De novo biosynthesis of Asperosaponin VI in Saccharomyces cerevisiae

Asperosaponin VI (ASA VI), the primary bioactive triterpenoid saponin marker of <i>Dipsacus asper</i> 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 <i>Saccharomyces cerevisiae</i> using a modular synthetic biology strategy. The pathway included in situ UDP-arabinose (UDP-Ara) biosynthesis via heterologous expression of <i>AtUGDH3</i>, <i>GuUXS2</i>, and <i>GuUXE1</i>; triterpenoid scaffold generation through <i>ERG9</i>, <i>ERG1</i>, <i>CqBAS1</i>, and <i>CqCYP716A78</i> for oleanolic acid (OA) production; and downstream modifications including C-23 hydroxylation by multicopy-expressed <i>CaCYP714E19</i>, stepwise glucosylation at C-28 by <i>CaUGT73AD1</i> and <i>CaUGT73C8</i>, and C-3 arabinosylation by <i>AsUGT99D1</i> 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.