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© 2015 Ryan Ellis Cobb © 2015 Ryan Ellis Cobb NATURAL PRODUCT DISCOVERY AND ENGINEERING AT THE PROTEIN, PATHWAY, AND GENOME SCALES BY RYAN ELLIS COBB DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Engineering in the Graduate College of the University of Illinois at Urbana-Champaign, 2015 Urbana, Illinois Doctoral Committee: Professor Huimin Zhao, Chair Professor William W. Metcalf Professor Satish K. Nair Professor Christopher V. Rao Abstract For centuries, mankind has turned to natural sources for cures, remedies, and ways to improve its quality of life. It was not until the discovery of the antibiotic penicillin in 1928, however, that natural products – small molecule secondary metabolites produced by a variety of organisms for a variety of purposes – were truly appreciated as the source of these beneficial properties. In the ensuing decades, research in the field of natural products boomed, and a number of discoveries were made that revolutionized global health. In the 1970s and 1980s, however, these discoveries started to become fewer and farther between, prompting pharmaceutical companies to turn away from natural products research in favor of synthetic chemistry approaches to drug discovery. Nevertheless, despite diminishing returns from traditional natural product discovery methods, modern genomics has in fact revealed that vast numbers of natural product gene clusters exist across all domains of life, far in excess of the number of known natural products and far surpassing any previous predictions. Thus, natural product discovery efforts to date have only scratched the surface of Nature’s true capabilities. In this work, we sought to leverage modern techniques for natural product discovery at the protein, pathway, and genome scales to tap into this biosynthetic potential. The past several years have seen the development of many tools for the manipulation of DNA with ease and precision far exceeding the standards set by traditional methods. These methods, combined with the ever-increasing number of putative natural product pathways revealed by genome sequencing efforts, open the door for new platforms of natural product discovery and engineering. At the protein level, we demonstrate the synthesis of novel derivatives of the antimalarial natural product FR-900098 by leveraging the substrate promiscuity of the native biosynthetic machinery. ii Through structural and biochemical characterization of the N-acetyltransferase FrbF and the corresponding target for FR-900098 inhibition, Dxr from Plasmodium falciparum, we show that the novel FR-900098 derivatives can serve as more potent inhibitors. A platform for their biosynthesis is also established in Escherichia coli. We additionally demonstrate a genome-mining platform for fungal polyketide synthases via the one-step assembly of expression-ready plasmids by homologous recombination in yeast. Through the evaluation of previously uncharacterized endogenous promoters from Saccharomyces cerevisiae, we demonstrate the heterologous production of polyketides from the dimorphic fungus Talaromyces marneffei. Extension of this approach to the pathway level is also demonstrated to assemble both a 6-gene resorcylic acid lactone cluster and a 13-gene phosphonic acid biosynthetic cluster. The latter case, in which all 13-genes from a phosphonic acid non-producing strain were placed under individual strong promoters, enabled the production of novel phosphonic acid compounds when heterologously expressed in Streptomyces lividans. At the genome scale, we developed a versatile system from genome engineering in a broad range of Streptomyces species. As the genus Streptomyces is by far the most important producer of pharmaceutical natural products to date, tools to facilitate their genetic engineering are of significant interest to aid discovery and improve production. Here, we show that the CRISPR/Cas9 system of S. pyogenes can be reconstituted in S. lividans for high-efficiency, multiplex editing of genomic loci. We show that deletions ranging from 20 bp to 31 kb can be successfully introduced in one step, and extend this approach to additional Streptomyces strains. iii To my parents, for all of their love and support iv Acknowledgements “We don’t learn. We just get older, and we know.” Leslie Caron, “Lili” (1953, Metro-Goldwyn-Mayer Studios) First and foremost, I thank my advisor, Prof. Huimin Zhao, for granting me the opportunity to perform graduate research in his lab. His patience and trust in my judgment enabled me to pursue my goals to the best of my ability. Further, I am grateful for the many opportunities he presented me to develop my love of writing. I also thank my committee, Profs. Bill Metcalf, Satish Nair, and Chris Rao, for the valuable feedback they have given me, along with Profs. Wilfred van der Donk and Neil Kelleher of the MMG theme. For technical assistance, I am grateful to Dr. Lucas Li and Dr. Alex Ulanov of the Roy J. Carver Metabolomics Center; Dr. Jiangtao Gao, Dr. Brad Evans and Dr. Jaeheon Lee of the IGB MMG theme; Dr. Furong Sun of the SCS Mass Spectrometry Facility; and Dr. Xudong Guan of the IGB Core Facilities. For financial support, I am grateful for the NIH Chemistry-Biology Interface Training Grant and the SCS Harry Drickamer Fellowship. And finally, to all the rest: this process is long and sometimes arduous, with many twists, turns, false starts, and dead ends along the way. Certainly we are different in leaving than we were upon entering, but by the end it can be easy to lose sight of the journey as chronology is sacrificed for the benefit of the narrative. Over time, that which has become known– detailed in the pages that follow– becomes the story, supplanting the learning process from which it came. Nevertheless, know that I am indebted to all of those who have helped to shape my journey and, in doing so, myself. Over the years, I have learned so much from so many that any attempt to list them all would surely fall short of its mark, to the chagrin of the author and the disappointment of those omitted. Thus will I acquiesce to brevity with a simple “thank you.” v Table of Contents CHAPTER 1. Natural product discovery and engineering ...................................................... 1 1.1 Natural Products: Invaluable Natural Resources ............................................................ 1 1.2 Methods for Natural Product Discovery ........................................................................... 3 1.2.1 Pathway-independent approaches .............................................................................. 3 1.2.1.1 Environmental sampling ......................................................................................... 3 1.2.1.2 Culture manipulation .............................................................................................. 6 1.2.1.3 Global genetic and epigenetic manipulation .......................................................... 8 1.2.2 Pathway-specific approaches ...................................................................................... 9 1.2.2.1 Bioinformatic tools to identify natural product gene clusters ................................ 9 1.2.2.2 Manipulation of native strains .............................................................................. 15 1.2.2.3 Heterologous expression ....................................................................................... 17 1.3 Enabling Technologies for Natural Product Engineering ............................................. 20 1.3.1 Modern DNA assembly methods .............................................................................. 20 1.3.2 Directed evolution ...................................................................................................... 26 1.3.2.1 Random evolution strategies ................................................................................. 28 1.3.2.2 Targeted evolution strategies ................................................................................ 30 1.3.3 Genome engineering .................................................................................................. 33 1.3.3.1 Genome evolution ................................................................................................. 33 1.3.3.2 Genome tailoring .................................................................................................. 37 1.4 Project Overview ............................................................................................................... 39 1.5 References .......................................................................................................................... 42 CHAPTER 2. Characterization and engineering of FrbF for the synthesis of novel FR- 900098 analogs ............................................................................................................................. 67 2.1 Introduction ....................................................................................................................... 67 2.1.1 The ongoing malaria threat....................................................................................... 67 2.1.2 Antimalarial phosphonic acids fosmidomycin and FR-900098 ............................. 69 2.1.2.1 Initial discovery and studies ................................................................................. 69 2.1.2.2 Renewed interest and clinical trials ...................................................................... 70 2.1.2.3
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