Genetically encoded biosensor enabled mining, characterisation and engineering of aromatic acid MFS transporters

Area of Science:

• Cellular Biology
• Biotechnology
• Biochemistry

Background:

• Active transport across cell membranes is crucial for cellular function and biotechnology.
• Major Facilitator Superfamily (MFS) transporters are vital for importing nutrients and exporting toxins.
• Characterizing membrane transport proteins is challenging due to difficulties in production and assaying.

Purpose of the Study:

• To screen TphK and PcaK homologs for aromatic acid uptake using genetically encoded biosensors.
• To assess structure-activity relationships of transporter-biosensor constructs with various aromatic acid effectors.
• To evaluate the potential for protein engineering of MFS transporters by creating chimeric constructs.

Main Methods:

• Syntenic analysis was used to identify TphK and PcaK homologs.
• Genetically encoded biosensors were employed to screen transporter activity.
• A library of aromatic acid effectors was used to assess structure-activity relationships.
• Chimeric transporter-biosensor constructs were created to study protein engineering feasibility.

Main Results:

• A library of 11 TphK and 10 PcaK homologs was screened for protocatechuic acid and terephthalic acid uptake.
• Structure-activity relationships revealed effector recognition plasticity in PcaK and TphK transporters.
• Chimeric constructs demonstrated the modularity of core transmembrane domains.
• Validated TphK and PcaK homologs were identified.

Conclusions:

• Genetically encoded biosensors are valuable tools for characterizing and engineering MFS transporters.
• The study provides a validated library of TphK and PcaK homologs for biotechnological applications.
• Engineered MFS transporters exhibit plasticity, offering potential for pathway optimization in microbial hosts.