Purification and characterization of Taxol and 10-Deacetyl baccatin III from the bark, needles, and endophytes of Taxus baccata by preparative high-performance liquid chromatography, ultra-high-performance liquid chromatography-mass spectrometry, and nuclear magnetic resonance.

Taxol and 10-Deacetyl baccatin III are major taxanes in the bark, needles, and endophytes of Taxus baccata. The current study aimed to develop a process for their separation from different matrices. Crude taxoid was prepared by extraction of samples with methanol, followed by partitioning with dichloromethane and precipitation with hexane. Analytical high-performance liquid chromatography involved isocratic elution on C18 column (4.6 × 250 mm, 5 µm) with methanol-water (70:30 v/v) at a flow rate of 1 ml/min. Injection volume was 20 µl and detection was carried out at 227 nm. The content of Taxol and 10-Deacetyl baccatin III in bark, needles and endophytic culture broth was 11.19 and 1.75 µg/mg; 11.19 and 1.75 µg/mg; and 2.80 and 0.22 µg/L, respectively. Preparative high-performance liquid chromatography was done on C18 column (10 × 250 mm, 5 µm) at a flow rate of 10 ml/min. About 20 g crude taxoid was processed in < 3 h with a recovery of about 90% for both the analytes. The purity of recovered Taxol and 10-Deacetyl baccatin III determined by ultra-high-performance liquid chromatography-mass spectrometry was found to be 95.78 ± 3.63% and 99.72 ± 0.18%, respectively. The structure of recovered Taxol was confirmed by nuclear magnetic resonance. The method can find use in biotransformation studies.

A simple and efficient sample preparation for taxanes in Taxus chinensis needles with natural menthol-based aqueous deep eutectic solvent.

Taxanes are natural anticancer constituents, and the sample preparation from matrix normally depends on organochlorine solvents. In this study, green and natural menthol-based aqueous deep eutectic solvent was synthesized and used for sample preparation for taxanes. Five key parameters were optimized and the optimal preparation conditions were as follows: menthol/1-propanol ratio 1:1 (mol/mol), solid-liquid ratio 1:30 g/mL, extraction time 30 min, ultrasonic power 250 W, and water content 80%. Under the above conditions, the total extraction efficiency of seven main taxanes was 1.25- to 1.44-fold to the conventional methods. In addition, a high-performance liquid chromatography method with C18 column was established for quantitation of seven main taxanes in <25 min, which had excellent linearity (R2  > 0.9986), precision (relative standard deviation < 3.00%), repeatability (relative standard deviation < 3.69%), and recovery (90.26-109.00%). This method performed the extraction, and enrichment processes simultaneously, and it had advantages such as high extraction efficiency, simple operation, low cost, and eco-friendliness. This work indicated that the natural menthol-based deep eutectic solvent aqueous could be an excellent alternative to the sample preparation from Taxus or other plants.

Detection of Taxuyunnanin C in Suspension Cell Culture of Taxuscanadensis.

This is the first study to isolate the taxoid taxuyunnanin C (group of 14-hydroxylated taxoids) from the biomass of suspension cell culture of the Canadian yew (Taxus canadensis). According to available data, this is the first report of the presence of nonpolar (polyacylated) forms of 14-hydroxylated taxoids, including taxuyunnanin C, in T. canadensis.

Comparative metabolomics reveals the metabolic variations between two endangered Taxus species (T. fuana and T. yunnanensis) in the Himalayas.

BACKGROUND: Plants of the genus Taxus have attracted much attention owing to the natural product taxol, a successful anti-cancer drug. T. fuana and T. yunnanensis are two endangered Taxus species mainly distributed in the Himalayas. In our study, an untargeted metabolomics approach integrated with a targeted UPLC-MS/MS method was applied to examine the metabolic variations between these two Taxus species growing in different environments. RESULTS: The level of taxol in T. yunnanensis is much higher than that in T. fuana, indicating a higher economic value of T. yunnanensis for taxol production. A series of specific metabolites, including precursors, intermediates, competitors of taxol, were identified. All the identified intermediates are predominantly accumulated in T. yunnanensis than T. fuana, giving a reasonable explanation for the higher accumulation of taxol in T. yunnanensis. Taxusin and its analogues are highly accumulated in T. fuana, which may consume limited intermediates and block the metabolic flow towards taxol. The contents of total flavonoids and a majority of tested individual flavonoids are significantly accumulated in T. fuana than T. yunnanensis, indicating a stronger environmental adaptiveness of T. fuana. CONCLUSIONS: Systemic metabolic profiling may provide valuable information for the comprehensive industrial utilization of the germplasm resources of these two endangered Taxus species growing in different environments.

Comparative Transcriptome Analysis Revealed the Tissue-Specific Accumulations of Taxanes among Three Experimental Lines of Taxus yunnanensis.

Taxus yunnanensis (Yew) is known for natural anticancer metabolite paclitaxel (Taxol) and its biosynthesis pathway in yew species still needs to be completely elucidated. In the current study, productions of paclitaxel and 10-DAB III from three different tissues (needle, branch, and root) of T. yunnanensis wild type (WT) and two new cultivars Zhongda-1 (Zd1) and Zhongda-2 (Zd2) were determined, and significant tissue differences in contents of the taxanes were observed among the three experimental lines. The much higher 10-DAB III and lower paclitaxel contents in needle of Zd2 when compared with that of Zd1 indicated the low conversion from 10-DAB III to paclitaxel in the needle of Zd2. In order to uncover the mechanisms of the tissue-specific biosynthesis of the taxanes, transcriptome analysis of cultivar Zd2 was conducted, and the previously reported transcriptome data of Zd1 and WT were used to perform a comparison. The enhancement of TDAT and T10ßH side biosynthetic pathway in roots of Zd2 in early taxane synthesis might lead to the biosynthesis of other toxoids, while the preference of T13αH route in the needle and branch of Zd2 was mainly responsible for the tissue-specific reinforced biosynthesis of 10-DAB III and paclitaxel in Zd2. Different from Zd1, the tissue-specific pattern of paclitaxel biosynthesis genes in Zd2 was similar to WT. However, the lower transcript abundance of final steps genes (TBT, DBAT, BAPT, and DBTNBT) of the paclitaxel biosynthesis pathway in Zd2 than in Zd1 might further promote 10-DAB III accumulation in Zd2.

UV spectroscopy and least square matching for high throughput discrimination of taxanes in commercial formulations and compounded bags.

The need for high-throughput quality control of pharmaceuticals after compounding is often required before the treatment of the patients. Ultra-fast analysis using flow injection analysis coupled to UV spectroscopy and least square matching was assessed for the simultaneous quantification and identification of three therapeutic taxanes after dilution in physiological saline (cabazitaxel, docetaxel, and paclitaxel). In-depth preliminary analysis of the zero and first order UV spectra of the taxanes using principal component analysis (PCA) allowed us focusing on relevant spectral range with very low formulation influence. Least square-matching algorithm available on basic HPLC software was applied to these spectra yielding very high match scores (>999) with significant difference (P < 0.0001). The approach was qualitatively assessed through specificity and sensitivity which were excellent for the three taxanes (100%, n = 378), irrespective of their formulation. In terms of quantification, satisfactory linearity and accuracy were achieved for each of the taxanes according to their therapeutic range (0.05 to 1.02 mg·mL-1). The RSD (%) of the precision was satisfactory (<3%). Finally, the suitability of the approach for the taxanes QC has been demonstrated under routine application.

A Critical Review of Properties and Analytical Methods for the Determination of Docetaxel in Biological and Pharmaceutical Matrices.

Docetaxel (DTX) is an antineoplastic agent of the second generation of the taxoid family. It is a semi-synthetic drug prepared from a precursor extracted of the plant Taxus baccata. The commercial formulation of DTX, Taxotere®, employs the surfactant polysorbate 80, due to the low water solubility of the drug, causing several side effects. Therefore, there is a need to develop delivery systems to reduce the side effects of DTX. In addition, this drug has been qualitative and quantitatively analyzed in pharmaceutical formulations and biological samples. Thus, several techniques and analytical methods have been reported with the aim of optimizing the analytical signal, increasing sensitivity, selectivity and reducing the effects of interference. Herein, we highlight immunoassay, capillary electrophoresis and chromatographic methods. This review presents a summary of physicochemical and pharmacokinetics properties, mechanisms of action, drug delivery systems and analytical methods used in quantification of DTX in diverse matrices such as blood, plasma, oral fluid, urine, carcinoma cells, pharmaceutical formulations and delivery systems.

Application of liquid chromatography-tandem mass spectrometry to study the effect of docetaxel on pharmacokinetics and tissue distribution of apatinib in mice.

Apatinib, a highly selective small-molecule inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2), has attracted many attentions due to its anticancer activity in various malignancies containing non-small-cell lung cancer (NSCLC). Our previous preclinical study confirmed the enhanced anti-tumor efficacy of combined treatment between apatinib and docetaxel for NSCLC. However, the effects of docetaxel on pharmacokinetics and tissue distribution of apatinib are not clear. In present study, a reliable HPLC-MS/MS method was established for determination of apatinib. This method had a good linearity in the range of 1-5000 ng/mL, and the recovery and matrix effect were 100.1-103.5%, 77.6-83.5%, respectively. Plasma exposure level of apatinib and the values of Cmax, AUC0-12h, T1/2, and MRT were not affected by multi-dose of docetaxel. The tissue distributions (kidney, heart, lung, spleen) of apatinib in combined treatment group were lower at 0.25 h but higher at 2 h, and that in intestine and liver were not significantly changed compared with control group. However, pre-treatment with docetaxel had no significant effect on AUC0-4h of apatinib in tissues in mice. In conclusion, plasma and tissues exposure levels of apatinib were not affected by long-termed treatment with docetaxel, indicating that docetaxel is less likely to increase the side effect of apatinib such as hypertension, hand-foot syndrome and so on.

Laser Desorption/Ionisation Mass Spectrometry Imaging of European Yew (Taxus baccata) on Gold Nanoparticle-enhanced Target.

INTRODUCTION: European yew (Taxus baccata) is a plant known to man for centuries as it produces many interesting and important metabolites. These chemical compounds were repeatedly analysed by various analytical techniques, but none of the methods used so far allowed the localisation of the chemical compounds within the tissue and also correlation between plant morphology and its biochemistry. OBJECTIVE: Visualisation of the spatial distribution of yew metabolites with nanoparticle-based mass spectrometry imaging. METHODOLOGY: Compounds occurring on cross-section of a one-year yew sprig has been transferred to gold nanoparticle-enhanced target (AuNPET) by imprinting. The imprint was then subjected to mass spectrometry imaging analysis. RESULTS: Nanoparticle-enhanced mass spectrometry imaging made it possible to study the distribution of selected compounds in the European yew tissue, including taxanes - terpene alkaloids characteristic for the Taxus genus. Results prove that aspartate, taxinine M, baccatin IV and taxine B are located mainly in the cortex. Taxuspine W was located in the vascular tissue. Maleate was found to be located mainly in the phloem tissue. In contrast, the proton adduct of chlorophyll b was found in the external layer of twigs. CONCLUSION: The results presented a high correlation between the location of compounds and the morphology of the plant, thus giving the opportunity to see the selected details of chemical structure of the analysed tissue for the first time. Copyright © 2017 John Wiley & Sons, Ltd.

Development of a Multichannel Dialysis Microchip for Bioassay of Drug Efficacy and Retention.

Here, we developed a multichannel dialysis microchip having three sets of dialysis systems, which consisted of three independent circulation channels with a common micropump. Each dialysis system was composed of a pneumatic micropump (heart), dialysis unit (renal corpuscle), and cell culture chamber (drug target) as well as circulation and dialysate channels to mimic the circulation system. Small molecules were successfully separated in parallel from macromolecules at the dialysis components. Anticancer tests using this microchip showed results that considered both serum protein-binding nature and drug activity. In the anticancer bioassay, the multichannel chip showed similar reproducible and reliable results as those of the single-channel system but with higher throughput.

A lyophilized sterically stabilized liposome-containing docetaxel: in vitro and in vivo evaluation.

OBJECTIVES: In this study, an improved lyophilized PEGylated liposomal formulation of docetaxel (DOC) has been developed. METHODS: PEGylated docetaxel liposome (PL-DOC) was prepared by thin-film evaporation method and lyophilization. The effect of various components of the lipids and their compatibility with DOC on the entrapment efficiency (EE) of liposome was investigated. The lyophilized PL-DOC was characterized by morphology, particle size, zeta potential, EE, release in vitro and stability. Pharmacokinetics and biodistribution in vivo of lyophilized PL-DOC were also investigated. RESULTS: The optimal liposome formulation was egg phosphatidylcholine (EPC):cholesterol (CH):DSPE-PEG2000:DOC = 56:40:4:4 (molar ratio). Sucrose and mannitol were chosen as cryoprotectant in the lyophilization (cryoprotectant-to-lipid (C/L) mass ratio = 8:1). The size of lyophilized PL-DOC was 152.3 ± 1.0 nm with negative charge and the EE was 89.75 ± 1.79%. Compared with nonlyophilized PL-DOC, the lyophilized PL-DOC was more stable at 4 °C for six months. The lyophilized PL-DOC also showed the good stability after reconstituted by 5% glucose injection. In vitro release study of PL-DOC showed that PL-DOC had a sustained release effect. After i.v. administration at the dose of 10 mg/kg in rats, a significant increase in the AUC0-∞, MRT0-∞ and t1/2 was observed in PL-DOC group compared with conventional docetaxel liposome (CL-DOC) and DOC injection (DOC-I) group. Biodistribution studies in mice showed that PL-DOC significantly decreased the uptake by the organs of mononuclear phagocytic system (MPS), such as liver and spleen, while prolonging the retention time of DOC in the plasma. CONCLUSION: Our PEGylated liposome formulation reported in this study could potentially produce viable clinical strategies for improved delivery of DOC for the treatment of human cancer.

Seasonal Dynamics of Metabolites in Needles of Taxus wallichiana var. mairei.

Seasonal variations of the phytochemicals contents in needles of T. wallichiana var. mairei due to the effects of growth meteorological parameters were investigated in this study. The needles of T. wallichiana var. mairei were collected from different months and the contents of taxoids (paclitaxel, 10-deacetylbaccatin III (10-DAB), baccatin III, cephalomannine, 10-deacetyltaxol (10-DAT)), flavones (ginkgetin, amentoflavone, quercetin) and polysaccharides were quantified by ultra performance liquid chromatography (UPLC) and the resonance light scattering (RIL) method. The content of taxoids gave the highest level of 1.77 ± 0.38 mg·g-1 in January, and the lowest value of 0.61 ± 0.08 mg·g-1 in September. Unlike taxoids, the content of flavonoids was the highest in August. The content of polysaccharides reached peak value of 28.52 ± 0.57 mg·g-1 in September, which was two times higher than the lowest content of 9.39 ± 0.17 mg·g-1 in January. The contents of paclitaxel, 10-DAB, 10-DAT and polysaccharides significantly depended on meteorological parameters. The mean of minimum temperature (R = -0.61) and length of daylight (R = -0.60) were significantly correlated to 10-DAB content, while 10-DAT level showed significant correlation with length of daylight (R = -0.70) and relative humidity (R = 0.70). In addition, temperature had significantly negative effect on the content of paclitaxel and a significantly positive effect on that of polysaccharides. This study enriched the knowledge on the accumulation pattern of metabolites and could help us to determine the collecting time of T. wallichiana var. mairei for medicinal use.

UPLC-MS/MS method for simultaneous determination of cyclophosphamide, docetaxel, doxorubicin and 5-fluorouracil in surface samples.

INTRODUCTION: There is certainly a great benefit to treatment with antineoplastic drugs for cancer patients with a life-threatening disease. However, for the workers who are exposed to these agents as part of their work practice, precautions should be taken to eliminate or reduce exposure as much as possible. OBJECTIVE: The aim of this study is to develop and validate a wipe sampling procedure followed by liquid chromatographic separation with electrospray ionization and a tandem mass spectrometric detection method for the simultaneous determination of cyclophosphamide (CP), docetaxel (DOC), doxorubicin (DOXO) and 5-fluorouracil (5-FU). METHOD: The chromatographic separation was carried out in 15min by applying a gradient elution of 0.1% formic acid and acetonitrile. MS/MS was performed on a triple quadrupole instrument in the multiple reaction monitoring (MRM) mode. RESULTS: The analytical range was linear between 3.5 to 300ng/mL for CP, 4 to 300ng/mL for DOC and DOXO and 2 to 300ng/mL for 5-FU. The present method offers a high sensitivity, with detection limits of 1.0ng/mL for CP, DOXO and DOC and 0.5ng/mL for 5-FU. The selectivity, accuracy (relative standard error between 82.3 and 113.9%) and precision (relative standard deviation between 1.2 and 14.2%) make the method suitable for the routine determination of these drugs to estimate the occupational exposure of personnel handling. CONCLUSIONS: The developed method allows a reliable assessment of exposure, which is one of the steps for evaluation the risk inherent to workers in contact with antineoplastic drugs.

Understanding taxanes in prostate cancer; importance of intratumoral drug accumulation.

BACKGROUND: Resistance to docetaxel is common in metastatic castration-resistant prostate cancer (mCRPC) and may be caused by sub-therapeutic intratumoral drug concentrations. Cabazitaxel demonstrated survival benefit in docetaxel-pretreated and docetaxel-refractory patients. In this study, we investigated whether the superior antitumor activity of cabazitaxel in mCRPC is explained by higher intratumoral cabazitaxel levels. Since recent studies suggest a reduced efficacy of docetaxel following treatment with novel androgen receptor (AR)-targeted agents, we also investigated taxane efficacy in an enzalutamide-resistant tumor model. METHODS: Intratumoral concentrations of docetaxel and cabazitaxel were correlated with antitumor activity in docetaxel-naïve, docetaxel-resistant, and enzalutamide-resistant patient-derived xenografts (PDXs) of prostate cancer. RESULTS: Intratumoral drug levels were negatively related to intrinsic and acquired resistance to docetaxel. Also, the observed stronger antitumor activity of cabazitaxel was associated with increased cumulative exposure and higher intratumoral of cabazitaxel concentrations in all PDXs. CONCLUSIONS: The superior antitumor activity of cabazitaxel in docetaxel- and enzalutamide-resistant tumors can be partly attributed to higher intratumoral drug concentrations. Especially for patients who are intrinsically resistant to docetaxel resulting from suboptimal intratumoral docetaxel concentrations, cabazitaxel may be the preferred chemotherapeutic agent. Prostate 76:927-936, 2016. © 2016 Wiley Periodicals, Inc.

P-glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain Disposition and Oral Availability of the Novel Taxane Cabazitaxel (Jevtana) in Mice.

We aimed to clarify the roles of the multidrug-detoxifying proteins ABCB1, ABCG2, ABCC2, and CYP3A in oral availability and brain accumulation of cabazitaxel, a taxane developed for improved therapy of docetaxel-resistant prostate cancer. Cabazitaxel pharmacokinetics were studied in Abcb1a/1b, Abcg2, Abcc2, Cyp3a, and combination knockout mice. We found that human ABCB1, but not ABCG2, transported cabazitaxel in vitro. Upon oral cabazitaxel administration, total plasma levels were greatly increased due to binding to plasma carboxylesterase Ces1c, which is highly upregulated in several knockout strains. Ces1c inhibition and in vivo hepatic Ces1c knockdown reversed these effects. Correcting for Ces1c effects, Abcb1a/1b, Abcg2, and Abcc2 did not restrict cabazitaxel oral availability, whereas Abcb1a/1b, but not Abcg2, dramatically reduced cabazitaxel brain accumulation (>10-fold). Coadministration of the ABCB1 inhibitor elacridar completely reversed this brain accumulation effect. After correction for Ces1c effects, Cyp3a knockout mice demonstrated a strong (six-fold) increase in cabazitaxel oral availability, which was completely reversed by transgenic human CYP3A4 in intestine and liver. Cabazitaxel markedly inhibited mouse Ces1c, but human CES1 and CES2 only weakly. Ces1c upregulation can thus complicate preclinical cabazitaxel studies. In summary, ABCB1 limits cabazitaxel brain accumulation and therefore potentially therapeutic efficacy against (micro)metastases or primary tumors positioned wholly or partly behind a functional blood-brain barrier. This can be reversed with elacridar coadministration, and similar effects may apply to ABCB1-expressing tumors. CYP3A4 profoundly reduces the oral availability of cabazitaxel. This may potentially be greatly improved by coadministering ritonavir or other CYP3A inhibitors, suggesting the option of patient-friendly oral cabazitaxel therapy.

Changes in gene transcription and taxane production in elicited cell cultures of Taxus × media and Taxus globosa.

The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), supplied to the culture medium either separately or together, was studied. Two-stage Taxus globosa and Taxus media cell cultures were established and the elicitors were added at the beginning of the second stage. Growth, taxane production, and the expression of known taxol biosynthetic genes, including the recently characterized CoA ligase gene, were studied. Although CORO reduced the growth capacity of both cell lines, CD apparently counteracted this negative effect. Taxane production was significantly enhanced by the simultaneous addition of CD and CORO to the medium. The total taxane production in the T. media cell line was more than double that of T. globosa, but in the latter more than 90% of the taxanes produced were excreted to the medium. Individual taxane patterns also differed: at the height of production, the main taxanes in T. globosa cultures were cephalomannine and 10-deacetyltaxol, and in T. media, taxol and baccatin III. The low transcript levels of taxane biosynthetic genes found in T. globosa cells mirrored the lower taxane production in these cultures, while a high expression was strongly correlated with a high taxane production in T. media.

Infrared and Raman spectroscopic methods for characterization of Taxus baccata L.--Improved taxane isolation by accelerated quality control and process surveillance.

Different yew species contain poisonous taxane alkaloids which serve as resources for semi-synthesis of anticancer drugs. The highly variable amounts of taxanes demand new methods for fast characterization of the raw plant material and the isolation of the target structures during phyto extraction. For that purpose, applicability of different vibrational spectroscopy methods in goods receipt of raw plant material and in process control was investigated and demonstrated in online tracking isolation and purification of the target taxane 10-deacetylbaccatin III (10-DAB) during solvent extraction. Applying near (NIRS) and mid infrared spectroscopy (IRS) the amount of botanical impurities in mixed samples of two different yew species (R(2)=0.993), the leave-to-wood ratio for Taxus baccata material (R(2)=0.94) and moisture in dried yew needles (R(2)=0.997) can be quantified. By partial least square analysis (PCA) needles of different Coniferales species were successfully discriminated by Attenuated Total Reflectance-Fourier-Transform Infrared Spectroscopy (ATR-FT-IR). The analytical potential of ATR-FT-IR and Fourier Transform-Raman Spectroscopy (FT-RS) in process control of extraction and purification of taxanes is demonstrated for determination of the water content in methanolic yew extracts (R(2)=0.999) and for quantification of 10-DAB (R(2)=0.98) on a highly sophisticated level. The decrease of 10-DAB in the plant tissue during extraction was successfully visualized by FT-IR imaging of thin cross sections providing new perspectives for process control and design.

[Chemical constituents of Taxus chinensis var. mairei cell cultures].

The chemical constituents of Taxus chinensis var. mairei cell cultures were investigated by chromatographic methods, including silica gel column chromatography, Sephadex LH-20 and preparative HPLC. Thirteen compounds were isolated from the 80% ethanol extract of cultured cells and their structures were elucidated by spectral data and physicochemical properties, which were identified as 2α,4α,7ß,9α,10ß-pentaacetoxy-14ß-hydroxytax-11-ene (1), 2α,4α,7ß,9α,10ß-pentaacetoxytax-11-ene (2), 1ß-deoxybaccatin VI (3), 2α-acetoxytaxusin (4), taxuyunnanine C (5), yunnanxane (6), 2α,5α,10ß-triacetoxy-14ß-propionyloxy-4 (20), 11-taxadiene (7), 2α,5α,10ß-triacetoxy-14ß-isobutyryloxy-4 (20), 11-taxadiene (8), 2α,5α,10ß-triacetoxy-14ß-(2'-methyl)butyryloxy-4 (20), 11-taxadiene (9), 13-dehydroxylbaccatin III (10), 13-dehydroxy-10-deacetylbaccatin III (11), paclitaxel (12) and (13) ß-sitosterol. Among them, compound 1 is a new compound, and compounds 2, 4, 10 and 11 are isolated from the cell culture of Taxus chinensis var. mairei for the first time.

Pilot studies on scale-up biocatalysis of 7-ß-xylosyl-10-deacetyltaxol and its analogues by an engineered yeast.

Paclitaxel content in yew tree is extremely low, causing a worldwide shortage of this important anticancer drug. Yew tree can also produce abundant 7-ß-xylosyl-10-deacetyltaxol that can be bio-converted into 10-deacetyltaxol for semi-synthesis of paclitaxel. However, the bio-conversion by the screened natural microorganisms was inefficient. We have constructed the recombinant yeast with a glycoside hydrolase gene from Lentinula edodes and explored the bioconversion. Based on previously established reaction conditions, the bioconversion of 7-ß-xylosyl-10-deacetyltaxol or its extract was further optimized and scaled up with the engineered yeast harvested from 200-L scale high-cell-density fermentation. The optimization included the freeze-dried cell amount, dimethyl sulfoxide concentration, addition of 0.5% antifoam supplement, and substrate concentration. A 93-95% bioconversion and 83% bioconversion of 10 and 15 g/L 7-ß-xylosyltaxanes in 10 L reaction volume were achieved, respectively. The yield of 10-deacetyltaxol reached 10.58 g/L in 1 L volume with 15 g/L 7-ß-xylosyl-10-deacetyltaxol. The conversion efficiencies were not only much higher than those of other reports and our previous work, but also realized in 10 L reaction volume. A pilot-scale product purification was also established. Our study bridges the gap between the basic research and commercial utilization of 7-ß-xylosyl-10-deacetyltaxol for the industrial production of semi-synthetic paclitaxel.

Development of a hazel cell culture-based paclitaxel and baccatin III production process on a benchtop scale.

The growing demand for the antitumorous agent paclitaxel and the difficulty in increasing its production by genetic engineering has prompted a search for new sources of taxanes. It has been reported that taxanes can be extracted from the angiosperm Corylus avellana L. Our aim was to improve taxane production by scaling up the process from mL-level to benchtop bioreactors, optimizing culture conditions and comparing the effect of two elicitors, 1 µM coronatine (Cor) and 100 µM methyl jasmonate (MeJA). Orbitally shaken flask cultures achieved a maximum fresh cell weight of 11.54 gDCW/L under control conditions, and MeJA- and Cor-treatment produced a statistically significant reduction in growth to 4.28 gDCW/L and 5.69 gDCW/L, while increasing the taxane content 3- and 27-fold, respectively. The enhancing effect of these elicitors on taxane production, despite affecting growth, was confirmed in orbitally shaken TubeSpin Bioreactors 50, where the highest taxane content (8583.3 µg/L) was obtained when 1µM Cor was used and elicitation took place at a packed cell volume of 50%. Two benchtop stirred bioreactors, BIOSTAT B plus and UniVessel SU, were compared, the latter providing a higher biomass of C. avellana cell suspension cultures. Transferring the established optimum culture conditions for taxane production to the UniVessel SU resulted in a total taxane content of 6246.1 µg/L, a 10-fold increase compared with shake flask experiments.