Artificially Created\nUDP-Glucose 2‑Epimerase\nEnables Concise UDP/GDP-Mannose Production via the Synthase–Epimerase\nRoute

Uridine/guanosine diphosphate-mannose (UDP/GDP-Man) is\nthe major\nmannosyl donor in producing mannose-containing oligo/polysaccharides.\nIts acquisition is greatly limited by its complex and costly synthetic\nprocess, which requires multiple substrates and enzymes. The natural\nUDP/GDP-glucose 2-epimerase functioning C2 epimerization between UDP/GDP-Glc\nand UDP/GDP-Man remains unreported which is the main hurdle to realize\nconcise production of UDP/GDP-Man. Here, the UDP-glucose 2-epimerase\n(Glc2E), which behaves like a naturally evolved enzyme, is created\nand exhibits high-efficient catalysis in producing UDP-Man. Multidimensional\nengineering, including redesigning the nucleobase recognition region,\ndisplacement of the substrate tunnel entrance, and expansion of space\nfor sugar ring rotation, is employed to develop Glc2E from CDP-tyvelose\n2-epimerase. Glc2E converts 55.63% of UDP-Glc to UDP-Man, a trace\nvalue for the initial enzyme, <i>st</i>TyvE, and its aptitude\nfor GDP-Glc epimerization evolves from unobserved activity to 23.94%\nconversion. Coupling sucrose synthase with Glc2E achieves the theoretical\nsynthase–epimerase route for UDP/GDP-Man production from inexpensive\nsucrose. The space-time-yield of UDP-Man is maximized to 8.05 g/L/h\nwithin 2.5 h, with a final titer of 22.54 g/L, demonstrating competitive\napplication potential. Moreover, the GDP-Man is synthesized successfully\nat a titer of 3.49 g/L. Our work inspires the enzyme engineering for\nepimerases and glycosyltransferases that catalyze nucleotide sugars.\nThe application of Glc2E in the synthase–epimerase route unlocks\na concise and feasible synthetic approach for producing cost-competitive\nmannosyl donors.