SUMMARY Astragaloside IV is a significant chemical component derived from the medicinal plant Astragalus membranaceus . Despite the characterization of several glycosyltransferases from A. membranaceus , the complete biosynthetic pathway of astragaloside IV has not been fully elucidated. In this study, we propose a biosynthetic pathway for astragaloside IV that involves a sequence of oxidation–reduction reactions. The biosynthesis pathway from cycloastragenol to astragaloside IV encompasses four key steps: C‐3 oxidation, 6‐ O ‐glucosylation, C‐3 reduction, and 3‐ O ‐xylosylation. We identified a hydroxysteroid dehydrogenase AmHSD1 from A. membranaceus . AmHSD1 catalyzes the C‐3 oxidation of cycloastragenol, yielding cycloastragenol‐3‐one, and the C‐3 reduction of cycloastragenol‐3‐one‐6‐ O ‐glucoside, resulting in cycloastragenol‐6‐ O ‐glucoside. Additionally, the glycosyltransferases AmGT8 and AmGT1, previously reported by our groups, were identified as catalyzing the 6‐ O ‐glucosylation and 3‐ O ‐xylosylation steps, respectively. Astragaloside IV was successfully synthesized in transient expression in Nicotiana benthamiana using the combination of AmHSD1, AmGT8 and AmGT1. These results support the proposed four‐step biosynthetic pathway and suggest that AmHSD1 probably plays a crucial role in the biosynthesis of astragaloside IV within A. membranaceus .

A four‐step biosynthetic pathway involving C‐3 oxidation–reduction reactions from cycloastragenol to astragaloside <scp>IV</scp> in <i>Astragalus membranaceus</i>

A four‐step biosynthetic pathway involving C‐3 oxidation–reduction reactions from cycloastragenol to astragaloside IV in Astragalus membranaceus

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