Optimization of a

Metabolic engineering is a promising approach for the synthesis of valuable compounds. Transcriptional factor-based biosensors are efficient tools to regulate the metabolic pathway dynamically. Here, we engineered the p-coumaric acid responsive regulator PadR from Bacillus subtilis. We found that yveF and yveG, two previously uncharacterized components in the sensor system, showed positive impacts on the regulation of PadR-PpadC sensor system, mostly on assisting the release of the repression by PadR. By site directed PadR engineering, we obtained two mutants, K64A and H38A, which exhibited increased dynamic range and superior sensitivity. To increase the promoter strength of the sensor system and investigate whether the PadR binding boxes can function in a "plug-and-play" manner, a series of hybrid promoters were constructed. Four of them, P1, P2, P7, and P9, showed increased strength compared to PpadC and can be regulated by PadR and p-coumaric acid. The PadR variants and hybrid promoters obtained in this paper would expand the applicability of this sensor system in future metabolic engineering research.