Diversity of Taxol Producing Endophytic Fungi from Taxus Baccata and Process Optimization for Taxol Production

Abstract

Endophytic fungi represent an under explored resource of novel lead compounds and have the capacity to produce diverse classes of plant secondary metabolites. In the present study, we investigated the diversity of taxol-producing endophytic fungi from Taxus baccata L. subsp. wallichiana (Zucc.) Pilger. A total of 60 fungal endophytes were isolated from the inner bark (phloem-cambium) of T. baccata, collected from different locations of the northern Himalayan region of India. Two key genes, DBAT (10-deacetylbaccatin III-10-O-acetyl transferase) and BAPT (C-13 phenylpropanoid side chain-CoA acyltransferase), involved in taxol biosynthesis were used as molecular markers for screening of the taxol producing strains. Five different endophytic species gave positive amplification hits by molecular marker screening with the bapt gene. These fungi were characterized based on morphological characters and internal transcribed spacer (ITS) sequence analysis and identified as: Fusarium redolens (TBPJ-B), Gibberella avenacea (C-1), Paraconiothyrium brasiliense (TBPJ-13), Microdiplodia sp. (TBPJ-A) and Fusarium tricinctum (B-7), The taxol-producing capability of these endophytic fungi was validated by HPLC-MS. The highest yield of taxol was found to be 66.25 μg/L by Fusarium redolens compared to other four strains. The amount of taxol produced by other four strains in S-7 liquid medium are TBPJ-A (27.40 µg/L), B-7 (23.47 µg/L), TBPJ-13 (19.60 µg/L) and C-1 (11.03 µg/L). The antitumour activity of the fungal taxol was tested by potato disc tumor induction assay using Agrobacterium tumefaciens as the tumor induction agent. This assay depicted that fungal taxol from all the endophytes successfully inhibited tumor formation in potato discs just like authentic taxol while it did not affect bacterial viability in any case. These results confirmed that all fungal extracts tested in this study had similar antineoplastic activity as that of paclitaxel. The medium components and different growth parameters were optimized for production of taxol from the endophytic fungus Fusarium redolens. Optimization of medium components was performed using Plackett-Burman (PB) design and response surface methodology (RSM). Different carbon and nitrogen sources were compared and then PB design was employed for screening the important trace elements. Results showed that NH4NO3, MgSO4 and NaOAc were the most important components which were further investigated by Box-Behnken design. Optimal concentrations of NH4NO3, MgSO4 and NaOAc achieved for maximum taxol production were 6.25, 0.63 and 1.25 g/L, respectively. The predicted response in Box-Behnken experimental design for taxol production gave a value of 195 µg/L, while the actual experimental value was 198 µg/L, suggesting that experimental and predicted values were in good agreement. About three fold increase of taxol production was observed after optimization of fermentation conditions and medium components by RSM. These results suggested the success of RSM in enhancing the production of fungal taxol. In the present investigation, potential of pharmaceutically important endophytes has been investigated. This study has shed light on endophyte biotechnology for its applications in production of anticancerous drug taxol. PCR amplification of genes involved in taxol biosynthesis (DBAT and BAPT) is an efficient and reliable method for pre-screening the taxol-producing fungi. F. redolens and F. verticillioides are the first ever reports of endophytic fungi capable of taxol production obtained from Taxus baccata subsp. wallichiana. Paraconiothyrium brasiliense, Microdiplodia sp. and Phomopsis sp. reported from other Taxus plants but not reported from the Himalayan yew. This study offers important information and a new source for the production of the important anticancer drug taxol by endophytic fungus fermentation. The endophytic taxol was found to be equally effective as that of authentic paclitaxel which paves way for exploitation of these potent endophytic fungi for industrial bioprocesses. Enhanced production of fungal taxol by RSM encouraged the use of these statistical tools for large scale production of this bioactive compound. Though further research at the molecular level is requisite for better understanding of the host endophyte interactions involved in production of this immensely demanding antitumour drug, the current work is a step forward to encourage the use of these fungal endophytes as alternative to the conventional sources.