CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently

• Main Authors: Changchuan Ye, Xi Chen, Mengjie Yang, Xiangfang Zeng, Shiyan Qiao
• Format: Article
• Language: English
• Published: BMC 2021-08-01
• Series: Journal of Biological Engineering
• Subjects: E. coli; CRISPR/cas9; T7 Expression System; Fluorescent Protein; 5-Aminolevulinic Acid; promoter variants
• Online Access: https://doi.org/10.1186/s13036-021-00270-9
• ISSN: 1754-1611

Abstract T7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of the T7 RNA Polymerase gene under control of the lacUV5 promoter into Escherichia coli BW25113. The T7 Expression System worked efficiently in this mutant strain named BW25113-T7. We demonstrated that this mutant strain could satisfactorily produce 5-Aminolevulinic Acid via C5 pathway. A final study was designed to enhance the controllability of T7 Expression System in this mutant strain by constructing a T7 Promoter Variants Library. These efforts advanced E. coli BW25113-T7 to be a practical host for future metabolic engineering efforts.