Enriching intracellular macrolides in Escherichia coli improved the sensitivity of bioluminescent sensing systems

A repressor protein MphR and an enhanced green fluorescent protein (eGFP) were used to construct a bioluminescent sensing system for macrolide analysis in Escherichia coli host cells. We deleted TolC, an efflux pump for macrolides in E. coli, to promote the intracellular accumulation of macrolides. The binding constant (K_1/2) of the sensing system constructed in an E. coli strain was decreased up to 33-fold with deleted TolC, and its sensitivity to the macrolides erythromycin, azithromycin, roxithromycin, and pikromycin was increased. The limit of detection of the bioluminescent sensing system for serum azithromycin was 4.1 nM. The ability to detect serum azithromycin concentrations was confirmed by analyzing photographs using ImageJ software. We also developed a novel sensing system for the immune suppressor FK506, another macrolide that is frequently prescribed. Deleting TolC also significantly improved the sensitivity of this sensing system. Bioluminescent sensing systems constructed in TolC mutants were sensitive to various macrolides, indicating their potential for clinical application with hand-held devices.