Paclitaxel Production Research Articles

High-effective biosynthesis of baccatin Ⅲ by using the alternative acetyl substrate, N-acetyl-d-glucosamine.

Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17709677·4 USDmol-1 ), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin Ⅲ, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production. In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin Ⅲ-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30·95 USDmol-1 , about 572202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2·13-fold of that when using acetyl-CoA. In the in vivo system, the yield increased 24·17%, which may indicate its cooperation with acetyl-CoA. The success of open-whole-cell synthesis and in vivo synthesis of baccatin Ⅲ by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin Ⅲ. All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin Ⅲ semi-synthesis.

Agro-industrial wastes for production of paclitaxel by irradiated Aspergillus fumigatus under solid-state fermentation.

Paclitaxel is the most profitable drug ever developed in cancer chemotherapy; however, the yield of paclitaxel from microbial platforms is still far from the commercial purpose. Thus, this study was conducted to explore the possibility of solid-state fermentation (SSF) for production of paclitaxel by fungal fermentation. Different agro-industrial wastes were screened as solid substrates for production of paclitaxel by the endophytic Aspergillus fumigatus TXD105 under SSF. Sugarcane bagasse followed by wheat bran, and rice bran were the most suitable substrates for maximum production of paclitaxel. In the effort to increase the paclitaxel production, selection of the most proper moistening agent that supports the production of paclitaxel by the fungal strain was investigated. The effect of varying inoculum concentrations on the production of paclitaxel was also studied. Moreover, optimization of SSF conditions (moisture level, substrate concentrations and nutrients concentration) was adopted using response surface methodology. SSF carried out under the optimum conditions of 20g sugarcane bagasse, twofold nutrients concentration of the MM1D broth, 80% moisture level and inoculum concentration of 107 spores per ml intensified the paclitaxel concentration to 145·61mgkg-1 which represents a 10-fold increase. The production of paclitaxel by the fungal strain was further improved via exposure to UV and gamma radiation at specific doses. The paclitaxel concentrations were intensified following UV and gamma radiation to 209·91 and 351·82mgkg-1 . This is the first report on the production of paclitaxel using agro-industrial wastes as cheap source that may contribute in lowering the cost of producing paclitaxel. These findings offer new and alternate sources with excellent biotechnological potential for paclitaxel production by fungal fermentation.

Elicitors stimulate paclitaxel production by endophytic fungi isolated from ecologically altered Taxus baccata

The commercial production of paclitaxel; the most potent anti-cancer drug, is based on its extraction from Taxus spp (yews) or microbial fermentation from yews’ endophytes. The present study invest...

Strain improvement and immobilization technique for enhanced production of the anticancer drug paclitaxel by Aspergillus fumigatus and Alternaria tenuissima.

UV and gamma irradiation mutagenesis was applied on Aspergillus fumigatus and Alternaria tenuissima in order to improve their producing ability of paclitaxel. Among the screened mutants, two stable strains (designated TXD105-GM6 and TER995-GM3) showed the maximum paclitaxel production. Paclitaxel titers of the two respective mutants were dramatically intensified to 1.22- and 1.24-fold, as compared by their respective parents. Immobilization using five different entrapment carriers of calcium alginate, agar-agar, Na-CMC, gelatin, and Arabic gum was successfully applied for production enhancement of paclitaxel by the two mutants. The immobilized cultures were superior to free-cell cultures and paclitaxel production by the immobilized mycelia was much higher than that of the immobilized spores using all the tried carriers. Moreover, calcium alginate gel beads were found the most conductive and proper entrapment carrier for maximum production of paclitaxel. The feasibility of the paclitaxel production by the immobilized mycelia as affected by incubation period, medium volume, and number of beads per flask was adopted. Under the favorable immobilization conditions, the paclitaxel titers were significantly intensified to 1.31- and 1.88-fold by the respective mutants, as compared by their free cultures. The obtained paclitaxel titers by the immobilized mycelia of the respective mutants (694.67 and 388.65μgL-1) were found promising in terms of fungal production of paclitaxel. Hence, these findings indicate the future possibility to reduce the cost of producing paclitaxel and suggest application of the immobilization technique for the biotechnological production of paclitaxel at an industrial scale.

New synergistic co-culture of Corylus avellana cells and Epicoccum nigrum for paclitaxel production.

Paclitaxel is a main impressive chemotherapeutic agent with unique mode of action and broad-spectrum activity against cancers. Hazel (Corylus avellana) is a paclitaxel-producing species through bioprospection. Endophytic fungi have significant roles in plant paclitaxel production. This study evaluated the effect of co-culture of C. avellana cells and paclitaxel-producing endophytic fungus, Epicoccum nigrum strain YEF2 and also the effect of elicitors derived from this fungal strain on paclitaxel production. The results clearly revealed that co-culture of C. avellana cells and E. nigrum was more effective than elicitation of C. avellana cells by only cell extract or culture filtrate of this fungal strain. Co-culture of C. avellana cells and E. nigrum surpassed monocultures in terms of paclitaxel production designating their synergistic interaction potential. Fungal inoculum amount, co-culture establishment time and co-culture period were important factors for achieving the maximum production of paclitaxel in this co-culture system. The highest total yield of paclitaxel (404.5µgL-1) was produced in co-culture established on 13th day using 3.2% (v/v) of E. nigrum mycelium suspension, which was about 5.5 and 136.6 times that in control cultures of C. avellana cells and E. nigrum, respectively. This is the first report on positive effect of co-culture of paclitaxel-producing endophytic fungus and non-host plant cells for enhancing paclitaxel production.

Improved effects of polyethylene glycol on the growth, antioxidative enzymes activity and taxanes production in a Taxus baccata L. callus culture

Application of abiotic stress-inducing elicitors is one of the effective strategies in order to increase the production of secondary metabolites in plant tissues. Here, the effect of in vitro polyethylene glycol (PEG)-induced drought stress on morphological and biochemical changes as well as the production of paclitaxel and 10-deacetyl baccatin III in a Taxus baccata callus culture was investigated. The results showed that increased PEG concentration in the culture medium enhanced the levels of H2O2 and the membrane lipid peroxidation in the calli. The activity of catalase and guaiacol peroxidase as antioxidant responses were increased significantly in all PEG concentrations. At 6% of PEG concentration, the levels of fresh weight (FW) and dry weight (DW) in the calli were declined significantly by dropping in the amount of water content in the calli and rising the level of oxidative stress, and the morphological changes occurred following the increased browning index of the calli. However, the PEG at low to moderate concentrations reduced the accumulation and oxidation of phenolic compounds in the calli through the absorption of phenolic compounds and the reduction in the activity of the polyphenol oxidase. As a result, improved growth and viability of the calli resulted in the enhanced levels of FW and DW at the concentrations of 1% up to 3% (w/v) PEG. The highest contents of 10-deacetyl baccatin III and taxol were obtained at concentrations of 2% and 3% PEG, respectively. The concentration of 3% PEG increased the production of taxol by 2.5 times more than the control. In conclusion, PEG can be proposed as an effective elicitor in the production of taxanes in the Taxus cell and tissue cultures.

Bioprocess optimization using response surface methodology for production of the anticancer drug paclitaxel by Aspergillus fumigatus and Alternaria tenuissima: Enhanced production by ultraviolet and gamma irradiation

Abstract Paclitaxel is the most profitable anticancer drug in history; however, the yield of paclitaxel from fungal fermentation is still far from the commercial purpose. In this study, two local fungal endophytes of Aspergillus fumigatus TXD105 and Alternaria tenuissima TER995 were used as promising sources for the production of paclitaxel under submerged fermentation. In order to improve the paclitaxel magnitude, response surface methodology optimization program was applied to medium constituents that showed the high contribution to paclitaxel production. Moreover, improvement of paclitaxel production by UV and gamma irradiation was studied. The use of response surface methodology resulted in significant increases in the paclitaxel production of the two strains by 16.25 and 19.86%, respectively. Furthermore, the production of paclitaxel by the two strains was greatly enhanced by the application of UV and gamma irradiation. Using 0.75 KGy of gamma irradiation, paclitaxel concentrations were intensified to 495.31 and 195.41 μg L−1, respectively. The obtained paclitaxel concentrations by the two strains irradiated by UV for 30 min were 1.16 and 1.20 fold, respectively of their controls. These findings showed a great potential for production of paclitaxel by fungal cultures in industrial scale that indicate the future possibility to reduce the cost of producing fermentation-based drugs.

In vitro enzymatic synthesis of baccatin III with novel and cheap acetyl donors by the recombinant taxoid 10β-O-acetyl transferase

Despite the importance of baccatin III as a precursor to paclitaxel, a widely used chemotherapeutic agent, efficient enzymatic synthesis methods are lacking. Therefore, in this study, the recombinant taxoid 10β-O-acetyl transferase was prepared to produce baccatin III in vitro. The recombinant enzyme could use vinyl acetate, butyl acetate, sec-butyl acetate, isobutyl acetate, amyl acetate, and isoamyl acetate as novel and cheap alternative acetyl group donors to replace the expensive acetyl CoA for the enzymatic synthesis of baccatin III. A molecular docking study further confirmed that these acetyl donors could reasonably bind to the enzyme molecule. Using the aqueous two-phase bio-catalytic reaction system, hexane and ethyl acetate could increase the yield of product baccatin III by 2.8% and 1.1% respectively. This approach using novel and cheap acetyl donors is promising for the enzymatic synthesis of baccatin III for the future industrial production of paclitaxel.

Effect of Methyl Jasmonate on Production of Paclitaxel and Related Taxanes in a Hydrophobic Ionic Liquid-Medium Two Phase Culture System

Effect of Methyl Jasmonate on Production of Paclitaxel and Related Taxanes in a Hydrophobic Ionic Liquid-Medium Two Phase Culture System

A decision support framework and system for design of sustainable pharmaceutical supply chain network

With the issues of climate change continuously taking center stage, there is thus a pressing need for the pharmaceutical companies to assess the sustainability of their supply chains. However, the highly regulated nature of the industry has prompted various supply chain decisions such as material sourcing, siting of plants, and distribution network configuration to be locked into the registration of the new drug. This could consequently lead to inefficient supply chain operation during the drug commercial stage, yielding high greenhouse gas emissions per unit of drug product. Previously, we have proposed a systematic framework for design of a sustainable commercial-stage pharmaceutical supply chain network that can be driven from the early drug development stages. In this paper, we extend the framework by incorporating more functionalities. We also introduce a novel decision support system, called PharmaSC, which implements the framework. PharmaSC system has been developed by integrating two computer tools—an Excel-based analytic hierarchy process and LLamasoft’s Supply Chain Guru—into one coherent framework for selecting the most suitable suppliers and manufacturers, mapping a supply chain superstructure network, and optimizing the network to identify and resolve trade-offs between economic and environmental factor. To illustrate the system, an industrially motivated case study involving a paclitaxel production is solved.

Identification of rate-limiting enzymes involved in paclitaxel biosynthesis pathway affected by coronatine and methyl-β-cyclodextrin in Taxus baccata L. cell suspension cultures.

BackgroundPaclitaxel is a potent antitumor alkaloid widely used for the treatment of several cancer types. This valuable secondary metabolite naturally exists in the inner bark of Taxus species in very low amounts. The small-scale production of paclitaxel in Taxus cell cultures requires utilization of several elicitors.ObjectiveThe main objective of this work was to identify key genes that encode rate-limiting enzymes in paclitaxel biosynthesis pathway by investigating the possible relationship between paclitaxel production and a set of 13 involved genes’ relative expression in Taxus baccata L. cell suspension cultures affected by coronatine and methyl-β-cyclodextrin.MethodsIn the present research, the most important key genes were identified using gene expression profiling evaluation and paclitaxel production assessment in Taxus baccata L. cell cultures affected by mentioned elicitors.Results and ConclusionGene expression levels were variably increased using methyl-β-cyclodextrin, and in some cases, a synergistic effect on transcript accumulation was observed when culture medium was supplemented with both elicitors. It was revealed that DBAT, BAPT, and DBTNBT are the most important rate-limiting enzymes in paclitaxel biosynthesis pathway in Taxus baccata L. cell suspension cultures under coronatine and methyl-β-cyclodextrin elicitation condition. Moreover, PAM was identified as one of the important key genes especially in the absence of β-phenylalanine. In cell cultures affected by these elicitors, paclitaxel was found largely in the culture media (more than 90%). The secretion of this secondary metabolite suggests a limited feedback inhibition and reduced paclitaxel toxicity for producer cells. It is the result of the ABC gene relative expression level increment under methyl-β-cyclodextrin elicitation and highly depends on methyl-β-cyclodextrin’s special property (complex formation with hydrophobic compounds). Paclitaxel biosynthesis was obviously increased due to the effect of coronatine and methyl-β-cyclodextrin elicitation, leading to the production level of 5.62 times higher than that of the untreated cultures. Graphical abstractRate Limiting Enzymes in Paclitaxel Biosynthesis Pathway: DBAT, BAPT, DBTNBT and PAM.

A cellulase-supported two-phase in situ system for enhanced biosynthesis of paclitaxel in Taxus \xd7 media hairy roots

The effectiveness of two liquid-phase culture systems, in situ supported with perfluorodecalin (PFD), and elicited with methyl jasmonate (100 µM) and sodium nitroprusside (10 µM) (spiked with l-phenylalanine (100 µM) and sucrose (30 g/l) feeding), and additionally combined with cellulolytic enzyme application (Cellulase or Viscozyme® L at doses 0.01%, 0.1%, 0.5% or 1%), was investigated on the enhancement of paclitaxel release in Taxus × media harbouring taxadiene synthase transgene hairy root cultures. Neither elicitation nor in situ application of PFD significantly had an effect on root growth until enzyme addition; however, the hairy root response to the culture conditions varied depending on enzyme type and dosage. The highest paclitaxel total yield (intracellular + extracellular) was determined in the one-phase elicited culture systems without enzymes and amounted to 264.2 µg/flask (1 434.9 ± 516.6 µg/g DW) and 29.6 µg/flask in root biomass and medium phase, respectively. Although the application of cellulolytic enzymes did not enhance the total paclitaxel production, they intensified paclitaxel release in a dose-depending manner. Among two examined cellulolytic enzyme treatments, Viscozyme® L addition caused the highest paclitaxel release up to 59% of its total content when used at a concentration of 0.01%. Whereas in two-phase in situ systems, the combination of Viscozyme® L at dosage of 0.5% and PFD-aerated, resulted in the highest extracellular paclitaxel concentration up to 20%.

Elicitors derived from endophytic fungi Chaetomium globosum and Paraconiothyrium brasiliense enhance paclitaxel production in Corylus avellana cell suspension culture

Paclitaxel is an impressive chemotherapeutic agent that shows biological activity against a wide range of cancers. Cell suspension culture of Corylus avellana has been reported as an alternative source for production of the valuable secondary metabolite, paclitaxel. The fungal elicitors have been considered as the most impressive strategy for enhancement of secondary metabolites in plant cell culture. In this study, two endophytic fungal strains YEF20 and HEF114 were isolated from Taxus baccata and C. avellana, respectively. The isolates YEF20 and HEF114 were identified as Chaetomium globosum and Paraconiothyrium brasiliense, respectively by sequencing of ITS1-5.8S-ITS2 rDNA region. This is the first report of P. brasiliense on C. avellana tree. Also, this study presents the positive effect of fungal elicitors on paclitaxel production in C. avellana for the first time. The effect of fungal elicitors on paclitaxel production was dependent on fungal species, and also type, concentration and adding time of elicitors to cell culture. The highest total yield of paclitaxel in cell culture treated with cell extract (CE) of C. globosum (291.5 µg L−1) was obtained by using 10% (v/v) of this fungal elicitor on 17th day of cell culture cycle (late log-phase), which was about 4.1 times obtained in the control culture. The cell culture treated with C. globosum CE had an average growth rate of 0.491 g L−1 day−1, i.e. 12.3% lower than that in the control. Among the fungal elicitors, 10% (v/v) CE of C. globosum on 17th day of culture cycle showed the best results with respect to extracellular paclitaxel portion (44.0%). Paclitaxel secretion to culture medium is essential for large-scale steady production of paclitaxel.

Elicitors Derived from Hazel (Corylus avellana L.) Cell Suspension Culture Enhance Growth and Paclitaxel Production of Epicoccum nigrum

The microbial fermentation is considered as the potential source for large-scale production of paclitaxel. Since co-cultivation/mixed fermentation strategy has been reported as a yield enhancement strategy for paclitaxel production, investigation of fungal endophyte response to plant culture medium, plant cell extract (CE) and medium filtrate (MF) of plant cell suspension culture in terms of growth and paclitaxel production is interesting. In this study, 35 endophytic fungi were isolated from Taxus baccata and Corylus avellana grown in Iran. The analysis of high-performance liquid chromatography and mass spectrometry showed that one isolate (YEF2) produced paclitaxel. The isolate YEF2 was identified as Epicoccum nigrum by sequencing of ITS1-5.8S-ITS2 rDNA region and actin gene. YEF2 was slow-growing in Murashige and Skoog medium, but the synergistic interaction of gibberellic acid (GA3) and CE of C. avellana enhanced the growth of YEF2. The highest total yield of paclitaxel (314.7 µg/l; 11.5-folds) of E. nigrum strain YEF2 was obtained by using 28% (v/v) filter sterilized CE of C. avellana and 2 µg ml−1 GA3 that was significantly higher than the control. In this study, the effects of the plant cell extract on growth and paclitaxel production of paclitaxel producing endophytic fungus were studied for the first time.

Advances in the Regulation of In Vitro Paclitaxel Production: Methylation of a Y-Patch Promoter Region Alters BAPT Gene Expression in Taxus Cell Cultures.

Plant cell biofactories represent a promising solution to the increasing demand for plant-derived compounds, but there are still limiting factors that prevent optimal production, including the loss of yield during in vitro maintenance. Our results reveal a clear correlation between genomic methylation levels and a progressive decline in taxane production in Taxus spp. cell cultures. A comparative study of two cell lines, one 10 years old and low productive and the other new and high productive, revealed important differences in appearance, growth, taxane accumulation and expression levels of several taxane biosynthetic genes. Differences in taxane content and gene expression profile indicate an altered pathway regulation and that the BAPT gene, located in the center of the expression network of taxane biosynthetic genes, is active in a potentially flux-limiting step. The methylation patterns of the BAPT gene were studied in both cell lines by bisulfite sequencing, which revealed high rates of CHH methylated cytosines on the core promoter. Using a bioinformatics approach, this hotspot was identified as a Y-patch promoter element. The Y-patch may play a key role in the epigenetic regulation of the taxane biosynthetic pathway, which would open up novel genetic engineering strategies toward stable and high productivity.

Identification of early jasmonate-responsive genes in Taxus × media cells by analyzing time series digital gene expression data.

Jasmonate, an effective elicitor, can induce the biosynthesis of paclitaxel, a well-known anticancer drug, in Taxus cell culture. The jasmonate signaling pathway has been well studied in Arabidopsis, and many early jasmonate-responsive genes have beenfound to be involved in signaling pathway. In Taxus, only a few late jasmonate-responsive genes related to paclitaxel biosynthesis were identified. So, identification of early responsive genes and knowledge of the jasmonate signaling pathway are essential for understanding the effects of jasmonate on paclitaxel biosynthesis and for improving paclitaxel production in Taxus cells. In this study, total RNA of Taxus × media cells cultured in liquid medium was extracted after 0, 0.5, 3, and 24h of methyl jasmonate treatment. Three biological independent repetitions were performed. The 12 extracted RNA samples were integrated and sequenced on an Illumina HiSeq2500 platform using the paired-end method. A total of 45,583 transcript clusters were obtained by de novo assembly of the sequenced reads. Based on the transcriptome data, the digital gene expressions of each RNA sample were investigated. We found that after 0.5, 3, and 24h of methyl jasmonate treatment; 134, 1008, and 987 unigenes were differentially expressed. For the secondary metabolism pathways, phenylalanine pathway unigenes were responsive to jasmonate after 3h of treatment, while genes related to paclitaxel biosynthesis were induced after 0.5h of treatment. The digital gene expression levels of candidate genes related to paclitaxel biosynthesis were confirmed by qRT-PCR. Transcriptome sequencing and digital gene expression profiling identified early jasmonate-responsive genes in cultured Taxus × media cells. The comprehensive time series jasmonate-responsive gene expression data have provided transcriptome-wide information about the mechanism of paclitaxel biosynthesis regulation by jasmonate signaling.

Development of an Agrobacterium-mediated transformation method for Taxus 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.

A Novel Medium for Enhancing Callus Growth of Hazel (Corylus avellana L.)

Paclitaxel is a powerful antimitotic agent with excellent activity against a range of cancers. Hazel has been described as a paclitaxel-producing species among angiosperms. Fast-growing callus is a prerequisite for the success of callus production and then paclitaxel production. Therefore, optimizing the medium culture for enhancing callus growth is a crucial step for paclitaxel production. In this research, Murashige and Skoog (1962) (MS) medium was optimized for improving callus growth of hazel (Corylus avellana L.). The M10 medium (MS medium with pH 6.0 and supplemented with 1000 mg l−1 spirulina powder, 1000 mg l−1 casein hydrolysate and 3 g l−1 gelrite) significantly improved hazel callus growth. This modified MS medium increased callus fresh weight (55.8%) as compared to the control. M10 medium increased fatty acids yield of callus (66.7%) as compared to the control. Liquid M10 medium maintained growth over a longer period of time and also increased slightly, the paclitaxel production as compared to the control. This novel medium is promising for facilitating the mass production of hazel callus as a source of valuable metabolites including paclitaxel, linoleic and oleic acids.

Production of paclitaxel with anticancer activity by two local fungal endophytes, Aspergillus fumigatus and Alternaria tenuissima.

Among 60 fungal endophytes isolated from twigs, bark, and mature leaves of different plant species, two fungal isolates named TXD105 and TER995 were capable of producing paclitaxel in amounts of up to 84.41 and 37.92μg L-1, respectively. Based on macroscopic and microscopic characteristics, ITS1-5.8S-ITS2 rDNA sequence, and phylogenetic characteristic analysis, the two respective isolates were identified as Aspergillus fumigatus and Alternaria tenuissima. In the effort to increase paclitaxel magnitude by the two fungal strains, several fermentation conditions including selection of the proper fermentation medium, agitation rate, incubation temperature, fermentation period, medium pH, medium volume, and inoculum nature (size and age of inoculum) were tried. Fermentation process carried out in M1D medium (pH6.0) and maintained at 120rpm for 10days and at 25°C using 4% (v/v) inoculum of 5-day-old culture stimulated the highest paclitaxel production to attain 307.03μg L-1 by the A. fumigatus strain. In the case of the A. tenuissima strain, fermentation conditions conducted in flask basal medium (pH6.0) and maintained at 120rpm for 14days and at 25°C using 8% (v/v) inoculum of 7-day-old culture were found the most favorable to attain the highest paclitaxel production of 124.32μg L-1. Using the MTT-based assay, fungal paclitaxel significantly inhibited the proliferation of five different cancer cell lines with 50% inhibitory concentration values varied from 3.04 to 14.8μg mL-1. Hence, these findings offer new and alternate sources with excellent biotechnological potential for paclitaxel production by fungal fermentation.

Response and Defense Mechanisms of Taxus chinensis Leaves Under UV-A Radiation are Revealed Using Comparative Proteomics and Metabolomics Analyses.

Taxus chinensis var. mairei is a species endemic to south-eastern China and one of the natural sources for the anticancer medicine paclitaxel. To investigate the molecular response and defense mechanisms of T. chinensis leaves to enhanced ultraviolet-A (UV-A) radiation, gel-free/label-free and gel-based proteomics and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The transmission electron microscopy results indicated damage to the chloroplast under UV-A radiation. Proteomics analyses in leaves and chloroplasts showed that photosynthesis-, glycolysis-, secondary metabolism-, stress-, and protein synthesis-, degradation- and activation-related systems were mainly changed under UV-A radiation. Forty-seven PSII proteins and six PSI proteins were identified as being changed in leaves and chloroplasts under UV-A treatment. This indicated that PSII was more sensitive to UV-A than PSI as the target of UV-A light. Enhanced glycolysis, with four glycolysis-related key enzymes increased, provided precursors for secondary metabolism. The 1-deoxy-d-xylulose-5-phosphate reductoisomerase and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase were identified as being significantly increased during UV-A radiation, which resulted in paclitaxel enhancement. Additionally, mRNA expression levels of genes involved in the paclitaxel biosynthetic pathway indicated a down-regulation under UV-A irradiation and up-regulation in dark incubation. These results reveal that a short-term high dose of UV-A radiation could stimulate the plant stress defense system and paclitaxel production.