(590f) Chassis Engineering for Heterologous Protein Production in E. coli Nissle 1917

The candidate probiotic E. coli Nissle 1917 (EcN) has huge potential for use as a live biotherapeutic, as evidenced by its long history of human use and recent successes in phase 1 and 2 clinical trials by engineered versions of this strain. Wild-type EcN contains two cryptic plasmids, pMUT1 and pMUT2, which can sometimes interfere with heterologous protein expression via increased metabolic burden and plasmid incompatibility. We have developed a CRISPR-based method of rapidly curing these plasmids from EcN. We also report a method that enables cloning in these cryptic plasmids for for stable heterologous protein expression. Finally, we report the combination of integration of T7 RNA polymerase into the EcN genome and knockout of various proteases to maximize protein production in this important host. Taken together, this work represents a suite of tools for chassis engineering for optimal protein production in EcN.