An Escherichia coli Nissle 1917-based live therapeutics platform with integrated phage resistance and programmable temperature sensitivity

Abstract

Live bacterial therapeutics (LBT) represent a transformative modality for managing refractory chronic diseases. However, the absence of optimized microbial chassis systems is a significant barrier to clinical translation. To bridge this gap, we engineered Escherichia coli Nissle 1917 (EcN) into a versatile platform that meets the requirements for strain development and clinical application. First, to achieve precise transcriptional control, a constitutive promoter toolbox was constructed by hybridizing synthetic core promoters with engineered ribosome-binding sites. This library exhibited a 50.5-fold range in expression, enabling inducer-free fine-tuning of heterologous gene expression. Next, to ensure stable and antibiotic-free genetic maintenance, we repurposed the native plasmids pMUT1 and pMUT2 of EcN as modular shuttle vectors. This system was achieved through locus-specific integration, plasmid size minimization, and copy number amplification. The resulting plasmids, pMcol1 and pMrsf2, equipped with the high-strength hybrid promoter P_J23111-B₀₀₃₅, exhibited a 74- and 130-fold increase in reporter protein expression, respectively, compared to the pMUT1 and pMUT2 with the benchmark P_UTR4-driven system. Both plasmids had a performance similar to that of the commercial pET-28a(+) system, demonstrating their high potential for diagnosis and treatment using EcN strains. To enhance bacterial persistence in the intestine, we genomically integrated a phosphorothioate-based defense module, which increased bacterial survival by 2.7-fold. This integration provides EcN with a defense mechanism against phages, thereby improving its stability and efficacy as a therapeutic agent. We also designed a temperature-sensitive kill switch circuit to induce bacterial elimination in the feces. This optimized EcN platform addresses challenges related to genetic stability, enteric survival, and biocontainment. Its therapeutic performance was validated in a murine colitis model, where it reduced histological inflammation, evidenced by intact crypt architecture and low immune cell infiltration. These findings underscore the high potential of this EcN platform for clinical translation and LBT applications.