BIOSYNTHETIC GENE CLUSTERS GUIDE ANTIBIOTIC DISCOVERY PhD Thesis – E.J. Culp - McMaster University – Biochemistry and Biomedical Sciences BIOSYNTHETIC GENE CLUSTERS GUIDE RATIONAL ANTIBIOTIC DISCOVERY FROM ACTINOMYCETES By ELIZABETH J. CULP, B.Sc. A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy McMaster University © Copyright Elizabeth Jane Culp, October 2020 i PhD Thesis – E.J. Culp - McMaster University – Biochemistry and Biomedical Sciences DESCRIPTIVE NOTE McMaster University DOCTOR OF PHILOSOPHY (2020) Hamilton, Ontario (Biochemistry and Biomedical Sciences) TITLE: Biosynthetic gene clusters guide rational antibiotic discovery from Actinomycetes AUTHOR: Elizabeth J. Culp, B.Sc. SUPERVISOR: Gerard D. Wright, Ph.D. NUMBER OF PAGES: xv; 231 ii PhD Thesis – E.J. Culp - McMaster University – Biochemistry and Biomedical Sciences Foreword LAY ABSTRACT Antibiotics are essential for treating life-threatening infections, but the rise of antibiotic resistance renders them ineffective. To treat these drug-resistant infections, new antibiotics that work in new ways are required. A family of bacteria commonly isolated from soil called Actinomycetes produce most antibiotics we use today, but it has become increasingly difficult to find new antibiotics from this source. My work describes three techniques that can be applied to actinomycetes to help overcome the challenges associated with antibiotic discovery. Specifically, these techniques guide discovery efforts by making use of regions in actinomycete genomes called biosynthetic gene clusters that often encode antibiotics. In doing so, I describe ways to uncover rare antibiotics from actinomycete strains that produce common and uninteresting antibiotics, use antibiotic family trees to discover antibiotics that work in new ways, and apply antibiotic resistance to identify biosynthetic gene clusters likely to act on a certain bacterial target. iii PhD Thesis – E.J. Culp - McMaster University – Biochemistry and Biomedical Sciences ABSTRACT As the spread of antibiotic resistance threatens our ability to treat infections, avoiding the return of a pre-antibiotic era urgently requires the discovery of novel antibiotics. Actinomycetes, a family of bacteria commonly isolated from soil, are a proven source of clinically useful antibiotics. However, easily identifiable metabolites have been exhausted and the rediscovery of common antibiotics thwarts searches for rarer molecules. Sequencing of actinomycete genomes reveals that they contain far more biosynthetic gene clusters with the potential to encode antibiotics than whose products can be readily observed in the laboratory. The work presented in this thesis revolves around developing approaches to mine these previously inaccessible metabolites as a source of new antibiotics. First, I describe how inactivation of biosynthetic gene clusters for common antibiotics can uncover rare antibiotics otherwise masked in these strains. By applying CRISPR-Cas9 to knockout genes encoding nuisance antibiotics, I develop a simple strategy to reveal the hidden biosynthetic potential of actinomycete strains that can be used to discover rare or novel antibiotics. Second, I describe the use of the evolutionary history of biosynthetic gene clusters to prioritize divergent members of an antibiotic family, the glycopeptide antibiotics, that are likely to possess new biological activities. Using these predictions, I uncover a novel functional class of glycopeptide antibiotics that blocks the action of autolysins, essential peptidoglycan hydrolases required for remodelling the cell wall during growth. Finally, I apply target-directed genome mining, which makes use of target duplication as a predicted resistance mechanism within an antibiotic’s biosynthetic gene cluster. Using this approach, I discover the association of a family of gene clusters with the housekeeping protease ClpP and characterize the produced metabolite’s effect on ClpP function. These three research projects mine previously inaccessible chemical matter from a proven source of antibiotics, actinomycetes. The techniques and antibiotics described are required now more than ever to develop life-saving antibiotics capable of combatting multidrug-resistant pathogens. iv PhD Thesis – E.J. Culp - McMaster University – Biochemistry and Biomedical Sciences ACKNOWLEDGEMENTS To Gerry – I can’t thank you enough for the opportunities, support and advice you’ve provided. Your encouragement and excitement through the hills and valleys of science always kept me going. Your dedication to excellence inspires the type of scientist I want to be, and your belief in and motivation of your students is the type of mentor I will model myself after. To Marie and Brian, my committee members – thank you for making my committee meetings productive and motivational, and taking the time for thoughtful contemplation and helpful suggestions about my science. To the Wright lab members, past and present – I have you to thank for everything in this thesis, but also for making the journey so much fun. Thank you all. Especially, and in no particular order…Nick – most of my projects wouldn’t exist without your insight and work. Wen – thank you for teaching me everything I know about natural product purification, purifying countless compounds for me, and also endless unsolicited advice about real estate. Min – I trust no one more than you for advice about Streptomyces. I owe you many favours. Mike – I’ll miss our walks to get coffee; a lot of great science ideas came from those. Also a lot of puns. Linda – Thank you for keeping the lab together, from teaching us the ropes to running things smoothly. Kalinka – thanks for everything you do for the lab and being my early morning buddy. Grace – my CRISPR partner in crime. Thanks for spearheading that project, and also for imparting the way you say “OK” and “Oh boy” onto me. Dave – your enthusiasm for science is infectious. Thanks for all the helpful discussions and always having a willingness to lend expertise. Allison, Drew, Haley, Matt, Adam, Caitlyn, Andy, Christian, Emily, Vishwas – you guys are such great friends and made it all a blast. Thanks for all the ridiculous shenanigans. To my spin crew – Thanks for the sweat puddles, heavy beats, and always giving me somewhere to retreat to. To my awesome family – Thanks for listening to my complaints, fixing my broken stuff and 9+ years of visiting me in Hamilton. I could not have done this without your endless love and support. To so many others who made this work possible – Every time I thought I was finished writing this acknowledgments section I thought of more people who helped me along the way. Thank you. v PhD Thesis – E.J. Culp - McMaster University – Biochemistry and Biomedical Sciences Table of Contents Foreword ........................................................................................................................... iii Lay Abstract ................................................................................................................... iii Abstract ........................................................................................................................... iv Acknowledgements .......................................................................................................... v List of figures .................................................................................................................. ix List of tables ................................................................................................................. xiii List of abbreviations...................................................................................................... xiv Declaration of Academic Achievement ......................................................................... xv Chapter I – Introduction ................................................................................................... 1 Natural products as a source of antibiotics ...................................................................... 2 Biosynthetic gene clusters ................................................................................................ 3 Key natural product classes ......................................................................................... 5 Barriers to discovery: dereplication ................................................................................. 7 Easing the dereplication burden ................................................................................... 7 Exploring unchartered territory for new NPs ............................................................... 9 Barriers to discovery: Cryptic biosynthetic gene clusters .............................................. 11 Pleiotropic approaches to BGC activation ................................................................. 11 Targeted approaches to BGC activation .................................................................... 14 Purpose and goals of this thesis ..................................................................................... 20 Chapter II – Hidden antibiotics in actinomycetes can be identified by inactivation of gene clusters for common antibiotics ............................................................................. 21 Preface ............................................................................................................................ 22 Abstract .........................................................................................................................