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

Area of Science:

• Synthetic biology
• Metabolic engineering
• Biosensor development

Background:

• Genetically encoded biosensors are crucial for high-throughput screening but often have limited operating ranges.
• Transcription factor (TF)-based biosensors offer tunable performance through regulator expression modification.

Purpose of the Study:

• To engineer MphR-based erythromycin biosensors with modulated sensitivity and operating ranges.
• To develop a panel of biosensors for diverse screening applications in synthetic biology.

Main Methods:

• Ribosome-binding site (RBS) engineering to fine-tune TF expression.
• Iterative fluorescence-assisted cell sorting (FACS) to generate biosensors with varied sensitivities.
• Microfluidic-based fluorescence-activated droplet sorting (FADS) for high-throughput screening of mutant libraries.

Main Results:

• A panel of MphR-based erythromycin biosensors with distinct sensitivities was successfully generated.
• Engineered biosensors facilitated high-throughput screening of *Saccharopolyspora erythraea* mutants.
• Significant improvements in erythromycin production (up to 6.8-fold and over 100%) were achieved in engineered strains.

Conclusions:

• A straightforward strategy for engineering biosensor performance properties was demonstrated.
• The developed biosensors are valuable tools for stepwise strain engineering and optimizing antibiotic production.
• This approach advances the application of biosensors in metabolic engineering and synthetic biology.