Readily Adaptable Biosensor-Guided Platform Enables the Selection of Herbicide-Metabolizing CYP2B6 Variants

Directed evolution of small-molecule-modifying enzymes is limited by the availability of scalable functional assays. Systems that link biocatalysis to selectable microbial phenotypes often require ad hoc development, posing a major technical barrier. In principle, genetically encoded biosensors may guide enzyme engineering by relaying intracellular changes in substrate or product concentrations. Their coupling to versatile outputs such as gene expression or fluorescence makes biosensors comparatively amenable to directed evolution, enabling their adaptation to alternative ligands. Small-molecule biosensors can therefore serve as a modular link between enzyme activity and microbial selection. Here, we integrate biosensor evolution and enzyme screening within a unified microbial framework. The system builds upon a yeast two-hybrid platform compatible with fluorescence-activated cell sorting, which we adapted to support coexpression and screening of cytochrome P450 enzymes (CYPs). We leveraged PYRABACTIN RESISTANCE 1-like receptors and their agonist-triggered interaction with type 2C protein phosphatases as an evolvable biosensor module. As proof of principle, we applied this system to enhance CYP2B6-mediated metabolism of the herbicide alachlor. We resolved biosensor cross-reactivity and isolated receptor variants capable of discriminating between alachlor and its CYP2B6-derived <i>N</i>-dealkylated product. Four rounds of biosensor-guided CYP2B6 evolution validated the utility of this platform by successfully targeting both expression and catalytic efficiency. These results demonstrate how evolvable biosensors and modular strain design can be combined to accelerate biocatalyst development.