Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene <i>PAL</i> promotes ginsenoside Rg₃ accumulation in ginseng plant chassis

Abstract

Abstract The ginsenoside Rg 3 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 3 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 3 from Rh 2 . 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 3 . Overexpression of PAL accelerated the formation of the xylem structure, significantly improving ginsenoside Rg 3 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 3 accumulation. Finally, we produced ginsenoside Rg 3 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 3 commercially for pharmaceutical use.