Cloning and Functional Characterization of Farnesyl Diphosphate Synthase Gene in Biosynthesis of Terpenoid Components in Chinese Materia Medica
10.13422/j.cnki.syfjx.20250380
- VernacularTitle:中药萜类成分合成中法尼基二磷酸合成酶基因的克隆及功能表征
- Author:
Yue ZHANG
1
;
Feng ZHANG
1
;
Yue ZHANG
1
;
Chaoyue LIU
1
;
Bolin ZHANG
1
;
Jia LIU
1
;
Caixia WANG
1
Author Information
1. Institute of Chinese Materia Medica，China Academy of Chinese Medical Sciences，Beijing 100700，China
- Publication Type:Journal Article
- Keywords:
Saccharomyces cerevisiae;
sesquiterpenes;
β-elemene;
gene cloning;
farnesyl pyrophosphate（FPP）;
farnesyl diphosphate synthase（FPS）
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2025;31(3):175-183
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveThis study aims to enhance of the farnesyl pyrophosphate（FPP） pool in Saccharomyces cerevisiae by heterologously expressing different farnesyl diphosphate synthases（FPSs） from various plants， thereby increasing the production of terpenoid compounds by the engineered yeast. MethodsRNA from mixed samples of roots， stems， and leaves of seven plants including Arabidopsis thaliana， Rosa rugosa， Artemisia annua， Centella asiatica， Humulus lupulus， Medicago sativa， and Panax ginseng was extracted by column chromatography and reverse transcribed into the first strand of complementary DNA（cDNA）， and based on the transcriptome data of the seven species of plants， sequence-specific primers were designed for CaFPS， RrFPS， MsFPS， HiFPS， PgFPS， AtFPS， and AaFPS， the full-length of the genes was cloned， and the genes were analyzed for bioinformatics in order to construct a pESC yeast shuttle vector. These seven plant-derived FPSs were further heterologously expressed in the previous constructed β-elemene-producing yeast， and the yield of β-elemene was indicated for their catalytic acivities. ResultsThe coding sequences of CaFPS， RrFPS， MsFPS， HiFPS， PgFPS， AtFPS， and AaFPS were all of 1 021 bp in length and encoding 301 amino acids， all of which were similarly related to the endogenous FPS-encoding gene（ERG20） in S. cerevisiae. After heterologous expression， RrFPS was identified as the most effective in catalyzing the synthesis of FPP from isopentenyl pyrophosphate（IPP） and dimethylallyl pyrophosphate（DMAPP）. Compared to the control strains， the RrFPS overexpressed yeast strains YB-1-Rr and YB-3-Rr increased the production of β-elemene by 231.25% and 189.3%， respectively. ConclusionBy comparing the functions of FPS-encoding genes from seven different plant sources， it is determined that the protein encoded by the RrFPS from R. rugosa has the best catalytic ability， which can provide key genetic elements for the construction of engineered yeast strain constructs with high terpenoid production.
