Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. 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 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 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 and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.
Small molecules play essential roles in organisms, and so methods to sense these molecules within living cells could have wide-ranging uses in both biology and biotechnology. However, current methods for making new “biosensors” are limited and only a narrow range of small molecules can be detected.
One approach to biosensor design in yeast and other eukaryotic organisms uses proteins called ligand-binding domains, which bind to small molecules. Here, Feng, Jester, Tinberg, Mandell et al. have developed a new method to make biosensors from ligand-binding domains that could, in principle, be applied to any target small molecule. The new method involves taking a ligand-binding domain that is either engineered or occurs in nature and linking it to something that can be readily detected, such as a protein that fluoresces or that controls gene expression. This combined biosensor protein is then engineered, via mutations, such that it is unstable unless it binds to the small molecule. This means that, in the absence of the small molecule, these proteins are destroyed inside living cells. However, the binding of a target molecule to one of these proteins protects it from degradation, which allows the signal to be detected.
Feng, Jester, Tinberg, Mandell et al. use this method to create biosensors for a human hormone called progesterone and a drug called digoxin, which is used to treat heart disease. Further experiments used the biosensors to optimize the production of progesterone in yeast and to regulate the activity of a gene editing protein called Cas9 in human cells. The biosensors can be also used to produce long-term environmental sensors in plant cells.
This approach makes it possible to produce a wide variety of biosensors for different organisms. The next step is to continue to explore the ability of various proteins to be converted into biosensors, and to find out how easy it is to transfer a biosensor produced in one species to another.