枯草芽孢杆菌表面活性素合成及调控研究

The low fermentation efficiency of the bio-surfactin restricts its industrial production and application, and the fundamental reason for this lies in the lack of high yield producers. After resolved the main problem (the expression of surfactin synthase) in biosurfactin synthesis, in this work, the mechanism of synthesis and regulation of surfactin will be studied based on the molecular structure of surfactin and the metabolic network of Bacillus subtilis. This research includes the following key points: (1) study the effect of 3-hydroxy fatty acid on the synthesis of surfactin by knocking down/off or overexpression of some genes; (2) study the effect of cofactor on the synthesis of surfactin by interference the intracellular cofactor, and optimize the intracellular cofactor content and redox status for surfactin production; (3) complementary the synthesis and regulation metabolic network of surfacin in Bacillus subtilis, and construct a high yield engineered strain. This work will help people to understand the synthesis and regulating mechanism of biosurfactin in Bacillus subtilis, and it will substantially increase the surfactin titer of genetic engineered Bacillus subtilis. This work has theoretical and practical meaning.

生物表面活性素发酵产率低限制了其工业化生产和应用，根本原因在于菌株合成产物能力不足。本申请在解决表面活性素合成关键因素（表面活性素合酶的表达）后，立足于枯草芽孢杆菌代谢网络，从表面活性素分子结构及组分模块合成途径出发，研究表面活性素合成及调控机制。核心内容：（1）以THY7-pg3（目前已报道最高产量生产菌株）为研究对象，基于基因组测序结果，采用asRNA干扰、基因敲除和过表达等方法，解析β-羟基脂肪酸合成及调控对表面活性素合成的影响规律；（2）干扰胞内辅因子代谢，解析辅因子调控对表面活性素合成的影响规律，探究表面活性素合成的最适能量及氧化还原状态；（3）完善表面活性素合成调控网络，综合β-羟基脂肪酸和辅因子最优调控方案构建表面活性素高产菌株。本研究成果有助于理解枯草芽孢杆菌表面活性素合成及调控机制，有望构建表面活性素高产菌株，具有重要的理论和现实意义。

表面活性素是一种具有优良应用前景的芽孢杆菌脂肽，菌株发酵产率低限制了其工业化生产和应用。申请人所在课题组前期通过强化产物运输、增强表面活性素合酶表达等手段构建高产表面活性素基因工程菌。本项目在前期工作基础上，从表面活性素合成的代谢路径出发，通过反义RNA策略研究了用于表面活性素合成的 β-羟基脂肪酸生物合成路径及其中的关键限速步骤，过表达该关键酶，表面活性素产量提高了43%，经培养优化后表面活性素产量可达17.4 g/L；进一步研究了微生物细胞内辅因子变化对表面活性素合成的影响；此外，还初步研究了产表面活性素的枯草芽孢杆菌芽孢形成过程以及用于表面活性素合成的无芽孢枯草芽孢杆菌构建策略。此研究构建的高产表面活性素基因工程菌有望实现工业化生产和应用。