RESUMO
Taxol, which is a widely used important chemotherapeutic agent, was originally isolated from Taxus stem barks. However, little is known about the precise distribution of taxoids and the transcriptional regulation of taxoid biosynthesis across Taxus stems. Here, we used MALDI-IMS analysis to visualize the taxoid distribution across Taxus mairei stems and single-cell RNA sequencing to generate expression profiles. A single-cell T. mairei stem atlas was created, providing a spatial distribution pattern of Taxus stem cells. Cells were reordered using a main developmental pseudotime trajectory which provided temporal distribution patterns in Taxus stem cells. Most known taxol biosynthesis-related genes were primarily expressed in epidermal, endodermal, and xylem parenchyma cells, which caused an uneven taxoid distribution across T. mairei stems. We developed a single-cell strategy to screen novel transcription factors (TFs) involved in taxol biosynthesis regulation. Several TF genes, such as endodermal cell-specific MYB47 and xylem parenchyma cell-specific NAC2 and bHLH68, were implicated as potential regulators of taxol biosynthesis. Furthermore, an ATP-binding cassette family transporter gene, ABCG2, was proposed as a potential taxoid transporter candidate. In summary, we generated a single-cell Taxus stem metabolic atlas and identified molecular mechanisms underpinning the cell-specific transcriptional regulation of the taxol biosynthesis pathway.
Assuntos
Taxoides , Taxus , Taxoides/metabolismo , Transcriptoma , Taxus/genética , Taxus/metabolismo , Paclitaxel , Espectrometria de MassasRESUMO
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.
Assuntos
Sistema Enzimático do Citocromo P-450 , Taxus , Sistema Enzimático do Citocromo P-450/metabolismo , Paclitaxel/metabolismo , Éteres Cíclicos , Catálise , Taxus/genética , Taxus/metabolismoRESUMO
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.
Assuntos
Código de Barras de DNA Taxonômico , Filogenia , Folhas de Planta , Taxus , Taxus/genética , Taxus/anatomia & histologia , Taxus/classificação , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , China , DNA de Plantas/genética , FenótipoRESUMO
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.
Assuntos
Carbono , Metabolômica , Nitrogênio , Folhas de Planta , Taxus , Taxus/metabolismo , Nitrogênio/metabolismo , Carbono/metabolismo , Folhas de Planta/metabolismo , Casca de Planta/metabolismo , Casca de Planta/químicaRESUMO
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.
Assuntos
Paclitaxel , Taxus , Paclitaxel/metabolismo , Taxus/genética , Taxus/metabolismo , Células Cultivadas , Taxoides/farmacologia , Taxoides/metabolismoRESUMO
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.
Assuntos
Alternaria , Endófitos , Paclitaxel , Taxus , Taxus/microbiologia , Paclitaxel/biossíntese , Endófitos/genética , Endófitos/metabolismo , Endófitos/isolamento & purificação , Endófitos/classificação , Alternaria/genética , Alternaria/metabolismo , Alternaria/classificação , Alternaria/isolamento & purificação , Filogenia , Fungos/genética , Fungos/metabolismo , Fungos/classificação , Fungos/isolamento & purificação , DNA Fúngico/genética , DNA Espaçador Ribossômico/genéticaRESUMO
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.
Assuntos
Micorrizas , Simbiose , Taxus , Micorrizas/fisiologia , Taxus/microbiologia , Raízes de Plantas/microbiologiaRESUMO
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.
Assuntos
Altitude , Florestas , Micorrizas , Compostos Fitoquímicos , Folhas de Planta , Taxus , Taxus/microbiologia , Taxus/química , Micorrizas/química , Micorrizas/classificação , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/química , Índia , Folhas de Planta/química , Folhas de Planta/microbiologia , Biodiversidade , Microbiologia do Solo , Alcaloides/análise , Alcaloides/química , Flavonoides/análise , Terpenos/análise , Extratos Vegetais/química , Raízes de Plantas/microbiologia , Raízes de Plantas/química , Saponinas/análise , Saponinas/químicaRESUMO
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.
Assuntos
Taxus , Humanos , Filogenia , Taxus/genética , Aciltransferases , Cromossomos Humanos Par 1 , PaclitaxelRESUMO
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.
Assuntos
Taxus , Taxus/química , Humanos , Química Farmacêutica/métodos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Animais , Antioxidantes/farmacologia , Antioxidantes/químicaRESUMO
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.
Assuntos
Regulação da Expressão Gênica de Plantas , Taxoides , Taxus , Raios Ultravioleta , Taxus/metabolismo , Taxus/genética , Taxoides/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Paclitaxel , Folhas de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Ácido Abscísico/metabolismo , AlcaloidesRESUMO
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.
Assuntos
Taxaceae , Taxus , Taxus/química , ChinaRESUMO
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.
Assuntos
Extratos Vegetais , Taxus , Taxus/química , Extratos Vegetais/química , Humanos , Composição de Medicamentos/métodos , Extração em Fase Sólida/métodosRESUMO
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.
Assuntos
Biotransformação , Taxoides , Taxus , Taxus/metabolismo , Taxus/química , Taxoides/metabolismo , Alcaloides/biossíntese , Alcaloides/metabolismo , Alcaloides/química , Folhas de Planta/metabolismo , Folhas de Planta/química , Acetiltransferases/metabolismo , Acetiltransferases/genéticaRESUMO
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.
Assuntos
Taxus , Humanos , Diálise Renal , Polissacarídeos/farmacologia , Polissacarídeos/química , Monossacarídeos/análise , Citocinas , GlucoseRESUMO
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.
Assuntos
Sistema Enzimático do Citocromo P-450 , Éteres Cíclicos , Paclitaxel , Sistema Enzimático do Citocromo P-450/metabolismo , Paclitaxel/biossíntese , Paclitaxel/química , Paclitaxel/metabolismo , Éteres Cíclicos/química , Éteres Cíclicos/metabolismo , Taxus/enzimologia , Taxus/metabolismo , Biocatálise , Nicotiana/metabolismo , Nicotiana/enzimologia , Estrutura MolecularRESUMO
BACKGROUND: Taxaceae, is a class of dioecious and evergreen plant with substantial economic and ecology value. At present many phytochemical analyses have been performed in Taxus plants. And various biological constituents have been isolated from various Taxus species. However, the difference of compounds and antioxidant capacity of different tissues of T. media is not clear. RESULTS: In the present study, we investigated the metabolites and antioxidant activity of four tissues of T. media, including T. media bark (TB), T. media fresh leaves (TFL), T. media seeds (TS), T. media aril (TA). In total, 808 compounds, covering 11 subclasses, were identified by using UPLC-MS/MS. Paclitaxel, the most popular anticancer compound, was found to accumulate most in TS, followed by TB, TFL and TA in order. Further analysis found that 70 key differential metabolites with VIP > 1.0 and p < 0.05, covering 8 subclasses, were screened as the key differential metabolites in four tissues. The characteristic compounds of TFL mainly included flavonoids and tanninsis. Alkaloids and phenolic acids were major characteristic compounds of TS and TB respectively. Amino acids and derivatives, organic acids, saccharides and lipids were the major characteristic compounds of TA. Additionally, based on FRAP and ABTS method, TS and TFL exhibited higher antioxidant activity than TB and TA. CONCLUSION: There was significant difference in metabolite content among different tissues of T. media. TFL and TS had higher metabolites and antioxidant capacity than other tissues, indicating that TFL and TS were more suitable for the development and utilization of T. media in foods and drinks.
Assuntos
Antioxidantes , Taxus , Antioxidantes/metabolismo , Taxus/metabolismo , Extratos Vegetais/análise , Cromatografia Líquida , Espectrometria de Massas em Tandem , Metabolômica/métodos , Flavonoides/metabolismoRESUMO
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.
Assuntos
Taxus , Filogenia , Taxus/genética , Herança Paterna , China , Cloroplastos/genéticaRESUMO
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.
Assuntos
Paclitaxel , Taxus , Fungos/genética , Fungos/metabolismo , Taxus/microbiologiaRESUMO
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.