放线菌全局性调控蛋白GlnR调控细胞代谢的分子机理

Nitrogen metabolism in actinomycetes is governed by an orphan global regulator, GlnR, different from that of Escherichia coli, which is controlled by a typical two component system of NtrB/NtrC. Till now, most GlnR associated studies are conducted in Streptomyces coelicolor, a model species of actinomycete, focusing on the characterization of GlnR target genes related to nitrogen metabolism. However, the function of GlnR as well as its operating mechanism, particularly, that regarding the modulation of GlnR activities are far more complicated than what we have ever imagined before. GlnR has been found to involve in the regulation of physiological processes other than nitrogen metabolism, e.g. carbon and secondary metabolisms. Besides, as an orphan response regulator, it is still unclear how GlnR is regulated either at the transcriptional level or by posttranslational modification, both in response to different extracellular nitrogen sources. In addition, it is also important to learn how many GlnR targets exist in pathways other than nitrogen metabolism. To answer these scientific questions, this project is designed to conduct a systematic study on GlnR of S. coelicolor. The upstream regulator for glnR will be identified along with delineating its regulatory mechanisms. The probable posttranslational modification of GlnR will be characterized with respect to its regulatory function upon GlnR activities. The novel GlnR target genes will be screened and the GlnR regulon will be further explored with respect to different metabolic conditions. In summary, we hope to construct the global regulatory network of GlnR in regulating both primary and secondary metabolisms and provide a theoretical guidance for genetic engineering of industrial actinomycete strains.

放线菌的氮代谢因与次级代谢密切相关而备受关注。与受经典双组分系统调控的肠杆菌氮代谢不同，放线菌的氮代谢调控由孤儿应答调控蛋白GlnR负责。目前对GlnR的研究多集中于鉴定其与氮代谢相关的靶标基因。但是，GlnR除参与氮代谢调控外，还参与调控碳代谢和次级代谢等生理过程，预示其调控网络的复杂性。另外，作为非典型的孤儿应答调控蛋白，GlnR蛋白的活力及其编码基因glnR的转录受到何种分子机理调控以及其如何调控氮代谢以外的其它代谢途径等重要科学问题都尚未被真正认识。为此，本项目将在现有工作基础上，以模式放线菌天蓝色链霉菌GlnR为主要研究对象，寻找参与调控glnR转录的上游调控因子并揭示其调控机制，鉴定GlnR翻译后修饰类型及其对GlnR调控功能的影响，筛选并鉴定GlnR新的下游靶标基因，最终解析GlnR介导的初级代谢与次级代谢全局性调控网络，为放线菌的遗传改造等重要实践课题提供理论指导。

作为放线菌全局性氮代谢调控蛋白， GlnR调控了氮代谢过程中的所有关键步骤，包括铵转运、硝酸盐同化以及谷氨酰胺的合成，等等。在鉴定了GlnR的转录调控元件后，越来越多非氮代谢通路的靶标基因被发现，预示着GlnR功能不仅仅局限于氮代谢调控。为了解析GlnR全局性调控细胞代谢的分子机理，本项目对GlnR蛋白的翻译后修饰、glnR基因自身的转录调控以及GlnR调控次级代谢的机制都做了深入的研究。本项目借助于放线菌内源表达纯化的GlnR蛋白，利用蛋白质质谱鉴定了GlnR受含甲基化、乙酰化、琥珀酰化、类泛素化及磷酸化修饰，并且这些修饰是通过影响GlnR与靶标DNA的结合力来影响GlnR的转录调控活力。通过研究放线菌目分枝杆菌科的耻垢分枝杆菌（Mycolicibacterium smegmatis）glnR的转录调控，本项目证明了glnR基因受其自身的转录调控，且这种调控也存在于同科的致病结核分枝杆菌（Mycobacterium tuberculosis）中；而在研究该GlnR调控靶标基因的过程中，又发现并鉴定了一种全新类型的同化型硝酸还原酶NasN。在研究GlnR全局性调控网络方面，本项目进一步鉴定了GlnR结合元件序列，挖掘了参与抗生素生物合成基因簇的新型靶标位点，证明了GlnR通过协同调控菌体的初级和次级代谢来应对外界氮源供给的胁迫。此外，本项目还开发了DNA片段和放线菌基因组编辑系统，为今后更便利地开展代谢的调控机制研究提供了新的工具，也为代谢调控分子机制指导下的工业生产菌株高效改造打下了基础；其中CRISPR-Cas12a的研究及开发工作，还为新型分子诊断技术开拓了新的方向。