Research Collection Doctoral Thesis Reprogramming Nonribosomal Peptide Synthesis Author(s): Niquille, David L. Publication Date: 2018 Permanent Link: https://doi.org/10.3929/ethz-b-000252186 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH No. 24827 Reprogramming Nonribosomal Peptide Synthesis A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by DAVID LAURENT NIQUILLE MSc ETH Zurich born on 29.06.1988 citizen of Val-de-Charmey (Fribourg) accepted on the recommendation of Prof. Dr. D. Hilvert, examiner Prof. Dr. J. Piel, co-examiner 2018 And if you don't know, now you know. The Notorious B.I.G. Acknowledgements This thesis would not have been possible without the support of the excep- tional people in and around the Hilvert lab. In particular, I want to thank my supervisor Don Hilvert for being everything I could hope for in a supervisor, for making me the scientist I am, for teaching me the peculiarities of English language, and for being a good sport when losing the Schmutzliparty (again). Your sermon on the amenities of a mullet will not be forgotten. Similarly, I want to thank Peter Kast for accepting his victories with stoic composure and thereby contributing significantly to my staying in the group. Also, your door has always been open to discuss biochemistry. I am grateful to J¨orn Piel for refereeing this thesis and helpful discussions. Furthermore, I would like to thank Hans-Martin Fischer and Anette Schutz¨ for technical support with radiochemistry and FACS, respectively. Anita Lussi-¨ Meier and Antonella Toth have been a great help with administrative matters and Leyla Hernandez has not only made lab-life more fun but has also been extremely helpful in making sure that no mutants leave the lab. During my PhD, I have benefitted tremendously from working with Hajo Kries and Doug Hansen who have been great mentors and scientific role- models. Thank you Hajo for showing me the beauty of natural product re- search and for many good moments at conferences, although we cannot visit Frankfurt any time soon. Doug, your grand-fatherly rambling about the his- tory of antibiotics has inspired the first Chapter of this thesis. I also appreciate your taking care of my macros. ACKNOWLEDGEMENTS I was fortunate to supervise and work alongside excellent students who have contributed significantly to my research. Thank you David Fercher for your persistence in displaying things on yeast, Sophie Basler for maudlin mo- ments, Simon Burgener and Claire Lin for holding up the flag of the iridium capsid project, Anna Camus for insights into the \Regenbogenmaschine", and Ines Folger for interminable monologs. It is a joy to see you continue your scientific career in the Hilvert lab or elsewhere. The list of Hilvert lab members I am indebted to, past and present, is long and I thank you all - in particular (but with no particular order) Shiksha Mantri for showing me the value of shaking things at very low RPM, my swim- ming companion Takahiro Mori, Clement Dince for inaugurating me into the Hilvert lab and being a great party mentor, Clemens Mayer for his chemistry support and many laps around H¨onggerberg, Cindy Schulenburg and An Van- demoulebroucke for always listening to my sorrows and advice in all aspects of life, Christoph Giese and Tom van Mele for good times when the women were away, Richard Obexer for showing me what a head can be useful for, Reinhard Zschoche for being my Samichlaus and recitation of his renowned lion joke, Aaron Debon \high/low five", Stephan Tetter for reservations in Italy, Xavier Garrabou for his optimism, Matthias Tinzl for creative swearing in Austrian, Tom Edwardson for delicious craft beers, Nathalie Preiswerk for brutal hon- esty, Takahiro Hayashi and Isabelle Heinzmann for generous donations from the shower basket and a lot of sushi, Moritz Pott and Jana Kanellopoulos for nearly making it to Garbicz and good times in Zurich and abroad, Susanne Mailand for our Uetliberg¨ runs, Yusuke Azuma and Mai Matsushita for bring- ing Japanese culture into our home, and Raphael Frey for gentlemen poker nights. VI I am proud to have captained our immensely successful SOLA team and am grateful for all the effort and sweat, especially the valiant efforts of Marcel Grogg to conquer mount Uetliberg¨ and to Cathleen Zeymer for cracking the whip on training days. Hajo, Doug, Cindy, Tom, and Sabine Studer possessed the heroic patience to proof-read this thesis for which I am extremely thankful. In the hustle and bustle of a PhD thesis, a solid base is invaluable. I would like to say thank you to all friends and family. In particular I am grateful to Heinz Stark, Tobias Burge,¨ and Joris Muller¨ for exploring the cities of Europe with me, to Marcus Textor for igniting my passion for science, to Manuela, Alfons, Sarah, and Florian Studer for being my second family in Wallis and bestowing the honorary title of racletteur to me, to my brother for not instigating the next world-wide financial meltdown (so far), and to my parents for their unconditional love and support. Finally, I want to say thank you to you Sabine for being with me through all the ups and downs and making life so enjoyable. VII VIII Parts of this thesis have been published Nonribosomal biosynthesis of backbone-modified peptides Niquille, D.L., Hansen, D.A., Mori, T., Fercher, D., Kries, H., and Hilvert, D. (2018) Nature Chemistry 10, 282-287. Related publicatons A subdomain swap strategy for reengineering nonribosomal peptides Kries, H.*, Niquille, D.L.*, and Hilvert, D. (2015) Chemistry & Biology 22, 640-648. (*denotes co-first authorship) Reprogramming nonribosomal peptide synthetases for \clickable" amino acids Kries, H., Wachtel, R., Pabst, A., Wanner, B., Niquille, D., and Hilvert, D. (2014) Angewandte Chemie International Edition 53, 10105-10108. IX X Contents Abstract XV Zusammenfassung XIX 1 Introduction 1 1.1 The antibiotic era . .1 1.1.1 Antibiotics from natural products . .4 1.1.2 Antimicrobial resistance . 11 1.1.3 Next-generation antibiotics . 14 1.1.4 Biosynthetic assembly lines . 17 1.2 Nonribosomal peptide synthetases . 20 1.2.1 The core machinery . 20 Building block selection and loading . 20 Amide bond formation . 22 Product offloading . 24 1.2.2 Peptide tailoring . 27 1.2.3 Structure & dynamics . 28 1.3 Biosynthetic access to novel NRPs . 29 1.3.1 Combinatorial biosynthesis of NRPs . 31 Rewiring NRPSs via COM domains . 32 XI CONTENTS Molecular lego with NRPS modules . 32 Directed evolution of chimeric NRPSs . 33 1.3.2 A-domain engineering . 34 1.4 Aims of this thesis . 37 2 Installing bioorthogonal handles on NRP antibiotics 41 2.1 Introduction . 41 2.2 Results . 44 Mapping the substrate scope of TycApY ......... 44 E and C domain tolerance . 46 Reprogramming gramicidin S biosynthesis . 48 Biosynthetic access to tyrocidine A (11) analogs . 49 2.3 Discussion . 54 3 Biosynthesis of backbone-modified peptides 59 3.1 Introduction . 59 3.2 Results . 61 A high-throughput A-domain assay . 61 Reprogramming TycA for (S)-β-Phe . 63 Structural analysis of the α/β-switch . 66 Downstream processing of β-amino acids . 67 Engineering TycA for N -alkylated Phe analogs . 72 Biosynthesis of N -methylated peptides . 75 3.3 Discussion . 78 XII 4 Perspectives 83 5 Appendix 91 5.1 Materials and methods . 91 5.1.1 Chemical synthesis . 91 5.1.2 Biocatalysis . 102 5.1.3 Cloning . 109 5.1.4 Protein production . 122 5.1.5 Protein purification . 123 5.1.6 Adenylation kinetics . 125 5.1.7 High-throughput assay . 126 5.1.8 Crystallization . 129 5.1.9 Dipeptide synthetase reactions . 132 5.1.10 In vitro gramicidin S biosynthesis . 134 5.1.11 In vivo NRP production . 136 5.1.12 Tyrocidine biosynthesis . 137 5.2 LC-MS . 138 5.3 Yeast cell surface display . 156 XIII XIV Abstract In light of the antibiotic crisis, expansion of our therapeutic arsenal is im- perative to keep multi-resistant pathogens at bay. The preeminent source for clinical antibiotics are microbial secondary metabolites including vital pep- tide drugs such as vancomycin and the penicillins. However, bioactive natural products are not drugs per se and often require improved therapeutic proper- ties before clinical application. At present, natural product tailoring is accom- plished via semisynthesis, where the periphery of an isolated natural product is chemically modified in order to optimize pharmacological properties. In- herent limitations to this approach have motivated efforts to alter natural product composition through manipulating the biosynthetic machinery. Nonribosomal peptides (NRPs) represent an important class of natural products that are produced in assembly-line fashion by NRP synthetases (NRPSs) with dedicated modules responsible for incorporating amino acid building blocks. Harnessing this inherent modularity for biosynthetic en- gineering promises to become a powerful and sustainable approach to ac- cess next-generation medicines. Key to the reprogramming of NRPSs are adenylation (A) domains, that select and incorporate building blocks from a plethora of cytosolic metabolites. For example, a single tryptophan-to-serine (W239S) mutation in TycAF, the initiation module of tyrocidine synthetase, afforded a dramatic switch in substrate specificity for the \clickable" amino acid O-propargyl-L-Tyr that allows for selective reactions in complex mix- tures. We show that the expanded A-domain recognition pocket of W239S XV ABSTRACT TycA (TycApY) exhibits remarkable plasticity, accommodating a range of bioorthogonal conjugation handles. When introduced into reconstituted as- sembly lines, the single W239S mutation enabled efficient incorporation of unique reactivity into the gramicidin S and tyrocidine antibiotics, providing new entry points for derivatization.