Taxol Production Research Articles

Increased Production of Taxoids in Suspension Cultures of Taxus globosa after Elicitation

The objectives of this study were to investigate whether elicitors induce the production of taxoids in Taxus globosa by testing the hypothesis that the cells induce a greater accumulation of taxoids depending on the type and concentration of the elicitor treatment tested. Cell cultures were initiated from Taxus globosa friable calli for producing taxoids using the Gamborg medium supplemented with different initial combinations of growth regulators as follows: naphthaleneacetic acid, benzylaminopurine, picloram, and polyvinylpyrrolidone. The cell suspension cultures were then used for evaluating taxoid production through different elicitor treatments, such as methyl jasmonate, ethanol, buthionine sulphoximine, and hydrogen peroxide. The cell suspension cultures showing the best growth characteristics were found in the medium supplemented with 10.74 µM of naphthaleneacetic acid and 3.33 µM of picloram. The highest biomass for the cell cultures was obtained in the EtOH-2 treatment (0.12 ± 0.005 mg·g−1 of dry weight) after 8 days, while the biomass in the control treatment at that time was 0.095 ± 0.2 mg·g−1 of dry weight. The exogenous application of a combination of elicitors buthionine and hydrogen peroxide in the cell suspension cultures significantly increased the concentration of the 10-deacetylbaccatin (1662 µg·g−1 of dry weight), cephalomannine (334.32 µg·g−1 of dry weight), and the production of taxol (157.0 µg·g−1 of dry weight).

A Review on Taxol producing endophytes isolated from medicinal plants and their potential applications

Taxol, commercially available as palcitaxel is a potent anticancer drug which has been naturally produced by pacific yew tree. This molecule has been included in the WHO’s list of essential medicines. This paper summarises the different sources of Taxol production, starting from initial isolations from Pacific yew tree which was followed by hairy root cultures and during the last decade, much focus was on endophytes as potential sources for Taxol production. Further, this review specifically talks about the medicinal plant Terminalia arjuna and its medicinal values with regard to its endophytes and their capability of producing Taxol. The process of initial screening, purification and media optimization for increasing the yields of secondary metabolites is also discussed.

Endophytic fungus Pseudodidymocyrtis lobariellae KL27 promotes taxol biosynthesis and accumulation in Taxus chinensis

BackgroundTaxol from Taxus species is a precious drug used for the treatment of cancer and can effectively inhibit the proliferation of cancer cells. However, the growth of Taxus plants is very slow and the content of taxol is quite low. Therefore, it is of great significance to improve the yield of taxol by modern biotechnology without destroying the wild forest resources. Endophytic fungus which symbiosis with their host plants can promote the growth and secondary metabolism of medicinal plants.ResultsHere, an endophytic fungus KL27 was isolated from T. chinensis, and identified as Pseudodidymocyrtis lobariellae. The fermentation broth of KL27 (KL27-FB) could significantly promote the accumulation of taxol in needles of T. chinensis, reaching 0.361 ± 0.082 mg/g·DW (dry weight) at 7 days after KL27-FB treatment, which is 3.26-fold increase as compared to the control. The RNA-seq and qRT-PCR showed that KL27-FB could significantly increase the expression of key genes involved in the upstream pathway of terpene synthesis (such as DXS and DXR) and those in the taxol biosynthesis pathway (such as GGPPS, TS, T5OH, TAT, T10OH, T14OH, T2OH, TBT, DBAT and PAM), especially at the early stage of the stimulation. Moreover, the activation of jasmonic acid (JA) biosynthesis and JA signal transduction, and its crosstalk with other hormones, such as gibberellin acid (GA), ethylene (ET) and salicylic acid (SA), explained the elevation of most of the differential expressed genes related to taxol biosynthesis pathway. Moreover, TF (transcriptional factor)-encoding genes, including MYBs, ethylene-responsive transcription factors (ERFs) and basic/helix-loop-helix (bHLH), were detected as differential expressed genes after KL27-FB treatment, further suggested that the regulation of hormone signaling on genes of taxol biosynthesis was mediated by TFs.ConclusionsOur results indicated that fermentation broth of endophytic fungus KL27-FB could effectively enhance the accumulation of taxol in T. chinensis needles by regulating the phytohormone metabolism and signal transduction and further up-regulating the expression of multiple key genes involved in taxol biosynthesis. This study provides new insight into the regulatory mechanism of how endophytic fungus promotes the production and accumulation of taxol in Taxus sp.

Purification and Biochemical Characterization of Taxadiene Synthase from Bacillus koreensis and Stenotrophomonas maltophilia

Taxadiene synthase (TDS) is the rate-limiting enzyme of Taxol biosynthesis that cyclizes the geranylgeranyl pyrophosphate into taxadiene. Attenuating Taxol productivity by fungi is the main challenge impeding its industrial application; it is possible that silencing the expression of TDS is the most noticeable genomic feature associated with Taxol-biosynthetic abolishing in fungi. As such, the characterization of TDS with unique biochemical properties and autonomous expression that is independent of transcriptional factors from the host is the main challenge. Thus, the objective of this study was to kinetically characterize TDS from endophytic bacteria isolated from different plants harboring Taxol-producing endophytic fungi. Among the recovered 23 isolates, Bacillus koreensis and Stenotrophomonas maltophilia achieved the highest TDS activity. Upon using the Plackett–Burman design, the TDS productivity achieved by B. koreensis (18.1 µmol/mg/min) and S. maltophilia (14.6 µmol/mg/min) increased by ~2.2-fold over the control. The enzyme was purified by gel-filtration and ion-exchange chromatography with ~15 overall folds and with molecular subunit structure 65 and 80 kDa from B. koreensis and S. maltophilia, respectively. The chemical identity of taxadiene was authenticated from the GC-MS analyses, which provided the same mass fragmentation pattern of authentic taxadiene. The tds gene was screened by PCR with nested primers of the conservative active site domains, and the amplicons were sequenced, displaying a higher similarity with tds from T. baccata and T. brevifolia. The highest TDS activity by both bacterial isolates was recorded at 37–40 °C. The Apo-TDSs retained ~50% of its initial holoenzyme activities, ensuring their metalloproteinic identity. The activity of purified TDS was completely restored upon the addition of Mg2+, confirming the identity of Mg2+ as a cofactor. The TDS activity was dramatically reduced upon the addition of DTNB and MBTH, ensuring the implementation of cysteine-reactive thiols and ammonia groups on their active site domains. This is the first report exploring the autonomous robust expression TDS from B. koreensis and S. maltophilia with a higher affinity to cyclize GGPP into taxadiene, which could be a novel platform for taxadiene production as intermediary metabolites of Taxol biosynthesis.

Isolation of Taxol and Flavin-like fluorochrome from Endophytic Fungi of Mangifera indica

Scouting for novel and plant-derived biomolecules from endophytic microbial sources draws greater focus on the discovery of novel bioactive metabolites. With this rationale, we scouted the endophytic fungi for taxol, an anticancer diterpenoid and fluorescent biomolecules. In the present study, about 31 endophytic fungal isolates recovered from the Mangifera indica leaves were screened for taxol production in M1D medium. About five isolates were shortlisted based on the thin layer chromatographic analysis of the fungal extracts. Among them Colletotrichum sp. MIP-5 has been identified as a producer of fungal taxol based on UV, FTIR, TLC and HPLC analysis. The partially purified fungal taxol showed similar spectral and chromatographic features of commercially available paclitaxel. In addition to this, we also report the production of a fluorescent compound by Penicillium sp. MIP-3. The Flavin-like compound exhibited a bright greenish-yellow fluorescence with an emission maximum in the range of 505 – 545nm. GC-MS analysis showed the occurrence of Latia luciferin, primarily associated with the bioluminescence of freshwater limpet Latia neritoides. This is the first report of this compound from Penicillium sp. In addition, therapeutically active steroid (β-Sitosterol, Stigmasterol, Campesterol), quinones (Benzo[h]quinoline, 2,4-dimethyl-) and phloroglucinol (Aspidinol) derivatives were also identified from Penicillium sp. MIP-3 based on GC-MS analysis. These molecules could potentially be used in biological and pharmaceutical applications in future.

Taxus yunnanensis genome offers insights into gymnosperm phylogeny and taxol production

Taxol, a natural product derived from Taxus, is one of the most effective natural anticancer drugs and the biosynthetic pathway of Taxol is the basis of heterologous bio-production. Here, we report a high-quality genome assembly and annotation of Taxus yunnanensis based on 10.7 Gb sequences assembled into 12 chromosomes with contig N50 and scaffold N50 of 2.89 Mb and 966.80 Mb, respectively. Phylogenomic analyses show that T. yunnanensis is most closely related to Sequoiadendron giganteum among the sampled taxa, with an estimated divergence time of 133.4−213.0 MYA. As with most gymnosperms, and unlike most angiosperms, there is no evidence of a recent whole-genome duplication in T. yunnanensis. Repetitive sequences, especially long terminal repeat retrotransposons, are prevalent in the T. yunnanensis genome, contributing to its large genome size. We further integrated genomic and transcriptomic data to unveil clusters of genes involved in Taxol synthesis, located on the chromosome 12, while gene families encoding hydroxylase in the Taxol pathway exhibited significant expansion. Our study contributes to the further elucidation of gymnosperm relationships and the Taxol biosynthetic pathway.

Integrated system for paclitaxel production from endophytic fungus, Neopestalotiopsis clavispora ASU1 and subsequent extraction of chitosan from fungal biomass wastes

<p>Nowadays, breast cancer is considered to be one of the most prevalent diseases worldwide, this initiated interest and growing demand for taxol production in large quantities. The limited availability of traditional taxol production from the Pacific yew trees has encouraged research into the development of taxol production from alternative sources. Thus, the current study aimed to investigate the chemopreventive effect of paclitaxel derived from endophytic fungus Neopestalotiopsis clavispora ASU1. The endophytic fungus Neopestalotiopsis clavispora ASU1(KY624416) showed potent productivity of paclitaxel recording 100.6 µg/l which confirmed by HPLC, LC/Ms-Ms and FTIR analyses. In vitro, the extracted fungal taxol exerted a significant cytotoxic effect (P< 0.05) at 300 nM, revealed that the increase in paclitaxel concentration induces increased cell death. Furthermore, the present study provides a promising approach for coupling paclitaxel production technology by endophytic fungus N. clavispora with chitosan production from residual fungal biomass resulting from taxol extraction and therefore improve the feasibility and commercialization of taxol production. The chitosan yield represented 5.94% of residual fungal dry biomass. Also, fungal chitosan was characterized for the degree of deacetylation (DD) (54.60%), FTIR spectroscopy, reducing power activity (0.263±0.051 mg/ml) may be attributed to hydroxyl groups (OH), and amine groups. These results confirmed that fungi are promising alternative sources for chitosan with superior physiochemical characteristics for food and medical prospective applications.</p>

Enhancement of Taxol Production by Endophytic fungi from Hibiscus and Moringa plant using Gamma irradiation

Enhancement of Taxol Production by Endophytic fungi from Hibiscus and Moringa plant using Gamma irradiation

Optimization of Taxol Extraction Process Using Response Surface Methodology and Investigation of Temporal and Spatial Distribution of Taxol in Taxus mairei.

Taxus mairei is an important source for industrial extraction of taxol in China. However, the standard and steps of extraction are currently not uniform, which seriously affects the taxol yield. In the present study, the influence of four factors (methanol concentration, solid-liquid ratio, ultrasonic extraction temperature, and ultrasonic extraction time) on the taxol yield was successively explored in T. mairei. A response surface methodology (RSM) was used to optimize the extraction process based on the single-factor experiments above. The optimal conditions were as follows: methanol concentration was 90%, solid-liquid ratio was 1:15 (g/mL), ultrasonic extraction temperature was 40 °C and ultrasonic extraction time was 60 min. Moreover, the twigs and needles from T. mairei with different tree ages were treated by the optimum extraction process, which further revealed temporal and spatial distribution of taxol in the reproducible tissues. Interestingly, the taxol content was relatively higher in needles of T. ‘Jinxishan’ (a cultivar from T. mairei with yellow aril, FY), but was less in FY twigs. The accumulation of taxol in twigs and leaves of females (with red aril, FR) was significantly higher than that of males (M); however, the content showed a decreasing trend with the increasing tree ages. Therefore, it is suitable to increase the proportion of female trees especially the FY leaves as raw materials for the industrial production of taxol from T. mairei, and the tree ages should be better controlled at 3–7 years.

Exploiting endophytic microbes as micro-factories for plant secondary metabolite production.

Plant secondary metabolites have significant potential applications in a wide range of pharmaceutical, food, and cosmetic industries by providing new chemistries and compounds. However, direct isolation of such compounds from plants has resulted in over-harvesting and loss of biodiversity, currently threatening several medicinal plant species to extinction. With the breakthrough report of taxol production by an endophytic fungus of Taxus brevifolia, a new era in natural product research was established. Since then, the ability of endophytic microbes to produce metabolites similar to those produced by their host plants has been discovered. The plant "endosphere" represents a rich and unique biological niche inhabited by organisms capable of producing a range of desired compounds. In addition, plants growing in diverse habitats and adverse environmental conditions represent a valuable reservoir for obtaining rare microbes with potential applications. Despite being an attractive and sustainable approach for obtaining economically important metabolites, the industrial exploitation of microbial endophytes for the production and isolation of plant secondary metabolites remains in its infancy. The present review provides an updated overview of the prospects, challenges, and possible solutions for using microbial endophytes as micro-factories for obtaining commercially important plant metabolites.Key points• Some "plant" metabolites are rather synthesized by the associated endophytes.• Challenges: Attenuation, silencing of BGCs, unculturability, complex cross-talk.• Solutions: Simulation of in planta habitat, advanced characterization methods.

Priming with CuO Nanoparticles and Ultrasound Enhanced Antioxidant Potential and Total Taxol in the Cell Suspension Culture of Corylus avellana L.

The main superiority of taxol production in hazelnuts compared to the yew tree is that hazelnut is broadly accessible, grows much faster in nature, and is easier to establish in the in vitro systems. The aim of this experiment was to improve taxol production and evaluate other characteristics of cell suspension culture treated with 4 concentrations (0, 2, 4, 6 mg L-1) of copper oxide nanoparticles (CuO NPs), ultrasonication (30 and 60 seconds) in two sampling times (9 and 18 days after treatment). Interaction of all three factors was significant on fresh weight (FW), protein content, H2O2 generation, and taxol content. The highest fresh and dry weights (DW) and protein content were obtained from non-application treatments of CuO NPs. The content of anthocyanins, proline, antioxidant activity, and catalase and peroxidase enzymes increased with the application of silver nitrate nanoparticles. Comparing means for interaction effects showed that H2O2 and taxol content have nearly similar trend, indicating the stimulation of taxol production pathway by reactive oxygen species (ROS). Ultrasonication (US) enhanced most of the traits studied in this experiment and stimulated the antioxidant system without negatively affecting on cellular growth. The antioxidant response and taxol content evoked by stress treatments i.e. US and CuO NPs is partially relieved by increasing the duration after treatment (the 18th day after treatment).

Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases.

sTaxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10β-hydroxylase (T10βOH) and a newly putative β-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.

Production, bioprocess optimization and γ-irradiation of Penicillium polonicum, as a new Taxol producing endophyte from Ginko biloba

Twenty-eight fungal endophytes were recovered from the different parts of Ginkgo biloba and screened for their Taxol producing potency. Among these isolates, Penicillium polonicum AUMC14487 was reported as the potent Taxol producer (90.53 μg/l). The chemical identity of the extracted Taxol was verified from the TLC, HPLC, NMR, EDX, and FTIR analyses. The extracted Taxol displayed a strong antiproliferative activity against HEPG2 (IC50 4.06 μM) and MCF7 (IC50 6.07 μM). The yield of Taxol by P. polonicum was optimized by nutritional optimization with the Response Surface Methodology (RSM) using Plackett-Burman and Central Composite Designs. In addition to nutritional optimization, the effect of γ-irradiation of the spores of P. polonicum on its Taxol producing potency was evaluated. The yield of Taxol by P. polonicum was increased via nutritional optimization by response surface methodology with Plackett-Burman and FCCD designs, and γ-irradiation by about 4.5 folds, comparing to the control culture.The yield of Taxol was increased by about 1.2 folds (401.2 μg/l) by γ -irradiation of the isolates at 0.5−0.75 kGy, comparing to the control cultures (332.2 μg/l).The highest Taxol yield was obtained by growing P. polonicum on modified Czapek’s- Dox medium (sucrose 40.0 g/l, malt extract 20.0 g/l, peptone 2.0 g/l, K2PO4 2.0 g/l, KCl 1.0 g/l, NaNO3 2.0 g/l, MgSO4. 5H2O 1.0 g/l) of pH 7.0 at 30.0 °C for 7.0 days. From the FCCD design, sucrose, malt extract and incubation time being the highest significant variables medium components affecting the Taxol production by P. polonicum.

Development of hairy root culture in Taxus baccata sub sp wallichiana as an alternative for increased Taxol production

The Himalayan Yew or Taxus baccata sub sp. wallichiana is the native of the Temperate belt of Himalayas known widely for its ethnomedical properties and especially for the production of an anti-cancerous compound Taxol but since the plant is an understory and in low population size, the production of Taxol in nature is very poor so it cannot meet the high demand of Taxol for medical use. Hairy root culture is known for its genetic and physiochemical stability and increased production of secondary metabolites as compared to cell suspension culture and with the use of low frequency sonication for 1–2 min, the rate of transformation can be increased thereby induing more hairy roots. In our study, when the combination of low frequency sonication for production of hairy roots was used it helped in development of the hairy root culture of Taxus baccata sub sp. wallichiana as an alternative for increased Taxol production. It was seen that the hairy root induction from the explants emerged when an effective co– infection method in presence of acetosyringone with sonication for 60 s was done. The increase in hairy root biomass was further done for 16 weeks in root induction media that was without acetosyringone. The methanolic extract of the biomass when quantified for total Taxol content produced around 12 mg of Taxol per gram of hairy root biomass of Taxus baccata sub sp. wallichiana which is 79% more than the natural production of 0.15 mg per gram of bark stating a possible alternative for Taxol production commercially in a large amount.

Machine learning tools can now help with improving Taxol production in plant cell cultures

Chemotherapeutic intervention for cancer care is an important step. One of the most effective chemotherapy agents in use today is Paclitaxel (PTX), sold under the common name Taxol and Oxanol. Due to its ability to inhibit microtubule formation in cells, PTX is effective at all stages of the cancer and is FDA approved for treatment of many types of cancer (ovarian cancer, esophageal cancer, breast cancer, lung cancer, Kaposi sarcoma, cervical cancer, and pancreatic cancer). PTX is a plant alkaloid in the taxane family of compounds obtained from bark of the Pacific Yew tree (Taxus brevifolia) [1]. Adequate market supply of PTX has remained a challenge, as paclitaxel represents only a minor proportion of the total taxoid content of the Taxus species. Over the years, research into finding an alternate to cutting down Yew trees for PTX harvesting has been on the forefront. It is estimated that up to 60 trees may need to be harvested for the treatment of one patient.

Current State and Future Directions of Genetics and Genomics of Endophytic Fungi for Bioprospecting Efforts.

The bioprospecting of secondary metabolites from endophytic fungi received great attention in the 1990s and 2000s, when the controversy around taxol production from Taxus spp. endophytes was at its height. Since then, hundreds of reports have described the isolation and characterization of putative secondary metabolites from endophytic fungi. However, only very few studies also report the genetic basis for these phenotypic observations. With low sequencing cost and fast sample turnaround, genetics- and genomics-based approaches have risen to become comprehensive approaches to study natural products from a wide-range of organisms, especially to elucidate underlying biosynthetic pathways. However, in the field of fungal endophyte biology, elucidation of biosynthetic pathways is still a major challenge. As a relatively poorly investigated group of microorganisms, even in the light of recent efforts to sequence more fungal genomes, such as the 1000 Fungal Genomes Project at the Joint Genome Institute (JGI), the basis for bioprospecting of enzymes and pathways from endophytic fungi is still rather slim. In this review we want to discuss the current approaches and tools used to associate phenotype and genotype to elucidate biosynthetic pathways of secondary metabolites in endophytic fungi through the lens of bioprospecting. This review will point out the reported successes and shortcomings, and discuss future directions in sampling, and genetics and genomics of endophytic fungi. Identifying responsible biosynthetic genes for the numerous secondary metabolites isolated from endophytic fungi opens the opportunity to explore the genetic potential of producer strains to discover novel secondary metabolites and enhance secondary metabolite production by metabolic engineering resulting in novel and more affordable medicines and food additives.

Annulohypoxylon sp. strain MUS1, an endophytic fungus isolated from Taxus wallichiana Zucc., produces taxol and other bioactive metabolites.

The online version contains supplementary material available at 10.1007/s13205-021-02693-z.

Recent Advances in Metabolic Engineering, Protein Engineering, and Transcriptome-Guided Insights Toward Synthetic Production of Taxol.

The diterpenoid paclitaxel (Taxol®) is a blockbuster anticancer agent that was originally isolated from the Pacific yew (Taxus brevifolia) five decades ago. Despite the wealth of information gained over the years on Taxol research, there still remains supply issues to meet increasing clinical demand. Although alternative Taxol production methods have been developed, they still face several drawbacks that cause supply shortages and high production costs. It is highly desired to develop biotechnological production platforms for Taxol, however, there are still gaps in our understanding of the biosynthetic pathway, catalytic enzymes, regulatory and control mechanisms that hamper production of this critical drug by synthetic biology approaches. Over the past 5 years, significant advances were made in metabolic engineering and optimization of the Taxol pathway in different hosts, leading to accumulation of taxane intermediates. Computational and experimental approaches were leveraged to gain mechanistic insights into the catalytic cycle of pathway enzymes and guide rational protein engineering efforts to improve catalytic fitness and substrate/product specificity, especially of the cytochrome P450s (CYP450s). Notable breakthroughs were also realized in engineering the pathway in plant hosts that are more promising in addressing the challenging CYP450 chemistry. Here, we review these recent advances and in addition, we summarize recent transcriptomic data sets of Taxus species and elicited culture cells, and give a bird’s-eye view of the information that can be gleaned from these publicly available resources. Recent mining of transcriptome data sets led to discovery of two putative pathway enzymes, provided many lead candidates for the missing steps and provided new insights on the regulatory mechanisms governing Taxol biosynthesis. All these inferences are relevant to future biotechnological production of Taxol.

Evaluation of spore inoculum and confirmation of pathway genetic blueprint of T13\u03b1H and DBAT from a Taxol-producing endophytic fungus

Taxol (paclitaxel), a plant-derived anticancer drug, has been among the most successful anticancer drugs of natural origin. Endophytic fungi have been proposed as a prominent alternative source for Taxol and its intermediate Baccatin III, however the very low yields remain a hinderance to their commercial utilization. Significant research efforts towards this end are underway globally. Here, we report the results on our earlier reported Taxol-producing endophytic fungus, Fusarium solani from the standpoint of spores as seed inoculum and media selection for enhanced Taxol and baccatin III yields. Spores produced on M1D medium with 94.76% viability were used for further media optimization for Taxol and Baccatin III production in five different liquid media under static and shaker condition at different cultivation days. Taxol and Baccatin III when quantified through competitive inhibition enzyme immunoassay (CIEIA), showed maximum production at 136.3 µg L−1 and 128.3 µg L−1, respectively in the modified flask basal broth (MFBB) under shaking condition. Further, two important genes of this pathway, namely taxane 13α-hydroxylase (T13αH) and 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) have been identified in this fungus. These findings are hoped to assist in further manipulation and metabolic engineering of the parent F. solani strain towards the enhanced production of Taxol and baccatin III.

Antimicrobial activity and cytotoxicity of endophytic fungi associated with four medicinal plants from Sudan

In this study four medicinal plants, used in Sudan to cure different types of health problems, were selected for isolation of endophytic fungi, fungi reside within the plant host tissues without causing disease symptoms. A total of 20 distinct endophytic fungi species were isolated from surface- sterilized parts of the plants, Kigelia africana, Cymbopogon proximus, Balanites egyptiaca and Solenostemma argel. Nine of the isolated fungi were identified according to their microscopic and macroscopic characteristics. The remaining strains which did not have spores or have sterile mycelium were given codes as unidentified species (UID). The ethyl acetate extracts of the pure isolated endophytic fungi were prepared from fungal material and culture media. Antimicrobial activity using agar diffusion well against different pathogenic microbes at 500 µg/ml concentration and MTT cytotoxicity bioassay using African green monkey kidney normal cell line (vero cell line) were performed for all extracts. The extracts were then screened for the presence of Taxol, a diterpene anticancer drug, using different chromatographic and spectroscopic techniques. The antimicrobial activity against the most common pathogenic bacteria and fungi revealed significant activity with inhibition zone range between 14- 37 mm compare to antimicrobial controls 17- 26 mm at 40µg/ml. The cytotoxicity assay results showed that the IC50 values were quite diverse, ranging from 3.5 μg/ml to more than 1000 μg/ml, whereas that of the positive control podophyllotoxin, a wellknown cytotoxic lignan, was 2.72 μg/ml. Taxol was detected in reference to standard one from the extract of unidentified fungi No. 10 of Kigelia africana,. Future work will focus on the bioassay guidedfractionation of potent bioactive extracts to isolate the active compounds responsible for their activities. Improvement of culture techniques and the application of genetic engineering to increase taxol production will be carried out, followed by isolation of pure Taxol from the active crude extract.