Single-Step Chromosomal Engineering Integrates Biocontainment and Therapeutic Function for Regulatory-Oriented Live Biotherapeutic Chassis Design

Background: Live biotherapeutic products (LBPs) require robust genetic stability and effective biocontainment to support safe clinical translation and regulatory acceptance. Aim: This study presents a single-step chromosomal engineering strategy that integrates auxotrophy-mediated biocontainment with therapeutic gene insertion to support regulatory-oriented live biotherapeutic chassis design. Methods: A no-SCAR genome-editing approach combining CRISPR/Cas9 and λ-Red recombineering was used to generate an Escherichia coli MG1655 ΔilvC::hlyA strain by replacing ilvC with the hlyA gene encoding listeriolysin O. Chromosomal and episomal expression systems were compared for auxotrophy, growth, haemolytic activity, plasmid stability, and intracellular DNA delivery to RAW 264.7 macrophages. Results: Auxotrophy was successfully established and restored by branched-chain amino acid supplementation. Chromosomal integration preserved haemolytic activity and bacterial growth while improving long-term genetic stability and plasmid maintenance compared with episomal expression. Both systems supported intracellular DNA delivery, whereas the chromosomal construct showed improved host-cell preservation under higher bacterial challenge. Conclusions: This proof-of-concept study supports the feasibility of using a single-step chromosomal engineering strategy to combine intrinsic biocontainment with therapeutic-gene integration in an engineered bacterial chassis.