Comparative study of the antibacterial effects of wound secretions of different cultivars of Chinese fir.

Background: The bark of Chinese fir (Cunninghamia lanceolata), the largest afforestation tree species in the forest areas of southern China, is susceptible to injuries and bites from small animals. The population of small animals has recently increased owing to improvements in the ecological environment across various forested areas, thus increasing the incidence of injuries in the bark of Chinese fir. Following such injuries, the bark secretes light yellow or milky white secretions, the function of which remains unclear. The present study aimed to reveal the antibacterial effect of exudates of different Chinese fir cultivars on five bacterial species. Methods: The research involved three-year-old plantations of Taxus chinensis var. koraiensis and Yangkou3 and three-year-old container plantations of Taxus chinensis var. pendula, Yang 061, and Yang 020. The antibacterial effects of exudates were analyzed using the filter paper diffusion method. The minimum inhibitory concentration for each secretion and the bacterial inhibition zone were determined. Results: The exudates of the different Chinese fir bark exhibited notable antibacterial effects on Bacillus subtilis, Salmonella paratyphi B, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. However, the extent of these antibacterial effects varied among the different Chinese fir cultivars, as the minimum inhibitory concentrations (MICs) of the exudates against the five bacterial species varied. The mean MIC of Pseudomonas aeruginosa was lower potency, whereas that of Escherichia coli was the lowest. Notably, the antibacterial efficacy of the exudates was mainly influenced by the composition of the secretions rather than the number of secretions, with organic acid compounds and terpenoids potentially contributing to the antibacterial effects against E. coli and Bacillus subtilis, respectively. Conclusion: This study demonstrates the antibacterial effect of wound secretion of different Chinese fir cultivars, highlighting their varying efficacy on different bacterial species. Moreover, the antibacterial ability of the exudates of the strains was mainly determined by the composition of the wound secretions, and there was no noticeable relationship with the number of wound secretions. The results of this study offers a theoretical basis for screen Chinese fir cultivars with high-disease-resistant.

Fungal endophytes of Taxus species and regulatory effect of two strains on taxol synthesis.

BACKGROUND: Taxol, derived from Taxus trees, is a valuable natural resource for the development of anticancer drugs. Endophytic fungi from Taxus trees are a promising alternative source of Taxol. However, the impact of plant-endophytic microbial interaction on the host's Taxol biosynthesis is largely unknown. RESULTS: In the current study, the diversity of endophytic fungi in three different Taxus species was analyzed using Internal Transcribed Spacer sequencing. A total of 271 Operational Taxonomic Units (OTUs) were identified, grouping into 2 phyla, 8 classes, 16 orders, 19 families, and 19 genera. Alpha and beta diversity analysis indicated significant differences in endophytic fungal communities among the various Taxus trees. At the genus level, Alternaria and Davidiella were predominantly found in T. mairei and T. media, respectively. By utilizing a previously published dataset, a Pearson correlation analysis was conducted to predict the taxol biosynthesis-related fungal genera. Following screening, two isolates of Alternaria (L7 and M14) were obtained. Effect of inoculation with Alternaria isolates on the gene expression and metabolite accumulation of T. mairei was determined by transcriptomic and untargeted metabolomic studies. The co-inoculation assay suggests that the two Alternaria isolates may have a negative regulatory effect on taxol biosynthesis by influencing hormone signaling pathways. CONCLUSION: Our findings will serve as a foundation for advancing the production and utilization of Taxus and will also aid in screening endophytic fungi related to taxol production.

Taxus chinensis var. mairei (Lemée et Lévl) Cheng et L.K. Fu overcomes the resistance to osimertinib in EGFR-mutant non-small-cell lung cancer via suppression of ERK1/2-related cholesterol biosynthesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Acquired resistance to osimertinib limits its clinical efficacy in non-small cell lung cancer (NSCLC) with EGFR mutations. The widespread recognition of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu (Chinese yew) as a natural anti-cancer medication is well-established. However, the specific contribution of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu in addressing resistance to osimertinib is still uncertain. AIM OF THE STUDY: Based on the biological behaviors and lipid metabolism, we investigated whether aqueous extract of Taxus chinensis var. Mairei (Lemée et Lévl) Cheng et L.K. Fu (AETC) could enhance the antitumor effect of osimertinib in NSCLC with an investigation on the precise mechanisms. MATERIALS AND METHODS: The effect of AETC on enhancing osimertinib sensitivity was assessed via cell viability measurements, levels of reactive oxygen species (ROS), apoptosis, and lipid levels. Western blotting was used to verify the mechanisms of AETC responsible for overcoming the resistance to osimertinib via ERK1/2 overexpression and knockdown models. In vivo validation was conducted using subcutaneous xenografts from osimertinib-resistant cells in nude mice. RESULTS: Osimertinib-resistant cells exhibited altered cholesterol biosynthesis, which was induced by ERK1/2 activation. The combination of AETC and osimertinib can synergistically decrease the levels of ROS in cells, enhance apoptosis, and inhibit the growth of osimertinib-resistant cells. Mechanistic experiments demonstrated that AETC can downregulate the key regulators of cholesterol biosynthesis by regulating ERK1/2, inhibiting the endogenous synthesis rate of cholesterol, and suppressing the level of lipids in osimertinib-resistant cells and xenograft tumors when combined with osimertinib, ultimately reversing resistance to osimertinib. CONCLUSIONS: The resistance to osimertinib is significantly influenced by cholesterol biosynthesis, highlighting its pivotal role in this context. AETC can enhance osimertinib sensitivity via ERK/SREBP-2/HMGCR-mediated cholesterol biosynthesis. These results provide a promising therapeutic target and potential treatment option for resistance to osimertinib.

Identification and characterization of a new species of Taxus - Taxus qinlingensis by multiple taxonomic methods.

BACKGROUND: The taxonomy of Taxus Linn. remains controversial due to its continuous phenotypic variation and unstable topology, thus adversely affecting the formulation of scientific conservation strategies for this genus. Recently, a new ecotype, known as Qinling type, is mainly distributed in the Qinling Mountains and belongs to a monophyletic group. Here, we employed multiple methods including leaf phenotype comparison (leaf shapes and microstructure), DNA barcoding identification (ITS + trnL-trnF + rbcL), and niche analysis to ascertain the taxonomic status of the Qinling type. RESULTS: Multiple comparisons revealed significant differences in the morphological characters (length, width, and length/width ratio) among the Qinling type and other Taxus species. Leaf anatomical analysis indicated that only the Qinling type and T. cuspidata had no papilla under the midvein or tannins in the epicuticle. Phylogenetic analysis of Taxus indicated that the Qinling type belonged to a monophyletic group. Moreover, the Qinling type had formed a relatively independent niche, it was mainly distributed around the Qinling Mountains, Ta-pa Mountains, and Taihang Mountains, situated at an elevation below 1500 m. CONCLUSIONS: Four characters, namely leaf curvature, margin taper, papillation on midvein, and edges were put forward as primary indexes for distinguishing Taxus species. The ecotype Qingling type represented an independent evolutionary lineage and formed a unique ecological niche. Therefore, we suggested that the Qingling type should be treated as a novel species and named it Taxus qinlingensis Y. F. Wen & X. T. Wu, sp. nov.

Research on the Medicinal Chemistry and Pharmacology of Taxus × media.

Taxus × media, belonging to the genus Taxus of the Taxaceae family, is a unique hybrid plant derived from a natural crossbreeding between Taxus cuspidata and Taxus baccata. This distinctive hybrid variety inherits the superior traits of its parental species, exhibiting significant biological and medicinal values. This paper comprehensively analyzes Taxus × media from multiple dimensions, including its cultivation overview, chemical composition, and multifaceted applications in the medical field. In terms of chemical constituents, this study delves into the bioactive components abundant in Taxus × media and their pharmacological activities, highlighting the importance and value of these components, including paclitaxel, as the lead compounds in traditional medicine and modern drug development. Regarding its medicinal value, the article primarily discusses the potential applications of Taxus × media in combating tumors, antibacterial, anti-inflammatory, and antioxidant activities, and treating diabetes. By synthesizing clinical research and experimental data, the paper elucidates the potential and mechanisms of its primary active components in preventing and treating these diseases. In conclusion, Taxus × media demonstrates its unique value in biological research and tremendous potential in drug development.

Altitudinal Impact on Phytochemical Composition and Mycorrhizal Diversity of Taxus Contorta Griff in the Temperate Forest of Shimla District.

Taxus contorta (family Taxaceae) is a native plant of temperate region of western Himalaya. The current study investigated the effect of altitude on the phytochemical composition and mycorrhizal diversity, associated with distribution of T. contorta in Shimla district, Himachal Pradesh, India. Quantitative phytochemical analysis of the leaf extracts indicated that alkaloid levels decreased with altitude, with the highest value in Himri's methanol extracts (72.79 ± 1.08 mg/g) while phenol content increased with altitude, peaking in Nankhari's methanol extracts (118.83 ± 5.90 mg/g). Saponin content was higher in methanol extracts (78.13 ± 1.66 mg/g in Nankhari, 68.06 ± 1.92 mg/g in Pabbas, and 56.32 ± 1.93 mg/g in Himri). Flavonoid levels were notably higher in chloroform extracts, particularly in Nankhari (219.97 ± 2.99 mg/g), and positively correlated with altitude. Terpenoids were higher in chloroform extracts at Himri (11.34 ± 0.10 mg/g) and decreased with altitude. Taxol content showed minimal variation between solvents and altitudes (4.53-6.98 ppm), while rutin was only detected in methanol extracts (1.31-1.46 ppm). Mycorrhizal spore counts in T. contorta's rhizosphere varied with altitude: highest at Himri (77.83 ± 2.20 spores/50 g soil), decreasing to Pabbas (68.06 ± 1.96 spores/50 g soil) and lowest at Nankhari (66.00 ± 2.77 spores/50 g soil), with 17 AMF species identified overall, showing significant altitudinal influence on spore density. The rhizosphere of T. contorta was shown to be dominated by the Glomus species. The rhizospheric soil of the plant was found to be slightly acidic. Organic carbon and available potassium content decreased contrasting with increasing available nitrogen and phosphorus with altitude. Correlation data showed strong negative links between organic carbon (-0.83), moderate positive for nitrogen (0.46) and phosphorus (0.414), and moderate negative for potassium (-0.56) with the altitude. This study provides a comprehensive insight into changes in phytochemical constituents, mycorrhizal diversity and soil composition of T. contorta along a range of altitude.

Dynamic response of allelopathic potency of Taxus cuspidata Sieb. et Zucc. mediated by allelochemicals in Ficus carica Linn. root exudates.

In a mixed forest, certain plants can release allelochemicals that exert allelopathic effects on neighboring plants, thereby facilitating interspecific coexistence of two species. Previous studies have demonstrated that allelochemicals released from Ficus carica Linn. roots in mixed forest of F. carica and Taxus cuspidata Sieb. et Zucc. has phase characteristics over time, which can improve the soil physicochemical properties, enzyme activity and microbial diversity, thus promoting the growth of T. cuspidata. Based on the irrigation of exogenous allelochemicals, changes in soil fertility (soil physical and chemical properties, soil enzyme activity and soil microelement content) were observed in response to variations in allelochemicals during five phases of irrigation: initial disturbance phase (0-2 d), physiological compensation phase (2-8 d), screening phase (8-16 d), restore phase (16-32 d) and maturity phase (32-64 d), which was consistent with the response of soil microorganisms. The allelopathic response of growth physiological indexes of T. cuspidata, however, exhibited a slight lag behind the soil fertility, with distinct phase characteristics becoming evident on the 4th day following irrigation of allelochemicals. The findings demonstrated that the allelochemicals released by the root of F. carica induced a synergistic effect on soil fertility and microorganisms, thereby facilitating the growth of T. cuspidata. This study provides a comprehensive elucidation of the phased dynamic response-based allelopathic mechanism employed by F. carica to enhance the growth of T. cuspidata, thus establishing a theoretical basis for optimizing forest cultivation through allelopathic pathways.

Effects of Different Doses of sUV-B Exposure on Taxane Compounds' Metabolism in Taxus wallichiana var. Mairei.

UV-B is an important environmental factor that differentially affects plant growth and secondary metabolites. The effects of supplemental ultraviolet-B (sUV-B) exposure (T1, 1.40 kJ·m-2·day-1; T2, 2.81 kJ·m-2·day-1; and T3, 5.62 kJ·m-2·day-1) on the growth biomass, physiological characteristics, and secondary metabolites were studied. Our results indicated that leaf thickness was significantly (p < 0.05) reduced under T3 relative to the control (natural light exposure, CK); The contents of 6-BA and IAA were significantly reduced (p < 0.05); and the contents of ABA, 10-deacetylbaccatin III, and baccatin III were significantly (p < 0.05) increased under T1 and T2. The paclitaxel content was the highest (0.036 ± 0.0018 mg·g-1) under T3. The cephalomannine content was significantly increased under T1. Hmgr gene expression was upregulated under T1 and T3. The gene expressions of Bapt and Dbtnbt were significantly (p < 0.05) upregulated under sUV-B exposure, and the gene expressions of CoA, Ts, and Dbat were significantly (p < 0.05) downregulated. A correlation analysis showed that the 6-BA content had a significantly (p < 0.05) positive correlation with Dbat gene expression. The IAA content had a significantly (p < 0.05) positive correlation with the gene expression of Hmgr, CoA, Ts, and Dbtnbt. The ABA content had a significantly (p < 0.05) positive correlation with Bapt gene expression. Dbat gene expression had a significantly (p < 0.05) positive correlation with the 10-deacetylbaccatin content. Hmgr gene expression was positively correlated with the contents of baccatin III and cephalomannine. Bapt gene expression had a significantly (p < 0.01) positive correlation with the paclitaxel content. A factor analysis showed that the accumulation of paclitaxel content was promoted under T2, which was helpful in clarifying the accumulation of taxane compounds after sUV-B exposure.

An Integrated Strategy Based on 10-DAB Extraction and In Situ Whole-Cell Biotransformation of Renewable Taxus Needles to Produce Baccatin III.

Baccatin III is a crucial precursor in the biosynthesis pathway of paclitaxel. Its main sources are extraction from Taxus or chemical synthesis using 10-deacetylbaccatin III (10-DAB) as substrate. However, these preparation approaches exhibit serious limitations, including the low content of baccatin III in Taxus and the complicated steps of chemical synthesis. Heterologous expression of 10-deacetylbaccatin III-10-O-acetyltransferase (TcDBAT) in microbial strains for biotransformation of 10-DAB is a promising alternative strategy for baccatin III production. Here, the promotion effects of glycerol supply and slightly acidic conditions with a low-temperature on the catalysis of recombinant TcDBAT strain were clarified using 10-DAB as substrate. Taxus needles is renewable and the content of 10-DAB is relatively high, it can be used as an effective source of the catalytic substrate 10-DAB. Baccatin III was synthesized by integrating the extraction of 10-DAB from renewable Taxus needles and in situ whole-cell catalysis in this study. 40 g/L needles were converted into 20.66 mg/L baccatin III by optimizing and establishing a whole-cell catalytic bioprocess. The method used in this study can shorten the production process of Taxus extraction for baccatin III synthesis and provide a reliable strategy for the efficient production of baccatin III by recombinant strains and the improvement of resource utilization rate of Taxus needles.

Metabolomics analysis of different diameter classes of Taxus chinensis reveals that the resource allocation is related to carbon and nitrogen metabolism.

Taxus chinensis (Taxus cuspidata Sieb. et Zucc.) is a traditional medicinal plant known for its anticancer substance paclitaxel, and its growth age is also an important factor affecting its medicinal value. However, how age affects the physiological and metabolic characteristics and active substances of T. chinensis is still unclear. In this study, carbon and nitrogen accumulation, contents of active substances and changes in primary metabolites in barks and annual leaves of T. chinensis of different diameter classes were investigated by using diameter classes instead of age. The results showed that leaves and barks of small diameter class (D1) had higher content of non-structural carbohydrates and C, which were effective in enhancing defense capacity, while N content was higher in medium (D2) and large diameter classes (D3). Active substances such as paclitaxel, baccatin III and cephalomannine also accumulated significantly in barks of large diameter classes. Moreover, 21 and 25 differential metabolites were identified in leaves and barks of different diameter classes, respectively. The differential metabolites were enhanced the TCA cycle and amino acid biosynthesis, accumulate metabolites such as organic acids, and promote the synthesis and accumulation of active substances such as paclitaxel in the medium and large diameter classes. These results revealed the carbon and nitrogen allocation mechanism of different diameter classes of T. chinensis, and its relationship with medicinal components, providing a guidance for the harvesting and utilization of wild T. chinensis.

Japanese Yew (Taxus) poisoning of wild ungulates in Utah during the winter of 2022-2023.

Taxus is a genus of coniferous shrubs and trees, commonly known as the yews, in the family Taxaceae. All species of yew contain taxine alkaloids, which are ascribed as the toxic principles. Anecdotally, free ranging ruminants such as antelope, deer, elk, and moose have been regarded as tolerant to yew. Herein several cases of intoxication of deer, elk, and moose by yew from the state of Utah in the winter of 2022-2023 are documented. Ingestion of yew was documented by three means among the poisoned cervids; plant fragments consistent with yew were visually observed in the rumen contents, chemical analysis, and subsequent detection of the taxines from rumen and liver contents, and identification of exact sequence variants identified as Taxus species from DNA metabarcoding. Undoubtedly, the record snowfall in Utah during the winter of 2022-2023 contributed to these poisonings.

[Advances in paclitaxel biosynthesis and transcriptional regulation mechanisms].

Paclitaxel, a rare diterpene extracted from the bark of Chinese yew (Taxus chinensis), is renowned for its anti-cancer activity and serves as a primary drug for treating cancers. Due to the exceptionally low content of paclitaxel in the bark, a semi-synthetic method that depletes Chinese yew resources is used in the production of paclitaxel, which, however, fails to meet the escalating clinical demand. In recent years, researchers have achieved significant progress in heterologous biosynthesis and metabolic engineering for the production of paclitaxel. This article comprehensively reviews the advancements in paclitaxel production, encompassing chemical synthesis, heterologous biosynthesis, and cell engineering. It provides an in-depth introduction to the biosynthetic pathway and transcriptional regulation mechanisms of paclitaxel, aiming to provide a valuable reference for further research on paclitaxel biosynthesis.

Research Progress on Taxus Extraction and Formulation Preparation Technologies.

Taxus, as a globally prevalent evergreen tree, contains a wealth of bioactive components that play a crucial role in the pharmaceutical field. Taxus extracts, defined as a collection of one or more bioactive compounds extracted from the genus Taxus spp., have become a significant focus of modern cancer treatment research. This review article aims to delve into the scientific background of Taxus extracts and their considerable value in pharmaceutical research. It meticulously sifts through and compares various advanced extraction techniques such as supercritical extraction, ultrasound extraction, microwave-assisted extraction, solid-phase extraction, high-pressure pulsed electric field extraction, and enzymatic extraction, assessing each technology's advantages and limitations across dimensions such as extraction efficiency, extraction purity, economic cost, operational time, and environmental impact, with comprehensive analysis results presented in table form. In the area of drug formulation design, this paper systematically discusses the development strategies for solid, liquid, and semi-solid dosage forms based on the unique physicochemical properties of Taxus extracts, their intended medical uses, and specific release characteristics, delving deeply into the selection of excipients and the critical technical issues in the drug preparation process. Moreover, the article looks forward to the potential directions of Taxus extracts in future research and medical applications, emphasizing the urgency and importance of continuously optimizing extraction methods and formulation design to enhance treatment efficacy, reduce production costs, and decrease environmental burdens. It provides a comprehensive set of preparation techniques and formulation optimization schemes for researchers in cancer treatment and other medical fields, promoting the application and development of Taxus extracts in pharmaceutical sciences.

A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis.

Oxetane synthase (TmCYP1), a novel cytochrome P450 enzyme from Taxus×media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2α,5α,7ß,9α,10ß,13α-hexaacetoxytaxa-4(20),11(12)-diene (1) as a substrate led to the production of a major oxetane derivative, 1ß-dehydroxybaccatin IV (1 a), and a minor 4ß,20-epoxide derivative, baccatin I (1 b). However, feeding the substrate decinnamoyltaxinine J (2), a 5-deacetylated derivative of 1, yielded only 5α-deacetylbaccatin I (2 b), a 4ß,20-epoxide. A possible reaction mechanism was proposed on the basis of substrate-feeding, 2H and 18O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation-acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4ß,20-epoxide might be a shunt product. In this process, the C5-O-acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)-epoxy ring formation by TmCYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.

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.

Mycorrhizal symbiosis in Taxus: a review.

Taxus, a genus of conifers known for its medicinal significance, faces various conservation challenges with several species classified under different threat categories by the IUCN. The overharvesting of bark and leaves for the well-known chemotherapy drug paclitaxel has resulted in its population decline. Exploring the mycorrhizal relationship in Taxus is of utmost importance, as mycorrhizal fungi play pivotal roles in nutrition, growth, and ecological resilience. Taxus predominantly associates with arbuscular mycorrhizal fungi (AM), and reports suggest ectomycorrhizal (EM) or dual mycorrhizal associations as well. This review consolidates existing literature on mycorrhizal associations in Taxus species, focusing on structural, physiological, and molecular aspects. AM associations are well-documented in Taxus, influencing plant physiology and propagation. Conversely, EM associations remain relatively understudied, with limited evidence suggesting their occurrence. The review highlights the importance of further research to elucidate dual mycorrhizal associations in Taxus, emphasizing the need for detailed structural and physiological examinations to understand their impact on growth and survival.

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.

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.

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.