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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 EC 50 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.

ABSTRACT 10-deacetylbaccatin III-10-β-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. Biochemical insights into the biotechnologically important fungal recombinant 10-deacetylbaccatin III-10-I2-O-acetyltransferase enzyme.

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.

The present study highlights/demonstrates facile synthesis of β-Glucan nanoparticles (β-GluNPs) that can be used in the prevention of breast cancer and other infectious diseases. Moreover, this method is inexpensive and shows effectivity towards different biological applications. Further, the characterization of synthesized β-GluNPs was exclusively confirmed through UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), zeta potential, scanning electron microscopy (SEM), high resolution-transmission electron microscopy (HR-TEM), and X-ray powder diffraction (XRD) analysis. The synthesized β-GluNPs were further confirmed by FT-IR spectroscopy. The HR-TEM results demonstrated that the formation of polydispersed nanoparticles with a mean size of 20 ± 5 nm. The hydrostatic zeta potential was − 22.7 mV, which indicated their colloidal stability. The XRD pattern revealed the crystalline nature of the nanoparticles. Besides, β-GluNPs showed better antibacterial activity against the tested pathogens. The apoptosis and DNA fragmentation observed to be IC50 42.5 µg/ml of the β-GluNPs. The DNA fragmentation assay indicated the selective inhibition of the MCF-7 cell line by DNA damage. Hence, the study reports that the β-GluNPs have a potential to be used as a promising alternative drug against human breast cancer.

Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp--CrP14, obtained from stem tissues, and Talaromyces radicus--CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 μg/ml and 20 μg/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus--CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus--CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 μg/l) in modified M2 medium and of vinblastine (70 μg/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns.

Drug resistance to the classically used chemotherapeutic drugs is the major challenge in their treatment of cancer needing the discovery of novel anticancer drugs. In terms of finding novel therapeutics, endophytes are quite promising as they are an excellent source of novel structures, which exhibit bioactivity. The present study demonstrated a dose-dependent antiproliferative activity of mycelial-derived secondary metabolites from a macroalgae associated endophyte Aspergillus unguis AG 1.1(G). The antiproliferative activity of A. unguis mycelial extract (AUME) was observed on different human cancer cell lines. PI live/dead assay further confirmed the cytotoxic potential of the mycelial extract. Furthermore, A. unguis mycelial extract caused mitochondrial membrane aberration and generated ROS production as well indicating its potential to induce cell death by apoptosis. The metabolic profiling of the mycelial extract using GC-MS and LC-MS/MS revealed the presence of fatty acids, a benzoquinolinone derivative, imidazolidinedione derivative, diethyl phthalate and phthalate acid ester, a difuraxanthone, two prenylxanthone analogues and a phthalide derivative and some unknown metabolites. Presence of 4-(4-Hydroxy-3,5-dimethoxy-phenyl)-3,4-dihydro-1H-benzo[h]quinolin-2-one, 1-hydroxy-3,5-dimethoxy-2-prenylxanthone, 1,6-dihydroxy-3-methoxy-2-prenylxanthone and 3-butylidene-7-hydroxyphthalide in AUME could be correlated to the notable cytotoxicity exhibited by the endophyte. The additional presence of many unidentified compounds heightened the prospects of finding some novel bioactive metabolites. Our results indicated that secondary metabolites produced by A. unguis AG 1.1 (G) have therapeutic potential as anticancer agents.

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.

Eighteen endophytic fungi were isolated from various tissues of Datura metel and genes encoding for putrescine N-methyltransferase (PMT), tropinone reductase 1 (TR1) and hyoscyamine 6β-hydroxylase (H6H) were used as molecular markers for PCR-based screening approach for tropane alkaloids (TAs) producing endophytic fungi. These fungi were identified taxonomically by sequence analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) and also based on morphological characteristics of the fungal spore as Colletotrichum boninense, Phomopsis sp., Fusarium solani, Colletotrichum incarnatum, Colletotrichum siamense and Colletotrichum gloeosporioides. The production of TAs hyoscyamine and scopolamine by the fungi has been ascertained using chromatography and spectroscopy methods by comparison with the standards. Among the fungi, the highest yields of hyoscyamine (3.9 mg/L) and scopolamine (4.1 mg/L) were found in C. incarnatum culture. This is the first report of endophytic fungi possess the PMT, TR1 and H6H genes and produces TAs. These endophytic fungi have significant potential to be applied in fermentation technology to meet the demands for TAs economically.

Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported here. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in V(max) of the enzyme activity by these phospholipids significantly decreased the K(m) indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K(½) = 114 nM) compared to PA (K(½) = 335 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response.

•Fungal endophytes have been shown to produce Taxol® (paclitaxel).•Industrial-scale Taxol® production using fungal endophytes has not yet been achieved.•Further research on biosynthesis and regulation of Taxol® production is required.•Improved strategies for sustained Taxol® supply using endophytes must be developed. Taxol® (generic name paclitaxel) represents one of the most clinically valuable natural products known to mankind in the recent past. More than two decades have elapsed since the notable discovery of the first Taxol®-producing endophytic fungus, which was followed by a plethora of reports on other endophytes possessing similar biosynthetic potential. However, industrial-scale Taxol® production using fungal endophytes, although seemingly promising, has not seen the light of the day. In this opinion article, we embark on the current state of knowledge on Taxol® biosynthesis focusing on the chemical ecology of its producers, and ask whether it is actually possible to produce Taxol® using endophyte biotechnology. The key problems that have prevented the exploitation of potent endophytic fungi by industrial bioprocesses for sustained production of Taxol® are discussed.