Systematic Engineering of Escherichia coli for Efficient Production of Pseudouridine from Glucose and Uracil

In this study, we proposed a biological approach to efficiently produce pseudouridine (psi) from glucose and uracil in vivo using engineered Escherichia coli. By screening host strains and core enzymes, E. coli MG1655 overexpressing psi monophosphate (psi MP) glycosidase and psi MP phosphatase was obtained, which displayed the highest psi concentration. Then, optimization of the RBS sequences, enhancement of ribose 5-phosphate supply in the cells, and overexpression of the membrane transport protein UraA were investigated. Finally, fed-batch fermentation of psi in a 5 L fermentor can reach 27.5 g/L with a yield of 89.2 mol % toward uracil and 25.6 mol % toward glucose within 48 h, both of which are the highest to date. In addition, the psi product with a high purity of 99.8% can be purified from the fermentation broth after crystallization. This work provides an efficient and environmentally friendly protocol for allowing for the possibility of psi bioproduction on an industrial scale.