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

The current study focuses on the isolation and in vitro characterization of bioactive metabolites produced by endophytic fungi isolated from the Himalayan yew (Taxus wallichiana Zucc.). The endophytic fungi were isolated on artificial media from inner tissues of bark and needles. Antimicrobial and antioxidant activity, along with total phenolic- and flavonoid-content assays were used in the evaluation of bioactivity of the fermented crude extracts. The ability of the endophytes to produce the anticancer compound Taxol was also analyzed using thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (RP-HPLC). A total of 16 fungal morphotypes were obtained from asymptomatic inner tissues of the bark and needles of T. wallichiana. Among the 16 isolates, the ethyl acetate (EA) fraction of isolate MUS1, showed antibacterial and antifungal activity against all test-pathogens used (Streptococcus faecalis ATCC 19433, Staphylococcus aureus ATCC 12600, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Salmonella enterica ATCC 13076, Pseudomonas aeruginosa ATCC 27853, and Candida albicans). MUS1 showed significant inhibition against Pseudomonas aeruginosa ATCC 27853 (minimum inhibitory concentration (MIC): 250 mu g/ml) and the pathogenic yeast, Candida albicans (MIC: 125 mu g/ml). Antioxidant activity, total phenolic, and total flavonoid content as well as in vitro Taxol production were evaluated for EA fraction of isolate MUS1. Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. At a concentration of 100 mu g/ml, the % DPPH radical scavenging activity was 83.15 +/- 0.40, 81.62 +/- 0.11, and 62.36 +/- 0.29, for ascorbic acid, butylated hydroxytoluene (BHT), and the EA fraction of MUS1, respectively. The DPPH-Half maximal inhibitory concentration (DPPH-IC50) value for the EA fraction was 81.52 +/- 0.23 mu g/ml, compared to BHT (62.87 +/- 0.08 mu g/ml) and ascorbic acid (56.15 +/- 0.19 mu g/ml). The total phenolic and flavonoid content in the EA fraction were 16.90 +/- 0.075 mu g gallic acid equivalent (GAE) and 11.59 +/- 0.148 mu g rutin equivalent (RE), per mg of dry crude extract, respectively. TLC and RP-HPLC analysis showed that the isolate MUS1 also produces Taxol (282.05 mu g/l of fermentation broth). Isolate MUS1 was identified as Annulohypoxylon sp. by internal transcribed spacer (ITS) sequencing. Having the ability to produce antimicrobial and antioxidant metabolites, as well as the anticancer compound Taxol, makes Annulohypoxylon sp. strain MUS1, a promising candidate for further study of naturally occurring bioactive metabolites.