Engineering <i>Gypsophila elegans</i> hairy root cultures to produce endosomal escape‐enhancing saponins

The limited cytosolic delivery of DNA and protein-based therapeutics due to endosomal entrapment reduces drug efficacy, increasing treatment costs and possible side effects in human and veterinary medicine as a consequence of higher administered dosages. Plant-derived triterpenoid saponins, specifically those with endosomal escape-enhancing (EEE) properties, have shown promise in overcoming this limitation by disrupting endosomal membranes. QS-21, a well-known EEE saponin, has been used as an adjuvant in vaccines, and recent studies have elucidated its biosynthetic pathway. However, EEE saponins are typically present as minor compounds in plants, posing challenges for their large-scale production and purification. Here we investigated the possibility of engineering SO1861 production, an EEE saponin from Saponaria officinalis, using heterologous gene expression in Gypsophila elegans hairy roots, a plant species known to synthesize structurally related saponins. Via S. officinalis transcriptomics, we identified jasmonate-responsive saponin biosynthetic genes, and three cytochrome P450s (CYP450s) involved in C23, C28 and C16 oxidations were characterised. Heterologous expression of these CYP450s in G. elegans hairy roots successfully altered the saponin profile, with notable increases in SO1861 precursors in lines expressing the C23-oxidases SoCYP72A984 and SoCYP72A1003. Interestingly, expression of only SoCYP72A1003, a non-canonical C23 oxidase, resulted in the accumulation of a compound matching the SO1861 standard, suggesting the activation of a potentially latent pathway and of silent enzymes in a novel combination. This work underscores the potential of engineering strategies in heterologous plant systems to steer triterpenoid saponin biosynthetic pathways and suggests new avenues for producing high-value EEE saponins.