A General Method for Sensing Small Molecules in Eukaryotes

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. We describe a method to create biosensors starting with a computationally designed ligand-binding domain (LBD) that, in principle, can be generated for any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to cells expressing a biosensor activates transcription in yeast, mammalian cells and plants, with a dynamic range of up to ~100-fold.

We use the biosensors to improve the biotransformation of pregnenolone to progesterone and to regulate CRISPR activity. In concert with computational LBD design approaches, this method should enable the generation of biosensors for a broad range of molecules.