枯草芽孢杆菌高效表达系统的构建

Construction of high-level expression system in Bacillus subtilis

【目的】优化枯草芽孢杆菌(Bacillus subtilis)启动子和表达宿主,构建其高效的表达系统,为重组枯草芽孢杆菌的研究和应用奠定基础。【方法】以β-半乳糖苷酶编码基因为报告基因,以诱导型的枯草芽孢杆菌麦芽糖操纵元启动子为调控元件、大肠杆菌-枯草芽孢杆菌穿梭质粒为载体骨架,构建枯草芽孢杆菌高效表达载体pGJ222,将其电转化到B.subtilis 1A747菌株后进行诱导表达试验和葡萄糖抑制试验。利用同源重组的方法,用枯草芽孢杆菌组成型启动子P43替换了B.subtilis野生型菌株1A747麦芽糖操纵元的上游调控序列,得到优化重组表达宿主菌B.subtilis BCYL,将pGJ222转入B.subtilis BCYL后,对优化的表达系统进行诱导表达试验和葡萄糖抑制试验。分别用PCR扩增大肠杆菌和枯草芽孢杆菌维生素B12合成前期途径的谷氨酰-tRNA合成酶编码基因hemA,构建其表达载体,并将其转化B.subtilis BCYL中进行诱导表达检测。【结果】成功构建了枯草芽孢杆菌高效表达载体pGJ222,并实现了β-半乳糖苷酶的高效表达,其表达量占总可溶性蛋白的18%;质量分数5%的麦芽糖诱导24hβ-半乳糖苷酶活达到16U/mL。成功获得了优化重组表达宿主菌B.subtilis BCYL,其可使β-半乳糖苷酶活性有大幅度提高,在质量分数5%麦芽糖诱导24hβ-半乳糖苷酶活达到21U/mL,葡萄糖的抑制作用明显减弱。【结论】通过对启动子和表达宿主的优化,获得了枯草芽孢杆菌高效表达系统,为枯草芽孢杆菌基因工程研究提供了有力工具。

【Objective】 In order to facilitating the genetic manipulation in Bacillus subtilis,high-level expression system as efficient tool was developed in this study,in which the inducible promoter Pglv and expression host were reconstructed and optimized respectively.【Method】 The inducible maltose promoter and E.coli-B.subtilis shuttle vector were employed to construct the expression vector pGJ222,in which the bgaB coding for β-galactosidase was used as reporter.The pGJ222 was electro-transformed into B.subtilis 1A747 to examine the expression efficiency and repression caused by glucose.By means of homologous recombination,the expression host was reconstructed through replacement of maltose operon regulatory element by constitutive promoter P43 in chromosomal DNA of wild type B.subtilis strain 1A747,yielding B.subtilis BCYL.The resultant strain B.subtilis BCYL was transformed by pGJ222 and the improvement expression system was used to determine the expression efficiency and repression caused by glucose.In Another experiment,the hemA genes coding for glutamyl-tRNA synthetase,which involved in the synthesis pathway of vitamin B12,were PCR amplified and used to construct expression vectors.The resultant expression vectors were transformed into B.subtilis BCYL to examine expression efficiency.【Result】 The high-level expression vector pGJ222 was successfully constructed in B.subtilis and the β-galactosidase driven by pGJ222 was successfully expressed in this study.SDS-PAGE showed that expression amount of β-galactosidase accounts for 18% of total soluble protein.The production β-galactosidase achieved 16 U/mL after induction（5% maltose） of 24 h.By means of the optimized host system B.subtilis BCYL,the production of β-galactosidase was improved and reached 21 U/mL after maltose induction of 24 h;meanwhile,the repression caused by glucose was significantly alleviated.【Conclusion】 Thus,a high-level expression system was developed,which provides a potential tool for genetic engineering of B.subtilis.