Genetic Transformation System for Woody Plant Tripterygium wilfordii and Its Application to Product Natural Celastrol

Zhao, Yujun; Zhang, Yifeng; Su, Ping; Yang, Jian; Huang, Luqi; Gao, Wei

doi:10.3389/fpls.2017.02221

Cited by 30 publications

(25 citation statements)

References 67 publications

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“…Each transformation was carried out two times. The bombarded suspension cells were cultured for another 7 days before harvesting for qRT-PCR and UPLC analysis 73 .…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis

Zhang

et al. 2020

Nat Commun

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123

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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.

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“…Each transformation was carried out two times. The bombarded suspension cells were cultured for another 7 days before harvesting for qRT-PCR and UPLC analysis 73 .…”

Section: Methodsmentioning

confidence: 99%

“…Vector pH7WG2D (Invitrogen) was used to overexpress TwCYP728B70 in suspension cells following the protocol mentioned above. The resulting recombinant cells were harvested for qRT-PCR and UPLC analysis after being cultured for 3, 5, 7, and 9 days 73 .…”

Section: Methodsmentioning

confidence: 99%

Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis

Zhang

et al. 2020

Nat Commun

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123

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“…To further investigate the Tw OSC responsible for the biosynthesis of celastrol, Tw OSC1, Tw OSC2 and Tw OSC3 were selected as candidate genes for the RNA interference (RNAi) study. In our early study, a stable and highly efficient transformation system suitable for T. wilfordii cell suspensions was developed based on particle bombardment (Zhao et al ., ). Here, we used this transformation system to transform the candidate genes.…”

Section: Resultsmentioning

confidence: 97%

“…Resulting vectors were transformed into T. wilfordii suspension cells according to Zhao et al . (). Suspension cells in the logarithmic growth phase were plated on MS solid medium (pH = 5.8) and grown for 7 d before transformation.…”

Section: Methodsmentioning

confidence: 97%

Friedelane‐type triterpene cyclase in celastrol biosynthesis from Tripterygium wilfordii and its application for triterpenes biosynthesis in yeast

Zhou

Gao

et al. 2019

New Phytologist

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Summary 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.

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“…In this study, indirect selection strategy was used as this selection method allowed active proliferation of putative transformed tissues or cells to produce sufficient resistant cell lines to tolerate the toxicity of hygromycin. According to Zhao et al (2017), the timing of selection post bombardment may also influence the transformation efficiency. This was also supported by da Silva and Tanaka (2011) which stated that putative transformants should be grown on selection agent free medium in order to allow the explants to recover from the damages caused during the particle bombardment process.…”

Section: Discussionmentioning

confidence: 99%

Development of an Efficient Particle Bombardment Transformation System for the Endemic Orchid, Phalaenopsis bellina

Chew¹,

Abdullah²,

Kok³

et al. 2019

JSM

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Phalaenopsis bellina is an important indigenous orchid with high commercial value. In this study, we established an efficient particle bombardment transformation system for P. bellina using the protocorm-like-bodies (PLBs) as target tissues. Leaf derived PLBs were proliferated on ½ strength Murashige and Skoog (MS) medium supplemented with 0.8 µM 2,4 dichlorophenoxyacetic acid (2, 4-D). Both physical and biological parameters affecting the transformation system were optimised using the green-fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter systems. Optimal bombardment conditions with 6 cm target tissues distance, 1100 psi acceleration pressure, 1.0 μm gold particle size, 27 mmHg chamber vacuum pressure, single bombardment time, spermidine as DNA precipitation agent, 72 h post bombardment incubation time, 2 μg plasmid DNA in 0.15:0.12 pmol ratio (pSMCHS:p35SGFP) were successfully determined. Surviving PLBs transformants were successfully recovered from the hygromycin selection medium and verified using genomic PCR analysis. The established system is not only useful for a simple and reliable transient gene analysis but as well as generating stable transformants for selective traits improvement in orchids. ABSTRAKPhalaenopsis bellina ialah salah suatu orkid yang mempunyai nilai komersial yang tinggi. Dalam kajian ini, kami telah membentuk sistem transformasi pembedilan zarah yang berkesan untuk P. bellina menggunakan jasad seperti protokorm (PLB) sebagai tisu sasaran. Daun daripada PLBs bercambah pada ½ kekuatan medium Murashige dan Skoog (MS) yang mengandungi 0.8 μM 2,4 asid d diklorofenok siasetik (2, 4-D). Parameter biologi dan fizikal yang mempengaruhi sistem transformasi telah dioptimumkan dengan menggunakan protein fluoresen hijau (GFP) dan β-glukuronidase (GUS) sebagai sistem pelapor. Keadaan pembedilan optimum dengan jarak tisu 6 cm, tekanan pecutan 1100 psi, saiz zarah 1.0 μm, tekanan vakum 27 mmHg, pembedilan tunggal, spermidine sebagai agen pemendapan DNA, 72 jam tempoh inkubasi selepas pembedilan, 2 μg DNA plasmid dalam 0.15:0.12 pmol nisbah (pSMCHS:p35SGFP) telah ditentukan. Transforman PLB telah berjaya dipulihkan selepas inkubasi di dalam medium pemilihan higromisin dan disahkan menggunakan analisis PCR genom. Sistem yang ditubuhkan ini tidak hanya berguna untuk analisis gen sementara yang boleh dipercayai dan mudah tetapi juga mampu menjana transformasi stabil bagi peningkatan ciri-ciri terpilih dalam orkid.Kata kunci: β-glukuronidase (GUS); jasad seperti protokorm (PLB); Phalaenopsis bellina; pembedilan zarah; protein fluoresen hijau (GFP)

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Genetic Transformation System for Woody Plant Tripterygium wilfordii and Its Application to Product Natural Celastrol

Cited by 30 publications

References 67 publications

Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis

Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis

Friedelane‐type triterpene cyclase in celastrol biosynthesis from Tripterygium wilfordii and its application for triterpenes biosynthesis in yeast

Development of an Efficient Particle Bombardment Transformation System for the Endemic Orchid, Phalaenopsis bellina

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