Lighting Up Gut Dysbiosis: Red-Shifted Whole-Cell Bioluminescence Enables Functional Sensing of Bile Salt Hydrolase Activity in Human Feces

Bile salt hydrolase (BSH) activity is an emerging functional biomarker of gut microbiota dysbiosis and altered bile acid (BA) metabolism, but its direct quantification in feces remains challenging because stool components strongly attenuate optical signals. Here, we report a red-shifted whole-cell bioluminescent (BL) bioassay for sensitive and accurate quantitative BSH sensing in human feces. The platform couples amino-luciferin chenodeoxycholic acid (aLuc-CDCA), a BSH-cleavable caged luciferin probe, with Caco-2 cells expressing the engineered far-red-emitting luciferase PxRE-R215K (lambda max approximate to 640 nm). In cell-free assays, purified choloylglycine hydrolase (CH) generated a linear BL response with a limit of detection (LOD) of 0.010 +/- 0.002 U/mL; however, analysis of fecal extracts was severely affected by matrix interference. Intracellular luciferase expression mitigated these effects, and PxRE-R215K outperformed the green-emitting AmyLuc reporter in diluted stool suspensions (1 mg/mL), providing LOD and limit of quantification (LOQ) values of 0.02 +/- 0.01 and 0.06 +/- 0.04 U/mL, respectively. Spike-and-recovery experiments confirmed higher accuracy for PxRE-R215K than AmyLuc (83.3-93.4% vs 66.7-87.5%) and improved interassay precision (CV: 14% vs 29.9%). In clinical samples, the PxRE-R215K assay detected significantly higher fecal BSH activity in colorectal cancer (CRC) than in hyperplastic polyp or adenoma groups. UHPLC-MS/MS BA profiling of matched serum samples corroborated the functional readout, revealing CRC-associated increases in total BA levels, secondary-to-primary BA ratio, and unconjugated secondary BA fractions. This red-shifted whole-cell platform offers a simple and scalable strategy for functional monitoring of microbiota-associated BA metabolism in fecal samples with minimal handling.