Development of a “turn off–on” whole‐cell biosensor for sulforaphane detection based on the ultrasensitive activator HrpRS

• Published in: Biotechnology and applied biochemistry 2023-04, Vol.70 (2), p.798-810
• Main Authors: Li, Renjie, Chen, Shengyan, Li, Yangguang, Chen, Xuan, Ye, Bang‐Ce
• Format: Article
• Language: English
• Subjects: Anticancer properties; Bacterial Proteins - genetics; Biosensors; dual‐color sensor; Fluorescence; genetic switch; Inflammation; Isothiocyanates - pharmacology; Medical treatment; Metabolites; promoter modification; Sulforaphane; sulforaphane detection; Sulfoxides; Transcription Factors - metabolism; whole‐cell biosensor
• ISSN: 0885-4513; 1470-8744
• DOI: 10.1002/bab.2400

Sulforaphane (SFN), a defense secondary metabolite, can be used to predict the health status of plants and also has pharmacological effects, including anticancer, antioxidant, and anti‐inflammatory properties. The detection of SFN is therefore of great significance for the prevention and treatment of diseases. In this study, a “turn off” whole‐cell biosensor that can rapidly and robustly respond to the presence of SFN was constructed based on the orthogonal genetic components (hrpR, hrpS, and PhrpL) of Pseudomonas syringae (PS). The final optimized biosensor, p114(30R‐30S), was able to inhibit 91.7% of the fluorescence intensity in the presence of 100‐μM SFN. Subsequently, a HrpRS‐regulated OFF–ON genetic switch was designed by reconstituting a reverse σ70 promoter on the σ54‐PhrpL promoter sequence; this was coupled with dual‐color reporter genes to construct a “turn off–on” whole‐cell SFN biosensor. The PhrpLB variant increased the expression of green fluorescence a factor of 11.9 and reduced the expression of red fluorescence by 85.8% compared with the system in the absence of SFN. Thus, a robust switching of signal output from “turn off” to “turn on” was realized. In addition, the biosensor showed good linearity in the SFN concentration ranges of 0.1–10 μM (R2 = 0.99429) and 10–100 μM (R2 = 0.99465) and a detection limit of ~0.1 μM.