Rewiring Quorum Sensing Circuitry for Recombinant Protein Production in E. coli

Title of Document: Rewiring Quorum Sensing Circuitry for Recombinant Protein Production in E. coli Chen-Yu Tsao, Doctor of Philosophy, 2007 Directed By: Professor William E. Bentley Department of Chemical and Biomolecular Engineering The global objective of this research is to rewire the circuitry of bacterial quorum sensing to facilitate recombinant protein production in bacteria. Previous research has shown that the activity of AI-2, the putative “universal” bacterial autoinducer, decreased in culture fluids when several proteins were overexpressed in E. coli W3110, suggesting bacteria communicate or possibly potentiate the “metabolic burden” associated with protein overexpression. Additionally, conditioned medium obtained from LuxS and LuxS strains was added to these cultures, resulting in a 2-4 fold increase in specific yield for both chloramphenicol acetyltransferase (CAT) and organophosphorus hydrolase (OPH). These simple observations set the stage for examining the role of quorum sensing in recombinant protein expression systems and also suggested that “rewiring” the quorum sensing circuitry would lead to significant improvement of yield. In this dissertation, we have inserted luxS into expression vectors (IPTG inducible) which can co-synthesize target recombinant proteins (arabinose inducible) to accomplish the modulation of the metabolic landscape for protein synthesis via altered AI-2 signaling. Our results show significant enhancement in both protein yield and activity, and reveal a strong linkage between bacterial cell communication and cellular processes involved in synthesis and folding of recombinant proteins in E. coli. Second, we have attempted to rewire the native quorum sensing signaling circuitry and couple it to the widely-used T7 expression system for constructing an autoinducible recombinant protein expression platform. We demonstrate, for the first time, true autoinduction of recombinant proteins in E. coli or, in fact, any expression system. That is, our results showed that GFPuv, CAT, and LacZ were all expressed using this innovative system without cell growth monitoring or inducer addition. REWIRING QUORUM SENSING CIRCUITRY FOR RECOMBINANT PROTEIN PRODUCTION IN E. COLI