An orthogonal mammalian gene circuit for selective sensing of taurine-conjugated bile acids.

Beyond their traditional role as dietary lipid emulsifiers, bile acids (BAs) function as pleiotropic signaling molecules that regulate metabolism, inflammation, and diverse disease processes. Taurine-conjugated bile acids (Tau-BAs) and the glycine-to-taurine conjugation ratio (G/T ratio) have emerged as sensitive biomarkers of multiple pathological conditions, including chronic liver diseases, metabolic disorders, and neurodegenerative diseases. However, existing bile acid-responsive sensors and synthetic circuits largely rely on endogenous receptors or signaling pathways, thereby lacking true orthogonality. Importantly, no engineered sensing module has been shown to selectively detect Tau-BAs over their unconjugated or glycine-conjugated counterparts. To address these challenges, we repurposed VC1372, a Vibrio cholerae-derived bile acid-inducible diguanylate cyclase (DGC), to engineer an orthogonal Tau-BA-responsive gene circuit in mammalian cells. The circuit coupled bile acid-triggered production of cyclic di-GMP (c-di-GMP), a bacterial second messenger absent in higher eukaryotes, with a synthetic BldD-dependent transcriptional module to control gene expression. The engineered system exhibited robust, dose-dependent activation in response to the major circulating Tau-BAs-taurocholic acid (TCA), taurodeoxycholic acid (TDCA), and taurochenodeoxycholic acid (TCDCA)-while showing minimal responsiveness to unconjugated and glycine-conjugated bile acids. Targeted mutagenesis of the GGDEF domain further reduced basal activity and improved circuit dynamic range, achieving low-micromolar sensitivity (1-10 μM) that is relevant to pathological Tau-BA elevations in human diseases. Collectively, this work established VC1372 as a versatile orthogonal metabolite sensor for mammalian synthetic biology and provided a modular platform for developing Tau-BA-responsive gene circuits for future diagnostic and therapeutic applications.