(479b) Investigating and Profiling the Engineering Potential of a Salicylic Acid Biosensor in Escherichia coli

Recent years have witnessed rapid advancement of applying transcriptional factor-based biosensors (TFBs) in dynamic metabolic pathways regulations or high-throughput screening. Understanding the properties of TFBs, such as their dynamic ranges, sensitivities and substrate specificities, is important on engineering and applying the TFBs into specific metabolic engineering scenarios. In this study, we systematically explored the engineering potential of a salicylic acid responsive biosensor system MarR-PmarO in Escherichia coli. We found that the elimination of endogenous MarR in E. coli genome would enhance the activity of PmarO promoter by 2.40-fold. To understand the function of marO boxes in the PmarO promoter, we constructed a series of hybrid promoters by placing the marO boxes in the constitutive pL promoter. The engineered hybrid promoters became responsive to MarR and salicylic acid, and one variant can reach a 3.58-fold increase of dynamic range compared with wild type PmarO promoter. Next, the dynamic modeling was introduced to simulate the molecular interaction between MarR and each nucleotide in the marO box. Based on the results of dynamic modeling simulation, site-directed mutagenesis was carried out to mutate the critical nucleotides with high binding energies. A variant 12A14T exhibited an 8.42-fold improvement of activation intensity. To sum up, we explored the engineering potential of MarR-PmarO sensor system, and extended its dynamic range and activation strength. The results in our study would be beneficial for future metabolic engineering applications.