One-pot, real-time monitoring and detection of chemical-producing capability in Escherichia coli using fluorophore-quencher-based aptasensor

Microbial engineering for the production of valuable chemicals has garnered global attention as a promising solution to environmental issues associated with the oil industry. Recent advances in chemical-producing cells have focused on technologies that regulate multiple genes and create extensive cell libraries. Therefore, there is a critical need for monitoring and screening technologies that can rapidly analyse large cell libraries. Here, we demonstrated the repurposing of a fluorophore-quencher (F-Q) and aptamer-based sensing strategy as a cell screening tool for the development of microbial cell factories. The FAM-labelled capture strand and the BHQ1-modified short strand form a structure-switching double-stranded DNA construct, transitioning from a fluorescence ‘off’ to an ‘on’ state when bacterial cells produce the target chemical. To ensure effective adaptation, we conducted extensive system optimization across various culture conditions. As practical applications, we analysed an E. coli strain library engineered to produce L-phenylalanine, demonstrating high correlation with HPLC results in both small well plate and flask cultures, as well as in real-time monitoring. The system also effectively monitored caffeine-producing strains, highlighting its capability in detecting small quantities of chemicals. The system proved efficient for analysing microbial cells producing high-value chemicals.