Bioprospecting for Anti- Tuberculosis Endophytic Natural Products from Indonesian Traditional Medicinal Plants

Bioprospecting for Anti- Tuberculosis Endophytic Natural Products from Indonesian Traditional Medicinal Plants A DISSERTATION SUBMITTED BY Alfonsus Alvin B.SC. HONS (UNSW) IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY (Ph.D.) AT THE SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES THE UNIVERSITY OF NEW SOUTH WALES SYDNEY, AUSTRALIA April 2016 Bioprospecting for Anti- Tuberculosis Endophytic Natural Products from Indonesian Traditional Medicinal Plants A DISSERTATION SUBMITTED BY Alfonsus Alvin B.SC. HONS (UNSW) IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY (Ph.D.) AT THE SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES THE UNIVERSITY OF NEW SOUTH WALES SYDNEY, AUSTRALIA SUPERVISORS PROFESSOR BRETT ANTHONY NEILAN SUPERVISOR DR. JOHN ALEXANDER KALAITZIS CO-SUPERVISOR SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES THE UNIVERSITY OF NEW SOUTH WALES SYDNEY, AUSTRALIA THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or family name: Alvin First name: Alfonsus Other name/s: - Abbreviation for degree as given in the University calendar: PhD School: Biotechnology and Biomolecular Sciences Faculty: Science Title: Bioprospecting for Anti-Tuberculosis Endophytic Natural Products from Indonesian Traditional Medicinal Plants Abstract 350 words maximum: The rapid emergence of antibiotic-resistant pathogens has driven the discovery of new drug leads from natural sources. Traditional medicinal plants have long been investigated as sources of bioactive molecules due to their rich ethnobotanical history. The vast biodiversity and empirical medicinal history of Indonesia make their flora an attractive target for the discovery of novel therapeutic compounds. A large proportion of bioactive natural products are non- ribosomal peptides and polyketides. Many of these compounds, which were originally isolated from plants, have since been found to be produced by their microbial endophytes. It is also understood that the genes encoding the biosynthesis of these bioactive molecules are useful for dereplication. Therefore, genetic and bioactivity screening of culturable endophytes from twelve traditional Indonesian medicinal plants used to treat symptoms of tuberculosis was conducted to identify strains capable of producing potential antitubercular polyketides and peptides. Phylogenetic analysis of the endophytes revealed a rich community of bacteria from the phyla Firmicutes, Actinobacteria, and Proteobacteria, and fungi from the phylum Ascomycota. A high proportion of these endophytes (83% of bacteria and 94% of fungi) contained either non-ribosomal peptide or polyketide biosynthetic genes. Preliminary antibacterial screening of the fungi against Gram-positive, Gram-negative, and mycobacterial strains showed most isolates exhibited antiproliferative activity against at least one of the test strains, and suggested a correlation between the biosynthetic genes and bioactivity. Two isolates which exhibited bactericidal activity against M. tuberculosis were selected for active compound isolation. Bioassay- and NMR-guided fractionation resulted in the discovery of four anti-M. tuberculosis polyketides: javanicin and anhydrofusarubin from Fusarium sp. 9RF2 (MIC of 25 μg mL-1 and 50 μg mL-1, respectively), and acropyrone and compound 11UF1.S-5D6B from Endothia sp. 11UF1 (MIC of 50 μg mL-1 and 100 μg mL-1, respectively). This investigation confirmed the hypothesis that traditional medicinal plants are valuable sources of endophytes that produce bioactive compounds. As the world continues to search for novel pharmaceuticals, prospecting of genetic resources, as described in this thesis, is a viable and productive approach that has the potential to be adapted in the context of exploiting traditional medicinal plants from around the world. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). .................................................. .................................................. .................................................. Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS iv ORIGINALITY STATEMENT I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged. Signed ............................................... Date ............................................... v COPYRIGHT STATEMENT I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International. I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation. Signed ............................................... Date ............................................... AUTHENTICITY STATEMENT I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format. Signed ............................................... Date ............................................... vi ABSTRACT The rapid emergence of antibiotic-resistant pathogens has driven the discovery of new drug leads from natural sources. Traditional medicinal plants have long been investigated as sources of bioactive molecules due to their rich ethnobotanical history. The vast biodiversity and empirical medicinal history of Indonesia make their flora an attractive target for the discovery of novel therapeutic compounds. A large proportion of bioactive natural products are non-ribosomal peptides and polyketides. Many of these compounds, which were originally isolated from plants, have since been found to be produced by their microbial endophytes. It is also understood that the genes encoding the biosynthesis of these bioactive molecules are useful for dereplication. Therefore, genetic and bioactivity screening of culturable endophytes from twelve traditional Indonesian medicinal plants used to treat symptoms of tuberculosis was conducted to identify strains capable of producing potential antitubercular polyketides and peptides. Phylogenetic analysis of the endophytes revealed a rich community of bacteria from the phyla Firmicutes, Actinobacteria, and Proteobacteria, and fungi from the phylum Ascomycota. A high proportion of these endophytes (83% of bacteria and 94% of fungi) contained either non-ribosomal peptide or polyketide biosynthetic genes. Preliminary antibacterial screening of the fungi against Gram-positive, Gram-negative, and mycobacterial strains showed most isolates exhibited antiproliferative activity against at least one of the test strains, and suggested a correlation between the biosynthetic genes and bioactivity. Two isolates which exhibited bactericidal activity against M. tuberculosis were selected for active compound isolation. Bioassay- and NMR-guided fractionation resulted in the discovery of four anti-M. tuberculosis polyketides: vii javanicin and anhydrofusarubin from Fusarium sp. 9RF2 (MIC of 25 μg mL-1 and 50 μg mL-1, respectively), and acropyrone and compound 11UF1.S-5D6B from Endothia sp. 11UF1 (MIC of 50 μg mL-1 and 100 μg mL-1, respectively). This investigation

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