GENETIC MANIPULATION OF SECONDARY METABOLITE PRODUCTION IN ACTINOMYCETES By:

........................................................................................................................................... iii Acknowledgements .......................................................................................................................... vi List of Figures ................................................................................................................................... x List of Tables .................................................................................................................................. xii Abbreviations ................................................................................................................................. xiii Declaration of Academic Achievement ......................................................................................... xiv Chapter 1: Introduction .................................................................................................................... 1 1.1 Need for Discovery of Novel Antibiotics .............................................................................. 1 1.2 Streptomyces: Brief History and Life Cycle ......................................................................... 2 1.3 Streptomyces as source of novel antibiotics ........................................................................... 3 1.4 Regulation of secondary metabolite production .................................................................... 6 1.5 Strategies to improve secondary metabolite production ...................................................... 10 1.5.1 Manipulation of pathway-specific regulators:............................................................... 12 1.5.2 Manipulation of pleiotropic regulators in native hosts: ................................................ 13 1.5.3 Heterologous expression of pleiotropic regulators to activate cryptic gene cluster: ..... 14 Chapter 2: Materials and Methods ................................................................................................. 21 2.1 Bacterial strains, vectors and growth conditions .................................................................. 21 2.2 Conjugation and screening ................................................................................................... 22 2.2.1 Medium-throughput conjugation .................................................................................. 22 2.2.2 PCR confirmation of the ex-conjugants ........................................................................ 23 2.2.3 Colony diffusion assays ................................................................................................ 24 2.3 Metabolite Profiling, Purification and Characterization of the Molecules .......................... 24 2.3.1 Preparation of crude extracts for comparative metabolite profiling.............................. 24 2.3.2 Preparation of large scale crude extracts for comparative metabolite profiling ............ 25 2.3.3 LC-MS specifications ................................................................................................... 25 2.3.4 Purification of the molecules from WAC00263 by HPLC ........................................... 25 2.4 Antimicrobial Activity Testing ............................................................................................ 27 2.4.1 Disk diffusion assay .......................................................................................................... 27