Directed Evolution of Herbicide Biosensors in a Fluorescence-Activated Cell-Sorting-Compatible Yeast Two-Hybrid Platform.

Developing sensory modules for specific molecules of interest represents a fundamental challenge in synthetic biology and its applications. A somewhat generalizable approach for this challenge is demonstrated here by evolving a naturally occurring chemically induced heterodimer into a genetically encoded sensor for herbicides. The interaction between PYRABACTIN-RESISTANT-like receptors and type-2C protein phosphatases is induced by abscisic acid─a small-molecule hormone in plants. We considered abscisic acid receptors as a potential scaffold for the development of biosensors because of past successes in their engineering, a structurally defined ligand cavity and the availability of large-scale assays for their activation. A panel of 475 receptor variants, mutated at ligand-proximal residues, was screened for activation by 37 herbicides from several classes. Twelve compounds activated at least one member of the mutant panel. To facilitate the subsequent improvement of herbicide receptors through directed evolution, we engineered a yeast two-hybrid platform optimized for sequential positive and negative selection using fluorescence-activated cell sorting. By utilizing this system, we were able to isolate receptors with low nanomolar sensitivity and a broad dynamic range in sensing a ubiquitous group of chloroacetamide herbicides. Aside from its possible applicative value, this work lays down conceptual groundwork and provides infrastructure for the future development of biosensors through directed evolution.

A Modified Yeast Two-Hybrid Platform Enables Dynamic Control of Expression Intensities to Unmask Properties of Protein-Protein Interactions.

The yeast two-hybrid (Y2H) assay is widely used for protein-protein interaction characterization due to its simplicity and accessibility. However, it may mask changes in affinity caused by mutations or ligand activation due to signal saturation. To overcome this drawback, we modified the Y2H system to have tunable protein expression by introducing a fluorescent reporter and a pair of synthetic inducible transcription factors to regulate the expression of interacting components. We found that the application of inducers allowed us to adjust the concentrations of interacting proteins to avoid saturation and observe interactions otherwise masked in the canonical Y2H assay, such as the abscisic acid-mediated increase in affinity of monomeric abscisic acid receptors to the coreceptor. When applied in future studies, our modified system may provide a more accurate characterization of protein-protein interactions.