Enhancing Centelloside Production in Hairy Root Lines through Metabolic Engineering of Triterpene Biosynthetic Pathway Early Genes.

is a medicinal plant with a rich tradition of use for its therapeutic properties. Among its bioactive compounds are centellosides, a group of triterpenoid secondary metabolites whose potent pharmacological activities have attracted significant attention. Metabolic engineering has emerged as a powerful biotechnological tool to enhance the production of target compounds. In this study, we explored the effects of overexpressing the squalene synthase () gene and transcription factor on various aspects of hairy root lines: the expression level of centelloside biosynthetic genes, morphological traits, as well as squalene, phytosterol, and centelloside content. Three distinct categories of transformed lines were obtained: LS, harboring ; LT, overexpressing ; and LST, simultaneously carrying both transgenes. These lines displayed noticeable alterations in morphological traits, including changes in branching rate and biomass production. Furthermore, we observed that the expression of T-DNA genes, particularly and genes, significantly modulated the expression of pivotal genes involved in centelloside biosynthesis. Notably, the LS lines boasted an elevated centelloside content but concurrently displayed reduced phytosterol content, a finding that underscores the intriguing antagonistic relationship between phytosterol and triterpene pathways. Additionally, the inverse correlation between the centelloside content and morphological growth values observed in LS lines was countered by the action of in the LST and LT lines. This difference could be attributed to the simultaneous increase in the phytosterol content in the -expressing lines, as these compounds are closely linked to root development. Overall, these discoveries offer valuable information for the biotechnological application of hairy roots and their potential to increase centelloside production.