De novo biosynthesis of Asperosaponin VI in Saccharomyces cerevisiae

ABSTRACT Asperosaponin VI (ASA VI), the primary bioactive triterpenoid saponin marker of Dipsacus asper Wall. (Chinese Pharmacopoeia 2020), possesses significant neuroprotective, anti-inflammatory, and osteogenic activities. However, its low natural abundance limits large-scale production. In this study, we reported the first complete biosynthetic reconstruction of ASA VI in Saccharomyces cerevisiae using a modular synthetic biology strategy. The pathway included in situ UDP-arabinose (UDP-Ara) biosynthesis via heterologous expression of AtUGDH3 , GuUXS2 , and GuUXE1 ; triterpenoid scaffold generation through ERG9 , ERG1 , CqBAS1 , and CqCYP716A78 for oleanolic acid (OA) production; and downstream modifications including C-23 hydroxylation by multicopy-expressed CaCYP714E19 , stepwise glucosylation at C-28 by CaUGT73AD1 and CaUGT73C8 , and C-3 arabinosylation by AsUGT99D1 to yield ASA VI. LC-MS analysis confirmed ASA VI biosynthesis and the accumulation of key intermediates (OA, HED, HED-28-Glc, and HED-28-Glc-Glc). Although production remained at trace levels (395 ng/L), pathway analysis suggested that the downstream glycosylation steps and UDP-Ara supply could be the major rate-limiting factors. This work established a microbial chassis for the sustainable synthesis of ASA VI and related arabinosylated saponins.