Mining UDP-Glucosyltransferases to Develop High-Efficiency Production Strategies for Emodin Glucosides

Emodin-6-<i>O</i>-β-d-glucoside (E6G) and Emodin-8-<i>O</i>-β-d-glucoside (E8G) are promising plant-derived bioactive compounds with significant pharmaceutical potential. However, their large-scale production is hindered by low natural abundance and complex extraction processes. In this study, the biosynthesis of emodin glucosides in filamentous fungi was comprehensively investigated using synthetic biology approaches. We evaluated ten highly efficient emodin UDP-glucosyltransferases (EUGTs) and individually integrated these EUGTs into an emodin-accumulating <i>Aspergillus terreus</i> strain. Among these, the ΔgedA-Ft73BE variant achieved the highest E6G titer of 217.5 mg/L. However, further optimization was impeded by challenges in expressing plant-derived proteins in filamentous fungi. To address this, we developed a whole-cell catalytic system in <i>Escherichia coli</i> harboring Ft73BE, achieving a high conversion rate of 78.9%. Sequence, structural, and phylogenetic analyses provided key insights into the enzymatic mechanisms of EUGTs, revealing OsUGT's dual-site glucosylation activity. This work advances our understanding of emodin glucosylation and plant protein expression in microbial hosts while establishing a sustainable platform for the large-scale production of these valuable compounds.