Metabolic Engineering of Chromosomally Integrated Bacillus licheniformis for the Efficient Biosynthesis of Pseudouridine

Pseudouridine (Ψ) is extensively applied in the medical domain, particularly for mRNA vaccines. However, the production titers of the existing engineered strains remain insufficient to meet market demands. In this study, a multilevel metabolic engineering strategy was implemented to achieve high-level Ψ production in Bacillus licheniformis. First, heterologous ΨMP glycosidase was introduced and engineered, resulting in Ψ production of 1.8 g/L. Subsequently, yqfW and yfnB with strong dephosphorylation for ΨMP were overexpressed, elevating Ψ production by 179.3%. Furthermore, carbon flux was redirected toward intracellular ribose-5-phosphate supply via attenuated pgi expression, increasing Ψ production to 12.1 g/L. Finally, scale-up cultivation in a 5 L bioreactor achieved a Ψ titer of 38.6 g/L, with molar conversion rates of 96.5% from uracil and 31.5% from glucose. These results represent the highest Ψ production level reported in Bacillus species to date, providing a robust foundation for the biosynthesis and industrial application of pyrimidine nucleosides.