(115d) Engineering the P-Coumaric Acid Responsive Biosensor System Padr-PpadC for Versatile Dynamic Properties

Transcriptional factors-based biosensors are efficient tools for high-throughput screening and dynamic pathway regulation. However, natural biosensors often exhibit limited dynamic ranges and unfavorable output strengths, which may limit their applicability in metabolic engineering. Here, a p-coumaric acid responsive biosensor system PadR-P_padC was engineered in Escherichia coli. We revealed that yveF and yveG, two putative genes behind the P_padC promoter in PadR-P_padC sensor system, showed positive effects on assisting the release of PadR inhibition. Through sited-directed mutagenesis, two PadR variants, H38A and K64A, 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 by placing the PadR binding boxes into the constitutive promoter PL. Four engineered promoters, P1, P2, P7, and P9, can not only be regulated by PadR and p-coumaric acid, but also displayed increased strength compared to P_padC. The PadR variants and hybrid promoters obtained in this study can benefit future metabolic engineering research.