Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene PAL promotes ginsenoside Rg accumulation in ginseng plant chassis.

The ginsenoside Rg found in Panax species has extensive pharmacological properties, in particular anti-cancer effects. However, its natural yield in Panax plants is limited. Here, we report a multi-modular strategy to improve yields of Rg in a Panax ginseng chassis, combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene, phenylalanine ammonia lyase (PAL). We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2, a glycosyltransferase that directly catalyzes the biosynthesis of Rg from Rh . Next, we used clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg . Overexpression of PAL accelerated the formation of the xylem structure, significantly improving ginsenoside Rg accumulation (to 6.19-fold higher than in the control). We combined overexpression of the ginsenoside aglycon synthetic genes squalene epoxidase, Pq3-O-UGT2, and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rg accumulation. Finally, we produced ginsenoside Rg at a yield of 83.6 mg/L in a shake flask (7.0 mg/g dry weight, 21.12-fold higher than with wild-type cultures). The high-production system established in this study could be a potential platform to produce the ginsenoside Rg commercially for pharmaceutical use.