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There is a dichotomy in the biosynthetic pathway of terpenoid precursor isopentenyl diphosphate (IPP) in higher plant. One is the classical mevalonate pathway in cytosol, and the other is non-mevalonate pathway in plastid. To know the origin of the taxane ring system of taxol in suspension culture of Taxus chinensis, lovastatin and fosmidomycin were used to block the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and 1-deoxy-D-xylulose-5-phosphate reducto-isomerase (DXR) in the mevalonate and non-mevalonate branch respectively of the terpenoid biosynthetic pathway. Methyl jasmonate (MJ) was used to improve the biosynthesis of taxol. Taxol content was determined by HPLC, the transcriptional expression of genes encoding DXR and HMGR were investigated by real time PCR. Taxol production was lowered by about 2/5 and 1/5 by fosmidomycin (200 mmol/L) and fosmidomycin (200 mmol/L)+MJ (100 mmol/L) treatment respectively, and was lowered by about 1/6 and 1/10 by lovastatin (1 mmol/L) and lovastatin (1 mmol/L) + MJ (100 mmol/L) respectively, which means that both mevalonate and non-mevalonate pathway contribute to taxol biosynthesis, and the latter is the main source of IPP. Inhibitors lovastatin and fosmidomycin both promoted the transcriptional expression of hmgr and dxr, which indicated a metabolic cross talk between cytosolic and plastidial pathways of taxol biosynthesis.

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. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:269-280, 2014 [ABSTRACT FROM AUTHOR]

Abstract: Yields of paclitaxel decreased with repeated subculturing of Taxus media cells. We used minimal growth conservation and manipulation of genome methylation to sustain paclitaxel production by Taxus media cell cultures. The subculture period of Taxus cells can be prolonged to 180d by incubating them at a low temperature (5°C). Paclitaxel levels increased in the cells after conservation and during the first recovery subculture cycle, and then decreased during the subsequent recovery subculture cycle. Analysis of genetic variations in these cultures using amplified fragment-length polymorphism (AFLP) technology identified only two polymorphic bands associated with the second and sixth recovery cycle cultures. However, the results of high-performance liquid chromatography indicated that DNA methylation increased during the course of repeated subculturing. A decrease in DNA methylation level caused by treatment with 5-Aza-2′-deoxycytidine coincided with an increase in paclitaxel levels. Simultaneous exposure to both methyl jasmonate and 5-Aza-2′-deoxycytidine increased paclitaxel levels to 320.43μgg−1 (dry weight), which is more than six times the paclitaxel content before conservation. To our knowledge, this is the first report about improving paclitaxel production by ensuring sustainable use of Taxus cells. [Copyright &y& Elsevier]