芽孢杆菌是重要的工业微生物，在基因来源及酶（全球60％的酶制剂）生产中起重要作用。虽然国内外开始重视芽孢杆菌启动子等表达元件的筛选，但至今国内没有成熟的商品化枯草表达系统，国外也没有启动子突变文库元件标准化及精细表达调控系统的构建研究。本项目以启动子元件库及芽孢杆菌精细调控系统为题，包括Pgrac启动子突变文库构建及元件标准化，启动子检测系统pNW-promoter筛选，高效可控组装的芽孢杆菌精细调控系统构建等研究。从酶制剂生产实际出发及经过标准化通用系统的构建是本申请最鲜明的特色。性质优良表达系统的获得及机理研究是改变我国酶制剂产业整体落后的重要途径，也是本申请的出发点。构建启动效率幅度变化并线性分布的系列枯草芽孢杆菌表达质粒不仅在科学问题上研究表达元件组装的基本原理，为多酶体系关键酶的代谢调控及合成生物学提供工具；而且在生产实践中能提高工业酶制剂和蛋白质药物等产品的生产效率与产品品质。

As an important industrial microbe, Bacillus sp. plays a key role in the enzyme gene sources and production enzymes (about 60% of the world's industrial enzymes). Although, the Bacillus subtilis expression element (like promoters) selection has attracted more and more attentions, there is few report on the mature commercial Bacillus subtilis expression system in domestic and no report on the promoter mutant library element standardization in foreign. There is also no fine regulation report about the Bacillus subtilis expression system in foreign including the system construction and application. In this project, we will focus on the promoter elements analysis and Bacillus fine control system. This project includes the following contents: the construction of the Pgrac promoter mutant library and the standardization of components, the selection of the promoter detection system using pNW-promoter plasmid and the construction of an efficient/controlled Bacillus subtilis expression system. In the project, the aspects of practical enzyme preparation and general system standardization are the most distinctive features. Besides, the constructing standardization in production process and the metabolic regulation mechanism research is also important research focus, and will supply a new biology tool in the fields of the enzyme production. Moreover, it’s also a significant route to improve the enzyme preparation industry in China by excellent expression system acquisition and mechanism research. In conclusion, the construction of a series of Bacillus subtilis expression plasmid with efficiency amplitude variation and linear distribution properties not only study the basic principle of expression and components assembled in scientific issues, but also provide tools for metabolic regulation of key enzymes in multienzyme system and synthetic biology. Finally, this research can improve the production efficiency and product quality in industrial enzymes, protein drugs and other products in practical production.