A Synthetic Protein Secretion System for Living Bacterial Therapeutics

Abstract

Bacteria species can thrive and colonize different parts of the human body. Those naturally residing at disease sites e.g., tumors and gut can be designed for targeted protein delivery which can provide better clinical profiles for protein-based therapies. Therefore, a generalizable, efficient, and safe protein secretion system would a be valuable tool to engineer therapeutically active microbes, especially for gram-negative species due to the presence of the second cell wall. Here, we propose an approach called iLOM-SS, an acronym forinducibleLeakyOuterMembrane basedSecretionSystem, to secrete proteins in gram-negative bacteria (GNB). In iLOM-SS, the outer membrane of GNB is made permeable by transient suppression of structural protein(s) to enable free diffusion of cargo proteins expressed at the periplasm. To validate this approach, an iLOM-SS is constructed inEscherichia coliNissle 1917 (EcN) strain. Proteins including enzymes and a human cytokine were proven to be secreted with iLOM-SS by EcNin vitro. Further characterizations of iLOM-SS in ECN showed that fast and titratable secretion, a stop switch design for secretion, and functional implementation of the secretion system in different genetic circuit architectures were possible. We foresee that this work will pave the way for designing GNB to secrete proteins for diverse arrays of applications including but not limited to the development of sentinel cells for therapeutic purposes.