Dynamic D-Allose Monitoring Via a Toggle-Programmed Microbial Fluorescent Biosensor

Recently, microbial fermentation for the synthesis of rare sugars has garnered considerable attention. d-Allose, a rare monosaccharide, has emerged as a significant bioproduct in the food and pharmaceutical industries due to its unique health benefits and physiological functions. However, the detection of d-allose in fermentation broth samples predominantly depends on complex chromatographic techniques. This study introduces a toggle-programmed microbial fluorescent biosensor designed for the dynamic monitoring of d-allose. The d-allose-sensing P_alsR promoter, located within the alsRBACEK operon, was initially characterized and used as a core component in the biosensor. The native P_T7 promoter and the P_alsR promoter were then utilized for the expression of green fluorescent protein (GFP) and T7 RNA polymerase (T7 RNAP), respectively. The constitutive P_pdc promoter was employed to overexpress the repressor protein AlsR in the cytoplasm, thereby ensuring effective regulation of P_alsR. More importantly, a fast-folding and fast-degrading mGFP-DAS replaced GFP as the reporter, facilitating a rapid response to d-allose during continuous microbial incubation. The results demonstrated that this novel biosensor exhibited high sensitivity to d-allose levels, with measurement deviation controlled within 15%. This approach offers significant advantages over traditional d-allose chromatography detection methods, particularly in terms of simplicity and cost-effectiveness.