An Antibiotic-Free Cryptic Plasmid Platform for Modular Pathway Coupling in Probiotic Escherichia Coli Nissle 1917

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Microbial Biotechnology

Background:

• Probiotic Escherichia coli Nissle 1917 (EcN) has safety advantages but limited industrial use due to genetic instability and antibiotic dependence.
• Existing expression systems often lead to plasmid instability and metabolic burden, hindering efficient metabolite production.

Purpose of the Study:

• To develop a genetically stable, antibiotic-free metabolic engineering strategy for vitamin B5 (VB5) biosynthesis in EcN.
• To engineer native cryptic plasmids as a robust platform for pathway gene expression and enhance VB5 production.

Main Methods:

• Integrated global regulatory rewiring (CRP, CRA deletion) with a cryptic plasmid-based pathway coupling platform for VB5 biosynthesis.
• Coordinated expression of AlsS, PanB, and PanC to enhance precursor supply and ketopantoate accumulation.
• Engineered native cryptic plasmids (pMUT1, pMUT2) for modular gene expression, ensuring genetic stability without antibiotic selection.

Main Results:

• Transcriptomic analysis revealed significant carbon metabolism shifts and enhanced pathway gene transcription upon CRP and CRA deletion.
• Engineered EcN produced 98 mg/L VB5 in shake-flask cultures with stable productivity over multiple passages.
• A 5-L fed-batch bioreactor achieved a VB5 titer of 1.15 g/L, an 11.8-fold increase over flask cultivation.

Conclusions:

• Established a robust cryptic plasmid-based pathway engineering framework for probiotic E. coli.
• Demonstrated the potential of EcN as a safe, stable, and scalable chassis for antibiotic-free production of value-added metabolites like VB5.
• The developed strategy overcomes limitations of genetic instability and antibiotic dependence in microbial cell factories.