Engineering a heme-dependent tryptophan hydroxylase pathway in E. coli for enhanced melatonin production

Melatonin is a high-value bioactive indoleamine broadly applied in the pharmaceutical, food and nutraceutical industries, yet its microbial production remains constrained by pathway complexity and low enzymatic efficiency. Here, a streamlined melatonin biosynthetic pathway was established in Escherichia coli by introducing the heme-dependent tryptophan hydroxylase Luz15. We further enhanced the intracellular heme availability and increased melatonin production by 111.45%. Fusion-tag engineering improved the solubility of all heterologous enzymes and boosted production by 50.64%. Structure-guided rational design of Luz15 subsequently yielded a beneficial D299S/W376H mutant, enhancing hydroxylation activity and contributing a 33.81% increase. To divert metabolic flux toward melatonin, a high-producing chassis was constructed via gene-editing and systematic sRNA library screening. The final strain produced 753.78 mg/L melatonin in a 5-L fed-batch bioreactor from glucose, a 38.78-fold improvement over the initial. This study establishes an efficient and scalable microbial platform for the sustainable biosynthesis of melatonin and other hydroxylated tryptophan-derived compounds.