University of Minnesota PhD dissertation. February 2018. Major: Chemical Engineering. Advisor: Wei-Shou Hu. 1 computer file (PDF): xi, 110 pages.Decades of overuse of antibiotics has led to the emergence of resistant infections across the globe. Healthcare professionals are running out of viable options, as clinical isolates have begun resisting treatment to even last resort therapies. The emergence of these ‘superbugs’, coupled with the lack of new drugs in the discovery pipeline, has led to the possibility of a ‘post-antibiotic’ era. With the primary driving force for resistance development being the overuse of antibiotics, technologies are being sought to limit their injudicious application within the clinical and agricultural sectors. For decades, antimicrobial peptides (AMPs) have been proposed as a promising contender in the fight against microbial resistance. AMPs are small peptides that are produced natively from organisms across all domains of life as a first line of defense against microbial challenge. However, despite their therapeutic potential, AMPs have widely failed in translational success due to delivery and synthesis challenges. In this work, we propose engineering probiotic bacteria as AMP-delivery vehicles to overcome the inherent transport barriers of AMP-therapy. We focus on developing engineered probiotics to target resident pathogens of the gastrointestinal tract. The success of this technology could aid in the resistance crisis by unlocking the antibiotic power of many otherwise unusable peptide antibiotics. We have developed several derivatives of the probiotic strain, E.coli Nissle 1917 (EcN), which are capable of eliciting antibiotic activity against clinical and foodborne pathogens. The foundation of this work lays in the reorganization of AMP biosynthetic gene clusters for functional utility. We describe our development of the engineered probiotic, EcN(J25), which led to the first in vivo success of AMP-producing probiotics. Treatment with EcN(J25) was capable of reducing Salmonella carriage in pre-harvest poultry by 97% just 14-days post-treatment. In a similar workflow, we then focused on the development of EcN(C7) for use in decolonizing multidrug resistant E.coli in human carriers. Along the way we studied mechanisms of resistance, applied bioinformatics techniques, and developed novel synthetic biology tools for use in future engineered bacteria. The work within describes many of the challenges and potential of engineered probiotics, laying a foundation for future work in the field
