Enhancement of UDP-Arabinose Supply and Engineering of Glycosyltransferase DaUGT121 from Dipsacus asperoides for Cauloside A Biosynthesis in Escherichia coli

Cauloside A (hederagenin 3- O -α- L -arabinopyranoside) is a bioactive triterpenoid saponin with demonstrated anti-inflammatory, antimicrobial, cytotoxic, hemolytic, and molluscicidal properties. However, its structurally complex nature and limited natural availability make both large-scale chemical synthesis and extraction from medicinal plants particularly challenging. Microbial conversion via heterologous expression of glycosyltransferases provides a convenient and sustainable approach to produce cauloside A. Consequently, the efficient supply of uridine diphosphate-arabinose (UDP-Ara) is a critical determinant of the microbial synthesis of glycosides. In this study, we first engineered Escherichia coli ( E. coli ) to express pathway enzymes, enabling the accumulation of UDP-glucose, UDP-glucuronic acid, UDP-xylose, and UDP-Ara. The biosynthesis of UDP-Ara was subsequently enhanced through pathway optimization and the implementation of a uridine triphosphate regeneration system. Additionally, a salvage pathway comprising arabinose kinase and UDP-sugar pyrophosphorylase was engineered to increase the supply of UDP-Ara in E. coli . Finally, the production of cauloside A was achieved for the first time by introducing the engineered glycosyltransferase DaUGT121 from Dipsacus asperoides into UDP-Ara-producing strains. Through fed-batch fermentation in a 5 L bioreactor, the concentration of cauloside A reached 435.6 mg∙L –1 . This study presents an efficient and scalable strategy for the biosynthesis of other high-value arabinose-derived natural products.