Directed Evolution of Membrane Transport Using Synthetic Selections

Abstract

Understanding and engineering solute transporters is important for metabolic engineering and the development of therapeutics. However, limited available experimental data on membrane transporters makes sequence-function relationships complex to predict. Here we apply ligand-responsive biosensor systems that enable selective growth of E. coli cells only if they functionally express an importer that is specific to the biosensor ligand. Using this system in a directed evolution framework, we successfully engineer the specificity of nicotinamide riboside transporters, PnuC, to accept thiamine as a substrate. Our results provide insight into the molecular determinants of substrate recognition of the PnuC transporter family and demonstrate how synthetic biology can be deployed to engineer the substrate spectrum of small molecule transporters.

Keywords: biosensors; directed evolution; membrane transport; selection; structural biology; synthetic biology.