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.DOI: http://dx.doi.org/10.7554/eLife.10606.001

We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.

BackgroundThere is an unmet need to monitor human and natural environments for substances that are intentionally or unintentionally introduced. A long-sought goal is to adapt plants to sense and respond to specific substances for use as environmental monitors. Computationally re-designed periplasmic binding proteins (PBPs) provide a means to design highly sensitive and specific ligand sensing capabilities in receptors. Input from these proteins can be linked to gene expression through histidine kinase (HK) mediated signaling. Components of HK signaling systems are evolutionarily conserved between bacteria and plants. We previously reported that in response to cytokinin-mediated HK activation in plants, the bacterial response regulator PhoB translocates to the nucleus and activates transcription. Also, we previously described a plant visual response system, the de-greening circuit, a threshold sensitive reporter system that produces a visual response which is remotely detectable and quantifiable.Methodology/Principal FindingsWe describe assembly and function of a complete synthetic signal transduction pathway in plants that links input from computationally re-designed PBPs to a visual response. To sense extracellular ligands, we targeted the computational re-designed PBPs to the apoplast. PBPs bind the ligand and develop affinity for the extracellular domain of a chemotactic protein, Trg. We experimentally developed Trg fusions proteins, which bind the ligand-PBP complex, and activate intracellular PhoR, the HK cognate of PhoB. We then adapted Trg-PhoR fusions for function in plants showing that in the presence of an external ligand PhoB translocates to the nucleus and activates transcription. We linked this input to the de-greening circuit creating a detector plant.Conclusions/SignificanceOur system is modular and PBPs can theoretically be designed to bind most small molecules. Hence our system, with improvements, may allow plants to serve as a simple and inexpensive means to monitor human surroundings for substances such as pollutants, explosives, or chemical agents.

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SummaryPlants have evolved elegant mechanisms to continuously sense and respond to their environment, suggesting that these properties can be adapted to make inexpensive and widely used biological monitors, or sentinels, for human threats. For a plant to be a sentinel, a reporting system is needed for large areas and widespread monitoring. The reporter or readout mechanism must be easily detectable, allow remote monitoring and provide a re-set capacity; all current gene reporting technologies fall short of these requirements.Chlorophyll is one of the best-recognized plant pigments with an already well-developed remote imaging technology. However, chlorophyll is very abundant, with levels regulated by both genetic and environmental factors. We designed a synthetic de-greening circuit that produced rapid chlorophyll loss on perception of a specific input. With induction of the de-greening circuit, changes were remotely detected within 2 h. Analyses of multiple de-greening circuits suggested that the de-greening circuit functioned, in part, via light-dependent damage to photosystem cores and the production of reactive oxygen species. Within 24 -48 h of induction, an easily recognized white phenotype resulted.Microarray analysis showed that the synthetic de-greening initiated a process largely distinct from normal chlorophyll loss in senescence. Remarkably, synthetically de-greened white plants re-greened after removal of the inducer, providing the first easily re-settable reporter system for plants and the capacity to make re-settable biosensors. Our results showed that the de-greening circuit allowed chlorophyll to be employed as a simple but powerful reporter system useful for widespread areas.