Modulating Sensitivity of an Erythromycin Biosensor for Precise High-Throughput Screening of Strains with Different Characteristics

Genetically encoded biosensors are powerful tools forproduct-drivenhigh-throughput screening in synthetic biology and metabolic engineering.However, most biosensors can only properly function in a limited concentrationcutoff, and the incompatible performance characteristics of biosensorswill lead to false positives or failure in screening. The transcriptionfactor (TF)-based biosensors are usually organized in modular architectureand function in a regulator-depended manner, whose performance propertiescan be fine-tuned by modifying the expression level of the TF. Inthis study, we modulated the performance characteristics, includingsensitivity and operating range, of an MphR-based erythromycin biosensorby fine-adjusting regulator expression levels via ribosome-bindingsite (RBS) engineering and obtained a panel of biosensors with variedsensitivities by iterative fluorescence-assisted cell sorting (FACS)in Escherichia coli to accommodatedifferent screening purposes. To exemplify their application potential,two engineered biosensors with 10-fold different sensitivities wereemployed in the precise high-throughput screening by microfluidic-basedfluorescence-activated droplet sorting (FADS) of Saccharopolysporaerythraea mutant libraries with different startingerythromycin productions, and mutants representing as high as 6.8folds and over 100% of production improvements were obtained startingfrom the wild-type strain and the high-producing industrial strain,respectively. This work demonstrated a simple strategy to engineerbiosensor performance properties, which was significant to stepwisestrain engineering and production improvement.