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UNIVERSITY of CALIFORNIA SAN DIEGO Heterologous Expression UNIVERSITY OF CALIFORNIA SAN DIEGO Heterologous expression and genetic manipulation of natural product biosynthetic gene clusters from marine bacteria A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Marine Biology by Jamie R. Zhang Committee in charge: Professor Bradley S. Moore, Chair Professor Rachel J. Dutton Professor William H. Gerwick Professor Susan S. Golden Professor Chambers C. Hughes Professor Kit Pogliano 2019 Copyright Jamie R. Zhang, 2019 All rights reserved. SIGNATURE PAGE The Dissertation of Jamie R. Zhang is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _______________________________________________________ _______________________________________________________ _______________________________________________________ _______________________________________________________ _______________________________________________________ _______________________________________________________ Chair University of California San Diego 2019 iii DEDICATION To Mr. Walter K. Erhardt, who taught me that education is about curiosity and persistence. iv EPIGRAPH Not all of us can do great things, but we can all do small things with great love. Mother Teresa v TABLE OF CONTENTS SIGNATURE PAGE ................................................................................................................... iii DEDICATION ............................................................................................................................. iv EPIGRAPH ................................................................................................................................. v TABLE OF CONTENTS ............................................................................................................. vi LIST OF FIGURES .................................................................................................................. viii LIST OF TABLES ..................................................................................................................... xiii ACKNOWLEDGEMENTS ........................................................................................................ xiv VITA .......................................................................................................................................... xv ABSTRACT OF THE DISSERTATION ..................................................................................... xvi CHAPTER 1. Genetic platforms for heterologous expression of microbial natural products. ...... 1 1.1 Abstract ............................................................................................................................ 2 1.2 Introduction ....................................................................................................................... 3 1.3 Cloning of microbial BGCs ................................................................................................ 6 1.3.1 Library-based methods............................................................................................... 6 1.3.2 Assembly methods ..................................................................................................... 8 1.3.3 Direct cloning methods ..............................................................................................10 1.4 Hosts for heterologous expression...................................................................................15 1.4.1 Actinobacteria ...........................................................................................................16 1.4.2 Firmicutes .................................................................................................................18 1.4.3 Proteobacteria ...........................................................................................................19 1.4.4 Cyanobacteria ...........................................................................................................24 1.4.5 Fungi .........................................................................................................................25 1.5 Genetic manipulation of cloned BGCs .............................................................................30 1.6 Conclusions and future perspectives ...............................................................................31 1.7 Acknowledgement ...........................................................................................................34 1.8 References ......................................................................................................................34 CHAPTER 2. Engineering Salinispora tropica for heterologous expression of natural product biosynthetic gene clusters. ........................................................................................................59 2.1 Abstract ...........................................................................................................................60 2.2 Reprint of “Engineering Salinispora tropica for heterologous expression of natural product biosynthetic gene clusters” ....................................................................................................61 2.3 Acknowledgement ...........................................................................................................79 vi CHAPTER 3. Broad-host-range heterologous expression reveals native and host regulatory elements that influence heterologous antibiotic production in Gram-negative bacteria. .............80 3.1 Abstract ...........................................................................................................................81 3.2 Reprint of “Broad-host-range heterologous expression reveals native and host regulatory elements that influence heterologous antibiotic production in Gram-negative bacteria.” ........82 3.3 Acknowledgement ......................................................................................................... 115 CHAPTER 4. Avant-garde assembly line biosynthesis expands diversity of cyclic lipodepsipeptide products. ...................................................................................................... 116 4.1 Abstract ......................................................................................................................... 117 4.2 Introduction .................................................................................................................... 118 4.3 Results .......................................................................................................................... 120 4.3.1 Cloning and heterologous expression of ttc and ttm. ............................................... 120 4.3.2 Reconstitution of thalassospiramide structural diversity........................................... 121 4.3.3 Characterization of non-assembly line genes. ......................................................... 125 4.3.4 Inactivation and testing of assembly line domains. .................................................. 125 4.3.5 Models for thalassospiramide biosynthesis. ............................................................ 129 4.4 Discussion ..................................................................................................................... 136 4.5 Methods ........................................................................................................................ 139 4.5.1 General methods..................................................................................................... 139 4.5.2 Cloning and heterologous expression of ttc and ttm ................................................ 139 4.5.3 Extraction and LC-MS analysis ............................................................................... 140 4.5.4 Gene deletion and complementation experiments ................................................... 141 4.5.5 Inactivation and testing of assembly line enzymatic domains .................................. 142 4.6 Data availability ............................................................................................................. 143 4.7 Supplementary Information ............................................................................................ 143 4.7.1 Supplementary Tables ............................................................................................ 143 4.7.2 Supplementary Figures ........................................................................................... 156 4.8 Acknowledgements ....................................................................................................... 186 4.9 References .................................................................................................................... 186 vii LIST OF FIGURES Figure 1.1 Graphical abstract of the chapter. ............................................................................. 2 Figure 1.2 General workflow for (A) cloning, (B) heterologous expression, and (C) genetic manipulation of microbial BGCs. .............................................................................................. 4 Figure 1.3 Chemical structures of natural products mentioned in this chapter. .......................... 5 Figure 1.4 Streamlined design of universal BGC expression platforms. (A) Modular expression vector system, leveraging knock-in of modular expression elements for BGC integration across many hosts. (B) Single vector system, compatible across several engineered
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