Taxane Production Research Articles

Corylus avellana as a potential alternative source for the production of taxol and taxanes

This paper demonstrates the increased interest that scientists have recently shown in the non-food components of the hazel plant, such as leaves, as well as hard and green shells, in addition to the nuts. The review discusses studies conducted to determine the qualitative and quantitative profiles of different parts of Corylus avellana. It has been demonstrated that extracts made from various Corylus avellana components possess a variety of biological properties, such as anticancer, antiproliferative, and antioxidant effects. It has been shown that secondary metabolites can be directly synthesized by in vitro cell culture, which allows the production of valuable secondary metabolites such as paclitaxel, one of the most important anticancer compounds synthesized by hazel. A brief review of existing literature data serves to emphasize C.avellana as a source of bioactive compounds. Further research is needed to explore the health benefits of C.avellana extracts and their use as food supplements. Keywords: Hazel, Corylus avellana, secondary metabolites, taxol, anticancer drug

Исследование химического состава и антибактериальной активности СО2 экстрактов Juniperus pseudosabina, Larix dahurica, Taxus cuspidata

Extracts of coniferous plants Juniperus pseudosabina, Larix dahurica, Taxus cuspidata were obtained by supercritical fluid extraction method. In the present study, it was found that polyphenols: isoflavonoids and flavonoids, and especially flavonoglycosides, which have P-vitamin activity, constitute the most extensive group of substances in the CO2 extracts of all three plants. The CO2 extract of Taxus cuspidata can be considered as a promising alternative to Taxus berry in the role of a source for the production of individual taxanes, such as paclitaxel and docetaxel. The presence of such groups of compounds as flavanoids, isoflavanoids in the composition of CO2 extracts increases their potential as sources of biologically active compounds suitable for use as components of biologically active and feed additives with immunomodeling antioxidant properties.

Overexpression of BAPT and DBTNBT genes in Taxus baccatain vitro cultures to enhance the biotechnological production of paclitaxel.

Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 μm coronatine plus 50 mm of randomly methylated-β-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line.

Enhancement of taxane production in cell suspension culture of Corylus avellana L. Kalınkara by elicitation and precursor feeding

Paclitaxel (trademark Taxol®) is the most effective chemotherapeutic agent against a wide range of cancers. Hazelnut (Corylus avellana L.) cell culture is a promising and inexpensive strategy for producing paclitaxel and related taxanes. This study evaluated the effect of elicitation and taxane precursor feeding for the enhancement of taxanes in a Turkish hazelnut (C. avellana cv. ‘Kalınkara’) cell suspension culture. Elicitor methyl jasmonate (100, 200 and 300 µmol L−1) and precursor phenylalanine (3μmol, 3 and 6 mmol L−1) were tested in cell suspension cultures. While methyl jasmonate increased the accumulation of taxanes, especially cephalomannine, via induction of the paclitaxel biosynthetic pathway, phenylalanine slightly induced the conversion of 10-deacetylbaccatin III and baccatin III to the end product paclitaxel (0.5349 µg g−1). The results showed that 200 µmol L−1 methyl jasmonate led to significantly greater induction of taxanes than 100 and 300 µmol L−1. The amount of cephalomannine (331.6 µg g−1) in cell suspension cultures elicited by 200 µmol L−1 methyl jasmonate was 8-fold higher than that in the control (41.61 µg g−1), while 10-deacetylbaccatin III (6.174 µg g−1) and baccatin III (3.956 µg g−1) increased almost twice as much as the control. The highest total paclitaxel yield was 5.453 µg g−1 in the same culture conditions. In conclusion, cell culture systems of C. avellana L. cv. ‘Kalınkara’ hazelnut, established for the first time, may become a potential candidate for industrial-level production of cephalomannine and other taxanes with further optimization.

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.

Polyploidy as a strategy to increase taxane production in yew cell cultures: Obtaining and characterizing a Taxus baccata tetraploid cell line

Novel approaches to optimize the production of plant specialized metabolites are crucial to reach maximum productivity of plant biofactories. Plant polyploidization frequently enhances protein synthesis and thereby increases the biosynthesis of specialized metabolites. Paclitaxel is a valuable anticancer agent scarcely produced in nature. Therefore, plant biofactories represent a sustainable alternative source of this compound and related taxanes. With the aim of improving the productivity of Taxus spp. cell cultures, we induced polyploidy in vitro by treating immature embryos of Taxus baccata with colchicine. To obtain the polyploid cell lines, calli were induced from T. baccata plantlets previously treated with colchicine and ploidy levels were accurately identified using flow cytometry. In terms of cell morphology, tetraploid cells were about 3-fold bigger than the diploid cells. The expression of taxane pathway genes was higher in the tetraploid cell line compared to the diploid cells. Moreover, taxane production was 6.2-fold higher and the production peak was achieved 8 days earlier than in the diploid cell line, indicating a higher productivity. The obtained tetraploid cell line proved to be highly productive, constituting a step forward towards the development of a bio-sustainable production system for this chemotherapeutic drug.

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.

Gene expression pattern and taxane biosynthesis in a cell suspension culture of Taxus baccata L. subjected to light and a phenylalanine ammonia lyase (PAL) inhibitor.

Taxus baccata L. cell culture is a promising commercial method for the production of taxanes with anti-cancer activities. In the present study, a T. baccata cell suspension culture was exposed to white light and 2-aminoindan-2-phosphonic acid (AIP), a phenylalanine ammonia lyase (PAL) inhibitor, and the effects of this treatment on cell growth, PAL activity, total phenol content (TPC), total flavonoid content (TFC), taxane production and the expression of some key taxane biosynthetic genes (DXS, GGPPS, T13OH, BAPT, DBTNBT) as well as the PAL were studied. Light reduced cell growth, whereas AIP slightly improved it. Light increased PAL activity up to 2.7-fold relative to darkness. The highest TPC (24.89mg GAE/g DW) and TFC (66.94mg RUE/g DW) were observed in cultures treated with light and AIP. Light treatment also resulted in the maximum content of total taxanes (154.78μg/g DW), increasing extracellular paclitaxel and cephalomannin (3.3-fold) and intracellular 10-deacetyl paclitaxel (2.5-fold). Light significantly increased the expression level of PAL, DBTNBT, BAPT, and T13αOH genes, whereas it had no effect on the expression of DXS, a gene active at the beginning of the taxane biosynthetic pathway. AIP had no significant effect on the expression of the target genes. In conclusion, the light-induced activation of PAL transcription and altered expression of relevant biosynthetic genes reduced cell growth and increased the content of total phenolic compounds and taxanes. These findings can be applied to improve taxane production in controlled cultures and bioreactors.

The Epigenetic Regulation in Plant Specialized Metabolism: DNA Methylation Limits Paclitaxel in vitro Biotechnological Production.

Environmental conditions are key factors in the modulation of the epigenetic mechanisms regulating gene expression in plants. Specifically, the maintenance of cell cultures in optimal in vitro conditions alters methylation patterns and, consequently, their genetic transcription and metabolism. Paclitaxel production in Taxus x media cell cultures is reduced during its maintenance in in vitro conditions, compromising the biotechnological production of this valuable anticancer agent. To understand how DNA methylation influences taxane production, the promoters of three genes (GGPPS, TXS, and DBTNBT) involved in taxane biosynthesis have been studied, comparing the methylation patterns between a new line and one of ~14 years old. Our work revealed that while the central promoter of the GGPPS gene is protected from cytosine methylation accumulation, TXS and DBTNBT promoters accumulate methylation at different levels. The DBTNBT promoter of the old line is the most affected, showing a 200 bp regulatory region where all the cytosines were methylated. This evidence the existence of specific epigenetic regulatory mechanisms affecting the last steps of the pathway, such as the DBTNBT promoter. Interestingly, the GGPPS promoter, a regulatory sequence of a non-specific taxane biosynthetic gene, was not affected by this mechanism. In addition, the relationship between the detected methylation points and the predicted transcription factor binding sites (TFBS) showed that the action of TFs would be compromised in the old line, giving a further explanation for the production reduction in in vitro cell cultures. This knowledge could help in designing novel strategies to enhance the biotechnological production of taxanes over time.

Enhancing Saccharomyces cerevisiae Taxane Biosynthesis and Overcoming Nutritional Stress-Induced Pseudohyphal Growth.

The recent technological advancements in synthetic biology have demonstrated the extensive potential socio-economic benefits at laboratory scale. However, translations of such technologies to industrial scale fermentations remains a major bottleneck. The existence and lack of understanding of the major discrepancies in cultivation conditions between scales often leads to the selection of suboptimal bioprocessing conditions, crippling industrial scale productivity. In this study, strategic design of experiments approaches were coupled with state-of-the-art bioreactor tools to characterize and overcome nutritional stress for the enhanced production of precursors to the blockbuster chemotherapy drug, Taxol, in S. cerevisiae cell factories. The batch-to-batch variation in yeast extract composition was found to trigger nutritional stress at a mini-bioreactor scale, resulting in profound changes in cellular morphology and the inhibition of taxane production. The cells shifted from the typical budding morphology into striking pseudohyphal cells. Doubling initial yeast extract and peptone concentrations (2YP) delayed filamentous growth, and taxane accumulation improved to 108 mg/L. Through coupling a statistical definitive screening design approach with the state-of-the-art high-throughput micro-bioreactors, the total taxane titers were improved a further two-fold, compared to the 2YP culture, to 229 mg/L. Filamentous growth was absent in nutrient-limited microscale cultures, underlining the complex and multifactorial nature of yeast stress responses. Validation of the optimal microscale conditions in 1L bioreactors successfully alleviated nutritional stress and improved the titers to 387 mg/L. Production of the key Taxol precursor, T5αAc, was improved two-fold to 22 mg/L compared to previous maxima. The present study highlights the importance of following an interdisciplinary approach combining synthetic biology and bioprocessing technologies for effective process optimization and scale-up.

Definitive screening accelerates Taxol biosynthetic pathway optimization and scale up in Saccharomyces cerevisiae cell factories.

Recent technological advancements in synthetic and systems biology have enabled the construction of microbial cell factories expressing diverse heterologous pathways in unprecedentedly short time scales. However, the translation of such laboratory scale breakthroughs to industrial bioprocesses remains a major bottleneck. In this study, an accelerated bioprocess development approach was employed to optimize the biosynthetic pathway of the blockbuster chemotherapy drug, Taxol. Statistical design of experiments approaches were coupled with an industrially relevant high-throughput microbioreactor system to optimize production of key Taxol intermediates, Taxadien-5α-ol and Taxadien-5α-yl-acetate, in engineered yeast cell factories. The optimal factor combination was determined via data driven statistical modelling and validated in 1 L bioreactors leading to a 2.1-fold improvement in taxane production compared to a typical defined media. Elucidation and mitigation of nutrient limitation enhanced product titers a further two-fold and titers of the critical Taxol precursors, Taxadien-5α-ol and Taxadien-5α-yl-acetate were improved to 34 and 11mg L-1 , representing a three-fold improvement compared to the highest literature titers in S. cerevisiae. Comparable titers were obtained when the process was scaled up a further five-fold using 5 L bioreactors. The results of this study highlight the benefits of a holistic design of experiments guided approach to expedite early stage bioprocess development.

Improved biotechnological production of paclitaxel in Taxus media cell cultures by the combined action of coronatine and calix[8]arenes

Paclitaxel (PTX), a widely used anticancer agent, is found in the inner bark of several Taxus species, although at such low levels that its extraction is ecologically unsustainable. Biotechnological platforms based on Taxus sp. cell cultures offer an eco-friendlier approach to PTX production, with yields that can be improved by elicitation. However, the also limited excretion of target compounds from the producer cells to the medium hampers their extraction and purification. In this context, we studied the effect of treating T. media cell cultures with the elicitor coronatine (COR) and calix[8]arenes (CAL), nanoparticles that can host lipophilic compounds within their macrocyclic scaffold. The highest taxane production (103.5 mg.L−1), achieved after treatment with COR (1 μM) and CAL (10 mg.L−1), was 15-fold greater than in the control, and PTX represented 82% of the total taxanes analyzed. Expression levels of the flux-limiting PTX biosynthetic genes, BAPT and DBTNBT, increased after the addition of COR, confirming its elicitor action, but not CAL. The CAL treatment significantly enhanced taxane excretion, especially when production levels were increased by COR; 98% of the total taxanes were found in the culture medium after COR + CAL treatment. By forming complexes with PTX, the nanoparticles facilitated its excretion to the medium, and by protecting cells from PTX toxicity, its intra-and extra-cellular degradation may have been avoided. The addition of COR and CAL to T. media cell cultures is therefore a bio-sustainable and economically viable system to improve the yield of this important anticancer compound.

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies.

Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyze the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation to taxadien-5α-yl-acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5α-ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography-mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien-5α-yl-acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7-fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.

Physiological, biochemical, and metabolic responses of a Taxus baccata L. callus culture under drought stress

The metabolic stimulation induced by abiotic stress is an efficient strategy for the production of secondary metabolites in sterile and controlled plant cell, tissue, and organ cultures. Paclitaxel (taxol), one of the most widely used therapeutic compounds for the treatment of various cancers, is mainly produced through cell culture of the European yew (Taxus baccata L.). In this work, a T. baccata callus culture was subjected to drought stress induced in vitro by mannitol and sorbitol (1, 2, 3, and 4%) and sucrose (6 and 8%) to evaluate its impact on physiological and biochemical traits as well as paclitaxel and 10-deacetyl baccatin III (10-DBA) production. It was observed that drought stress caused a significant increase (P < 0.05) in dry weight, proline, soluble sugars, hydrogen peroxide, lipid peroxidation, total phenolics, flavonoids, and flavonols, and a decrease in relative water content, fresh weight, relative growth rate, and cell viability. Constitutive activities of superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase were enhanced by the induced stress. The content of paclitaxel and 10-DBA was higher in stressed cultures than in the control. It can be concluded that T. baccata cells have a protection mechanism against oxidative damage involving induced activities of enzymatic and non-enzymatic antioxidants. The induction of moderate drought stress could therefore be an effective strategy for increasing taxanes production in T. baccata callus cultures.

A Novel Hydroxylation Step in the Taxane Biosynthetic Pathway: A New Approach to Paclitaxel Production by Synthetic Biology.

Engineered plant cell lines have the potential to achieve enhanced metabolite production rates, providing a high-yielding source of compounds of interest. Improving the production of taxanes, pharmacologically valuable secondary metabolites of Taxus spp., is hindered by an incomplete knowledge of the taxane biosynthetic pathway. Of the five unknown steps, three are thought to involve cytochrome P450-like hydroxylases. In the current work, after an in-depth in silico characterization of four candidate enzymes proposed in a previous cDNA-AFLP assay, TB506 was selected as a candidate for the hydroxylation of the taxane side chain. A docking assay indicated TB506 is active after the attachment of the side chain based on its affinity to the ligand 3′N-dehydroxydebenzoyltaxol. Finally, the involvement of TB506 in the last hydroxylation step of the paclitaxel biosynthetic pathway was confirmed by functional assays. The identification of this hydroxylase will contribute to the development of alternative sustainable paclitaxel production systems using synthetic biology techniques.

Dataset of Targeted Metabolite Analysis for Five Taxanes of Hellenic Taxus baccata L. Populations

Novel primary sources of one of the world’s leading anticancer agent, paclitaxel, as well as of other antineoplastic taxanes such as 10-deacetylbaccatin-III, are needed to meet an increasing demand. Among the Taxus species the promise of Taxus baccata L. (European or English yew) has been documented. In this study, the metabolite analysis of two marginal T. baccata populations in Greece (Mt. Cholomon and Mt. Olympus), located at the southeastern edge of the species natural distribution, are being explored. A targeted liquid chromatography – mass spectrometry (LC-MS/MS) analysis was used to determine the content of 10-deacetylbaccatin III, baccatin III, 10-deacetyltaxol, paclitaxel and cephalomannine in the needles of each of the populations from three sampling periods (spring, summer and winter). This is the first survey to generate a taxane targeted metabolite data set, since it derives from Hellenic natural populations that have not been explored before. Furthermore, it has used an extensive sample design in order to evaluate chemodiversity at the population level. The analysis revealed significant levels of chemodiversity within and among the investigated populations and significant seasonal variation that could be exploited for the selection of superior germplasm native to Greece, for yew plantations and further exploitation which is necessary for the production of important taxanes.

Transfecting Taxus � media Protoplasts to Study Transcription Factors BIS2 and TSAR2 as Activators of Taxane-Related Genes.

Taxane diterpenes are secondary metabolites with an important pharmacological role in the treatment of cancer. Taxus spp. biofactories have been used for taxane production, but the lack of knowledge about the taxane biosynthetic pathway and its molecular regulation hinders their optimal function. The difficulties in introducing foreign genes in Taxus spp. genomes hinder the study of the molecular mechanisms involved in taxane production, and a new approach is required to overcome them. In this study, a reliable, simple and fast method to obtain Taxus � media protoplasts was developed, allowing their manipulation in downstream assays for the study of physiological changes in Taxus spp. cells. Using this method, Taxus protoplasts were transiently transfected for the first time, corroborating their suitability for transfection assays and the study of specific physiological responses. The two assayed transcription factors (BIS2 and TSAR2) had a positive effect on the expression of several taxane-related genes, suggesting their potential use for the improvement of taxane yields. Furthermore, the results indicate that the developed method is suitable for obtaining T. � media protoplasts for transfection with the aim of unraveling regulatory mechanisms in taxane production.

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.

Comparison of elicitor-based effects on metabolic responses of Taxus\u2009\xd7\u2009media hairy roots in perfluorodecalin-supported two-phase culture system

Key messageTwo lines of Taxus × media hairy roots harbouring or not the TXS transgene demonstrated diverse gene expression and taxane yield during cultivation in PFD-supported two liquid-phase culture system.Two lines of Taxus × media hairy roots were subjected to single or twice-repeated supplementation with methyl jasmonate, sodium nitroprusside, l-phenylalanine, and sucrose feeding. One line harboured transgene of taxadiene synthase (ATMA), while the second (KT) did not. Both hairy root lines were cultured in two-phase culture systems containing perfluorodecalin (PFD) in aerated or degassed form. The relationship between TXS (taxadiene synthase), BAPT (baccatin III: 3-amino, 3-phenylpropanoyltransferase), and DBTNBT (3′-N-debenzoyl-2-deoxytaxol-N-benzoyltransferase) genes and taxane production was analysed. The ATMA and KT lines differed in their potential for taxane accumulation, secretion, and taxane profile. In ATMA biomass, both paclitaxel and baccatin III were detected, while in KT roots only paclitaxel. The most suitable conditions for taxane production for ATMA roots were found in single-elicited supported with PFD-degassed cultures (2 473.29 ± 263.85 µg/g DW), whereas in KT roots in single-elicited cultures with PFD-aerated (470.08 ± 25.15 µg/g DW). The extracellular levels of paclitaxel never exceeded 10% for ATMA roots, while for KT increased up to 76%. The gene expression profile was determined in single-elicited cultures supported with PFD-degassed, where in ATMA roots, the highest taxane yield was obtained, while in KT the lowest one. The gene expression pattern in both investigated root lines differed substantially what resulted in taxane yield characterized particular lines. The highest co-expression of TXS, BAPT and DBTNBT genes noted for ATMA roots harvested 48 h after elicitation corresponded with their higher ability for taxane production in comparison with the effects observed for KT roots.

The effects of salicylic acid and glucose on biochemical traits and taxane production in a Taxus baccata callus culture

The combined use of elicitors can be an effective way to increase the production of secondary metabolites (SMs) in plant cell, tissue and organ cultures. This study investigated the effects of a salicylic acid (SA) pretreatment and different glucose levels on the growth, biochemical traits and taxane production in a Taxus baccata callus culture. For this purpose, after a pretreatment with SA (5 μM), three-month-old calli were cultured on B5 medium fortified with different concentrations of glucose (0, 0.5, 1, 2 and 3%), and they were compared with calli cultured on a B5 medium supplemented only with glucose. When the calli were harvested at 21 days, their fresh weight (g), dry weight (g) and cell viability (%) had decreased significantly (p < 0.05) with the higher glucose concentrations. The glucose treatment increased the hydrogen peroxide (H2O2) and malondialdehyde (MDA) content, and caused oxidative stress in treated tissues. The lower H2O2 content and oxidative stress was associated with an increased antioxidant enzyme activity in the SA-pretreated samples, which resulted in less membrane damage and improved growth and cell viability under the glucose treatment compared to the control. By reducing the activity of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO), the SA pretreatment reduced the production and oxidation of phenolic compounds under the glucose treatment; this decrease was associated with less browning of tissues and higher viability. Increases in taxol (5.1-fold) and total taxanes (3.5-fold) in the SA-pretreated calli cultured on the medium containing 2% glucose, compared to the control, indicated that the two treatments had a significant effect on taxane production in the T. baccata callus culture.