UC San Diego Research Theses and Dissertations Title Biosynthesis and Engineering of Cyclomarin and Cyclomarazine: Prenylated, Non-Ribosomal Cyclic Peptides of Marine Actinobacterial Origin Permalink https://escholarship.org/uc/item/21b965z8 Author Schultz, Andrew W. Publication Date 2010 Peer reviewed eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Biosynthesis and Engineering of Cyclomarin and Cyclomarazine: Prenylated, Non-Ribosomal Cyclic Peptides of Marine Actinobacterial Origin A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Oceanography by Andrew William Schultz Committee in charge: Professor Bradley Moore, Chair Professor Eric Allen Professor Pieter Dorrestein Professor William Fenical Professor William Gerwick 2010 Copyright Andrew William Schultz, 2010 All rights reserved. The Dissertation of Andrew William Schultz is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ Chair University of California, San Diego 2010 iii DEDICATION To my wife Elizabeth and our son Orion and To my parents Dale and Mary Thank you for your never ending love and support iv TABLE OF CONTENTS Signature Page ..................................................................................................... iii Dedication ............................................................................................................ iv Table of Contents ................................................................................................. v List of Abbreviations ............................................................................................. ix List of Figures ...................................................................................................... xii List of Tables ..................................................................................................... xvii Acknowledgements .......................................................................................... xviii Vita ..................................................................................................................... xxi Abstract of the Dissertation ............................................................................... xxii Chapter 1: Introduction: The Chemical Diversity of Non-ribosomal Peptides .............................................................................................................. 1 1.1: Introduction to non-ribosomal peptides .............................................. 2 1.2: NRPs are assembled via non-ribosomal peptide synthetases ......... 12 1.3: Tailoring….. ..................................................................................... 16 1.3.1: Epimerization ..................................................................... 16 1.3.2: Oxidation ............................................................................ 16 1.3.3: Halogenation ...................................................................... 21 1.3.4: Methylation ......................................................................... 22 1.3.5: Prenylation ......................................................................... 23 1.3.6: Glycosolation ...................................................................... 24 1.3.7: Cyclization of serine, threonine, or cysteine to form oxazoline or thiazoline ........................................................ 24 1.3.8: Reductive offloading ........................................................... 25 1.4: Biosynthetic pathways to non-proteinogenic amino acids ................ 26 v 1.4.1: Direct intermediates of primary metabolic pathways .......... 28 1.4.2: Transaminaition of α-keto acids ......................................... 28 1.4.3: Polyketide synthase derived ............................................... 29 1.4.5: Shikimate pathway ............................................................. 30 1.4.6: Aldolase/dehydrogenase heterodimers .............................. 33 1.5: Interfacing non-ribosomal peptide synthetases with polyketide synthases adds further chemical diversity ......................................... 33 1.6: Engineering of nonribosomal peptides to increase nature’s peptide diversity ............................................................................................. 34 1.6.1: Precursor directed biosynthesis and mutasynthesis .......... 35 1.6.2: Combinatorial biosynthesis ................................................ 40 1.7: Conclusion ....................................................................................... 43 1.8: References ...................................................................................... 46 Chapter 2: Biosynthesis of Cyclomarins and Cyclomarazines, Prenylated Cyclic Peptides of Marine Actinobacterial Origin .......................................... 55 2.1: Abstract....... ...................................................................................... 56 2.2 Introduction... ..................................................................................... 57 2.2.1: Cyclomarin consists of several modified and non- proteinogenic amino acids of significant biosynthetic interest ............................................................. 57 2.2.2: Specific aims ....................................................................... 62 2.3: Results…… ...................................................................................... 63 2.2.1: Molecular basis for the biosynthesis of the cyclomarins ...... 63 2.2.2: 2-Amino-3,5-dimethyl-4-hexenoic acid (ADH) biosynthesis 69 2.2.3: Reverse N-prenylation of the tryptophan residue ................ 73 2.2.4: Oxidative tailoring ................................................................ 76 2.4: Discussion….. ................................................................................ …79 2.5: Experimental section ......................................................................... 83 2.5.1: General experimental procedures ....................................... 83 2.5.2: Bacterial strains, plasmids, culture conditions, and DNA manipulations ....................................................................... 84 2.5.3: Cultivation of S arenicola and isolation of 1, 8, and 9 .......... 85 2.5.4: Labeling experiments .......................................................... 87 2.5.5: DNA sequence analysis of the cyclomarin gene cluster (cym) ........................................................................ 87 vi 2.5.6: Conjugation protocol for S. arenicola .................................. 88 2.5.7: Inactivation of cymD, cymQ, cymS, cymV and cym W ........ 88 2.5.8: Analysis of the S. arenicola cym mutants ............................ 89 2.6: Appendix…… .................................................................................... 90 2.6.1: Phylogenetic analysis of the cytochrome P450s CymO, CymS, CymV to predict their role in cyclomarin and cyclomarazine biosynthesis ................................................. 90 2.7: References ..................................................................................... 120 2.8: Acknowledgements ......................................................................... 125 Chapter 3: Functional Characterization of the Cyclomarin/Cyclomarazine Prenyltransferase CymD Directs the Biosynthesis of Unnatural Cyclic Peptides .......................................................................................................... 126 3.1: Abstract…… ................................................................................... 127 3.2: Introduction ..................................................................................... 127 3.3: Results…….. ................................................................................... 132 3.4: Discussion……. .............................................................................. 137 3.5: Experimental section….. ................................................................. 138 3.5.1: General experimental procedures…. ................................ 138 3.5.2: Bacterial strains and culture conditions ............................. 138 3.5.3: CymD purification .............................................................. 139 3.5.4: CymD prenyltransferase assay ......................................... 140 3.5.5: Purification of N-(1,1-dimethyl-1-allyl)-tryptophan from S. arenicola CNS-205 ............................................................ 141 3.5.6: Chemical complementation of the cymD- mutant and the production of novel cyclomarin and cyclomarazine analogues via mutasynthesis ............................................. 142 3.6 Appendix ..................................................................................................... 144 3.7: References ................................................................................................ 160 3.8: Acknowledgment ....................................................................................... 162 Chapter 4: Efforts towards Functional Characterization and Biosynthetic Manipulation of the 2-Amino-3,5-dimethyl-4-hexenoic Acid Residue of Cyclomarin .......................................................................... 163