Depression exhibits a complex and multifaceted pathophysiology, accompanied by high rates of relapse and disability with current medication treatments. 5-Hydroxytryptophan (5-HTP) is a promising candidate for depression therapy, but its poor pharmacokinetics hinders its clinical application. To address this limitation, we introduced the hydroxylase XcP4H into Escherichia coli Nissle 1917 (EcN) to biosynthesize 5-HTP in vivo. To create a high-yielding EcN strain for 5-HTP production, we engineered XcP4H through enzyme-directed evolution using a novel genetic code expansion-based high-throughput screening method. The most effective XcP4H variant achieved a 22-fold increase in 5-HTP production, and molecular dynamic simulations elucidated the underlying mechanisms. After pathway engineering and gene editing, we further improved the 5-HTP yield in EcN. When the most robust strain, EcN@5-HTP, was employed as a live therapeutic, it alleviated depressive-like behaviors in mice by increasing 5-HT levels in both the gut and brain, repairing neurological abnormalities, inhibiting inflammation, elevating SCFAs concentrations, and modulating gut microbiota dysbiosis. By integrating synthetic biology with enzyme-directed evolution, we successfully addressed the pharmacokinetic limitations of 5-HTP through a live therapeutic approach. This proof-of-concept design clearly demonstrates that combining synthetic biology with probiotics has the potential to significantly revolutionize our strategies for disease detection, prevention, and treatment.