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

Abstract

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.

Agrochemical control of gene expression using evolved split RNA polymerase. II

“…The ABI-PYR1 pair has been recently expanded to be sensors of other agrochemicals (Zimran, 2022; Park, 2024). Our work will readily help develop new inducible gene expression systems of these agrochemicals.…”

“…Agrochemicals are key to health promotion and growth management in modern agriculture. The development of biosensors for agrochemicals opens new avenues to remote control of cell behavior (Park, 2015, Zimran, 2022, Park, 2024). One of the possible applications is to use agrochemicals to control gene expression of bacteria associated with plants or animals.…”

An orthogonalized PYR1-based CID module with reprogrammable ligand-binding specificity

“…We previously developed multiple agrochemical-regulated CID modules, including PYR1 MANDI , designed to enable agrochemical control of plant ABA signaling 25 . More recently, we used computationally guided mutagenesis to develop PYR1 sensors for 21 structurally diverse cannabinoids and organophosphates, and others reprogrammed a different PYL to sense 12 herbicides 23 , 24 . Thus, PYLs have malleable, reprogrammable ligand-binding pockets.…”

Computational structural-based GPCR optimization for user-defined ligand: Implications for the development of biosensors

“…The development of sensors able to sensitively and selectively detect environmental small molecules is of paramount importance for synthetic biology applications and it is still a difficult task [1] , [2] . Monitoring the presence of some molecules is important in many fields, such as environmental, medical, or food sample control [3] , [4] .…”