Artificially Created UDP-Glucose 2‑Epimerase Enables Concise UDP/GDP-Mannose Production via the Synthase–Epimerase Route

Uridine/guanosine diphosphate-mannose (UDP/GDP-Man) is the major mannosyl donor in producing mannose-containing oligo/polysaccharides. Its acquisition is greatly limited by its complex and costly synthetic process, which requires multiple substrates and enzymes. The natural UDP/GDP-glucose 2-epimerase functioning C2 epimerization between UDP/GDP-Glc and UDP/GDP-Man remains unreported which is the main hurdle to realize concise production of UDP/GDP-Man. Here, the UDP-glucose 2-epimerase (Glc2E), which behaves like a naturally evolved enzyme, is created and exhibits high-efficient catalysis in producing UDP-Man. Multidimensional engineering, including redesigning the nucleobase recognition region, displacement of the substrate tunnel entrance, and expansion of space for sugar ring rotation, is employed to develop Glc2E from CDP-tyvelose 2-epimerase. Glc2E converts 55.63% of UDP-Glc to UDP-Man, a trace value for the initial enzyme, stTyvE, and its aptitude for GDP-Glc epimerization evolves from unobserved activity to 23.94% conversion. Coupling sucrose synthase with Glc2E achieves the theoretical synthase–epimerase route for UDP/GDP-Man production from inexpensive sucrose. The space-time-yield of UDP-Man is maximized to 8.05 g/L/h within 2.5 h, with a final titer of 22.54 g/L, demonstrating competitive application potential. Moreover, the GDP-Man is synthesized successfully at a titer of 3.49 g/L. Our work inspires the enzyme engineering for epimerases and glycosyltransferases that catalyze nucleotide sugars. The application of Glc2E in the synthase–epimerase route unlocks a concise and feasible synthetic approach for producing cost-competitive mannosyl donors.