Endophytic fungi harbored in Camptotheca acuminata, Hypericum perforatum and Juniperus communis plants as promising sources of camptothecin, hypericin and deoxypodophyllotoxin D I S S E R T A T I O N Submitted for the degree of Dr. rer. nat. (rerum naturalium) to the Faculty of Chemistry Technische Universität Dortmund by Souvik Kusari 2010 Endophytic fungi harbored in Camptotheca acuminata, Hypericum perforatum and Juniperus communis plants as promising sources of camptothecin, hypericin and deoxypodophyllotoxin APPROVED DISSERTATION Doctoral Committee Chairman: Prof. Dr. Carsten Strohmann Reviewers: 1. Prof. Dr. Dr.h.c. Michael Spiteller 2. Prof. Dr. Oliver Kayser Date of defense examination: October 04, 2010 Chairman of the examination: Prof. Dr. Christof M. Niemeyer “The grand aim of all science is to cover the greatest number of empirical facts by logical deduction from the smallest number of hypotheses or axioms” Albert Einstein (March 14, 1879 – April 18, 1955) THIS THESIS IS DEDICATED TO MY PARENTS … i Declaration Declaration I hereby declare that this thesis is a presentation of my original research work, and is provided independently without any undue assistance. Wherever contributions of others are involved, every effort is made to indicate this clearly, with due reference to the literature(s), and acknowledgement of collaborative research and discussions. This work was done under the guidance and supervision of Professor Dr. Dr.h.c. Michael Spiteller, at the Institute of Environmental Research (INFU) of the Faculty of Chemistry, Chair of Environmental Chemistry and Analytical Chemistry, TU Dortmund, Germany. Dated: August 10, 2010 SOUVIK KUSARI Place: Dortmund, Germany In my capacity as supervisor of the candidate’s thesis, I certify that the above statements are true to the best of my knowledge. Prof. Dr. Dr.h.c. Michael Spiteller Dated: August 10, 2010 ii List of original contributions List of original contributions Parts of the work reported in this thesis have already been published, presented and/or are intended for publication. PUBLICATIONS PUBLISHED ARTICLES 1. Kusari, S.; Zühlke, S.; Spiteller, M. (2010): Chemometric evaluation of the anti-cancer pro-drug podophyllotoxin and potential therapeutic analogues in Juniperus and Podophyllum species. Phytochemical Analysis, in press. 2. Kusari, S.; Zühlke, S.; Košuth, J.; 5ellárová, E.; Spiteller, M. (2009): Light-independent metabolomics of endophytic Thielavia subthermophila provides insight into microbial hypericin biosynthesis. Journal of Natural Products, 72, 1825-1835. 3. Kusari, S.; Zühlke, S.; Borsch, T.; Spiteller, M. (2009): Positive correlations between hypericin and putative precursors detected in the quantitative secondary metabolite spectrum of Hypericum. Phytochemistry, 70, 1222-1232. 4. Kusari, S.; Lamshöft, M.; Spiteller, M. (2009): Aspergillus fumigatus Fresenius, an endophytic fungus from Juniperus communis L. Horstmann as a novel source of the anticancer pro-drug deoxypodophyllotoxin. Journal of Applied Microbiology, 107, 1019-1030. 5. Kusari, S.; Zühlke, S.; Spiteller, M. (2009): An endophytic fungus from Camptotheca acuminata that produces camptothecin and analogues. Journal of Natural Products, 72, 2-7. 6. Kusari, S.; Lamshöft, M.; Zühlke, S.; Spiteller, M. (2008): An endophytic fungus from Hypericum perforatum that produces hypericin. Journal of Natural Products, 71, 159-162. PUBLISHED BOOK CHAPTERS 1. Kusari, S.; Spiteller, M. (2010): Lessons from endophytes: Peering under the skin of plants. In Biotechnology – Its Growing Dimensions, Patro, L. R. (ed.), Sonali Publications, New Delhi, India, pp. 1-27. 2. Kusari, S.; Spiteller, M. (2010): Do endophyte metabolomics reflect the phytochemical diversity of the host plant? A test of the hypothesis with Hypericum species. In Biodiversity Conservation and Management, Patro, L. R. (ed.), Discovery Publishing House Pvt. Ltd., New Delhi, India, pp. 1-28. iii List of original contributions SUBMITTED/UNDER REVISION 1. Kusari, S.; Košuth, J.; 5ellárová, E.; Spiteller, M. (2010): Perspectives on the survival- strategies of endophytic Fusarium solani against indigenous camptothecin biosynthesis. Submitted/under review. PRESENTATIONS INVITED TALKS 1. An endophytic Fusarium solani as a novel source of antineoplastic camptothecin and its 9- methoxy and 10-hydroxy derivatives. At the University of Agricultural Sciences (UAS), Bangalore, India. Within the scope of Indo-German (DBT-BMBF/DLR) collaboration. (7–15 Nov 2008). 2. Lessons from endophytes: Peering under the skin of plants. At the International Scientific Colloquium (ISC) and Alumni Meeting for International Summer Schools, Center of Applied Spectroscopy, Faculty of Technology, University of Novi Sad, R. Serbia. Funded by the German Academic Exchange Service (DAAD), Germany. (2–4 Oct 2008). ORAL PRESENTATIONS 1. Endophytes as hidden treasures within the botanical richness of nature: Camptotheca acuminata as an example. At the European Science Foundation-European Cooperation in Science and Technology (ESF-COST) High-Level Research Conference-Natural Products Chemistry, Biology and Medicine II, Acquafredda di Maratea, Italy. (29 Aug–3 Sept 2009). 2. An endophytic fungus as a novel source of antineoplastic camptothecin and its methoxy and hydroxy analogues. At the International Conference on “Emerging Trends in Biological Sciences”, KIIT School of Biotechnology, KIIT University, Orissa, India. (24–25 Oct 2008).(**) CONTRIBUTED TALKS 1. Bioactive compounds from endophytic fungi: Isolation, analysis and possibilities for biotechnological production. GDCh-Colloquium, Universität Duisburg-Essen, Germany. (02 June, 2010). 2. Endophytes as a promising source of bioactive compounds used in traditional medicine: future prospects and challenges. In Cameroon. (30 Nov–16 Dec 2009). 3. Endophytes, a promising source of bioactive compounds used in traditional medicine: future prospects and challenges. At the Alexander Humboldt Colloquium, Beijing, China. (24 Sept–2 Oct 2009). iv List of original contributions 4. Formation and structural elucidation of biologically active compounds from endophytic fungi. At Budapest University of Technology and Economics, Department of Inorganic and Analytical Chemistry, Budapest, Hungary. (09 Feb 2009). 5. Formation and structural elucidation of biological active compounds from endophytic fungi. At Sofia University "St. Kl. Ohridski", Faculty of Chemistry, Sofia. (20 Jan 2009). POSTERS 1. Endophytic Thielavia subthermophila demonstrates light-independent metabolomics in axenic cultures: what can we say about microbial hypericin biosynthesis in planta? At the 3rd Tag der Chemie (Chemistry Day); the Faculty of Chemistry, TU Dortmund, and the JCF Dortmund. (5 Feb 2010). 2. Thielavia subthermophila, an endophytic fungus from Hypericum perforatum L. as a novel source of hypericin and its probable precursor emodin. At the International Conference on "Emerging Trends in Biological Sciences", KIIT School of Biotechnology, KIIT University, Bhubaneswar, Orissa, India. (24–25 Oct 2008).(**) 3. Attenuation of camptothecin and 9-methoxycamptothecin production by endophytic Fusarium solani over generations: a possible indication of a split biosynthetic pathway? At the International Conference on "Emerging Trends in Biological Sciences", KIIT School of Biotechnology, KIIT University, Bhubaneswar, Orissa, India. (24–25 Oct 2008).(**) (**) Received ‘Young Researcher Award 2008’ for best scientific contribution; awarded by panel of judges headed by Prof. Dr. Richard R. Ernst (Nobel Laureate in Chemistry, 1991), the chief-guest of the conference. v ACKNOWLEDGEMENTS vi Acknowledgements A journey is easier when we travel together. This thesis embodies the results of the last couple of years’ work whereby I have been accompanied and supported by many people. It is an honor and a very pleasant opportunity to be able to express my gratitude to all of them. It has been a privilege to work under the dynamic leadership and guidance of my supervisor Prof. Dr. Dr.h.c. Michael Spiteller (Institut für Umweltforschung, INFU, der Fakultät Chemie, Lehrstuhl für Umweltchemie und Analytische Chemie, Technische Universität Dortmund). Only at his first rendezvous with me several years ago, he foresaw my amateur scientific intentions. Those fleeting moments that featured his visage would never be erased from my heart by the winds of time. His unerring support, coherence of scientific thoughts and remarkable presence of mind propelled me through my scholarly tenure to move forward during the finest moments and the darkest hours. I bow down before him with great respect for showing me the fourth dimension of life called ‘Science’. I am deeply grateful to Prof. Dr. Oliver Kayser (Lehrstuhl Technische Biochemie, Fachbereich Bio- und Chemieingenieurwesen, Technische Universität Dortmund) for giving me the golden opportunity to present my doctoral work to him. Even before I met him in person, I was always inspired, encouraged and fascinated by reading his work. His clarity of thoughts and the superlative command on science that festooned his articles and books are ever admirable. He could not even realize how much I have learned from him. His ideas and thoughts are treasure for me and will remain so for the rest of my life. I wish to express my deep sense of appreciation and gratitude to my senior colleagues Dr. Sebastian Zühlke and Dr. Marc Lamshöft for their generous help, critical suggestions, able guidance, and for the realization and interpretation of mass spectrometric