A combined pseudouridine biomanufacturing platform enabled by a streamlined designer pathway

Le Yu, Ruyi Chen, Chenyue Zhang, Zhengyuan Wang, Zhuqing Wang, Xinyue Zeng, Han Liang, Yuanyuan He, Yixuan She, Yifei Wang, Rong Gong, Xuemin Song, Zixin Deng, Qiang Fei & Wenqing Chen

Nature Communications 2025

https://doi.org/10.1038/s41467-025-63906-0

Abstract

mRNA vaccines, featured by incorporated pseudouridine (Ψ), represent a milestone in combating diseases, thus highlighting Ψ importance in drug development. However, economic and environmental challenges have persisted in sustainable Ψ production. Here, we formulate a streamlined designer Ψ pathway, comprising UMP nucleosidase, ΨMP glycosidase, and ΨMP phosphatase, and realize its gram-scale production by targeted discovery of a prominent UMP-preferred nucleosidase (NmYgdH). The optimized pathway, containing NmYgdH, RjPsuG (ΨMP glycosidase), and HDHD1 (ΨMP-specific phosphatase) is cloned into E. coli and systematic evaluation of multiple strategies achieves a Ψ titer of 44.8 g·L−1. Moreover, a thyA-dependent, tunable, and eco-friendly strategy for sustainable Ψ production is demonstrated in a 5 L bioreactor achieving titer of 45.3 g·L−1. Finally, we establish a simplified-strategy for rapid Ψ purification with a recovery-rate of 71%, and techno-economic analysis is employed to validate the feasibility and advantages of this fermentation platform for Ψ biomanufacturing. Therefore, this study provides a blueprint for industrial-production of nucleoside-related molecules.