Plasmid-Stabilizing Strains for Antibiotic-Free Chemical Fermentation.

Area of Science:

• Biotechnology
• Microbial Engineering
• Synthetic Biology

Background:

• Plasmid-mediated fermentation is industrially important but challenged by plasmid instability and cell burden.
• Stable plasmid maintenance relies on complex genetic mechanisms, hindering rational strain design.
• Current methods for identifying plasmid-stabilizing factors are limited.

Purpose of the Study:

• To develop a high-throughput screening method for identifying plasmid-stabilizing strains.
• To engineer robust Escherichia coli and Bacillus subtilis strains for antibiotic-free fermentation.
• To characterize the genetic basis of enhanced plasmid stability and its impact on production.

Main Methods:

• A fluorescence-based high-throughput screening method was established.
• Genomic fragment-deletion strains of Escherichia coli MG1655 and Bacillus subtilis 168 were screened.
• Engineered strains were evaluated for plasmid stability and production of target compounds under antibiotic-free conditions.

Main Results:

• Screening identified effective plasmid-stabilizing strains: EcΔ50 for E. coli and BsΔ44 (BsΔyueB) for B. subtilis.
• EcΔ50 demonstrated a 2.9-fold increase in alanine titer due to improved plasmid maintenance and copy number.
• BsΔyueB showed a 61.99% increase in acetoin titer and an 80.63% increase in 2,3-butanediol titer, attributed to minimized sporulation and enhanced host adaptation.

Conclusions:

• A novel screening approach successfully generated plasmid-stabilizing chassis strains for E. coli and B. subtilis.
• The engineered strains exhibit significantly improved plasmid stability and production yields in antibiotic-free fermentation.
• These findings offer a valuable platform for metabolic engineering and the sustainable production of chemicals.