基于Pgrac启动子突变文库筛选及元件组装的芽孢杆菌精细调控表达系统的研究

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.

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。

项目的背景：芽孢杆菌属菌种是多种酶的基因来源微生物，同时也是一类重要的表达系统宿主及工业微生物，在酶制剂生产中起重要作用。国内芽孢杆菌主要研究集中于野生菌种及启动子的筛选工作，还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。至今国内没有成熟的商品化枯草表达系统。.主要研究内容：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系统构建等研究。利用定点诱变/易错PCR/全基因合成技术构建启动子的突变文库，使启动子的启动效率以野生型为标准产生幅度变化，并呈现由高到低的线性化分布。该启动子突变文库的筛选不仅可以筛选高效启动子从而提高异源蛋白的产量，而且作为工具，用于代谢工程中基因的精细调控表达。.重要结果：完成枯草芽孢杆菌组成型启动子P43 及诱导型启动子Pgrac 的启动子及相关序列优化，提供了一种有自主知识产权的用于在枯草芽孢杆菌（Bacillus subtilis）中表达外源蛋白的质粒载体及其基因工程菌株。该表达设备具体包括：（1）控制外源基因在枯草芽孢杆菌细胞内转录的自诱导及乳糖诱导启动子；（2）控制外源表达蛋白从枯草芽孢杆菌分泌到细胞外的信号肽的编码序列；（3）包含启动子、信号肽、筛选标记、多克隆位点及终止子等的酶切位点系统；（4）控制外源基因在枯草芽孢杆菌中终止转录的终止子。.科学意义：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。应用该表达系统，外源基因可以通过质粒构建和转化构建工程菌，同时可以通过酶切位点控制外源蛋白的细胞内及细胞外分泌表达，提高了异源蛋白表达的通用性。该枯草芽孢杆菌培养条件方便、生长迅速，适合工业生产发酵，并可以高效地向胞外分泌目的蛋白,从而使得目的蛋白的纯化相对简单，具有潜在的实用价值。得到了多种可在芽孢杆菌系统中表达的新型普鲁兰酶。