Paclitaxel Biosynthesis Research Articles

Comparative metabolomic analysis reveals the variations in taxoids and flavonoids among three Taxus species

BackgroundTrees of the genus Taxus are highly valuable medicinal plants with multiple pharmacological effects on various cancer treatments. Paclitaxel from Taxus trees is an efficient and widely used anticancer drug, however, the accumulation of taxoids and other active ingredients can vary greatly among Taxus species. In our study, the metabolomes of three Taxus species have been investigated.ResultsA total of 2246 metabolites assigned to various primary and secondary metabolic pathways were identified using an untargeted approach. Analysis of differentially accumulated metabolites identified 358 T. media-, 220 T. cuspidata-, and 169 T. mairei-specific accumulated metabolites, respectively. By searching the metabolite pool, 7 MEP pathway precursors, 11 intermediates, side chain products and derivatives of paclitaxel, and paclitaxel itself were detected. Most precursors, initiated intermediates were highly accumulated in T. mairei, and most intermediate products approaching the end point of taxol biosynthesis pathway were primarily accumulated in T. cuspidata and T. media. Our data suggested that there were higher-efficiency pathways to paclitaxel in T. cuspidata and T. media compared with in T. mairei. As an important class of active ingredients in Taxus trees, a majority of flavonoids were predominantly accumulated in T. mairei rather than T. media and T. cuspidata. The variations in several selected taxoids and flavonoids were confirmed using a targeted approach.ConclusionsSystematic correlativity analysis identifies a number of metabolites associated with paclitaxel biosynthesis, suggesting a potential negative correlation between flavonoid metabolism and taxoid accumulation. Investigation of the variations in taxoids and other active ingredients will provide us with a deeper understanding of the interspecific differential accumulation of taxoids and an opportunity to accelerate the highest-yielding species breeding and resource utilization.

Evolutionary Developments in Plant Specialized Metabolism, Exemplified by Two Transferase Families.

Plant specialized metabolism emerged from the land colonization by ancient plants, becoming diversified along with plant evolution. To date, more than 1 million metabolites have been predicted to exist in the plant kingdom, and their metabolic processes have been revealed on the molecular level. Previous studies have reported that rates of evolution are greater for genes involved in plant specialized metabolism than in primary metabolism. This perspective introduces topics on the enigmatic molecular evolution of some plant specialized metabolic processes. Two transferase families, BAHD acyltransferases and aromatic prenyltransferases, which are involved in the biosynthesis of paclitaxel and meroterpenes, respectively, have shown apparent expansion. The latter family has been shown to beinvolved in the biosynthesis of a variety of aromatic substances, including prenylated coumarins in citrus plants and shikonin in Lithospermum erythrorhizon. These genes have evolved in the development of each special subfamily within the plant lineage. The broadness of substrate specificity and the exon-intron structure of their genes may provide hints to explain the evolutionary process underlying chemodiversity in plants.

Regulation Mechanism of MYC Family Transcription Factors in Jasmonic Acid Signalling Pathway on Taxol Biosynthesis.

Paclitaxel is an important anticancer drug. The phytohormone jasmonic acid can significantly induce the biosynthesis of paclitaxel in Taxus, but the molecular mechanism has not yet been resolved. To establish the jasmonic acid signalling pathway of Taxus media, based on the gene of the jasmonic acid signalling pathway of Arabidopsis thaliana, sequence analysis was performed to isolate the jasmonic acid signal from the transcriptome, a transcriptional cluster of pathway gene homologs and the full length of 22 genes were obtained by RACE PCR at 5′ and 3′: two EI ubiquitin ligase genes, COI1-1 and COI1-2;7 MYC bHLH type transcription factor (MYC2, MYC3, MYC4, JAM1, JAM2, EGL3, TT8); 12 JAZ genes containing the ZIM domain; and MED25, one of the components of the transcriptional complex. The protein interaction between each were confirmed by yeast two hybridization and bimolecular fluorescence complementation based on similar genes interaction in Arabidopsis. A similar jasmonate signaling pathway was illustrated in T. media. All known paclitaxel biosynthesis genes promoters were isolated by genome walker PCR. To investigate the jasmonate signaling effect on these genes’ expression, the transcription activity of MYC2, MYC3 and MYC4 on these promoters were examined. There are 12, 10 and 11 paclitaxel biosynthesis genes promoters that could be activated by MYC2, MYC3 and MYC4.

Identification of rate-limiting enzymes involved in paclitaxel biosynthesis pathway affected by coronatine and methyl-β-cyclodextrin in Taxus baccata L. cell suspension cultures.

BackgroundPaclitaxel is a potent antitumor alkaloid widely used for the treatment of several cancer types. This valuable secondary metabolite naturally exists in the inner bark of Taxus species in very low amounts. The small-scale production of paclitaxel in Taxus cell cultures requires utilization of several elicitors.ObjectiveThe main objective of this work was to identify key genes that encode rate-limiting enzymes in paclitaxel biosynthesis pathway by investigating the possible relationship between paclitaxel production and a set of 13 involved genes’ relative expression in Taxus baccata L. cell suspension cultures affected by coronatine and methyl-β-cyclodextrin.MethodsIn the present research, the most important key genes were identified using gene expression profiling evaluation and paclitaxel production assessment in Taxus baccata L. cell cultures affected by mentioned elicitors.Results and ConclusionGene expression levels were variably increased using methyl-β-cyclodextrin, and in some cases, a synergistic effect on transcript accumulation was observed when culture medium was supplemented with both elicitors. It was revealed that DBAT, BAPT, and DBTNBT are the most important rate-limiting enzymes in paclitaxel biosynthesis pathway in Taxus baccata L. cell suspension cultures under coronatine and methyl-β-cyclodextrin elicitation condition. Moreover, PAM was identified as one of the important key genes especially in the absence of β-phenylalanine. In cell cultures affected by these elicitors, paclitaxel was found largely in the culture media (more than 90%). The secretion of this secondary metabolite suggests a limited feedback inhibition and reduced paclitaxel toxicity for producer cells. It is the result of the ABC gene relative expression level increment under methyl-β-cyclodextrin elicitation and highly depends on methyl-β-cyclodextrin’s special property (complex formation with hydrophobic compounds). Paclitaxel biosynthesis was obviously increased due to the effect of coronatine and methyl-β-cyclodextrin elicitation, leading to the production level of 5.62 times higher than that of the untreated cultures. Graphical abstractRate Limiting Enzymes in Paclitaxel Biosynthesis Pathway: DBAT, BAPT, DBTNBT and PAM.

A cellulase-supported two-phase in situ system for enhanced biosynthesis of paclitaxel in Taxus \xd7 media hairy roots

The effectiveness of two liquid-phase culture systems, in situ supported with perfluorodecalin (PFD), and elicited with methyl jasmonate (100 µM) and sodium nitroprusside (10 µM) (spiked with l-phenylalanine (100 µM) and sucrose (30 g/l) feeding), and additionally combined with cellulolytic enzyme application (Cellulase or Viscozyme® L at doses 0.01%, 0.1%, 0.5% or 1%), was investigated on the enhancement of paclitaxel release in Taxus × media harbouring taxadiene synthase transgene hairy root cultures. Neither elicitation nor in situ application of PFD significantly had an effect on root growth until enzyme addition; however, the hairy root response to the culture conditions varied depending on enzyme type and dosage. The highest paclitaxel total yield (intracellular + extracellular) was determined in the one-phase elicited culture systems without enzymes and amounted to 264.2 µg/flask (1 434.9 ± 516.6 µg/g DW) and 29.6 µg/flask in root biomass and medium phase, respectively. Although the application of cellulolytic enzymes did not enhance the total paclitaxel production, they intensified paclitaxel release in a dose-depending manner. Among two examined cellulolytic enzyme treatments, Viscozyme® L addition caused the highest paclitaxel release up to 59% of its total content when used at a concentration of 0.01%. Whereas in two-phase in situ systems, the combination of Viscozyme® L at dosage of 0.5% and PFD-aerated, resulted in the highest extracellular paclitaxel concentration up to 20%.

Comparative Transcriptome Analysis Revealed the Tissue-Specific Accumulations of Taxanes among Three Experimental Lines of Taxus yunnanensis

Taxus yunnanensis (Yew) is known for natural anticancer metabolite paclitaxel (Taxol) and its biosynthesis pathway in yew species still needs to be completely elucidated. In the current study, productions of paclitaxel and 10-DAB III from three different tissues (needle, branch, and root) of T. yunnanensis wild type (WT) and two new cultivars Zhongda-1 (Zd1) and Zhongda-2 (Zd2) were determined, and significant tissue differences in contents of the taxanes were observed among the three experimental lines. The much higher 10-DAB III and lower paclitaxel contents in needle of Zd2 when compared with that of Zd1 indicated the low conversion from 10-DAB III to paclitaxel in the needle of Zd2. In order to uncover the mechanisms of the tissue-specific biosynthesis of the taxanes, transcriptome analysis of cultivar Zd2 was conducted, and the previously reported transcriptome data of Zd1 and WT were used to perform a comparison. The enhancement of TDAT and T10βH side biosynthetic pathway in roots of Zd2 in early taxane synthesis might lead to the biosynthesis of other toxoids, while the preference of T13αH route in the needle and branch of Zd2 was mainly responsible for the tissue-specific reinforced biosynthesis of 10-DAB III and paclitaxel in Zd2. Different from Zd1, the tissue-specific pattern of paclitaxel biosynthesis genes in Zd2 was similar to WT. However, the lower transcript abundance of final steps genes (TBT, DBAT, BAPT, and DBTNBT) of the paclitaxel biosynthesis pathway in Zd2 than in Zd1 might further promote 10-DAB III accumulation in Zd2.

Activity Essential Residue Analysis of Taxoid 10β-O-Acetyl Transferase for Enzymatic Synthesis of Baccatin.

Taxoid 10β-O-acetyl transferase (DBAT) is a key enzyme in the biosynthesis of the famous anticancer drug paclitaxel, which catalyses the formation of baccatin III from 10-deacetylbaccatin III (10-DAB). However, the activity essential residues of the enzyme are still unknown, and the acylation mechanism from its natural substrate 10-deacetylbaccatin III and acetyl CoA to baccatin III remains unclear. In this study, the homology modelling, molecular docking, site-directed mutagenesis, and kinetic parameter determination of the enzyme were carried out. The results showed that the enzyme mutant DBATH162A resulted in complete loss of enzymatic activity, suggesting that the residue histidine at 162 was essential to DBAT activity. Residues D166 and R363 which were located in the pocket of the enzyme by homology modelling and molecular docking were also important for DBAT activity through the site-directed mutations. Furthermore, four amino acid residues including S31 and D34 from motif SXXD, D372 and G376 from motif DFGWG also played important roles on acylation. This was the first report of the elucidation of the activity essential residues of DBAT, making it possible for the further structural-based re-design of the enzyme for efficient biotransformation of baccatin III and paclitaxel.

Identification of early jasmonate-responsive genes in Taxus × media cells by analyzing time series digital gene expression data.

Jasmonate, an effective elicitor, can induce the biosynthesis of paclitaxel, a well-known anticancer drug, in Taxus cell culture. The jasmonate signaling pathway has been well studied in Arabidopsis, and many early jasmonate-responsive genes have beenfound to be involved in signaling pathway. In Taxus, only a few late jasmonate-responsive genes related to paclitaxel biosynthesis were identified. So, identification of early responsive genes and knowledge of the jasmonate signaling pathway are essential for understanding the effects of jasmonate on paclitaxel biosynthesis and for improving paclitaxel production in Taxus cells. In this study, total RNA of Taxus × media cells cultured in liquid medium was extracted after 0, 0.5, 3, and 24h of methyl jasmonate treatment. Three biological independent repetitions were performed. The 12 extracted RNA samples were integrated and sequenced on an Illumina HiSeq2500 platform using the paired-end method. A total of 45,583 transcript clusters were obtained by de novo assembly of the sequenced reads. Based on the transcriptome data, the digital gene expressions of each RNA sample were investigated. We found that after 0.5, 3, and 24h of methyl jasmonate treatment; 134, 1008, and 987 unigenes were differentially expressed. For the secondary metabolism pathways, phenylalanine pathway unigenes were responsive to jasmonate after 3h of treatment, while genes related to paclitaxel biosynthesis were induced after 0.5h of treatment. The digital gene expression levels of candidate genes related to paclitaxel biosynthesis were confirmed by qRT-PCR. Transcriptome sequencing and digital gene expression profiling identified early jasmonate-responsive genes in cultured Taxus × media cells. The comprehensive time series jasmonate-responsive gene expression data have provided transcriptome-wide information about the mechanism of paclitaxel biosynthesis regulation by jasmonate signaling.

Transcriptome profiling reveals specific patterns of paclitaxel synthesis in a new Taxus yunnanensis cultivar

The difference in contents of paclitaxel and 10-deacetylbaccatin III (10-DABIII) in needles between wildtype (WT) and a new cultivar (Zhongdayihao, ZD1) of Taxus yunnanensis was examined. Transcriptome profiling was conducted for different tissues of the ZD1 and WT to illustrate the regulation mechanism of paclitaxel biosynthesis. It was observed that average contents of paclitaxel and 10-DABIII in ZD1 were 4 folds and 32 folds higher than those in WT, respectively. More significant elevations of differential expressed genes (DEGs) from paclitaxel biosynthesis pathway were revealed in ZD1 rather than WT, which should be responsible for the higher contents of paclitaxel and 10-DABIII in the ZD1. Special tissues-dependent expression patterns of paclitaxel biosynthesis DEGs in ZD1 compared to WT were unraveled. The relative higher expressions of paclitaxel biosynthesis genes in needles than other tissues supported the higher content of paclitaxel and 10-DABIII content in needles of ZD1. Attenuation of plant hormone signal transduction pathway led to the lower expression of TFs in ZD1 rather than WT. Besides, the significant negative correlations between differential expressed TFs and DEGs from paclitaxel biosynthesis pathway displayed a possibly negative regulation pattern of these TFs on paclitaxel biosynthesis pathway genes. These results provided new insights into the molecular process of paclitaxel synthesis in Taxus.

Taxadiene‐5α‐ol is a minor product of CYP725A4 when expressed in Escherichia coli

CYP725A4 is a P450 enzyme from Taxus cuspidata that catalyzes the formation of taxadiene-5α-ol (T5α-ol) from taxadiene in paclitaxel biosynthesis. Past attempts expressing CYP725A4 in heterologous hosts reported the formation of 5(12)-oxa-3(11)-cyclotaxane (OCT) and/or 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) instead of, or in addition to, T5α-ol. Here, we report that T5α-ol is produced as a minor product by Escherichia coli expressing both taxadiene synthase and CYP725A4. The major products were OCT and iso-OCT, while trace amounts of unidentified monooxygenated taxanes were also detected by gas chromatography-mass spectrometry. Since OCT and iso-OCT had not been found in nature, we tested the hypothesis that protein-protein interaction of CYP725A4 with redox partners, such as cytochrome P450 reductase (CPR) and cytochrome b5, may affect the products formed by CYP725A4, possibly favoring the formation of T5α-ol over OCT and iso-OCT. Our results show that coexpression of CYP725A4 with CPR from different organisms did not change the relative ratios of OCT, iso-OCT, and T5α-ol, while cytochrome b5 decreased overall levels of the products formed. Although unsuccessful in finding conditions that promote T5α-ol formation over other products, we used our results to clarify conflicting claims in the literature and discuss other possible approaches to produce paclitaxel via metabolic and enzyme engineering.

Jasmonate-responsive expression of paclitaxel biosynthesis genes in Taxus cuspidata cultured cells is negatively regulated by the bHLH transcription factors TcJAMYC1, TcJAMYC2, and TcJAMYC4.

Taxus cell suspension culture is a sustainable technology for the industrial production of paclitaxel (Taxol®), a highly modified diterpene anti-cancer agent. The methyl jasmonate (MJ)-mediated paclitaxel biosynthetic pathway is not fully characterized, making metabolic engineering efforts difficult. Here, promoters of seven genes (TASY, T5αH, DBAT, DBBT, PAM, BAPT, and DBTNBT), encoding enzymes of the paclitaxel biosynthetic pathway were isolated and used to drive MJ-inducible expression of a GUS reporter construct in transiently transformed Taxus cells, showing that elicitation of paclitaxel production by MJ is regulated at least in part at the level of transcription. The paclitaxel biosynthetic pathway promoters contained a large number of E-box sites (CANNTG), similar to the binding sites for the key MJ-inducible transcription factor AtMYC2 from Arabidopsis thaliana. Three MJ-inducible MYC transcription factors similar to AtMYC2 (TcJAMYC1, TcJAMYC2, and TcJAMYC4) were identified in Taxus. Transcriptional regulation of paclitaxel biosynthetic pathway promoters by transient over expression of TcJAMYC transcription factors indicated a negative rather than positive regulatory role of TcJAMYCs on paclitaxel biosynthetic gene expression.

Natural and engineered production of taxadiene with taxadiene synthase.

Taxadiene synthase (TXS) is the rate-limiting enzyme in the biosynthesis of paclitaxel, an important anticancer compound. TXS catalyzes the conversion of the diterpene precursor geranylgeranyl pyrophosphate (GGPP) into the diterpene taxadiene. Due to the importance of taxadiene in the overall biosynthetic pathway of paclitaxel biosynthesis, the enzyme TXS has been the subject of intense scientific and engineering investigations. The crystal structure of TXS was recently elucidated, thereby providing an atomic blueprint for future protein engineering efforts. Metabolic engineering of TXS for taxadiene product in different microbial and plant organisms have also been extensively performed, culminating in the high-titer production in Escherichia coli. Additional aspects of taxadiene production by TXS will be discussed in the review, including metabolic regulation in native host and possible production by endophytic fungal hosts.

De Novo characterization of a Cephalotaxus hainanensis transcriptome and genes related to paclitaxel biosynthesis.

Cephalotaxus hainanensis, an endangered plant, is known to contain several metabolites with anti-cancer activity. Despite its clinical impact, the alkaloid metabolism of this species has remained largely uncharacterized. The potential of Cephalotaxus for metabolic engineering of medically interesting compounds has, so far, not been exploited, due to the almost complete lack of molecular information. We have therefore performed a high throughput RNA-seq analysis and assembled the transcriptome de novo. Raw reads comprising 4.3 Gbp were assembled de novo into 39,416 unique sequences (unigenes) with a mean length of 1,089.8 bp and a total assembly size of 45.8 Mbp, which equals to more than 50 times the number of Cephalotaxaceae sequences currently deposited in the GenBank (as of August 2013). As proof of principle for medically interesting pathways, gene fragments related to paclitaxel biosynthesis were searched and detected. To verify their functionality, the metabolic product paclitaxel, and its precursor baccatin III, were identified in the leaves of C. hainanensis by HPLC, and shown to be induced by MeJA. This finding demonstrates exemplarily the potential of the annotated transcriptome as information resource for the biotechnological exploitation of plant secondary metabolism.

Taximin, a conserved plant-specific peptide is involved in the modulation of plant-specialized metabolism.

Small peptides play important roles in the signalling cascades that steer plant growth, development and defence, and often crosstalk with hormonal signalling. Thereby, they also modulate metabolism, including the production of bioactive molecules that are of high interest for human applications. Yew species (Taxus spp.) produce diterpenes such as the powerful anticancer agent paclitaxel, the biosynthesis of which can be stimulated by the hormone jasmonate, both in whole plants and cell suspension cultures. Here, we identified Taximin, as a gene encoding a hitherto unreported, plant-specific, small, cysteine-rich signalling peptide, through a transcriptome survey of jasmonate-elicited T.baccata suspension cells grown in two-media cultures. Taximin expression increased in a coordinated manner with that of paclitaxel biosynthesis genes. Tagged Taximin peptides were shown to enter the secretory system and localize to the plasma membrane. In agreement with this, the exogenous application of synthetic Taximin peptide variants could transiently modulate the biosynthesis of taxanes in T.baccata cell suspension cultures. Importantly, the Taximin peptide is widely conserved in the higher plant kingdom with a high degree of sequence conservation. Accordingly, Taximin overexpression could stimulate the production of nicotinic alkaloids in Nicotiana tabacum hairy root cultures in a synergistic manner with jasmonates. In contrast, no pronounced effects of Taximin overexpression on the specialized metabolism in Medicago truncatula roots were observed. This study increases our understanding of the regulation of Taxus diterpene biosynthesis in particular and plant metabolism in general. Ultimately, Taximin might increase the practical potential of metabolic engineering of medicinal plants.

Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum NRRL 62431

BackgroundPaclitaxel (Taxol™) is an important anticancer drug with a unique mode of action. The biosynthesis of paclitaxel had been considered restricted to the Taxus species until it was discovered in Taxomyces andreanae, an endophytic fungus of T. brevifolia. Subsequently, paclitaxel was found in hazel (Corylus avellana L.) and in several other endophytic fungi. The distribution of paclitaxel in plants and endophytic fungi and the reported sequence homology of key genes in paclitaxel biosynthesis between plant and fungi species raises the question about whether the origin of this pathway in these two physically associated groups could have been facilitated by horizontal gene transfer.ResultsThe ability of the endophytic fungus of hazel Penicillium aurantiogriseum NRRL 62431 to independently synthesize paclitaxel was established by liquid chromatography-mass spectrometry and proton nuclear magnetic resonance. The genome of Penicillium aurantiogriseum NRRL 62431 was sequenced and gene candidates that may be involved in paclitaxel biosynthesis were identified by comparison with the 13 known paclitaxel biosynthetic genes in Taxus. We found that paclitaxel biosynthetic gene candidates in P. aurantiogriseum NRRL 62431 have evolved independently and that horizontal gene transfer between this endophytic fungus and its plant host is unlikely.ConclusionsOur findings shed new light on how paclitaxel-producing endophytic fungi synthesize paclitaxel, and will facilitate metabolic engineering for the industrial production of paclitaxel from fungi.

Taxoids profiling of suspension Taxus chinensis var. mairei cells in response to shear stress

To develop an optimal bioprocess for paclitaxel (Taxols) supply, taxoid biosynthetic pathway regulation must be better understood. The main taxoid metabolites (paclitaxel, baccatin III, taxol C, etc.) in Taxus cell culture showed great difference under shear stress. However, the regulating mechanism of taxoids metabolism under shear stress remained elusive. Here an efficient metabolic profiling approach combined with multivariate analysis was employed to profile taxoids changes of Taxus cells under laminar shear stress. A total of 21 taxoids were identified and quantified by ultra-performance liquid chromatography coupled with Q-TOF mass spectrometry. The result showed the contents of paclitaxel and baccatin III were reduced by shear stress, indicating the inhibitory effect of shear stress on paclitaxel biosynthesis. The levels of other taxoids uninvolved in paclitaxel biosynthesis were decreased except several metabolites. Further analysis of mapping measured taxoids concentrations onto paclitaxel biosynthesis pathway illustrating proposed intermediates and “off-pathway” metabolites revealed shear stress might disrupt the appropriate cyclization process of geranylgeranyl-pyrophosphate, aggravate the inappropriate order of hydroxylations and acylations, and not be good for functional group oxetane formation. These findings revealed the possible mechanism for shear stress limiting paclitaxel production and might have important biotechnological applications to increase the yields of paclitaxel and relevant precursors.

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

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.

Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate

Coronatine is a toxin produced by the pathogen Pseudomonas syringae. This compound has received much attention recently for its potential to act as a plant growth regulator and elicitor of plant secondary metabolism. To gain more insight into the mechanism by which elicitors can affect the biosynthesis of paclitaxel (Px) and related taxanes, the effect of coronatine (Cor) and methyl jasmonate (MeJA) on Taxus media cell cultures has been studied. For this study, a two-stage cell culture was established, in which cells were first cultured for 14 days in a medium optimised for growth, after which the cells were transferred to medium optimised for secondary metabolite production. The two elicitors were added to the medium at the beginning of the second stage. Total taxane production in the cell suspension was significantly enhanced by both elicitors, increasing from a maximum level of 8.14mg/L in control conditions to 21.48mg/L (day 12) with MeJA and 77.46mg/L (day 16) with Cor. Expression analysis indicated that the txs, t13oh, t2oh, t7oh, dbat, pam, bata and dbtnbt genes were variably induced by the presence of the elicitors. Genes encoding enzymes involved in the formation of the polihydroxylated hypothetical intermediate (TXS, T13OH, T2OH, T7OH) and the phenylalanoil CoA chain (PAM) were stronger induced than those encoding enzymes catalysing the last steps of the Px biosynthetic pathway (DBAT, BAPT and DBTNBT). Notably, although taxane accumulation differed qualitatively and quantitatively following MeJA- or Cor-elicitation, gene expression induction patterns were similar, inferring that both elicitors may involve distinct but yet uncharacterised regulatory mechanisms.

Liquid–liquid extraction for recovery of paclitaxel from plant cell culture: Solvent evaluation and use of extractants for partitioning and selectivity

A major challenge in the production of metabolites by plant cells is the separation and purification of a desired product from a number of impurities. An important application of plant cell culture is the biosynthesis of the anticancer agent paclitaxel. Liquid-liquid extraction plays a critical role in the recovery of paclitaxel and other valuable plant-derived products from culture broth. In this study, the extraction of paclitaxel and a major unwanted by-product, cephalomannine, from plant cell culture broth into organic solvents is quantified. Potential solvent mixtures show varying affinity and selectivity for paclitaxel over cephalomannine. The partition coefficient of paclitaxel is highest in ethyl acetate and dichloromethane, with measured values of 28 and 25, respectively; however, selectivity coefficients are less than 1 for paclitaxel over cephalomannine for both solvents. Selectivity coefficient increases to 1.7 with extraction in n-hexane, but the partition coefficient decreases to 1.9. Altering the pH of the aqueous phase results in an increase in both recovery and selectivity using n-hexane but does not change the results for other solvents significantly. The addition of extractants trioctylamine (TOA) or tributylphosphate (TBP) to n-hexane gives significantly higher partition coefficients for paclitaxel (8.6 and 23.7, respectively) but no selectivity. Interestingly, when 20% hexafluorobenzene (HFB) is added to n-hexane, the partition coefficient remains approximately constant, but the selectivity coefficient for paclitaxel over cephalomannine improves to 4.5. This significant increase in selectivity early in the purification process has the potential to simplify downstream processing steps and significantly reduce overall purification costs.

Taxol Biosynthesis: Tyrocidine Synthetase A Catalyzes the Production of Phenylisoserinyl CoA and Other Amino Phenylpropanoyl Thioesters

In Taxus plants the biosynthesis of the pharmaceutical paclitaxel includes the transfer of β-amino phenylpropanoyls from coenzyme A to the diterpenoid baccatin III by an acyl CoA-dependent acyltransferase. Several enzymes on the pathway are known, yet a few remain unidentified, including the putative ligase that biosynthesizes key β-amino phenylpropanoyl CoAs. The multienzyme, nonribosomal peptide synthetase that produces tyrocidines contains a tridomain starter module tyrocidine synthetase A that normally activates (S)-α-Phe to an adenylate anhydride in the adenylation domain. The Phe moiety is then thioesterified by the pendent pantetheine of the adjacent thiolation domain. Herein, the adenylation domain was found to function as a CoA ligase, making α-, β-phenylalanyl, and phenylisoserinyl CoA. The latter two are substrates of a phenylpropanoyltransferase on the biosynthetic pathway of the antimitotic paclitaxel.