Modularity in Enzyme Engineering Enables a High-Yielding Microbial Platform for Melatonin Biosynthesis

The limited availability of structurally specific hydroxylated tryptophan derivatives, whether they are obtained through natural extraction or chemical synthesis, presents challenges for industrial applications. In this study, we report a modular biosynthetic platform in Escherichia coli for producing melatonin and related indoleamines by functionally integrating an animal hydroxylase, an insect decarboxylase, and an avian N-acetyltransferase as well as a plant O-methyltransferase. Following the rational design of key enzymes as well as metabolic pathway optimization, the recombinant strains produced 22.8 g/L of 5-hydroxytryptophan (5-HTP) in a 70 L bioreactor and 6.2 g/L of melatonin in a 5 L bioreactor with exogenous tryptophan feeding. This engineered microbial system establishes an efficient route for manufacturing pharmaceutically relevant tryptophan derivatives and provides a versatile enzymatic toolkit for exploring hydroxylated indole compound biochemistry.