大肠杆菌合成丹参素的适配性调控机制研究

Creating mircrobes for high efficient prodution of targeting compounds is the frontier of synthetic microbiology. We previously constructed the artificial pathway of danshensu in E.coli, which has strong acetate overflow and accumulates competetive byproduct L-tyrosine. The fitness regulation between danshensu module and E. coli chassis cells is the key scientific issue to be probed in this project. Acetate switch will be studied by construction and characterization of the promoter library, the regulatory targets will be uncovered, and acetate metabolism of E. coli will be dynamically controlled. Mechanism of L-tyrosine accumulation will be elucidated by anti-sense inhibition with sRNA and transcriptional analysis of the pathway genes, tyrB transcription will be attenuatted in E. coli. Multivariate including promoters, ribosome binding sites, untranslation regions and coding sequences will be designed and combined, and danshensu module will regulated via fine tunning and optimizing expression of hpaBC and d-ldh. Interplay between danshensu module and E. coli chassis will be explored by symstems biology and redox balance, and mechanism of fitness regulation will be explained. This project has important roles in the fundamental research and practical application. New methods of fitness regulation would provide the knowledge of for our understanding synthetic microbiology. Engineered E. coli strain would enhance danshensu productivity and reduce acetate discharge, which aims to profitable bio-industry with energy concervation and emission reduction.

创造高效合成目标产物的工程微生物是微生物合成生物学研究前沿。我们前期在大肠杆菌中构建了丹参素人工途径，但乙酸溢流代谢强和竞争性副产物L-酪氨酸积累。丹参素模块与底盘细胞之间的适配性调控是本项目拟解决的关键科学问题。本项目通过人工启动子文库的构建和表征，研究底盘细胞乙酸开关，发现调控靶点，进行乙酸代谢动态调控。通过sRNA反义抑制和基因转录分析，研究L-酪氨酸积累的机理，对底盘细胞tyrB进行衰减调控。通过启动子、核糖体结合位点、非翻译区、编码序列的设计和组合，对hpaBC和d-ldh进行微调和优化，研究丹参素模块的表达调控。通过系统生物学、氧化还原平衡，研究丹参素模块与底盘细胞的相互作用关系，阐明适配性调控机制。一方面创新适配性调控方法，为微生物合成生物学积累新知识。另一方面，将提高丹参素发酵效率，减少乙酸排放，达到增效、节能、减排的目的。因此，本项目具有重大科学意义和重要应用价值。

在微生物中进行天然产物的异源生物合成途径重构，并实现高效合成是合成生物学的重要方向。异源途径导入底盘细胞后，多数情况下是表达差，合成效率低下，甚至难以合成目标产物。异源途径与底盘细胞之间的适配性是功能实现的关键科学问题。本项目以植物源治疗心血管疾病的化学药物丹参素为例，研究了异源途径与底盘细胞之间的适配性。设计和重构了丹参素前体酪氨酸的底盘细胞，解除了葡萄糖抑制效应，能高效木糖或同时利用葡萄糖和木糖，将适配的酪氨酸合成模块整合到染色体上，同时敲除芳香降解基因簇和阻遏基因，获得了组成型高效合成和积累酪氨酸的功能底盘细胞。研究了丹参素模块中的两个基因及相互间关系的微调和协同表达，优化了多种启动子与丹参素模块的单和双顺反子表达模式，确定了串联启动子双顺反子模式表达丹参素模块，并整合在染色体的表达热位点上，实现丹参素途径与底盘细胞的适配性，构建了丹参素的人工细胞微生物工厂的新菌株。该菌株发酵，无需添加诱导剂和抗生素，利用糖的发酵培养基，组成型高效合成丹参素，很少积累中间产物和副产物。在实验室小型发酵罐上，丹参素产量达到克级，具有良好的产业化应用前景。