检测脯氨酸代谢的遗传编码荧光探针

Proline, as one of the 20 basic amino acids, plays a key role in energy metabolism. It can scavenge ROS, serve as molecular chaperones, enhance the resistance of plants, which is also in close relationship with human diseases including tumors and cancer. In view of the importance of proline, high spatial, in vivo, real-time dynamic detection of proline in living cells plays an crucial role in related research. However, traditional biochemical detection methods require tissue or cell homogenization, and it is difficult to monitor the metabolism of proline in single cells, which seriously restricts the development of related fields. Therefore, to develop a real-time monitoring method has turned into an urgent problem remained to be solved. Based on synthetic biology, this study develops a high performance encoded fluorescent sensor for proline, which may shed some light on revealing the relationship between proline metabolism abnormalities and liver cancer, and exploring changes in proline synthesis and decomposition pathways. In addition, the fluorescent sensor can also be applied to study the underlying role of proline in stress resistance. This research provides flexible and powerful research technology for basic research of life sciences, which not only offers valuable tools for medical research and human health, but also serves as an alternative and effective means for research on drought resistance and salt and alkali resistance of plants, indicating great scientific significance and wide socio-economic value.

脯氨酸作为20种氨基酸之一，在能量代谢中起着关键的作用，能够清除活性氧，具有分子伴侣的功能，可以增强植物的抗逆性，也与肿瘤、癌症等人类疾病密切相关。鉴于脯氨酸的重要性，对活细胞内的脯氨酸进行高时空、原位、实时动态地检测对相关研究举足轻重。然而，传统的生物化学检测方法都需要组织或细胞均浆液，难以监测单细胞内脯氨酸的代谢，严重制约了相关领域的发展。因此发展实时动态的活细胞内脯氨酸监测技术成为亟待解决的问题。本研究基于合成生物学，开发高性能的脯氨酸遗传编码荧光探针，利用该探针揭示脯氨酸代谢异常与肝癌之间的联系，探究脯氨酸合成与分解途径的变化。此外，利用该探针还可研究脯氨酸在抗逆性中的重要作用。本研究不仅为生命科学基础研究提供一种灵活有力的研究技术，而且为医学研究与人类健康提供极具价值的工具，也为植物抗旱、耐盐碱方面的研究提供一种有效手段，展现出非常强的科学意义和社会经济价值。

本项目围绕脯氨酸代谢实时监测这一重要科学问题，创新研发检测技术。在青年基金的资助下，我们建立了微生物高通量筛选与定向进化体系，创建光激活调控新技术，可使人们以前所未有的精度来控制大肠杆菌生命活动，并构建复杂基因回路为合成生物学提供有价值的元器件和模块。基于此，我们通过理性设计和定向进化策略，筛选获得高特异、高响应、超灵敏的脯氨酸高性能荧光探针，实现了活细胞、亚细胞脯氨酸代谢的实时动态监测。我们还聚焦中心碳代谢创建了性质优异的乳酸探针FiLa，首次揭示哺乳动物细胞线粒体高度富集乳酸；高通量药物筛选表明乳酸是感知各种代谢活动的关键枢纽；建立临床样本即时检测体系，发现母系遗传性糖尿病(MIDD)患者尿液中乳酸水平显著高于健康人、I型或II型糖尿病，为临床诊断提供了重要依据。此外，我们还开发了丙酮酸荧光探针和柠檬酸荧光探针。上述相关工作发表在国际著名学术期刊Cell Metabolism和Nucleic Acids Research；授权中国发明专利3项，申请国际PCT发明专利8项和中国发明专利14项。上述创新技术为生命现象解析、疾病机制探索、创新药物发现、疾病快速诊断等生命医学领域提供了创新性的研究工具，助力人民生命健康。