Engineering Salvia miltiorrhiza hairy roots as a scalable platform for high-yield production of paclitaxel and ginsenoside precursors.

Plant-derived natural products offer a rich source of therapeutic agents. However, sustainable and high-yield production remains a grand challenge. We engineered Salvia miltiorrhiza hairy roots to produce taxadiene, a key precursor for the anticancer drug paclitaxel, and protopanaxadiol, a precursor for ginsenosides. The heterologous expression of two key biosynthetic genes, taxadiene synthase from Taxus wallichiana and protopanaxadiol synthase from Panax notoginseng, enabled the production of taxadiene and protopanaxadiol, respectively. Our strategy combined multiple approaches to enhance terpenoid production, including genome editing to redirect metabolic flux by eliminating a competing GGPP sink (via SmCPS1 disruption), transcriptional reprogramming through SmWRKY61 overexpression to enhance terpenoid precursor pathways (MVA/MEP), and optimization of cultivation conditions. This holistic approach yielded 65.17 ± 5.25 mg/kg fresh weight (FW) taxadiene in batch cultures, and the protopanaxadiol yield reached 50.04 ± 2.94 mg/kg dry weight (DW) without optimization. These results highlight the potential of this platform for industrial-scale production. Our findings demonstrate that S. miltiorrhiza hairy roots can serve as a robust and scalable platform to produce valuable plant-derived compounds. This work paves the way for future metabolic engineering efforts to achieve cost-effective and sustainable production of high-value natural products using medicinal plant systems, addressing critical supply bottlenecks for pharmaceutical compounds.