Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli

Area of Science:

• Microbiology
• Synthetic Biology
• Genetic Engineering

Background:

• Lactobacilli are vital in food fermentation and animal health, with growing interest in their use for human and animal healthcare.
• Engineering lactobacilli requires efficient and versatile genetic tools for high-level gene expression and stable plasmid maintenance.
• Existing genetic systems often rely on antibiotics or complex genomic modifications, limiting their application.

Purpose of the Study:

• To develop novel genetic modules for enhanced gene expression in Lactiplantibacillus plantarum.
• To establish an antibiotic-free plasmid retention system for engineering lactobacilli.
• To facilitate the creation of tuneable, transient genetically modified lactobacilli for diverse applications.

Main Methods:

• Identification and characterization of a novel promoter, P_tlpA, from Salmonella typhimurium for high-level gene expression.
• Implementation of multiple toxin-antitoxin systems for stable, antibiotic-free plasmid retention.
• Evaluation of gene expression levels and plasmid stability in Lactiplantibacillus plantarum.

Main Results:

• The P_tlpA promoter achieved up to 5-fold higher gene expression compared to previously reported promoters in Lactiplantibacillus plantarum.
• Toxin-antitoxin systems provided a self-contained and effective strategy for plasmid retention without selective pressure.
• These genetic modules enable the engineering of tuneable, transient genetically modified organisms.

Conclusions:

• Developed genetic modules significantly enhance the genetic programmability of lactobacilli.
• The novel promoter and plasmid retention system are valuable tools for advancing lactobacilli engineering for healthcare.
• These findings pave the way for broader applications of genetically modified lactobacilli in medicine and industry.