Engineering Escherichia coli Nissle as a Safe Chassis for the Production of Therapeutic Peptides

Synthetic biology enables the installation of programable genetic functions in microorganisms, positioning them as versatile platforms for therapeutic applications. However, clinical translation requires chassis organisms that integrate safety, genetic stability, and precise control over the activity. Here, we present an innovative engineering framework for the probiotic Escherichia coli Nissle (EcN) that transforms EcN into a safe, efficient, and non-proliferative chassis for therapeutic production. The native EcN plasmids pM1 and pM2 were cured and repurposed to encode two functional modules: a CRISPR-Cas12 chromosome-shredding device that prevents bacterial propagation and a programable circuit for payload expression, thereby generating a non-proliferative therapeutic-producing system. To broaden the range of therapeutic payloads, we developed an artificial-intelligence-guided bioinformatic pipeline to predict anticancer cell-penetrating peptides (ACCPPs). As a proof of concept, a candidate identified through this workflow was produced in the engineered EcN chassis and evaluated in human cell culture for anticancer activity. Collectively, this strategy established a modular platform that integrates computational therapeutic discovery with engineered probiotic production, paving the way for the future development of safer microbial therapies and their clinical applications.