A general strategy to construct small molecule biosensors

Abstract

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Despite their broad utility, it remains a longstanding challenge to rapidly create new small molecule biosensors that are robust and specific. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD may be fused to any protein-based reporter such 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 in Saccharomyces cerevisiae and demonstrating portability to both mammalian cell culture and multicellular plants. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast by creating a novel selection to enable directed evolution of a metabolic pathway and to regulate Cas9 activity in mammalian cells. We further propose to expand the biosensor development and deployment to new systems such as prokaryotes and cell-free lysates. Lastly, we propose an antibody fragment based scaffold that can have its target response readily switched with minimal effort to arbitrary molecules while retaining high levels of affinity and specificity.