Metabolic Engineering of Escherichia\ncoli for Efficient Production of Pseudouridine

Pseudouridine-incorporated\nmRNA vaccines can enhance protein expression\nand reduce immunogenicity, leading to a high demand for pseudouridine\nto be used in mRNA drug production. To achieve the low-cost production\nof pseudouridine, Escherichia coli was\nsystematically modified to utilize inexpensive raw materials to efficiently\nproduce pseudouridine. First, in the pyrimidine biosynthesis pathway,\ngenes related to the precursor competing pathway and the negative\nregulator were deleted, which increased pseudouridine production.\nSecond, two critical genes, pseudouridine-5′-phosphate glycosidase\n(<i>psuG</i>) and phosphatase genes from different bacteria,\nwere screened and employed in various genetic constructs, and the\npseudouridine yield of the optical strain increased to 599 mg/L. The\naccumulation of pseudouridine was further increased by the deletion\nof pseudouridine catabolism-related genes. Ultimately, the pseudouridine\ntiter in a 5 L bioreactor reached 7.9 g/L, and the yield of pseudouridine\non glucose was 0.15 g/g. Overall, a cell factory producing pseudouridine\nwas successfully constructed and showed potential for industrial production.