Modular reconstruction and multi-level regulation of Escherichia coli for efficient uridine production

Uridine is an important pyrimidine nucleoside with extensive applications in pharmaceuticals, food, and biomanufacturing. However, the microbial synthesis of uridine is still hindered by unsatisfactory titer, yield, and productivity. Here, modular reconstruction combined with multilevel metabolic engineering was employed to design and construct a high-yielding Escherichia coli platform strain for efficient uridine synthesis. Enhancing the pyrimidine biosynthetic capacity was initially achieved by introducing a pyrimidine operon derived from Bacillus subtilis and disrupting the uridine degradation pathway, thereby promoting de novo uridine formation. Further metabolic optimization focused on improving precursor availability and product accumulation by strengthening carbamoyl phosphate supply, increasing pyrimidine operon dosage, reducing uridine uptake, and optimizing precursor supply. To balance cellular growth and uridine synthesis, the Esa quorum sensing system was utilized for dynamic regulation of thrA expression, while overexpression of the 5'-nucleotidase YjjG substantially increased uridine production. Through the combination of these strategies, the engineered strain U20-2 achieved a uridine titer of 17.99 g/L in shake flask fermentation. An optimized two-stage temperature-controlled fed-batch fermentation process further increased uridine production to 105.68 g/L within 82 h, with a yield and productivity of 0.265 g/g and 1.28 g/L/h, respectively. This represents the highest titer reported to date for glucose-based uridine production in E. coli, and notably, the entire fermentation process was carried out without exogenous inducers or antibiotics. Overall, this study offers a highly promising and practical strategy for the efficient biomanufacturing of uridine and related pyrimidine nucleosides.