Structure-Guided Engineering of UGT94B1_M0 Enhances Thermostability and Enables Efficient Rebaudioside D Biosynthesis...

Enhancing the thermostability of UDP-glycosyltransferases is essential for their industrial application in natural product biosynthesis. Here, we developed a structure-guided, computation-assisted strategy to improve the stability and efficiency of UGT94B1_M0, which catalyzes the conversion of rebaudioside A to the high-value sweetener rebaudioside D. By integrating multiple computational tools and energy-based analyses, a focused mutation library was constructed and experimentally screened. The optimal variant, UGT94B1_M3, displayed an 8.40 °C higher melting temperature, a 20.65-fold longer half-life, and a 1.45-fold enhancement in catalytic efficiency relative to M0. Molecular dynamics simulations revealed that these improvements were associated with increased structural rigidity and favorable electrostatic interactions. When coupled with Arabidopsis thaliana sucrose synthase for UDP-glucose regeneration, M3-AtSuSy produced 33.87 mM Reb D within 1 h at a molar conversion rate of 84.67%, 2.81-fold higher than M0-AtSuSy. This work establishes a generalizable strategy for thermostability engineering of UDP-glycosyltransferases toward efficient and sustainable glycoside biosynthesis.