Total Synthesis of Celastrol, Development of a Platform to Access Celastroid Natural Products

Camelio, Andrew M.; Johnson, Trevor; Siegel, Dionicio

doi:10.1021/jacs.5b06261

Cited by 39 publications

(29 citation statements)

References 30 publications

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“…Despite considerable pharmaceutical interest, the low celastrol levels, slow growth rate and restricted habitat of this plant have all limited the further study and application of celastrol. To address the above issues, methods have been developed to obtain a sustainable and reliable supply such as cell culture systems and total synthesis (Su et al ., ; Camelio et al ., ). In recent years, metabolic engineering in microorganisms has been shown to be a promising method for the production of high‐value natural products (Zhou et al ., 2012a; Paddon et al ., ; Galanie et al ., ; Lau & Sattely, ).…”

Section: Introductionmentioning

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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Section: Introductionmentioning

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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“…Furthermore, the production is geographically restricted as the export of T. wilfordii from its natural habitat is regulated by the Nagoya protocol. Although total organic synthesis of celastrol has been reported, it is laborious and inefficient [22]. Thus, sourcing from nature or chemical synthesis does not provide sufficient amounts of pure compound.…”

Section: Introductionmentioning

confidence: 99%

Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol

et al. 2020

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Background: Celastrol is a promising anti-obesity agent that acts as a sensitizer of the protein hormone leptin. Despite its potent activity, a sustainable source of celastrol and celastrol derivatives for further pharmacological studies is lacking. Results:To elucidate the celastrol biosynthetic pathway and reconstruct it in Saccharomyces cerevisiae, we mined a root-transcriptome of Tripterygium wilfordii and identified four oxidosqualene cyclases and 49 cytochrome P450s as candidates to be involved in the early steps of celastrol biosynthesis. Using functional screening of the candidate genes in Nicotiana benthamiana, TwOSC4 was characterized as a novel oxidosqualene cyclase that produces friedelin, the presumed triterpenoid backbone of celastrol. In addition, three P450s (CYP712K1, CYP712K2, and CYP712K3) that act downstream of TwOSC4 were found to effectively oxidize friedelin and form the likely celastrol biosynthesis intermediates 29-hydroxy-friedelin and polpunonic acid. To facilitate production of friedelin, the yeast strain AM254 was constructed by deleting UBC7, which afforded a fivefold increase in friedelin titer. This platform was further expanded with CYP712K1 to produce polpunonic acid and a method for the facile extraction of products from the yeast culture medium, resulting in polpunonic acid titers of 1.4 mg/L. Conclusion:Our study elucidates the early steps of celastrol biosynthesis and paves the way for future biotechnological production of this pharmacologically promising compound in engineered yeast strains.

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“…The pharmaceutical potential of T. wilfordii diterpenoids has spurred interest in the development of methods for their sustainable and reliable production, particularly given the generally low content of these metabolites, slow growth rate, and the restricted habitat of this species (GBIF, 2016). While cell culture systems and total synthesis have been investigated as sources of T. wilfordii bioactive terpenoids (Kutney et al, 1981;Zhang et al, 2014;Camelio et al, 2015), an attractive alternative yielding access to bioactive terpenoids is the metabolic engineering of microorganisms for heterologous production (Paddon et al, 2013). This approach may ultimately offer economic and environmental benefits, but requires functional knowledge of the biosynthetic pathways of the target molecules.…”

Section: Introductionmentioning

confidence: 99%

The terpene synthase gene family in Tripterygium wilfordii harbors a labdane‐type diterpene synthase among the monoterpene synthase TPS‐b subfamily

et al. 2017

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SUMMARYTripterygium wilfordii (Celastraceae) is a medicinal plant with anti-inflammatory and immunosuppressive properties. Identification of a vast array of unusual sesquiterpenoids, diterpenoids and triterpenoids in T. wilfordii has spurred investigations of their pharmacological properties. The tri-epoxide lactone triptolide was the first of many diterpenoids identified, attracting interest due to the spectrum of bioactivities. To probe the genetic underpinning of diterpenoid diversity, an expansion of the class II diterpene synthase (diTPS) family was recently identified in a leaf transcriptome. Following detection of triptolide and simple diterpene scaffolds in the root, we sequenced and mined the root transcriptome. This allowed identification of the root-specific complement of TPSs and an expansion in the class I diTPS family. Functional characterization of the class II diTPSs established their activities in the formation of four C-20 diphosphate intermediates, precursors of both generalized and specialized metabolism and a novel scaffold for Celastraceae. Functional pairs of the class I and II enzymes resulted in formation of three scaffolds, accounting for some of the terpenoid diversity found in T. wilfordii. The absence of activity-forming abietane-type diterpenes encouraged further testing of TPSs outside the canonical class I diTPS family. TwTPS27, close relative of mono-TPSs, was found to couple with TwTPS9, converting normal-copalyl diphosphate to miltiradiene. The phylogenetic distance to established diTPSs indicates neo-functionalization of TwTPS27 into a diTPS, a function not previously observed in the TPS-b subfamily. This example of evolutionary convergence expands the functionality of TPSs in the TPS-b family and may contribute miltiradiene to the diterpenoids of T. wilfordii.

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Total Synthesis of Celastrol, Development of a Platform to Access Celastroid Natural Products

Cited by 39 publications

References 30 publications

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

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

Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol

The terpene synthase gene family in Tripterygium wilfordii harbors a labdane‐type diterpene synthase among the monoterpene synthase TPS‐b subfamily

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