Cloning and Direct Evolution of a Novel 7-<i>O</i>-Glycosyltransferase from <i>Cucurbita moschata</i> and Its Application in the Efficient Biocatalytic Synthesis of Luteolin-7-<i>O</i>-glucoside

Luteolin-7-<i>O</i>-glucoside(L7G), a glycosylation product of luteolin, is present in a variety of foods, vegetables, and medicinal herbs and is commonly used in dietary supplements due to its health benefits. Meanwhile, luteolin-7-<i>O</i>-glucoside is an indicator component for the quality control of honeysuckle in the pharmacopoeia. However, its low content in plants has hindered its use in animal pharmacological studies and clinical practice. In this study, a novel 7-<i>O</i>-glycosyltransferase CmGT from <i>Cucurbita moschata</i> was cloned, which could efficiently convert luteolin into luteolin-7-<i>O</i>-glucoside under optimal conditions (40 °C and pH 8.5). To further improve the catalytic efficiency of CmGT, a 3D structure of CmGT was constructed, and directed evolution was performed. The mutant CmGT-S16A-T80W was obtained by using alanine scanning and iterative saturation mutagenesis. This mutant exhibited a <i>k</i>_cat/<i>K</i>_m value of 772 s^-1·M^-1, which was 3.16-fold of the wild-type enzyme CmGT. Finally, by introducing a soluble tag and UDPG synthesis pathway, the strain BXC was able to convert 1.25 g/L of luteolin into 1.91 g/L of luteolin-7-<i>O</i>-glucoside under optimal conditions, achieving a molar conversion rate of 96% and a space-time yield of 27.08 mg/L/h. This study provides an efficient method for the biosynthesis of luteolin-7-<i>O</i>-glucoside, which holds broad application prospects in the food and pharmaceutical industry.