地衣芽孢杆菌胞外多糖无细胞表达平台构建

细菌胞外多糖赋予细胞重要生理功能，并展示出巨大的应用潜力。其复杂的代谢机制决定了多糖种类多，功能迥异。要明晰多糖合成的遗传控制原理进而促进其应用，尚有挖掘的空间和必要。本项目拟引入无细胞表达系统（Cell-free gene expression, CFE），基于前期对Bacillus licheniformis CGMCC 2876胞外多糖合成机制的系统工作基础，利用体外培养模式探究限制菌株多糖合成代谢通路总体性能的eps基因(簇）。通过构建iPROBE系统对菌株多糖代谢反应通路进行模拟，实现精确评估和控制代谢过程关键蛋白，揭示其最优合成路径。本项目旨在建立基于细菌多糖合成的非模式生物无细胞表达系统，以打破传统研究手段对生物细胞胞内反应难以控制的局限性。不仅为解决多糖类生物大分子的复杂酶蛋白反应提供更加有效、便捷的研究平台，亦为其他工业用非模式菌株的无细胞生物合成系统建立奠定重要基础。

Bacterial extracellular polysaccharide (EPS) endows cells with important physiological functions and shows great application potential. There is a wide variety of polysaccharides with different functions because of the complex metabolic mechanism. Therefore, it’s necessary to clarify the genetic control principles of polysaccharide synthesis so as to promote its application better. This project plans to introduce a cell-free gene expression (CFE) system based on our previous work of the mechanism of EPS from Bacillus licheniformis CGMCC 2876. The eps gene cluster will be further investigated by in vitro culture model, which will be accurately evaluated and controlled by constructing an iPROBE (in vitro prototyping and rapid optimization of biosynthetic enzymes) model. An optimal EPS synthesis pathway will then be hypothesized and verified. This project aims to establish a non-model bacterial CFE system based on exopolysaccharide synthesis. The CFE system will provide a more effective and convenient platform for research on complex metabolic networks of biomacromolecules such as polysaccharides, breaking the limitations of traditional in vivo research methods which are difficult to control the intracellular bioreactions. The construction of this Bacillus-CFE platform also lays an important foundation for the establishment of CFE system for other industrial non-model strains and promote its application in synthetic biology.