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

Area of Science:

• Synthetic Biology
• Molecular Biology
• Plant Science

Background:

• Plants utilize abscisic acid (ABA) signaling pathways involving receptors like PYR1 and phosphatases like HAB1.
• The PYR1 receptor system allows for reprogramming of ligand recognition, offering unique synthetic biology potential.
• Existing systems lack orthogonality and multi-channel capabilities for complex sensing applications.

Purpose of the Study:

• To design and validate an orthogonal chemical-induced dimerization (CID) module to expand the PYR1 receptor system.
• To create novel PYR1-based biosensors with high sensitivity to specific ligands.
• To demonstrate the application of these biosensors for detecting banned organophosphate contaminants and building genetic circuits.

Main Methods:

• Design of an orthogonal '*' CID module featuring a dimer interface salt bridge.
• X-ray crystallography, biochemical assays, and in vivo analyses to confirm module orthogonality.
• Construction and testing of PYR1*^MANDI/HAB1* and PYR1*^AZIN/HAB1* systems in Arabidopsis thaliana and Saccharomyces cerevisiae.

Main Results:

• The orthogonal '*' module was successfully designed and validated.
• Engineered PYR1*^MANDI/HAB1* and PYR1*^AZIN/HAB1* systems exhibited nanomolar ligand sensitivity.
• Demonstrated sensitive detection of organophosphate contaminants and construction of multi-input/output genetic circuits in living organisms.

Conclusions:

• The novel ligand-programmable CID modules significantly expand the capabilities of synthetic biology tools.
• These advancements enable the development of new plant-based and microbe-based sensing modalities.
• The developed system offers a versatile platform for multi-channel chemical sensing and genetic circuit construction.