Biosynthesis and bioactivity of anti-inflammatory triterpenoids in Calendula officinalis

Abstract Plants have been central to traditional medicine for millennia, yet the precise metabolites responsible for their therapeutic properties often remain unidentified. In this work, we investigate the reported anti-inflammatory properties of Calendula officinalis (pot marigold), an ancient medicinal herb. We confirm C16-hydroxylated triterpenoids as key contributors to the anti-inflammatory activity of C. officinalis floral extracts and uncover a mechanism by which they act in modulating interleukin 6 release. Through biosynthetic pathway elucidation, we demonstrate that the oxidosqualene synthase catalysing the first committed step emerged early in Asteraceae evolution and identify residues governing product specificity. Further, we functionally characterise cytochrome P450s and acyltransferases responsible for downstream modifications. By reconstructing the complete biosynthetic pathway in the plant chassis Nicotiana benthamiana , we provide a basis for the future bioproduction of the anti-inflammatory components. Our work highlights how integrated studies of bioactivity and biosynthesis can unlock the therapeutic potential of medicinal plants.