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

•Using a combination truncation strategy to remove unnecessary protein domains hindering enzyme activity, we obtained the double-truncated mutant M1 (NΔ143/CΔ26) with excellent enzymatic properties. In addition, we provide structural insights into terminus effects on the catalytic efficiency and thermal stability of mutant M1.•Using the enzyme coupling method, phosphoric dehydrogenase and formic dehydrogenase were introduced to achieve the cyclic regeneration of coenzymes NADPH and NADH in the system, significantly reducing production costs.•In this study, we developed a new in vitro one-pot biocatalytic system (MECCS) to convert L-tryptophan to 5-hydroxytryptamine efficiently. The process is simple, with a high substrate conversion rate and product yield, and has good industrial application prospects.