Discovery of Scrophularia nodosa harpagoside synthase, a novel BAHD cinnamoyltransferase, bridges a key gap in the iridoid biosynthetic pathway

Harpagoside, a high-value anti-inflammatory iridoid compound, is traditionally extracted from the roots of Harpagophytum procumbens (Pedaliaceae, Lamiales), a Southern African desert plant widely used in traditional medicine but currently threatened by overexploitation. Scrophularia nodosa (Scrophulariaceae, Lamiales) is a perennial annual plant widely distributed in Western Europe and accumulates several iridoid compounds with known biological activities, such as catalpol, aucubin, harpagide and particularly harpagoside. We gathered extensive genomic and transcriptomics resources for this species and aimed at deciphering the biosynthetic pathway leading to the most abundant iridoid in S. nodosa, harpagoside. We found that the early iridoid pathway is well conserved with other iridoid-producing plants and validated the enzyme activities by transient co-expression in N. benthamiana. Investigation into the large BAHD family showed subclade 6i expanding in Scrophulariaceae, with an atypical VYPWG motif instead of the canonical DFGWG. In this branch, we discovered and characterized harpagoside synthase, a BAHD-type cinnamoyl transferase enzyme showing unique high specificity to the uncommon cinnamoyl-CoA acyl donor and catalyzing the final step of harpagoside biosynthesis. These results establish S. nodosa as a new model to investigate unexplored branches of the iridoids metabolism, and are a first step towards sustainable harpagoside and high-value cinnamoyl-containing conjugates production.