Engineering a biosensor based high-throughput screening platform for high-yield caffeic acid production in Escherichia coli.

Caffeic acid (CA) is a valuable phenolic compound with wide applications in pharmaceuticals, food additives, and materials. However, its microbial production faces several challenges, including low heterologous enzyme activity and product toxicity. Here, we report the development of an integrated biosensor-driven high-throughput screening (HTS) platform for the efficient production of CA in Escherichia coli. We first identified and characterized CarR, a novel phenolic acid-responsive transcription factor from Acetobacterium woodii, and engineered it into a p-coumaric acid (p-CA) biosensor. Systematic optimization of the p-CA biosensor, resulting in reduced background, extended dynamic range and increased sensitivity. By coupling this biosensor with fluorescence-activated cell sorting, we established an efficient HTS platform that enabled the rapid selection of an improved FjTALG85S mutant with a 6.85-fold enhancement in catalytic activity and a robust p-CA-producing strain (M5) with enhanced tolerance to p-CA and CA. Subsequent bottom-up metabolic engineering in strain CA8 achieved a CA titer of 9.61 g L-1 in a 5-L bioreactor, the highest reported titer to date. Our work not only overcomes key bottlenecks in CA biosynthesis (low tyrosine ammonia-lyase activity, CA and p-CA cytotoxicity) but also provides a powerful tool to accelerate the engineering of microbial cell factories for the production of p-CA and other derived chemicals.