(387e) Engineering E. coli Nissle 1917 for Increased Transformation Efficiency, Heterologous Protein Production, and Robustness in the Gut Environment

Escherichia coli Nissle 1917 (EcN) has a long history of human use as a probiotic and is an exemplary chassis for live biotherapeutic engineering. However, its usefulness as a live biotherapeutic is limited by poor colonization of the human gut, low transformation efficiency compared with other E. coli strains, and limited heterologous protein expression and secretion. We have approached chassis engineering in EcN by attacking each of these angles. First, we used adaptive laboratory engineering to isolate mutant strains of EcN with up to 10-fold higher transformation efficiency by electroporation. The resulting strains were also more robust to a host of cellular stresses in addition to electroporation. Second, we engineered this strain to produce high amounts of heterologous protein using orthogonal transcription factors and genome engineering to alleviate metabolic and unfolded protein stress. Finally, we have engineered this strain for increased robustness in the gut environment by introducing the use of privileged substrates (prebiotics) to selectively enrich the population both in vitro and in vivo. Overall, we present a multi-faceted strategy for probiotic engineering with the eventual goal of therapeutic secretion of beneficial small molecules, peptides, and proteins in the human gut environment.