天蓝色链霉菌孤立应答调控蛋白OrrA参与次级代谢全局调控的信号传导机制研究

Many studies demonstrated that in the important industrial microbe-Streptomyces,two-component systems (TCS) plays global roles in the regulation of secondary metabolism. So far the studies are mainly centered on typical TCS, which is composed of a histidine kinase (HK) and a cognate response regulator (RR). Yet relatively little is known about the functions of untypical TCS, such as orphan HK and RR, especially for the orphan RRs, the following questions remain to be addressed, including i) whether there exist upstream HKs paired with the orphan RRs in the signal transduction pathway; ii) whether they exert global roles in secondary metabolism;iii) whether the mechanism underlying the function of orphan RRs is different from that of the RRs from typical TCS. Recently,a novel orphan RR designated as OrrA,was identified being involved in the global regulation of secondary metabolism in Streptomyces coelicolor. In this study, we hope to identify the upstream HK paired with OrrA, define the OrrA regulon and its regulatory mechanism, and finally determine the OrrA-mediated signal transduction pathway. Meanwhile, we will also carry out the functional studies of the homologous orrA and its paired HK gene from other different Streptomyces strains to reveal the conservation and diversity of the OrrA-mediated regulatory mechanism in Streptomyces. This work will elucidate the regulatory mechanism of orphan RR involved in the global regulation of secondary metabolism for the first time in Streptomyces, and will possibly reveal novel regulatory pathway for the regulation of secondary metabolism, furthermore provide theoretical guidance for metabolic enginneering in industrial Streptomyces.

研究证实，双组分系统(TCS)在重要工业微生物-链霉菌的次级代谢过程中发挥着全局性调控作用。但是，目前的研究主要关注由组氨酸激酶(HK)与应答调控蛋白(RR)配对组成的典型TCS，而关于非典型TCS如孤立HK和RR的研究则较少，尤其对孤立RR的信号通路上是否存在匹配的HK、是否参与次级代谢的全局调控、作用机制是否有别于典型TCS等亟待解决。最近，我们在天蓝色链霉菌中鉴定了一个新的参与次级代谢全局调控的孤立RR OrrA。本项目拟在前期工作的基础上,发现并鉴定与OrrA匹配的上游HK,确定其直接调控的下游靶基因群并解析其分子机制,进而阐明OrrA介导的信号传导通路。此外，将对其它链霉菌的orrA及其匹配HK的同源基因进行功能研究，以揭示OrrA调控机制的保守性及多样性。本研究将首次阐明链霉菌孤立RR参与次级代谢全局调控的分子机制，有望揭示新的次级代谢调控通路，为工业链霉菌的改造提供理论指导。

目前，关于链霉菌中双组分系统（TCS）来源的孤立应答调控蛋白（RR）的信号通路上是否存在与之匹配的上游组氨酸激酶（HK）、是否参与次级代谢的全局调控以及作用机制是否有别于典型TCS等科学问题亟待解决。在本研究中，我们在天蓝色链霉菌中鉴定了一个参与次级代谢与形态分化发育全局调控的孤立RR OrrA，其编码基因的缺失导致多种抗生素（如放线紫红素ACT与灵菌红素RED）过量产生，形态分化异常，包括菌落表明呈现粉红色、气生菌丝与孢子形成显著减少。随后，运用表型相似性与转录谱差异一致性分析结合细菌双杂交实验等技术手段，鉴定了与孤立RR OrrA相匹配的上游HK OhkA。这是首次在链霉菌中报道孤立RR与孤立HK匹配成对，组成TCS。orrA同源基因广泛存在于链霉菌中，对达托霉素产生菌-玫瑰孢链霉菌中的orrA同源基因（orrAsro）进行功能研究，证实orrAsro在该菌中行使类似的全局性调控，预示OrrA介导的次级代谢与形态分化的调控作用在链霉菌中是保守的。另外，本研究还通过对OrrA的DNA结合结构域进行了定点突变，筛选到只参与次级代谢而不参与形态分化发育调控的OrrA突变体，实现了该重要调控因子的功能优化。将其编码基因导入orrA缺失突变体，回补菌株的形态分化表型恢复成野生型，但仍保留抗生素过量产生的有利表型。由此，我们成功实现了OrrA调控的功能拆分，为后续链霉菌工业高产菌株的构建提供了很好的功能元件。最后，在ohkA突变体（ΔohkA）的基础上，运用组合代谢工程策略，包括其他抗生素合成基因簇（BGC）的缺失以及RED合成基因簇的多拷贝扩增，构建了一株高产RED的工程菌株。获得的工程菌株SBJ106（缺失了2个其他抗生素的合成基因簇，同时整合有3个RED合成基因簇）的RED产量可达到96.8 mg/g（细胞干重），相比野生型菌株M145提高了近12倍。部分研究结果已发表于Science China Life Sciences上。