(312e) Engineering Colicin Production and Secretion to Eradicate Biofilms (Industry Candidate)

Bacterial biofilm formation is frequently involved in the development of chronic infectious diseases. Inhibiting biofilms is challenging due to their tolerance against conventional antibiotics which are not effective to penetrating biofilm matrix to kill the cells residing in biofilms. Also, metabolically dormant cells (persisters) frequently found in biofilms are difficult to eradicate by antibiotic treatment. Therefore, novel antimicrobial drugs that can effectively kill persisters or inhibit biofilms are needed urgently. Bacteriocins, antimicrobial toxins, are considered as a viable alternative of conventional antibiotics due to their unique cell killing mechanisms that can damage cells by pore-forming on the cell membrane, nuclease activity, and cell wall synthesis inhibition. We previously reported that colicins, Escherichia coli-produced bacteriocins, can be synthesized using cell-free protein synthesis and kill persister cells very rapidly. In this study, we hypothesized that colicins production and secretion may be controlled in engineered microbes to eradicate target biofilm cells without affecting other non-target biofilms. We confirmed that cell-free produced colicins kill biofilm cells by more than 100-fold with strain-specific recognition. Next, we constructed genetic circuits encoding lysis, colicin, and immunity proteins to precisely control colicin production and secretion in E. coli. The controlled release of the colicin from the engineered microbes significantly repressed target biofilms in multi-species biofilm community without affecting non-target biofilms. This study provides insights that antimicrobial colicins may be used for a promising antibiotic alternative, and engineered microbes via synthetic biology may be developed as living bacterial therapeutics.