J Integr Plant Biol ›› 2022, Vol. 64 ›› Issue (9): 1739-1754.DOI: 10.1111/jipb.13315

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  • 收稿日期:2022-01-30 接受日期:2022-06-20 出版日期:2022-09-01 发布日期:2022-09-16

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

Lu Yao1,2,3†, Huanyu Zhang1,2,3†, Yirong Liu1, Qiushuang Ji1, Jing Xie1, Ru Zhang1, Luqi Huang4, Kunrong Mei1*, Juan Wang1,2,3* and Wenyuan Gao1,2,3*   

  1. 1 School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China
    2 Wenzhou Safety (Emergency) Institute of Tianjin University, Wenzhou 325000, China
    3 Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, China
    4 National Resource Center for Chinese Meteria Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

    † These authors contributed equally to this work.
    * Correspondences: Wenyuan Gao (pharmgao@tju.edu.cn; Dr. Gao is fully responsible for the distribution of all materials associated with this article); Juan Wang (drwangjuan@tju.edu.cn); Kunrong Mei (kmei@tju.edu.cn)
  • Received:2022-01-30 Accepted:2022-06-20 Online:2022-09-01 Published:2022-09-16

Abstract:

The ginsenoside Rg3 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 Rg3 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 Rg3 from Rh2. 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 Rg3. Overexpression of PAL accelerated the formation of the xylem structure, significantly improving ginsenoside Rg3 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 Rg3 accumulation. Finally, we produced ginsenoside Rg3 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 Rg3 commercially for pharmaceutical use.

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