Tuberculosis Endophytic Natural Products from Indonesian Traditional Medicinal Plants

250 Pages·2016·3.94 MB·English

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 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.

viii
DEDICATION

This thesis is firstly dedicated to my late father John and my mother Sisca. Mum and
Dad, you have supported me financially, emotionally, and spiritually throughout my
life. Father, I know you are watching me from up there. I finally made it.

This thesis is also dedicated to my partner in crime Inez. Your patience and selflessness
have helped me through my arduous journey. My dearest, I forever owe you my life.

From the bottom of my heart, I sincerely thank all of you. I love you all.
ix
ACKNOWLEDGMENTS

I would like to firstly thank my supervisor Professor Brett Neilan for allowing me to
work in his laboratory, giving me freedom to choose my project, funding my research,
and guiding me throughout my PhD. Thank you for the mentorship and friendship we
have built over the years. Aside from all the know-how about PKS and NRPS, without
doubt, the highlight of our interaction would be when you brought Eddy to visit my
hometown for the conference. On one of those very rare occasions of talking to you
sober on the bus to Herbarium Bogoriense, I finally understood your philosophy of
opening our eyes and minds in searching for scientific ideas from anyone around the
world without exception. I really hope and look forward to future collaborations with
you when I return to Indonesia. My mum and I are also proud for introducing you and
Eddy to Indonesian cuisine, particularly from our cultural heritage West Sumatra.
Apologies for the petai beans though.

I also thank my co-supervisor Dr. John Alexander Kalaitzis for imparting his scientific
knowledge to me throughout my degree, and for the friendship we have built especially
since I joined him as a cave dweller, or as I prefer to call it Wandercave. Thank you for
your patience in helping me to understand the chemistry side of my project. Thank you
also for sharing your hobbies and proud history of your family. You have inspired me to
uphold my family values and I shall forever treasure your quote, “The more lies they
say about me, the more truth I shall say about them”. And also perhaps one day, I will
be able to witness Parramatta Eels lift the Provan-Summons Trophy again. I think the
Wanderers will lift the A-League trophy again before that happens though.

I would also thank Dr. Rocky Chau, my unofficial co-supervisor. Your contribution to
my project, especially near my thesis submission, is immense. Your knowledge in
science technology is superb and your patience is admirable. Thank you for showing me
how to operate the NMR apparatus and how to interpret the data, and thank you for
checking my thesis chapters. Just to let you know, I still have those Albert Park Circuit
rocks you and John gave me two years ago and I am still not sure what to do with them.
At least I will remember the both of you whenever I see them.
x