Overexpression of the DXP reductoisomerase (DXR) gene increases the production of valuable biologically-active compounds in the transgenic hairy roots of Plectranthus venteri (van Jaarsv. & Hankey)

Transgenic hairy root cultures offer a sustainable alternative for producing high-value bioactive compounds. This study aimed to generate Plectranthus venteri hairy roots overexpressing the 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene of the methylerythritol phosphate pathway (MEP) to enhance diterpenoid biosynthesis. Stable integration of the DXR transgene was confirmed by molecular analyses. DXR-overexpressing hairy root clones showed increased accumulation of valuable diterpenoids compared with unmodified controls transformed with a pGFPGUSPlus vector. The best-growing clone was scaled up in a 10 L sprinkle bioreactor, resulting in markedly higher levels of selected metabolites. The averaged peak area of coleon U-quinone isomer 1 was 17.3-fold higher in the bioreactor culture and 10.5-fold higher in the Erlenmeyer flask culture relative to the unmodified sample, while coleon U isomer 2 levels were 3.66-fold and 5.19-fold higher in the bioreactor and Erlenmeyer cultures, respectively. High-resolution mass spectrometry also identified dihydroxyroyleanone, royleanone derivatives, horminone, and 7α-hydroxyroyleanone, compounds known to have anticancer, antimicrobial, and anti-inflammatory activities. These findings demonstrate that combining DXR overexpression with bioreactor cultivation significantly enhances diterpenoid production in P. venteri hairy roots and may serve as an efficient platform for sustainable production of pharmacologically relevant metabolites. • Confirm integration of the DXR transgene into hairy root genomes by molecular analyses. • Achieve DXR gene overexpression in Plectranthus venteri hairy root cultures. • Scale up hairy root cultures in a 10 L sprinkle bioreactor for diterpene production. • Identify a broader range of terpenoids in hairy roots using mass spectrometry.