Construction of tetrahydropyrimidine biosensor

Abstract

The aim of this study was to construct a novel tetrahydropyrimidine biosensor to modify the AraC protein in E. coli by site-specific mutation technology so that it can respond specifically to tetrahydropyrimidine rather than its natural ligand, L-arabinose. The successful introduction of the mutation was confirmed by PCR and DNA sequencing, and the effective expression of the modified AraC protein in E. coli was confirmed by Western blot analysis. Surface plasmonic resonance (SPR) technique showed that the modified AraC protein had a high affinity for tetrahydropyrimidine, while its affinity for L-arabinosewas significantly reduced, demonstrating its specificity. In addition, a dose-dependent increase in the expression level of reporter genes regulated by AraC protein was observed by progressively increasing the concentration of tetrahydropyrimidine, confirming its sensitivity in the detection of tetrahydropyrimidine. The response characteristics of the reporter gene EGFP indicate that the constructed biosensor can achieve quantitative detection of tetrahydropyrimidine through the change of fluorescence signal in the presence of different concentrations of tetrahydropyrimidine. At the same time, the synthetic strains of Escherichia coli with tetrahydropyrimidine biosensor can maintain relatively stable growth under different concentrations of tetrahydropyrimidine, indicating that the biosensor can be used to monitor the growth state of strains and the synthesis process of tetrahydropyrimidine in real time, without causing significant adverse effects on the growth of strains. This result provides a powerful tool for the construction of microbial cell factories of tetrahydropyrimidine and lays the foundation for the development of future metabolic engineering and biosensing technologies. Future research will further optimize the performance of biosensors and explore their potential applications in areas such as industrial fermentation and environmental monitoring.