Taxus Cell Research Articles

Exploring the Interplay between Metabolic Pathways and Taxane Production in Elicited Taxus baccata Cell Suspensions.

Taxus cell cultures are a reliable biotechnological source of the anticancer drug paclitaxel. However, the interplay between taxane production and other metabolic pathways during elicitation remains poorly understood. In this study, we combined untargeted metabolomics and elicited Taxus baccata cell cultures to investigate variations in taxane-associated metabolism under the influence of 1 µM coronatine (COR) and 150 µM salicylic acid (SA). Our results demonstrated pleiotropic effects induced by both COR and SA elicitors, leading to differential changes in cell growth, taxane content, and secondary metabolism. Metabolite annotation revealed significant effects on N-containing compounds, phenylpropanoids, and terpenoids. Multivariate analysis showed that the metabolomic profiles of control and COR-treated samples are closer to each other than to SA-elicited samples at different time points (8, 16, and 24 days). The highest level of paclitaxel content was detected on day 8 under SA elicitation, exhibiting a negative correlation with the biomarkers kauralexin A2 and taxusin. Our study provides valuable insights into the intricate metabolic changes associated with paclitaxel production, aiding its potential optimization through untargeted metabolomics and an evaluation of COR/SA elicitor effects.

Genetic approaches in improving biotechnological production of taxanes: An update.

Paclitaxel (PTX) and its derivatives are diterpene alkaloids widely used as chemotherapeutic agents in the treatment of various types of cancer. Due to the scarcity of PTX in nature, its production in cell cultures and plant organs is a major challenge for plant biotechnology. Although significant advances have been made in this field through the development of metabolic engineering and synthetic biology techniques, production levels remain insufficient to meet the current market demand for these powerful anticancer drugs. A key stumbling block is the difficulty of genetically transforming the gymnosperm Taxus spp. This review focuses on the progress made in improving taxane production through genetic engineering techniques. These include the overexpression of limiting genes in the taxane biosynthetic pathway and transcription factors involved in its regulation in Taxus spp. cell cultures and transformed roots, as well as the development and optimization of transformation techniques. Attempts to produce taxanes in heterologous organisms such as bacteria and yeasts are also described. Although promising results have been reported, the transfer of the entire PTX metabolic route has not been possible to date, and taxane biosynthesis is still restricted to Taxus cells and some endophytic fungi. The development of a synthetic organism other than Taxus cells capable of biotechnologically producing PTX will probably have to wait until the complete elucidation of its metabolic pathway.

Transcription level differences in Taxus wallichiana var. mairei elicited by Ce3+, Ce4+ and methyl jasmonate.

Taxol is a precious and effective anticancer drug. Cerium and methyl jasmonate (MJ) have been shown to increase the yield of taxol in taxus cells. However, the mechanisms of cerium-mediated and MJ-mediated taxol biosynthesis remain unknown. RNA-Seq was applied to study the overall regulation mechanism of cerium and MJ on taxol biosynthesis and analyze the differences among T. mairei cells elicited by Ce3+, Ce4+ and MJ on transcriptional level . Using sequence homology, 179 unigenes were identified as taxol synthesis genes. Under the condition of 100 μM MJ, taxol synthesis genes were up-regulated. Notably, taxol synthesis genes were down-regulated expression at 1 mM Ce3+ and 1 mM Ce4+. Differential expression genes involved in some related functions were analyzed, such as MAPK signaling pathway and plant-pathogen interaction. Sequence alignment and phylogenetic analysis of nine differentially expressed WRKYs in our data were carried out.

Switching cell fate by the actin–auxin oscillator in Taxus: cellular aspects of plant cell fermentation

Key messagePaclitaxel synthesis in Taxus cells correlates with a cell-fate switch that leads to vacuoles of a glossy appearance and vermiform mitochondria. This switch depends on actin and apoplastic respiratory burst.Plant cell fermentation, the production of valuable products in plant cell culture, has great potential as sustainable alternative to the exploitation of natural resources for compounds of pharmaceutical interest. However, the success of this approach has remained limited, because the cellular aspects of metabolic competence are mostly unknown. The production of the anti-cancer alkaloid Paclitaxel has been, so far, the most successful case for this approach. In the current work, we map cellular aspects of alkaloid synthesis in cells of Taxus chinensis using a combination of live-cell imaging, quantitative physiology, and metabolite analysis. We show evidence that metabolic potency correlates with a differentiation event giving rise to cells with large vacuoles with a tonoplast that is of a glossy appearance, agglomerations of lipophilic compounds, and multivesicular bodies that fuse with the plasma membrane. Cellular features of these glossy cells are bundled actin, more numerous peroxisomes, and vermiform mitochondria. The incidence of glossy cells can be increased by aluminium ions, and this increase is significantly reduced by the actin inhibitor Latrunculin B, and by diphenylene iodonium, a specific inhibitor of the NADPH oxidase Respiratory burst oxidase Homologue (RboH). It is also reduced by the artificial auxin Picloram. This cellular fingerprint matches the implications of a model, where the differentiation into the glossy cell type is regulated by the actin–auxin oscillator that in plant cells acts as dynamic switch between growth and defence.

Bottleneck removal of paclitaxel biosynthetic pathway by overexpression of DBTNBT gene under methyl-β-cyclodextrin and coronatine elicitation in Taxus baccata L.

Paclitaxel is a highly functionalized diterpenoid that is broadly used for the treatment of several cancer types. This valuable specialized metabolite naturally exists in the inner bark of Taxus species in low amounts. The limited-scale production of paclitaxel in Taxus cell cultures has necessitated the use of several elicitors. Recently, methyl-β-cyclodextrin (CD) and coronatine (COR) have been considered to be highly effective elicitors in producing plant specialized metabolites. Given the limited production of paclitaxel due to the rate limiting enzymes' function, bottleneck removal is conducive to the production of more significant amounts of paclitaxel. In the present study, the full length of DBTNBT coding sequence (CDS), as one of the paclitaxel pathway bottlenecks, was integrated downstream of the CaMV 35S promoter (pCAMBIA1304-DBTNBT) and transiently expressed in Taxus baccata leaves via Agrobacterium tumefaciens and vacuum infiltration method. Paclitaxel production and the expression level of several involved genes were evaluated through different treatments. The transient overexpression of the DBTNBT gene, associated with dual elicitation, resulted in 7.4-fold more paclitaxel production compared with the no-inoculation/no-elicitation control. These ratios were 2.1 and 1.8 in the CD + COR and pCAM treatments, respectively. Among T13αH, T14βH, DBAT, BAPT, DBTNBT, and ABC genes, the most increased expression level belonged to the DBTNBT gene, followed by ABC and BAPT genes. It seems as though in the near future, bottleneck removal could be used on a large scale in Taxus metabolic engineering, resulting in the relative removal of some other bottlenecks and an increase in the final paclitaxel production.

TcMYB29a, an ABA-Responsive R2R3-MYB Transcriptional Factor, Upregulates Taxol Biosynthesis in Taxus chinensis.

Paclitaxel (Taxol), a highly modified diterpene agent mainly obtained from Taxus species, is the most widely used anticancer drug. Abscisic acid (ABA) is a well-known stress hormone that plays important roles in the secondary metabolism of plants, and it can also induce the accumulation of taxol in Taxus cell suspension cultures. However, the mechanism behind the regulation of taxol biosynthesis by ABA remains largely unknown. In previous research, a R2R3 MYB transcription factor (TF) TcMYB29a was observed to show a significant correlation with taxol biosynthesis, indicative of its potential role in the taxol biosynthesis. In this study, the TcMYB29a encoded by its gene was further characterized. An expression pattern analysis revealed that TcMYB29a was highly expressed in the needles and roots. Overexpression of TcMYB29a in Taxus chinensis cell suspension cultures led to an increased accumulation of taxol, and upregulated expression of taxol-biosynthesis-related genes, including the taxadiene synthase (TS) gene, the taxane 5α-hydroxylase (T5OH) gene, and the 3′-N-debenzoyl-2′-deoxytaxol-N-benzoyltransferase (DBTNBT) gene as compared to the controls. Chromatin immunoprecipitation (ChIP) assays, yeast one-hybrid (Y1H) assays, electrophoretic mobility shift assays (EMSAs), and dual-luciferase reporter assays verified that TcMYB29a could bind and activate the promoter of TcT5OH. Promoter sequence analysis of TcMYB29a revealed that its promoter containing an AERB site from -313 to -319 was a crucial ABA-responsive element. Subsequently, the ABA treatment assay showed that TcMYB29a was strongly upregulated at 6 h after ABA pretreatment. Furthermore, TcMYB29a was strongly suppressed at 3 h after the methyl jasmonate (MeJA) treatment and was depressed to the platform at 12 h. Taken together, these results reveal that TcMYB29a is an activator that improves the accumulation of taxol in Taxus chinensis cells through an ABA-medicated signaling pathway which is different from JA-medicated signaling pathways for the accumulation of taxol. These findings provide new insights into the potential regulatory roles of MYBs on the expression of taxol biosynthetic genes in Taxus.

The Effect of The Combination of Precursor and Elicitors Enhance the Taxol Accumulation in Red Pine Cell Cultivation

The material introduced into the callus culture was the in vitro cloned red pine stem and leaves. Callus obtained through culture from leaves and stems were included in the study on proliferation on agar and liquid media. The suspension obtained after 45 days of culture was determined for biomass, used in proliferation culture, and studied taxol accumulation. Media for taxus cell cloning was MS supplemented with 3 mg/l 2.4D, 3 mg/l NAA, 0.5 mg/l kinetin, 0.1 mg/l BA, 10% CW. Selections were carried out through 8 steps in the year of 2010 with interval cultivation time of 45 days/each step. It’s could improve the taxol accumulation via cell suspension cultures sourced from leaves 170.1 mg/gDW and stems 27.3 mg/gDW. Picloram was not effect on taxol accumulation. On the basic media MS supplemented with 3 mg/l 2.4D, 3 mg/l NAA, 0.5 mg/l kinetin, 0.1 mg/l BA, 10% CW supplemented with precursor of 15mg/l phenyl alanine (PA) effects on the percentage of FW/DW (fresh weight/dried weight) was 9.815 and the taxol accumulation was 0.778%. The effects of elicitors supplemented to media with 10 mg/l methyl jasmonate (MJ) having 7.183 FW/DW and 0.273% taxol, 100 mg/l salysilic acid (SA) having 10.12 FW/DW and 0.094% taxol, 50mg/l chitosan having 10.09 FW/DW and 0.119% taxol, 5mg/l oligo-chitosan having 9.090 FW/DW and 0.778% taxol. The effects of the combination of precursor and elicitors (15 mg/l phenyl alanine, 10 mg/l methyl jasmonate, 5 mg/l O-chitosan, 100 mg/l salysilic acid) enhance the taxol accumulation to 1.003% in comparison of separately as PA having 0.114% taxol, PA+Ochi having 0.542% taxol, PA+MJ+Ochi having 0.564%. Cell cloning from leaves (1.701%) had the taxol accumulation more than from stem (0.273%).

The Research Progress of Taxol in Taxus.

Taxus is a valuable woody species with important medicinal value. The bark of Taxus can produce taxol, a natural antineoplastic drug that is widely used in the treatment of breast, ovarian and lung cancers. However, the low content of taxol in the bark of Taxus can not meet the growing clinical demands, so the current research aims at finding ways to increase taxol production. In this review, the research progress of taxol including the factors affecting the taxol content, biosynthesis pathway of taxol, production of taxol in vitro and the application of multi-omics approaches in Taxus as well as future research prospects will be discussed. The taxol content is not only dependent on the species, age and tissues but is also affected by light, moisture levels, temperature, soil fertility and microbes. Most of the enzymes in the taxol biosynthesis pathway have been identified and characterized. Total chemical synthesis, semi-synthesis, plant cell culture and biosynthesis in endophytic fungi have been explored to product taxol. Multi-omics have been used to study Taxus and taxol. Further efforts in the identification of unknown enzymes in the taxol biosynthesis pathway, establishment of the genetic transformation system in Taxus and the regulatory mechanism of taxol biosynthesis and Taxus cell growth will play a significant role in improving the yield of taxol in Taxus cells and plants.

Improved effects of polyethylene glycol on the growth, antioxidative enzymes activity and taxanes production in a Taxus baccata L. callus culture

Application of abiotic stress-inducing elicitors is one of the effective strategies in order to increase the production of secondary metabolites in plant tissues. Here, the effect of in vitro polyethylene glycol (PEG)-induced drought stress on morphological and biochemical changes as well as the production of paclitaxel and 10-deacetyl baccatin III in a Taxus baccata callus culture was investigated. The results showed that increased PEG concentration in the culture medium enhanced the levels of H2O2 and the membrane lipid peroxidation in the calli. The activity of catalase and guaiacol peroxidase as antioxidant responses were increased significantly in all PEG concentrations. At 6% of PEG concentration, the levels of fresh weight (FW) and dry weight (DW) in the calli were declined significantly by dropping in the amount of water content in the calli and rising the level of oxidative stress, and the morphological changes occurred following the increased browning index of the calli. However, the PEG at low to moderate concentrations reduced the accumulation and oxidation of phenolic compounds in the calli through the absorption of phenolic compounds and the reduction in the activity of the polyphenol oxidase. As a result, improved growth and viability of the calli resulted in the enhanced levels of FW and DW at the concentrations of 1% up to 3% (w/v) PEG. The highest contents of 10-deacetyl baccatin III and taxol were obtained at concentrations of 2% and 3% PEG, respectively. The concentration of 3% PEG increased the production of taxol by 2.5 times more than the control. In conclusion, PEG can be proposed as an effective elicitor in the production of taxanes in the Taxus cell and tissue cultures.

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.

Advances in the Regulation of In Vitro Paclitaxel Production: Methylation of a Y-Patch Promoter Region Alters BAPT Gene Expression in Taxus Cell Cultures.

Plant cell biofactories represent a promising solution to the increasing demand for plant-derived compounds, but there are still limiting factors that prevent optimal production, including the loss of yield during in vitro maintenance. Our results reveal a clear correlation between genomic methylation levels and a progressive decline in taxane production in Taxus spp. cell cultures. A comparative study of two cell lines, one 10 years old and low productive and the other new and high productive, revealed important differences in appearance, growth, taxane accumulation and expression levels of several taxane biosynthetic genes. Differences in taxane content and gene expression profile indicate an altered pathway regulation and that the BAPT gene, located in the center of the expression network of taxane biosynthetic genes, is active in a potentially flux-limiting step. The methylation patterns of the BAPT gene were studied in both cell lines by bisulfite sequencing, which revealed high rates of CHH methylated cytosines on the core promoter. Using a bioinformatics approach, this hotspot was identified as a Y-patch promoter element. The Y-patch may play a key role in the epigenetic regulation of the taxane biosynthetic pathway, which would open up novel genetic engineering strategies toward stable and high productivity.

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.

Advances and prospects of taxol biosynthesis by endophytic fungi

Taxol is one of the most important chemotherapeutic drugs against cancer. Taxol has been mainly extracted from the bark of yews for a long time. However, methods for the extraction of taxol from the bark of Taxus species were inefficient and environmentally costly. As a result of the high ecological toll exacted on trees with the potential for Pacific yew extinction, investigators began to look for other methods of taxol production. Recently, increasing efforts have been made to develop alternative means of taxol production, such as using complete chemical synthesis, semi-synthesis, Taxus spp. plant cell culture and microbe fermentation. Using microbe fermentation in the production of taxol would be a very prospective method for obtaining a large amount of taxol. Therefore, it is necessary to understand the molecular basis and genetic regulation mechanisms of taxol biosynthesis by endophytic fungi, which may be helpful to construct the genetic engineering strain with high taxol output. In this paper, the taxol biosynthesis pathway from Taxus cells and the advantages of taxol biosynthesis by endophytic fungi were discussed. The study on the isolation and biodiversity of taxol-producing endophytic fungi and the taxol biosynthesis related genes are also discussed.

ChemInform Abstract: Advances in Exploring Alternative Taxol Sources

AbstractReview: 75 refs.

Optimization of a broth conductivity controlling strategy directed by an online viable biomass sensor for enhancing Taxus cell growth rate and Taxol productivity

In this work, we investigated the effects of a constant broth conductivity controlling strategy directed by an online viable biomass sensor on Taxol productivity ofTaxus chinensisvar.maireiin suspension cultivation.

New taxane biosynthetic gene encoding a β-phenylalanine-CoA ligase identified in jasmonate-elicited Taxus cells

The data-generating power of “omics” technologies is increasingly being harnessed in studies of plant biology to shed light on aspects such as metabolite production patterns, gene sequences and protein expression. Taxol and related taxanes, produced by several Taxus species, are high-value secondary metabolites widely used in cancer therapy due to their antineoplastic activity. To date, several biosynthetic steps of these compounds remain undefined. After carrying out a genome-wide expression analysis of jasmonate-elicited Taxus baccata cell cultures using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP), we found that the increased taxane production in the target cultures was correlated with an extensive elicitor-induced genetic reprogramming. Subsequent in silico analysis allowed us to identify 15 genes with jasmonate-induced differential expression putatively involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene and its predicted 3D structure showed a strong homology with other genes previously reported to encode acyl-CoA ligases, suggesting it was involved in the formation of the taxol lateral chain. In vitro analysis confirmed that the TB768 gene encodes an acyl-CoA ligase, localized in the cytoplasm and able to convert b-phenylalanine into its CoA ester derivative. The latter is then attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. In determining that the TB768 gene encodes a cytosolic CoA ligase with an important role in the taxol biosynthetic pathway, we have corroborated that the combination of a cDNA-AFLP transcriptome profiling approach with bioinformatics analysis constitutes an efficient set of tools for the identification of new genes involved in the metabolism of valuable bioactive compounds. The newly described gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.

Two jasmonate-responsive factors, TcERF12 and TcERF15, respectively act as repressor and activator of tasy gene of taxol biosynthesis in Taxus chinensis

Methyl jasmonate (MeJA) is one of the most effective inducers of taxol biosynthetic genes, particularly the tasy gene. However, the mechanism underlying the regulation of tasy by MeJA is still unknown. In this study, a 550-bp 5'-flanking sequence was obtained and confirmed as the promoter of the tasy gene. Deletion analysis revealed that the fragment containing a GCC-box from -150 to -131 was the crucial jasmonate (JA)-responsive element, designated as JRE. Using JRE as bait, two binding proteins, namely TcERF12 and TcERF15, were discovered. Sequence alignment and phylogenetic analysis showed that TcERF12 was related to the repressor AtERF3, while TcERF15 was more related to the activator ORA59; these are typical GCC-box-binding ethylene-responsive factors. Both could significantly respond to MeJA for 10 and 4.5 times, respectively, in 0.5h. When the two TcERFs were overexpressed in Taxus cells, tasy gene expression decreased by 2.1 times in TcERF12-overexpressing cells, but increased by 2.5 times in TcERF15-overexpressing cells. Results indicated that TcERF12 and TcERF15 were negative and positive regulators, respectively, in the JA signal transduction to the tasy gene by binding the GCC-box in the JRE of the tasy promoter. Our results promote further research on regulatory mechanisms of taxol biosynthesis.

High-throughput sequencing reveals miRNA effects on the primary and secondary production properties in long-term subcultured Taxus cells.

Plant-cell culture technology is a promising alternative for production of high-value secondary metabolites but is limited by the decreased metabolite production after long-term subculture. The goal of this study was to determine the effects of miRNAs on altered gene expression profiles during long-term subculture. Two Taxus cell lines, CA (subcultured for 10 years) and NA (subcultured for 6 months), were high-throughput sequenced at the mRNA and miRNA levels1. A total of 265 known (78.87% of 336) and 221 novel (79.78% of 277) miRNAs were differentially expressed. Furthermore, 67.17% of the known differentially expressed (DE) miRNAs (178) and 60.63% of the novel DE-miRNAs (134) were upregulated in NA. A total of 275 inverse-related miRNA/mRNA modules were identified by target prediction analysis. Functional annotation of the targets revealed that the high-ranking miRNA targets were those implicated in primary metabolism and abiotic or biotic signal transduction. For example, various genes for starch metabolism and oxidative phosphorylation were inversely related to the miRNA levels, thereby indicating that miRNAs have important roles in these pathways. Interestingly, only a few genes for secondary metabolism were inversely related to miRNA, thereby indicating that factors other than miRNA are present in the regulatory system. Moreover, miR8154 and miR5298b were upregulated miRNAs that targeted a mass of DE genes. The overexpression of these miRNAs in CA increased the genes of taxol, phenylpropanoid, and flavonoid biosynthesis, thereby suggesting their function as crucial factors that regulate the entire metabolic network during long-term subculture. Our current studies indicated that a positive conversion of production properties from secondary metabolism to primary metabolism occurred in long-term subcultured cells. miRNAs are important regulators in the upregulation of primary metabolism.

Changes in gene transcription and taxane production in elicited cell cultures of Taxus × media and Taxus globosa

The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), supplied to the culture medium either separately or together, was studied. Two-stage Taxus globosa and Taxus media cell cultures were established and the elicitors were added at the beginning of the second stage. Growth, taxane production, and the expression of known taxol biosynthetic genes, including the recently characterized CoA ligase gene, were studied. Although CORO reduced the growth capacity of both cell lines, CD apparently counteracted this negative effect. Taxane production was significantly enhanced by the simultaneous addition of CD and CORO to the medium. The total taxane production in the T. media cell line was more than double that of T. globosa, but in the latter more than 90% of the taxanes produced were excreted to the medium. Individual taxane patterns also differed: at the height of production, the main taxanes in T. globosa cultures were cephalomannine and 10-deacetyltaxol, and in T. media, taxol and baccatin III. The low transcript levels of taxane biosynthetic genes found in T. globosa cells mirrored the lower taxane production in these cultures, while a high expression was strongly correlated with a high taxane production in T. media.

Transcript profiling of jasmonate-elicited Taxus cells reveals a β-phenylalanine-CoA ligase.

Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate-elicited Taxus baccata cell cultures by complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) indicated a correlation between an extensive elicitor-induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate-induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl-CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl-CoA ligase that localizes to the cytoplasm and is able to convert β-phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β-phenylalanyl-CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.