Bright Single-Cell Fluorescent Reporter Enables Ultrasensitive Target Detection for Microbial Cell-Based Biosensors

Whole-cell biosensors (WCBs) capable of sensitively detecting trace amounts of analytes hold great potential for in situ detection of pollutants, toxins, or synthetic products. As the terminal signal actuator, the reporter gene directly influences the ultimate sensitivity of WCBs. Although fluorescent proteins (FPs) have been widely used as reporters, their reporting sensitivity is generally lower than that of enzymatic reporters, which often limits the sensitivity and response speed of FP-based sensors in practical applications. Here, we developed an ultrasensitive FP reporter via a noninvasive N-terminal peptide fusion strategy. By adding an N-terminal decapeptide obtained from a high-throughput screening, we constructed an NGFP4 variant that retains the inherent advantages of sfGFP while exhibiting superior reporter gene characteristics, such as rapid expression and robust intracellular stability. These properties enhanced the single-cell fluorescence intensity of NGFP4 by 6.4- to 28-fold in four typical microbial hosts, including E. coli (28-fold), Bacillus subtilis (15.5-fold), Pichia pastoris (9.1-fold), and Saccharomyces cerevisiae (6.4-fold). When applied to WCBs, the NGFP4 reporter greatly shortened the detection time to 1 h for salicylic acid (LOD of 0.36 μM) and 2-chlorobiphenyl (LOD of 18.2 μM), representing the fastest detection time for such sensors. Therefore, our work provides a cross-species compatible FP reporter that enables sensitive detection of microbial cell-based biosensors and other bioanalytical systems, facilitating their field deployment with minimal genetic manipulation and shorter detection time.