Engineering Programmable Tryptophan-Responsive Biosensors Based on RNA-Binding Attenuation Protein for Strain Optimization

Biosensors have been widely applied for high-throughput strain screening and dynamic regulation of metabolic networks. However, existing tryptophan sensors based on transcription factors or riboswitches often suffer from a narrow dynamic range and limited response threshold. In this study, we developed a series of tryptophan-responsive biosensors in <i>Escherichia coli</i> using the tryptophan-activated RNA-binding attenuation protein (TRAP) as the sensing module. First, we validated TRAP functionality and engineered a functional biosensor by fine-tuning its expression. Subsequently, screening of TRAP variants and optimization of TRAP-leader sequence interactions yielded two biosensors that exhibited distinct dynamic ranges (up to 22.1-fold) and response thresholds of 0-2.2 g/L, respectively. Using these biosensors, we screened two beneficial variants of key rate-limiting enzymes in the tryptophan biosynthetic pathway and further investigated their catalytic mechanisms through molecular dynamics simulations. Collectively, this study provides tools for engineering high tryptophan-producing strains and new strategies for biosensor design.