Metabolic engineering of Escherichia coli for N-methylserotonin biosynthesis.

N-methylserotonin (NMS) is a valuable indole alkaloid with therapeutic potential for psychiatric and neurological disorders, and it is used in health foods, cosmetics, and weight loss supplements. However, environmental challenges and low reaction efficiencies significantly hinder cost-effective, large-scale production of NMS in plants or through chemical synthesis. Herein, we have successfully engineered Escherichia coli strains to enhance NMS production from L-tryptophan using whole-cell catalysis. We developed multiple biosynthesis pathways incorporating modules for serotonin (5-hydroxytryptamine, 5-HT), tetrahydromonapterin (MH₄), and S-adenosylmethionine (SAM) synthesis. To enhance MH₄ availability, we employed a high-activity Bacillus subtilis FolE and minimized carbon flux loss through targeted gene knockouts in competitive metabolic pathways, improving 5-HT production. Additionally, we constructed a comprehensive SAM biosynthesis module to facilitate transmethylation by a selected N-methyltransferase fused with ProS2. These engineered modules were coexpressed in two plasmids within the optimized strain NMS-19, producing 128.6 mg/L of NMS in a 5-L bioreactor using fed-batch cultivation-a 92-fold increase over the original strain. This study introduces a viable strategy for NMS production and provides insights into the biosynthesis of SAM-dependent methylated tryptamine derivatives.