Screening strong anaerobic E. coli promoters to enhance therapeutical protein expression in tumors.

Engineered bacterial biotherapeutics have shown increasingly promising potential for cancer treatment, yet they are hindered by unresolved challenges regarding off-target toxicity and suboptimal therapeutic efficacy. In this study, we report the first systematic screening of strong constitutive anaerobic promoters in the probiotic Escherichia coli Nissle 1917 (EcN). Via RNA-seq analysis, we initially identified forty candidate promoters from EcN. Subsequent functional characterization using a firefly luciferase reporter assay yielded six robust constitutive anaerobic promoters, whose activity was 1.18- to 1.85-fold higher than that of Ptac-a strong constitutive promoter traditionally leveraged for therapeutic protein expression in EcN-based cancer biotherapeutics. Further functional validation confirmed that these promoters enable efficient production of immune checkpoint nanobodies, PD-L1nb and CTLA-4nb, in EcN under anaerobic conditions. Notably, in MC38 murine tumor models, EcN-based therapy incorporating these potent constitutive anaerobic promoters achieved significantly enhanced antitumor efficacy compared to conventional promoter-based systems. This not only reinforces the safety profile of engineered EcN but also boosts its therapeutic potency when employed as a tumor-targeted delivery platform in cancer immunotherapy. Collectively, our work establishes a precision delivery framework for potent yet highly toxic anticancer agents, facilitating the development of bacterial biotherapeutics.