Directed Evolution of a Macrolide-Sensing Transcription Factor Biosensor for the Detection of Macrolactone Aglycones via “Effector Walking” and Efflux Pump Deletion

Glycosylated macrolactones (macrolides) often display broad and potent biological activities and are targets for drug development and discovery. The modular genetic organization of macrolide polyketide synthases (PKSs) and various polyketide tailoring enzymes has inspired the combinatorial biosynthesis of new-to-nature macrolides. However, most engineered PKS and macrolide biosynthetic pathways are ineffective and produce reduced or negligible product titers. Directed evolution could improve the activity of engineered PKSs and associated pathways but critically requires a high-throughput screen to identify active variants from large libraries. Transcription factor-based biosensors can be used for this purpose. However, the effector specificity of the only known macrolide-sensing transcription factor MphR is limited to macrolides modified with the sugar, desosamine. The potential applications of MphR are subsequently limited, ruling out the possibility of leveraging MphR to screen libraries of pathway variants that make macrolactones that lack sugars (i.e., macrolide aglycones) such as the direct products of PKSs. In this study, we aimed to engineer the effector specificity of the MphR biosensor strain for detecting macrolide aglycones. By developing an "effector walking" strategy coupled with efflux pump deletion, the effector profile of MphR was dramatically broadened to include several erythronolide macrolactones. This work sets the stage for applying directed evolution and other high-throughput screening approaches to various PKSs. Our results suggest a broadly applicable approach to developing biosensors that detect ligands that are very different in structure from the native effector.