Engineering the Stability of Sucrose Synthase to Enable Efficient Coupled Production of UDP‐Glucuronic Acid

Abstract

The biocatalytic synthesis of UDP‐glucuronic acid (UDPGA), a key precursor for glucuronides and high‐value carbohydrates, is limited by an unstable UDP‐glucose (UDPG) supply in coupled systems. In this study, through an extensive screening of 10 sucrose synthases derived from both eukaryotic and prokaryotic microorganisms, we identified a broad‐spectrum, highly efficient enzymatic scaffold suitable for the synthesis of UDP‐sugars. A rational design integrating PROSS and ThermoMPNN algorithms with molecular dynamics (MD) analysis yielded the V353L mutant, which retained 97% of wild‐type activity and extended the half‐life at 55°C from 0.4 to 5.5 h. Tetrameric MD simulations indicated that V353L strengthens hydrophobic stacking in the GT‐B linker region, reducing conformational fluctuations and driving the enzyme toward a stable low‐energy state. In a dual‐enzyme cascade synthesizing UDPG and UDPGA, the mutant sustained UDP consumption and increased the accumulated UDPGA concentration from 0.60 to 2.09 mM after 8 h. Engineering sucrose synthase thermostability thus improves UDPG supply in cascade biocatalysis and offers a viable enzymatic strategy for UDPGA‐centered synthesis of high‐value carbohydrate derivatives.