Site-directed mutagenesis of ent-kaurane diterpenoid C-19 oxidase TwKO in Tripterygium wilfordii.
10.19540/j.cnki.cjcmm.20240918.101
- Author:
Rong-Feng WANG
1
;
Zheng LIU
1
;
Xin-Meng WANG
1
;
Wei GAO
1
;
Jia-Dian WANG
1
;
Ya-Ting HU
1
;
Lu-Qi HUANG
2
Author Information
1. School of Traditional Chinese Medicine, Capital Medical University Beijing 100069, China.
2. National Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences Beijing 100700, China.
- Publication Type:Journal Article
- Keywords:
ent-kaurane diterpenoids;
ent-kaurane oxidase;
site-directed mutagenesis;
tripterifordin
- MeSH:
Tripterygium/chemistry*;
Mutagenesis, Site-Directed;
Diterpenes, Kaurane/chemistry*;
Plant Proteins/chemistry*;
Cytochrome P-450 Enzyme System/chemistry*;
Saccharomyces cerevisiae/metabolism*
- From:
China Journal of Chinese Materia Medica
2024;49(24):6667-6675
- CountryChina
- Language:Chinese
-
Abstract:
Tripterifordin and neotripterifordin are important ent-kaurane diterpenoids in the Chinese medicinal herb Tripterygium wilfordii, possessing significant anti-HIV(human immunodeficiency virus) activity. On the basis of elucidating the natural biosynthetic pathways of these compounds, heterologous production with microbial cell factories can help to alleviate the reliance on plant resources and provide abundant raw materials for sustainable production. TwKO is the first CYP450 enzyme involved in the biosynthesis of tripterifordin and neotripterifordin. This study aimed to enhance the catalytic activity of TwKO by site-directed mutagenesis to benefit the production of tripterifordin and neotripterifordin in yeast. The AlphaFold DB established based on the AlphaFold 2 was employed to obtain the protein model of TwKO. According to multiple sequence alignments and principles of natural evolution, the key residues influencing the binding of TwKO to the substrate were identified. Subsequently, functional characterization of the mutants were conducted in Saccharomyces cerevisiae. A total of 71 mutants were obtained, among which 11 and 11 mutants had the abilities of enhancing the production of 16α-hydroxy-ent-kaurenol and 16α-hydroxy-ent-kaurenoic acid, respectively. In addition, 10 mutants could increase the proportion of the oxidation product of 16α-hydroxy-ent-kaurenol. In particular, R304 was identified as a key residue affecting the catalytic specificity of TwKO, the mutation of which led to the specific prodiction of 16α-hydroxy-ent-kaurenol. This study was the first to reveal the key residue affecting the catalytic activity of TwKO and obtained the mutants with increased TwKO activity, lay a foundation for the biosynthesis of tripterifordin and neotripterifordin.
