UriR/RbsR参与谷氨酸棒杆菌抵御高温压力的机制研究

Microorganisms often face high temperature stress that has higher temperature than the physiological one. Studying the mechanism of high temperature stress resistance will benefit the understanding of microbial environmental adaptation and improve the industrial fermentation economy. It was discovered that inactivation of transcriptional regulator UriR and RbsR improved the high temperature stress resistance of Corynebacterium glutamicum. Since this phenomenon has not been reported, a postulated mechanism was proposed. Inactivation of UriR and RbsR activates transcription of uri and rbs operons. The transport and catabolism of uridine and ribose are enhanced, which could provide templates and energy for new protein synthesis and promote trehalose synthesis. In consequence, new proteins will be synthesized and the existing proteins will be stabilized, which will help cells resist high temperature stress. In this project, a series of gene deactivated and overexpressed mutants of uri and rbs operons will be first constructed to resolve the molecular basis of UriR and RbsR-mediated high temperature stress resistance. Second, isotope labelling-based metabolomics analysis will be conducted for different mutants under different cultivation temperatures to determine the metabolic flux change of uridine and ribose, especially the changes of trehalose and protein synthesis pathway. Finally, mutants will be constructed by deactivating its trehalose synthesis pathway and key heat shock proteins. These mutants will be used to test the effects of UriR and RbsR inactivation on high temperature stress resistance and protein synthesis. Based on the results, the mechanism of UriR/RbsR-mediated high temperature stress resistance will be unveiled.

微生物经常面临高于其生理温度的环境压力，研究高温抵御机制对理解微生物环境适应性和提高发酵工业经济性具有重要意义。项目组发现失活调控因子UriR或RbsR可显著增强谷氨酸棒杆菌的高温抵御能力，该现象未见报道。本项目提出其作用机制假设：失活该调控因子激活uri和rbs操纵子，促进细胞转运利用尿嘧啶核苷和核糖，为新生蛋白合成提供mRNA和ATP，同时推动海藻糖合成，从增强新生蛋白合成和稳定已有蛋白两方面协助细胞对抗高温压力。本项目首先对uri和rbs操纵子进行基因失活和表达调控，解析其参与细胞抵御高温压力的分子基础。其次使用同位素标记的代谢组分析，示踪不同突变株在不同温度下尿嘧啶核苷和核糖的代谢流变化，特别是海藻糖和蛋白质合成途径的变化。最后测试海藻糖合成途径和关键热激蛋白缺失时，UriR和RbsR失活对细胞耐热和蛋白合成的影响。综合实验结果揭示UriR和RbsR参与棒杆菌抵御高温压力的机制。

微生物经常面临高于其生理温度的环境压力，研究高温抵御机制对理解微生物环境适应性和提高发酵工业经济性具有重要意义。项目组前期发现一株敲除uri操纵子的谷氨酸棒杆菌突变菌株具有显著增强的高温耐受性，结合文献调研推测uri和rbs操纵子及协同调控转录因子UriR和RbsR与谷氨酸棒杆菌抵御高温压力相关。为验证该假设，首先构建了多株uri和rbs操纵子的单基因和多基因敲除突变菌株，并检测这些菌株在不同温度下的生长表型，结果表明UriR和RbsR及所在操纵子相关基因与菌株抵御高温压力无关。之后，通过对先前获得的uri操纵子敲除突变菌株的全基因组测序，发现除uri操纵子缺失外，在cgl1525和cgl1526基因间区有一段171 bp片段的意外缺失，是导致菌株高温耐受性增强的原因。因此，本项目没有继续针对UriR和RbsR的作用机制开展研究。为重新挖掘谷氨酸棒杆菌中参与抵御高温压力的转录调控因子，研究其作用机制，项目组使用碱基编辑技术，对谷氨酸棒杆菌中已报道及预测的共计182个转录因子构建了包含1.5万条gRNA的文库，通过碱基编辑构建了全部转录因子的随机突变文库，随后通过该文库在正常生理温度及高温压力下的培养筛选，以及gRNA文库丰度测序，筛选到一些与高温压力耐受相关的转录调控因子及突变体。针对其中一个可显著提升高温耐受性的突变体ClgRE71K，通过转录组学分析发现分子伴侣蛋白、压力响应、能量代谢等相关基因的表达水平发生显著变化，从而使菌株具有抵御高温压力的能力，从而解析了突变体ClgRE71K的功能机制。这是首次在基因组规模对C. glutamicum的全部转录因子进行系统研究，建立了多个未知功能转录因子与压力耐受表型的关联，为该菌株环境压力响应与耐受机制研究提供了丰富的资源。项目成果在Nature Communications等杂志发表论文7篇（均标注该项目资助），基因组规模转录因子文库构建与CglRE71K耐热机制的部分成果正在整理，准备投稿；此外，申请和授权中国发明专利3项；培养硕士研究生2名，博士研究生1名。