Multiple Metabolic Strategies for Green and Efficient 5-Hydroxytryptamine Biosynthesis in Escherichia coli

5-Hydroxytryptamine (5-HT), also known as serotonin, plays a critical role in mood regulation, insomnia treatment, and free radical scavenging. Currently, 5-HT production primarily relies on plant extraction, which is challenged by limited raw materials and environmental concerns, emphasizing the need for innovative green production strategies. In this study, we explored a high-performance producer for biosynthesizing 5-HT in Escherichia coli. First, we constructed and optimized the 5-HT biosynthetic pathway to convert L-tryptophan (l-Trp) to 5-HT, avoiding the accumulation of tryptamine and 5-hydroxytryptophan. Moreover, precursor enrichment eliminated the need for l-Trp supplementation and dynamic downregulation of pyruvate kinase balanced growth and production. Further, the metabolic flux between the tetrahydrobiopterin (BH4) module and the hydroxylation-decarboxylation module was optimized, reducing l-Trp accumulation. A novel “GXAA” system was implemented to enhance intracellular NAD(P)H supply, including (1) expression of gdhbsu (glucose dehydrogenase) from Bacillus subtilis, gapXfbr (mutated glyceraldehyde-3-phosphate dehydrogenase) from Corynebacterium glutamicum, and pntAB (pyridine nucleotide transhydrogenase); (2) knockout of ldhA (lactate dehydrogenase). Finally, transcriptomic analysis under high-5-HT stress revealed a potential 5-HT transporter, YhjX. The resulting strain WW15 produced 15.5 g/L 5-HT and a yield of 0.063 g/g glucose through a two-stage fed-batch fermentation, representing the highest titer and yield reported to date.