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

ABSTRACT Escherichia coli Nissle 1917 (EcN) is a well‐established probiotic chassis with an excellent safety profile; however, its application as an industrial microbial cell factory remains limited, largely due to genetic instability and antibiotic‐dependent expression systems. In this study, we developed an antibiotic‐free and genetically stable metabolic engineering strategy for vitamin B 5 (VB 5 ) biosynthesis in EcN by integrating global regulatory rewiring with a cryptic plasmid–based pathway coupling platform. To enhance precursor supply, the VB 5 biosynthetic pathway was reinforced by coordinated expression of AlsS, PanB, and PanC, leading to improved ketopantoate accumulation. Deletion of the global transcriptional regulators CRP and CRA significantly reshaped carbon metabolism, as revealed by transcriptomic analysis, resulting in enhanced transcription of key pathway genes and increased specific VB 5 productivity despite reduced cellular growth. To overcome plasmid instability and metabolic burden associated with conventional expression systems, native cryptic plasmids (pMUT1 and pMUT2) were engineered as modular expression platforms. Positional integration of pathway genes into cryptic plasmids enabled tunable gene expression and ensured long‐term genetic stability without antibiotic selection. The optimized strain harboring the complete VB 5 pathway on engineered cryptic plasmids produced 98 mg/L VB 5 in shake‐flask cultures with stable productivity over multiple passages. In a 5‐L fed‐batch bioreactor, VB 5 titer reached 1.15 g/L, representing an 11.8‐fold improvement compared with flask cultivation. This work establishes a robust cryptic plasmid–based pathway engineering framework for probiotic E. coli , highlighting its potential as a safe, stable, and scalable chassis for antibiotic‐free production of value‐added metabolites.