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Engineered Stable, Antibiotic-Free, High-Level Protein Expression in the Probiotic Chassis Escherichia coli Nissle
Halimatun Sakdiah Zainuddin1, Sanjeeva Kumar Murali2, Thomas J Mansell2
1Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, Serdang, Malaysia.
Biotechnology and Bioengineering
|December 24, 2025
Summary
This study engineered the probiotic Escherichia coli Nissle 1917 (EcN) for high-level, antibiotic-free protein expression. This platform enables in situ drug delivery for enhanced therapeutic effects in the gut.
Area of Science:
- Microbiology
- Biotechnology
- Genetic Engineering
Background:
- Engineered live biotherapeutic products (LBPs) require efficient heterologous protein expression for therapeutic benefits.
- Challenges include targeted protein delivery, antibiotic-free systems, and achieving therapeutic protein titers.
- Escherichia coli Nissle 1917 (EcN) is a promising probiotic chassis but requires optimization for protein production.
Purpose of the Study:
- To engineer Escherichia coli Nissle 1917 (EcN) as an in situ drug delivery platform.
- To develop stable, antibiotic-free protein expression systems in EcN.
- To enhance recombinant protein titers for therapeutic applications.
Main Methods:
- Engineered EcN using native cryptic plasmids for antibiotic-free expression.
- Integrated T7 RNA polymerase for high-level protein production.
- Knocked out OmpT protease activity to improve expression levels.
Main Results:
- Achieved stable, antibiotic-free protein expression in EcN.
- Demonstrated high-level protein expression comparable to industry standards.
- Successfully expressed antimicrobial peptide microcin L (MccL) in engineered EcN.
Conclusions:
- Established an antibiotic-free, high-level protein expression platform in EcN.
- The platform is expandable for in situ delivery of various therapeutic proteins.
- Engineered EcN shows potential for delivering therapeutics to combat gut pathogens and enhance probiotic fitness.
