Metabolic engineering of Escherichia coli for high-yield dopamine production via optimized fermentation strategies.

Dopamine (DA) is a high-value metabolic product; however, its biosynthesis has multiple limitations due to metabolic regulation and fermentation strategies. This study aimed to construct a high-yield dopamine-producing Escherichia coli strain devoid of plasmids and defects using E. coli W3110 as the chassis strain. We constitutively expressed the DmDdC gene from Drosophila melanogaster in E. coli, which was combined with the hpaBC gene from E. coli BL21 (DE3), successfully constructed a dopamine biosynthesis module, and achieved preliminary dopamine synthesis in E. coli. By optimizing the promoters of the key enzyme genes, we achieved a coordinated balance between the generation and utilization of intermediate metabolites. Subsequently, we used metabolic engineering strategies, such as increasing the carbon flux through the dopamine synthesis pathway, elevating the gene copy number of key enzymes, and constructing an FADH(2)-NADH supply module to create a high-yield strain, DA-29. In this study, a two-stage pH fermentation strategy was developed to enhance fermentation. The first stage ensures the normal growth of the strain, whereas the second stage reduces dopamine degradation by maintaining a low pH. Finally, using a combined Fe(2+) and ascorbic acid feeding strategy, we obtained 22.58 g/L of dopamine in a 5 L bioreactor, demonstrating that the constructed strain DA-29 possesses high dopamine production capacity, providing strong support for the industrial-scale dopamine production. IMPORTANCE: In this study, we developed a plasmid-free, defect-free Escherichia coli strain with high dopamine production. We further optimized the fermentation process for this strain by applying the dual-stage pH fermentation strategy developed in this research, combined with an Fe²⁺-ascorbic acid co-feeding strategy. This approach significantly increased dopamine yield and addressed the issue of dopamine oxidation during fermentation. The yield reached 22.58 g/L, marking the highest known yield to date and laying a solid foundation for future scale-up production. This research explores the metabolic pathway of dopamine and the efficient fermentation methods for its production, providing a novel fermentation strategy. It offers new insights into microbial production of aromatic amino acid derivatives, advancing research in this field.