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UNIVERSITY of CALIFORNIA, SAN DIEGO Discovery And UNIVERSITY OF CALIFORNIA, SAN DIEGO Discovery and characterization of calcium-dependent antibiotics via activation of a silent natural product gene cluster A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Chemistry by Kirk Alan Reynolds Committee in charge: Professor Bradley Moore, Chair Professor Pieter Dorrestein, Co-Chair Professor Michael Burkart Professor William Fenical Professor Judy Kim 2016 i ii The dissertation of Kirk Alan Reynolds is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ Co-Chair ________________________________________________________________ Chair University of California, San Diego 2016 iii Dedication This dissertation is dedicated to my beautiful wife Hannah. Throughout our years together she has endured the late nights, cancelled trips, and the challenges of graduate student life with a grace and poise beyond what I have deserved. While there are teachers, colleagues, friends, and parents who have invested in me throughout my life, it is by Hannah’s generosity of heart that I continue to find my inspiration to endure the most challenging times. This dissertation is dedicated to Hannah whose unconditional love, support, and sacrifice has made it possible for me to achieve the successes that are described herein. iv Table of Contents Signatures Page: .................................................................................................. iii Dedication ............................................................................................................iv List of Figures .......................................................................................................vi List of Tables ........................................................................................................ix Acknowledgements .............................................................................................. x Vita ...................................................................................................................... xii Abstract of the Dissertation ................................................................................ xiii Chapter 1: Introduction to Natural Products ........................................................ 1 Chapter 2: Direct cloning and refactoring of a silent lipopeptides biosynthetic gene cluster yields the antibiotic taromycin A ..................................................... 39 Chapter 3: Isolation and Structure Elucidation of Taromycin B.......................... 68 Chapter 4: Future Growth of Tar Cloning and the Taromycin Series ............... 100 v List of Figures Figure 1.1: Historically relevant terrestrial natural products. ................................ 2 Figure 1.2: Marine natural products and analogues that are currently used in the clinic or are in phase I/II clinical trials ................................................................. 3 Figure 1.3: Examples of aminoglycoside antibiotics isolated from actinobacterial sources ................................................................................................................. 7 Figure 1.4: Peptides offer a diverse collection of antibiotics ............................... 10 Figure 1.5: Sequenced genome of Streptomyces coelicolor .............................. 16 Figure 1.6: Gene cluster organization for the expression and production of Epothilone A (R=H) and B (R=CH3) .................................................................... 19 Figure 1.7: (A) Coelichelin (17) one of the earliest secondary metabolites isolated from genome mining of S. coelicolor. (B) Oxidation of epi-isozizaene (18) into albaflavenol by cytochrome P-450. ...................................................... 21 Figure 1.8: Gene cluster organization for the putative lodopyridone biosynthetic gene cluster ........................................................................................................ 25 Figure 1.9: Examples of proposed β -carbon chemistry .................................... 28 Figure 1.10: Proposed biosynthesis of lodopyridone. ........................................ 29 Figure 1.10: Proposed biosynthesis of lodopyridone. ........................................ 30 Figure 2.1. Design and strategy of TAR-based cloning and expression ............. 41 Figure 2.2. Gene organization of lipopeptide biosynthetic gene clusters ............ 42 Figure 2.3. Physical maps of the TAR-cloned tar gene cluster and the pKY01- based complementation vectors ......................................................................... 43 Figure 2.4. HPLC analysis of the taromycins produced heterologously by S. coelicolor mutants and structures of daptomycin and taromycin A. .................... 43 Figure S2.1. Tar cloning of the taromycin biosynthetic gene cluster (tar) ........... 55 Figure S2.2. Restriction maps of the TAR-cloned tar gene cluster ..................... 56 Figure S2.3. HPLC-MS analysis of the taromycins produced by S. coelicolor M1146/pCAP01-tarM2 ........................................................................................ 57 Figure S2.4 Effect of genetic complementation with tar regulatory genes in S. coelicolor M1146 / pCAP01-tarM1 (∆tar19 sarp, ∆tar20 luxR ............................. 58 Figure S2.5. Effect of genetic complementation of the tar regulatory genes in S. coelicolor M1146 / pCAP01-tarM2 (∆tar20 luxR) ................................................ 58 vi Figure S2.6 Schematic diagram for construction of the codon redressed tar20 luxR regulatory gene .......................................................................................... 59 Figure S2.7 Effect of genetic complementation of the codon redressed tar20 luxR regulatory gene. .................................................................................................. 59 Figure S2.8 HSQC spectra of daptomycin aromatic region (d6-DMSO, 600 MHz) ........................................................................................................................... 61 Figure S2.9 HSQC spectra of taromycin A aromatic region (d6-DMSO, 600 MHz) ........................................................................................................................... 62 Figure S2.10. MS and MSn analysis of taromycin A. MS 2 .................................. 63 Figure S2.11 MS and MSn analysis of taromycin ............................................... 64 Figure S2.12 HPLC analysis of the marinopyrrole A produced by S. coelicolor mutants ............................................................................................................... 65 Figure 3.1: lipopeptides showcasing fatty acid side chain diversity as well as diverse amino acid sequences. .......................................................................... 68 Figure 3.2: Chromatograph of the taromycin series produced by M1146-M1 showing 10 distinct taromycin compounds ......................................................... 71 Figure 3.3: MS and MSn analysis of taromycin B ............................................... 73 Figure 3.4: Selected 2D NMR correlations for taromycin B side chain. ............. 75 1 Figure S3.1: H NMRspectrum of taromycin B (d6-DMSO, 600 MHz)................ 82 Figure S3.2: COSY NMR spectrum of taromycin B (d6-DMSO, 600 MHz). ....... 83 Figure S3.3: TOCSY NMR spectrum of taromycin B (d6-DMSO, 600 MHz). .... 84 Figure S3.4: HSQC NMR spectrum of taromycin B (d6-DMSO, 600 MHz). ...... 85 Figure S3.5: HMBC NMR spectrum of taromycin B (d6-DMSO, 600 MHz) ....... 86 Figure S3.6: HSQC spectra of daptomycin aromatic region (d6-DMSO, 600 MHz) ........................................................................................................................... 87 Figure S3.7: HSQC spectra of taromycin B aromatic region (d6-DMSO, 600 MHz) ................................................................................................................... 88 Figure S3.8: HPLC chromatogram showing rotomeric nature of the taromycin A molecule ............................................................................................................. 89 Figure S3.9: HPLC chromatogram showing rotomeric nature of the taromycin B molecule ............................................................................................................. 91 vii Figure 4.1: Deacylation of N-Orn-Boc-taromycin series to N-Orn-Boc-taromycin nucleus ............................................................................................................. 101 Figure 4.2: Direct production of taromycin nucleus .......................................... 101 viii List of Tables Table 1.1: Evolution of resistance to clinical antibiotics. .................................... 14 Table 1.2: AntiSMASH gene cluster assignment for Saccharomonospora sp. CNQ-490. ........................................................................................................... 24 Table 1.3: Detailed analysis of putative lodopyridone genes: Size, proposed role, and known similar protein ........................................................................... 26 Table S2.1. Strains and Plasmids Used in this study .......................................
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