Kinetics of two-liquid-phase Taxus cuspidata cell culture for production of Taxol

The effects of different organic solvents (paraffin, organic acid, alcohol and ester) and their volumetric fractions on the cell growth and Taxol production were studied in two-liquid-phase and the two-stage culture. A kinetic model, incorporated the effects of the toxicity of organic solvents was developed for two-liquid-phase culture of Taxus cuspidata in the two-stage Taxol production. The results showed that the proposed kinetic model could fit the experimental data satisfactorily. The results also showed that Taxol production could reach the optimal value when 10-logP was in the range of 2 to 5 and the volumetric fraction of the organic solvents at the corresponding the highest Taxol production should be lower when 10-logP was high.     

Catechin production in cultured cells of Taxus cuspidata and Taxus baccata

The main polyphenols in callus and cell suspension cultures of Taxus cuspidata and T. baccata were (+)-catechin and (−)-epicatechin, while lignans, such as (+)-taxiresinol, (+)-isotaxiresinol, (+)-isolariciresinol and (−)-secoisolariciresinol, were present in trace amounts. T. cuspidata cells contained 1.7% (+)-catechin and 2.4% (−)-epicatechin on dry wt basis but when stimulated with methyl jasmonate produced 3.4% catechin and 5.2% epicatechin. These are the highest levels of these metabolites obtained in plant cell cultures.     

Supercritical fluid extraction of taxol and baccatin III from needles of Taxus cuspidata

Summary Taxol and baccatin III were extracted from the ground needles of Taxus cuspidata using supercritical carbon dioxide mixed with 3 wt % ethanol as a cosolvent. The pressure and temperature ranges used to attain supercritical fluid condition are 100300 bar and 4070 °C, respectively. However, the amount of taxol and baccatin III in the extract obtained at 100 bar was not noticeable, while the major portion of extract was found to be the waxy compounds. The highest selectivity of taxol and baccatin III were about 0.094 and 0.158 wt %, respectively, at 40 °C and 300 bar. At the same pressure and temperature condition, taxol and baccatin III selectivities in the extract obtained from the ground seeds of Taxus cuspidata was about 0.198 and 0.157 wt %, respectively.     

红豆杉资源与紫杉醇生产概况

红豆杉属（ＴａｘｕｓＬ．）植物全世界共１１种,分布于北半球的温带至亚热带地区。我国有４种１变种,即：东北红豆杉（Ｔ．ｃｕｓｐｉｄａｔａＳｉｅｂ．ｅｔＺｕｃ．）、云南红豆杉（Ｔ．ｙｕｎｎａｎｅｎｓｉｓＣｈｅｎｇｅｔＬ．Ｋ．Ｆｕ）、西藏红豆杉（Ｔ．ｗａｌｉｃｈｉａｎａＺｕｃｃ．）、中国红豆杉〔Ｔ．ｃｈｉｎｅｎｓｉｓ（Ｐｉｌｇｅｒ）Ｒｅｈｄ．〕和南方红豆杉〔Ｔ．ｃｈｉｎｅｎｓｉｓｖａｒ．ｍａｉｒｅｉ（ＬｅｍｅｅｅｔＬéｖｌ．）ＣｈｅｎｇｅｔＬ．Ｋ．Ｆｕ〕,分布于我国大部分地区,较为集中的产区为东北、西南和华东地区。云南省是我国红豆杉资源的主要分布区域。红豆杉树皮中含有新型抗癌药物紫杉醇,近年来,由于过度砍剥致使我国红豆杉野生资源遭到严重破坏。因此必须制定保护措施,进行人工种植。由于紫杉醇在红豆杉中含量甚微,结构异常复杂,所以研究紫杉醇的新来源成为世界性的热点,主要研究内容包括：非树皮部分的提取,人工大量种植,衍生物的寻找,化学合成和生物技术等。我国在以上研究领域内已取得多项成果,但与世界先进国家相比还有一定的差距,有待深入研究     

Isolation of differential genes in suspension cultures of Taxus cuspidata induced by additional taxol

Addition of taxol into suspension cultures of Taxus cuspidata induced cell apoptosis, which was confirmed by gel electrophoresis of the DNA ladders indicating the progressive delineation of fragmented nuclear DNA (nDNA) into distinct bodies. The additional taxol not only changed the microtubule assembly of cells, but also affected the gene expression. Fourteen cDNA fragments, named as TIGT9-22, were isolated after addition of taxol and their GenBank accession numbers were given as BF704560-BF704573, respectively. Among them, TIGT13 and TIGT21 were apparently homogeneous with apbE and carbamoylphosphate synthetase, respectively. Other cDNA fragments showed no significant analogy with the known sequences in GenBank.     

红豆杉细胞两相培养生产紫杉醇的研究

研究了两相培养系统中红豆杉细胞的生长与代谢规律,经４０天培养,细胞生物量为１７．８５ｇ／Ｌ（干重）,紫杉醇产量为３０．１９ｍｇ／Ｌ。     

Elicitation of Taxus sp. cell cultures for production of taxol

Summary The addition of cell extracts and cultures filtrate of Pencillium minioluteum, Botrytis cinerea, Verticillium dahliae, and Gilocladium deliqucescens on the tenth day after transferring Taxus sp. (RO1-M28) cell suspensions into an induction medium, further improved the production of Taxol and total taxanes. Arachidonic acid (1mg/L) addition at the time of inoculation increased Taxol production by 150%. Oxidative stress induction and copper sulphate or sodium orthovanadate addition had no effect on Taxol production. Three categories of elicitors; those specifically stimulating Taxol production, those specifically stimulating the producion of other taxanes, and those stimulating taxane production uniformly, could be identified. The biosynthetic site of action of these elicitors is currently not known.

---

     

Taxane diterpenoids from Taxus yunnanensis and Taxus cuspidata

Chemical examination of the seeds of the Chinese yew, Taxus runnanensis Cheng et L. K. Fu and the Japanese yew, Taxus cuspidata Sieb et Zucc, resulted in the isolation of four taxane diterpenoids. The structures of these taxoids were established as (12alpha)-2alpha-acetoxy-5alpha,9alpha, 10beta-trihydroxy-3,11-cyclotax-4(20)-en-13-one; 2alpha,7beta,13alpha-triacetoxy-5alpha, 9alpha-dihydroxy-2(3-->20)abeotaxa-4(20),11-dien-10-one; 9alpha,10beta-diacetoxy-5alpha-cinnamoyloxytaxa- 4(20),11-dien-13alpha-ol and the known 2alpha,7beta,9alpha,10beta,13-pentaacetoxytax a-4(20),12-diene-5alpha,11beta-diol on the basis of spectral analysis.     

Characterization of aggregate size in Taxus suspension cell culture

Plant cells grow as aggregates in suspension culture, but little is known about the dynamics of aggregation, and no routine methodology exists to measure aggregate size. In this study, we evaluate several different methods to characterize aggregate size in Taxus suspension cultures, in which aggregate diameters range from 50 to 2,000 μm, including filtration and image analysis, and develop a novel method using a specially equipped Coulter counter system. We demonstrate the suitability of this technology to measure plant cell culture aggregates, and show that it can be reliably used to measure total biomass accumulation compared to standard methods such as dry weight. Furthermore, we demonstrate that all three methods can be used to measure an aggregate size distribution, but that the Coulter counter is more reliable and much faster, and also provides far better resolution. While absolute measurements of aggregate size differ based on the three evaluation techniques, we show that linear correlations are sufficient to account for these differences (R ² > 0.99). We then demonstrate the utility of the novel Coulter counter methodology by monitoring the dynamics of a batch process and find that the mean aggregate size increases by 55% during the exponential growth phase, but decreases during stationary phase. The results indicate that the Coulter counter method can be routinely used for advanced process characterization, particularly to study the relationship between aggregate size and secondary metabolite production, as well as a source of reliable experimental data for modeling aggregation dynamics in plant cell culture.     

Statistical optimization of single-cell production from Taxus cuspidata plant cell aggregates

Flow-cytometric characterization of plant cell culture growth and metabolism at the single-cell level is a method superior to traditional culture average measurements for collecting population information. Investigation of culture heterogeneity and production variability by obtaining information about different culture subpopulations is crucial for optimizing bio-processes for enhanced productivity. Obtaining high yields of intact and viable single cells from aggregated plant cell cultures is an enabling criterion for their analysis and isolation using high-throughput flow cytometric methods. The critical parameters affecting the enzymatic isolation of single cells from aggregated Taxus cuspidata plant cell suspensions were optimized using response-surface methodology and factorial central composite design. Using a design of experiments approach, the output response single-cell yield (SCY, percentage of cell clusters containing only a single cell) was optimized. Optimal conditions were defined for the independent parameters cellulase concentration, pectolyase Y-23 concentration, and centrifugation speed to be 0.045% (w/v), 0.7% (w/v), and 1200?×?g, respectively. At these optimal conditions, the model predicted a maximum SCY of 48%. The experimental data exhibited a 72% increase over previously attained values and additionally validated the model predictions. More than 99% of the isolated cells were viable and suitable for rapid analysis through flow cytometry, thus enabling the collection of population information from cells that accurately represent aggregated suspensions. These isolated cells can be further studied to gain insight into both growth and secondary metabolite production, which can be used for bio-process optimization.     

Expression of arabinogalactan proteins involved in Taxol production by immobilized Taxus cuspidata cells

The expression of arabinogalactan-proteins (AGPs), known as extracellular signal molecules, in immobilized T. cuspidata cells was investigated by immunofluorescence localization and Western blot analysis. It was found that the relative intensity of JIM13-reactive AGPs and Taxol production by T. cuspidata cells was increased 1.43-fold and 2.2-fold by immobilized cultures on day 25, respectively. Particularly, the expression levels of JIM13-reactive AGPs were much higher in the cells located in central and middle zones of the immobilized support matrices than these in the outer zone or in the suspension. Whether in immobilized T. cuspidata cells or in suspended T. cuspidata cells, the expression level of JIM13-reactive AGPs and Taxol production after two to three subcultures had no significant changes, but the immobilized cells always kept high-level expression of JIM13-reactive AGP and Taxol production during subcultures. Moreover, the enhancement of Taxol production was accompanied with a high-level expression of JIM13-reactive AGPs by T. cuspidata cells after treatment with 200 microM methyl jasmonate. Taken together, these results implicate that the AGPs in T. cuspidata cells may be taken as potential signal molecular involved in regulating the Taxol production by immobilized T. cuspidata cells.     

稀土元素对红豆杉细胞悬浮培养及紫杉醇合成的影响

研究了在250mL摇瓶中,不同浓度的硝酸镧、硫酸铈铵、硝酸亚铈3种稀土化合物对细胞生长及紫杉醇分泌和释放的影响。结果表明,在培养初期加入稀土元素。3种不同稀土化合物对细胞生长影响强弱不同,但趋势相似,均使细胞的延迟期缩短。1ppm的Ce^4 促进细胞生长的效果最明显。细胞干重第17d达到10.9g/L。在指数期加入稀土元素。10ppmCe^3 刺激细胞生长的效果最明显,细胞干重最高值达到11.5g/dL,比对照高1.5g/L,而10ppm的La^3 抑制细胞的生长。经稀土元素处理后,细胞胞内和胞外紫杉醇含量都有大幅度的提高,其中以10ppmCe^3 处理,胞外紫杉醇释放率最大,达37.7%。     

Taxol (paclitaxel) production using free and immobilized cells of Taxus cuspidata

The production characteristics for Taxol (paclitaxel) using free and immobilized cells of Taxus cuspidata were investigated in a perfusion culture bioreactor. Although the cell growth was inhibited by higher dilution rates, the specific production rate of Taxol was increased by perfusion compared with that using batch operation. Perfusion cultures using a nylon-mesh cell separator for free suspension cells showed similar production profiles to those obtained using immobilized cells. Continuous Taxol production was successfully obtained at an approximate specific production rate of 0.3 mg/g DCW (dry cell weight) per day for up to 40 days. (c) 1997 John Wiley & Sons, Inc.     

Cell aggregate suspension culture for large-scale production of biomolecules

Summary A method is described for the rapid reversible conversion of a number of continuous cell lines from anchorage-dependent growth to growth as aggregates of cells in suspension culture. Employing this technique, an inoculum of three 75-cm² flasks of BALB/c SV3T3 cells was grown to 60 liters of aggregate suspension in 14 days. This yielded 120 ml of packed cells or 9.1×10¹⁰ cells. Similar results were obtained for other cell lines. Biomolecules such as migration-inhibition factor (MIF) and plasminogen activator were produced from these cultures.     

Taxus callus cultures: Initiation,growth optimization,characterization and taxol production

Callus was induced from Taxus baccata cv. Repandens Parsons ex Rehd., T. brevifolia Nutt., T. cuspidata Sieb. & Zucc., and T. x media cvs. Hicksii and Densiformis Rehd. using different concentrations of 2,4-d-(2,4-dichlorophenoxyacetic acid), IBA (indole-3-butyric acid), or NAA -naphthalene acetic acid in combination with kinetin. All cultures grew slowly following the first subculture, and a majority turned brown and ceased growth within the next six to twelve months. The callus cultures which lived, continued to grow very slowly for one to two years before the growth rate improved. Initiation of roots and shoot primordia-like structures occurred on some cultures maintained in the dark, and 16 h light/8 h dark, respectively. A fast-growing, habituated callus line (CR-1) derived from T. x media Rehd. cv. Hicksii was established from callus initiated in 1986. Supplementing the medium with casein hydrolysate and both fructose and glucose enhanced the growth rate. A great deal of heterogeneity was found among and within the callus, with respect to the amount of taxol produced. The callus exhibited levels of taxol ranging from 0.1 to 13.1 mg kg^-1 (0.0001 to 0.0131%) on a dry weight basis. Overall, the older brown-colored callus produced more taxol than the younger pale yellow-colored callus. The presence of taxol in callus samples was established by high performance liquid chromatography, its biological activity confirmed by a microtubule-stabilizing bioassay and its structure confirmed using one-and two-dimensional ¹H and ¹³C nuclear magnetic resonance spectroscopy.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - kinetin 6-furfurylaminopurine - 2iP 6-(,-dimethylallylamino)purine     

Enhanced production of taxol in suspension cultures of Taxus chinensis by controlling inoculum size

Inoculum size (1.5-6.0g dry weight/l) significantly affected cell growth and accumulation of intracellular and extracellular taxol in Taxus chinensis. A shorter cultivation time and a higher biomass productivity were achieved using inoculum size of 6.0g DW/l. Both the intracellular content and total production of taxol were increased almost 30% with an increase of inoculum size from 1.5 to 3.0g DW/l, while an even higher inoculum size decreased taxol formation. The extracellular taxol concentration was relatively higher (0.091mg/l) at low inoculum sizes of 1.5 and 2.0g DW/l; and in various cases it was less than 25% of the total amount of taxol produced.     

A phytoecdysteroid,taxisterone, from Taxus cuspidata

A new phytoecdysteroid, taxisterone, was isolated from Taxus cuspidata and the structure was deduced to be 22-deoxyecdysterone by spectral data.     

东北红豆杉的化学成分和药理作用研究进展

本文综述了东北红豆杉(Taxus cuspidata)在化学成分和药理学方面的研究进展,着重强调了近年来紫杉烷类化合物的化学研究和生理活性。     

Protoplast culture and paclitaxel production by Taxus yunnanensis

Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. The planting density was 2.5–3.0×10⁵ protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating efficiency was about 7%. Protoplast-derived colonies showed significant variations in both growth and paclitaxel content. A negative correlation existed between paclitaxel accumulation in colonies and their growth to some extent (r = −0.4485). Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content. Paclitaxel accumulation in colony TY-7 was not great enough to produce paclitaxel for commercial purposes, however, success in inducing colony formation from T. yunnanensis protoplasts provides an opportunity to obtain cell lines with high paclitaxel productivity from mutagenized protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitric oxide mediates inactivation of glutathione S-transferase in suspension culture of Taxus cuspidata during shear stress

The importance of nitric oxide (NO) in regulating plant cell responses to environmental stresses is becoming evident. Here the possible role of NO in suspension cultures of Taxus cuspidata under shear stress was investigated in a Couette-type shear reactor. It was found that shear stress with 190 s(-1) caused NO generation in 8 h. NO formation can be inhibited by N-nitro-L-arginine, a nitric oxide synthase inhibitor. Moreover, the activity of glutathione S-transferase (GST), a principal enzyme responsible for detoxification, decreased during shear stress. This inactivation partially recovered when NOS inhibitor or NO scavenger was added into cell cultures during shear stress. Treatment with reactive nitrogen species (RNS) also caused inactivation of GST in cells. The results indicate that NO plays a crucial role in GST inactivation in Taxus cuspidata cells under shear stress.