title: Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis.
abstract: A strong promoter and highly efficient signal peptides are essential for the secretory overproduction of recombinant proteins in Bacillus subtilis. To enhance the limited overexpression capability of natural promoters, various strategies for promoter engineering have been developed and used to construct gene expression systems in B. subtilis and other hosts. By applying a semi-rational approach for promoter engineering, a series of expression plasmids containing single and dual promoters were constructed using aminopeptidase (AP) with an intrinsic signal peptide as the reporter protein. Of the single and dual promoters investigated, the dual promoter PgsiB-PHpaII gave the best performance. To optimize secretion efficiency, the signal peptide YncM was selected after screening a library containing 19 different Sec-type signal peptides. The AP activity detected in the supernatants of a recombinant strain containing the plasmid pBSG24-YncM was as high as 88.86U/mL. The capacity of the expression plasmid pBSG24-YncM was also evaluated with batch fermentation in a 5-L fermentor. Increased production of AP (205U/mL, equal to 1.7g/L) was achieved after 45h of fermentation. These results suggest that this expression system can be used for high-level protein expression in B. subtilis.
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Chengran
authors.initials: C
authors.lastname: Guan
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China; Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Jintao
authors.initials: J
authors.lastname: Cheng
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Liu
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China; Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Zhongmei
authors.initials: Z
authors.lastname: Liu
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China; Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China; Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, Wuxi, Jiangsu, China. Electronic address: zhmzhou@jiangnan.edu.cn.
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000626:Aminopeptidases / Q000378:metabolism; D001412:Bacillus subtilis / Q000254:growth & development / Q000378:metabolism*; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D005285:Fermentation; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals
chemical_list: D021382:Protein Sorting Signals; D000626:Aminopeptidases