Integration of Multiple Phage Attachment Sites System to Create the Chromosomal T7 System for Protein Production in Escherichia coli Nissle 1917

Escherichia coli Nissle 1917 (EcN) is a probiotic used to treat gastrointestinal diseases. The probiotic and endotoxinfree characteristics of EcN support its potential to be developed into a microbial expression system. With this aim, in this study, the powerful T7 expression system was constructed in the cryptic plasmid-free EcN (EcNP) to generate the T7 expression host ENL6P. The concept of multiple copies of gene expression cassettes regulated by the chromosomal T7 promoter was promoted due to plasmid instability issues with protein production in ENL6P. The integration of multiple phage attachment sites (IMPACT) system, which combined Cre-lox72, CRIM, and lambda red recombinase systems, was designed to simplify the manipulation and achieve the multiple phi 80 bacterial attachment sites (attB) in ENL6P to generate the new strain ENL6PP4 with four phi 80 attB sites. The strain can simultaneously integrate four copies of gene expression cassettes in the chromosome to produce recombinant proteins. The IMPACT systems incorporated several tools in gene editing to rapidly achieve more robust and stable microbial strains for research and various industrial applications.