酰基化修饰及微生物合成代谢调控研究

The key scientific problem to be solved in synthetic biology is to elucidate the crosstalk and adaptive mechanism between the engineered synthetic pathway and the whole cellular metabolic network. Our studies found that the protein post-translational modifications (PTMs) might play a significant role in nutrition metabolism, supply of precursors, regulation of metabolic enzymatic activities and metabolic flux distribution in microorganisms. In this project, we will analyze the enzymatic and non-enzymatic acylation modification and regulatory signaling network in three strains: artificial engineered E. coli for pinosylvin production, Saccharopolyspora erythraea for erythromycin production, and Clostridium acetobutylicum for butanol biosynthesis. We will systematically investigate the occurrence, regulation, and dynamic change of acylation modification on metabolic enzymes in biosynthesis pathway, and their effects on enzymatic activities and metabolic flux distribution. Further, we will elucidate the acylation feedback regulation mechanism on microbial biosynthesis resulted from acyl-CoA accumulation, and also clarify the crosstalk between the artificial engineered pathways and whole cellular metabolic network at the post-translational level of protein acylation modifications. These studies will provide the theoretical principle for novel strategy of metabolic engineering and synthetic biology: ME and SynBio based on the protein acylation modifications for designing and optimizing the artificial engineered pathways through controlling acylation levels of enzymes.

揭示工程化设计的生物合成体系与细胞整体代谢网络的交互作用及适配性机制是合成生物学研究需要解决的关键科学问题。我们发现微生物固有的翻译后修饰系统在营养代谢及前体供应、合成代谢酶活性调节及代谢流分布调控中可能有重要作用。本项目拟以嵌入赤松素合成途径的工程化大肠杆菌、红霉糖孢菌（红霉素合成菌）及丙酮丁醇梭菌（丁醇生产菌）为研究对象，解析微生物酰基化修饰系统（酶、非酶）及调控信号通路，系统研究合成代谢相关酶酰基化修饰的发生、调节和动态变化，探讨酰基化修饰对代谢酶活性的调节效应机制及其对代谢流分布的影响，阐明酰基CoA积累对微生物合成代谢的多重作用及酰基化反馈调控机理，从酰基CoA平衡及蛋白质翻译后修饰角度揭示微生物内源或嵌入的人工生物合成体系与细胞整体代谢网络的互作机制，通过调节代谢酶酰基化修饰提高合成代谢的适配性及相对代谢流，为建立基于翻译后修饰调控的代谢工程及合成生物学设计提供理论基础。

本项目圆满完成了既定任务，以常用微生物底盘工业放线菌和大肠杆菌为主要研究对象，发现了基于营养代谢的酰基CoA积累诱发翻译后修饰对微生物合成代谢的反馈抑制效应，解析了酰基化修饰平衡营养代谢与合成代谢的分子机制，建立了翻译后修饰代谢工程（PTM-ME）策略，应用红霉素、丁醇、赤松素等高产菌株的改造实例，证实了PTM-ME策略能够消除酰基化修饰引起的反馈抑制，提升合成代谢途径（内源与外源）与底盘细胞的适配性，强化产物合成；阐明了第二信使与酰基化修饰互作调控合成代谢的分子机制，建立第二信使代谢工程策略，实现了红霉素高产菌株的代谢工程改造；解析了蛋白质酰基化修饰调控全局性蛋白质翻译以及CRISPR基因编辑效率的分子机制，开发了新型翻译调控元件和基因编辑元件；拓展研究了致病性放线菌分枝杆菌中酰基化修饰调控信号通路以及酰基化修饰代谢调控新功能，揭示了基于翻译后修饰的理论与方法研究对于高效合成生物系统工程化设计以及结核病防治都具有重要意义。成果在Cell Chem Biol, Mol Cell Proteomics, Mol Microbiol, ACS Chem Biol, ACS Synth Biol, J Bacteriol, PNAS Nexus等期刊发表标注论文52篇；申请发明专利6项；培养博士后2名，培养博士研究生17名，已毕业11名，上海市优秀毕业生1名；硕士研究生15名，已毕业8名；项目组成员在国内外学术大会报告10余次，在微生物翻译后修饰代谢调控的研究和合成生物学应用中发挥了重要作用。