Highly multiplexed design of an allosteric transcription factor to sense novel ligands.

Designing biosensors to detect novel molecules is important in biotechnology. Allosteric transcription factors (aTF), which are widely used as biosensors, have proven challenging to design because mutating ligand-binding residues often disrupt allostery. Moreover, existing screening methods lack the sensitivity to identify rare, functional variants amidst a large pool of non-functional designs. We introduce Sensor-seq, a platform for designing and screening aTF biosensors with non-native ligand specificity. Sensor-seq uses phylogeny-guided library design, implicitly capturing allostery, and employs an RNA barcoding system for sensitive, highly multiplexed screening. From a library of 17,737 variants of aTF TtgR, Sensor-seq identified novel biosensors for six non-native ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design showed shape complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we developed cell-free detection systems with our designed biosensors. Sensor-seq enables rapid, scalable design of new biosensors, overcoming constraints of natural biosensors.