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KAKKAR-DISSERTATION-2018.Pdf EXPANDING THE STRUCTURAL DIVERSITY OF THE RiPP CLASS OF NATURAL PRODUCTS BY NIDHI KAKKAR DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2018 Urbana, Illinois Doctoral Committee: Professor Wilfred A. van der Donk, Chair Professor John A. Gerlt Professor Yi Lu Associate Professor Douglas A. Mitchell Abstract Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a diverse and rapidly expanding class of natural products. Despite starting out as linear chains of amino acids, RiPP natural products acquire structural diversity ranging from small molecules like microcin C7 to 48-mer polytheonamides with varying post-translational modifications installed by biosynthetic enzymes. These conformational restrictions allow these natural products to exhibit a wide range of therapeutic activities like antifungal, antibacterial, allelopathic, and antiviral. Despite the structural and functional disparities, they all share a common feature: the presence of amino acid sequence in the precursor peptide called the ‘leader peptide’ that helps guide the biosynthetic enzymes towards the remaining precursor peptide termed the ‘core peptide’ to act on specific residues and install different PTMs. This leader-peptide guided biosynthetic route provides tremendous opportunity to bioengineer libraries of analogues of RiPPs. Exploiting this feature, in Chapter 2, I report my investigations on a strategy wherein by swapping the leader peptides from unrelated RiPP biosynthetic pathways and modifying the core peptide sequences, different biosynthetic enzymes can perform their characteristic chemistries on the core peptide residues. In Chapter 3, I report the design and usage of chimeric leader peptides that can be bound by multiple enzymes from unrelated RiPP pathways to create new-to-nature hybrid RiPPs. This strategy involves the combination of a cyclodehydratase with modification enzymes from different lanthipeptide RiPP classes. These hybrids provide insight into enzyme promiscuity and compatibility, and the biosynthetic order of events, and establish a general platform for the engineering additional hybrid RiPPs. ii In Chapter 4, I develop two different strategies for non-proteinogenic amino acid incorporation in the class I lantibiotic nisin and the class II lantibiotic lacticin 481 using amber stop codon technology. iii To my family and my teachers iv Acknowledgements "If I have seen further, it is by standing upon the shoulders of giants" -Sir Issac Newton First of all, I want to thank my mom, Raj, and my sister, Swati, for their unconditional love and support throughout my life. Their belief in me led me on the course that brought me seven thousand miles away from home, but despite the physical distance, I have always felt their presence with me. Mom, it is because of your love, hard work and prayers that I have accomplished anything and everything in my life, and try as I will, would not be able to pay for it. I love you, and am forever grateful to you. Apart from my family, the one person that I have been privileged to observe up close is my advisor Professor Wilfred van der Donk. Anyone who has heard of him in the scientific community knows how brilliant a scientist he is but not many would know that he is such a kind- hearted, respectful and humble person. I was fortunate enough to attend his graduate class twice, first as the student and then as the TA, and watching him teach with ever-lasting patience is truly remarkable. I really want to thank him for his constructive guidance, patience and honest advice in projects and life in general. He is not just a great scientific advisor but also a fabulous mentor. His support encouraged me immensely through the good and specially the bad times. I owe him my deepest gratitude and not just for the PhD training! I would also like to thank my committee members, Professor Douglas A. Mitchell, Professor Yi Lu and Professor John A. Gerlt for their suggestions and advice during the graduate studies. Special thanks to Prof. Satish K. Nair for providing his valuable input on my Original Research Paper. I want to convey my thanks to our collaborators, Professor Wenshe Liu, Professor v Michael C. Jewett and Professor Douglas A. Mitchell for generously providing several bacterial strains and plasmids and for sharing their expertise in different techniques with me. I am eternally grateful to so many of the past and present members of van der Donk laboratory for their friendship and wisdom. Right from my first year till this day, I found a great friend and mentor in Dr. Ayse Okesli. Her pleasant personality always brightened up the bay. I want to thank Dr. Yanxiang Shi, Dr. Noah Bindman, Dr. Subha Mukherjee and other senior students for their generous help. I would also like to express my sincere gratitude towards the post- doctoral fellows, Dr. Mark C. Walker, Dr. Lindsay Repka, Dr. Ian Bothwell, Dr. Liujie Huo, Dr. Debapriya Dutta, Dr. Tania Caetano, Dr. Qi Zhang, Dr. Rebecca Splain, Dr. Julian Hegemann and Dr. Chi Pan Ting for all the useful discussions and the friendly banter in lab. Mark, you are a blessing that Wilfred brought into the lab in my second year and I want to specially thank you for your kind and helpful attitude, and your encyclopedic knowledge about cloning techniques that has saved my life so many times. Thank you, Marc R. Gancayco and Subhanip Biswas, for making this journey entertaining. Your witty remarks, pranks and unintentional humor kept the fun alive at work. I also want to thank Nan Holda for our interesting conversations and her ever uplifting attitude. I also want to thank my friends Rohini Singh, Tejas Ghirnikar and Abhishek Sheshadri for their love, compassion and often unsolicited sarcasm. Lastly, I want to thank all the teachers and professors that taught me in school, and during bachelors and masters for their love, support and unwavering encouragement. This day would not have been possible without them. Beloved Ma’ams and Sirs, I respect you and I applaud you for all your efforts. ‘A teacher takes a hand, opens a mind and touches a heart’ and you have done all three for me. Dhanyawad. vi Table of Contents Chapter 1: Gifts from Microorganisms............................................................................................1 1.1 Introduction....................................................................................................................1 1.2 Ribosomally synthesized and post-translationally modified peptides (RiPPs)..............4 1.3 Lanthipeptides................................................................................................................7 1.4 Linear azole-containing peptides (LAPs).....................................................................13 1.5 Discussion....................................................................................................................16 1.6 References ...................................................................................................................18 Chapter 2: Leader-Peptide Guided Generation of Non-Natural Hybrid RiPP Products – Swapping the Leader Peptide Strategy...........................................................................................28 2.1 Introduction..................................................................................................................28 2.2 Sortase-mediated swapping of leader peptides............................................................30 2.3 Design strategy.............................................................................................................31 2.4 Results and discussion..................................................................................................37 2.5 Methods........................................................................................................................48 2.6 References....................................................................................................................60 Chapter 3: Leader-Peptide Guided Generation of Non-Natural Hybrid RiPP Products – Chimeric Leader Peptide Strategy..................................................................................................67 3.1 Introduction..................................................................................................................67 3.2 Results..........................................................................................................................70 vii 3.3 Discussion....................................................................................................................87 3.4 Methods........................................................................................................................89 3.5 References....................................................................................................................99 Chapter 4: Incorporation of Non-Proteinogenic Amino Acids in Class I and II Lantibiotics.....107 4.1 Introduction................................................................................................................107 4.2 Results and discussion................................................................................................108 4.3 Conclusion..................................................................................................................118 4.4 Methods......................................................................................................................118
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