A fluorescence-based high-throughput assay enabling engineering of BsPPS, a unique phenolic compound–phosphorylating enzyme from Bacillus subtilis

An assay enables high-throughput screening of phosphorylation activity. • Umbelliferone enables activity readout via phosphorylation-induced fluorescence quenching. • The assay was validated with high accuracy, precision, and reproducibility. • Directed evolution using the HTS platform identified a thermostable Bs PPS variant (G145D). To date, enzymatic systems capable of phosphorylating phenolic compounds are rare, and phenolic phosphate synthetase from Bacillus subtilis ( Bs PPS) is distinguished by its ability to regioselectively phosphorylate a broad range of structurally diverse natural polyphenols, a transformation valuable for improving the aqueous solubility and bioavailability of phytochemicals. However, the lack of an efficient high-throughput screening (HTS) platform has limited its engineering and application. Here, we developed a fluorescence-based HTS assay for rapid estimation of Bs PPS activity using umbelliferone as a fluorogenic probe. Phosphorylation of umbelliferone causes pronounced fluorescence quenching, providing an inverse correlation between fluorescence intensity and catalytic activity. The assay was systematically optimized and validated, demonstrating high accuracy, precision, and reproducibility. Application to an error-prone PCR library identified Bs PPS variants with improved thermostability while maintaining catalytic activity. Overall, this assay fills a critical methodological gap and provides a practical foundation for future exploration of related phenolic compound–phosphorylating enzymes. Download: Download high-res image (211KB) Download: Download full-size image