biostudies-literatureJavanpour AANational Institute of General Medical SciencesNIGMS NIH HHS2705-2714https://www.ebi.ac.uk/biostudies/studies/S-EPMC8818326biostudies-literatureUnknown10(10)Genetically encoded biosensors are valuable for the optimization of small-molecule biosynthesis pathways, because they transduce the production of small-molecule ligands into a readout compatible with high-throughput screening or selection in vivo. However, engineering biosensors with appropriate response functions and ligand preferences remains challenging. Here, we show that the continuous hypermutation system, OrthoRep, can be effectively applied to evolve biosensors with a high dynamic range, reprogrammed activity toward desired noncognate ligands, and proper operational range for coupling to biosynthetic pathways. In particular, we encoded the allosteric transcriptional factor, BenM, on OrthoRep such that the propagation of host yeast cells resulted in BenM's rapid and continuous diveACS synthetic biologyEvolving Small-Molecule Biosensors with Improved Performance and Reprogrammed Ligand Preference Using OrthoRep.PMC8818326R35GM136297DP2 GM119163R35 GM1362971DP2GM119163Liu CCJavanpour AAfalseEvolving Small-Molecule Biosensors with Improved Performance and Reprogrammed Ligand Preference Using OrthoRep.Genetically encoded biosensors are valuable for the optimization of small-molecule biosynthesis pathways, because they transduce the production of small-molecule ligands into a readout compatible with high-throughput screening or selection in vivo. However, engineering biosensors with appropriate response functions and ligand preferences remains challenging. Here, we show that the continuous hypermutation system, OrthoRep, can be effectively applied to evolve biosensors with a high dynamic range, reprogrammed activity toward desired noncognate ligands, and proper operational range for coupling to biosynthetic pathways. In particular, we encoded the allosteric transcriptional factor, BenM, on OrthoRep such that the propagation of host yeast cells resulted in BenM's rapid and continuous dive2021-01-01T00:00:00Z2021 Oct2025-04-19T04:46:53.072Z2025-04-19T04:46:53.072ZS-EPMC88183263459750210.1021/acssynbio.1c00316