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

Active transport of chemical species across the cell membrane represents a critical biological and biotechnological function, allowing the cell to selectively import compounds of nutritional value whilst exporting potentially toxic compounds. Major facilitator superfamily (MFS) transporters represent a ubiquitous class able to uptake and export an array of different chemical species. When designing biosynthetic pathways within microbial hosts, for production or remediation, transport is often critical to the efficiency of the resulting engineered strain. However, transport is a commonly neglected node for characterisation and engineering given difficulties in producing, purifying and assaying membrane transport proteins outside of their native environment. Here, using syntenic analysis and genetically encoded biosensors a library of 11 TphK and 10 PcaK homologs were screened for their ability to uptake the aromatic acids, protocatechuic acid and terephthalic acid. The structure activity relationships of the corresponding PcaK and TphK transporter-biosensor constructs, were then assessed with a library of aromatic acid effectors. Finally, the feasibility of protein engineering was assessed, by the creation of chimeric MFS transporter-biosensor constructs, revealing a degree of effector recognition plasticity and the modularity of core transmembrane domains. This study provides a library of validated TphK and PcaK homologs and demonstrates the value of employing genetically encoded biosensors in the characterisation and engineering of this important transport function.