Taxol Production Research Articles

Enhancement of Taxol production by applying amino acid complex along with chitosan in suspension culture of Taxus baccata L.

Enhancement of Taxol production by applying amino acid complex along with chitosan in suspension culture of Taxus baccata L.

Isolation Of Anticancer Drug Taxol Producing Endophytic Fungus Alternaria Alternata By Using HPLC And LC- MS

Abstract Introduction/Objective In the exploration of an alternative source, optimum and numerous solvents with different percentage were exploited to extract Taxol from plant palm branche. The fungal endophytes producing secondary metabolites that are effective against the human infections have fascinated many researchers across the world. Among these, the exploration for novel cancer therapeutic agents is of countless reputation due to upsurge in the number of cancer deaths, high worth of the drugs and the side effects concomitant with cancer treatments. Accordingly, in the current study, an endophytic fungal strain was isolated from its host plant palm branche and identified as Alternaria Alternata based on its morphological features and internal transcribed spacer sequence analysis. Methods Firstly, palm branche were collected for the isolation of endophytic fungi. The samples were placed on PDA plates and after 72 h, the mycelium produced were isolated and sub-cultured. Secondly, further analysis for the presence of Taxol by using chromatographic and spectrometric techniques. Illustratively, ethanol or acetone and 50% and 20% of ethanol. Then, the residues were dissolved in 5 ml of methanol and filtered. By using the internal standard, the resulting solution was subjected to the HPLC to determine the extraction efficiencies of tested solvents. Finally, filtrate was also subjected to the LC-MS – ESI using water/acetonitrile (10/90) as mobile phase to identify the authenticity of Taxol Results HPLC analysis revealed that Taxol could be quantified by UV detection using standard curve. Furthermore, the standard curve covering the concentration was linear (r2= 0.9992) and CV% ranged from 0.52 to 15.36. The peaks of fraction 5 obtained by HPLC analysis were quite similar to that of standard taxol confirming the presence of taxol. A parent ion peak of m/z 851.85 was observed in the LC–ESI–MS spectrum which was similar to paclitaxel with reported m/z of 854 [M+H] + ion. A. Alternata produced about 113.193 mg/L taxol as quantified through HPLC Conclusion we are recording for the first time the production of taxol by an endophytic fungus Alternaria Alternata. In a final word, present study results advocate that the endophytic fungus A.Alternata aids as a potential source for the production of taxol isolated from Palm branche plant

Detection, Purification and Elucidation of Chemical Structure and Antiproliferative Activity of Taxol Produced by Penicillium chrysogenum.

Penicillium chrysogenum has been reported as a potent taxol producer based on quantitative analysis by TLC and HPLC. The biosynthetic potency of taxol has been validated from PCR detection of rate-limiting genes of taxol synthesis such as taxadienesynthase and 10-de-acetylbaccatin III-O-acetyltransferase (DBAT), which catalyzes the immediate diterpenoid precursor of the taxol substance, as detected by PCR. Taxol production by P. chrysogenum was assessed by growing the fungus on different media. Potato dextrose broth (PDB) was shown to be the best medium for obtaining the higher amount of taxol (170 µg/L). A stepwise optimization of culture conditions necessary for production of higher amounts of taxol was investigated. The substance taxol was produced optimally after 18 d of incubation at 30 °C in PDB adjusted initially at pH 8.0 with shaking (120 rpm) (250 µg/L). The P. chrysogenum taxol was purified successfully by HPLC. Instrumental analyzes such as Fourier transform infrared spectroscopy (FTIR), ultraviolet (UV) spectroscopy, 1HNMR and 13C NMR approved the structural formula of taxol (C47H51NO14), as constructed by ChemDraw. The P. chrysogenum taxol showed promising anticancer activity.

Correction to: Polypore fungi of Caucasian alder as a source of antioxidant and antitumor agents

As determined through 11 subcultures of Taxus cuspidata callus, the growth in FW and level of Taxol production were stable by the ninth generation.

Production of the anticancer drug taxol by the endophytic fungus Epicoccum nigrum TXB502: enhanced production by gamma irradiation mutagenesis and immobilization technique.

Taxol, a phyto-extracted diterpenoid, is the most commercially needed drug in cancer chemotherapy. In spite of the microbial production of taxol being successful and prospective, the reported yields are still not sufficient for large-scale production. Thus, the discovery of new taxol-producing microbial strains and production enhancement methodologies such as process optimization, strain improvement, and immobilization technique are the main objectives. In this paper, a taxol-producing start strain Epicoccum nigrum TXB502 (initial yield 61.35 μg L-1) was isolated from Taxus baccata and identified by morphological and molecular tools. The optimum cultivation and nutritional conditions were assessed by testing one parameter at a time approach that resulted in 88.59% significant production increase. In addition, a stable mutant with improved productivity (40.07% yield increase in comparison with the parent strain) was successfully developed after gamma irradiation mutagenesis of the start strain. The taxol titer was further improved via testing different immobilization carriers for both spores and mycelia of this mutant. Over taxol production was achieved using alginate-immobilized mycelia with the feasibility of conducting six successive production cycles in a semi-continuous form. The final total concentration reached 8187.77 μg taxol 6 L-1 which represents approximately 22-fold increase, as compared to the initial titer of the start strain. These findings can pave the way for the prospective industrial manufacturing of taxol, as the achieved taxol production in this study is the highest reported by academic laboratories for microbial cultures. KEY POINTS: • Discovery of a new taxol-producing endophytic fungus E. nigrum TXB502 strain. • Taxol yield was successfully improved via bioprocess optimization and strain mutagenesis. • Alginate-immobilized mycelia were efficient for a semi-continuous production of taxol. • The final total concentration of taxol showed approximately 22-fold increase as compared to the initial titer.

Exploiting the Biosynthetic Potency of Taxol from Fungal Endophytes of Conifers Plants; Genome Mining and Metabolic Manipulation.

Endophytic fungi have been considered as a repertoire for bioactive secondary metabolites with potential application in medicine, agriculture and food industry. The biosynthetic pathways by fungal endophytes raise the argument of acquisition of these machineries of such complex metabolites from the plant host. Diterpenoids “Taxol” is the most effective anticancer drug with highest annual sale, since its discovery in 1970 from the Pacific yew tree, Taxus brevifolia. However, the lower yield of Taxol from this natural source (bark of T. brevifolia), availability and vulnerability of this plant to unpredicted fluctuation with the ecological and environmental conditions are the challenges. Endophytic fungi from Taxus spp. opened a new avenue for industrial Taxol production due to their fast growth, cost effectiveness, independence on climatic changes, feasibility of genetic manipulation. However, the anticipation of endophytic fungi for industrial Taxol production has been challenged by the loss of its productivity, due to the metabolic reprograming of cells, downregulating the expression of its encoding genes with subculturing and storage. Thus, the objectives of this review were to (1) Nominate the endophytic fungal isolates with the Taxol producing potency from Taxaceae and Podocarpaceae; (2) Emphasize the different approaches such as molecular manipulation, cultural optimization, co-cultivation for enhancing the Taxol productivities; (3) Accentuate the genome mining of the rate-limiting enzymes for rapid screening the Taxol biosynthetic machinery; (4) Triggering the silenced rate-limiting genes and transcriptional factors to activates the biosynthetic gene cluster of Taxol.

Comparative transcriptome analysis of a taxol-producing endophytic fungus, Aspergillus aculeatinus Tax-6, and its mutant strain

Taxol is a rare but extremely effective antitumor agent extracted from Taxus yew barks. Taxus plants are valuable and rare species, and the production of taxol from them is a complex process. Therefore, taxol-producing endophytic fungi seem to be a promising alternative because of their high practical value and convenient progress. In this study, the transcriptome of an endophytic fungus, Aspergillus aculeatinus Tax-6 was analyzed in order to understand the molecular mechanisms of producing fungal taxol. The results showed that genes involved in the mevalonate (MVA) pathway and non-mevalonate (MEP) pathway were expressed, including isopentenyl pyrophosphate transferase, geranyl pyrophosphate transferase, and geranylgeranyl pyrophosphate synthetase. However, those downstream genes involved in the conversion of taxa-4(5)-11(12)-diene from geranylgeranyl pyrophosphate were not expressed except for taxane 10-beta-hydroxylase. Additionally, a mutant strain, A. aculeatinus BT-2 was obtained from the original strain, A. aculeatinus Tax-6, using fungicidin as the mutagenic agent. The taxol yield of BT-2 was 560 µg L−1, which was higher than that of Tax-6. To identify the mechanism of the difference in taxol production, we compared the transcriptomes of the two fungi and explored the changes in the gene expression between them. When compared with the original strain, Tax-6, most genes related to the MVA pathway in the mutant strain BT-2 showed upregulation, including GGPPS. Moreover, most of the downstream genes were not expressed in the mutant fungi as well. Overall, the results revealed the pathway and mechanism of taxol synthesis in endophytic fungi and the potential for the construction of taxol-producing genetic engineering strains.

The Research Progress of Taxol in Taxus.

Taxus is a valuable woody species with important medicinal value. The bark of Taxus can produce taxol, a natural antineoplastic drug that is widely used in the treatment of breast, ovarian and lung cancers. However, the low content of taxol in the bark of Taxus can not meet the growing clinical demands, so the current research aims at finding ways to increase taxol production. In this review, the research progress of taxol including the factors affecting the taxol content, biosynthesis pathway of taxol, production of taxol in vitro and the application of multi-omics approaches in Taxus as well as future research prospects will be discussed. The taxol content is not only dependent on the species, age and tissues but is also affected by light, moisture levels, temperature, soil fertility and microbes. Most of the enzymes in the taxol biosynthesis pathway have been identified and characterized. Total chemical synthesis, semi-synthesis, plant cell culture and biosynthesis in endophytic fungi have been explored to product taxol. Multi-omics have been used to study Taxus and taxol. Further efforts in the identification of unknown enzymes in the taxol biosynthesis pathway, establishment of the genetic transformation system in Taxus and the regulatory mechanism of taxol biosynthesis and Taxus cell growth will play a significant role in improving the yield of taxol in Taxus cells and plants.

Establishment of regenerative callus, cell suspension system, and molecular characterization of Taxus wallichiana Zucc. for the in vitro production of Taxol

Taxus wallichiana, an endangered Taxus species native to Nepal, is a source of Taxol (paclitaxel), an anticancer drug, which if produced via plant cell culture in vitro not only reduces the pressure on natural habitat but also allows continuous production throughout the seasons. Gamborg’s B5 medium was the culture medium to develop callus and suspension culture. Quantification of Taxol was carried out via high performance liquid chromatography (HPLC) using C18 column, water-acetonitrile mobile phase, and 1 ml/minute flowrate at 227 nm wavelength. Genetic variation among population was determined using the POPGEN32, version 1.31. Best callus response was in medium with 1 mg/l 2,4-D for both stem and needle explants. For cell suspension culture, callus from needle explants showed better response compared to stem. Fructose and sucrose proved to be the best carbon sources. Cell count increased as carbon concentration increased. 2,4-D at low concentration (5 mg/l) showed higher cell growth rate. Inoculum volume was directly proportional to cell count. Extra addition of macronutrients had inhibitory effect on cell count, but the effect was reversed for MgSO4. Using HPLC, for bark, the highest Taxol yield was 0.057 mg/g distilled water (DW), while for needle was 0.056 mg/g DW. For suspension cultured HPLC samples, Taxol yield ranged at 0.004%. Size range of amplified Deoxyribonucleic acids was between 200 and 2,000 bp. Thus, this investigation generated a concrete data for Taxus population of Nepal providing efficient protocols for establishment of callus and cell suspension culture with the aim of producing Taxol in in vitro conditions.

Biochemical characterization of acyl activating enzymes for side chain moieties of Taxol and its analogs

Taxol (paclitaxel) is a very widely used anticancer drug, but its commercial sources mainly consist of stripped bark or suspension cultures of members of the plant genus Taxus. Taxol accumulates as part of a complex mixture of chemical analogs, termed taxoids, which complicates its production in pure form, highlighting the need for metabolic engineering approaches for high-level Taxol production in cell cultures or microbial hosts. Here, we report on the characterization of acyl-activating enzymes (AAEs) that catalyze the formation of CoA esters of different organic acids relevant for the N-substitution of the 3-phenylisoserine side chain of taxoids. On the basis of similarities to AAE genes of known function from other organisms, we identified candidate genes in publicly available transcriptome data sets obtained with Taxus × media. We cloned 17 AAE genes, expressed them heterologously in Escherichia coli, purified the corresponding recombinant enzymes, and performed in vitro assays with 27 organic acids as potential substrates. We identified TmAAE1 and TmAAE5 as the most efficient enzymes for the activation of butyric acid (Taxol D side chain), TmAAE13 as the best candidate for generating a CoA ester of tiglic acid (Taxol B side chain), TmAAE3 and TmAAE13 as suitable for the activation of 4-methylbutyric acid (N-debenzoyl-N-(2-methylbutyryl)taxol side chain), TmAAE15 as a highly efficient candidate for hexanoic acid activation (Taxol C side chain), and TmAAE4 as suitable candidate for esterification of benzoic acid with CoA (Taxol side chain). This study lays important groundwork for metabolic engineering efforts aimed at improving Taxol production in cell cultures.

Mycosynthesis of anticancer drug taxol by Aspergillus oryzae, an endophyte of Tarenna asiatica, characterization, and its activity against a human lung cancer cell line

Abstract Taxol is a member of the taxane family of chemotherapy drugs, which includes cabazitaxel and docetaxel that have a promising role as anti-cancer drugs for specific human cancers. Hence, in order to meet the requirement, an alternative source for Taxol production, such as filamentous fungi is needed. In this study, a different endophytic fungus was obtained from a particular medicinal plant, Tarenna asiatica, and was selected for Taxol production. Among 28 screened fungi, only six fungal species produced Taxol in the modified liquid medium. The novel Taxol-producing endophytic fungi were identified through morphological and molecular identification using DNA sequencing and phylogenetic tree construction as Aspergillus oryzae. The structure of Taxol obtained from A. oryzae was identified with the appropriate control using an ultraviolet–visible (UV–Vis) spectrophotometer (235 nm), thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), and Fourier-transform infrared (FTIR) analysis. The fungal Taxol (95.04 μg/L) from the A. oryzae exhibits potent in vitro activity against selected cell line (Human Lung Cancer Cell Line- NCI–H460).

Semi-continuous production of the anticancer drug taxol by Aspergillus fumigatus and Alternaria tenuissima immobilized in calcium alginate beads.

Taxol is the most profitable drug ever developed in cancer chemotherapy; however, the market demand for the drug greatly exceeds the supply that can be sustained from its natural sources. In this study, Aspergillus fumigatus TXD105-GM6 and Alternaria tenuissima TER995-GM3 were immobilized in calcium alginate beads and used for the production of taxol in shake flask cultures. In an effort to increase the taxol magnitude, immobilization conditions were optimized by response surface methodology program (RSM). The optimum levels of alginate concentration, calcium chloride concentration, and mycelium fresh weight were 5%, 4%, and 15% (w/v), respectively. Under these conditions, taxol production by the respective fungal strains was intensified to 901.94μg L-1 and 529.01μgL-1. Moreover, the immobilized mycelia of both strains were successfully used in the repeated production of taxol for six different fermentation cycles. The total taxol concentration obtained in all cycles reached 4540.14μgL-1 by TXD105-GM6 and 2450.27μgL-1 by TER995-GM3 strain, which represents 7.85- and 6.31-fold increase, as compared to their initial titers. This is the first report on the production of taxol in semi-continuous fermentation. To our knowledge, the taxol productivity achieved in this study is the highest reported by academic laboratories for microbial cultures which indicates the future possibility to reduce the cost of taxol production.

Hitherto Unknown Terpene Synthase Organization in Taxol-Producing Endophytic Bacteria Isolated from Marine Macroalgae.

Taxol is a successful anti-cancer drug, which extensively studied in Taxus spp. However, microbial endophytes also reported as taxol producers, and especially fungal endophytes extensively studied for the taxol biosynthesis pathway. Although it was well considered, the taxol biosynthesis pathway remains undisclosed since its discovery in bacteria. To decipher this gap, we isolated and identified the endophytic bacteria such as Bacillus flexus strain DMTMMB08, Bacillus licheniformis strain DMTMMB10, and Oceanobacillus picturae strain DMTMMB24, which are unprecedented for taxol production. Subsequently, the genome annotation of these bacteria exhibited the isoprene biosynthesis pathway and terpene synthase profile. Feasibly, this is the very first report on taxol-producing endophytic bacteria from the non-Taxus host and solitary investigation on its genome analysis. The genomic insight into the bacterial system for taxol biosynthesis leads to understanding the terpene synthesis and evolution. This piece of work could expand our perception of the diversity of terpenes and their related natural products.

Conjugation of Aspergillus flavipes Taxol with Porphyrin Increases the Anticancer Activity of Taxol and Ameliorates Its Cytotoxic Effects.

Taxol is one of the potential anticancer drugs; however, the yield of Taxol and its cytotoxicity are common challenges. Thus, manipulating the Taxol biosynthetic pathway from endophytic fungi, in addition to chemical modification with biocompatible polymers, is the challenge. Four fungal isolates, namely, Aspergillus flavipes, A. terreus, A. flavus, and A. parasiticus, were selected from our previous study as potential Taxol producers, and their potency for Taxol production was evaluated in response to fluconazole and silver nitrate. A higher Taxol yield was reported in the cultures of A. flavipes (185 µg/L) and A. terreus (66 µg/L). With addition of fluconazole, the yield of Taxol was increased 1.8 and 1.2-fold for A. flavipes and A. terreus, respectively, confirming the inhibition of sterol biosynthesis and redirecting the geranyl phosphate pool to terpenoids synthesis. A significant inhibition of ergosterol biosynthesis by A. flavipes with addition of fluconazole was observed, correlating with the increase on Taxol yield. To increase the Taxol solubility and to reduce its cytotoxicity, Taxol was modified via chemical conjugation with porphyrin, and the degree of conjugation was checked from the Thin layer chromatography and UV spectral analysis. The antiproliferative activity of native and modified Taxol conjugates was evaluated; upon porphyrin conjugation, the activity of Taxol towards HepG2 was increased 1.5-fold, while its cytotoxicity to VERO cells was reduced 3-fold.

Production and Characterization of Taxol as Anticancer Agent from Aspergillus terreus

Production and Characterization of Taxol as Anticancer Agent from Aspergillus terreus

Hyper-production of taxol from Aspergillus fumigatus, an endophytic fungus isolated from Taxus sp. of the Northern Himalayan region

Taxol® (generic name Paclitaxel) is a chemotherapeutic drug, effective against head, neck, breast, lung, bladder, ovary, and cervix cancers. Rising demands in chemotherapy and limited supply of natural taxol have ultimately increased the cost of the drug. Semi synthesis using taxol precursors is not able to meet the global supply and has intensified the need to find alternative ways of taxol production. In the present study, 34 different endophytes were isolated from Taxus sp. collected from Shimla, Himachal Pradesh (India). Primary screening of taxol-producing fungi was carried out based on the presence of dbat gene, essential for the taxol biosynthetic pathway. A fungal isolate TPF-06 was screened to be a taxol-producing strain based on the PCR amplification results. It was characterized and identified as Aspergillus fumigatus by 18S rRNA (Accession No. KU-837249). Multiple sequence alignment (MSA) of nuclear ribosomal internal transcribed spacer (ITS) region and phylogenetic analysis confirmed that strain belonged to A. fumigatus clade (Accession No. MF-374798) and is endophytic in nature. Presence of taxol was detected and quantified by High-Performance Liquid Chromatography (HPLC) and characterized by using Thin Layer Chromatography (TLC), Ultraviolet (UV) spectroscopy, Mass spectrometry (MS), Fourier-Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopy. Microbial fermentation in the S7 medium yielded 1.60 g/L of taxol, which to the best of our knowledge is the highest taxol production from an endophytic fungus. Findings of the present study suggest that the A. fumigatus is an excellent alternate source for taxol supply, and it may become a highly potent strain on a commercial scale. The involvement of dbat gene in A. fumigatus KU-837249 strain further suggested a way of increasing taxol yield in fungi by medium engineering and recombinant DNA technology in the future.

Chloroplastic metabolic engineering coupled with isoprenoid pool enhancement for committed taxanes biosynthesis in Nicotiana benthamiana

Production of the anticancer drug Taxol and its precursors in heterologous hosts is more sustainable than extraction from tissues of yew trees or chemical synthesis. Although attempts to engineer the Taxol pathway in microbes have made significant progress, challenges such as functional expression of plant P450 enzymes remain to be addressed. Here, we introduce taxadiene synthase, taxadiene-5α-hydroxylase, and cytochrome P450 reductase in a high biomass plant Nicotiana benthamiana. Using a chloroplastic compartmentalized metabolic engineering strategy, combined with enhancement of isoprenoid precursors, we show that the engineered plants can produce taxadiene and taxadiene-5α-ol, the committed taxol intermediates, at 56.6 μg g−1 FW and 1.3 μg g−1 FW, respectively. In addition to the tools and strategies reported here, this study highlights the potential of Nicotiana spp. as an alternative platform for Taxol production.

Restoring the Taxol biosynthetic machinery of Aspergillus terreus by Podocarpus gracilior Pilger microbiome, with retrieving the ribosome biogenesis proteins of WD40 superfamily

Attenuating the Taxol yield of Aspergillus terreus with the subculturing and storage were the technical challenges that prevent this fungus to be a novel platform for industrial Taxol production. Thus, the objective of this study was to unravel the metabolic machineries of A. terreus associated with attenuation of Taxol productivity, and their restoring potency upon cocultivation with the Podocarpus gracilior microbiome. The Taxol yield of A. terreus was drastically reduced with the fungal subculturing. At the 10th subculture, the yield of Taxol was reduced by four folds (78.2 µg/l) comparing to the original culture (268 µg/l), as authenticated from silencing of molecular expression of the Taxol-rate limiting enzymes (GGPPS, TDS, DBAT and BAPT) by qPCR analyses. The visual fading of A. terreus conidial pigmentation with the subculturing, revealing the biosynthetic correlation of melanin and Taxol. The level of intracellular acetyl-CoA influx was reduced sequentially with the fungal subculturing, rationalizing the decreasing on Taxol and melanin yields. Fascinatingly, the Taxol biosynthetic machinery and cellular acetyl-CoA of A. terreus have been completely restored upon addition of 3% surface sterilized leaves of P. gracilior, suggesting the implantation of plant microbiome on re-triggering the molecular machinery of Taxol biosynthesis, their transcriptional factors, and/or increasing the influx of Acetyl-CoA. The expression of the proteins of 74.4, 68.2, 37.1 kDa were exponentially suppressed with A. terreus subculturing, and strongly restored upon addition of P. gracilior leaves, ensuring their profoundly correlation with the molecular expression of Taxol biosynthetic genes. From the proteomic analysis, the restored proteins 74.4 kDa of A. terreus upon addition of P. gracilior leaves were annotated as ribosome biogenesis proteins YTM and microtubule-assembly proteins that belong to WD40 superfamily. Thus, further ongoing studies for molecular cloning and expression of these genes with strong promotors in A. terreus, have been initiated, to construct a novel platform of metabolically stable A. terreus for sustainable Taxol production. Attenuating the Taxol yield of A. terreus with the multiple-culturing and storage might be due to the reduction on main influx of acetyl-CoA, or downregulation of ribosome biogenesis proteins that belong to WD40 protein superfamily.

Iso-Seq analysis of the Taxus cuspidata transcriptome reveals the complexity of Taxol biosynthesis

BackgroundTaxus cuspidata is well known worldwide for its ability to produce Taxol, one of the top-selling natural anticancer drugs. However, current Taxol production cannot match the increasing needs of the market, and novel strategies should be considered to increase the supply of Taxol. Since the biosynthetic mechanism of Taxol remains largely unknown, elucidating this pathway in detail will be very helpful in exploring alternative methods for Taxol production.ResultsHere, we sequenced Taxus cuspidata transcriptomes with next-generation sequencing (NGS) and third-generation sequencing (TGS) platforms. After correction with Illumina reads and removal of redundant reads, more than 180,000 nonredundant transcripts were generated from the raw Iso-Seq data. Using Cogent software and an alignment-based method, we identified a total of 139 cytochrome P450s (CYP450s), 31 BAHD acyltransferases (ACTs) and 1940 transcription factors (TFs). Based on phylogenetic and coexpression analysis, we identified 9 CYP450s and 7 BAHD ACTs as potential lead candidates for Taxol biosynthesis and 6 TFs that are possibly involved in the regulation of this process. Using coexpression analysis of genes known to be involved in Taxol biosynthesis, we elucidated the stem biosynthetic pathway. In addition, we analyzed the expression patterns of 12 characterized genes in the Taxol pathway and speculated that the isoprene precursors for Taxol biosynthesis were mainly synthesized via the MEP pathway. In addition, we found and confirmed that the alternative splicing patterns of some genes varied in different tissues, which may be an important tissue-specific method of posttranscriptional regulation.ConclusionsA strategy was developed to generate corrected full-length or nearly full-length transcripts without assembly to ensure sequence accuracy, thus greatly improving the reliability of coexpression and phylogenetic analysis and greatly facilitating gene cloning and characterization. This strategy was successfully utilized to elucidate the Taxol biosynthetic pathway, which will greatly contribute to the goals of improving the Taxol content in Taxus spp. using molecular breeding or plant management strategies and synthesizing Taxol in microorganisms using synthetic biological technology.

Isolation of taxol producing endophytic fungus Alternaria brassicicola from non-Taxus medicinal plant Terminalia arjuna.

In the present study, an endophytic fungal strain was isolated from its non-Taxus host plant Terminalia arjuna and identified as Alternaria brassicicola based on its morphological characteristics and internal transcribed spacer sequence analysis. This fungus was grown in potato dextrose broth and analyzed for the presence of taxol by using chromatographic and spectrometric techniques. The ethyl acetate extract of A.brassicicola was subjected to column chromatography. Among the different fractions, the fraction 7 showed positive to taxol, which was further confirmed by UV absorption, HPLC, FTIR spectra and LC-ESI-MS by comparing with the authentic taxol (Paclitaxel). The peaks of fraction 7 obtained by UV spectroscopy, FTIR and HPLC analysis were quite similar to that of standard taxol confirming the presence of taxol. A parent ion peak of m/z 854.95 was observed in the LC-ESI-MS spectrum which was similar to paclitaxel with reported m/z of 854 [M+H]+ ion. A. brassicicola produced about 140.8μg/l taxol as quantified through HPLC. Present study results suggest that the endophytic fungus A.brassicicola serves as a potential source for the production of taxol isolated from non-Taxus plant.