Development of a highly efficient -coumaric acid-responsive biosensor in .

Developing biosensors to monitor and regulate intracellular biosynthesis pathways can significantly enhance natural product yields in microbial cell factories. This study created a novel biosensor in to respond to -coumaric acid, a critical precursor in the biosynthesis of polyphenols and flavonoids. This biosensor was constructed by expressing the BsPadR repressor from and engineering hybrid promoters. Notably, the P hybrid promoter exhibited tight regulation by BsPadR and enhanced activity in response to -coumaric acid. However, excessive BsPadR expression negatively impacted yeast growth, which was mitigated by using weaker promoters, P and P . Furthermore, the impact of nuclear localization signal (SV40-NLS) positioning on BsPadR functionality was explored, revealing that fusion of an SV40-NLS at the C-terminus of BsPadR enhanced the biosensor's performance. To validate its utility, we applied this system to dynamically regulate (geranylgeranyl pyrophosphate synthase), a key enzyme in lycopene biosynthesis. By coupling -coumaric acid production with lycopene biosynthesis, we enabled high-throughput colorimetric screening for enzyme evolution and strain selection. This novel biosensor serves as a valuable tool for future studies aimed at optimizing the production of -coumaric acid and its derivatives in , thereby advancing the efficiency of biosynthetic processes in microbial cell factories.