Unsaturated fatty acid, Nonacosenoic acid isolated from an endophyte Chaetomium nigricolor inhabiting the stem of Catharanthus roseus and its bioactivity.

The endophytic fungus Chaetomium nigricolor culture filtrate's hexane extract was used to identify a cytotoxic very long-chain fatty acid. Based on multiple spectroscopic investigations, the structure of the compound was predicted to be an unsaturated fatty acid, Nonacosenoic acid (NA). Using the MTT assay, the compound's cytotoxic potential was evaluated against MCF-7, A-431, U-251, and HEK-293 T cells. The compound was moderately cytotoxic to breast carcinoma cell line, MCF-7 cells and negligibly cytotoxic to non-cancerous cell line HEK-293 T cells. The compound exhibited mild cytotoxic activity against A-431 and U-251 cells. The compound also induced ROS generation and mitochondrial depolarization in MCF-7 cells when assessed via the NBT and JC-1 assays, respectively. This is the first report on the production of nonacosenoic acid from the endophytic fungus Chaetomium nigricolor and the assessment of its bioactivity.

Insights into Taxol® biosynthesis by endophytic fungi.

There have been two hundred reports that endophytic fungi produce Taxol®, but its production yield is often rather low. Although considerable efforts have been made to increase Taxol/taxanes production in fungi by manipulating cocultures, mutagenesis, genome shuffles, and gene overexpression, little is known about the molecular signatures of Taxol biosynthesis and its regulation. It is known that some fungi have orthologs of the Taxol biosynthetic pathway, but the overall architecture of this pathway is unknown. A biosynthetic putative gene homology approach, combined with genomics and transcriptomics analysis, revealed that a few genes for metabolite residues may be located on dispensable chromosomes. This review explores a number of crucial topics (i) finding biosynthetic pathway genes using precursors, elicitors, and inhibitors; (ii) orthologs of the Taxol biosynthetic pathway for rate-limiting genes/enzymes; and (iii) genomics and transcriptomics can be used to accurately predict biosynthetic putative genes and regulators. This provides promising targets for future genetic engineering approaches to produce fungal Taxol and precursors. KEY POINTS: • A recent trend in predicting Taxol biosynthetic pathway from endophytic fungi. • Understanding the Taxol biosynthetic pathway and related enzymes in fungi. • The genetic evidence and formation of taxane from endophytic fungi.

Diversity of Marine Macro-Algicolous Endophytic Fungi and Cytotoxic Potential of Biscogniauxia petrensis Metabolites Against Cancer Cell Lines.

Hypersaline environments are known to support diverse fungal species from various orders. The production of secondary metabolites is one of the strategies that fungi adopt to thrive under such extreme environments, bringing up the stress tolerance response. Some such unique secondary metabolites also exhibit clinical significance. The increasing prevalence of drug resistance in cancer therapy demands further exploration of these novel bioactive compounds as cancer therapeutics. In the present study, a total of 31 endophytic fungi harboring inside red, green, and brown marine algae have been isolated and identified. The maximum likelihood analysis and diversity indices of fungal endophytes revealed the phylogenetic relationship and species richness. The genus Aspergillus was found to be the dominating fungus, followed by Cladosporium spp. All the isolated endophytic fungal extracts were tested for their cytotoxicity against HeLa and A431 cancer cell lines. Nine isolates were further analyzed for their cytotoxic activity from the culture filtrate and mycelia extract. Among these isolates, Biscogniauxia petrensis showed potential cytotoxicity with CC50 values of 18.04 and 24.85 µg/ml against HeLa and A431 cells, respectively. Furthermore, the media and solvent extraction optimization revealed the highest cytotoxic active compounds in ethyl acetate extract from the potato dextrose yeast extract broth medium. The compound-induced cell death via apoptosis was 50-60 and 45% when assayed using propidium iodide-live/dead and loss of mitochondrial membrane potential assay, respectively, in HeLa cells. Four bioactive fractions (bioassay-based) were obtained and analyzed using chromatography and spectroscopy. This study reports, for the first time, the cytotoxic activity of an endophytic fungal community that was isolated from marine macro-algae in the Rameswaram coastal region of Tamil Nadu, India. In addition, B. petrensis is a prominent apoptotic agent, which can be used in pharmaceutical applications as a therapeutic.

A novel synergistic anticancer effect of fungal cholestanol glucoside and paclitaxel: Apoptosis induced by an intrinsic pathway through ROS generation in cervical cancer cell line (HeLa).

In the search for efficient therapeutics with economically viable for cancer treatment, combination therapy has developed as a keystone in the pursuit of novel approaches for drug discovery. In this regard, we confirmed the presence of cholestanol glucoside (CG) in Lasiodiplodia theobromae culture filtrate and its production was estimated to be 20.01 mg/l. The purified fungal CG was obtained with a molecular mass of 550.18 m/z. The combination of CG and paclitaxel (PTX) was found to have potent cytotoxicity against HeLa cells. We revealed that the synergistic effect of CG and PTX induced apoptosis through the formation of nuclear fragments, DNA fragmentation and sub G1 cell cycle arrest. Further, it was proven that apoptosis took place by loss of the mitochondrial membrane potential (MMP) through reactive oxygen species (ROS) production and caspase 3/7 activity. Moreover, the data suggests that the synergistic effect of CG and PTX played a role in a mitochondrial intrinsic pathway through the apoptotic gene expression of Bax, caspase-9 and caspase-3. In addition, the down-regulation of Bcl-2 strongly described the induced apoptosis through an intrinsic pathway using the Western blot analysis. The conclusion of this study is that a combination of CG and PTX has synergistic apoptotic effects in HeLa cells, which provides a possible therapeutic strategy for cancer therapy in the future.

Evaluation of the anticancer activity of enzymatically synthesized Baccatin III: an intermediate precursor of Taxol®.

Baccatin III is an important precursor for the synthesis of clinically important anticancer drug Taxol. Previously, we have characterized a key enzyme of 10-deacetylbaccatin III-10-ß-O-acetyltransferase (DBAT) which catalyses the 10-deacetylbaccatin III into baccatin III in taxol biosynthesis. Here, in the present study, we have evaluated and compared the cytotoxic properties of the enzymatically synthesized baccatin III (ESB III) with standard baccatin III in different human cancer cell lines, namely human cervical cancer (HeLa), human lung cancer (A549), human skin cancer (A431) and human liver cancer cells (HepG2). Among the various cancer lines tested, HeLa was more susceptible to ESB III with IC50 of 4.30 µM while IC50 values for A549, A431 and HepG2 ranged from 4 to 7.81 µM. Further, it showed G2/M phase cell cycle arrest, production of reactive oxygen species and depolarised mitochondrial membrane potential. In addition, annexin V-FITC staining was performed which showed the apoptotic cell death of HeLa cells, when treated with ESB III. Hence, ESB III was capable to show anticancer activities by inducing apoptotic cell death which could further be used for the semisynthesis of taxol in future.

Pleiotropic Functions and Biological Potentials of Silver Nanoparticles Synthesized by an Endophytic Fungus.

In recent years, the biological synthesis of silver nanoparticles (AgNPs) from microorganisms has become an emerging trend for developing biocompatible nanomaterials that finds applications in nano and biomedical sectors. In the present study, we demonstrated a facile, green and eco-friendly method for AgNPs synthesis using the endophytic fungi (Colletotrichum incarnatum DM16.3) isolated from medicinal plant Datura metel and its in vitro antithrombin and cytotoxic activity. At first, biosynthesis of colloidal AgNPs was predicted by visual observation of color change and UV-visible spectra demonstrated specific surface plasmon resonance peak at 420 nm which confirmed the presence of nanoparticles. Microscopic analyses revealed the structure of highly aggregated, spherical and crystalline AgNPs in the diameter range of 5-25 nm. Transform infrared spectroscopy (FT-IR) spectral analysis confirmed the presence of probable biomolecules required for the reduction of silver ions. In vitro evaluation of thrombin activity demonstrates that AgNPs could exert strong inhibition against both thrombin activity (87%) and thrombin generation (84%), respectively. Further, in silico based mechanistic analysis yielded a better insight in understanding the probable amino acids responsible for AgNPs binding with thrombin protein. Similarly, in vitro cytotoxicity of synthesized AgNPs on human epithelial cells using MTT assay did not produce any substantial effects after 24 h exposure which indicates excellent biocompatibility nature, whereas notable toxicity was observed on human cancerous (HeLa) cells at 50 µg/mL (IC50 value). In addition, assessment of AgNPs at 10 µg/mL concentration via crystal violet method on biofilm forming Gram-positive (Vibrio cholerae) and Gram-negative bacteria (Bacillus cereus) revealed inhibition up to 85 and 46%, respectively. Overall, this study showed the possibility of microbially synthesized AgNPs as a potent inhibitor for managing acute thrombosis and highlighted their role for other biomedical applications.

Biochemical insights into the recombinant 10-deacetylbaccatin III-10-ß-O-acetyltransferase enzyme from the Taxol-producing endophytic fungus Lasiodiplodia theobromae.

10-deacetylbaccatin III-10-ß-O-acetyltransferase (DBAT) is a key rate-limiting enzyme of the Taxol biosynthetic pathway, which is uncharacterized in Taxol-producing endophytic fungi. Here, an open reading frame of DBAT was cloned from the Taxol-producing endophytic fungus Lasiodiplodia theobromae (LtDBAT). The LtDBAT enzyme was heterologously expressed and purified by the affinity and gel filtration chromatography methods. The molecular weight of the purified protein was 49 kDa and its identity was confirmed by western blot. The purified LtDBAT enzyme was capable of catalyzing 10-deacetylbaccatin III into baccatin III, as shown by liquid chromatography-mass spectroscopy. The mass spectra of baccatin III were identical to the authentic baccatin III. The LtDBAT enzyme was characterized and the kinetic parameters of catalysis were determined. In addition, localization of LtDBAT was performed by using confocal microscopy and the result showed that the enzyme was localized in lipid droplets. Together, this study provides biochemical insights into the fungal recombinant DBAT enzyme that is involved in the Taxol biosynthetic pathway. In the near future, engineering of the LtDBAT enzyme and the Taxol biosynthetic pathway in endophytic fungi could be an eco-friendly and economically feasible alternative source for production of Taxol and its precursors.

Salicylic acid as an effective elicitor for improved taxol production in endophytic fungus Pestalotiopsis microspora.

Salicylic acid (SA) is an effective elicitor to increase taxol production in Pestalotiopsis microspora. Addition of SA at the concentration of 300 µM yielded taxol 625.47 µg L-1, 45- fold higher than that of the control. Elicitation of the role of SA in the fungal taxol biosynthetic pathway revealed that SA enhanced reactive oxygen species and lipid peroxidation of unsaturated fatty acids of P. microspora mycelia. This oxidative process stimulates isoprene biosynthetic pathway by triggering expression of the geranylgeranyl pyrophosphate synthase gene leading to improved biosynthesis of taxol in P. microspora.

Diversity and biological activities of endophytic fungi associated with Catharanthus roseus.

BACKGROUND: The present study involves diversity and bioactivity of the endophytic fungal community from Catharanthus roseus inhabiting the coastal region. This study has been conducted hypothesizing that the microbial communities in the coastal regions would tolerate a range of abiotic stress such as salinity, humidity, temperature and soil composition, and it may produce new metabolites, which may possess bioactive property. Therefore in the current study, the cytotoxicity and free radical scavenging potential of the fungal organic extracts have been investigated. Moreover, the apoptotic and the antioxidant potential of the fungus that exhibited the best activity in preliminary screening has also been demonstrated. RESULTS: Twenty endophytic fungal isolates were obtained from different parts of the plant, and identified using internal transcribed spacer region analysis. Based on the colonization frequency, the dominant genera were found to be Colletotrichum, Alternaria and Chaetomium with colonization frequency % of 8.66, 7.00 and 6.33, respectively. It was observed that the species diversity and richness was the highest in bark followed by leaf and stem regions of the plant. On screening the fungal ethyl acetate extracts for cytotoxicity against the HeLa cells, the Chaetomium nigricolor extract exhibited potent cytotoxic activity of 92.20% at 100 µg mL- 1 concentration. Comparison between the different organic extracts (ethyl acetate, chloroform, dichloromethane and hexane) of Chaetomium nigricolor mycelial and culture filtrate, it was observed that the mycelial as well the culture filtrate ethyl acetate extracts and the culture filtrate hexane extract showed significant cytotoxic potential against the HeLa and MCF-7 cells, respectively. The apoptotic- and mitochondrial membrane depolarisation-induction potential of the Chaetomium nigricolor ethyl acetate extract has also been demonstrated in this study. Further the screening of antioxidant potential of the ethyl acetate fungal extracts using DPPH scavenging assay showed that Chaetomium nigricolor extract exhibited potential activity with a significant EC50 value of 22 µg mL- 1. The ethyl acetate extract of Chaetomium nigricolor also exhibited superoxide radical scavenging potential. CONCLUSION: These results indicated that diverse endophytic fungal population inhabits Catharanthus roseus. One of the fungal isolate Chaetomium nigricolor exhibited significant cytotoxic, apoptotic and antioxidant potential.

Cloning and sequence analysis of 10-deacetylbaccatin III-10-O-acetyl transferase gene and WRKY1 transcription factor from taxol-producing endophytic fungus Lasiodiplodia theobromea.

In this study, we have isolated an endophytic fungal strain Lasiodiplodia theobromae from non-Taxus host plant Piper nigrum. The strain L. theobromae identity was confirmed by morphological characteristics and internal transcribed spacer sequence analysis. Taxol produced by L. theobromae was observed to be identical to the authentic taxol as analyzed by chromatography and spectroscopy methods. The quantity of taxol produced by the fungus was estimated to be 247 µg L-1, and fungal taxol showed potent cytotoxic activity towards cancer cell line. Evidence to support the independent production of taxol by L. theobromea, the gene encoding 10-deacetylbacccation-III-O-acetyltransferase (DBAT), as well as, for the first time, open reading frame (ORF) of WRKY1 transcription factor (TF) were cloned and sequenced. The predicted amino sequence of L. theobromae dbat gene shared high homology with the taxol-producing plant and fungal dbat gene. Not only dbat gene, ORF of WRKY1 TF too shared high homology with Taxus chinensis WRKY1 TF ORF. To the best of our knowledge, this is the first report on cloning of dbat gene and its transcription factor from endophytes of non-Taxus host plant.

Fungal 7-epi-10-deacetyltaxol produced by an endophytic Pestalotiopsis microspora induces apoptosis in human hepatocellular carcinoma cell line (HepG2).

BACKGROUND: Paclitaxel (taxol) is a potent anticancer drug that is used in the treatment of a wide variety of cancerous. In the present study, we identified a taxol derivative named 7-epi-10-deacetyltaxol (EDT) from the culture of an endophytic fungus Pestalotiopsis microspora isolated from the bark of Taxodium mucronatum. This study was carried out to investigate the effects of fungal EDT on cell proliferation, the induction of apoptosis and the molecular mechanisms of apoptosis in human hepatoma HepG2 cells in vitro. METHODS: The endophytic fungus was identified by traditional and molecular taxonomical characterization and the fungal EDT was purified using column chromatography and confirmed by various spectroscopic and chromatographic comparisons with authentic paclitaxel. We studied the in vitro effects of EDT on HepG2 cells for parameters such as cell cycle distribution, DNA fragmentation, reactive oxygen species (ROS) generation and nuclear morphology. Further, western blot analysis was used to evaluate Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), p38-mitogen activated protein kinase (MAPK) and poly [ADP-ribose] polymerase (PARP) expression. RESULTS: We demonstrate that the fungal EDT exhibited significant in vitro cytotoxicity in HepG2 cells. We investigated cytotoxicity mechanism of EDT in HepG2 cells. The results showed nuclear condensation and DNA fragmentation were observed in cells treated with fungal EDT. Besides, the fungal EDT arrested HepG2 cells at G2/M phase of cell cycle. Furthermore, fungal EDT induced apoptosis in HepG2 cells in a dose-dependent manner associated with ROS generation and increased Bax/Bcl-2 ratio, p38 MAPKs and PARP cleavage. CONCLUSIONS: Our data show that EDT induced apoptotic cell death in HepG2 cells occurs through intrinsic pathway by generation of ROS mediated and activation of MAPK pathway. This is the first report for 7-epi-10-deacetyltaxol (EDT) isolated from a microbial source.

A novel antibacterial and antifungal phenolic compound from the endophytic fungus Pestalotiopsis mangiferae.

A novel phenolic compound, 4-(2,4,7-trioxa-bicyclo[4.1.0]heptan-3-yl) phenol (1), was isolated from Pestalotiopsis mangiferae, an endophytic fungus associated with Mangifera indica Linn. The structure of the compound was elucidated on the basis of comprehensive spectral analysis (UV, IR, ¹H-, ¹³C- and 2D-NMR, as well as HRESI-MS). Compound (1) shows potent antibacterial and antifungal activity against Bacillus subtilis, Klebsiella pneumoniae, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa and Candida albicans. The transmission electron microscope study for the mode of inhibition of compound (1) on bacterial pathogens revealed the destruction of bacterial cells by cytoplasm agglutination with the formation of pores in cell wall membranes.