产聚富马酸丁二醇酯大肠杆菌的代谢工程改造研究

本项目拟用微生物直接合成生物可降解非天然聚合物聚富马酸-1,4-丁二醇酯(PBF)，生物合成结构性能优良的聚合物，推动绿色生物制造合成生物基材料的产业目标。PBF不仅具有优良的化学结构和热力学性能，而且由于其链上的碳碳双键具有较强的加工性能，可用于生产各种具有不同支链的化工产品。本项目拟在大肠杆菌共积累富马酸及1,4-丁二醇的基础上，采用生物信息学手段分析现有的富马酸酰基连接酶，筛选表达能够活化富马酸羧基的酰基连接酶，构建富马酸酰基-CoA或富马酸酰基-AMP的合成途径；基于链霉菌来源的富马酸酰基转移酶以及项目组前期研究的来源于不动杆菌的双功能酰基转移酶，筛选表达能够合成PBF的酰基转移酶，构建新的PBF生物合成途径；根据离子泵的响应机制构建生物传感器，使工程菌能够自主调控富马酸模块和1,4 -丁二醇积累模块中碳流的分布平衡，研究细胞工厂内代谢与调控的适配机制，促进PBF的高效合成。

In this project, the biodegradable non-natural polymer polyfumarate-1, 4-butanediol ester (PBF) was synthesized directly by microorganism to biosynthesis polymers with excellent structure and performance, to promote green bio-manufacturing for bio-materials. PBF not only has excellent chemical structure and thermodynamic properties, but also can be used to produce a variety of chemical products with different branched chains, including polymers with comb and brush structures, due to its strong processing properties of the carbon-carbon double bond on the chain. In this project, we plan to use bioinformatics methods for protein sequence alignment and homology analysis, based on the existing fumaric acid acyl ligase, to screen an optimum CoA ligase/AMP ligase function on fumaric acid to construct fumaric acid CoA/AMP synthesis pathway. On the foundation of it, based on the source of Streptomyces fumaric acid acyltransferase and the acyltransferase from Acinetobacter researched in our previous work, we screen an optimum acyltransferase using the same method above to construct PBF synthesis pathway; regulation of the balance of carbon flow by pH responsive biosensor: according to the response mechanism of the ion pump, the constructed engineering bacteria can independently regulate the distribution balance of carbon flow in the fumaric acid module and 1, 4-butanediol accumulation module by pH responsive biosensor, studying the adaptation mechanism and regulation metabolism in the cell factory, which lay a foundation for further promoting the efficient synthesis of PBF.