This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Bioprospecting indigenous actinomycetes for natural product discovery Pang, Li Mei 2018 Pang, L. M. (2018). Bioprospecting indigenous actinomycetes for natural product discovery. Doctoral thesis, Nanyang Technological University, Singapore. http://hdl.handle.net/10356/75687 https://doi.org/10.32657/10356/75687 Downloaded on 30 Sep 2021 11:26:30 SGT Bioprospecting Indigenous Actinomycetes for Natural Product Discovery Pang Li Mei SCHOOL OF BIOLOGICAL SCIENCES 2018 Bioprospecting Indigenous Actinomycetes for Natural Product Discovery Pang Li Mei SCHOOL OF BIOLOGICAL SCIENCES A thesis submitted to the Nanyang Technological University in partial fulfilment of the requirement for the degree of Doctor of Philosophy 2018 ii Acknowledgements First and foremost, I would like to express my gratitude to my supervisor, Assoc. Prof Liang Zhao-Xun for the opportunity to pursue a PhD programme in his laboratory. I am thankful for his patience, support and guidance throughout the past four years allowing me to experience different aspects of research life. His inspirational and motivational talks have brought me through the darkest moments of my research life. I would also like to thank my co-supervisor, Dr Tan Lik Tong (NIE) and thesis advisory committee, Prof James P Tam, Assoc. Prof Liu Chuan Fa and Assoc. Prof Newman Sze for their invaluable advice throughout my PhD. I am thankful for the research platform that NTU and MOE had provided for me to pursue my research. Next, I would like to thank National Parks Board for their assistance in our sample collection from Sungei Buloh Wetland Reserve. I would like to thank Assoc. Prof Cao Bin (CEE) and team for the providing samples from Pulau Ubin Quarry Lake. I would like to thank Asst. Prof Yang Liang (SCELSE) and his student Ding Yichen for the draft genome assembly of actinomycete strains. I would also like to thank our collaborators Dr Yoganathan K. (BII) and Assoc. Prof Valerie Lin for structure elucidation and estrogen response element assay for 4’,5-dihydroxy-7-methoxy-3-methylflavanone. Special thanks to Low Zhen Jie, Ye Hong and Gary Ding for providing the NMR spectra of 4’,5-dihydroxy- 7-methoxy-3-methylflavanone and echinomycin. This project would not be made possible without the help of a special team member, Low Zhen Jie, who fought extremely hard to get things to work. I am glad to be able to meet my fiancé during my PhD journey. His strengths complement my weaknesses which allowed me to pull through certain hurdles in experiments and in life. In addition, I am also grateful for the help and support from other lab members, Ye Rui Juan, Hoa Tran, Howard Saw, Xin Lingyi, Cheang Qing Wei, Sheng Shuo, and former FYP/attachment students. Lastly, I would like to thank my friends and families for their support and encouragement throughout my PhD programme. iii Table of contents Acknowledgements....................................................................................................... iii Table of contents ...........................................................................................................iv List of figures ................................................................................................................ vii List of tables ................................................................................................................... xi Abbreviations .............................................................................................................. xiii Abstract .......................................................................................................................... 1 CHAPTER 1: Background ........................................................................................... 3 1.1 Actinomycetes as a rich source of bioactive natural product .................... 3 1.2 Actinomycetes from unique environments ................................................. 5 1.3 Phenomenon of unculturable bacteria ........................................................ 7 1.4 Drug resistance and the need for new drug discovery ............................... 8 1.5 Discover bioactive compounds from actinomycetes in the post-genomics era ....................................................................................................................... 11 1.6 Overview of thesis ....................................................................................... 13 CHAPTER 2: Isolation of actinomycetes from mangrove and lake sediment ...... 15 2.1 Introduction ................................................................................................. 15 2.2 Materials and methods ............................................................................... 17 2.2.1 Source and sample collection ............................................................. 17 2.2.2 Pre-treatment methods ....................................................................... 19 2.2.3 Isolation media for actinomycetes ..................................................... 21 2.2.4 Assembly of in-situ cultivation disk ................................................... 25 2.2.5 Maintenance of strains ........................................................................ 26 2.2.6 Isolation of genomic DNA for 16S rDNA PCR amplification ......... 26 2.2.7 16S rDNA PCR amplification ............................................................ 27 2.2.8 Phylogenetic analysis .......................................................................... 27 2.3 Results .......................................................................................................... 28 2.3.1 Summary of strains from mangrove and lake sediment .................. 28 2.3.2 Comparison between direct plating and in-situ cultivation method ............................................................................................................... 31 2.3.3 Strain identification by 16S rDNA sequencing ................................. 32 2.4 Discussion ..................................................................................................... 36 2.5 Conclusion.................................................................................................... 39 CHAPTER 3: Strain prioritization by bioactivity screening .................................. 40 3.1 Introduction ................................................................................................. 40 iv 3.2 Materials and methods ............................................................................... 41 3.2.1 Cross streak assay ............................................................................... 41 3.2.2 Overlay assay ....................................................................................... 42 3.2.3 Fermentation of strains for bioactivity assays .................................. 43 3.2.4 Microtiter plate based antibacterial assay ........................................ 43 3.2.5 Microtiter plate based antifungal assay ............................................ 44 3.2.6 Anti-biofilm assay ............................................................................... 45 3.3 Results .......................................................................................................... 47 3.3.1 Antibacterial activities from Streptomyces sp. SW24, SD24 and SD50 ............................................................................................................... 48 3.3.2 Antibacterial activities from Streptomyces sp. P19 ........................... 49 3.3.3 Antibacterial and antifungal activities from Streptomyces sp. P9 ... 50 3.3.4 Antibacterial activities from Streptomyces sp. P46 ........................... 51 3.3.5 Antibacterial activities from Micromonospora sp. MD118 .............. 53 3.3.6 Antibacterial activities from Streptomyces sp. MD100 .................... 53 3.3.7 Antifungal activities from Streptomyces sp. MD102 and P7 ............ 54 3.3.8 Anti-biofilm activities from Streptomyces sp. SD9, SD35 and SD48 54 3.5 Conclusion.................................................................................................... 58 CHAPTER 4: Strain prioritization by genome sequencing and metabolite profiling ........................................................................................................................ 59 4.1 Introduction ................................................................................................. 59 4.2 Materials and methods ............................................................................... 60 4.2.1 Isolation of genomic DNA ................................................................... 60 4.2.2 Genome sequencing and assembly ..................................................... 61 4.2.3 Genome visualization of Micromonospora sp. MD118 with DNAplotter .......................................................................................................... 62 4.2.4 Analytical HPLC of crude extracts.................................................... 62 4.2.5 Fermentation of Streptomyces sp. P19 ............................................... 62 4.2.6 Fermentation of Streptomyces sp. SD50............................................. 63 4.3 Results .......................................................................................................... 63 4.3.1 Micromonospora sp. MD118 is a high potential strain that contains many novel biosynthetic gene