Programming a bacterial biosensor for directed evolution of tryptophan hydroxylase via high-throughput droplet sorting

The modification of tryptophan hydroxylase (TPH) for the biosynthesis of 5-hydroxytryptophan (5-HTP) has recently become a focus of research. In this study, we established a droplet-based ultrahigh-throughput microfluidic screening platform (DTSP) to improve the industrial properties of TPH, whereas a bacterial biosensor for L-tryptophan (L-Trp) detection was engineered to improve sensitivity. The promoter pJ23111 achieved a strong negative correlation between the L-Trp concentration and the fluorescence output of the biosensor. The optimized DTSP platform was then utilized to screen superior TPH mutants by employing iterative saturation mutagenesis (ISM). The M4-1 variant (D129L/Q132M/P103A/T236K) demonstrated a 4.25-fold increase of catalytic activity compared to the parental CviPAH. Moreover, it exhibited excellent thermostability, with a 3.2-fold increase of half-life at 45 °C. Our study provides a reference for the development of bacterial biosensors for microdroplet-based screening platforms.