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Area of Science:

  • Biotechnology
  • Synthetic Biology
  • Biochemical Engineering

Background:

  • Pseudouridine (Ψ) is crucial for mRNA vaccine efficacy and drug development.
  • Existing methods for pseudouridine production face economic and environmental sustainability challenges.

Purpose of the Study:

  • To develop a streamlined and sustainable biomanufacturing platform for pseudouridine (Ψ) production.
  • To engineer an optimized metabolic pathway in E. coli for high-titer pseudouridine (Ψ) synthesis.

Main Methods:

  • Designed a synthetic pathway involving UMP nucleosidase, ΨMP glycosidase, and ΨMP phosphatase.
  • Identified and utilized a UMP-preferred nucleosidase (NmYgdH) for pathway construction.
  • Optimized the pathway in E. coli and employed a thyA-dependent strategy for scalable, eco-friendly production in a 5L bioreactor.

Main Results:

  • Achieved a pseudouridine (Ψ) titer of 44.8 g·L⁻¹ in E. coli using the optimized pathway.
  • Demonstrated a sustainable production titer of 45.3 g·L⁻¹ in a 5L bioreactor.
  • Developed a simplified purification strategy with a 71% recovery rate and validated the platform's economic feasibility.

Conclusions:

  • The engineered pathway and fermentation platform provide a viable blueprint for industrial-scale pseudouridine (Ψ) biomanufacturing.
  • This study offers a sustainable and cost-effective solution for producing pseudouridine (Ψ) and related nucleoside molecules.