Li Xu, Liu Yanghao, Ma Ling, Jiang Wenjing, Shi Tangen, Li Lanxiao, Li Changgeng, Chen Zhichao, Fan Xiaoguang, Xu Qingyang
College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China.
Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science and Technology, Tianjin, China.
Appl Environ Microbiol. 2025 Jun 18;91(6):e0015925. doi: 10.1128/aem.00159-25. Epub 2025 May 8.
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 strain devoid of plasmids and defects using W3110 as the chassis strain. We constitutively expressed the gene from in , which was combined with the gene from BL21 (DE3), successfully constructed a dopamine biosynthesis module, and achieved preliminary dopamine synthesis in . 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-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 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.
In this study, we developed a plasmid-free, defect-free 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.
多巴胺（DA）是一种高价值的代谢产物；然而，由于代谢调控和发酵策略，其生物合成存在多种限制。本研究旨在以W3110为底盘菌株构建一株无质粒且无缺陷的高产多巴胺生产菌株。我们在大肠杆菌中组成型表达来自嗜热栖热菌的基因，并与来自BL21(DE3)的基因相结合，成功构建了一个多巴胺生物合成模块，并在大肠杆菌中实现了初步的多巴胺合成。通过优化关键酶基因的启动子，我们实现了中间代谢物生成与利用之间的协调平衡。随后，我们采用代谢工程策略，如增加通过多巴胺合成途径的碳通量、提高关键酶的基因拷贝数以及构建FADH-NADH供应模块，以创建高产菌株DA-29。在本研究中，开发了一种两阶段pH发酵策略来提高发酵效果。第一阶段确保菌株的正常生长，而第二阶段通过维持低pH值减少多巴胺降解。最后，采用铁和抗坏血酸联合补料策略，我们在5L生物反应器中获得了22.58g/L的多巴胺，表明构建的菌株DA-29具有高多巴胺生产能力，为工业规模的多巴胺生产提供了有力支持。
在本研究中，我们开发了一种无质粒、无缺陷且多巴胺产量高的菌株。我们通过应用本研究中开发的双阶段pH发酵策略，并结合Fe²⁺-抗坏血酸共补料策略，进一步优化了该菌株的发酵过程。这种方法显著提高了多巴胺产量，并解决了发酵过程中多巴胺氧化的问题。产量达到22.58g/L，是迄今为止已知的最高产量，为未来的扩大生产奠定了坚实基础。本研究探索了多巴胺的代谢途径及其高效发酵生产方法，提供了一种新颖的发酵策略。它为芳香族氨基酸衍生物的微生物生产提供了新的见解，推动了该领域的研究。
