Reprogramming AraC-type transcriptional factor to respond to new ligands 1,3-propanediol and 1,4-butanediol via directed evolution.

Biosensors play a critical role in metabolic regulation and high-yield strain development in synthetic biology. Here, we developed a biosensor-specific screening platform based on growth-coupled selection and fluorescence-activated cell sorting (FACS), and engineered the AraC-family regulator PocR from Klebsiella pneumoniae to reprogram its response to 1,3-propanediol (1,3-PDO) and 1,4-butanediol (1,4-BDO). Structural analysis revealed that the D152N mutant possesses an enlarged ligand-binding pocket. Using this variant as a template, specificity toward 1,3-PDO and 1,4-BDO was optimized through a kinetics-guided semi-rational strategy and a non-rational approach, resulting in more than a 3.5-fold increase in dynamic range. The optimized biosensor was integrated into a co-culture droplet microfluidic system, enabling high-throughput screening of 1.6 million droplets and successfully isolating a high-producing 1,3-PDO K. pneumoniae strain with a titer of 11.5 g/L, twice that of the wild type. This platform provides a scalable, high-throughput strategy to accelerate biosensor-guided metabolic engineering and the development of high-yield industrial microbial strains.