Triptolide is a trace natural product of Tripterygium wilfordii. It has antitumor activities, particularly against pancreatic cancer cells. Identification of genes and elucidation of the biosynthetic ...pathway leading to triptolide are the prerequisite for heterologous bioproduction. Here, we report a reference-grade genome of T. wilfordii with a contig N50 of 4.36 Mb. We show that copy numbers of triptolide biosynthetic pathway genes are impacted by a recent whole-genome triplication event. We further integrate genomic, transcriptomic, and metabolomic data to map a gene-to-metabolite network. This leads to the identification of a cytochrome P450 (CYP728B70) that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis. We think the genomic resource and the candidate genes reported here set the foundation to fully reveal triptolide biosynthetic pathway and consequently the heterologous bioproduction.
Celastrol, a triterpene compound derived from the traditional Chinese medicine Tripterygium wilfordii, has been reported to possess potential antitumor activity towards various malignancies. However, ...the effect of celastrol on glioma cells and the underlying molecular mechanisms remain elusive.
Glioma cells, including the U251, U87-MG and C6 cell lines and an animal model were used. The effects of celastrol on cells were evaluated by flow cytometry, confocal microscopy, reactive oxygen species production assay and immunoblotting after treatment of celastrol. Fisher's exact test, a one-way ANOVA and the Mann-Whitney U-test were used to compare differences between groups. All data were analyzed using SPSS version 21.0 software.
Here, we found that exposure to celastrol induced G2/M phase arrest and apoptosis. Celastrol increased the formation of autophagosomes, accumulation of LC3B and the expression of p62 protein. Celastrol-treated glioma cells exhibited decreased cell viability after the use of autophagy inhibitors. Additionally, autophagy and apoptosis caused by celastrol in glioma cells inhibited each other. Furthermore, celastrol induced JNK activation and ROS production and inhibited the activities of Akt and mTOR kinases. JNK and ROS inhibitors significantly attenuated celastrol-trigged apoptosis and autophagy, while Akt and mTOR inhibitors had opposite effects.
In conclusion, our study revealed that celastrol caused G2/M phase arrest and trigged apoptosis and autophagy by activating ROS/JNK signaling and blocking the Akt/mTOR signaling pathway.
Panax notoginseng, a perennial herb of the genus Panax in the family Araliaceae, has played an important role in clinical treatment in China for thousands of years because of its extensive ...pharmacological effects. Here, we report a high-quality reference genome of P. notoginseng, with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb, produced with third-generation PacBio sequencing technology. This is the first chromosome-level genome assembly for the genus Panax. Through genome evolution analysis, we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis. We performed a detailed transcriptional analysis of P. notoginseng and explored gene-level mechanisms that regulate the formation of characteristic tubercles. Next, we studied the biosynthesis and regulation of saponins at temporal and spatial levels. We combined multi-omics data to identify genes that encode key enzymes in the P. notoginseng terpenoid biosynthetic pathway. Finally, we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P. notoginseng. The genetic information obtained in this study provides a resource for further exploration of the growth characteristics, cultivation, breeding, and saponin biosynthesis of P. notoginseng.
This study uses PacBio sequencing and Hi-C-assisted assembly technology to construct a high-quality, chromosome-level reference genome for the famous medicinal plant Panax notoginseng. Furthermore, this study investigates the biosynthesis of saponins in P. notoginseng and characterizes key UGT enzymes.
Squalene synthase (SQS), squalene epoxidase (SE), and oxidosqualene cyclase (OSC) are encoding enzymes in downstream biosynthetic pathway of triterpenoid in plants, but the relationship between three ...genes and celastrol accumulation in
Tripterygium wilfordii
still remains unknown. Gene transformation system in plant can be used for studying gene function rapidly. However, there is no report on the application of cambial meristematic cells (CMCs) and dedifferentiated cells (DDCs) in genetic transformation systems. Our aim was to study the effects of individual overexpression of
TwSQS
,
TwSE
, and
TwOSC
on terpenoid accumulation and biosynthetic pathway related gene expression through CMCs and DDCs systems. Overexpression vectors of
TwSQS
,
TwSE
, and
TwOSC
were constructed by Gateway technology and transferred into CMCs and DDCs by gene gun. After overexpression, the content of celastrol was significantly increased in CMCs compared with the control group. However, there was no significant increment of celastrol in DDCs. Meanwhile, the relative expression levels of
TwSQS
,
TwSE
,
TwOSC
, and terpenoid biosynthetic pathway related genes were detected. The relative expression levels of
TwSQS
,
TwSE
, and
TwOSC
were increased compared with the control group in both CMCs and DDCs, while the pathway-related genes displayed different expression trends. Therefore, it was verified in
T. wilfordii
CMCs that overexpression of
TwSQS
,
TwSE
, and
TwOSC
increased celastrol accumulation and had different effects on the expression of related genes in terpenoid biosynthetic pathway, laying a foundation for further elucidating the downstream biosynthetic pathway of celastrol through
T. wilfordii
CMCs system.
Hook. f. (
) is an important medicinal plant with anti-inflammatory, immunosuppressive and anti-tumor activities. The main bioactive ingredients are diterpenoids and triterpenoids, such as ...triptolide, triptophenolide and celastrol. However, the production of terpenoids from original plants, hairy roots and dedifferentiated cells (DDCs) are not satisfactory for clinical applications. To find a new way to further improve the production of terpenoids, we established a new culture system of cambial meristematic cells (CMCs) with stem cell-like properties, which had strong vigor and high efficiency to produce large amounts of terpenoids of
.
CMCs of
were isolated and cultured for the first time. CMCs were characterized consistent with stem cell identities based on their physiological and molecular analysis, including morphology of CMCs, hypersensitivity to zeocin, thin cell wall and orthogonal partial least square-discriminant analysis, combination of transcriptional data analysis. After induction with methyl jasmonate (MJ), the maximal production of triptolide, celastrol and triptophenolide in CMCs was 312%, 400% and 327% higher than that of control group, respectively. As for medium, MJ-induced CMCs secreted 231% triptolide and 130% triptophenolide at the maximum level into medium higher than that of control group. Maximal celastrol production of induced CMCs medium was 48% lower than that of control group. Long-term induction significantly enhanced the production of terpenoids both in cells and medium. The reason for increasing the yield of terpenoids was that expression levels of
-
-d-
-
-
(
),
-
-d-
-
-
(
) and
-
(
) were upregulated in CMCs after induction.
For the first time, CMCs of
were isolated, cultured, characterized and applied. Considering the significant enrichment of terpenoids in CMCs of
, CMCs could provide an efficient and controllable platform for sustainable production of terpenoids, which can be a better choice than DDCs.
With the advent of the "Internet plus" era, there is a huge gap in the demand for "mass entrepreneurship and innovation" talents in the field of fine arts. Therefore, the government has put forward ...the concept of "mass entrepreneurship and innovation" to put forward the direction for solving the employment problem of graduates. Based on the characteristics of fine arts majors, this study analyzes the opportunities and challenges from the perspective of "Internet plus", which is of practical significance for strengthening students' entrepreneurial ability, building entrepreneurial platforms, innovating educational concepts and promoting the three-dimensional reform of entrepreneurship education in colleges and universities. In this paper, the fine arts class specialized students' innovative undertaking education present situation and the status quo of the double gen talent training and analyzes the existing problems, in view of the double and the ways for the cultivation of the talents were discussed, and the "Internet +" thinking and "double gen" oriented puts forward the optimized university fine arts class specialized personnel training mode of specific measures. Through the "Internet +" self-employment questionnaire survey and individual interviews of art majors, reliable and real data are collected, and the advantages and problems of art majors in "Internet +" entrepreneurship are found out by combining literature analysis with empirical research.
Celastrol is a promising bioactive compound isolated from Tripterygium wilfordii and has been shown to possess many encouraging preclinical applications. However, the celastrol biosynthetic pathway ...is poorly understood, especially the key oxidosqualene cyclase (OSC) enzyme responsible for cyclisation of the main scaffold.
Here, we report on the isolation and characterisation of three OSCs from T. wilfordii: TwOSC1, TwOSC2 and TwOSC3. Both TwOSC1 and TwOSC3 were multiproduct friedelin synthases, while TwOSC2 was a β-amyrin synthase.
We further found that TwOSC1 and TwOSC3 were involved in the biosynthesis of celastrol and that their common product, friedelin, was a precursor of celastrol. We then reconstituted the biosynthetic pathway of friedelin in engineered yeast constructed by the CRISPR/Cas9 system, with protein modification and medium optimisation, leading to heterologous production of friedelin at 37.07 mg l-1 in a shake flask culture.
Our study was the first to identify the genes responsible for biosynthesis of the main scaffold of celastrol and other triterpenes in T. wilfordii. As friedelin has been found in many plants, the results and approaches described here have laid a solid foundation for further explaining the biosynthesis of celastrol and related triterpenoids. Moreover, our results provide insights for metabolic engineering of friedelane-type triterpenes.
Celastrol, a potent anticancer and anti-obesity drug, was first isolated from Tripterygium wilfordii Hook. f. and it is produced in small quantities in many members of the Celastraceae family. The ...heterologous reconstitution of celastrol biosynthesis could be a promising method for the efficient production of celastrol and natural and unnatural derivatives thereof, yet only part of the biosynthetic pathway is known. Here, we report a cytochrome P450 monooxygenase (TwCYP712K1) from T. wilfordii that performs the three-step oxidation of friedelin to polpunonic acid in the celastrol pathway. Heterologous expression of TwCYP712K1 showed that TwCYP712K1 catalyses not only the transformation of friedelin to polpunonic acid but also the oxidation of β-amyrin or α-amyrin. The role of TwCYP712K1 in the biosynthesis of celastrol was further revealed via RNA interference. Some key residues of TwCYP712K1 were also screened by molecular docking and site-directed mutagenesis. Our results lay a solid foundation for further elucidating the biosynthesis of celastrol and related triterpenoids.
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•Several cytochrome P450 monooxygenase was mined from T. wilfordii.•The role of TwCYP712K1 in celastrol biosynthesis was revealed.•The catalyst performance of TwCYP712K1 was improved by site-directed mutagenesis.•New function of MtCYP72A63 was found.
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