Development of a highly efficient p-coumaric acid-responsive biosensor in Saccharomyces cerevisiae

Abstract

Developing biosensors to monitor and regulate intracellular biosynthesis pathways can significantly enhance natural product yields in microbial cell factories. This study created a novel biosensor in Saccharomyces cerevisiae to respond to p-coumaric acid, a critical precursor in the biosynthesis of polyphenols and flavonoids. This biosensor was constructed by expressing the BsPadR repressor from Bacillus subtilis and engineering hybrid promoters. Notably, the P _BS1-CCW12 hybrid promoter exhibited tight regulation by BsPadR and enhanced activity in response to p-coumaric acid. However, excessive BsPadR expression negatively impacted yeast growth, which was mitigated by using weaker promoters, P _BST1 and P _ERG9 . Furthermore, the impact of nuclear localization signal (SV40-NLS) positioning on BsPadR functionality was explored, revealing that fusion of an SV40-NLS at the C-terminus of BsPadR enhanced the biosensor's performance. To validate its utility, we applied this system to dynamically regulate CrtE (geranylgeranyl pyrophosphate synthase), a key enzyme in lycopene biosynthesis. By coupling p-coumaric acid production with lycopene biosynthesis, we enabled high-throughput colorimetric screening for enzyme evolution and strain selection. This novel biosensor serves as a valuable tool for future studies aimed at optimizing the production of p-coumaric acid and its derivatives in S. cerevisiae, thereby advancing the efficiency of biosynthetic processes in microbial cell factories.

Keywords: Biosensor; BsPadR transcriptional repressor; Hybrid promoter; Saccharomyces cerevisiae; p-Coumaric acid.

Graphical abstract

Fig. 1

Impact of inserting various BsPadR-specific operator sequences on the activity of P_CCW12 promoter and a synthetic P_TEF1-GAL1 hybrid promoter. A. Design of the synthetic promoters. The BsPadR-specific operator sequence was inserted 7 bp downstream of the TATA box in P_CCW12 as well as 7 bp downstream of the TATA box in P_TEF1-GAL1 synthetic promoter; B. Comparison of the fluorescence intensity driven by various hybrid promoter.

Fig. 2

Evaluation of growth characteristics and promoter strength in recombinant yeast strains. A. Growth curves of engineered yeast strains expressing BsPadR ∼ NLS under different promoters. strains were cultured in YPD medium; B. Quantitative analysis of promoter activity. Fluorescence intensity was measured after 8 h and 26 h of growth in YPD medium. Error bars represent the standard deviation from three replicates.

Fig. 3

Design and construction of p-coumaric acid biosensor system. A. The genetic circuit of p-coumaric acid biosensor system; B. Comparison of fluorescence intensity between biosensor systems containing BsPadR ∼ NLS under the control of P_BST1 or P_ERG9 promoters and control systems without BsPadR ∼ NLS expression.

Fig. 4

Effects of the location of SV40-NLS on the performance of BsPadR. Error bars represent standard deviations of three independent experiments.

Fig. 5

The impact of BsPadR variants on biosensor performance. Error bars represent standard deviations of three independent experiments.

Fig. 6

Dynamic responsive range and effector specificity of the p-coumaric acid biosensor. To release the inhibition by BsPadR ∼ NLS in the Y-P_BS1-CCW12-1 strain, 0–800 mg/L of p-coumaric acid, ferulic acid and caffeic acid were used as inducers. p-HCA: p-coumaric acid; FA: ferulic acid; CA: caffeic acid.

Fig. 7

Biosensor-mediated regulation of CrtE enables colorimetric screening of p-coumaric acid-producing yeast colonies. A. Schematic of the p-coumaric acid-responsive biosensor regulating CrtE expression; B. Colony phenotype of YPCA-lyc-2 without p-coumaric acid supplementation; C. Colony phenotype of YPCA-lyc-2 supplemented with 500 mg/L exogenous p-coumaric acid; D. Colony phenotype of YPCA-lyc-20 (YPCA-lyc-2 with integrated empty vector pUMRI-11), serving as a negative control; E. The Colony phenotype of YPCA-lyc-21 ((YPCA-lyc-2 with integrated Sfi I- linearized pUMRI-11-ORgTAL), demonstrating biosensor activation via endogenous p-coumaric acid production.