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Bioactive Actinobacteria Associated with Two South African Medicinal Plants, Aloe Ferox and Sutherlandia Frutescens

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Bioactive Actinobacteria Associated with Two South African Medicinal Plants, Aloe Ferox and Sutherlandia Frutescens Bioactive actinobacteria associated with two South African medicinal plants, Aloe ferox and Sutherlandia frutescens Maria Catharina King A thesis submitted in partial fulfilment of the requirements for the degree of Doctor Philosophiae in the Department of Biotechnology, University of the Western Cape. Supervisor: Dr Bronwyn Kirby-McCullough August 2021 http://etd.uwc.ac.za/ Keywords Actinobacteria Antibacterial Bioactive compounds Bioactive gene clusters Fynbos Genetic potential Genome mining Medicinal plants Unique environments Whole genome sequencing ii http://etd.uwc.ac.za/ Abstract Bioactive actinobacteria associated with two South African medicinal plants, Aloe ferox and Sutherlandia frutescens MC King PhD Thesis, Department of Biotechnology, University of the Western Cape Actinobacteria, a Gram-positive phylum of bacteria found in both terrestrial and aquatic environments, are well-known producers of antibiotics and other bioactive compounds. The isolation of actinobacteria from unique environments has resulted in the discovery of new antibiotic compounds that can be used by the pharmaceutical industry. In this study, the fynbos biome was identified as one of these unique habitats due to its rich plant diversity that hosts over 8500 different plant species, including many medicinal plants. In this study two medicinal plants from the fynbos biome were identified as unique environments for the discovery of bioactive actinobacteria, Aloe ferox (Cape aloe) and Sutherlandia frutescens (cancer bush). Actinobacteria from the genera Streptomyces, Micromonaspora, Amycolatopsis and Alloactinosynnema were isolated from these two medicinal plants and tested for antibiotic activity. Actinobacterial isolates from soil (248; 188), roots (0; 7), seeds (0; 10) and leaves (0; 6), from A. ferox and S. frutescens, respectively, were tested for activity against a range of Gram-negative and Gram-positive human pathogenic bacteria. These isolates were mass screened for antibiotic activity using a perpendicular streak method followed by screening using a standard overlay technique. Twenty isolates from each plant with high antibacterial activity were subjected to further antibiotic screening and revealed activity against B. cereus (80%), S. aureus (85%) and P. aeruginosa (55%). Investigation into the genomes of two Streptomyces strains, strain A81 and strain S149, revealed that these isolates have the genetic potential to produce a wide range of natural products, including genes involved in the production of antibiotics, anti-tuberculosis, anti-HIV, and anti-tumour compounds. Interestingly, strain A81 additionally had genes that are involved in the production of a typical plant metabolite, naringenin, which is also produced by the strain’s host plant, A. ferox. The genome of strain S149 contained genetic machinery for the production of auxin, another typical plant hormone. This endophyte was isolated from the leaves of S. frutescens and production of auxin may indicate its involvement with plant growth and development. iii http://etd.uwc.ac.za/ Findings from this study thus highlights that actinobacteria associated with medicinal plants, and unique environments in general, have great potential to produce a wide range of pharmaceutically important compounds. Furthermore, in addition to antibiotics the actinobacteria isolated in this study have the potential to produce anti-tuberculosis, anti-HIV and anti-tumour compounds. August 2021 iv http://etd.uwc.ac.za/ Declaration I declare that “Bioactive actinobacteria associated with two South African medicinal plants, Aloe ferox and Sutherlandia frutescens.” is my own work, that it has not been submitted for any degree or examination in any other university, and that all the sources I have used or quoted have been indicated and acknowledged by complete references. Full name: ..............................................................Maria Catharina King Date: ......................2021-08-13............. Signed: ......................................... v http://etd.uwc.ac.za/ Acknowledgements First, I would like to thank my supervisor, Dr Bronwyn Kirby-McCullough, for her guidance, support and advice throughout this process. Thank you for teaching me how to be a great scientist and for believing in my abilities. Thanks to all the researchers and staff at IMBM, with special thanks to Prof M Trindade, Dr L van Zyl and Dr A Burger for their further guidance and insights into the project. Mr Leo Blackway, Ms Robin Karelse and Ms Carmen Cupido are thanked for their technical and administrative support in the laboratory and for making sure that everything ran smoothly. The study would not have been possible without the funding from the National Research Fund (NRF) and the Institute for Microbial Biotechnology and Metagenomics (IMBM). A special thanks to Organic Aloe (Pty) Ltd in Albertinia and Parceval (Pty) Ltd in Wellington for sampling of plant and soil material from their sites. Thanks for being willing to help and showing us around your beautiful farms. I would also like to thank my fellow students from IMBM for their continuous support during the years, especially the Actino group. To all my loved ones and friends, thanks for believing in me and supporting me through the hard times and sharing in my happiness. Thanks to my family members, especially my husband, parents, and in-laws, for your love and support throughout my academic career. And finally, I am thankful to the Lord for everything I have in this life and the continuous blessings I receive. vi http://etd.uwc.ac.za/ Content Title Page ............................................................................................................................... i Keywords ...............................................................................................................................ii Abstract................................................................................................................................. iii Declaration ............................................................................................................................ v Acknowledgements ............................................................................................................... vi Chapter 1: Literature review .................................................................................................. 1 1.1 Natural product discovery ............................................................................................ 1 1.1.1 Introduction ........................................................................................................... 1 1.1.2 A Brief History of NP’s and drug discovery ............................................................ 2 1.1.3 Discovering NPs today .......................................................................................... 4 1.1.4 NP discovery and the future ................................................................................ 12 1.2 Actinobacteria ............................................................................................................ 13 1.2.1 Lifecycle .............................................................................................................. 13 1.2.2 Actinobacterial Taxonomy ................................................................................... 14 1.2.3 Genomic classification......................................................................................... 15 1.2.4 Physiological and Biochemical Characteristics .................................................... 16 1.2.5 Morphological Characteristics ............................................................................. 16 1.2.6 Chemotaxonomy of Actinobacteria ...................................................................... 17 1.3 Genetic and Metabolic potential of Actinobacteria ..................................................... 21 1.3.1 Antibiotics............................................................................................................ 23 1.3.2 Anti-tumour compounds ...................................................................................... 24 1.3.3 Antifungals .......................................................................................................... 25 1.3.4 Antivirals ............................................................................................................. 26 1.3.5 Other bioactive compounds and activities ........................................................... 26 1.4 Plant-microbe interactions ......................................................................................... 28 1.4.1 Rhizobacteria ...................................................................................................... 29 1.4.2 Epiphytes and Endophytes .................................................................................. 30 vii http://etd.uwc.ac.za/ 1.4.3 Actinobacterial interactions with Medicinal Plants................................................ 32 1.5 Medicinal plants ......................................................................................................... 34 1.5.1 Medicinal plants of South Africa .......................................................................... 35 1.5.2 Aloe ferox ............................................................................................................ 37 1.5.3 Sutherlandia frutescens......................................................................................
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