Development of Highly Sensitive and Specific and Genetically Encoded Biosensors for Calcifediol and Calcitriol Detection

1α,25-Dihydroxyvitamin D₃ (calcitriol) and 25-hydroxyvitamin D₃ (calcifediol) are essential steroid metabolites that serve as key indicators of vitamin D₃ status in the human body. However, conventional detection methods, such as high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA), are costly, time-consuming, and labor-intensive, limiting their accessibility for routine analysis. Here, we engineered genetically encoded biosensors by leveraging the human vitamin D receptor (VDR) and retinoid X receptor alpha (RXRA) to enable precise detection of calcitriol and calcifediol in <i>Saccharomyces cerevisiae</i>. The biosensors were designed as two-component modules by fusing the VDR ligand-binding domain to the <i>S. cerevisiae</i> GAL4 DNA-binding domain and RXRA to the VP16 activation domain, facilitating ligand-dependent transcriptional activation. To enhance sensitivity, we increased the copy number of the reporter module and incorporated a transcriptional amplifier, improving the half-maximal effective concentration (EC₅₀) to 23.3 nM for calcitriol and 219.8 nM for calcifediol. Furthermore, biosensor specificity for calcitriol was optimized by screening VDR mutations using a URA3-mediated dual-selection system, yielding the VDR_CT mutant, which exhibited no response to 10 μM calcifediol. The biosensors demonstrated high accuracy in measuring calcifediol in dietary supplements and calcitriol in pharmaceutical formulations, showing strong concordance with HPLC results. These findings suggest that the proposed biosensors may provide a cost-effective, rapid, and reliable alternative for food safety inspection and clinical diagnostics.