Paclitaxel Production Research Articles

Enhancement of paclitaxel production by Neopestalotiopsis vitis via optimization of growth conditions.

Accessibility of paclitaxel and other taxoids from natural resources is restricted. Endophytic fungi are novel, rapidly growing resources for producing these compounds. Neopestalotiopsis vitis (N. vitis) has been recently isolated from Corylus avellana, and its ability to produce a variety of taxoids has been detected and confirmed by analytical methods. Simultaneous growth and high production of taxoids by application of different sorts and concentrations of carbon and nitrogen were targeted in the present research. These criteria were assessed in different acidities (pH 4.0-7.0), carbon sources (sucrose, fructose, glucose, mannitol, sorbitol, and malt extract), and nitrogen forms (urea, ammonium nitrate, potassium nitrate, ammonium phosphate, and ammonium sulfate) by testing one parameter at a time approach. The first analysis introduced pH 7.0 as the best acidity of the medium for N. vitis, where the highest paclitaxel yield was generated. Further analysis introduced 3% Malt extract as the best carbon-providing medium. In the next step, the effects of nitrogen forms on the growth rate, paclitaxel yield, alkaloids, and amino acid contents were evaluated. Based on the results of this experiment, 5 mM ammonium sulfate was selected as the best nitrogen source to obtain the maximum biomass and paclitaxel yield. Overall, the results introduce a medium containing 3% (w/v) malt extract and 5 mM ammonium sulfate at pH 7.0 as the best medium in which N. vitis produces the highest paclitaxel yield coincident with rapid and sustainable growth. The findings pave the way for industrial manufacturing of taxoids.

Paclitaxel production from endophytic Mucor circinelloides isolated from Taxus sp. of the Northern Himalayan region.

The online version contains supplementary material available at 10.1007/s13205-024-04091-7.

A novel step towards the heterologous biosynthesis of paclitaxel: Characterization of T1βOH taxane hydroxylase

In the quest for innovative cancer therapeutics, paclitaxel remains a cornerstone in clinical oncology. However, its complex biosynthetic pathway, particularly the intricate oxygenation steps, has remained a puzzle in the decades following the characterization of the last taxane hydroxylase. The high divergence and promiscuity of enzymes involved have posed significant challenges. In this study, we adopted an innovative approach, combining in silico methods and functional gene analysis, to shed light on this elusive pathway. Our molecular docking investigations using a library of potential ligands uncovered TB574 as a potential missing enzyme in the paclitaxel biosynthetic pathway, demonstrating auspicious interactions. Complementary in vivo assays utilizing engineered S. cerevisiae strains as novel microbial cell factory consortia not only validated TB574's critical role in forging the elusive paclitaxel intermediate, T5αAc-1β,10β-diol, but also achieved the biosynthesis of paclitaxel precursors at an unprecedented yield including T5αAc-1β,10β-diol with approximately 40 mg/L. This achievement is highly promising, offering a new direction for further exploration of a novel metabolic engineering approaches using microbial consortia. In conclusion, our study not only furthers study the roles of previously uncharacterized enzymes in paclitaxel biosynthesis but also forges a path for pioneering advancements in the complete understanding of paclitaxel biosynthesis and its heterologous production. The characterization of T1βOH underscores a significant leap forward for future advancements in paclitaxel production using heterologous systems to improve cancer treatment and pharmaceutical production, thereby holding immense promise for enhancing the efficacy of cancer therapies and the efficiency of pharmaceutical manufacturing.

Endophytic Aspergillus fumigatiaffinis: Novel paclitaxel production and optimization insights

This study investigated the potential of endophytic fungi to produce paclitaxel (Taxol®), a potent anticancer compound widely employed in chemotherapy. This research aimed to identify, confirm, and characterize endophytic fungi capable of paclitaxel (PTX) production and assess their paclitaxel yield. Additionally, it aimed to investigate factors influencing paclitaxel production. A total of 100 endophytic fungal isolates were collected and identified from the roots of Artemisia judaica. Aspergillus fumigatiaffinis exhibited the highest PTX production (26.373 μg L−1) among the isolated endophytic fungi. The strain was identified as A. fumigatiaffinis (Accession No. PP235788.1). Molecular identification confirmed its novelty, representing the first report of PTX production by A. fumigatiaffinis, an endophyte of Artemisia judaica. Optimization through full factorial design of experiments (DOE) and response surface methodology (RSM) significantly enhanced PTX production to 110.23 μg L−1 from 1 g of dry weight of the fungal culture under optimal conditions of pH 8.0, 150 μg L−1 becozyme supplementation, and 18 days of fermentation in potato dextrose broth. The presence of paclitaxel was confirmed using thin layer chromatography, high performance liquid chromatography, and gas chromatography–mass spectrometry. These findings maximize the role of endophytic fungus to produce a secondary metabolite that might be able to replace the chemically produced PTX and gives an opportunity to provide a sustainable source of PTX eco-friendly at high concentrations.Key points• Endophytic fungi, like A. fumigatiaffinis, show promise for eco-friendly paclitaxel production• Optimization strategies boost paclitaxel yield significantly, reaching 110.23 μg L−1• Molecular identification confirms novelty, offering a sustainable PTX source

Improved taxadiene production by optimizing DXS expression and fusing short-chain prenyltransferases

This study highlights the significance of overexpressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from the MEP (methylerythritol 4-phosphate) pathway, in addition to short-chain prenyltransferase fusions for the improved production of the diterpene, taxa-4,11-diene, the first committed intermediate in the production of anti-cancer drug paclitaxel. The results showed that the strain which has (i) the taxadiene synthase (txs) gene integrated into the genome, (ii) the MEP pathway genes overexpressed, (iii) the fpps-crtE prenyltransferases fusion protein and (iv) additional expression of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), yielded the highest production of taxa-4,11-diene at 390 mg/L (26 mg/L/OD600). This represents a thirteen-fold increase compared to the highest reported concentration in B. subtilis. The focus on additional overexpression of DXS and utilizing short-chain prenyltransferase fusions underscores their pivotal role in achieving significant titer improvements in terpene biosynthesis.

HPLC/ESI-QTOF-MS/MS based untargeted metabolomics authentication of Taxus × media six tissues.

Taxus media (Taxus × media Rehder) is renowned for its high paclitaxel content, serving as a major source for industrial paclitaxel production. In addition to paclitaxel, T. media contains a diverse range of metabolites, including flavonoids, alkaloids, and terpenoids, which have been shown to possess antioxidant, antibacterial, anti-inflammatory, and immunomodulatory effects. However, these compounds have not been thoroughly studied as key metabolites in T. media. The untargeted metabolomics analysis of six T. media tissues provides new insights into the development and utilization of T. media metabolites. The extracts from six tissues of T. media were analyzed and subjected to analysis using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) and chemometric techniques. Using a reliable HPLC-Q-TOF-MS/MS method, we identified 312 compounds in six T. media tissues, including 214 previously unreported in T. media. To identify characteristic compounds across different tissues, 34 metabolites were further screened. KEGG metabolic pathway analysis revealed that these compounds primarily occur in the metabolic pathways of terpene glycosides, flavans, and O-methylated flavonoids. This study initially utilized an HPLC-QTOF-MS/MS-based metabolomics approach to assess the metabolites in different tissues of T. media, providing a basis for their utilization and management.

Optimization of the fermentation media, mathematical modeling, and enhancement of paclitaxel production by Alternaria alternata after elicitation with pectin

Alternaria alternata fungus is a potent paclitaxel producer isolated from Corylus avellana. The major challenge is the lack of optimized media for endophytic fungi productivity. In the effort to maximize the production of taxoids by A. alternata, several fermentation conditions, including pH (pH 4.0–7.0), different types and concentrations of carbon (fructose, glucose, sucrose, mannitol, sorbitol, and malt extract), and nitrogen (urea, ammonium nitrate, potassium nitrate, ammonium phosphate, and ammonium sulfate) were applied step by step. Based on the results, A. alternata in a medium containing sucrose 5% (w/v) and ammonium phosphate 2.5 mM at pH 6.0 showed a rapid and sustainable growth rate, the highest paclitaxel yield (94.8 µg gFW−1 vs 2.8 µg gFW−1 in controls), and the maximum content of amino acids. Additionally, the effect of pectin was evaluated on fungus, and mycelia harvested. Pectin significantly enhanced the growth and taxoid yield on day 21 (respectively 171% and 116% of their corresponding on day 7). The results were checked out by mathematical modeling as well. Accordingly, these findings suggest a low-cost, eco-friendly, and easy-to-produce approach with excellent biotechnological potential for the industrial manufacture of taxoids.

Advances in paclitaxel biosynthesis and transcriptional regulation mechanisms

Paclitaxel, a rare diterpene extracted from the bark of Chinese yew (Taxus chinensis), is renowned for its anti-cancer activity and serves as a primary drug for treating cancers. Due to the exceptionally low content of paclitaxel in the bark, a semi-synthetic method that depletes Chinese yew resources is used in the production of paclitaxel, which, however, fails to meet the escalating clinical demand. In recent years, researchers have achieved significant progress in heterologous biosynthesis and metabolic engineering for the production of paclitaxel. This article comprehensively reviews the advancements in paclitaxel production, encompassing chemical synthesis, heterologous biosynthesis, and cell engineering. It provides an in-depth introduction to the biosynthetic pathway and transcriptional regulation mechanisms of paclitaxel, aiming to provide a valuable reference for further research on paclitaxel biosynthesis.

Regulatory microRNAs and phasiRNAs of paclitaxel biosynthesis in Taxus chinensis.

Paclitaxel (trade name Taxol) is a rare diterpenoid with anticancer activity isolated from Taxus. At present, paclitaxel is mainly produced by the semi-synthetic method using extract of Taxus tissues as raw materials. The studies of regulatory mechanisms in paclitaxel biosynthesis would promote the production of paclitaxel through tissue/cell culture approaches. Here, we systematically identified 990 transcription factors (TFs), 460 microRNAs (miRNAs), and 160 phased small interfering RNAs (phasiRNAs) in Taxus chinensis to explore their interactions and potential roles in regulation of paclitaxel synthesis. The expression levels of enzyme genes in cone and root were higher than those in leaf and bark. Nearly all enzyme genes in the paclitaxel synthesis pathway were significantly up-regulated after jasmonate treatment, except for GGPPS and CoA Ligase. The expression level of enzyme genes located in the latter steps of the synthesis pathway was significantly higher in female barks than in male. Regulatory TFs were inferred through co-expression network analysis, resulting in the identification of TFs from diverse families including MYB and AP2. Genes with ADP binding and copper ion binding functions were overrepresented in targets of miRNA genes. The miRNA targets were mainly enriched with genes in plant hormone signal transduction, mRNA surveillance pathway, cell cycle and DNA replication. Genes in oxidoreductase activity, protein-disulfide reductase activity were enriched in targets of phasiRNAs. Regulatory networks were further constructed including components of enzyme genes, TFs, miRNAs, and phasiRNAs. The hierarchical regulation of paclitaxel production by miRNAs and phasiRNAs indicates a robust regulation at post-transcriptional level. Our study on transcriptional and posttranscriptional regulation of paclitaxel synthesis provides clues for enhancing paclitaxel production using synthetic biology technology.

Transcriptomics and Physiological Analyses Reveal Changes in Paclitaxel Production and Physiological Properties in Taxus cuspidata Suspension Cells in Response to Elicitors.

In this research, the cell growth, physiological, and biochemical reactions, as well as the paclitaxel production, of Taxus cuspidata suspension cells after treatment with polyethylene glycol (PEG), cyclodextrin (CD), or salicylic acid (SA) (alone or in combination) were investigated. To reveal the paclitaxel synthesis mechanism of T. cuspidata suspension cells under elicitor treatment, the transcriptomics of the Control group and P + C + S group (PEG + CD + SA) were compared. The results show that there were no significant differences in cell biomass after 5 days of elicitor treatments. However, the content of hydrogen peroxide (H2O2) and malondialdehyde (MDA), and the activities of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) after elicitor combination treatments were decreased compared with the single-elicitor treatment. Meanwhile, the antioxidant enzyme activity (superoxide dismutase (SOD), catalase (CAT), and peroxidase (PO)) and the contents of soluble sugar and soluble protein were increased after combination elicitor treatments. Additionally, the paclitaxel yield after treatment with the combination of all three elicitors (P + C + S) was 6.02 times higher than that of the Control group, thus indicating that the combination elicitor treatments had a significant effect on paclitaxel production in T. cuspidata cell suspension culture. Transcriptomics analysis revealed 13,623 differentially expressed genes (DEGs) between the Control and P + C + S treatment groups. Both GO and KEGG analyses showed that the DEGs mainly affected metabolic processes. DEGs associated with antioxidant enzymes, paclitaxel biosynthesis enzymes, and transcription factors were identified. It can be hypothesized that the oxidative stress of suspension cells occurred with elicitor stimulation, thereby leading to a defense response and an up-regulation of the gene expression associated with antioxidant enzymes, paclitaxel synthesis enzymes, and paclitaxel synthesis transcription factors; this ultimately increased the production of paclitaxel.

Research Advances in Clinical Applications, Anticancer Mechanism, Total Chemical Synthesis, Semi-Synthesis and Biosynthesis of Paclitaxel.

Paclitaxel, a natural secondary metabolite isolated and purified from the bark of the Taxus tree, is considered one of the most successful natural anticancer drugs due to its low toxicity, high potency and broad-spectrum anticancer activity. Taxus trees are scarce and slow-growing, and with extremely low paclitaxel content, the contradiction between supply and demand in the market is becoming more and more intense. Therefore, researchers have tried to obtain paclitaxel by various methods such as chemical synthesis, artificial culture, microbial fermentation and tissue cell culture to meet the clinical demand for this drug. This paper provides a comprehensive overview of paclitaxel extraction, combination therapy, total synthesis, semi-synthesis and biosynthesis in recent years and provides an outlook, aiming to provide a theoretical basis and reference for further research on the production and application of paclitaxel in the future.

Synthetic biology identifies the minimal gene set required for paclitaxel biosynthesis in a plant chassis

The diterpenoid paclitaxel (Taxol) is a chemotherapy medication widely used as a first-line treatment against several types of solid cancers. The supply of paclitaxel from natural sources is limited. However, missing knowledge about the genes involved in several specific metabolic steps of paclitaxel biosynthesis has rendered it difficult to engineer the full pathway. In this study, we used a combination of transcriptomics, cell biology, metabolomics, and pathway reconstitution to identify the complete gene set required for the heterologous production of paclitaxel. We identified the missing steps from the current model of paclitaxel biosynthesis and confirmed the activity of most of the missing enzymes via heterologous expression in Nicotiana benthamiana. Notably, we identified a new C4β-C20 epoxidase that could overcome the first bottleneck of metabolic engineering. We used both previously characterized and newly identified oxomutases/epoxidases, taxane 1β-hydroxylase, taxane 9α-hydroxylase, taxane 9α-dioxygenase, and phenylalanine-CoA ligase, to successfully biosynthesize the key intermediate baccatin III and to convert baccatin III into paclitaxel in N. benthamiana. In combination, these approaches establish a metabolic route to taxoid biosynthesis and provide insights into the unique chemistry that plants use to generate complex bioactive metabolites.

Overexpression of BAPT and DBTNBT genes in Taxus baccatain vitro cultures to enhance the biotechnological production of paclitaxel.

Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 μm coronatine plus 50 mm of randomly methylated-β-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line.

Increased paclitaxel recovery from Taxus baccata vascular stem cells using novel in situ product recovery approaches

In this study, several approaches were tested to optimise the production and recovery of the widely used anticancer drug Taxol® (paclitaxel) from culturable vascular stem cells (VSCs) of Taxus baccata, which is currently used as a successful cell line for paclitaxel production. An in situ product recovery (ISPR) technique was employed, which involved combining three commercial macro-porous resin beads (HP-20, XAD7HP and HP-2MG) with batch and semi-continuous cultivations of the T. baccata VSCs after adding methyl jasmonate (Me-JA) as an elicitor. The optimal resin combination resulted in 234 ± 23 mg of paclitaxel per kg of fresh-weight cells, indicating a 13-fold improved yield compared to the control (with no resins) in batch cultivation. This resin treatment was further studied to evaluate the resins’ removal capacity of reactive oxygen species (ROS), which can cause poor cell growth or reduce product synthesis. It was observed that the ISPR cultivations had fourfold less intracellular ROS concentration than that of the control; thus, a reduced ROS concentration established by the resin contributed to increased paclitaxel yield, contrary to previous studies. These paclitaxel yields are the highest reported to date using VSCs, and this scalable production method could be applied for a diverse range of similar compounds utilising plant cell culture.Graphical

Green synthesized Ag nanoparticles stimulate gene expression and paclitaxel production in Corylus avellana cells.

Synthesis of nanoparticles (NPs) through plant extracts has been suggested as an effective and nature-friendly method. Paclitaxel is one of the most valuable secondary metabolites with therapeutic uses, and hazelnut has been suggested as one of the sustainable resources for producing this metabolite. In the present study, we synthesized Ag NPs using the ethanolic extract of C. avellana leaves and were characterized using UV-visible, FTIR, XRD, EDX, DLS, SEM, and TEM analyses. In addition, we investigated the effect of green synthesized Ag (GS Ag) NPs (5 and 10 mg/L), para-aminobenzoic acid (PABA) (20 mg/L), and AgNO3 (10 mg/L) on cell viability, physiological characteristics, gene expression, and biosynthesis of secondary metabolites in hazelnut cell cultures. The results showed that 10 mg/L Ag NPs and AgNO3 significantly affected the cell viability, the content of ROS, peroxidation of lipids, antioxidant capacity, secondary metabolite production, and expression pattern of the genes involved in the taxanes biosynthesis pathway in the hazelnut cells. The cytotoxicity increased by increasing the GS Ag NPs concentration from 5 to 10 mg/L, which was associated with reduced membrane integrity and cell viability. Elicitation of the cells with 10 mg/L Ag NPs combined with 20 mg/L PABA (as a precursor) remarkably excited the expression of TAT and GGPPS genes and the production of secondary metabolites as well as paclitaxel. So that the highest expression of TAT and GGPPS genes (3.71 and 3.69) and the highest amount of taxol (230.21 μg g-1 FW) and baccatin (1025.8 μg g-1 FW) were observed in this treatment. KEY POINTS: • For the first time, we assessed and reported the molecular and physiological responses of C. avellana cells to GS Ag NPs, AgNO3, and PABA. • In hazel cells, GS Ag NPs stimulate several physiological and molecular responses. • In addition to increasing antioxidant activity, GS Ag NPs significantly increased the expression of genes involved in the paclitaxel biosynthesis pathway and the production of secondary metabolites.

Exploring the Interplay between Metabolic Pathways and Taxane Production in Elicited Taxus baccata Cell Suspensions.

Taxus cell cultures are a reliable biotechnological source of the anticancer drug paclitaxel. However, the interplay between taxane production and other metabolic pathways during elicitation remains poorly understood. In this study, we combined untargeted metabolomics and elicited Taxus baccata cell cultures to investigate variations in taxane-associated metabolism under the influence of 1 µM coronatine (COR) and 150 µM salicylic acid (SA). Our results demonstrated pleiotropic effects induced by both COR and SA elicitors, leading to differential changes in cell growth, taxane content, and secondary metabolism. Metabolite annotation revealed significant effects on N-containing compounds, phenylpropanoids, and terpenoids. Multivariate analysis showed that the metabolomic profiles of control and COR-treated samples are closer to each other than to SA-elicited samples at different time points (8, 16, and 24 days). The highest level of paclitaxel content was detected on day 8 under SA elicitation, exhibiting a negative correlation with the biomarkers kauralexin A2 and taxusin. Our study provides valuable insights into the intricate metabolic changes associated with paclitaxel production, aiding its potential optimization through untargeted metabolomics and an evaluation of COR/SA elicitor effects.

Screening and characterization of paclitaxel-producing fungus \(\textit{Talaromyces wortmannii}\) WQF18 isolated from \(\textit{Cephalotaxus mannii }\)Hook. f..

Cephalotaxus mannii Hook. f. is a rare evergreen conifer native listed in the International Union for Conservation of Nature (IUCN) Red List, which is utilized for leukemia treatment. Although endophytic fungi from C. mannii was reported before, their cytotoxic property has not been revealed yet. In the present study, a total of 7 endophytic fungi were isolated from C. mannii collected in Ha Giang province, Vietnam, among which the isolate WQF18 was active against 5 tested pathogens with inhibition zones ranging from 18.0 ± 0.7 to 25.0 ± 0.4 mm. In addition, only ethyl acetate extract of isolate WQF18 showed cytotoxicity on A549 and MCF7 cell lines with IC50 values of 69.6 ± 2.3 µg/mL and 78.6 ± 1.6 µg/mL, respectively. PCR-based molecular screening revealed that the positive hits for both 10-deacetylbaccatin III-10-O-acetyl transferase (dbat) and taxadiene synthase (ts) genes involved in paclitaxel biosynthesis were only observed in the WQF18 isolate. Based on morphological and molecular identification, the WQF18 isolate was identified as Talaromyces wortmannii WQF18. The presence of paclitaxel in T. wortmannii WQF18 was further confirmed by dbat sequence alignment, phenotypic, and HPLC-DAD analysis. To the best of our knowledge, this is the first report demonstrating the paclitaxel-producing capability of endophytic fungi from C. mannii. These findings provide a new platform for deciphering paclitaxel biosynthesis of endophytic fungi from non-Taxus plants and further paclitaxel production.

Developed network between taxoid and phenylpropanoid pathways in Cryptosporiopsis tarraconensis, taxan-producing endophytic fungus by Debiased Sparse Partial Correlation (DSPC) algorithm.

Although bioproduction of Paclitaxel by endophytic fungi is highly considered as an alternative promising source, but its yield is usually very low in comparison with other taxoids. Different strategies i.e., chemical and physical elicitations have been developed in order to overcome the shortage of Paclitaxel production. Paclitaxel biosynthesis is started with terpenoid pathway followed by phenylpropanoid metabolism where a benzoylphenylisoserine moiety is attached to C13 of baccatin III skeleton. This point which is catalyzed by the function of PAM seems to be a bottleneck that limits the rate of Paclitaxel production. Whether phenylpropanoids pathway regulates the taxanes biosynthesis in Cryptosporiopsis tarraconensis endophytic fungus elicited with benzoic acid (BA) was hypothesized in the present paper. The involvement of certain signal molecules and key enzymes of terpenoid and phenylpropanoid metabolism were investigated. According to the results, application of BA promoted a signaling pathway which was started with increase of H2O2 and ABA and continued by increase of NO and MJ, and finally resulted in increase of both phenylpropanoids and taxanes. However, again the rate of Paclitaxel production was lower than other taxoids, and the latter was much lower than phenolics. Therefore, supplying benzoic acid provided the precursor for the common taxan ring production. It is unlikely that Paclitaxel production is merely controlled by side chain production stage. It is more likely that in C. tarraconensis endophytic fungus, similar to Taxus sp., the competition between phenylpropanoid and taxoid pathways for substrate ended in favor of the former. The interaction network which was constructed based on DSPC algorithm confirmed that most compounds with close proximity have shared metabolic pathway relationships. Therefore, it is unlikely that the feeding with a given precursor directly result in increase of a desired metabolite which is composed of different merits.

Production of Taxol by Endophytic Fungi Isolated from Roots of Himalayan Yew (Taxus wallichiana Zucc.)

Taxol® (generic name – Paclitaxel), the most promising chemotherapeuticagent was isolated from bark of different Taxus sp. As Taxus species arethreatened with extinction (endangered in Himalaya), thus it is imperativeto develop alternate and sustainable method for commercialization and scaleup production of paclitaxel. In this respect, physical and chemical parametersare effective and important key points for active compound production par-ticularly by using endophytic microbes. In the present study, five endophyticfungi isolated from the roots of Taxus wallichiana, are tested for paclitaxelproduction using biochemical and molecular methods. Subsequently, effectof physico-chemical parameters like temperature, pH, incubation time, andmedium constituents i.e., salt concentration, carbon and nitrogen sources onpaclitaxel production were also analyzed. Among isolates, two of the fungiviz. GBPI TWR F1 (Penicillium sp.) and GBPI TWR F5 (Aspergillus sp.)were found to be paclitaxel producing. The genomic DNA samples were sequenced to confirm the presence of two genes viz. 10-deacetylbaccatinIII-10-O-acetyl transferase (DBAT) and C-13 phenylpropanoid side chain-CoA acyltransferase (BAPT), implicated in paclitaxel biosynthesis. Boththe endophytes showed the amplicons of DBAT and BAPT genes. Resultsrevealed that after optimization of medium components and physical condi-tion, paclitaxel production was increased in both the endophytes, maximumpaclitaxel production i.e., 5.45 ± 0.98 mg/L was obtained by GBPI TWR F5(Aspergillus sp.) following 10 days of incubation at 15◦C in optimized S7liquid medium composition

Mining natural products related to paclitaxel reveals the possible biosynthetic pathway of paclitaxel

Paclitaxel is a widely used anti-tumor drug. Currently, paclitaxel can only be extracted from plants or synthesized by chemical semi-synthesis, which cause environmental damage and cannot meet the growing demand. However, the complete biosynthetic pathway of paclitaxel is still not clear, which greatly limits the production of paclitaxel using methods such as synthetic biology. Here, we deduced the paclitaxel biosynthetic pathway by searching all possible intermediates in the paclitaxel synthesis pathway from the natural product databases. In addition, we performed the transcriptome sequencing of Taxus brevifolia and performed co-expression analysis of the identified genes in the paclitaxel synthesis pathway. All these results laid a solid foundation for the elucidation of paclitaxel biosynthetic pathway.