Optimization of a p-Coumaric Acid Biosensor System for Versatile Dynamic Performance

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.

Automated summary

Metabolic engineering has been enhanced by the development of a p-coumaric acid responsive regulator PadR. Novel components yveF and yveG were discovered to positively impact the regulation of PadR-PpadC sensor system. Mutants K64A and H38A showed increased dynamic range and sensitivity, while hybrid promoters P1, P2, P7, and P9 exhibited stronger strength than PpadC, expanding the applicability of the sensor system in future research.