Engineering the probiotic Escherichia coli Nissle 1917 as a host organism for coliphage production

Bacteriophage therapy emerges as a pivotal alternative in the ongoing battle against antibiotic-resistant bacteria. Employing probiotic bacteria as hosts for bacteriophage production bestows several notable advantages compared to traditional pathogenic hosts. These advantages encompass an enhanced safety profile, streamlined downstream processing, and greater regulatory acceptance. In this study, we engineered Escherichia coli Nissle 1917 (EcN), a non-pathogenic probiotic strain, to serve as a safe chassis host for efficient coliphage production. EcN is resistant to T1, T3, T5, and T7 phages and the T4 phage can only infect it at high titers. By eliminating all known phage defense systems in the genome, we constructed the EcN-9 strain that is susceptible to T3 and T7 phages. The infection efficiency of the T4 phage on the EcN-9 strain was 1000 times higher than that on the EcN-WT strain. Introduction of the appropriate FhuA receptor in EcN-9 rendered it susceptible to T1 and T5 phages. Optimizing the expression levels of FhuA led to a remarkable enhancement in the efficiency of plating, with increases of 3872-fold and 2593-fold for T1 and T5, respectively. Building upon this foundation, we further refined the phage production process by developing a fed-batch fermentation strategy, which not only substantially reduced the multiplicity of infection for inoculation to 0.1, but also resulted in high phage titers, reaching 4.47 × 10¹⁰ PFU mL^-1 for T1, 9.21 × 10¹⁰ PFU mL^-1 for T4, and 7.22 × 10¹⁰ PFU mL^-1 for T5. This study provides a strategic framework for adapting preferred bacterial host systems to produce target phages.