ABSTRACT KASEY, CHRISTIAN MAYFIELD. Genetically-Encoded Biosensors for High-throughput Engineering and Synthetic Biology of Polyketide Biosynthesis. (Under the direction of Gavin Williams). Polyketide synthases (PKSs) are responsible for the production of diverse and complex natural products that often contain clinically relevant properties. Rationally engineering PKSs to produce higher yields or analogues of a target compound in vivo is often hampered by the complexity of the biosynthetic machinery and lack of high-throughput polyketide screens. The MphR protein naturally detects erythromycin in the context of a microbial resistance mechanism. Here we describe engineering MphR as a transcription factor-based biosensor to respond to erythromycin with increasing levels of sensitivity by employing multiple mutagenic strategies to alter the MphR gene and its upstream RBS. Additionally, the selectivity of MphR for closely-related macrolides is described and the ability to fine tune ligand recognition between these macrolides demonstrated. By demonstrating the ability to detect erythromycin production in a parallel and high-throughput fashion, MphR-based biosensors hold promise as a means to alleviate the bottlenecks that plague PKS engineering. Investigations into the specificity determinants of erythromycin biosynthesis towards small molecule building blocks revealed the promiscuous nature of building block selection and incorporation. Mutagenesis of key enzyme residues revealed the plasticity of building block preference of the acyltransferase domains responsible for selection and incorporation of malonyl coenzyme A molecules (malonyl-CoA). This study demonstrated a complete reversal of building block preference in the preferred biosynthesis for a polyketide which incorporates a non-canonical malonyl-CoA versus the natural building block. This demonstration shows promise for the engineering of polyketide synthases with optimized function and opens the door for unbiased approaches to polyketide synthase engineering that may leverage high-throughput screening. Studies of the methyltransferase EryG and the erythromycin-producing microbe Aeromicrobium erythreum highlight further the scope of polyketide synthetic biology engineering aims. The application of biosensor-guided high-throughput screening is demonstrated in the detection of erythromycin biosynthesized by A. erythreum. © Copyright 2017 Christian Mayfield Kasey All Rights Reserved Genetically-Encoded Biosensors for High-throughput Engineering and Synthetic Biology of Polyketide Biosynthesis by Christian Mayfield Kasey A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Chemistry Raleigh, North Carolina 2017 APPROVED BY: _______________________________ ______________________________ Gavin Williams Reza Ghiladi Committee Chair ________________________________ ________________________________ Christian Melander David Muddiman DEDICATION I would like to dedicate this work to my loving wife, Christie, and awesome daughter, Joon, who have kept me on an even keel and been the balance my life has needed. This would not have been possible without you both. ii BIOGRAPHY Christian was born in Louisville, Kentucky and raised by his fantastic parents John and Judy Kasey, along with his brother, Jed, and sister, Kimmy. He grew up with an aptitude for science and art, and loved learning about things by taking them apart and usually putting them back together. He attended the University of Louisville where he studied history and chemistry. It was also there that he met his future wife, Christie Cheng. Christie, Christian and their daughter Joon moved to Raleigh, North Carolina where Christian is pursuing a PhD in chemistry at NC State University. iii ACKNOWLEDGMENTS I would first like to acknowledge my advisor Dr. Gavin Williams. His mentorship, support and encouragement have made me the scientist I am today. I would also like to thank the rest of my committee Dr. Reza Ghiladi, Dr. Christian Melander, Dr. David Muddiman, and Dr. Jason King. I also would like to acknowledge the Williams lab. The support, advice, and camaraderie of past and present group members have had a hugely positive effect on my success as a graduate student. Finally, I thank my family who have supported me and made me the person I am today. Having my wife and daughter to come home to each day has given my life and work balance and provided clarity for my goals. My parents always encouraged my curiosity and encouraged me to follow my dreams, while also instilling the values of hard work, honesty, and kindness. iv TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………………………………ix LIST OF FIGURES………………………………………………………………………………………………x CHAPTER 1 ........................................................................................................................................... 1 1.1 Polyketides and drug discovery…………………………………………………………………….1 1.1.1 Polyketide optimization ...................................................................................................... 1 1.2 Polyketide biosynthesis ............................................................................................................ 4 1.2.1 Polyketide synthase organization ..................................................................................... 5 1.2.2 Post-PKS enzymes ............................................................................................................. 6 1.3 Production of non-natural polyketides by biosynthetic engineering ................................... 7 1.3.1 Pathway engineering .......................................................................................................... 8 1.3.2 Mutasynthesis and chemobiosynthesis ........................................................................... 8 1.3.3 Domain and module swapping .......................................................................................... 9 1.3.4 Acyltransferase promiscuity ............................................................................................. 9 1.4 Post-PKS analoging ................................................................................................................. 12 1.4.1 P450 engineering .............................................................................................................. 12 1.4.2 Glycosyltransferase engineering .................................................................................... 13 1.4.3 Methyltransferase engineering ........................................................................................ 13 1.5 Polyketide synthetic biology ................................................................................................... 14 1.5.1 Library design and high-throughput screening ............................................................ 15 1.5.2 Transcription-factor biosensors ..................................................................................... 16 1.5.3 Biosensor successes ....................................................................................................... 17 1.6 The scope of this dissertation ................................................................................................ 20 1.7 References ................................................................................................................................ 21 CHAPTER 2…………….……………………………………………………………………………………...28 v 2.1 Introduction .............................................................................................................................. 28 2.2 Results and Discussion ........................................................................................................... 33 2.2.1 Identification of Ery6 mutants with altered extender unit specificities ....................... 33 2.2.2 Mutant Ery6TE-catalyzed generation of 10-deoxymethynolide analogues ................ 35 2.2.3 DEBS3-catalyzed generation of 10-deoxymethynolide analogues .............................. 39 2.2.4 Extender unit promiscuity of wild-type and engineered DEBS3 ................................. 41 2.3 Conclusion ................................................................................................................................ 42 2.4 Materials and Methods ............................................................................................................ 46 2.5 References ................................................................................................................................ 50 CHAPTER 3 ......................................................................................................................................... 58 3.1 Introduction .............................................................................................................................. 58 3.2 Results and Discussion ........................................................................................................... 61 3.2.1 Characterization of a prototype two-plasmid macrolide detection system in E. coli 61 3.2.2 Enhancing the sensitivity of MphR to erythromycin via random mutagenesis and multi-site saturation mutagenesis ........................................................................................... 63 3.2.3 Random Mutagenesis of the RBS of MphR .................................................................... 67 3.2.4 Narrowing the Macrolide Inducer