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<p>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 (<i>Nm</i>YgdH). The optimized pathway, containing <i>Nm</i>YgdH, <i>Rj</i>PsuG (ΨMP glycosidase), and HDHD1 (ΨMP-specific phosphatase) is cloned into <i>E. coli</i> and systematic evaluation of multiple strategies achieves a Ψ titer of 44.8 g·L<sup>−1</sup>. Moreover, a <i>thyA</i>-dependent, tunable, and eco-friendly strategy for sustainable Ψ production is demonstrated in a 5 L bioreactor achieving titer of 45.3 g·L<sup>−1</sup>. 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.</p>

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A combined pseudouridine biomanufacturing platform enabled by a streamlined designer pathway

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月6日

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  • 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

摘要

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.