Thermo-amplifier circuit in probiotic E. coli for stringently temperature-controlled release of a novel antibiotic

Peptide drugs have seen rapid advancement in biopharmaceutical development, with over 80 candidates approved globally. Despite their therapeutic potential, the clinical translation of peptide drugs is hampered by challenges in production yields and stability. Engineered bacterial therapeutics is a unique approach being explored to overcome these issues by using bacteria to produce and deliver therapeutic compounds at the body site of use. A key advantage of this technology is the possibility to control drug delivery within the body in real time using genetic switches. However, the performance of such genetic switches suffers when used to control drugs that require post-translational modifications or are toxic to the host. In this study, these challenges were experienced when attempting to establish a thermal switch for the production of a ribosomally synthesized and post-translationally modified peptide antibiotic, darobactin, in probiotic E. coli. These challenges were overcome by developing a thermo-amplifier circuit that combined the thermal switch with a T7 RNA Polymerase. Due to the orthogonality of the Polymerase, this strategy overcame limitations imposed by the host transcriptional machinery. This circuit enabled production of pathogen-inhibitory levels of darobactin at 40 °C while maintaining leakiness below the detection limit at 37 °C. Furthermore, the thermo-amplifier circuit sustained gene expression beyond the thermal induction duration such that with only 2 h of induction, the bacteria were able to produce pathogen-inhibitory levels of darobactin. This performance was maintained even in physiologically relevant simulated conditions of the intestines that include bile salts and low nutrient levels.