Engineering Escherichia coli Nissle 1917 to scavenge lactate enhances anti-tumor immunity

Bacterial therapy represents a promising approach for cancer treatment, with the probiotic Escherichia coli Nissle 1917 (EcN) being a particularly attractive candidate due to its inherent tumor-targeting capability and established safety profile. However, the underlying mechanisms of its tumor-colonization and anti-tumor activity remain unclear. Lactate, a key immunosuppressive metabolite generated abundantly in tumors via the Warburg effect, critically shapes the tumor microenvironment (TME) and promotes immune evasion. Given EcN's innate capacity to metabolize lactate, we hypothesize that lactate utilization is pivotal for its therapeutic activity and could be enhanced to potentiate anti-tumor immunity. To test this, we first constructed a lactate-utilization-deficient EcN strain (EcN-ΔLA) via CRISPR-Cas9, which exhibited severely impaired tumor colonization and abrogated anti-tumor effects, establishing lactate metabolism as a non-redundant mechanism for EcN's anti-tumor function. We then engineered a stable, plasmid-free strain with enhanced lactate-scavenging capacity (BELAC) by genomically integrating the lactate transporter gene lldP and dehydrogenase lldD to achieve hyper-scavenging of tumor-derived lactate. BELAC demonstrated superior lactate scavenging and robust tumor suppression across multiple syngeneic mouse models accompanied by increased infiltration of CD8⁺ T cells and significant remodeling of the immunosuppressive TME. Notably, in the cold tumor 4T1 breast cancer model, BELAC colonized tumors and reduced lactate levels but failed to elicit significant anti-tumor activity as a monotherapy, underscoring the limitations of lactate depletion alone in some low-immunogenic tumors. Critically, BELAC preserved EcN's tumor-targeting specificity and safety while maintaining high genomic stability. Collectively, this work establishes lactate metabolism as a fundamental requirement for EcN's anti-tumor activity and presents BELAC as a clinically translatable microbial therapeutic that exploits lactate depletion to reprogram the TME and enhances anti-tumor immunity in immunoresponsive contexts.