Engineered Stable, Antibiotic‐Free, High‐Level Protein Expression in the Probiotic Chassis <i>Escherichia coli</i> Nissle 1917

The application of engineered live biotherapeutic products (LBPs) to secrete small molecules, peptides, or proteins to benefit a human or animal host, relies on heterologous protein expression. Key challenges in this area include expressing protein in a targeted location, the use of antibiotic-free platforms, and expressing recombinant proteins at titers capable of the desired therapeutic effect. In this study, we sought to engineer the promising candidate probiotic chassis Escherichia coli Nissle 1917 (EcN) as an in situ drug delivery platform. Despite its long history of safe human use and general probiotic characteristics, wild-type EcN is not optimal for routine protein expression. In this work, we present several approaches to improve protein production in this host. First, we enable stable antibiotic-free protein expression system via native cryptic plasmids. Next, we integrate the T7 RNA polymerase for high level protein expression. Finally, we knock out OmpT protease activity, enabling expression levels comparable to the industry standard E. coli BL21 (DE3). To demonstrate its application, the above system was adapted to express antimicrobial peptide microcin L (MccL) from EcN, which can potentially reduce gut related pathogens and enhance fitness of the probiotic in the competitive niche of the gut. Overall, this study establishes an antibiotic free and high level protein expression platform in EcN, expandable for in situ delivery of therapeutic proteins.