氧自由基在ε-聚赖氨酸生物合成中的生理功能研究

ε-聚赖氨酸（ε-PL）生物合成严格依赖于外界酸环境和高强度细胞呼吸代谢，故极易引发ROS的大量积累，而目前ROS在ε-PL生物合成中的生理功能尚不清楚。申请人前期研究发现，一定浓度范围ROS能大幅强化ε-PL合成，而过高浓度ROS又将削弱ε-PL发酵水平，这说明ROS极有可能在ε-PL合成中发挥重要的调控作用。本课题拟采用恒化培养手段，构建具有不同ROS梯度的细胞样本；基于此，从ROS介导的基因转录调控角度解析其积极效应，从氧化损伤、细胞膜机能、ε-PL合成关键酶活性、能量和碳代谢的角度解析其消极效应。由此通过细胞生理、合成ε-PL代谢和基因转录三个层面的研究，系统阐明小白链霉菌对不同水平ROS的生理响应机制及其与ε-PL合成之间的关联。研究结果不仅可揭示ROS在ε-PL生物合成中的生理作用，还将为后续基于自由基水平调控的代谢工程改造和生化工程优化提供理论指导，具有一定的理论和实践意义。

The fermentation process of ε-poly-L-lysine (ε-PL) is heavily dependent on external acid environment and intensive cell respiration, which can easily cause excessive accumulation of reactive oxygen species (ROS). However, there is a lack of mechanism understanding of the effect of ROS to cell physiology and ε-PL biosynthesis currently. Our previous study indicates that, ROS at an appropriate level promotes the cell physiological performance for ε-PL biosynthesis, while ROS at extremely high concentration decreases the ε-PL productivity, which indicates that ROS might be a crucial factor in ε-PL biosynthesis. In this program, chemostat cultures at different ROS levels are first established to obtain cells samples for following physiological assays; Based on this, the positive effects of ROS are analyzed at the genes transcription level, while the negative parts are studied at enzymes and metabolism levels, including antioxidant damage, biomembrane status and enzymes activities, energy and central carbon metabolism for ε-PL biosynthesis. Finally, the physiological mechanisms of Streptomyces albulus responding to ROS and its correlation to ε-PL biosynthesis were systematically studied at the levels of cell physiology, metabolism for ε-PL biosynthesis and gene transcription. The excepted results will not only disclose the physiological effects of ROS in ε-PL production, but also lay the foundation for the regulation of cellular redox state through metabolic engineering and biochemical engineering thereafter, which are of great theoretical and practical significances.