Genome-Wide Identification and Expression Pattern Analysis of BAHD Acyltransferase Family in Taxus mairei.

BAHD acyltransferases are involved in catalyzing and regulating the secondary metabolism in plants. Despite this, the members of BAHD family and their functions have not been reported in the Taxus species. In this study, a total of 123 TwBAHD acyltransferases from Taxus wallichiana var. mairei genome were identified and divided into six clades based on phylogenetic analysis, of which Clade VI contained a Taxus-specific branch of 52 members potentially involved in taxol biosynthesis. Most TwBAHDs from the same clade shared similar conserved motifs and gene structures. Besides the typical conserved motifs within the BAHD family, the YPLAGR motif was also conserved in multiple clades of T. mairei. Moreover, only one pair of tandem duplicate genes was found on chromosome 1, with a Ka/Ks ratio < 1, indicating that the function of duplicate genes did not differentiate significantly. RNA-seq analysis revealed different expression patterns of TwBAHDs in MeJA induction and tissue-specific expression experiments. Several TwBAHD genes in the Taxus-specific branch were highly expressed in different tissues of T. mairei, suggesting an important role in the taxol pathway. This study provides comprehensive information for the TwBAHD gene family and sets up a basis for its potential functions.

Structural Characterization and Immunoenhancing Properties of Polysaccharide CPTM-P1 from Taxus media.

Polysaccharides extracted from Taxus media hrough an aqueous method were further refined by removing proteins via the Sevag technique and purified by dialysis. The separation of these polysaccharides was accomplished using a DEAE-cellulose chromatog-raphy column, yielding two distinct fractions, named CPTM-P1 and CPTM-P2. Notably, CPTM-P1 emerged as the primary polysaccharide component within Taxus media. Consequently, a comprehensive analysis focusing exclusively on CPTM-P1 was undertaken. The molecular weight of CPTM-P1 was established through gel permeation chromatography (GPC), and its monosaccharide composition was deciphered using HPLC-MS. The structure was further elucidated through nuclear magnetic resonance (NMR) spectroscopy. The molecular weight of CPTM-P1 was determined to be 968.7 kDa. The monosaccharide composition consisted of galactose (Gal), arabinose (Ara), galacturonic acid (Gal-UA), glucose (Glc), rhamnose (Rha), xylose (Xyl), mannose (Man), fucose (Fuc), glucuronic acid (Glc-UA), and ribose (Rib). The proportional distribution of these components was 30.53%, 22.00%, 5.63%, 11.67%, 11.93%, 1.69%, 8.50%, 1.23%, 5.63%, and 1.17%, respectively. This confirmed CPTM-P1 as an acidic heteropolysaccharide with a glycuronic acid backbone. Moreover, CPTM-P1 showed immunoenhancing properties, effectively augmenting the secretion of nitric oxide and cytokines (TNF-α, IL-1ß, and IL-6). Additionally, it significantly enhances the phagocytic capacity of RAW264.7 cells. These findings underscore the potential application of these polysaccharides in functional foods and pharmaceuticals, providing a solid scientific basis for further exploration and utilization of Taxus media polysaccharides.

Advancements in the Cultivation, Active Components, and Pharmacological Activities of Taxus mairei.

Taxus mairei (Lemée and H.Lév.) S.Y.Hu, indigenous to the southern regions of China, is an evergreen tree belonging to the genus Taxus of the Taxaceae family. Owing to its content of various bioactive compounds, it exhibits multiple pharmacological activities and has been widely applied in clinical medicine. This article comprehensively discusses the current state of cultivation, chemical constituents, applications in the pharmaceutical field, and the challenges faced by T. mairei. The paper begins by detailing the ecological distribution of T. mairei, aiming to provide an in-depth understanding of its origin and cultivation overview. In terms of chemical composition, the article thoroughly summarizes the extracts and monomeric components of T. mairei, unveiling their pharmacological activities and elucidating the mechanisms of action based on the latest scientific research, as well as their potential as lead compounds in new drug development. The article also addresses the challenges in the T. mairei research, such as the difficulties in extracting and synthesizing active components and the need for sustainable utilization strategies. In summary, T. mairei is a rare species important for biodiversity conservation and demonstrates significant research and application potential in drug development and disease treatment.

In Silico Insights into Micro-Mechanism Understanding of Extracts of Taxus Chinensis Fruits Against Alzheimer's Disease.

BACKGROUND: The taxus chinensis fruit (TCF) shows promises in treatment of aging-related diseases such as Alzheimer's disease (AD). However, its related constituents and targets against AD have not been deciphered. OBJECTIVE: This study was to uncover constituents and targets of TCF extracts against AD. METHODS: An integrated approach including ultrasound extractions and constituent identification of TCF by UPLC-QE-MS/MS, target identification of constituents and AD by R data-mining from Pubchem, Drugbank and GEO databases, network construction, molecular docking and the ROC curve analysis was carried out. RESULTS: We identified 250 compounds in TCF extracts, and obtained 3,231 known constituent targets and 5,326 differential expression genes of AD, and 988 intersection genes. Through the network construction and KEGG pathway analysis, 19 chemicals, 31 targets, and 11 biological pathways were obtained as core compounds, targets and pathways of TCF extracts against AD. Among these constituents, luteolin, oleic acid, gallic acid, baicalein, naringenin, lovastatin and rutin had obvious anti-AD effect. Molecular docking results further confirmed above results. The ROC AUC values of about 87% of these core targets of TCF extracts was greater than 0.5 in the two GEO chips of AD, especially 10 targets with ROC AUC values greater than 0.7, such as BCL2, CASP7, NFKBIA, HMOX1, CDK2, LDLR, RELA, and CCL2, which mainly referred to neuron apoptosis, response to oxidative stress and inflammation, fibroblast proliferation, etc.Conclusions:The TCF extracts have diverse active compounds that can act on the diagnostic genes of AD, which deserve further in-depth study.

Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III.

Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.

Overexpression of BAPT and DBTNBT genes in Taxus baccata in 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-ß-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.

Oxetane Ring Formation in Taxol Biosynthesis Is Catalyzed by a Bifunctional Cytochrome P450 Enzyme.

Taxol is a potent drug used in various cancer treatments. Its complex structure has prompted extensive research into its biosynthesis. However, certain critical steps, such as the formation of the oxetane ring, which is essential for its activity, have remained unclear. Previous proposals suggested that oxetane formation follows the acetylation of taxadien-5α-ol. Here, we proposed that the oxetane ring is formed by cytochrome P450-mediated oxidation events that occur prior to C5 acetylation. To test this hypothesis, we analyzed the genomic and transcriptomic information for Taxus species to identify cytochrome P450 candidates and employed two independent systems, yeast (Saccharomyces cerevisiae) and plant (Nicotiana benthamiana), for their characterization. We revealed that a single enzyme, CYP725A4, catalyzes two successive epoxidation events, leading to the formation of the oxetane ring. We further showed that both taxa-4(5)-11(12)-diene (endotaxadiene) and taxa-4(20)-11(12)-diene (exotaxadiene) are precursors to the key intermediate, taxologenic oxetane, indicating the potential existence of multiple routes in the Taxol pathway. Thus, we unveiled a long-elusive step in Taxol biosynthesis.

Parameter Optimization of Ultrasonic-Microwave Synergistic Extraction of Taxanes from Taxus cuspidata Needles.

Taxanes are the best-known compounds in Taxus cuspidata owing to their strong anticancer effects. However, the traditional taxanes extraction method is the solid-liquid extraction method, which is limited by a large energy consumption and low yield. Therefore, it is urgent to find an efficient method for taxanes extraction. The ultrasonic microwave synergistic extraction (UME) method integrates the cavitation effect of ultrasound and the intensifying heat transfer (ionic conduction and dipole rotation of molecules) effect of microwave to accelerate the release of intracellular compounds and is used in active ingredient extractions. This study aimed to evaluate the performance of UME in extracting taxanes from T. cuspidata needles (dichloromethane-ethanol as extractant). A single-factor experiment, Plackett-Burman design, and the response surface method showed that the optimal UME parameters for taxanes extraction were an ultrasonic power of 300 W, a microwave power of 215 W, and 130 sieve meshes. Under these conditions, the taxanes yield was 570.32 µg/g, which increased by 13.41% and 41.63% compared with the ultrasound (US) and microwave (MW) treatments, respectively. The reasons for the differences in the taxanes yield were revealed by comparing the physicochemical properties of T. cuspidata residues after the UME, US, and MW treatments. The cell structures were significantly damaged after the UME treatment, and numerous tiny holes were observed on the surface. The absorption peaks of cellulose, hemicellulose, and lignin increased significantly in intensity, and the lowest peak temperature (307.40 °C), with a melting enthalpy of -5.19 J/g, was found after the UME treatment compared with the US and MW treatments. These results demonstrate that UME is an effective method (570.32 µg/g) to extract taxanes from T. cuspidata needles by destroying cellular structures.

Research Advances in Clinical Applications, Anticancer Mechanism, Total Chemical Synthesis, Semi-Synthesis and Biosynthesis of Paclitaxel.

Paclitaxel, a natural secondary metabolite isolated and purified from the bark of the Taxus tree, is considered one of the most successful natural anticancer drugs due to its low toxicity, high potency and broad-spectrum anticancer activity. Taxus trees are scarce and slow-growing, and with extremely low paclitaxel content, the contradiction between supply and demand in the market is becoming more and more intense. Therefore, researchers have tried to obtain paclitaxel by various methods such as chemical synthesis, artificial culture, microbial fermentation and tissue cell culture to meet the clinical demand for this drug. This paper provides a comprehensive overview of paclitaxel extraction, combination therapy, total synthesis, semi-synthesis and biosynthesis in recent years and provides an outlook, aiming to provide a theoretical basis and reference for further research on the production and application of paclitaxel in the future.

Integrated bioinformatics and network pharmacology to explore the therapeutic target and molecular mechanisms of Taxus chinensis against non-small cell lung cancer.

Taxus chinensis (TC) has tremendous therapeutic potential in alleviating non-small cell lung cancer (NSCLC), but the mechanism of action of TC remains unclear. Integrated bioinformatics and network pharmacology were employed in this study to explore the potential targets and molecular mechanism of TC against NSCLC. Data obtained from public databases were combined with appropriate bioinformatics tools to identify the common targets for TC and NSCLC. Common targets were uploaded to the Metascape database for gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathway analyses. A protein-protein interaction network was established, and topological analysis was performed to obtain hub genes. The expression of the hub genes in NSCLC tissues and their consequent effects on the prognosis of patients with NSCLC were confirmed using the Human Protein Atlas database and appropriate bioinformatics tools. Molecular docking was used to verify the binding affinity between the active ingredients and hub targets. We found 401 common targets that were significantly enriched in the cancer, MAPK signaling, and PI3K/Akt signaling pathways. Proto-oncogene tyrosine-protein kinase Src (SRC), mitogen-activated protein kinase 1, phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1), AKT serine/threonine kinase 1 (AKT1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), and lymphocyte-specific protein tyrosine kinase were identified as the hub genes. Immunohistochemical results confirmed that the expression of SRC, mitogen-activated protein kinase 1, PIK3R1, AKT1, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha was upregulated in the NSCLC tissues, while survival analysis revealed the expression of SRC, AKT1, PIK3R1, and lymphocyte-specific protein tyrosine kinase was closely related to the prognosis of patients with NSCLC. Molecular docking results confirmed all bioactive ingredients present in TC strongly bound to hub targets. We concluded that TC exhibits an anti-NSCLC role through multi-target combination and multi-pathway cooperation.

Computational redesign of taxane-10ß-hydroxylase for de novo biosynthesis of a key paclitaxel intermediate.

Paclitaxel (Taxol®) is the most popular anticancer diterpenoid predominantly present in Taxus. The core skeleton of paclitaxel is highly modified, but researches on the cytochrome P450s involved in post-modification process remain exceedingly limited. Herein, the taxane-10ß-hydroxylase (T10ßH) from Taxus cuspidata, which is the third post-modification enzyme that catalyzes the conversion of taxadiene-5α-yl-acetate (T5OAc) to taxadiene-5α-yl-acetoxy-10ß-ol (T10OH), was investigated in Escherichia coli by combining computation-assisted protein engineering and metabolic engineering. The variant of T10ßH, M3 (I75F/L226K/S345V), exhibited a remarkable 9.5-fold increase in protein expression, accompanied by respective 1.3-fold and 2.1-fold improvements in turnover frequency (TOF) and total turnover number (TTN). Upon integration into the engineered strain, the variant M3 resulted in a substantial enhancement in T10OH production from 0.97 to 2.23 mg/L. Ultimately, the titer of T10OH reached 3.89 mg/L by fed-batch culture in a 5-L bioreactor, representing the highest level reported so far for the microbial de novo synthesis of this key paclitaxel intermediate. This study can serve as a valuable reference for further investigation of other P450s associated with the artificial biosynthesis of paclitaxel and other terpenoids. KEY POINTS: • The T10ßH from T. cuspidata was expressed and engineered in E. coli unprecedentedly. • The expression and activity of T10ßH were improved through protein engineering. • De novo biosynthesis of T10OH was achieved in E. coli with a titer of 3.89 mg/L.

Multiple paternally inherited chloroplast capture events associated with Taxus speciation in the Hengduan Mountains.

Mountainous regions provide a multitude of habitats and opportunities for complex speciation scenarios. Hybridization leading to chloroplast capture, which can be revealed by incongruent phylogenetic trees, is one possible outcome. Four allopatric Taxus lineages (three species and an undescribed lineage) from the Hengduan Mountains, southwest China, exhibit conflicting phylogenetic relationships between nuclear and chloroplast phylogenies. Here, we use multi-omic data at the population level to investigate their historical speciation processes. Population genomic analysis based on ddRAD-seq data revealed limited contemporary inter-specific gene flow involving only populations located close to another species. In a historical context, chloroplast and nuclear data (transcriptome) consistently showed conflicting phylogenetic relationships for T. florinii and the Emei type lineage. ILS and chloroplast recombination were excluded as possible causes, and transcriptome and ddRAD-seq data revealed an absence of the mosaic nuclear genomes that characterize hybrid origin scenarios. Therefore, T. florinii appears to have originated when a lineage of T. florinii captured the T. chinensis plastid type, whereas plastid introgression in the opposite direction generated the Emei Type. All four species have distinct ecological niche based on community investigations and ecological niche analyses. We propose that the origins of both species represent very rare examples of chloroplast capture events despite the paternal cpDNA inheritance of gymnosperms. Specifically, allopatrically and/or ecologically diverged parental species experienced a rare secondary contact, subsequent hybridization and reciprocal chloroplast capture, generating two new lineages, each of which acquired a unique ecological niche. These events might have been triggered by orogenic activities of the Hengduan Mountains and an intensification of the Asian monsoon in the late Miocene, and may represent a scenario more common in these mountains than presently known.

Constraints on the maintenance of wild Taxus cuspidata populations based on regression conservation.

To clarify the key factors constraining the maintenance of wild Taxus cuspidata populations and to develop conservation strategies and technical links for current populations, we investigated the renewal status and distribution patterns of wild T. cuspidata populations in the main distribution areas of China. We analyzed the effects of stand factors and human disturbance on population renewal and maintenance. The results showed that the overall regeneration of wild T. cuspidata populations was poor. The basal diameter and height class structure of renewed individuals showed an unhealthy state. 19% of the area was well regenerated. There were three types of regeneration, including poor regeneration with few adult trees, poor regeneration with many adult trees, and good regeneration with few adult trees. The communities in which T. cuspidata was found could be classified into Abies nephrolepis + Tilia amurensis forest, spinney forest, and Picea jezoensis var. microsperma + A. nephrolepis forest. The renewal number of A. nephrolepis + T. amurensis forest was significantly higher than that of spinney forest. Increased stand density and moderate human disturbance contributed to the regeneration of T. cuspidata. The regenerating T. cuspidata seedlings increased significantly when stand density increased from low to medium. The number of regenerating populations in moderately disturbed habitats was significantly higher than those in lightly disturbed habitats. Human disturbance and habitat were currently critical constraints to maintaining and regenerating wild T. cuspidata populations. The conservation of T. cuspidata should consider current status of population regeneration in each habitat patch to develop corresponding in situ conservation and regression conservation measures and focus on the influence of critical factors such as disturbances and habitat conditions.

Neuroprotective α-pyrones from Nigrospora oryzae, an endophytic fungus residing in Taxus chinensis var. mairei.

Endophytes coevolve with plant hosts and thus are more probable to acquire the character (in favor) of producing undescribed bioactive metabolites. Consequently, the topic has been intensely investigated for over two decades, but endophytic metabolites with neuroprotective effect remain scarce. The study presents the discovery of eight undescribed (named solanapyrones U-Z and prosolanapyrones A and B) and six known pyrones (solanapyrones A-C and E-G) from the culture of Nigrospora oryzae, an endophytic fungus associated with Taxus chinensis var. mairei. The structures and absolute configurations of undescribed pyrones were elucidated by extensive spectroscopic analysis, modified Mosher's method, and induced circular dichroism (ICD) spectrum. Solanapyrones A and B and an undescribed pyrone (solanapyrone U) were demonstrated to be more neuroprotective than clenbuterol in inducing bone marrow mesenchymal stem cells (bMSCs) to secret nerve growth factor (NGF). The work updates the pyrone chemodiversity in nature and extends the biofunction repertoire of solanapyrone-related polyketides.

Insights from multi-omics integration into seed germination of Taxus chinensis var mairei.

The transition from deep dormancy to seed germination is essential for the life cycle of plants, but how this process occurs in the gymnosperm Chinese yew (Taxus chinensis var mairei), the natural source of the anticancer drug paclitaxel, remains unclear. Herein, we analyse the transcriptome, proteome, spatial metabolome, and spatial lipidome of the Chinese yew and present the multi-omics profiles of dormant and germinating seeds. Our results show that abscisic acid and gibberellic acid 12 homoeostasis is closely associated with gene transcription and protein translation, and the balance between these phytohormones thereby determines if seeds remain dormant or germinate. We find that an energy supply of carbohydrates from glycolysis and the TCA cycle feed into the pentose phosphate pathway during seed germination, and energy supplied from lipids are mainly derived from the lipolysis of triacylglycerols. Using mass spectrometry imaging, we demonstrate that the spatial distribution of plant hormones and phospholipids has a remarkable influence on embryo development. We also provide an atlas of the spatial distribution of paclitaxel C in Chinese yew seeds for the first time. The data from this study enable exploration of the germination mechanism of Chinese yew seeds across several omics levels.

Insights into Taxol® biosynthesis by endophytic fungi.

There have been two hundred reports that endophytic fungi produce Taxol®, but its production yield is often rather low. Although considerable efforts have been made to increase Taxol/taxanes production in fungi by manipulating cocultures, mutagenesis, genome shuffles, and gene overexpression, little is known about the molecular signatures of Taxol biosynthesis and its regulation. It is known that some fungi have orthologs of the Taxol biosynthetic pathway, but the overall architecture of this pathway is unknown. A biosynthetic putative gene homology approach, combined with genomics and transcriptomics analysis, revealed that a few genes for metabolite residues may be located on dispensable chromosomes. This review explores a number of crucial topics (i) finding biosynthetic pathway genes using precursors, elicitors, and inhibitors; (ii) orthologs of the Taxol biosynthetic pathway for rate-limiting genes/enzymes; and (iii) genomics and transcriptomics can be used to accurately predict biosynthetic putative genes and regulators. This provides promising targets for future genetic engineering approaches to produce fungal Taxol and precursors. KEY POINTS: • A recent trend in predicting Taxol biosynthetic pathway from endophytic fungi. • Understanding the Taxol biosynthetic pathway and related enzymes in fungi. • The genetic evidence and formation of taxane from endophytic fungi.

miR5298b regulated taxol biosynthesis by acting on TcNPR3, resulting in an alleviation of the strong inhibition of the TcNPR3-TcTGA6 complex in Taxus chinensis.

Taxol, a valuable but rare secondary metabolite of the genus Taxus, is an effective anticancer drug. Understanding the regulation of taxol biosynthesis may provide a means to increase taxol content. The microRNA miR5298b was found to promote the accumulation of taxol and upregulate several taxol biosynthesis genes, including DBAT, TASY, and T5H, as demonstrated by experiments using the overexpression and mimicry of transient leaves. Moreover, miR5298b cleaves the mRNA sequence of TcNPR3, a homolog of the salicylic acid receptor AtNPR3/4. Overexpression and knockdown by RNA interference of TcNPR3 confirmed that it repressed taxol biosynthesis. These results indicate that miR5298b enhances taxol biosynthesis via the cleavage of TcNPR3. Yeast two-hybrid bimolecular fluorescence complementation and pull-down assays revealed that TcTGA6, a TGA transcription factor, physically interacted with TcNPR3. Functional experiments showed that TcTGA6 negatively regulates taxol biosynthesis by directly combining with the TGACG motif in the promoters of TASY, T5H, and T10H. TcNPR3 enhances TcTGA6 inhibition Luciferase assays showed that miR5298b alleviated the repression of the TcNPR3-TcTGA6 complex. In summary, miR5298b can cleave TcNPR3, thereby alleviating the inhibition of the TcNPR3-TcTGA6 complex to upregulate taxol biosynthesis genes.

Effect of Methyl Jasmonate on the Growth and Biosynthesis of C13- and C14-Hydroxylated Taxoids in the Cell Culture of Yew (Taxus wallichiana Zucc.) of Different Ages.

The effects of methyl jasmonate (MeJ) on growth and taxoid formation in the cell culture of Taxus wallichiana were investigated to elucidate the specifics of phytohormone action in dedifferentiated plant cells in vitro. The characteristics of the same suspension cell culture were compared in 2017 (the «young¼ culture) and in 2022 (the «old¼ culture)-1.5 or 6 years after culture induction, respectively. MeJ (100 µM) is added to the cell suspension at the end of the exponential growth phase. Cell culture demonstrated good growth (dry weight accumulation 10-18 g/L, specific growth rate µ = 0.15-0.35 day-1) regardless of its «age¼, cultivation system, and MeJ addition. UPLC-ESI-MS analysis revealed the presence of C14-hydroxylated taxoids (yunnanxane, taxuyunnanine C, sinenxane C, and sinenxane B) in the cell biomass. The content of C14-OH taxoids increased from 0.2-1.6 mg/gDW in «young¼ culture to 0.6-10.1 mg/gDW in «old¼ culture. Yunnanxane was the main compound in «young¼ culture, while sinenxane C predominated in «old¼ culture. Without elicitation, small amounts of C13-OH taxoids (<0.05 mg/gDW) were found only in «young¼ cultures. MeJ addition to «young¼ culture had no effect on the content of C14-OH taxoids but caused a 10-fold increase in C13-OH taxoid production (up to 0.12-0.19 mg/gDW, comparable to the bark of yew trees). By contrast, MeJ added to «old¼ culture was not beneficial for the production of C13-OH taxoids but notably increased the content of C14-OH taxoids (1.5-2.0 times in flasks and 5-8 times in bioreactors). These findings suggest that hormonal signaling in dedifferentiated yew cells grown in vitro is different from that in plants and can be affected by the culture's age. This might be a result of the high level of culture heterogeneity and constant auto-selection for intensive proliferation, which leads to the predominant formation of C14-OH taxoids versus C13-OH taxoids and a modified cell response to exogenous MeJ treatment.