Production of Taxol by Endophytic Fungi Isolated from Roots of Himalayan Yew (Taxus wallichiana Zucc.)

Priyanka  Adhikari; Mithilesh  Singh; Anita  Pandey

doi:10.13052/jgeu0975-1416.1028

Priyanka Adhikari G.B. Pant National Institute of Himalayan Environment, Kosi-Katarmal, Almora-263643, Uttarakhand, India
Mithilesh Singh G.B. Pant National Institute of Himalayan Environment, Kosi-Katarmal, Almora-263643, Uttarakhand, India
Anita Pandey 1)G.B. Pant National Institute of Himalayan Environment, Kosi-Katarmal, Almora-263643, Uttarakhand, India 2)Department of Biotechnology, Graphic Era Deemed to be University, Bell Road, Clement Town, Dehradun-248002, Uttarakhand, India

DOI: https://doi.org/10.13052/jgeu0975-1416.1028

Keywords: Taxus wallichiana, endophytes, taxol, Aspergillus, Penicillium

Abstract

Taxol® (generic name – Paclitaxel), the most promising chemotherapeutic agent was isolated from bark of different Taxus sp. As Taxus species are threatened with extinction (endangered in Himalaya), thus it is imperative to develop alternate and sustainable method for commercialization and scale up production of paclitaxel. In this respect, physical and chemical parameters are effective and important key points for active compound production particularly by using endophytic microbes. In the present study, five endophytic fungi isolated from the roots of Taxus wallichiana, are tested for paclitaxel production using biochemical and molecular methods. Subsequently, effect of physico-chemical parameters like temperature, pH, incubation time, and medium constituents i.e., salt concentration, carbon and nitrogen sources on paclitaxel production were also analyzed. Among isolates, two of the fungi viz. GBPI_TWR F1 (Penicillium sp.) and GBPI_TWR F5 (Aspergillus sp.) were found to be paclitaxel producing. The genomic DNA samples were sequenced to confirm the presence of two genes viz. 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) and C-13 phenylpropanoid side chain-CoA acyltransferase (BAPT), implicated in paclitaxel biosynthesis. Both the endophytes showed the amplicons of DBAT and BAPT genes. Results revealed that after optimization of medium components and physical condition, paclitaxel production was increased in both the endophytes, maximum paclitaxel production i.e., 5.45 ± 0.98 mg/L was obtained by GBPI_TWR F5 (Aspergillus sp.) following 10 days of incubation at 15∘C in optimized S7 liquid medium composition.

Downloads

Download data is not yet available.

Author Biographies

Priyanka Adhikari, G.B. Pant National Institute of Himalayan Environment, Kosi-Katarmal, Almora-263643, Uttarakhand, India

Priyanka Adhikari is Research Associate at National Institute of Pharmaceutical Education and Research (NIPER), Guwahati-Assam, India. Before Joining NIPER, Dr. Adhikari worked as Junior and Senior Project Fellow in National Mission on Himalayan Studies (NMHS) Program of Ministry of Environment, Forest and Climate Change, Govt. of India at G.B. Pant National Institute of Himalayan Environment, Almora – Uttarakhand, India. Dr. Adhikari has M.Sc. in Microbiology and Ph.D. in Biotechnology from Kumaun University, Nainital-Uttarakhand, India. Her research interests include plant-microbe interactions and characterization, and identification of microbe and plant based bioactive compounds and drug formulation. Dr Adhikari has been recognized for National Award (twice) from MoEF & CC Himalayan Researchers Consortium and National Medicinal Plant Board (NMPB) Govt. of India for her work on microbiological and biochemical aspects of Taxus wallichiana. Later, she received the Young Scientist Travel Grant to present her work in 8th Conference “FEMS 2019” at Glasgow Scotland, United-Kingdom. She has published more than 20 peer reviewed papers in National and International Journals.

Mithilesh Singh, G.B. Pant National Institute of Himalayan Environment, Kosi-Katarmal, Almora-263643, Uttarakhand, India

Mithilesh Singh is working as Scientist at Centre for Environmental Assessment and Climate Change, G.B. Pant National Institute of Himalayan Environment (GBP-NIHE), Almora, Uttarakhand. She has done post graduation in Botany from Banaras Hindu University and PhD in Biotechnology from IIT Guwahati, Assam. Her field of specialization includes plant biotechnology and bioprospection. She has published 55 research papers, chapters in edited books/proceedings and scientific popular articles having over 520 citation and 12 h index. Dr. Singh has received DBT BioCARe Women Scientist Award in 2014.

Anita Pandey, 1)G.B. Pant National Institute of Himalayan Environment, Kosi-Katarmal, Almora-263643, Uttarakhand, India 2)Department of Biotechnology, Graphic Era Deemed to be University, Bell Road, Clement Town, Dehradun-248002, Uttarakhand, India

Anita Pandey is presently affiliated with the Department of Biotechnology, Graphic Era (Deemed to be University, Dehradun, Uttarakhand, India. Previously, she was Scientist at G.B. Pant National Institute of Himalayan Environment, Almora, India. Dr. Pandey has extensively worked on Bioprospection of Microbial Diversity of Indian Himalayan region (IHR). Her research areas include- Extremophiles, Rhizosphere, Biodegradation, Fermented Foods, and Antimicrobials.

Her scientific contributions have been recognized at State, National, and International levels. She has been recipient of National Bioscientist Award by Department of Biotechnology, Govt. of India and Vishisht Mahila Vaigyanik Sammaan (Eminent Woman Scientist Recognition) by Uttarakhand Science, Education and Research Center, Department of Science and Technology, Govt. of Uttarakhand, India.

Dr. Pandey has hosted CV Raman International Fellowship for African Researchers twice (Morocco and Egypt). A new archeal species (Nantronococcus pandeyae sp. nov.) has been named in her honour for her extensive contribution to microbial diversity of IHR (Current Microbiology, vol. 79, Article number 51, 2022).

References

Abdel-Fatah, S.S., El-Batal, A.I., El-Sherbiny, G.M., Khalaf, M.A., El-Sayed, A.S. (2021). Production, bioprocess optimization and g-irradiation of Penicillium polonicum, as a new paclitaxel producing endophyte from Ginkgo biloba. Biotechnology Report. 30, e00623.

Adhikari, P., Pandey, A. (2019). Phosphate solubilization potential of endophytic fungi isolated from Taxus wallichiana Zucc. roots. Rhizosphere. 9, 2–9.

Adhikari, P., Pandey, A. (2018). Diversity of endophytic fungi associated with Himalayan yew (Taxus wallichiana Zucc.) roots. Proceedings of Himalayan Researchers Consortium. 1, 165–173.

Afshari, M., Shahidi, F., Mortazavi, S.A., Tabatabai, F., Eshaghi, Z. (2015). Investigating the influence of pH, temperature and agitation speed on yellow pigment production by Penicillium aculeatum ATCC 10409. Natural Products Research. 29, 1300–1306.

Andualem, B., Gessesse, A. (2013). Production of microbial medium from defatted brebra (Milletia ferruginea) seed flour to substitute commercial peptone agar. Asian Pacific Journal of Tropical Biomedical. 3, 790–797.

Barbuti, A.M., Chen, Z.S. (2015) Paclitaxel through the ages of anticancer therapy: Exploring its role in chemoresistance and radiation therapy. Cancers. 7, 2360–2371.

Choi, H.K., Kim, S.I., Son, J.S., Hong, S.S., Lee, H.S., Chung, I.S., Lee, H.J. (2000) Intermittent maltose feeding enhances paclitaxel production in suspension culture of Taxus chinensis cells. Biotechnology Letters. 22, 1793–1796.

Costa, E., Teixido, N., Usall, J., Atares, E., Vinas, I. (2002). The effect of nitrogen and carbon sources on growth of the biocontrol agent Pantoea agglomerans strain CPA-2. Letters in Applied Microbiology. 25, 117–120.

Chen, Y.J., Zhang, Z., Wang, Y., Su, Y., Zhang, R. (2003). Screening endophytic fungus to produce paclitaxel from Taxus yunnanensis. Biotechnology. 13, 10–11.

El-Bialy, H.A., El-Bastawisy, H. (2020). Elicitors stimulate paclitaxel production by endophytic fungi isolated from ecologically altered Taxus baccata. Journal of Radiation Research and Applied Sciences. 13, 79–87.

El-Sayed, A.S.A., Safan, S., Mohamed, N.Z., Shaban, L., Ali, G.S., Sitohy, M.Z. (2018) Induction of paclitaxel biosynthesis by Aspergillus terreus, endophyte of Podocarpus gracilior Pilger. upon intimate interaction with the plant endogenous microbes. Process Biochemistry. 71, 31–40.

El-Sayed, R., Ahmed, A.S., Hassan, I.A., Ismail, A.A., El-Din, A.Z.A.K., (2020). Semi-continuous production of the anticancer drug paclitaxel by Aspergillus fumigatus and Alternaria tenuissima immobilized in calcium alginate beads. Bioprocess and Biosystems Engineering. 43, 997–1008.

Kumar, P., Singh, B., Thakur, V., Thakur, A., Thakur, N., Pandey, D., Chand, D. (2019). Hyper production of paclitaxel from Aspergillus fumigatus, an endophytic fungus isolated from Taxus sp. of the northern Himalayan region. Biotechnology Reports. 24, 1–13.

Kusari, S., Singh, S., Jayabaskaran, C. (2014). Rethinking production of paclitaxel (paclitaxel) using endophyte biotechnology. Trends in Biotechnol. 32, 304–311.

Li, D., Fu, D.W., Zhang, Y., Ma, X., Gao, L., Wang, X., Zhau, D., Zhao, K. (2017). Isolation, purification, and identification of paclitaxel and related taxanes from paclitaxel-producing fungus Aspergillus niger subsp. taxi. Journal of Microbiology and Biotechnology. 27, 1379–1385.

Nadeem, M., Rikhari, H.C., Kumar, A., Palni, L.M.S., Nandi, S.K. (2002). Paclitaxel content in the bark of Himalayan Yew in relation to tree age and sex. Phytochemistry. 60, 627–631.

Naik, B.S. (2018). Developments in paclitaxel production through endophytic fungal biotechnology: A review. Oriental Pharmacy and Experimental Medicine. 19, 1–13.

NATA, Technical note 17 (2013). Guidelines for the validation and verification of quantitative and qualitative test methods. National Association of Testing Authorities, Australia. pp. 17–18.

Pandey, N., Jain, R., Pandey, A., Tamta, S. (2018). Optimisation and characterisation of the orange pigment produced by a cold adapted strain of Penicillium sp. (GBPI_P155) isolated from mountain ecosystem. Mycology. 9, 81–92.

Poupat, C., Hook, I., Gueritte, F., Ahond, A., Guenard, D., Adeline, M.T., Wang, X.P., Dempsey, D., Breuillet, S., Potier, P. (2000). Neutral and basic taxoid contents in the needles of Taxus species. Planta Medica. 66, 580–584.

Qiao, W., Ling, F., Yu, L., Huang, Y., Wang, T. (2017). Enhancing paclitaxel production in a novel endophytic fungus, Aspergillus aculeatus Tax-6, isolated from Taxus chinensis var. mairei. Fungal Biology. 121, 1037–1044.

Qiao, W., Tang, T., Ling, F. (2021). Comparative transcriptome analysis of a paclitaxel-producing endophytic fungus, Aspergillus aculeatinus Tax-6, and its mutant strain. Scientific Reports. 10, 10558. https://doi.org/10.1038/s41598-020-67614-1.

Roopa, G., Madhusudhan. M.C., Sunil, K.C.R., Lisa, N., Calvin, R., Poornima, R., Zeinab, N., Kini, K.R., Prakash, H.S., Geetha, N. (2015). Identification of paclitaxel producing endophytic fungi isolated from Salacia oblonga through genomic mining approach. Journal of Genetic Engineering and Biotechnology. 13, 119–127.

Saghai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A., Allard, R.W. (1984). Ribosomal DNA spacer-length polymorphism in barley: Mendelian inheritance, chromosomal location and population dynamics. Proceedings of National Academy of Science USA. 81, 8014–8018.

Sanchez, S., Chavez, A., Ferero, A., Huante, Y.G., Romero, A., Sanchez, M., Richa, D., Sanchez, B., Avalos, M., Trampe, S.G., Sanoja, R.R., Langley, E., Ruiz, B. (2010). Carbon source regulation of antibiotic production. Journal of Antibiotics. 63, 442–459.

Sengul, U. (2016). Comparing determination methods of detection and quantification limit for oflatoxin analysis in hazulnut. Journal of Food Drug and Analysis. 24, 56–62.

Sharma, B., Jha, D.K. (2015). Role of nitrogen sources in regulation of fungal secondary metabolism. In: Gupta VK, Mach RL, Sreenivasaprasad S (eds) Fungal biomolecules: Sources, applications and recent developments, 1st edn. John Wiley & Sons, Ltd., pp. 213–224.

Shreshta, K., Strobel, G.A., Shrivastava, S.P., Gewali, M.B. (2001). Evidence for paclitaxel from three new endophytic fungi of Himalayan Yew of Nepal. Planta Medica. 67, 374–376.

Shu, C.H. (2007). Fungal fermentation for medicinal plants. In: Yang ST (ed) Bioprocessing for value-added products from renewable resources, 1st edn. Elsevier, pp. 447–463.

Somjaipeng, S., Median, A., Kwasna, H., Ordaz-Ortiz, J., Magan, N. (2015). Isolation, identification and ecology of growth and paclitaxel production by an endophytoc strain of Paraconiothyrium variabile from English Yew tree (Taxus baccata). Brazilian Mycological Society. 119, 1022–1031.

Somjaipeng, S., Medina, A., Magan, N. (2016). Environmental stress and elicitors enhance paclitaxel production by endophytic strains of Paraconiothyrium variabile and Epicoccum nigrum. Enzyme and Microbial Technology. 90, 69–75.

Sun, D., Ran, X., Wang, J. (2008). Isolation and identification of a paclitaxel-producing endophytic fungus from Podocarpus. Acta Microbiologica Sinica. 48, 589–595.

Thomas, P., Farjon, A. (2011). Taxus wallichiana. The IUCN Red List of Threatened 2011:e.T46171879A9730085.

Stierle, A., Strobel, G., Stierle, D., 1993. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Sci. 260, 214–216.

Strobel, G.A., Yang, X., Sears, J., Kramer, R., Sidhu, R., Hess, W.M. (1996). Paclitaxel from Pestalotiopsis microspora, an endophytic fungus of Taxus wallichiana. Microbiology. 142, 435–440.

Wani, M.C., Taylor, H.L., Wall, M.E., Coggon, O., McPhail, A.T. (1971). Plant antitumor agents VI. The isolation and structure of paclitaxel, a novel antileukemic and antitumor agent from Taxus brevifolia. Journal of American Chemical Society. 92, 2325–2327.

Walker, K.D., Croteau, R.B. (2000). Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli. Proceedings of National Academic Science. 97, 583–587.

Yang, Y., Zhao, H., Barrero, R.A., Zhang, B., Sun, G., Wilson, L.W., Xie, F., Walker, K.D., Parks, J.W., Bruce, R., Guo, G., Chen, L., Zhang, Y., Huang, X., Tang, Q., Liu, H., Bellgard, M.I., Qiu, D., Lai ,J., Hoffman, A. (2014). Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum NRRL 62431. BMC Genomics. 69. https://doi.org/~10.1186/1471-2164-15-69

Zaiyou, J., Li, M., Xiqiao, H. (2017). An endophytic fungus efficiently producing paclitaxel isolated from Taxus wallichiana var. mairei. Medicine. 96, e7406. https://doi.org/10.1097/MD.0000000000007406