Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells

Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC–MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.     

Taxus associated fungal endophytes: anticancerous to other biological activities

Endophytes are the plant microorganisms that flourish inside plants (endosphere) and support the growth under normal and challenging environmental circumstances. Besides biological activities, microbial endophytes are important for their contribution in adaptation and survival of the host plants under stressed environments. While various species of Taxus have been globally recognized mainly due to their anticancer property, emphasis has also been on isolation of taxol producing endophytes in view of the taxonomic (endangered) status of the species. Furthermore, Taxus associated endophytes are also being recognized for the biological activities other than taxol production. The present study reviews the diversity of Taxus associated endophytes along with their biological activities. Available literature on Taxus associated endophytes revealed that T. chinensis has received maximum attention (19.5 %), followed by T. wallichiana (18.5 %), T. baccata (16.4 %), T. cuspidata (11.3 %), and T. mairei (10.3 %); with a minimum number of reports accounted for T. globose (1.0 %), and T. celebica (2.0 %). Most fungal endophytes have been identified as ascomycetes. Basidiomycetous endophytes have been reported in a limited number of studies. No endophytes have yet been identified from three species, namely T. controta (West Himalayan yew), T. floridana (Florida yew), and T. canadensis (Canadian yew). A total of 70 diverse endophytic species have been reported from different Taxus species. Fusarium sp. (25.2 %), Aspergillus sp. (17.6 %), Alternaria sp. (11.3 %), Penicillium sp. and Cladosporium sp. (10.0 %) are the five dominant genera. Furthermore, 59.6 % of studies from the reported literature are related to taxol production by fungal endophytes, 9.6 % related to antimicrobial activity, and a combined 15.3 % related to other biological activities, i.e., antifeeding, antimitogenic, antioxidant and cytotoxic activities. Additionally, 3.8 % of fungal endophytes were not studied for any of their biological activities. Only three bacterial endophytes namely Burkholderia sp., Enterobacter sp., and Pseudomonas sp., have been recorded from Taxus along with their contribution in plant growth, antagonistic, and antioxidant activities. Isolation and characterization of microbial endophytes from Taxus plant parts is important for the associated ecology and medicinally important biological activities. It will support the much-required conservation strategy of Taxus species. Further, isolation of plant growth promoting microorganisms from Taxus will help in propagation of the species.     

南方红豆杉枝叶中6种紫杉烷类化合物含量季节变化

紫杉醇(taxol)是主要来源于红豆杉属植物的一种天然抗肿瘤药物,紫杉烷(taxane)是紫杉醇的代谢前体或支路代谢产物,同样具有开发成为抗肿瘤新药的潜质。本文采用高效液相色谱法研究了紫杉醇(Taxol)、10-去乙酰巴卡亭Ⅲ(10-DAB)、7-木-10-去乙酰紫杉醇(7-xyl-10-DAT)、10-去乙酰紫杉醇(10-DAT)、三尖杉宁碱(CE)和7-表-10-去乙酰紫杉醇(7-epi-10-DAT)6种紫杉烷类化合物在南方红豆杉枝叶中含量的季节变化,结果显示Taxol和10-DAT在8、9月份含量最低,10-DAB和7-xyl-10-DAT在8、9月份含量相对最高,Taxol含量季节变化和10-DAB呈负相关,与CE呈显著正相关。7-xyl-10-DAT含量季节变化和10-DAB、10-DAT分别呈正相关。本文为研究南方红豆杉中紫杉醇及相关紫杉烷的代谢、积累规律提供了依据,不但有助于阐明紫杉醇的生物合成的关键步骤及调控的生理机制,而且对红豆杉资源的深度开发具有指导意义。     

Effect of the culture medium and biotic stimulation on taxane production in Taxus globosa Schltdl in vitro cultures

Taxus globosa is the only species of the Taxus genus that grows in Mexico. In this study, callus cultures from leaves and young shoots of T. globosa were established in Gamborg’s B5 medium supplemented with 2,4-dichlorophenoxiacetic acid (2 mg/L), kinetin (0.5 mg/L) and gibberellic acid (0.25 mg/L). Callus growth and taxane production were evaluated using two culture media: Woody Plant Medium and Gamborg’s B5 supplemented with picloram (2 mg/L), kinetin (0.1 mg/L) and gibberellic acid (0.5 mg/L). The effect of the inoculum size (50, 100 and 150 g FW/L) and culture media (Woody Plant Medium and Gamborg’s B5) with and without the presence of methyl jasmonate (100 μM) on T. globosa cell suspensions was assessed. Taxane analysis revealed that the calli in Gamborg’s B5 produced taxol (50 μg/g DW), baccatin III, 10-deacetyl baccatin III and 10-deacetyl taxol. Woody Plant Medium also induced the production of taxol, although to a lesser extent. The optimum inoculum size was 50 g FW/L. In cell suspension cultures, both media had a significant effect on taxane production when supplemented with methyl jasmonate. In Woody Plant Medium, at day 14, a total concentration of 197.999 μg/L of taxol, 160.622 μg/L of baccatin III, 633.724 μg/L of 10-deacetyl baccatin III and 229.611 μg/L 10-deacetyl taxol were obtained, with total excretion of baccatin III and 10-deacetyl taxol to the culture medium. In Gamborg’s B5, cephalomanine was obtained at a concentration of 91.428 μg/L without elicitation, and all taxanes were excreted to the medium to a variable extent.     

Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

Expression of recombinant alpha and beta tubulins from the yew Taxus cuspidata and analysis of the microtubule assembly in the presence of taxol

Taxol was originally isolated from the yew Taxus brevifolia. Because taxol inhibits the depolymerization of microtubules, the presence of a self-resistance mechanism in Taxus spp. was hypothesized. The cloning of the cDNA for alpha and beta tubulins from Taxus cuspidata and those from the human embryonic kidney cell line HEK293T revealed that the ²⁶Asp, ³⁵⁹Arg, and ³⁶¹Leu residues in the human beta tubulin, which are important for taxol binding, were replaced with Glu, Trp, and Met in the beta tubulin of T. cuspidata, respectively. The microtubule assembly of the recombinant alpha and beta tubulins was monitored turbidimetrically, and the results clearly demonstrated that the microtubule from T. cuspidata is less sensitive to taxol than that from HEK293T cells. The Taxus microtubule composed of the wild-type alpha tubulin and the beta tubulin with the E26D mutation restored the sensitivity to taxol. We thus postulated that the mutation identified in the beta tubulin of T. cuspidata plays a role in the self-resistance of this species against taxol.     

Source of isopentenyl diphosphate for taxol and baccatin III biosynthesis in cell cultures of Taxus baccata

To achieve a better understanding of the metabolism and accumulation of taxol and baccatin III in cell cultures of Taxus, three cell lines (I, II and III) of T. baccata were treated (on day 7) with several concentrations of fosmidomycin (100, 200 and 300 μM), an inhibitor of the non-mevalonate branch of the terpenoid pathway, or mevinolin (1, 3 and 5 μM), an inhibitor of the mevalonate branch, in both cases in presence and absence of 100 μM methyl jasmonate (MeJ). They were compared with lines treated only with the elicitor MeJ as well as an untreated control with respect to growth, viability and production of taxol and baccatin III. The results show that the cell line type was an important variable, mainly for taxane accumulation. The blocking effect of fosmidomycin on taxane production was significantly greater than that of mevinolin in all the cell lines, clearly suggesting that the isopentenyl diphosphate (IPP) used for the taxane ring formation was mainly formed via the non-mevalonate pathway. However, the significant reduction in the content of taxol (on average 3.8-fold) and baccatin III (on average 4.3-fold) in line I when treated with the elicitor together with mevinolin concentrations of 5 and 1 μM, respectively, also suggests that both non-mevalonate and mevalonate pathways are involved in the biosynthesis of the two taxanes as a result of cytosolic IPP and/or other prenyl diphosphate transport to the plastids. The observation that the inhibitory effect of fosmidomycin or mevilonin on taxol and baccatinn III yield does not interfere with methyl jasmonate elicitation is discussed.     

Baseline Survey of Root-Associated Microbes of Taxus chinensis (Pilger) Rehd

Taxol (paclitaxel) a diterpenoid is one of the most effective anticancer drugs identified. Biosynthesis of taxol was considered restricted to the Taxus genera until Stierle et al. discovered that an endophytic fungus isolated from Taxus brevifolia could independently synthesize taxol. Little is known about the mechanism of taxol biosynthesis in microbes, but it has been speculated that its biosynthesis may differ from plants. The microbiome from the roots of Taxus chinensis have been extensively investigated with culture-dependent methods to identify taxol synthesizing microbes, but not using culture independent methods.,Using bar-coded high-throughput sequencing in combination with a metagenomics approach, we surveyed the microbial diversity and gene composition of the root-associated microbiomefrom Taxus chinensis (Pilger) Rehd. High-throughput amplicon sequencing revealed 187 fungal OTUs which is higher than any previously reported fungal number identified with the culture-dependent method, suggesting that T. chinensis roots harbor novel and diverse fungi. Some operational taxonomic units (OTU) identified were identical to reported microbe strains possessing the ability to synthesis taxol and several genes previously associated with taxol biosynthesis were identified through metagenomics analysis.     

In vitro culture of <Emphasis Type="Italic">Taxus</Emphasis> sp.: strategies to increase cell growth and taxoid production

Interest in Taxus species has increased since paclitaxel, an anticancer drug, was isolated from the bark of Taxus brevifolia (Pacific yew) in the 1960’s. Great effort has been carried out to establish an efficient callus cultures of Taxus species. Culture media must be optimized for each Taxus species, and in general, there is no one method that guarantees success for Taxus cultures. The source of explant, culture medium composition and the growth regulators used appear to affect callus initiation and maintenance. Research effort has focused on obtaining a cell culture that exhibits good growth and a high rate of taxoid accumulation. In this sense, many strategies have been employed to stimulate taxoid production without affecting cell growth. In an attempt to scale-up cell culture, problems such us shear stress, oxygen supply and gas composition have been studied. A more detailed knowledge of the pathway and the fluxes of intermediates towards taxane accumulation will be key factors to obtain cell lines with increased taxane accumulation through metabolic engineering.     

Taxoids profiling of suspension Taxus chinensis var. mairei cells in response to shear stress

To develop an optimal bioprocess for paclitaxel (Taxols) supply, taxoid biosynthetic pathway regulation must be better understood. The main taxoid metabolites (paclitaxel, baccatin III, taxol C, etc.) in Taxus cell culture showed great difference under shear stress. However, the regulating mechanism of taxoids metabolism under shear stress remained elusive. Here an efficient metabolic profiling approach combined with multivariate analysis was employed to profile taxoids changes of Taxus cells under laminar shear stress. A total of 21 taxoids were identified and quantified by ultra-performance liquid chromatography coupled with Q-TOF mass spectrometry. The result showed the contents of paclitaxel and baccatin III were reduced by shear stress, indicating the inhibitory effect of shear stress on paclitaxel biosynthesis. The levels of other taxoids uninvolved in paclitaxel biosynthesis were decreased except several metabolites. Further analysis of mapping measured taxoids concentrations onto paclitaxel biosynthesis pathway illustrating proposed intermediates and “off-pathway” metabolites revealed shear stress might disrupt the appropriate cyclization process of geranylgeranyl-pyrophosphate, aggravate the inappropriate order of hydroxylations and acylations, and not be good for functional group oxetane formation. These findings revealed the possible mechanism for shear stress limiting paclitaxel production and might have important biotechnological applications to increase the yields of paclitaxel and relevant precursors.     

Development of the male reproductive structures in Taxus yunnanensis

Taxus yunnanensis Cheng et L. K. Fu is a rare species endemic to Southeast Asia. The development of the male reproductive structures with emphasis on microsporogenesis and microgametogenesis are described for the first time. Our results showed that T. yunnanensis exhibits several features that are very different from other species of Taxus, such as pollination occurring in the same year as pollen cone initiation; production of several forms of tetrads including isobilateral, T-shaped or tetrahedron; shorter duration of microsporogenesis; longer duration of microgametogenesis; and production of more microsporophylls per pollen cone suggesting faster growth rate. Reports on the details of male reproductive development in Taxus spp. have focused on the activities of generative nuclei, while the activities of the sterile and tube nuclei are rarely mentioned. This paper provides a few more details about the sterile and tube nuclei, particularly, their relative position during the division of the generative nucleus to form two sperm. Furthermore, as our knowledge of the phenology of Taxus is confined to species that grow on high latitude areas, this paper provides information on the phenology of Taxus in low latitude areas. Overall, this paper provides several new insights about pollen cone development, microsporogenesis, and microgametogenesis in Taxus.     

红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展

紫杉醇是一种四环二萜酰胺类化合物,是从红豆杉科红豆杉属植物中提取分离出来的次生代谢物,是世界公认广谱、活性强的天然抗癌新药。但直接从植物中提取紫杉醇的传统生产方式,不仅产量低,且会对野生红豆杉资源造成严重破坏,同时紫杉醇的化学全合成也由于其结构复杂而不具备商业价值。与之相反,细胞培养技术具有受外界影响少、生产成本低、次生代谢产物多、细胞生长周期短的优势,是目前最具前景的紫杉醇生产方式。近年来随着科研水平的不断提升,紫杉醇无论在生理代谢调控、关键基因挖掘,还是新药物制剂与剂型及其类似物的开发和运用等方面,都取得了进展,但要建立紫杉醇商业化高产体系,还必须和前人的研究经验相结合。该文对红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展进行了综述,主要包括前人对红豆杉属植物组织与细胞培养相关的外植体、培养基、激素、培养条件、褐化等问题的研究,以及从代谢调节、培养方式、基因工程等多方面提高紫杉醇含量的最新进展,最后总结了当前研究的不足,并对今后通过多种组合方式来提高紫杉醇含量的生产途径进行了展望。以期促进红豆杉组织培养技术的进步,为药用资源保护和利用提供一定的理论基础与生产指导。     

Taxol production in suspension cultures of Taxus baccata

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol^® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1^–1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1^–1) and low (<0.1 mg 1^–1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1^–1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1^–1.®|Taxol is a registered trademark of Bristol Meyer Squibb for paclitaxel     

Involvement of nitric oxide in cerebroside-induced defense responses and taxol production in <Emphasis Type="Italic">Taxus yunnanensis</Emphasis> suspension cells

This work was to characterize the generation of nitric oxide (NO) in Taxus yunnanensis cells induced by a fungal-derived cerebroside and the signal role of NO in the elicitation of plant defense responses and taxol production. (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine at 10 μg/ml induced a rapid and dose-dependent NO production in the Taxus cell culture, reaching a maximum within 5 h of the treatment. The NO donor sodium nitroprusside (SNP) potentiated cerebroside-induced H₂O₂ production and cell death. Inhibition of nitric oxide synthase activity by phenylene-1,3-bis(ethane-2-isothiourea) dihydrobromide or scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide partially blocked the cerebroside-induced H₂O₂ production and cell death. Moreover, NO enhanced cerebroside-induced activation of phenylalanine ammonium-lyase and accumulation of taxol in cell cultures. These results are suggestive of a role for NO as a new signal component for activating the cerebroside-induced defense responses and secondary metabolism activities of plant cells. Taxol is a trademark of Bristol-Myers Squibb, Madison, NJ.     

Interactions of Taxol-producing endophytic fungus with its host (Taxus spp.) during Taxol accumulation

Endophytic fungi (Fusarium mairei) culture broth (EFCB) was added to cell suspension cultures of Taxus cuspidata. After 5 days, cultures of T. cuspidata given 4 ml of EFCB produced a maximal yield of 6.11 mg/l paclitaxel, with a release ratio of 75%, 2- and 6.8-fold, respectively, greater than the controls. The active element in EFCB is an exopolysaccharide of ∼79 kD. Endophytic fungi produced 0.19 mg/l of paclitaxel in its producing medium. However, when the supernatant of Taxus cell suspension cultures from day 20 was added to the paclitaxel-producing medium, the biomass of fungi decreased by 24% and the yield of paclitaxel by 45%. In a co-culture system of plant and fungus, the yield of paclitaxel (12.8 mg/l) was >2-fold higher than that in the EFCB-treatment system.     

Viability characterization of Taxus chinensis plant cell suspension cultures by rapid colorimetric- and image analysis-based techniques

For the commercially established process of paclitaxel production with Taxus chinensis plant cell culture, the size of plant cell aggregates and phenotypic changes in coloration during cultivation have long been acknowledged as intangible parameters. So far, the variability of aggregates and coloration of cells are challenging parameters for any viability assay. The aim of this study was to investigate simple and non-toxic methods for viability determination of Taxus cultures in order to provide a practicable, rapid, robust and reproducible way to sample large amounts of material. A further goal was to examine whether Taxus aggregate cell coloration is related to general cell viability and might be exploited by microscopy and image analysis to gain easy access to general cell viability. The Alamar Blue assay was found to be exceptionally eligible for viability estimation. Moreover, aggregate coloration, as a morphologic attribute, was quantified by image analysis and found to be a good and traceable indicator of T. chinensis viability.     

Sustainable use of Taxus media cell cultures through minimal growth conservation and manipulation of genome methylation

Yields of paclitaxel decreased with repeated subculturing of Taxus media cells. We used minimal growth conservation and manipulation of genome methylation to sustain paclitaxel production by Taxus media cell cultures. The subculture period of Taxus cells can be prolonged to 180 d by incubating them at a low temperature (5 °C). Paclitaxel levels increased in the cells after conservation and during the first recovery subculture cycle, and then decreased during the subsequent recovery subculture cycle. Analysis of genetic variations in these cultures using amplified fragment-length polymorphism (AFLP) technology identified only two polymorphic bands associated with the second and sixth recovery cycle cultures. However, the results of high-performance liquid chromatography indicated that DNA methylation increased during the course of repeated subculturing. A decrease in DNA methylation level caused by treatment with 5-Aza-2′-deoxycytidine coincided with an increase in paclitaxel levels. Simultaneous exposure to both methyl jasmonate and 5-Aza-2′-deoxycytidine increased paclitaxel levels to 320.43 μg g^?1 (dry weight), which is more than six times the paclitaxel content before conservation. To our knowledge, this is the first report about improving paclitaxel production by ensuring sustainable use of Taxus cells.     

Yew (Taxus x media Rehd.) cell suspension cultures as a source of taxanes

Natural resources of paclitaxel, an effective anticancer compound, were threatened with extinction soon after the discovery of this valuable substance. Cell suspension cultures derived from different Taxus species have rapidly become an alternative source of paclitaxel and other taxanes. In this paper we provide some insight into cell growth characteristics in cell suspension culture of Taxus x media cv. Hicksii, with emphasis on the effects of jasmonic acid (JA) on taxane production in cell lines with different initial taxane content. Additionally cell growth characteristics of two cell lines was followed during cultivation of cell suspension culture of Taxus x media cv. Hicksii. Packed cell volume (PCV) was shown to be a reliable and efficient alternative for measuring cell growth instead of fresh and dry weight. The initial total taxane content was screened in a number of cell lines, followed by observing the effect of JA on cell mass and total taxane production of selected lines. We showed a great variability in initial taxane content in different cell lines, which decreased during cell suspension maintenance. JA was shown to inhibit cell growth and increase total taxane production (14 to 106 fold).