代谢流和代谢组技术研究大肠杆菌碳中心代谢系统氧化应激适应时的动力学过程和稳态机制

The project aims at testing a hypothesis, which is based on our previous work about the behavior of Escherichia coli's central carbon metabolism in response to paraquat induced oxidative stress. The hypothesis supposes that the metabolites level and enzyme activity in the metabolism system would undergo a chaos state after paraquat induced oxidative stimulus. As the cell gradually adapted to the extreme oxidative environment, the metabolism system would enter a steady state and exhibit an unchanged metabolic flux distribution. The hypothesis also assumed that the transcriptional network in oxidative stress and carbon metabolism had played a major role in controlling the recovery of the metabolism system from the chaos state. To test this hypothesis, we planned to monitor the metabolome and enzyme activity of central carbon metabolism at continuous and fixed time points after the stimulation of the Escherichia coli by paraquat, and compare the metabolites concentration, enzyme activity and metabolic flux distribution between the cell before and long after the oxidative stress. In addition, the behaviors of Escherichia coli mutants for the relevant transcriptional factors and sRNAs will be observed in parallel.The results of this project would helpful in elucidating the counteraction and active adaptation mechanism of the Escherichia coli to the oxidative stress, and would broaden our knowledge about the coordination in the regulation of central carbon metabolism. Thus, this project has been of interest and significance to both the systems biology and pathology communities.

本课题假设大肠杆菌受到paraquat引起的超氧化应激之后，中心碳代谢系统中关键途径的酶活和中间代谢物的水平,因而代谢流量,会产生发生迅速改变。随着细胞适应氧化胁迫，代谢流量回到胁迫前的水平。还猜测转录/翻译调控网络对于代谢系统回复稳态起到重要作用。为验证这些猜想，本课题在给予大肠杆菌超氧化刺激后，在连续，固定的时间点上检测碳中心代谢系统代谢物浓度和酶活，刻画氧化应激适应过程中代谢系统动力学轨迹。同时，通过氧化应激适应前后，其酶活，代谢物浓度和代谢流分布的对比阐明代谢流稳态的机制。针对不同转录因子/sRNAs缺陷型菌株进行类似实验，能够分析和筛选对于代谢系统回复稳态起到主要作用的转录因子和调控通路。本课题帮助阐明细菌对于氧化应激的反作用和主动适应机制，阐明碳中心代谢系统各途径的协同调控和代谢稳态机制，能够深入了解细胞和环境的相互作用。因此本课题在系统生物学和病理学的双重意义。

本课题假定大肠杆菌受到paraquat或者双氧水引起的氧化应激之后，中心碳代谢系统中关键途径的酶活和中间代谢物的水平,因而代谢流量,会产生发生迅速改变。还认为不同层面数据的动态过程存在协同调控，并最终回归一定的稳态。本课题在给予大肠杆菌瞬时的超氧化刺激和过氧化刺激1分钟内，在连续，固定的时间点上初步检测碳中心代谢系统代谢酶的mRNA浓度，部分酶活和代谢物的13C标记谱。因而，部分刻画了氧化应激适应过程极短时内代谢系统的动力学轨迹。此外，还构建了世界上首个代谢流量数据储存，可视化与比较分析平台；通过基元模式分析揭示环形反应途径是中心碳代谢的NADPH再生的有力途径；通过代谢通量比率分析和多目标优化揭示了大肠杆菌对超氧化应激时中心碳代谢系统内的此消彼长关系。本课题帮助阐明细菌对于氧化应激的反作用和主动适应机制，阐明碳中心代谢系统各途径的协同调控和代谢稳态机制。