Metabolic Engineering of Tobacco for Heterologous Production of Rare Ginsenosides CK and Rh2

Rare ginsenosides, such as compound K (CK) and Rh2, possess potent pharmacological activities but are present at extremely low levels in natural Panax ginseng . Here, we report the successful heterologous biosynthesis of CK and Rh2 in tobacco ( Nicotiana benthamiana and N. tabacum K326) through metabolic engineering. Three pivotal genes— DDS , CYP716A47 , and either UGTPg1 or UGTPg45 —were introduced into tobacco using a multi-gene overexpression construct. Transgenic lines were generated and screened, and ginsenoside accumulation was quantified via LC–MS. N. benthamiana exhibited superior CK production, reaching up to 47.87 µg/g DW in roots, whereas K326 showed higher Rh2 accumulation, up to 8.11 µg/g DW in roots. Across both species, roots consistently contained greater ginsenoside levels than leaves. Topping significantly enhanced yield, and callus cultures demonstrated the potential for scalable in vitro production. These results establish tobacco as a robust plant chassis for the heterologous production of rare ginsenosides.