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

Area of Science:

• Biotechnology
• Synthetic Biology
• Enzyme Engineering

Background:

• Directed evolution of enzymes is hindered by the lack of scalable functional assays.
• Linking biocatalysis to microbial phenotypes typically requires complex, custom system development.
• Genetically encoded biosensors offer a modular approach to link enzyme activity to selectable phenotypes.

Purpose of the Study:

• To develop an integrated platform for biosensor evolution and enzyme screening within a unified microbial framework.
• To adapt a yeast two-hybrid system for coexpression and screening of cytochrome P450 enzymes (CYPs).
• To demonstrate the platform's utility by enhancing CYP2B6-mediated metabolism of the herbicide alachlor.

Main Methods:

• Utilized a yeast two-hybrid platform coupled with fluorescence-activated cell sorting.
• Employed PYRABACTIN RESISTANCE 1-like receptors and type 2C protein phosphatases as an evolvable biosensor module.
• Performed four rounds of biosensor-guided evolution targeting CYP2B6 expression and catalytic efficiency.

Main Results:

• Successfully adapted the yeast two-hybrid system for CYP screening.
• Isolated biosensor variants capable of discriminating between alachlor and its metabolite.
• Achieved significant improvements in CYP2B6 expression and catalytic efficiency through directed evolution.

Conclusions:

• The developed platform integrates evolvable biosensors and modular strain design for accelerated biocatalyst development.
• This system provides a powerful tool for engineering small-molecule-modifying enzymes, particularly CYPs.
• The approach facilitates the creation of custom biocatalysts by linking enzyme function to selectable microbial traits.