Efficient biosynthesis of high-value 5‑Hydroxytryptophan using a multienzyme cascade

5-Hydroxytryptophan (5-HTP) is a precursor of serotonin. The increasing demand for 5-HTP is driving the search for a more efficient and cost-effective production method. In this study, a new in vitro multi-enzyme cascade system (MECCS) was developed to convert L-tryptophan (L-trp) to 5-HTP in a single container while achieving the cycling and regeneration of cofactors and coenzymes. The rate-limiting enzyme tryptophan hydroxylase (TPH) was truncated to improve the overall efficiency of the cascade. After screening, the mutant M1 (NΔ143/CΔ26) showed excellent enzymatic performance, with a 2.3-fold increase in catalytic activity. Molecular dynamics simulations showed that the increase in active center binding force and the improvement of substrate tunnel in M1 improved the overall catalytic efficiency. In addition, the rigidity enhancement of the ligand binding pocket surrounding the region improved the thermal stability of the enzyme. Under optimized conditions, the MECCS converted 50 mM L-trp to 36.9 mM 5-HTP, with a conversion rate of 73.8%. This study provides an alternative pathway for the efficient production of 5-HTP.