Developing PhoR-PhoB-Based Biosensor by Directed Evolution for Application in Ultralow Inorganic Phosphorus Detection

Inorganic phosphorus (Pi) is vital for all living organisms, but abnormal Pi levels in water and serum pose significant eutrophication and health risks. This study developed a highly sensitive PhoR-PhoB-based biosensor for ultralow-level Pi detection in environmental and serum samples. The <i>Escherichia coli</i> PhoR-PhoB system comprising the histidine kinase PhoR and the response regulator PhoB functions as a Pi-sensing two-component system. However, the limited dynamic range and suboptimal detection range of the native PhoR-PhoB biosensor restrict its practical application. To address these limitations, the expression levels of PhoR and PhoB were optimized, increasing the dynamic range to 97-fold. Directed evolution on the kinase domain of PhoR yielded the mutant PhoR (L222F/N307S/Q344H), which exhibited a 232-fold dynamic range and a linear detection range of 0.012-0.07 mM. This range covers normal serum Pi levels and meets international wastewater discharge standards. The optimized biosensor was successfully applied to Pi detection in serum and environmental water samples, demonstrating its potential for precise measurement of ultralow Pi levels in diverse environments.