Administering cultured Taxus cells with early precursors reveals bifurcations in the taxoid biosynthetic pathway

Ketchum, Raymond E.B.; Horiguchi, Tohru; Qiu, Deyou; Williams, Robert M.; Croteau, Rodney

doi:10.1016/j.phytochem.2006.10.019

Cited by 22 publications

(18 citation statements)

References 19 publications

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“…Similarly, 26 unique sequences with increased transcript changes in T. × media were also up-regulated in T. cuspidata , which contained 29 genes. These include TBT in paclitaxel biosynthesis [28], 1-aminocyclopropane-1-carboxylate oxidase ( ACO ) in ethylene biosynthesis [22], 12-oxophytodienoate reductase possible in jasmonate biosynthesis [29], cationic peroxidase in pathogen incompatible interaction [30], Chalcone synthases involve in phenylpropanoid biosynthesis which is inducible by pathogens [31], and taxane 14b-hydroxylase participating in the bifurcation in paclitaxel biosynthesis [32]. Notably several genes between the different studies were identified as having opposite transcript changes in response to elicitation with MeJA: 15 genes (include one of two ACC oxidases) in T. × media with lower transcript levels were up-regulated in T. cuspidata ,while 7 genes(e.g., dihydroflavonol-4-reductase in anthocyanin biosynthesis [33]) in T. × media with increased transcript levels were identified as down-regulated in T. cuspidata .…”

Section: Resultsmentioning

confidence: 99%

Deep Sequencing Reveals Transcriptome Re-Programming of Taxus × media Cells to the Elicitation with Methyl Jasmonate

et al. 2013

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BackgroundPlant cell culture represents an alternative source for producing high-value secondary metabolites including paclitaxel (Taxol®), which is mainly produced in Taxus and has been widely used in cancer chemotherapy. The phytohormone methyl jasmonate (MeJA) can significantly increase the production of paclitaxel, which is induced in plants as a secondary metabolite possibly in defense against herbivores and pathogens. In cell culture, MeJA also elicits the accumulation of paclitaxel; however, the mechanism is still largely unknown.Methodology/Principal FindingsTo obtain insight into the global regulation mechanism of MeJA in the steady state of paclitaxel production (7 days after MeJA addition), especially on paclitaxel biosynthesis, we sequenced the transcriptomes of MeJA-treated and untreated Taxus × media cells and obtained ∼ 32.5 M high quality reads, from which 40,348 unique sequences were obtained by de novo assembly. Expression level analysis indicated that a large number of genes were associated with transcriptional regulation, DNA and histone modification, and MeJA signaling network. All the 29 known genes involved in the biosynthesis of terpenoid backbone and paclitaxel were found with 18 genes showing increased transcript abundance following elicitation of MeJA. The significantly up-regulated changes of 9 genes in paclitaxel biosynthesis were validated by qRT-PCR assays. According to the expression changes and the previously proposed enzyme functions, multiple candidates for the unknown steps in paclitaxel biosynthesis were identified. We also found some genes putatively involved in the transport and degradation of paclitaxel. Potential target prediction of miRNAs indicated that miRNAs may play an important role in the gene expression regulation following the elicitation of MeJA.Conclusions/SignificanceOur results shed new light on the global regulation mechanism by which MeJA regulates the physiology of Taxus cells and is helpful to understand how MeJA elicits other plant species besides Taxus.

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Section: Resultsmentioning

confidence: 99%

Deep Sequencing Reveals Transcriptome Re-Programming of Taxus × media Cells to the Elicitation with Methyl Jasmonate

et al. 2013

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“…The increase in the proportion of the TAX group suggests that MJ‐induction could enhance one‐step or multiple‐step functionalization towards taxol biosynthetic pathways. TC and its derivatives (YN, TPT, TIBT, and TMBT) are unlikely to reside on the route to taxol because of inappropriate acylation patterns on the taxane ring system . Thus, the biosynthetic pathway of MAT is an important bypass to taxol.…”

Section: Resultsmentioning

confidence: 99%

“…The results from both the selected gene expression levels and PCA showed that the genes of OHX1, T10βH, and TαH contribute main variables to the samples induced by MJ, whereas T10βH and TαH seem to be responsible for the hydroxylation at C‐10 and C‐13 of the taxol (taxane) core. Ketchum reported that C‐13 hydroxyl taxanes are easier to generate when the taxanes bear the hydroxyl group at C‐10. Moreover, the variation in B‐III content is the most significant among the identified taxanes after MJ‐induction (Figures and ), correlating well with the variations in the above genes.…”

Section: Resultsmentioning

confidence: 99%

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Correlation analysis of the taxane core functional group modification, enzyme expression, and metabolite accumulation profiles under methyl jasmonate treatment

Song

Zhao

Zhang

et al. 2014

Biotechnology Progress

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Metabolomic and transcriptomic profiling data were obtained and integrated to elucidate the crucial network controls on taxol and its precursor biosynthesis during the taxane core functionalization within methyl jasmonate (MJ)-induced Taxus chinensis cells. Twelve metabolites were identified using liquid chromatography-electrospray ionization-mass spectrometry. These metabolites contain taxol (paclitaxel), baccatin III (B-III) and its analogs, a group structurally bearing multiple free hydroxyls (TAX), and another group of multiple acyl taxanes (MAT), including taxuyunnanine C (TC) and its analogs. The metabolomic profile showed a higher increase in TAX than in MAT. Particularly, the ratio of B-III and taxol to the total taxane content increased more significantly in TAX than in MAT. The MAT proportion did not significantly change, although they are predominant components in cell cultures compared with TAX. Quantitative real-time polymerase chain reaction (qRT-RCR) was used to determine the transcription level of 20 genes, among which 11 were reported responsible for taxol biosynthesis and 9 were obtained from our previous transcriptomic data. The total expression levels of hydroxylase after 24 h and 6 days were higher than those of acylase. The principal component analysis (PCA) results validated the metabolomic analysis data, indicating that hydroxylation was more crucial than acylation for controlling the flux toward TAX biosynthesis. Furthermore, the PCA contribution comparison showed that two undefined genes of OHX1 and ACX3 might have good potential in TAX upregulation and MAT downregulation. To the best of our knowledge, this study provides the first experimental evidence on the contribution of total hydroxylation to taxane biosynthesis.

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“…134 Rather, there is a softening of substrate specificity among the P450 enzymes that are each designed to oxygenate the taxoid core at a specific position. The net result, is that each oxygenated product, can function as a substrate of altering efficiency, for each P450 resulting in a complex matrix of oxygenated polyols, many of which finding their way all the way to baccatin III and taxol.…”

Section: Introductionmentioning

confidence: 99%

Natural Products Synthesis: Enabling Tools To Penetrate Nature’s Secrets of Biogenesis and Biomechanism

Williams

2011

J. Org. Chem.

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Selected examples from our laboratory of how synthetic technology platforms developed for the total synthesis of several disparate families of natural products was harnessed to penetrate biomechanistic and/or biosynthetic queries is discussed. Unexpected discoveries of biomechanistic reactivity and/or penetrating the biogenesis of naturally occurring substances were made possible through access to substances available only through chemical synthesis. Hypothesis-driven total synthesis programs are emerging as very useful conceptual templates for penetrating and exploiting the inherent reactivity of biologically active natural substances. In many instances, new enabling synthetic technologies were required to be developed. The examples demonstrate the often un-tapped richness of complex molecule synthesis to provide powerful tools to understand, manipulate and exploit Nature’s vast and creative palette of secondary metabolites.

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Administering cultured Taxus cells with early precursors reveals bifurcations in the taxoid biosynthetic pathway

Cited by 22 publications

References 19 publications

Deep Sequencing Reveals Transcriptome Re-Programming of Taxus × media Cells to the Elicitation with Methyl Jasmonate

Deep Sequencing Reveals Transcriptome Re-Programming of Taxus × media Cells to the Elicitation with Methyl Jasmonate

Correlation analysis of the taxane core functional group modification, enzyme expression, and metabolite accumulation profiles under methyl jasmonate treatment

Natural Products Synthesis: Enabling Tools To Penetrate Nature’s Secrets of Biogenesis and Biomechanism

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