(32f) Chassis Engineering of E. coli Nissle 1917 for Live Biotherapeutic Delivery

The non-pathogenic strain E. coli Nissle 1917 (EcN) has a long history of safe use as an over-the-counter probiotic. Recently, it has come into the spotlight as a live biotherapeutic, delivering enzymes and other biomolecules to the human gut in situ. Here we present an overall approach to optimizing biomolecule production in EcN. First, we examine the role of cryptic plasmids in recombinant protein production and plasmid stability (Zainuddin et al., in revision). Next, we exploit CRISPR-Cas9-based genome engineering protocols to add a butyrate production and sensing pathway to the genome of this host (Bai et al., in revision). In addition, we examine the role of engineered carbon source usage in favoring the survival of this organism in the harsh and competitive gut environment. Finally, we present a unique co-culture method to establish EcN in the lumen of "minigut" organoids, serving as a model system for protein secretion and delivery in the context of intestinal epithelial cells. Our goal is to establish a toolbox to optimize the production of therapeutic small molecules, peptides, and proteins in EcN by optimizing the chassis for ideal performance and dosing of these biomolecules.