遗传互补、凝胶阻滞、足迹实验、转录调控分析、蛋白与信号分子互作等研究，以揭示十字孢碱生物合成的调控机制，为十字孢碱产生菌的优化改造和高产工程菌株的构建奠定基础。

十字孢碱属于吲哚咔唑类化合物，对多种蛋白激酶都有抑制作用，被认为是抗肿瘤药物的优良先导化合物。本研究在弗氏链霉菌CGMCC 4.576中鉴定出两个独立的基因簇(基因簇A和簇B)，分别对应K-252c (糖基苷元配体)和糖基部分的生物合成，并在天蓝色链霉菌M1146中分别或一起进行异源表达。StaR是一个簇内LAL家族调控子，通过结合staO-staC和staG-staN的启动子区域激活十字孢碱的生物合成。通过逐步截短staO和staG的启动子，发现保守序列GGGGG和GCGCG，并通过突变实验进一步确定。在GYM培养基中，通过添加0.01 g/L-1 FeSO4过表达staR，与野生型弗氏链霉菌CGMCC 4.576相比，十字孢碱的产量增加到5.2倍。本研究结果为激活子介导的十字孢碱产量提高和为具有临床应用价值的吲哚咔唑类化合物调控机制的深入解析奠定基础。