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Biosynthetic Engineering of Plantazolicin Natural Products

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Biosynthetic Engineering of Plantazolicin Natural Products BIOSYNTHETIC ENGINEERING OF PLANTAZOLICIN NATURAL PRODUCTS BY CAITLIN D. DEANE 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, 2016 Urbana, Illinois Doctoral Committee: Associate Professor Douglas A. Mitchell, Chair Professor Wilfred A. van der Donk Professor William W. Metcalf Professor Huimin Zhao ABSTRACT Plantazolicin (PZN) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that exhibits extraordinarily narrow-spectrum antibacterial activity towards the causative agent of anthrax, Bacillus anthracis. During PZN biosynthesis, a cyclodehydratase catalyzes cyclization of cysteine, serine, and threonine residues in the PZN precursor peptide (BamA) to azolines. Subsequently, a dehydrogenase then oxidizes most of these azolines to thiazoles and (methyl)oxazoles. The final biosynthetic steps consist of leader peptide removal and dimethylation of the nascent N-terminus. To simultaneously establish the structure−activity relationship of PZN and the substrate tolerance of the biosynthetic pathway, an Escherichia coli expression strain was engineered to heterologously produce PZN analogs. 72 variant BamA peptides were screened by mass spectrometry to assess post-translational modification and export by E. coli, from which 29 PZN variants were detected. The modifying enzymes were exquisitely selective, installing heterocycles only at predefined positions within the precursor peptides while leaving neighboring residues unmodified. Nearly all substitutions at positions normally harboring heterocycles prevented maturation of a PZN variant. No variants containing additional heterocycles were detected, although several peptide sequences yielded multiple PZN variants as a result of varying oxidation states of select residues. Eleven PZN variants were produced in sufficient quantity to facilitate purification and assessment of their antibacterial activity, providing insight into the structure- activity relationship of PZN. Using heterologously expressed and purified heterocycle synthetase, the BamA peptide was processed in vitro concordant with the pattern of post-translational modification found in the naturally occurring compound. Using a suite of BamA-derived peptides, including amino acid substitutions as well as contracted and expanded substrate variants, the substrate tolerance of the heterocycle synthetase was elucidated in vitro, and the residues crucial for leader peptide binding were identified. Despite increased promiscuity compared to what was previously observed in E. coli, the synthetase retained selectivity in cyclization of unnatural peptides only at positions which correspond to those cyclized in the natural product. A cleavage site was subsequently introduced to facilitate leader peptide removal, yielding mature PZN variants after enzymatic or chemical dimethylation. In addition, we report the isolation and characterization of two novel PZN-like natural products whose existence was predicted by genome sequencing. ii ACKNOWLEDGEMENTS This undertaking would not have been possible without the help and support of many people. First and foremost, thank you to my adviser, Doug Mitchell, for accepting me into his lab, supplying direction and encouragement, keeping me on track, sending me to conferences, and celebrating my (and others’) accomplishments. Thank you to my thesis committee for their guidance and support. Special thanks to Wilfred van der Donk for his leadership of the CBI program and for extremely valuable career assistance. In addition, thank you to the UIUC Department of Chemistry for generous financial support. Utmost thanks to the entire Mitchell lab, past and present, for assistance in all manner of activities ranging from chemistry to cat-sitting. Special thanks to all my co-authors for invaluable scientific expertise and experimental contributions. I literally could not have done this without you! To the undergraduates I mentored, Agnieszka Maniak and Alice Lin, thank you both for being such a pleasure to work with. Thanks to Kyle Dunbar, Courtney Cox, Joel Melby, and Katie Molohon for so ably paving the way for those who came after them. Thanks to Team PZN, Katie Molohon and Patricia Blair, for always making subgroups interesting. Thanks to Tucker Maxson for being there throughout this whole crazy process, starting with day one of our summer rotation. Extra-special thanks to Brandon Burkhart for innumerable contributions, both scientific and otherwise. Your willingness to celebrate or commiserate as needed made all that time in lab more enjoyable (especially on Saturdays). Thanks to all my teachers throughout the years, notably Matt Hall, Brian Seed, and Brian Gilbert, for their inspiration and help in getting me to where I am today. Finally, the greatest of thanks to Brett Bobysud, Alysia Painter, and my parents, Bob and Norma Deane, for their unflagging love, support, and encouragement, even when (perhaps especially when) they didn’t understand what exactly I was doing. iii To Dad iv TABLE OF CONTENTS CHAPTER 1: Lessons learned from the transformation of natural product discovery to a genome-driven endeavor............................................................. 1 1.1 Introduction.......................................................................................................... 1 1.2 Lesson 1: Structure prediction............................................................................. 8 1.3 Lesson 2: Accurate sequence annotation............................................................. 9 1.4 Lesson 3: Continued study of model organisms.................................................. 11 1.5 Lesson 4: Avoiding genome size bias.................................................................. 11 1.6 Lesson 5: Genetic manipulation...........................................................................12 1.7 Lesson 6: Heterologous expression..................................................................... 12 1.8 Lesson 7: Potential engineering of natural product analogs................................ 13 1.9 Outlook................................................................................................................ 14 1.10 Conclusion......................................................................................................... 15 1.11 References.......................................................................................................... 16 CHAPTER 2: Engineering unnatural variants of plantazolicin through codon reprogramming........................................................................ 28 2.1 Introduction.......................................................................................................... 28 2.2 Heterologous production of PZN......................................................................... 31 2.3 Design and evaluation of BamA mutants............................................................ 33 2.4 Substrate tolerance at heterocyclized positions................................................... 54 2.5 Substrate tolerance at non-cyclized positions...................................................... 56 2.6 Pathway tolerance for variations in substrate length........................................... 58 2.7 Production of other PZN-class natural products.................................................. 59 2.8 Antibacterial activity of PZN analogs.................................................................. 62 2.9 Methods............................................................................................................... 64 2.10 References.......................................................................................................... 71 v CHAPTER 3: In vitro biosynthesis and substrate tolerance of the plantazolicin family of natural products............................................................................. 75 3.1 Introduction.......................................................................................................... 75 3.2 In vitro reconstitution of the PZN heterocycle synthetase................................... 78 3.3 Substrate tolerance of the PZN synthetase in vitro.............................................. 86 3.4 Leader peptide recognition and binding by the PZN synthetase......................... 110 3.5 Production of novel PZN analogs........................................................................ 118 3.6 Methods............................................................................................................... 125 3.7 Sequences............................................................................................................. 136 3.8 References............................................................................................................ 137 APPENDIX A: HIV protease inhibitors block streptolysin S production............................. 142 A.1 Introduction......................................................................................................... 142 A.2 Evidence for the role of SagE as a protease........................................................ 145 A.3 Aspartyl protease inhibitors block SagA proteolysis.......................................... 147 A.4 HIV protease inhibitors block SLS production................................................... 148 A.5 Structure-activity relationships..........................................................................
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