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Doctoral Thesis

Reprogramming Nonribosomal Peptide Synthesis

Author(s): Niquille, David L.

Publication Date: 2018

Permanent Link: https://doi.org/10.3929/ethz-b-000252186

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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.

Despite the successes with W239S and a few other examples, A-domain engineering is generally challenging, limited to relatively modest changes in side-chain properties, and usually accompanied by substantial losses in cat- alytic efficiency. To address current limitations, we established a fluorescence activated cell sorting (FACS) assay for A-domain catalysis on the surface of yeast. The ability to search large sequence space enabled us to explore biosynthetic backbone modification as a means to strategically modulate the structure and properties of NRPs. Using the W239S mutation as a stepping stone for the introduction of a fluorescence readout, we reprogrammed TycAF to accept and process the backbone-modified amino acid (S)-β-Phe with near- native specificity and activity. A co-crystal structure with a non-hydrolyzable aminoacyl-AMP analog revealed the origins of the 40,000-fold α/β-specificity switch, illuminating subtle but precise remodeling of the active site. When the engineered catalyst was paired with downstream module(s), (S)-β-Phe- containing peptides were produced at preparative scale in vitro (∼1 mmol) and high titers in vivo (∼100 mg/L), highlighting the potential of biosynthetic pathway engineering for the construction of backbone-modified nonribosomal frameworks.

To extend the applicability of our high-throughput assay, we repro- grammed TycAF for N -propargyl-L-Phe. Accommodation of an alternative

“click” handle in the resulting variant TycAN pF,1 not only underscores the

XVI malleability of A-domains but also supports a general anchoring strategy for bioorthogonal handles as all amino acid substrates possess an amino group. By freeing the side-chain, activation of N -propargylated amino acids opens the door to more general reshaping of the A-domain binding pocket. Fur- thermore, it should be possible to extend high-throughput screening to other important NRPS domains/modules. In a proof-of-principle experiment, for example, we detected condensation (C) domain-catalyzed peptide bond for- mation on yeast. Given the shared thiotemplate mechanism and precedence for activating “clickable” substrate analogs, even related polyketide pathways are conceivably amenable to this methodology.

Customizing the assembly line biosynthesis of natural products has been a long-standing goal. This thesis establishes high-throughput screening methods that exert unprecedented control over activity and substrate scope of NRPSs, promising to finally make their biosynthetic tailoring a routine task. These efforts are expected to significantly expand microbial chemistry in the search for life-saving therapeutics.

XVII XVIII Zusammenfassung

Angesichts der bestehenden Antibiotika-Krise ist die Erweiterung des vorhan- denen therapeutischen Arsenals zur Bek¨ampfung von multi-resistenten Krank- heitserregern essentiell. Bei den meisten klinisch genutzten Antibiotika handelt es sich um sekund¨are mikrobielle Metaboliten, so zum Beispiel lebenswichti- ge Peptidwirkstoffe wie Vancomycin und Penicilline. Allerdings sind bioaktive Naturstoffe nicht per se Arzneimittel und ben¨otigen oftmals verbesserte thera- peutische Eigenschaften bevor sie klinisch eingesetzt werden k¨onnen. Um die pharmakologischen Eigenschaften zu verbessern, werden Naturstoffe derzeit semisynthetisch optimiert, indem der isolierte sekund¨are Metabolit nachtr¨ag- lich chemisch modifiziert wird. Grundlegende Einschr¨ankungen dieses Verfah- rens haben Bemuhungen¨ die chemische Struktur des Naturstoffs direkt uber¨ die biosynthetische Maschinerie zu modifizieren verst¨arkt.

Nichtribosomale Peptide (NRPs) sind ein wichtige Gruppe von Naturstof- fen, die von NRP-Synthetasen (NRPSs) fliessbandartig hergestellt werden, wobei einzelne Module der Synthetase fur¨ den Einbau einzelner Aminos¨au- rebausteine verantwortlich sind. Das Ausnutzen¨ dieser intrinsischen Modula- rit¨at verspricht eine vielversprechende und nachhaltige Methode zu werden, um Medikamente der n¨achsten Generation herzustellen. Entscheidend fur¨ die Umprogrammierung von NRPSs sind die Adenylierungsdom¨anen (A), die die korrekten Bausteine aus einer Vielzahl von zytosolischen Metaboliten aus- w¨ahlen und einbauen. Eine einzelne Tryptophan-zu-Serin Mutation (W239S) im Initiierungsmodul der Tyrocidinsynthetase TycAF hat zum Beispiel zu

XIX ZUSAMMENFASSUNG einer drastischen Anderung¨ der Substratspezifit¨at fur¨ die klickbare“ Amino- ” s¨aure O-propargyl-L-Tyr gefuhrt,¨ welche selektive Reaktionen in komplexen Mischungen erlaubt. Wir konnten zeigen, dass die Erweiterung der Erken- nungstasche der A-Dom¨ane von W239S TycA (TycApY) zu einer erh¨ohten Plastizit¨at fuhrt,¨ die die Unterbringung einer Vielzahl von bioorthogonalen Konjugierungshenkeln erlaubt. Integriert in die Fertigungslinie erlaubte diese einzelne W239S Mutation den effizienten Einbau von einzigartigen Reaktivi- t¨aten in Gramcidin S- und Tyrocidin-Antibiotika und erm¨oglicht somit neue Ans¨atze zur Derivatisierung.

Trotz des Erfolgs der W239S Mutation sowie einigen anderen Beispielen, ist der Umbau von A-Dom¨anen generell anspruchsvoll und auf relativ geringe Anderungen¨ der Seitenketteneigenschaften limitiert, die meist mit einer erheb- lichen Abnahme der katalytischen Effizienz einhergehen. Um die bestehenden Einschr¨ankungen aufzuheben, haben wir einen Fluoreszenz-aktivierten Zell- sortierungsassay (FACS) fur¨ die A-Dom¨anenkatalyse auf der Oberfl¨ache von Hefezellen entwickelt. Dies erm¨oglichte uns einen immensen Sequenzraum zu durchk¨ammen und so biosynthetische Modifikationen des Peptidruckgrats¨ zu untersuchen, um so die Struktur und Eigenschaften von NRPs strategisch zu ver¨andern. Mit der W239S Mutation als Startpunkt fur¨ eine fluoreszenzbasier- te Auslese, haben wir TycAF so umprogrammiert, dass die ruckgratmodifizier-¨ te Aminos¨aure (S)-β-Phe mit fast naturlicher¨ Spezifit¨at und Aktivit¨at akzep- tiert und verarbeitet wird. Eine Kristallstruktur mit einem unhydrolisierbaren aminoacyl-AMP Analog zeigt einen subtilen aber pr¨azisen Umbau des akti- ven Zentrums und erkl¨art damit den 40,000-fachen α/β-Spezifizit¨atswechsels. Die Paarung dieses massgeschneiderten Katalysators mit den nachgeschal- teten Modulen erm¨oglichte die Herstellung von (S)-β-Phe-haltigen Peptiden

XX im pr¨aparativen Massstab in vitro (1 mmol) sowie mit hohen Titern in vi- vo (100 mg/L). Dies unterstreicht das hohe Potential des Umbaus von bio- synthetischem Fertigungslinien fur¨ die Erzeugung von ruckgratmodifizierten,¨ nichtribosomalen Grundgerusten.¨

Um die Anwendbarkeit unseres Hochdurchsatz-Assays zu erweitern, haben wir TycAF fur¨ N -propargyl-L-Phe umprogrammiert. Die Unterbringung eines alternativen Klick“-Henkels in der resultierenden TycA Variante best¨a- ” N pF,1 tigt nicht nur die hohe Plastizit¨at von A-Dom¨anen, sondern erm¨oglicht eine generelle Verankerungsstrategie fur¨ bioorthogonale Henkel, da die Aminogrup- pe ein Bestandteil aller Aminos¨auresubstrate ist. Da die Seitenkette nun frei zug¨anglich ist, erlaubt die Aktivierung von N -propargylierten Aminos¨auren einen allgemeinen Ansatz zum Umbau der A-Dom¨anenbindungstasche. Des Weiteren sollte es m¨oglich sein, das Hochdurchsatz-Screening auf andere wich- tige NRPS Dom¨anen und Module zu erweitern. In einem ersten Experiment detektierten wir zum Beispiel die Kondensationsdom¨anen-katalysierte Bildung von Peptidbindungen auf Hefe. In Anbetracht des gemeinsamen thioabh¨angi- gen Mechanismus und der Pr¨azedenz fur¨ die Aktivierung von klickbaren“ ” Substratanalogen k¨onnten sogar die verwandten Polyketid-Fertigungslinien mit dieser Methode zug¨anglich gemacht werden.

Das Manipulieren von biosynthetischen Fertigungslinien war ein langj¨ah- riges Ziel. Diese Doktorarbeit etabliert Hochdurchsatz-Screenings, die eine bisher unerreichte Kontrolle uber¨ die Aktivit¨at und den Substratumfang von NRPSs erm¨oglichen und in Aussicht stellen, dass deren biosynthetische Op- timierung eine Routineaufgabe werden k¨onnte. Es wird erwartet, dass diese Werkzeuge eine signifikante Erweiterung der mikrobiellen Chemie auf der Su- che nach neuen Medikamenten erlauben werden.

XXI XXII .

1 Introduction

1.1 The antibiotic era

Throughout the history of mankind, pathogenic microbes have been respon- sible for devastating pandemics leading to the demise of cities and entire nations. Still today, microbial infections are one of the leading causes of death worldwide [1]. However, rapid advances in the chemical and biological sciences during the late 19th and early 20th century led to the discovery of antimicrobial compounds, marking the beginning of the antibiotic era.

Traditional medicines around the globe rely on the antimicrobial proper- ties of plants, molds, and bacteria, with the first evidence of human exposure to antibiotics going back as many as 1,500 years [2, 3]. Red sand from Jor- dan, for example, is a historic remedy against skin infections that is still used today as an inexpensive alternative to antibiotics [4]. It was not before the seminal work of Louis Pasteur and Robert Koch in the second half of the 19th century, however, that pathogenic microorganisms were established as the causal agent of infectious diseases [5]. This profound paradigm shift in modern medicine redoubled efforts to search for novel therapies. At the newly founded Institute for Infectious Diseases in Berlin (now the Robert Koch In- stitute), Emil von Behring and Paul Ehrlich developed an antiserum for the bacterial toxins causing diphtheria, a breakthrough that was awarded the first Nobel Prize in Physiology or Medicine in 1901 [6]. Beyond immunology, Ehrlich also pioneered the field of chemotherapy [7]. Inspired by his stud-

1 1. INTRODUCTION ies on the differential staining of cells with organic dyes, he formulated the chemotherapeutic principle corpora non agunt nisi fixata, “agents do not work unless they are bound” [8]. In 1904, Ehrlich and colleagues embarked on the first systematic drug screening program to find a treatment for syphilis based on derivatives of the highly cytotoxic drug atoxyl (1, Fig. 1a) [9]. This work culminated in the discovery of salvarsan (2), “the arsenic that saves”, the first hypothesis-driven, antimicrobial compound (Fig. 1a) [7]. Now, more than 100 years since its discovery, the exact mode of action of salvarsan (2) still remains unknown and the solution structure was only solved recently (Fig. 1b) [10].

Although a huge commercial success, salvarsan (2) was accompanied by severe side effects and its use remained limited to the treatment of syphilis and sleeping sickness. Nonetheless, the compound screening approach introduced by Ehrlich and coworkers set the stage for a century of pharmacological re- search that led to the discovery of the first clinical broad-spectrum antibiotic, prontosil (5, Fig. 2) [11]. The red azo compound 5 was synthesized in 1932 by

a O b OH

As OH OH NH 2 OH NH 2 H2N NH 2 H N 2 HO OH atoxyl (1) As As As OH As As As H2N As As NH 2 H2N As As NH 2 H2N NH 2 HO OH HO salvarsan (2) 3 HO 4 OH

Figure 1. The first antibiotics. a, Screening of compound libraries based on atoxyl (1) for activity against spirochetes in mouse models afforded salvarsan (2), the first effective treatment for syphilis (structure as proposed by Ehrlich). b, Recent electron spray ioniza- tion mass spectrometry (ESI-MS) studies suggest the presence of cyclic species 3 and 4 in solution [10].

2 1.1. The antibiotic era

Josef Klarer and Fritz Mietzsch and tested by Gerhard Domagk at the Bayer laboratories in an effort to identify synthetic dyes with antimicrobial activity. It showed excellent activity in Streptococcus-infected mice but was completely inactive when tested in vitro. By the time the discovery was finally reported in 1935, a research team at the Institut Pasteur had found a biochemical ra- tionale for Domagk’s observation [12]: Prontosil (5) acts as a prodrug that is reductively cleaved in vivo to release the active pharmaceutical ingredient, sulfanilamide (6). Sulfanilamide 6 inhibits folic acid biosynthesis by mimick- ing the precursor molecule 4-aminobenzoic acid (PABA, 7) and competitively blocking the key pathway enzyme dihydropteroate synthase [13]. Since folic acid (8) is essential for DNA metabolism, its deficiency leads to growth arrest in bacteria and fungi (bacteriostasis) but not in higher eukaryotes that lack the folic acid pathway and rely on dietary uptake instead.

Eventually, sulfanilamide (6) was introduced to the market in 1936 and earned Domagk the 1939 Nobel Prize in Physiology or Medicine (which he could not accept until the end of world war II). The compound, however,

O O O O DNA S S biosynthesis HO O NH 2 NH 2 NH 2 N N H2N O sulfanilamide (6) O H2N O O N prontosil (5) H N OH OH HN N H

H2N H2NNN PABA ( 7) folic acid (vitamin B9, 8)

Figure 2. The discovery of sulfa drugs. The first broad-spectrum antibiotic prontosil (5) acts as a prodrug for the 4-aminobenzoic acid (PABA, 7) antagonist sulfanilamide (6). The biological activity of sulfanilamide (6) is derived from competitive inhibition of dihydropteroate synthase involved in folic acid (8) biosynthesis.

3 1. INTRODUCTION had already been synthesized by Austrian chemist Paul Gelmo in 1908 [14] and was therefore not patentable. While crushing any monetary aspirations of Klarer and Mietzsch who held the patent on the synthesis of prontosil (5), the ready availability of sulfanilamide (6) facilitated rapid distribution to research labs across the globe and the development of an entire family of sulfonamide- based (sulfa) drugs. Unprecedented in its ability to cure bacterial infections, this novel class of miracle drugs revolutionized pharmaceutical research and launched the beginning of the antibiotic era [15].

1.1.1 Antibiotics from natural products

Since the introduction of sulfa drugs, only two major antibiotic classes have been developed from synthetic compounds, despite enormous efforts to design drugs rationally and screen large compound libraries in higher throughput (Fig. 3). While modern compound libraries dwarf the comparably small col- lection of organic dyes that afforded prontosil (5), they still cover only a small fraction of chemical space and are often biased according to empirical rules for desirable drug properties (e.g. Lipinski’s rule of five for oral bioavailabil- ity [16]) that do not reflect the main challenge of antibiotics: penetrating prokaryotes. In contrast, microbial secondary metabolites evolved over more than three billion years to exhibit potent biological activities. Indeed, the term antibiotic was first used by Jean Paul Vuillemin to describe the antagonistic behavior of microorganisms that is governed by such natural products [17]. Despite (or due to?) their often complex and three-dimensional molecular structures that typically defy Lipinski’s rule of five, natural products have proven a treasure trove for antibiotic drug discovery.

4 1.1. The antibiotic era

O O H O F O OH N

NO O N N F HN NH

ciprofloxacin (9) linezolid (10 )

Figure 3. Modern synthetic antibiotics. The (fluoro)quinolones (e.g. ciprofloxacin, 9 [18]) and the oxazolidinones (e.g. linezolid, 10 [19]) are two important antibiotic classes derived from synthetic compound libraries.

Throughout history, the therapeutic properties of natural products have been appreciated. However, the discovery and characterization of penicillin marks the beginning of modern natural product research. In September 1928, the Scottish biologist Alexander Fleming noticed inhibition of bacterial growth in cultures that were contaminated with the fungus Penicillium notatum (to- day Penicillium chrysogenum). While similar observations had been made before, Fleming went on to use pure P. chrysogenum cultures to kill bacte- ria and concluded that the mold must secrete a bioactive compound that he called penicillin [20]. This seminal discovery received little attention until Ren´eDubos at Rockefeller University isolated tyrothricin from the soil bac- terium Bacillus brevis (today Brevibacillus parabrevis) a decade later [21–23]. Tyrothricin is a mixture of linear pentadecapeptides and cyclic decapeptides [gramicidin A-C and tyrocidine A-D (11)] that disrupt cell membranes and are therefore highly bactericidal. Unfortunately, this membrane activity also lyses red blood cells, allowing only topical applications [24].

Motivated by Dubos’ findings, a research team at Oxford University in- cluding Walter Florey and Boris Chain managed to isolate and characterize

5 1. INTRODUCTION

Fleming’s mystery substance only one year later, a compound that would be- come famous under the name penicillin G (12, Fig. 4) [25]. In 1949, Dorothy Hodgkin reported the X-ray crystal structure of 12, revealing a tripeptidic molecule with a central β-lactam ring. This core motif is essential for peni- cillin’s inhibition of bacterial cell wall biosynthesis by targeting the enzyme DD-transpeptidases (also known as penicillin-binding proteins, PBPs). The overall structure of penicillin G 12 mimics D-Ala-D-Ala recognition motifs in peptidoglycan cell wall precursors that recruit PBP for cross-linking. Conse- quently, 12 binds to PBP where the central β-lactam acylates and thereby inactivates the catalytically active Ser residue. Since peptidoglycan cross- linking is vital for bacteria, penicillin exhibits excellent bactericidal activity and, importantly, no toxicity in humans. The superior drug properties com- pared to sulfonamides quickly made penicillin the first-line treatment for bac- terial infections. To meet the enormous demand in times of war, the then small Brooklyn-based chemical company Pfizer developed deep-tank fermen- tation technology enabling the mass production of penicillin [26].

This success story triggered a gold rush for natural products between the 1940s and 1960s, which resulted in the discovery of most of the antibiotic classes used in the clinic today (Fig. 4). In addition to compounds with modes of action similar to that of tyrothricin [colistin A, lipopeptides] [27] and penicillin [cephalosporin C (13, cephalosporins) or vancomycin (14, gly- copeptides)] [28–30], these screening programs afforded the bacterial RNA polymerase inhibitor rifamycin B (15) [31] and a range of antibiotics target- ing the prokaryotic ribosome, including streptomycin (16, aminoglycosides) [32], chlortetracycline (17, tetracyclines) [33], chloramphenicol (18) [34], pleu- romutilin (19) [35],

6 1.1. The antibiotic era

H H OH NO 2 N S O Cl O N N chloramphenicol (18 ) O H Cl OH 1947, prokaryotic ribosome penicillin G (12 ) OH 1928, DD-transpeptidase (cell wall) O

pleuromutulin (19 ) OH O 1951, prokaryotic ribosome

O O NH 2 O HO H N O O H NH HN HN NH 2 O O OH O O NH O

N O H2N O OH HO N O NH 11 OH NH tyrocidine A ( ) HO H 1939, membranes O N O N O O H O O O O

20 erythromycin A ( ) O OH 1952, prokaryotic ribosome

OH H2N HO OH NH 2 HO HO N O OH NH streptogramin A ( 21 ) O O N NH 2 1953, prokaryotic ribosome OHC streptomycin (16 ) OH H2N O 1943, prokaryotic ribosome HO N

O O N chlortetracycline (17 ) 1945, prokaryotic ribosome Cl OH N O O H H OH O N H O OH OH OH O O NH 2 N O N O N H

H2N NH OH cephalosporin C (13 ) O 1945, DD-transpeptidase (cell wall) NO O H O H N H S O O N N H HN O O N O O streptogramin B (22 ) O HO O 1953, prokaryotic ribosome HO O

7 1. INTRODUCTION

HO rifamycin B (15 ) HO OH 1957, proakyrotic ribosome HO HO

H2N O vancomycin (14 ) AcO OH O O OH OH O 1953, lipid II (cell wall) MeO NH O O O Cl O Cl HO OH O O CO 2H O O O O H H H O N N N O OH N N N O H H H H H HN O O O HOOC O N H NH 2 O NH HN OH HO OH OH H2N O NH O NH O NH O O HN O HN thienamycin (23 ) O NH 2 O OH 1976 H , DD-transpeptidase N OH (cell wall) N N HN O HH H H O O NH O OH O O S N O O NH O

O H2N NH 2 O 24 OH HO daptomycin ( ) 1986, membrane

Figure 4. Natural product antibiotics. History of antibiotic discovery from natural products (with year of first isolation and molecular target).

erythromycin A (20, macrolides) [36], and streptogramin [a mixture of strep- togramin A (21) and streptogramin B (22)] [37].

By the end of the 1960s, however, a marked decrease in discovery rate and daunting frequencies of rediscovery, indicated a depletion of the natural product pool. Since the golden era of discovery, only two major additions to the antibiotic armamentarium have been made, namely the carbapenems [thienamycin (23, 1976], a subfamily of the β-lactams) [38] and membrane active lipopeptides [daptomycin (24), 1984)] [39].

8 1.1. The antibiotic era

Notably, these diverse natural product antibiotics were discovered in a small number of talented producers - primarily fungi (ascomycota & ba- sidiomycota) and soil bacteria (bacilli & ascomycetes, in particular Strepto- mycetes). With diminishing returns from established microorganisms, novel ecological niches could potentially provide an untapped reservoir of chemical diversity. Marine microorganisms, for example, are prolific producers of nat- ural products [40]. Recently, the human microbiome, bacteria in and on the human body, has also been identified as a potential source of antibiotics [41]. Starting in the 1980s, phylogenetic analysis of ribosomal RNA led to the real- ization that the known biodiversity represents but a minuscule fraction of the microbial cosmos [42]. The vast majority of bacteria (∼99 %) cannot be cul- tured under standard laboratory conditions [43]. Novel culturing technologies that mimic natural habitats can unlock this hidden biosynthetic potential, as shown by the isolation of the promising peptide antibiotic teixobactin from a previously unknown proteobacterium [44].

In recent years, enormous progress in DNA sequencing and manipulation has enabled a novel approach to antibiotic discovery. Instead of screening bacterial extracts for bioactive compounds, genetic data can now be searched directly for characteristic biosynthetic enzymes thus eliminating two main hurdles: the need for (1) cultivated, clonal cultures and (2) a compound that is produced at sufficient quantities for detection. Sequencing of the first Strep- tomyces genomes at the beginning of the 21st century revealed that even this excessively mined genus still holds many secrets. The number of encoded path- ways in these microorganisms exceeds the known natural product palette by a factor of ∼10 [45]. The most gifted actinomycetes encode up to 50 different natural products, devoting >20% of their genome to the production of sec-

9 1. INTRODUCTION ondary metabolites. However, many of these pathways are cryptic, meaning that they are not actively transcribed, and must be unlocked by physiological triggers (e.g. chemical elicitors or co-culturing with other microorganisms) or metabolic engineering (e.g. removing endogenous gene regulation) [46]. The availability of more than 1,800 sequenced bacterial genomes to date [47] provides an invaluable resource for pathway discovery. Additionally, mixed genetic material can be directly extracted and sequenced from environmen- tal samples providing a metagenomic inventory of the respective biosynthetic diversity. Although novel drugs have yet to be brought to the clinic, this new era in natural product research augurs well for the future of antibiotic discovery.

1.1.2 Antimicrobial resistance

Continuous development of novel antibiotics is vital due to the ongoing threat of antimicrobial resistance. Introduction of all major classes of antibiotics was followed (or preceded) by the emergence of resistance. Most producers encode self-resistance mechanisms that can rapidly disseminate by horizon- tal gene transfer. Novel, synthetic compounds do not represent insurmount- able obstacles for bacterial adaptability either, as illustrated by the rapid de- velopment of fluoroquinolone resistance by Staphylococcus aureus [48]. This problem is exacerbated by horizontal gene transfer of plasmid-borne resis- tance genes leading to rapid distribution among microbial communities once selection pressure is applied. A recent review on behalf of the British govern- ment and the Wellcome Trust estimates up to 10 million deaths per year by 2050 due to our inability to treat bacterial infections [49]. The small group of “ESKAPE bugs” (Enterococcus faecium, Staphylococcus aureus, Klebsiella

10 1.1. The antibiotic era pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enter- obacter species) represents a particular threat, since it is responsible for the majority of nosocomial infections and immune to the existing antibiotic arse- nal [50].

How do microbes escape antibiotics? Modes of resistance are manifold and complex, but typically include: (A) decreased membrane permeability and active efflux, (B) antibiotic inactivation or modification, (C) pathway bypassing, (D) target modification, and/or (E) amplification (Fig. 5). Resis- tance to penicillins and cephalosporins, for instance, is typically associated with β-lactamases that hydrolyze and thus inactivate the β-lactam nucleus. Modern β-lactam drugs therefore often pair the antibiotic with an irreversible β-lactamase inhibitor, such as the natural product clavulanic acid.

B D P

A gene

C E transfer

A + B C

Figure 5. Antibiotic resistance. Bacterial strategies for escaping antibiotics (red) include: (A) Reduced permeability and drug efflux, (B) antibiotic modification, (C) pathway bypassing, (D) target modification or (E) amplification. Acquired resistance mechanism are rapidly disseminated by horizontal gene transfer. This figure is adapted from [43].

11 1. INTRODUCTION

Not all antibiotics are equally susceptible to bacterial resistance. For in- stance, vancomycin resistance was first reported in 1988, 30 years after its clinical introduction [51]. It has been argued that this resilience is due to the multi-enzyme biosynthesis of peptidoglycan which does not allow for tar- get alteration by simple point mutation [52]. Additionally, vancomycin acts extracellularly and is therefore immune to resistance mechanisms based on membrane permeability and efflux pumps. Nevertheless, growing antimicro- bial resistance to this class of last-resort antibiotics is becoming increasingly alarming. Staphylococcus aureus strains with intermediate vancomycin resis- tance (VISA) were already reported in the 1990s [53]. VISA are associated with an unusually thick and poorly cross-linked peptidoglycan wall that not only blocks vancomycin penetration but also displays excess D-Ala-D-Ala tar- gets to trap vancomycin in external layers. Even worse, S. aureus can adopt the defense strategy of natural producers and become completely resistant to vancomycin (VRSA) by modification of its peptidoglycan termini to D-Ala-

D-Lac, thereby eliminating a critical hydrogen bond interaction needed for vancomycin binding [52, 54].

The enormous challenge of antimicrobial resistance requires a concerted ef- fort by policy makers, the pharmaceutical industry, and academia. Although inevitable, resistance development could be decelerated significantly with a responsible stewardship of antibiotics. Today, therapeutic application in hu- mans accounts for less than 50% of antibiotic usage [55]. Also, antibiotics are often used without clear evidence for bacterial infection or at suboptimal doses that do not eradicate all pathogens. A possible solution lies in combination therapy with different antibiotics that allows for a more radical and shorter treatment, decreasing the likelihood that resistance emerges. However, only a

12 1.1. The antibiotic era minority of human commensals are pathogenic, while most are beneficial. A harsh and broad-spectrum treatment can therefore also have adverse effects by damaging the human microbiome, as observed in opportunistic Clostridium difficile infections [56].

While preservation of the existing arsenal is essential, new antibiotic drugs will be indispensable. Unfortunately, antibiotic drug development provides poor return on investment. Not only has it proven extremely difficult to find novel antibiotics, but once developed, new compounds usually remain shelved to be used only as a last resort [57]. Additionally, antibiotics are typically used for a relatively short period of time, unlike blockbuster drugs for chronic diseases. Public incentives are thus required to encourage investment and rejuvenate antibiotic research.

1.1.3 Next-generation antibiotics − semisynthetic tailoring

Natural product antibiotics have not evolved for human applications. Ac- cordingly, tailoring the periphery of a bioactive scaffold can be successful at optimizing pharmacokinetic properties, expanding or narrowing the microbi- ological spectrum, and/or circumventing resistance mechanisms - a strategy that has also helped to keep fully synthetic antibiotics clinically relevant. In fact, 73% of the novel antibacterial chemical entities filed between 1981 and 2005 are semisynthetic derivatives of the penicillin, cephalosporin, quinolone, and macrolide scaffolds [58].

Penicillin G (12), for example, has undergone four rounds of semisyn- thetic tailoring by reacylation of the 6-aminopenicillanic acid nucleus [60]. Introduction of sterically demanding substituents, such as dimethoxybenzoyl

13 1. INTRODUCTION

HO HO OH HO

B HN O O Cl O O O O Cl Cl HO OH O O O H H H O N N N N N N H H H A H H C H HN O N N O

O NH 2 HO OH OH 25

OH OH OH OH OH OH OH OH O O O O O O O O O O O O OO O OO O O O O HO HO HO O HO NHAc AcHNNHAc AcHN NHAc AcHN NHAc AcHN O O A: trans-

L-Ala peptidation D-Lac L-Ala O D γ-D-Glu D-Lac -Ala γ-D-Glu L-Lys L-Lys L-Lys NH 2 D-Ala N D-Ala D D-Ala L-Lys γ- -Glu D-Ala H L-Lys L D-Lac γ-D-Glu -Ala D-Lac γ-D-Glu L-Ala L-Ala B: trans- OH OH OH OH glycosylation O OH OH O O O O HO OO O O HO O HO OO NHAc AcHN HO OO HO HO NHAc AcHN O O NHAc O O P O O P O O O P O cell wall O P O O O

C: membrane

Figure 6. Next-generation glycopeptides. Total synthesis of 25 afforded modified vancomycin and reinstated activity against VRSA (A) as well as expanding the mode of ac- tion to inhibition of transglycosylases (B) and induction of membrane permeability (C) [59].

14 1.1. The antibiotic era in methicillin, afforded the second generation of penicillins that was no longer subject to β-lactamase degradation enabling effective treatment of penicillin G(12)-resistant S. aureus. Nevertheless, heavy use of antistaphylococcal penicillins led to methicillin-resistant S. aureus (MRSA) that produced al- tered PBP targets. Subsequently, next-generation aminopenicillins (3rd, e.g. ampicillin, carboxypenicillins (4th, e.g. ticarcillin, and ureidopenicillins (5th, e.g. piperacillin) significantly expanded the activity spectrum of penicillins, especially to gram-negative pathogens such as Pseudomonas aeruginosa and various Enterobacteriaceae [57].

While the relatively simple structure of β-lactams has facilitated derivati- zation, many natural products have complex architectures with a multitude of functional groups. As a consequence, semisynthetic modification is much less accessible. For instance, semisynthetic glycopeptides have only been in- troduced during the last decade [52]. Inspired by the natural glycopeptide teicoplanin, these second-generation inhibitors of cell wall biosynthesis fea- ture a characteristic lipophilic tail that has been proposed to add a second mode of action by targeting the transglycosylases responsible for lipid II poly- merization [61]. These appendages can be introduced in a single step by reductive alkylation of the vancosamine sugar. Additionally, unique reactiv- ities at dihydroxyphenylglycine 7 and the C-terminus have been exploited for modifications aimed at increasing solubility or conferring extra membrane activity with a positively charged amine. Amidation of the C-terminus has also enabled installation of a Zn(II)-chelator to localize vancomycin at Zn(II)- lipid II-rich bacterial membranes and thus improve activity against VISA and VRSA significantly [62].

15 1. INTRODUCTION

Although not trivial, semisynthetic tailoring of natural products can be an important source of novel antibiotics, provided that adequate functional groups are available. Similarly, modification of the core scaffold could afford new drugs and, no longer limited to peripheral sites, more directly modu- late the primary biological activity. For example, removal of an amide car- bonyl at the core of vancomycin (14) reinstates binding to the modified D-

Ala-D-Lac target while retaining activity against the native target D-Ala-D- Ala (Fig. 6) [63]. Combined with two peripheral appendages, this core modifi- cation affords the prototype third-generation lipoglycopeptide 25 with potent activity against VRSA and a minimized risk of resistance due to its three- pronged mechanism of action [59]. Nevertheless, removal of a single atom in 25 required a total of 26 chemical reactions, highlighting the challenges of natural product tailoring by chemical means.

1.1.4 Biosynthetic assembly lines

Biosynthetic pathway engineering is a potentially powerful, green, and scalable alternative to chemical methods for accessing modified or even novel bioactive compounds [64]. Microorganisms are not only a source for invaluable natural products but also harbor fascinating enzymology dedicated to the biosynthesis of these secondary metabolites. Understanding this biosynthetic logic is key for the reprogramming of natural product pathways.

Secondary metabolism comprises enzymes involved in all three stages of natural product biosynthesis: (1) building block supply, (2) core scaffold as- sembly, and (3) post-assembly tailoring. It draws on primary metabolites, such as amino acids [nonribosomal peptides (NRPs), ribosomally synthesized

16 1.1. The antibiotic era and post-translationally modified peptides (RIPPs), alkaloids], malonyl-CoA & derivatives [polyketides (PKs)], isoprenoids (terpenes), and carbohydrates (aminoglycosides), as direct building blocks or precursor molecules. The ma- jority of antibiotic natural products belong to the classes of NRPs and PKs that are assembled by multifunctional enzyme complexes called nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), respectively.

NRPSs and PKSs share a modular biosynthetic logic with functional do- mains organized into separate modules responsible for building block incorpo- ration and tailoring (Fig. 7). The individual modules can function iteratively or in concert to form biosynthetic assembly lines that covalently tether sub- strates and intermediates to the enzyme complex via thioesters. A major dif- ference between NRPSs and PKSs lies in the respective building block reper- toires. Whereas PKSs draw on a small set of malonyl-CoA derivatives, more than 500 building blocks form the basis for structural diversity in NRPs [65]. On the other hand, PKS modules typically contain multiple auxiliary domains for reductive processing and the installation of stereocenters, whereas online tailoring in NRPSs is less frequent. To ensure fidelity, PKS pathways often possess multiple internal selectivity filters with strict requirements even for distal groups [66, 67]. From an engineering standpoint, this multilayered speci- ficity makes PKSs challenging targets for biosynthetic engineering as changes to one active site often entail problems with most downstream domains. Al- though NRPS specificity has not been studied to the same extent, the large substrate repertoire and the lower degree of online modification suggest higher plasticity. The principles of NRPS biochemistry and their implications for en- gineering will be discussed in the following sections.

17 1. INTRODUCTION

TycA F (120kDa) TycB PFF (410 kDa)

S S S S Phe Pro Phe Phe

DPhe Pro Phe

DPhe Pro

DPhe TycC NQYVOL (730 kDa)

S S S S S S Asn Gln Tyr Val Orn Leu

DPhe Asn Gln Tyr Val Orn

Phe DPhe Asn Gln Tyr Val

Pro Phe DPhe Asn Gln Tyr

DPhe Pro Phe DPhe Asn Gln

DPhe Pro Phe DPhe Asn

DPhe Pro Phe DPhe

O DPhe Pro Phe DPhe Pro O NH 2 DPhe O N O H NH HN HN NH 2 O OH O O NH

N O H2N O

NH NH O H tyrocidine A ( 11 ) N O N H O

Figure 7. Assembly-line biosynthesis. Tyrocidine synthetase, encompassing the three subunits TycAF, TycBPFF, and TycCNQYVOL, illustrates the modular biosynthetic logic of NRP natural products. Each of the ten modules is responsible for incorporating one amino acid into the cyclic decapeptide antibiotic tyrocidine A (11).

18 1.2. Nonribosomal peptide synthetases

1.2 Nonribosomal peptide synthetases

The plethora of NRP structures is reflected in manifold NRPS architectures that vary from freestanding domains to megaenzymes of enormous size, with the largest continuous synthetases comprising up to 15 separate modules [68, 69]. Typically, modules are distributed over several proteins (subunits) that interact selectively via short communication-mediating (COM) domains [70]. Additonal diversity is added by the ability of NPRSs to interface other path- ways, especially PKSs, to form chimeric assembly lines. Despite this structural heterogeneity, all NRPSs employ the same basic principles for the assembly of peptidic natural products.

1.2.1 The core machinery

The NRPS thiotemplate mechanism requires a minimal set of four core do- mains to activate and tether building blocks, form peptide bonds, and release the final product [71, 72].

Building block selection and loading. Adenylation (A) domains act as gatekeepers to NRPS assembly lines by selectively loading building blocks in a two-step process (Fig. 8a). The cognate substrate binds in the active site at the interface of a large N-terminal (AN, ∼50 kDa) and a smaller C-terminal

(AC, ∼10 kDa) subdomain, where it is adenylated by ATP with Mg(II) acting as a cofactor. Upon substrate activation, the A domain undergoes a major ◦ conformational rearrangement involving a ∼140 rotation of AC relative to

AN. This second conformation allows the A domain to catalyze nucleophilic attack of a phosphopantetheine (ppant) cofactor anchored on a thiolation (T)

19 1. INTRODUCTION

a b

OH O H H N N P TycA F HS O O ppant O O O

ATP PP i HSS AMP

OH O S H2N H2N AMP H2N

26 O O O

Figure 8. Loading amino acids onto NRPSs. a, The A domain of TycAF catalyzes selective adenylation of L-Phe (26) and subsequent transfer to the ppant arm attached post-translationally to the T domain. b, Structure of the A-domain active site with bound L-Phe (26) (yellow), AMP (green), and Mg(II) (green sphere). The specificity-conferring residues are shown as orange sticks (PDB 1AMU [74]). domain on the activated substrate to effect covalent substrate tethering. This domain alternation mechanism is supported by point mutations that reduce the main-chain flexibility in the hinge region between AN and AC locking the A domain in an adenylation competent state [73]. A domain catalysis thus results in covalent loading of the following T domain (∼10 kDa) that tethers and shuttles substrates and reaction intermediates between active sites using the ∼20 A˚ ppant cofactor as a swinging arm. Ppant is post-translationally loaded from coenzyme A (CoASH) to a conserved Ser residue in the T domain by dedicated phosphopantetheine transferases (PPTases).

Combined with sequence alignments, several X-ray crystal structures have pinpointed catalytic residues in the A domain. First insights into sub- strate recognition and activation were provided by the structure of the Phe- activating A domain (GrsAF-A) from gramicidin S biosynthesis (PDB 1AMU) with bound AMP and L-Phe (Fig. 8b) [74]. AMP is bound in a polar pocket

20 1.2. Nonribosomal peptide synthetases

flanked by the invariant residues Arg428 and Lys517 (1AMU numbering is used for A domains throughout this thesis [74]) as well as the Mg(II) ion that is proposed to stabilize the negative charge of the leaving group. The key residue Lys517 is donated by AC and also binds the carboxyl group of the substrate. Like all amino acid activating A domains, GrsAF-A additionally contains the conserved residue Asp235 to bind the amino group of L-Phe. The substrate side chain is buried in a hydrophobic pocket formed by eight residues that are diagnostic for substrate specificity and have been termed the “nonribosomal code” [75, 76]. Together with 24 surrounding residues, this code enables rapid and precise computational prediction of specificity. Not all A domains recognize a single substrate, however. The mixture of tyro- cidines from Brevibacillus parabrevis, for example, is produced by bispecific A domains that activate either L-Phe or L-Tyr and L-Trp.

A domains can be accompanied by small proteins called MbtH-like proteins (MLPs, ∼70 residues). While strictly required for activity by some, others depend only partially or not at all on these auxiliary factors. The nature of this activation, however, remains enigmatic despite co-crystal structures that illuminate the binding of MLPs to their partner AN subdomains [77].

Amide bond formation. Loaded substrates and intermediates are shuttled to the pseudodimeric, V-shaped condensation (C) domain (∼50 kDa), where they undergo peptide bond formation in what represents the first committed step of nonribosomal peptide synthesis (Fig. 9) [78]. The two lobes of the C domain flank a tunnel that allows the donor and acceptor T domains to position the thioester electrophile and amine nucleophile in a central active site (the terms donor and acceptor describe modules that donate or accept the growing peptide chain). The binding pocket of C domains is characterized by

21 1. INTRODUCTION a conserved His residue. Based on mutational studies, this His is important but not essential for catalysis [79]. A recent crystal structure reveals an H- bond with the incoming acceptor amine, suggesting substrate positioning as the predominant catalytic mechanism [80]. C domains are not limited to amines but can also catalyze reactions with hydroxyl nucleophiles to give depsipeptides [81].

Masked by the specificity of flanking A domains, C domain substrate speci- ficity is difficult to elucidate experimentally. Efforts to bypass A domains, have provided some insight into a possible editing function of the C domain, espe- cially with regard to the amine nucleophile. This was achieved with laborious single turnover experiments using radioactive aminoacyl-CoA derivatives that are loaded by the promiscuous PPTase Sfp from Bacillus subtilis [82] or with small-molecule surrogates that float into the C domain active site and capture

T

O S T HN acceptor

S NH 3 donor O N

HN C

Figure 9. Proposed mechanism for peptide bond formation. A conserved His residue in the C-domain active site of TycB1P positions the donor thioester for attack by the downstream amino acid.

22 1.2. Nonribosomal peptide synthetases the donor thioester [83]. However, it is not clear to what extent small-molecule probes can mimic the covalent delivery of substrates by the T domain. Novel approaches are needed for a more holistic picture of C domain specificity, es- pecially since X-ray structures have not provided any clues regarding possible selectivity determinants.

In addition to possible editing functions, C domains are crucial for the in- corporation of D-amino acids into NRPs by selecting the D-enantiomer from a racemic mixture generated by an upstream epimerization (E) domain [82–84]. Some C domains even exhibit dual E/C functionality catalyzing epimerization as well as condensation [85].

Upon amide bond formation, the resulting peptide is translocated by the T domain to the donor site of the downstream C domain, where it is presented for condensation with the next amino acid. The nascent peptide is thus elongated until it reaches the terminal module.

Product offloading. The full-length peptide is typically released by a ter- minal thioesterase (TE) domain (∼30 kDa) from the α/β-hydrolase family (Fig. 10) [86]. Of all NRPS domains, thioesterases display the greatest het- erogeneity, reflecting the structural diversity of their substrates. They employ a canonical catalytic triad (Ser/His/Asp, rarely Cys/His/Asp) and oxyanion stabilization to transfer the assembled peptide chain from the terminal T do- main and activate it for release by nucleophilic attack (Fig. 10). Often, TE domains use an internal nucleophile from the substrate to effect macrocy- clization — a hallmark of many NRPs. The cyclic core structure is not only more resistant to proteolysis but also provides better membrane permeability and conformational rigidity to lower the entropic barrier for target binding.

23 1. INTRODUCTION

TycTE TycTE TycTE O HN TycTE

S OH SH O O O O O S O OH Leu Leu Leu O Orn Orn Orn Leu Val Val Val Orn Tyr Tyr Tyr Val Gln Gln Gln Tyr Asn Asn Asn Gln DPhe DPhe DPhe Asn Phe Phe Phe DPhe Pro Pro Pro Phe DPhe DPhe DPhe O Pro NH NH NH 2 2 DPhe 2 O NH 2 NH O 2 N O H NH HN HN NH 2 O OH O O NH

N O H2N O

NH NH O H N tyrocidine A ( 11 ) O N H O

Figure 10. TE-mediated product offloading. The fully assembled decapeptide is transferred to the catalytic Ser residue of TycTE where it undergoes head-to-tail macro- cyclization to afford tyrocidine A (11) (left). Peptide delivery by the T domain-anchored ppant arm can be mimicked with SNAC surrogates (right). This figure is adapted from [89].

Structural analysis of TE domains that catalyze lipopeptide macrolactoniza- tion has revealed a large, hydrophobic substrate pocket with a trough for substrate delivery by the docked T domain [87, 88]. A flexible lid shields the active site from bulk solvent. Once loaded, the substrate presumably adopts a cyclic conformation that is poised for release by ring closure.

24 1.2. Nonribosomal peptide synthetases

Mapping of TE specificity has been facilitated by the use of N - acetylcysteamine (SNAC) probes as surrogate peptidyl donors for the active site Ser residue (Fig. 10). A set of linear tyrocidine A-SNAC analogs was used to map the tyrocidine TE domain (TycTE) in an alanine scan [90], for instance. TycTE natively catalyzes head-to-tail cyclization of the decapep- tide antibiotic tyrocidine A. Of all ten positions, only the initial D-Phe and the penultimate L-Orn residue proved crucial for cyclization. [90] TycTE even cyclized peptides with altered length, modified backbones, or an N-terminal hydroxyl group [89]. However, substitutions that abrogated internal hydro- gen bonds were typically not tolerated, suggesting that the intrinsic propen- sity for a cyclization-competent conformation is a key requirement. Whether substrate preorganization is additionally induced by a templating TE active site is not known. The promiscuity with regard to the acyl donor and the substrate makes TE domains interesting biocatalysts for the preparation of macrocycles. Compatible linkers even enable coupling of solid phase pep- tide synthesis (SPPS) with TE-mediated macrocyclization to rapidly generate NRP libraries [91].

Despite their prevalence, TE domains are not the only solution for peptide offloading. Alternative mechanisms include reductive domains that afford terminal aldehydes and aminoglycoside N -acetyltransferase-like domains that recruit and couple an external amine nucleophile. Also, not all TE domains catalyze peptide offloading. NRPS clusters additionally encode type II TE domains that act as proofreaders by deacylating misprimed ppant cofactors.

25 1. INTRODUCTION

1.2.2 Peptide tailoring

A multitude of enzymes are dedicated to the decoration of the peptide scaf- fold during and post nonribosomal peptide synthesis [72]. Many of the on- line tailoring enzymes are descendants of the C domain. These range from epimerases to cyclization (Cy) domains for the installation of thiazoline or oxazoline rings and more exotic pictet-spenglerases and β-lactam-forming do- mains. Additionally, the A domain can be equipped with additional function- ality, for example with an integrated methylation (M) domain. While tailoring domains may exhibit substrate specificity themselves, they often entail addi- tional checkpoints in subsequent domains to ensure correct peptide processing. Examples of such logic gates include the previously discussed stereoselective C domains that follow epimerases and the TE domain responsible for release of the β-lactam-containing antibiotic nocardicin A that exclusively accepts the modified precursor [92].

Prominent examples for NRPs that are modified in trans are the glycopep- tides, arguably one of the most elaborate NRP families [93]. Formation of the characteristic, bowl-shaped aglycon of vancomycin (14), for example, requires sequential action of at least four P450 oxygenases for oxidative cross-linking of the aromatic side chains. The oxygenases are recruited by a cryptic C do- main in the terminal module and act on the NRPS-bound precursor [94]. The aromatic side chains are additionally chlorinated by flavin-dependent haloge- nases, likely after the first cross-link has been installed. Once released, the heptapeptide scaffold is further decorated by glycosylation, acylation, and/or sulfation to afford the mature antibiotics. Although such tailoring domains represent an additional hurdle for the incorporation of novel building blocks,

26 1.2. Nonribosomal peptide synthetases they also hold great potential for the diversification of NRP scaffolds. For example, trans-acting gylcosyltransferases have been shown to enable facile de- and re-glycosylation for tailoring of the glycopeptide periphery [95].

1.2.3 Structure & dynamics

Despite significant progress toward understanding how NRPS domains func- tion, their dynamic assembly into multimodular assembly lines remains elu- sive. Structures for all core domains and snapshots of unimodular assemblies have provided a model for module architecture and dynamics (Fig. 11) [96–

98]. The module core consists of the C and AN domains that share an exten- sive protein interface (>1000 A˚2) that serves as a rigid catalytic platform [96]. The two lobes of the C domain have been observed in open and closed con- formations suggesting subtle dynamics for acyl-thioester binding and release. Although not understood mechanistically, hydrophobic interactions between the C domain acceptor site and the T domain appear to govern directionality in NRPSs, as C domain removal enables elongation modules to initiate pep- tide synthesis [99]. This acceptor site and the A domain binding pocket are both accessible from the same face of the CAN platform, some 70 A˚ apart, and require significant translocation of the T domain for substrate delivery. The necessary driving force for this rearrangement is provided by rotation of the AC subdomain relative to the CAN “working bench”, making the A do- main not only a key player in module selectivity but also dynamics. While the loaded substrate is presented in the C domain, the A domain must rear- range back to its adenylation state to activate the next substrate and ensure efficient building block supply [100]. Recently, the X-ray crystal structure of the initiation module LgrA for linear gramicidin biosynthesis revealed that

27 1. INTRODUCTION

Figure 11. Module structure. X-ray crystal structure of the termination module SrfCL showing the “working bench” formed by the C (green) and the AN domain (dark blue). The

A domain is in the thiolation state with the extended AC domain delivering the T domain (orange) to the C domain acceptor site. The terminal TE domain (purple) can be seen in the background (PDB 2VSQ [96]).

even greater A domain flexibility is necessary to access additional tailoring domains [97]. In the LgrA structure, the AC subdomain assumes a novel, fully extended conformation to deliver the T domain to an N-terminal formy- lation domain. Analogous to the C domain, the formylation domain recruits the T domain via a hydrophobic patch.

Interactions across module borders have proven more recalcitrant to struc- tural elucidation. The first insights into the intermodular interactions within NRPSs were provided this year by a combination of X-ray crystallography and electron microscopy (EM) [101]. These structures show a lack of strong,

28 1.3. Biosynthetic access to novel NRPs conserved contacts between consecutive module cores pointing to a highly flexible overall NRPS architecture.

1.3 Biosynthetic access to novel NRPs

Elucidation of the principles underlying NRP biosynthesis opens the door to biosynthetic engineering to access new products that would be difficult to prepare by alternative chemical strategies. Importantly, reprogrammed pathways would allow the sustainable and scalable production of such non- natural products. As modified NRPs are genetically encoded, their properties can, in principle, be honed by Darwinian evolution.

Biosynthetic modification of NRPs can target either the peptide assembly or decoration stage. While post-NRPS enzymes are often relatively promiscu- ous and can be harnessed to engineer the periphery of a molecule, modifica- tion of the NRPS-derived core scaffold has proven more challenging. Although NRPS enzymes do not exert the same level of control as their counterparts

A CA TE A CA TE A T T T T

S S S S

NH NH NH NH 2 2 A

H N H N 2 2

Figure 12. NRPSs engineering. Module specificity can be altered by A-domain ex- change or reprogramming. This figure is adapted from [102].

29 1. INTRODUCTION in primary metabolism, precursor-directed biosynthesis [103] and mutasyn- thesis [104] strategies that aim to feed NRPS pathways with building block analogs typically only allow for modest changes. More drastic modifications of the core scaffold require engineering of NRPS assembly lines (Fig. 12).

1.3.1 Combinatorial biosynthesis of NRPs

With their modular biosynthetic logic and a large building block repertoire, NRPSs lend themselves to engineering by combinatorial module (re)assembly. Indeed, evolutionary remnants in biosynthetic gene clusters suggest that nature has harnessed pathway modularity for diversification by recombina- tion [105]. However, extracting general guidelines for the rational construction of designer assembly lines in a “plug-and-play” fashion has not been possible (yet). Although chimeric NRPSs usually suffer from low activity, diversi- fication of the daptomycin lipopeptide scaffold by Cubist Pharmaceuticals arguably represents the most successful and comprehensive effort providing important lessons for NRPSs engineering [106].

Rewiring NRPSs via COM domains. For example, replacement of the terminal bimodular synthetase in the daptomycin pathway with compatible subunits from related biosynthetic clusters afforded derivatives with novel terminal dipeptides [107]. Understanding this selective, noncovalent interac- tion of NRPSs holds great potential for combinatorial engineering. Subunit cross-talk is governed by short, C- and N-terminal COM domains that are both necessary and sufficient for communication [70]. As demonstrated for daptomycin, compatible COM domains enable the interaction of non-partner

30 1.3. Biosynthetic access to novel NRPs subunits, even from different clusters, and can therefore provide an important tool for the assembly of chimeric assembly lines [108, 109].

Molecular lego with NRPS modules. Most NRPS modules are linked co- valently like “beads on a string”. Their exchange [110], deletion [111], and/or insertion [112, 113] therefore requires identification of boundaries that allow module recombination without disrupting enzyme integrity. Often, such cut and fusion sites are arbitrarily chosen in the less conserved linker regions, guided by sequence alignments [114]. Based on lessons from daptomycin, transplantation of an A domain alone is not a promising engineering strategy, likely due to its close interaction with the adjacent C domain and/or mis- matched specificity in the C domain acceptor site. Cubist Pharmaceuticals instead relied on the exchange of entire CA(T) units between daptomycin- related pathways to generate over 120 novel lipopetide analogs [106, 115]. Nevertheless, this extensive NRPS engineering campaign was not rewarded with antibiotics superior to daptomycin. It is tempting to speculate that, while fostering combinatorial biosynthesis, the homology of the lipopeptide pathways did not provide sufficient chemical novelty beyond the lipopeptide pool that had already been sampled by nature [106].

Directed evolution of chimeric NRPSs. Given the poorly understood NRPS dynamics, defining the boundaries between functional domains is chal- lenging. As a consequence, a general solution to transplant specificity with- out sacrificing activity seems unlikely. Nature uses evolution, i.e. iterative rounds of diversification, selection, and amplification, to hone its champion catalysts. Emulation of this process on a laboratory time scale has become the most successful strategy to engineer novel biocatalysts [116]. Evolution does not require a priori assumptions about possible solutions nor an in depth

31 1. INTRODUCTION understanding of the system, making it ideal for the engineering of complex and poorly understood enzymes such as NRPSs. Indeed, directed evolution has been able to partially restore function in chimeric assembly lines result- ing from A domain swap experiments in the enterobactin and andrimid syn- thetases [117]. Despite the substitution of A domains exhibiting the same or very similar specificity, both NRPSs suffered a ∼ 30-fold reduction in product formation. Relatively small libraries generated by error-prone gene amplifi- cation sufficed to identify improved variants in bioactivity assays. Beneficial mutations were distributed throughout the A domain structure, highlight- ing the importance of distal positions and unbiased libraries that allow their identification.

Although an enticing prospect, directed evolution on a full pathway level is experimentally limited by the genetic tractability of producing organisms and a lack of strategies for efficient selection. Both previously described examples relied on the model organism Escherichia coli and plasmid-borne synthetases for library generation. While the iron-scavenging natural product enterobactin is native to E. coli, the comparably simple architecture of andrimid synthetase, which comprises multiple, lone-standing proteins, enabled heterologous recon- stitution. For the enrichment of beneficial mutations, cells encoding improved variants must display phenotypes that allow for selection. In the best case, pathway activity can be coupled to cell survival. Enterobactin, for example, becomes vital under iron limiting conditions. However, antibiotic pathways like andrimid are not essential and therefore require probing of each variant, which limits the searchable sequence space drastically.

32 1.3. Biosynthetic access to novel NRPs

1.3.2 A-domain engineering

Complementary to recombination and rescue, direct active site engineering provides a surgical alternative to afford amino acid specificities beyond the existing repertoire. In its gatekeeping function, the A domain represents the foremost engineering target for the introduction of novel building blocks. Elu- cidation of the first A domain structure [74] and deduction of the specificity conferring code encompassing eight binding pocket residues [75, 76] raised the hope that A domains could be reprogrammed by rational permutation of the nonribosomal code. Indeed, conservative changes in substrate specificity were possible (e.g. Glu → Gln / Asp → Asn) [118, 119]. However, the assumption of an isolated, transferable specificity code proved too simplistic for more gen- eral A-domain redesign, as larger changes in substrate specificity could not be achieved. Nevertheless, these eight binding pocket residues can provide a basis for directed A-domain evolution.

The unique architecture of the andrimid pathway, which utilizes a free- standing AT bidomain, and the genetic tractability of the native producer

Pantoea agglomerans have enabled in vivo engineering of the L-Val-specific AdmK A domain [120]. An AdmK knockout strain was complemented with a small, plasmid-encoded A-domain library that targeted three positions in the substrate binding pocket simultaneously and screened for production of andrimid analogs by liquid chromatography-mass spectrometry (LC-MS). To minimize the screening effort, a reduced set of amino acid substitutions was used. Still, mutant andrimid pathways were identified that efficiently incor- porate L-Leu and L-Ile instead of L-Val. Some variants even produced low amounts of Ala- or Phe-containing peptides.

33 1. INTRODUCTION

Although screening for product formation ensures A domain functional- ity in a pathway context, it is limited to specificity changes that are tol- erated by all downstream domains. In contrast, direct engineering of the A domain would allow more drastic reprogramming and can uncover addi- tional pathway bottlenecks to inform future engineering efforts. However, probing A domain catalysis is not trivial. The initial adenylation half re- action can be monitored indirectly by reversible incorporation of radioac- 32 32 tive P–pyrophosphate ( P-PPi) into ATP [121]. In a microtiter plate for- 32 mat, this P-PPi/ATP exchange assay enabled reprogramming of the L-Phe- activating initiation module TycAF from tyrocidine biosynthesis for smaller proteinogenic amino acids [122]. Limited to small A domain libraries, this as- say only allowed the interrogation of single binding pocket positions in TycA and resulted in catalysts with poor activity and selectivity.

Single point mutations can, however, be sufficient for dramatic effects on substrate specificity. Analogous screening identified a Trp-to-Ser mutation (W239S) that enlarged the substrate binding pocket favoring activation of p-substituted L-Phe derivatives, including analogs with azide or alkyne sub- stituents for “click” chemistry [123]. Importantly, the resulting variants re- tained high activity and selectivity enabling in vivo production of “clickable” diketopiperazine products.

While W239S highlights the potential of A domain engineering, genuine reprogramming of the nonribosomal code beyond single point mutations re- quires novel approaches that allow for a more thorough and facile sampling of sequence space. To this end, A domain engineering by yeast cell surface dis- play and fluorescence activated cell sorting (FACS) has been explored [124], a technology that has been extensively used for the affinity maturation of anti-

34 1.4. Aims of this thesis bodies [125]. To couple A-domain specificity to a fluorescence readout, a stable sulfamate acyl-AMP analog with a biotin handle for fluorescent labeling was used. However, only modest improvements for conservative substrate changes were possible despite the large throughput of FACS suggesting that screening for acyl-AMP binding is not a viable strategy for the directed evolution of novel A domains.

1.4 Aims of this thesis

Controlling A-domain specificity is key to the reprogramming of NRPS path- ways. Accordingly, this gatekeeper domain has been the subject of intense engineering efforts. Nevertheless, achieved changes in substrate specificity have typically been conservative and often resulted in low catalytic activ- ity [118–120, 122, 124, 126, 127]. Furthermore, engineered A domains have rarely been tested in full NRPSs obscuring any potential relevance for path- way engineering. Reprogramming of TycAF for “clickable” amino acids by a single W239S mutation represents a notable exception, as the resulting vari- ant retained high activity for a range of p-substituted analogs and functioned in a bimodular dipeptide synthetase [123]. In Chapter 2 of this thesis, we explore biosynthetic incorporation of novel functionality into NRP antibiotics via the W239S mutation. To this end, reprogrammed gramicidin S and tyro- cidine synthetases were reconstituted and tested for the incorporation of novel substrates with bioorthogonal reactivity. In Chapter 3, a high-throughput as- say for A-domain catalysis is established to allow drastic changes in substrate specificity. We used this platform to reprogram archetypal NRPS machinery for backbone-modified amino acids as challenging but powerful building blocks for the strategic modulation of NRP backbones. The resulting variants were

35 1. INTRODUCTION characterized thoroughly and downstream processing of backbone-modified amino acids was assessed in reprogrammed assembly lines.

In establishing high-throughput methodology for the reprogramming of nonribosomal peptide synthesis, this thesis provides valuable insights into NRPS malleability and represents an important step towards the ultimate goal of custom-made biosynthetic assembly lines.

36 37 Author contributions Chapter 2: The author was in- volved in all aspects of the project. Anna Camus and Ines Folger performed some experiments on the biosynthesis and modification of gramicidin S and tyrocidine A as part of their Master theses. Sophie Basler performed some kinetic and DKP assays during her semester thesis.

38 2 Installing bioorthogonal handles on nonribosomal peptide antibiotics

2.1 Introduction

Chemoselective reactions orthogonal to the functionality found in nature have become an indispensable tool for chemists and biologists alike, enabling site- specific modification of biomolecules [128]. Ideally, such transformations are not only selective, but also fast and high-yielding under physiological condi- tions — criteria that are met by the copper(I)-catalyzed “click” reaction of azides and alkynes [129, 130]. Ketones [131] and halogens [132] represent ad- ditional bioorthogonal handles for selective oxime formation with hydrazide and aminooxy reagents or palladium-catalyzed cross-coupling with alkynes, respectively. Useful transformations are not limited to selective conjugation reactions. Photoreactive groups, such as benzophenone, have become powerful tools to identify molecular targets by photo-induced cross-linking of binding partners [133]. These successes have fueled efforts to endow a wide range of biomolecules with unique reactivity.

Expansion of the genetic code has been particularly instrumental in this regard, allowing novel chemistries in peptides and proteins [134]. Site-specific incorporation of bioorthogonal amino acids into proteins has enabled myr- iad applications and is becoming increasingly important for the engineer- ing of therapeutic peptides and proteins [135]. For example, incorporating p-acetyl-L-Phe into human growth hormone allowed precise installation of

39 2. INSTALLING BIOORTHOGONAL HANDLES ON NRP ANTIBIOTICS polyethylene glycol polymers via an oxime linkage to improve serum stabil- ity [136]. Such strategies not only enable production of chemically homoge- neous therapeutic agents but are also highly scalable. In the aforementioned example, fermentation on a >1000 L scale was possible to support clinical trials.

Given that semisynthetic modification of natural products accounts for 1/5 of all approved drugs since 1981 [137], the availability of bioorthogonal han- dles would similarly offer a welcome reprieve to medicinal chemists. Unique functionality in a complex secondary metabolite allows facile diversification of the bioactive core scaffold. For example, an N-terminal azide group enabled facile “click” chemistry-based preparation and testing of teicoplanin aglycon libraries [138, 139]. Also the phase III antibiotic solithromycin is prepared by a Cu(I)-catalyzed 1,3-dipolar cycloaddition and benefits from the metabolic stability of the resulting 1,2,3-triazole ring [140]. Nonetheless, installation of bioorthogonal groups by chemical synthesis is tedious and ultimately limited to the complement of available functional groups.

In analogy to a reprogrammed genetic code, engineering of biosynthetic assembly lines promises to be a powerful and, importantly, scalable alterna- tive to total synthesis for equipping bioactive natural products with functional handles. Indeed, alkaloid and NRPS pathways can be coerced into accepting halogenated substrates to allow subsequent bioorthogonal modification [141, 142]. Engineering of gatekeeper acyltransferase domains has further enabled incorporation of alkynes into macrolide antibiotics [143, 144]. However, over- riding the inherent selectivity of natural product pathways has proven chal- lenging, typically resulting in low yields of the modified natural products.

40 2.1. Introduction

Table 1. Catalytic parameters for pyrophosphate exchange.a

-1 -1 -1 Substrate k cat (min ) K M (mM) k cat/K M (min mM )

GrsAF-A

L-Phe 300±30 0.012±0.002 25,000±3000

L-Tyr 240±70 2.3±0.3 100±20

p-Cl-L-Tyr 78±9 0.5±0.8 160±40

O-methyl-L-Tyr 54±1 15±1 3.7±0.2

O-ethyl-L-Tyr 60±10 8±2 8±4

O-propargyl-L-Tyr 30±10 18±9 2±1

p-azido-L-Phe 200±60 8.7±0.9 25±9

GrsApY-A

L-Phe 200±100 1.1±0.4 190±70

L-Tyr 230±90 0.38±0.03 600±300

p-Cl-L-Tyr 190±20 0.126±0.002 1500±100

O-methyl-L-Tyr 210±20 0.09±0.01 2300±100

O-ethyl-L-Tyr 300±100 0.0054±0.0001 50,000±20,000

O-propargyl-L-Tyr 190±70 0.030±0.005 7,000±3,000

p-azido-L-Phe 200±100 0.023±0.002 9,000±4,000

TycAF

L-Phe 300±20 0.011±0.002 27,000±8,000

O-propargyl-L-Tyr 25±5 10±3 3±2

TycApY

L-Phe 260±10 2.6±0.3 100±20

O-propargyl-L-Tyr 280±10 0.33±0.03 800±100

a This table is adapted from [145].

41 2. INSTALLING BIOORTHOGONAL HANDLES ON NRP ANTIBIOTICS

We recently identified a single tryptophan-to-serine mutation (W239S) in the L-Phe-specific GrsAF-A domain as a promising gateway for incorporation of “clickable” amino acids into NRPs (Table 1) [123]. GrsAF is the initia- tion module of gramicidin S synthetase and contains an additional E domain for incorporation of D-Phe into the decapeptide antibiotic. In the GrsAF-A domain, a hydrophobic substrate binding pocket with excellent shape com- plementarity to L-Phe forms the basis for high substrate specificity [74]. By enlarging the bottom of this recognition pocket, the W239S mutation enabled efficient activation of a range of p-substituted L-Phe analogs, in particular the

“clickable” amino acids O-propargyl-L-Tyr and p-azido-L-Phe. However, the p-substituted amino acids were not efficiently processed by the downstream domains resulting in slow incorporation of the L-enantiomers. The W239S mu- tation was therefore introduced into the homologous initiation module TycAF from tyrocidine biosynthesis where it enabled efficient incorporation of O- propargyl-L-Tyr into a cyclic dipeptide. Here we show that implementation of the W239S mutation on a pathway level provides an efficient strategy for imparting NRPs with diverse functionalities.

2.2 Results

Mapping the substrate scope of TycApY. To map the potential of the expanded substrate binding pocket of TycApY to accommodate amino acids with bioorthogonal handles, we determined adenylation ki- 32 netics for a set of p-substituted L-Phe analogs using a P-PPi/ATP ex- change assay (Table 2) [121]. TycApY exhibited a catalytic efficiency of

700±150 min-1mM-1 for the “click” amino acid O-propargyl-L-Tyr (27) in accordance with the previously reported value [145]. Beyond O-propargyl-

42 2.2. Results

Table 2. Substrate scope of TycApY. Michaelis-Menten parameters for TycAF (Trp239) or TycApY (Ser239), respectively, with a set of p-substituted Phe analogs determined with 32 a a P-PPi/ATP exchange assay [121].

b Substrate TycA k cat K M k cat/K M Selectivity (min-1) (mM) (min-1mM)

Trp239 300±20 0.011±0.002 27,000±8,000 1.0 x 100 26c

Ser239 260±10 2.6±0.3 100±20 1.0 x 100

Trp239 4.5±0.7 2.1±0.1 2.2±0.5 8.0 x 10-5 27

Ser239 325±4 0.5±0.1 700±150 7.0 x 100

Trp239 180±10 0.8±0.4 240±100 9.0 x 10-3 28

Ser239 350±20 0.058±0.001 5,800±200 6.0 x 101

Trp239d 30 0.4 80 2.8 x 10-3 29

Ser239 370±50 0.025±0.004 14,900±400 1.5 x 102

Trp239 190±20 18±1 10.74±0.06 4 x 10-4 30

Ser239 280±10 2.4±0.2 120±7 1.2 x 100

Trp239 0.8±0.2 2±1 0.35±0.09 1.3 x 10-5 31

Ser239 170±50 1.236±0.008 140±40 1.4 x 100

a Errors are given as the standard deviation of two independent measurements. b c k cat/K M (target substrate) / k cat/K M (L-Phe). Values are taken from [145]. d Experiment was only performed once.

43 2. INSTALLING BIOORTHOGONAL HANDLES ON NRP ANTIBIOTICS

L-Tyr (27), the tested substrates set included p-bromo-L-Phe (28) and p-iodo-L-Phe (29) for palladium-catalyzed cross-coupling reactions [132], p-acetyl-L-Phe (30) for oxime conjugation [131], as well as the photoreac- tive amino acid p-benzoyl-L-Phe (31) [133]. Similar to W239S GrsAF-A,

TycApY was found to exhibit broad substrate tolerance for the p-substituted

L-Phe analogs with highest catalytic efficiencies for substituents of intermedi- ate size, such as p-bromo-L-Phe (28, 5,800 min-1mM-1) and p-iodo-L-Phe (29, -1 -1 15,000 min mM ). The high selectivity of TycApY for these substrates is especially notable for in vivo applications in the presence of competing endogenous amino acids. Also the sterically more challenging substrates p-acetyl-L-Phe (31) and p-benzoyl-L-Phe (31) were efficiently activated by

TycApY, albeit with higher K M values. Importantly, the k cat values for all tested substrates were ∼2 orders of magnitude above the rates for DKP for- mation and should therefore not be rate limiting for peptide formation. Incor- porated into NRPs, the bioorthogonal handles would facilitate semisynthetic tailoring or, in the case of p-benzoyl-L-Phe (31), target elucidation by photo- induced cross-coupling.

E and C domain tolerance for p-substituted L-Phe analogs. For ef- ficient incorporation into peptides, however, the bioorthogonal amino acids must be processed by downstream domains. Initial peptide bond formation can be assessed in a DKP assay by pairing TycAF with the acceptor module

TycB1P (Fig. 13a) [146]. L-Phe is loaded and epimerized by TycAF and subse- quently undergoes peptide bond formation with the downstream L-Pro residue activated by TycB1P. The high propensity of the resulting L-Pro-containing dipeptide thioester for cyclization results in spontaneous offloading of a DKP product.

44 2.2. Results

To test E and C domain tolerance for L-Phe analogs with bioorthogonal handles, TycB1P was paired with TycApY and supplemented with ATP, L-Pro, and the respective amino acid substrate (Fig. 13b). As previously reported,

O-propargyl-L-Tyr was efficiently incorporated into the respective DKP [123]. Product formation was monitored by HPLC and quantified by substrate de- pletion as no significant side products were apparent. The thus calculated k obs of 0.7 min-1 corresponds well to the published value (0.83±0.07 min-1). All other tested amino acids were similarly incorporated into the respective DKPs.

However, k obs values decreased with increasing size of the substituent, ranging from 1.7 min-1 for p-bromo-L-Phe (28) to 0.3 min-1 for p-benzoyl-L-Phe (31).

A notable exception to this trend was p-acetyl-L-Phe (30) with a k obs of

a b TycA X TycB1 P 3 TycA F

TycA pY

2 ) -1

SH S (min

L-Phe (analog) obs k L 1 -Pro O ATP NH N 2 O O R 0 NH N R = H Br I O O O

O R

Figure 13. Downstream processing of amino acids with bioorthogonal sub- stituents. a, To test peptide bond formation, TycApY (green) was paired with TycB1P in HEPES buffer, pH 8, supplemented with ATP, L-Pro, and the respective amino acids. b,

Initial rates of DKP formation with TycAF (blue) or TycApY (green), respectively, mea- sured after 30 min at 37 ◦C. DKP formation was quantified by amino acid depletion as no significant amounts of side products were formed. The k obs for TycAF and L-Phe (26) corresponds well to the published value of 1.4±0.2 min-1 [145].

45 2. INSTALLING BIOORTHOGONAL HANDLES ON NRP ANTIBIOTICS

2.6 min-1 despite the comparably bulky substituent. These results show that while the E and C domains impose steric constraints, diverse p-substituents are tolerated.

Reprogramming gramicidin S biosynthesis. The modularity of NRPSs allows pairing of disparate subunits by compatible COM-domains. Tetramod- ular GrsBPVOL, for example, contains a COM-domain that should enable cross-talk with TycAF [108]. Natively, GrsBPVOL interacts with the initiation module GrsAF to assemble two pentapeptides, which are dimerized and cy- clized on the terminal GrsTE domain to give the cyclic decapeptide antibiotic gramicidin S (32). Substitution of GrsAF with TycApY could therefore enable incorporation of bioorthogonal handles into 32.

For in vitro reconstitution of TycApY with GrsBPVOL, we cloned the gene encoding GrsBPVOL by Gibson assembly [147] from the natural producer Aneurinibacillus migulanus into an IPTG-inducible vector under the control of a strong Ptrc promoter [148, 149]. The resulting plasmid encoding a C- terminal His6-tag was transformed into E. coli strain HM0079, genomically encoding Sfp 4’-phosphopantetheine transferase from Bacillus subtilis [149]. Indeed, the enormous protein (510 kDa) could be produced recombinantly and purified by NiNTA affinity chromatography to enable in vitro studies (Fig. 14).

In vitro gramicidin S formation was previously shown by combining frac- tionated protein precipitates from the natural producer Aneurinibacillus migu- lanus [150]. When we paired recombinantly produced GrsBPVOL with GrsAF in bis-Tris buffer, pH 8, supplemented with amino acid substrates and ATP, 32 -1 was produced with a k obs of 1.7±0.4 min after a short lag time of ∼10 min.

46 2.2. Results

M1 234M 4 5 6

kDa kDa

220 220 170 170

116 116

76 76

Figure 14. NRPS reconstitution. SDS-PAGE analysis of gramicidin S and tyrocidine synthetase subunits after NiNTA (M: marker, 1: TycApY, 2: GrsBPVOL, 3: TycBPFF, 4:

TycCNQYVOL, 5: TycBPFpY, 6: TycAF).

Increasing concentrations of GrsAF had no effect on the rate of product for- mation, indicating GrsBPVOL to be rate determining.

Reconstitution of gramicidin S biosynthesis allowed us to test TycApY specificity in the context of a full synthetase. When supplemented with p-substituted Phe analogs, gramicidin S derivatives were produced by the engineered synthetase as determined by HPLC and LC-MS analysis (Fig. 15). Besides the dimerized and cyclized decapeptide, we also observed formation of shunt products, mainly DKP and hydrolyzed pentapeptide. The ratio of modified full-length gramicidin S to shunt products decreased markedly with increasing size of the substituent. For DKP formation, the amount of product formed depended to a lesser extent on the size of the substituent, suggest- ing steric problems in the GrsTE domain. Due to this size constraint, we also tested the smaller substrate p-chloro-L-Phe (33), which was efficiently incorporated into the corresponding gramicidin S analog 34. When E. coli

HM0079 cultures co-producing TycApY and GrsBPVOL were supplemented with p-chloro-L-Phe, we could detect formation of 34 by LC-MS analysis of

47 2. INSTALLING BIOORTHOGONAL HANDLES ON NRP ANTIBIOTICS

substrate 30 * ** 27 29 28 33 26

3 4 5 retention time (min) 38

R O NH 2 37 O

O O H H 36 N N I N N H H O O O N 35 Br N O O O H H N N N N 34 H H Cl O O 32 H H2N R

Figure 15. Biosynthetic modification of gramicidin S (32). HPLC chromatograms for the in vitro biosynthesis of gramicidin S (32) and analogs (34-38). When the engi- neered synthetase consisting of TycApY and GrsBPVOL was supplemented with L-Phe (26), p-chloro-L-Phe (33), p-bromo-L-Phe (28), p-iodo-L-Phe (29), O-propargyl-L-Tyr (27), or p-acetyl-L-Phe (30), the respective gramicidin S analogs (**) were produced. However, the ratio of cyclized product to hydrolyzed pentapeptide and DKP side products (*) decreased with increasing size of the substituent suggesting steric problems in the terminal GrsTE domain. The identity of the gramicidin S analogs was confirmed by LC-HRMS (Appendix Fig. 32).

the crude cell lysate (Appendix Fig. 33), demonstrating the potential of the reprogrammed gramicidin S synthetase for in vivo applications.

48 2.2. Results

Biosynthetic access to tyrocidine A (11) analogs. Although the re- programmed TycApY/ GrsBPVOL synthetase afforded modified gramicidin S analogs, GrsTE specificity for the initial amino acid [151] proved limiting for

L-Phe analogs possessing larger substituents. Conceivably, such modifications would be better tolerated at internal positions of the peptide that are not subject to the same level of scrutiny by the TE domain [90]. TycB3F from ty- rocidine biosynthesis, for example, represents a tempting engineering target, as it encodes the fourth position of tyrocidine A (11), which can be sub- stituted to improve therapeutic properties [91, 152–155]. Furthermore, the

A-domain recognition pocket of TycB3F is delimited by a Trp residue making it amenable to the W239S mutation.

Given the remarkable ability of E. coli to produce GrsBPVOL, we set out to reconstitute the even larger tyrocidine pathway, which encompasses the initiation module TycAF (120 kDa), trimodular TycBPFF (410 kDa), and hexamodular TycCNQYVOL (730 kDa). The genes encoding TycBPFF and

TycCNQYVOL were cloned by Gibson assembly [147] from the natural producer Brevibacillus parabrevis and introduced into two separate IPTG-inducible vec- tors under the control of strong Ptrc promoters [149, 156]. Indeed, recom- binant production of the resulting constructs with C-terminal His6-tags in E. coli strain HM0079 and subsequent NiNTA purification afforded the enor- mous subunits (Fig. 14).

To test incorporation of p-substituted Phe analogs, we assembled two engi- neered tyrocidine A synthetases with the W239S mutation either in the first or fourth module and monitored product formation by LC-MS (Fig. 16, see 5.2). In general, fewer side products were observed for the reprogrammed tyroci- dine synthetases. With the TycApY initiation module, p-bromo-L-Phe (28)

49 2. INSTALLING BIOORTHOGONAL HANDLES ON NRP ANTIBIOTICS

R OH OH

NH 3 NH 3

O O O O H H H H N N N N N N N N O H H O H H O O NH O O NH N O O N O O H H O H H O N N N N N N N N H H H H O O O O O O O O H N H N 2 NH 2 2 NH 2 Phe R Phe Tyr Tyr bAA bAA EIC areatargetcompoundEIC for areatargetcompoundEIC for n.a. n.a.

26 28 29 27 31 26 28 29 27 31

R = H Br I O O R = H Br I O O R R NH 2 NH 2

HOOC HOOC

Figure 16. Reprogramming tyrocidine biosynthesis. In vitro reconstitution of the engineered tyrocidine pathways TycApY/ TycBPFF/ TycCNQYVOL (left) and

TycAF/ TycBPFpY/ TycCNQYVOL (right) with the W239S mutation in the first or fourth module, respectively, enabled efficient biosynthesis of the corresponding tyrocidines when supplemented with the native substrate L-Phe (26) or the halogenated analogs p-bromo-L-Phe (28) and p-iodo-L-Phe (29). For the more challenging substrates O- propargyl-L-Tyr (27) and p-benzoyl-L-Phe (31), bypassing of TycTE specificity by sub- stitution at the fourth position resulted in better incorporation. TycAF/ TycBPFpY/

TycCNQYVOL, in particular, incorporated the bioorthogonal amino acids (bAAs) selec- tively into the respective tyrocidines (green) with little formation of wild type (blue) or L-Tyr-containing (gray) tyrocidine A (11). Data represent the average peak area of the elected ion chromatogram (EIC) for the target compounds where n = 2 (n. a. = not applicable). For EICs see Appendix 5.2. 50 2.2. Results was incorporated efficiently but not very selectively, as significant amounts of tyrocidine A (11) and a Tyr-containing analog were also formed. Unlike the reprogrammed gramicidin S synthetase, TycApY/ TycBPFF/ TycCNQYVOL also efficiently incorporated p-iodo-L-Phe (26) and O-propargyl-L-Tyr (27) and only encountered problems with p-benzoyl-L-Phe (31), suggesting higher flexibility of the TycTE domain. The full potential of the W239S mutation, however, was revealed when it was introduced into the TycB3F-A domain. All tested substrates were efficiently incorporated into the respective tyroci- dine analogs with low incorporation of competing L-Phe and L-Tyr, even for p-benzoyl-L-Phe (31).

Biosynthesis of the “clickable” tyrocidine A analog 39 was scaled up for isolation affording the pure compound. We harnessed the bioorthogonal han- dle of 39 for derivatization with the commercial fluorophore sulfo-Cy5 and a Zn(II)-chelator to give tyrocidine conjugates 40 and 41, respectively (Fig. 17). Modification with the Zn(II)-chelator has been shown to improve the thera- peutic properties of vancomycin (14) by targeting the antibiotic to bacterial cell walls at inflammatory sites rich in Zn(II) ions [62]. Conceivably a tar- geting group could similarly improve the selectivity of tyrocidine A (11) to remedy its hemolytic effect. Biological testing of tyrocidine analog 41 is under- way. While preliminary, these results showcase the potential of bioorthogonal handles for the tailoring of NRP scaffolds.

51 2. INSTALLING BIOORTHOGONAL HANDLES ON NRP ANTIBIOTICS

NH 3

O O H H N N OH N N O H H O O NH N O O H H O N N N N H H O O O O H N 2 NH 2 O labeling 39

NH 3 targeting O O H H N N OH N N O H H O O NH NH 3 N O O H H O N N N H O O O O H H OH O N N O N N O H H H N O O NH 2 NH 2 N O O H H O N O N N N N N N H H H N O O O O 40 O H N O 2 NH 2 O O O S O N N S O N O N H 41 N N N N N O O

S O O

Figure 17. “Click” functionalization of tyrocidine analog 39. Cu(I)-catalyzed Huisgen cycloaddtion harnessing the propargyl group of 39 enables facile modification of the tyrocidine scaffold as illustrated by the preparation of fluorescent derivative 40 and analog 41, which has an appended Zn(II)-chelator that reportedly targets bacterial cell walls [62].

2.3 Discussion

As gatekeepers to nonribosomal peptide synthesis, A domains continue to be a prime target for biosynthetic engineering. The W239S mutation provides a surgical and powerful means to reprogram A domains for a broad range of

52 2.3. Discussion chemical functionality — notably without appreciable loss of activity. How- ever, the impact of amino acid substitutions on a pathway level was not clear at the outset of this study. To address these issues, we reconstituted the entire gramicidin S and tyrocidine synthetases in vitro providing unique op- portunities for dissecting NRPS catalysis in vitro. The ease with which these gigantic enzymes could be produced in E. coli was totally unexpected and might also foster efforts to elucidate the multimodular structure of NRPSs by X-ray crystallography and cryo-electron microscopy.

Overall, our results showcase the remarkable malleability of the grami- cidin S and tyrocidine pathways. The ability of the TycB2F-C domain to pro- cess unnatural substrates is particularly notable, as C domains are reported to exert stricter specificity at the acceptor site [82, 83]. Efficient reprogramming of not only initiation, but also elongation (and termination) modules is key for full control over natural product composition. Nevertheless, incorporation of large modifications at the initial peptide positions proved problematic, un- derscoring the importance of TE selectivity [90, 151]. Given the ability of TycTE to produce gramicidin S [90], it might provide a more robust alter- native for replacement of the GrsTE domain. In general, however, a better understanding of TE structure and function is needed to guide future engi- neering efforts. SNAC-based LC-MS assays could be exploited as a screening platform to eliminate potential steric roadblocks by mutagenesis [90].

While in vitro reconstitution is ideal for characterizing and engineering NRPSs, the biosynthetic potential of reprogrammed pathways lies in the sus- tainable and scalable fermentation of designer products [64]. The biosyn- thesis of halogenated gramicidin S analogs in the heterologous host E. coli HM0079 demonstrates that in vivo implementation of the W239S mutation

53 2. INSTALLING BIOORTHOGONAL HANDLES ON NRP ANTIBIOTICS is possible. Scalable access represents a prerequisite for combinatorial diver- sification and screening of natural products. Despite their hemolytic activity, amphiphilic peptides such as gramicidin S and tyrocidine represent interest- ing scaffolds with potent bioactivities against a broad range of bacteria and a general mechanism of membrane disruption reducing the risk of antimicrobial resistance [157]. Efforts to endow the amphiphilic β-sheet of tyrocidine with better selectivity for bacterial membranes have identified Phe4 as a hotspot for reducing hemolytic activity [91, 152–155]. Fermentation of “clickable” ty- rocidine analog 39 could enable combinatorial optimization to reinvigorate one of the first reported antibiotics.

The W239S mutation is not limited to PheA domains in gramicidin S and tyrocidine. Many Phe-activating A domains contain a Trp residue at position

H2N O Natural product Nonribosomal code HN O O NH O tyrocidine A A W T I A A I C NH O O A W T I A G V C O N NH O gramicidin S A W T I A A I C O O thaxtomin A W T V A A V C O NH 3 glycopeptidolipid A W T A V A I C NH barbamide A W T V A A V C O NH 3 O HN HN virginiamycin A W T V A A V C N mannopeptimycin G W T G A I L S O O HN OH aureobasidin A A W V L A G I Q O HN O N-Me-D-Phe 5 NH OH A W V L A G I Q O lotilibcin A W T I A A V C lotilibcin (42 ) OH NH 2

Figure 18. Trp239 — a recurring theme. Representative L-Phe-specific A domains containing Trp239 including the fifth A domain from lotilibcin synthetase. The nonriboso- mal code specifies the amino acids at position 236, 239, 278, 299, 301, 322, 330, and 331, respectively. This table is adapted from [123].

54 2.3. Discussion

239 [123], including the fifth A domain in lotilibcin synthetase. Lotilibcin (42) is a cyclic depsipeptide ten times more active against MRSA infections than vancomycin (14) and currently in phase I clinical trials (Fig. 18) [158]. The introduction of novel functionality is also not limited to the W239S mutation. In Chapter 3, we present a high-throughput screening platform that should allow efficient reprogramming of A-domain specificity for any target. General strategies to equip NRPs with bioorthogonal reactivity can be expected to greatly enhance our ability to characterize and modulate this important class of therapeutics.

55 Author contributions for Chapter 3: The author was involved in all aspects of the project. Dr. Douglas A. Hansen performed chemical synthesis of substrates and au- thentic standards and optimized the production of β-amino acid-containing peptides. Dr. Takahiro Mori performed crys- tallography. Some initial yeast surface display experiments were performed by David Fercher as part of his semester thesis.

Engineering of TycAF for N -propargyl-L-Phe was performed with Ines Folger as part of her Master thesis. Anna Camus helped with initial testing of N -methyl-Phe-incorporation.

56 3 Biosynthesis of backbone-modified peptides

3.1 Introduction

Despite outstanding biological activities, peptides generally make poor drug scaffolds due to proteolytic degradation, low membrane permeability, and high flexibility that renders target binding entropically unfavorable [159]. Incorpo- ration of backbone-modified amino acids can remedy such shortcomings while preserving side-chain display, making them privileged building blocks for the synthesis of therapeutic peptides [160–163]. It is therefore not surprising, that nature has learned to harness backbone modifications for peptidic secondary metabolites. Seven out of the eleven peptide bonds in cyclosporin A (43), for instance, are N -methylated (Fig. 19) [164]. Also, many NRPs con- tain D-configured amino acids as illustrated by the two D-Phe residues in tyrocidine A (11), which are crucial for proper formation of the amphiphilic β-sheet structure.

For synthesis of backbone modifications, NRPSs contain auxiliary domains responsible for online modification of proteinogenic precursors. For exam- ple, cyclosporin A synthetase contains seven M domains to transfer methyl groups from S-adenosylmethionine cofactors to loaded amino acids prior to peptide bond formation. More rarely, microbes also use β-amino acids as integral building blocks in bioactive secondary metabolites, in particular in PKS-NRPS hybrid pathways [165]. Unlike other backbone modifications, β- amino acids are generated before A-domain selection by dedicated mutases

57 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES containing either 4-methylidene-5-imidazole-5-one or pyridoxal phosphate / adenoysl-B12 cofactors for the interconversion of α- and β-amino acids. Ac- cordingly, A domains must have evolved to selectively recognize the expanded backbone of β-amino acids. First insights were provided by X-ray crystal structures of free-standing A domains from the vicenistatin family of polyke- tide macrolactams [166]. Overall, these A domains exhibit similar substrate recognition modes as their α-counterparts, including conserved Asp and Lys residues. However, the side-chain binding pockets are characterized by a one amino acid shorter loop connecting β-sheets 13 and 14 (β13β14 loop) and large, hydrophobic residues on the opposite side of the pocket [167].

Reprogramming A domains for backbone-modified amino acids represents a potentially powerful strategy to modulate the structure and properties of

O O H H N N N N NH O O O O N O O O N N N N N H HO O O

cyclosporin A ( 43 )

Figure 19. Backbone methylation in cyclosporin A (43). Architecture of cyclosporin A synthetase comprising seven M domains (orange) and the molecular structure of 43 [164, 168].

58 3.2. Results

NRP antibiotics. However, such drastic changes in substrate specificity are well beyond the scope of current methodology. Here, we establish a high- throughput platform for NRPS engineering to emulate the possible natu- ral evolution of β-A domains and use it to reprogram the archetypal initi- ation module TycAF for (S)-β-Phe. Furthermore, we explore M domain- independent loading of N -alkylated amino acids. When the engineered cat- alysts are paired with downstream modules, backbone-modified peptides are produced efficiently in vitro and in vivo, highlighting the potential of biosyn- thetic pathway engineering for the construction of novel nonribosomal frame- works.

3.2 Results

A high-throughput assay for adenylation and thioesterification.

TycAF exhibits low promiscuous adenylation activity for (S)-β-Phe, but a

200:1 preference for its native substrate L-Phe (Table 3). Because previous attempts to alter this substrate specificity in favor of (S)-β-Phe by computa- tional engineering and microtiter plate screening of small libraries were unsuc- cessful [145], we established a high-throughput assay for both A-domain cat- alyzed reactions — adenylation and thioesterification — to sample sequence space more effectively. To that end, a minimal TycAF module (lacking the E domain) was displayed on the surface of yeast cells to enable screening by fluorescence activated cell sorting (FACS) (Fig. 20a) [125]. Flow cytometry after fluorescein isothiocyanate (FITC)-based immunofluorescence labelling of a C-terminal c-myc tag showed that minimal TycAF was produced and dis- played by yeast strain EBY100 (Fig. 20b). The displayed T domains were subsequently primed with the ppant arm using Sfp 4’-phosphopantetheinyl

59 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES transferase and coenzyme A (CoASH). To link A-domain catalysis to a fluores- cent readout, we used the previously identified point mutant TycApY [123] to incorporate O-propargyl-L-Tyr. The alkyne moiety allowed coupling of an R- phycoerythrin (R-PE) fluorophore via bioorthogonal “click” chemistry to label yeast cells encoding active TycA variants [129, 130]. Upon incubation of yeast cells with equimolar amounts of L-Phe and O-propargyl-L-Tyr, flow cytome-

a SH b CoASH 10 4 Sfp

10 3

yeast EBY100 O 0 aaloading a.u.) / (R-PE S 0 10 3 10 4

NH 2 surface display (FITC / a.u.) O

c

10 4

O N N 10 3 N S

NH 2 O 0 aaloading a.u.) / (R-PE

0 10 3 10 4 surface display (FITC / a.u.)

Figure 20. A high-throughput assay for adenylation and thioesterification. a, Schematic representation of the assay. Minimal modules (blue) were displayed on the surface of yeast cells [125] and post-translationally modified with a ppant arm using Sfp. Display was quantified by immunofluorescent FITC (green) labeling of a C-terminal c-myc epitope tag. The cells were incubated with the “clickable” O-propargyl-L-Tyr and ATP and active variants were labeled with an R-PE fluorophore (orange) by a Cu(I)-catalyzed azide-alkyne Huisgen cycloaddition [129, 130]. b, Flow cytometry of yeast EBY100 cells [125] displaying minimal TycAF (lacking the E domain) loaded with an equimolar mixture (20 µm each) of L-Phe and O-propargyl-L-Tyr. A typical sorting gate is indicated by the orange square. c,

Flow cytometry of identically treated yeast EBY100 cells displaying minimal TycApY

60 3.2. Results

try of TycApY-displaying cells showed a signal for both fluorophores (FITC and R-PE), indicating display and amino acid loading (Fig. 20c), whereas cells displaying wild-type TycAF only exhibited FITC fluorescence (Fig. 20b).

Reprogramming TycA for (S)-β-Phe. To demonstrate the feasibility of reprogramming NRPS modules for backbone-modified building blocks, we set out to engineer TycAF for (S)-β-Phe using VinNMD as a template. The high throughput of FACS allowed simultaneous randomization of several active site positions. Accordingly, we replaced the tetrapeptide Thr328-Ser329-Ile330-

Cys331 in the β13β14 loop of TycApY with a fully randomized tripeptide. Additionally, Ala236, adjacent to the conserved residue Asp235, was random- ized to allow for structural adjustments opposite the loop [167]. The result- ing library of ∼106 variants was screened for O-propargyl-(S)-β-Tyr-loading in the presence of competing 4-methoxy-L-Phe, affording significant enrich- ment in β-amino acid-specific TycA variants (Fig. 21a, b). Of ten sequenced variants, all contained an A236V mutation (Fig. 22). The fully randomized β13β14 loop converged to a relatively conserved Xaa-Leu-Val motif (where Xaa stands for Ala, Thr, Cys, Val, or Leu) with the greatest variability in the first position, farthest from the active site.

We chose variant TycAβpY, with the A236V mutation and a Cys-Leu- Val β13β14 loop sequence, in addition to the auxiliary W239S substitution, for further characterization (Fig. 21c, Table 3). Steady-state kinetics for the adenylation half-reaction determined by a pyrophosphate exchange assay [121] showed that these changes inverted the substrate preference in favor of the β- amino acid, increasing the apparent second-order rate constant (k cat/K M) for

O-propargyl-(S)-β-Tyr 330-fold and concomitantly decreasing k cat/K M for

O-propargyl-L-Tyr 380-fold. Subsequent reversion of the W239S mutation

61 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES

a b 10 5 15000 330 ) -1 10 4 mM 10000 220 -1 OH H2N 26 (min M 5000 110 O K 3 / 10 cat

k O 0 0 0 26 27 44 45 26 27 44 45 aaloading a.u.) (R-PE/

0 10 3 10 4 10 5 TycA W239S TycA F pY OH surface display (FITC / a.u.) H2N 27 O FACS O

c 105 S239W TycA βF TycA βpY O 4 3000 330 10 )

-1 H2N 44 OH

mM 2000 220 -1 10 3 (min M 1000 110 0 K aaloading a.u.) (R-PE/ / O cat k 0 0 0 10 3 10 4 10 5 H N OH 26 27 44 45 26 27 44 45 2 45 surface display (FITC / a.u.)

d pY F F pY β β

TycA TycA TycA TycA kDa kDa NiNTA FPLC 220 220 170 170 116 116 76

76 53

Figure 21. Engineering of TycAF. a, Engineering strategy for TycAβF with adenyla- tion kinetics from Table 3. b, Flow cytometry of a yeast cell library encoding ∼106 TycA variants loaded with 100 µM racemic O-propargyl-β-Tyr. Using FACS, the library was enriched in doubly-labeled yeast cells (orange square, 1.5% of total population) encoding TycA variants that selectively load β-amino acids. c Flow cytometry of the enriched yeast library loaded with 1 µM O-propargyl-(S)-β-Tyr in the presence of 1 mM 4-methoxy-L-Phe after three rounds of FACS (42.2% doubly-labeled cells). d, SDS-PAGE analysis of all four TycA variants after NiNTA affinity chromatography (left) and FPLC (MonoQ) purification of TycAβF (right).

62 3.2. Results

afforded variant TycAβF, which exhibited a 220:1 preference for (S)-β-Phe over L-Phe. Notably, the 40,000-fold change in α/β-specificity relative to

TycAF was achieved while maintaining high catalytic efficiency. The steady- -1 state parameters for this variant (k cat = 71±8 min , k cat/K M = 2,400±500 mM-1 min-1) are only two and fourfold lower, respectively, than the values determined for the wild-type catalyst towards L-Phe (Table 3).

Table 3. Adenylation steady-state parameters. Michaelis-Menten parameters for the 32 a four TycA variants determined with a P-PPi/ATP exchange assay [121].

TycAF TycApY TycAβpY TycAβF

b k cat 142±34 95±39 n.d. 29±4 (min-1) b L-Phe (26) K M 0.013±0.001 2.3±1.3 n.d. 2.6±0.3 (mM)

k cat/K M 11,100±2,600 39±11 0.009±0.007 11±1 (min-1mM-1)

b b b k cat n.d. 132±21 n.d. n.d. (min-1) b b b O-propargyl- K M n.d. 0.6±0.2 n.d. n.d. L-Tyr (27) (mM)

k cat/K M 0.5±0.2 228±87 0.6±0.3 0.76±0.09 (min-1mM-1)

b b k cat n.d. 0.6±0.4 31±4 n.d. (min-1) b b O-propargyl- K M n.d. 1.8±0.9 0.19±0.02 n.d. (S)-β-Tyr (44) (mM)

k cat/K M 0.12±0.07 0.5±0.6 167±31 0.5±0.1 (min-1mM-1)

b k cat 1.7±0.5 1.4±0.4 n.d. 71±8 (min-1) b (S)-β-Phe (45) K M 0.03±0.01 4.7±1.5 n.d. 0.030±0.005 (mM)

k cat/K M 54±14 0.2±0.1 4.2±0.6 2,400±500 (min-1mM-1)

a Data are reported as the mean ± standard deviation of three separate measurements with different batches of protein. b Not determined due to the absence of substrate saturation.

63 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES

**** TEQDRIGLFASMSFDASVSEMFMALLSGASLYILSKQTIHDFAAFEHYLSENELTIITLPPTYLT TycApY ...... V...... variant1 ...... V...... variant2 ...... V...... variant3 ...... V...... variant4 ...... V...... variant5 ...... V...... variant6 ...... V...... variant7 ...... V...... variant8 ...... V...... variant9 ...... V...... variant10 230 240 250 260 270 280

***** HLTPERITSLRIMITAGSASSAPLVNKWKDKLRYINAYGPTETSICATIWEAPSNQLSVQ TycApY ...... ALV-...... variant1 ...... LV-...... variant2 ...... TV-...... variant3 ...... CLA-...... variant4 ...... CTV-...... variant5 ...... CLV-...... variant6 ...... VLV-...... variant7 ...... VLV-...... variant8 ...... LLV-...... variant9 ...... LLV. -...... variant10 290 300 310 320 330 340

Figure 22. Sequences of selected O-propargyl-β-Tyr-specific TycA variants. Se- quence alignment of TycApY and ten variants obtained after three rounds of FACS screening for loading of O-propargyl-β-Tyr. Engineered positions are boxed while matching residues are abbreviated with dots. Binding pocket residues according to Stachelhaus [75] and Chal- lis [76] are highlighted with asterisks.

Structural analysis of the α/β-switch. To understand the molecular basis for the altered specificity, we determined X-ray crystal structures of the C- terminally truncated A domains of TycAβpY (TycAβpY-AN, PDB 5N81) and

TycAβF (TycAβF-AN, PDB 5N82) to 1.6 A˚ and 1.7 A˚ resolution (Table 7). The proteins were complexed with their cognate nonhydrolyzable aminoacyl-

AMP analogs 46 (TycAβF-AN) and 47 (TycAβpY-AN) (Fig. 23a,b). The backbones of TycAβpY-AN and TycAβF-AN are nearly superimposable with a root-mean square deviation (r.m.s.d) of 0.4 A˚ for all Cα-atoms and closely resemble those of the homologous GrsA and VinN A domains (r.m.s.d. values of 0.9 and 2.0 A,˚ respectively) [74, 167].

64 3.2. Results

a d

b NH 2 N NH 2 O O O N S N O N N H O R OH OH R = 5 6 H O

c e

D235 D235

W239 S239

Figure 23. Structural analysis of the engineered β-A domains. a, X-ray crystal structure of the N-terminal subdomain TycAβF-AN (PDB 5N82), comprising residues 1-

427, in complex with 46 (yellow sticks). The F O-F C omit map for the ligand is contoured at 3 σ. b, Nonhydrolyzable aminoacyl-adenylate analogs 46 and 47 used for co-crystallization with the engineered β-A domains TycAβF-AN and TycAβpY-AN, respectively. c, Cut- away view of the active sites of TycAβF-AN (left) and TycAβpY-AN (right, PDB 5N81) complexed with 46 and 47 (yellow sticks), respectively. The invariant residue Asp235 and the auxiliary position Trp/Ser239, used to enlarge the binding pocket, are shown as green sticks. d, Superimposed (top) and individual (bottom) structures of TycAβF-AN (green) and GrsA-A (blue) [74] with their respective ligands 46 (yellow, showing only the β-amino acid moiety of the ligand) and L-Phe (blue). Engineering of position 236 and β13β14 (positions 328-331) resulted in a reshaped substrate binding pocket that accommodates the rotated aryl group of 46. e, Notably, the β13β14 loops in TycAβF-AN (green) and VinN (white) as well as ligand 46 (yellow) and (2S,3S)-3-methyl-Asp (gray) are nearly superimposable.

65 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES

The β-aminoacyl-AMP analogs 46 and 47 adopt identical binding modes at the TycAβF-AN and TycAβpY-AN active sites, respectively, with the AMP moiety bound in the conserved A domain nucleotide pocket and the appended β-amino acid docked in the remodeled substrate recognition site. The latter is hydrophobic and exhibits excellent shape complementarity to the aryl group

(Fig. 23c). The W239S mutation at the bottom of the TycAβpY-AN pocket 3 enlarges the recognition site by 220 A˚ compared to TycAβF-AN, providing space for the propargyl substituent used for screening. The β-amine of (S)- β-Phe engages in a conserved salt bridge with Asp235, which orients it syn- periplanar to the sulfamate NH. Analogous placement of the amine within hydrogen bonding distance of the phosphate leaving group during catalysis might facilitate acyl transfer from AMP to the ppant prosthetic group. To- gether with the A236V mutation, shortening and tailoring of the β13β14 loop made this binding mode possible (Fig. 23d,e). Indeed, it is notable that the C1-C3 backbone atoms of (S)-β-Phe assume the same conformation as the backbone of (2S,3S)-3-methylaspartate in VinN, suggesting that these fea- tures may be sufficient to accommodate expanded backbones of disparate β-amino acids by other α-synthetases [166].

Downstream processing of β-amino acids. For successful pathway en- gineering, a repurposed NRPS module must function in an intra- and inter- modular context. We therefore tested TycAβF in increasingly challenging settings.

Initially, we reconstituted the first two modules of tyrocidine biosynthesis in vitro (Fig. 24a) [149]. The second module, TycB1, loads L-Pro and sub- sequently catalyses peptide bond formation via aminolysis of the upstream thioester. TycB1 was equipped with a variant of the surfactin thioesterase

66 3.2. Results

(TE) domain that contains a P26G mutation (SrfTEP26G) [169] to promote peptide offloading. When TycAβF and TycB1P-SrfTEP26G were incubated with (S)-β-Phe, L-Pro, and ATP, efficient formation of cyclic βα-dipeptide

51 was observed. The initial rate of peptide bond formation (k obs = 2.7±0.3

a R = 48 49 O H O N O HN R N SH SH O

R HN 50 51 TycA O H X TycB1-SrfTE P26G O

b variant TycA F TycA pY TycA βpY TycA βF

k -1 obs (min ) 4.7±0.4 3.2±0.2 1.2±0.1 2.7±0.3 k -1 obs,comp (min ) 4.3±0.3 2.2±0.2 1.5±0.1 2.7±0.3 TTN 3690±70 1820±10 510±20 1960±50

c

48 49 50 51

TycA F TycA pY TycA βpY TycA βF

2.9 3.4 3.9 2.9 3.4 3.9 2.9 3.4 3.9 2.9 3.4 3.9 time (min) time (min) time (min) time (min)

Figure 24. Biosynthesis of βα-dipeptide analogs. a, Pairing of TycAF and variants with TycB1P-SrfTEP26G in bis-Tris propane buffer, pH 9, containing ATP and L-Phe (26), O-propargyl-L-Tyr (27), O-propargyl-(S)-β-Tyr (44), or (S)-β-Phe (45) at 37 ◦C afforded cyclic dipeptides 48, 49, 50, or 51, respectively. b, Rate constants for formation of the dipeptide product from the cognate substrate (k obs) or equimolar mixtures (k obs,comp) and total turnover numbers (TTNs) with the cognate substrate. c, HPLC chromatograms of the authentic standards for the cyclic dipeptides (top) and the products obtained when the dif- ferent TycA variants were incubated with TycB1P-SrfTEP26G under competitive conditions with equimolar mixtures of substrates (bottom). Data are reported as the mean ± standard deviation, where n = 3.

67 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES min-1) was within a factor of two of the rate observed for the analogous reaction of wild-type TycAF with L-Phe, showing that the C domain does not discriminate against (S)-β-Phe (Fig. 24b). The bimodular NRPS ex- hibits notable specificity for (S)-β-Phe, strongly favoring the corresponding mixed βα-product in the presence of equimolar amounts of competing L-

Phe, O-propargyl-L-Tyr, and O-propargyl-β-Tyr (Fig. 24c). Furthermore, the artificial cyclo-βα-dipeptide synthetase is robust, performing 1960±50 to- tal turnovers over 24 h. Consequently, preparative (∼1 mmol) scale-up was straightforward, providing ∼250 mg of the cyclic βα-dipeptide 51 in 59% isolated yield.

To test β-amino acid incorporation in the context of a full NRPS path- way, we reconstituted gramicidin S synthetase with TycAβF (Fig. 25a). Due to the strict specificity of GrsTE for the first amino acid [151], hydrolytic off-loading of a modified pentapeptide rather than macrocyclization was an- ticipated. Indeed, when incubated with amino acid substrates and ATP in vitro, the engineered synthetase afforded the backbone-modified pentapeptide -1 (S)-β-Phe-L-Pro-L-Val-L-Orn-L-Leu (52) with a k obs of 1.1±0.3 min , compa- rable to that observed for production of gramicidin S by the wild-type system (Fig. 25b), demonstrating that (S)-β-Phe is well tolerated in all downstream condensation steps catalyzed by GrsBPVOL.

Finally, the engineered NRPS consisting of TycAβF and GrsB was recon- stituted in the heterologous host E. coli HM0079, genomically encoding Sfp 4’-phosphopantetheinyl transferase [149]. Intracellular peptide biosynthesis constitutes a particularly stringent test for an engineered pathway because of competing endogenous amino acids and lack of control over cytosolic reaction conditions. When the cells were incubated with all five amino acid substrates,

68 3.2. Results

a (S)-β-Phe-L-Pro-L-Val-L-Orn-L-Leu (52) titer of 120±20 mg/L was deter- mined by quantitative LC-MS analysis of the crude cell culture 48 h after induction (Fig. 25c). Of note, this system was not optimized with respect

a GrsB (510 kDa)

OH O

H2N H2N S S S S S HN O O O O O O H2N N NH H2N HN NH HN O O O O O

H2N N HN NH HN TycA (120 kDa) O O βF O O N H2N N HN O O O H2N H2N N O

H2N

52 b c ] ] 0 0 50 100 k -1 k -1 300 obs = 0.5±0.2 min obs = 1.1±0.3 min ] / [E / ] ] / [E / ] 75 52 32 200 25 50 100 25 (mg/L) titer 11

0 0 0 [gramicidinS 0 25 50 75 100 [pentapeptide 0 25 50 75 100 0 3 6 9 12 time (min) time (min) EIC (295±2 m/z) area (a.u.)

Figure 25. Biosynthesis of a β-amino acid-containing pentapeptide. a,

Schematic representation of the engineered pentapeptide synthetase comprising TycAβF and GrsBPVOL. b, Pairing of the two subunits in bis-Tris propane buffer, pH 8, containing ATP and the five substrate amino acids at 37 ◦C afforded the (S)-β-Phe-containing pen- tapeptide 52 at a comparable rate (right) to that of the wild-type synthetase under identical conditions (left). c, When TycAβF and GrsBPVOL were paired in the heterologous host E. coli and supplemented with (S)-β-Phe, pentapeptide 52 titers of 56±5 (orange dot) and 120±20 mg/L (blue dot), respectively, were detected 24 h and 48 h after induction by comparison to an authentic standard (black dots).

69 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES to plasmid copy number, promoter strength, or host organism; presumably even higher titers could be attained with further development, highlighting the potential of engineered NRPS pathways for the fermentation of novel non- ribosomal frameworks.

Engineering TycA for N -alkylated Phe analogs. While high- throughput FACS screening enabled the efficient reprogramming of TycAF for the backbone-modified amino acid (S)-β-Phe, the need for a bioorthogo- nal handle on the side-chain places inherent constraints on engineering of the binding pocket. To address this limitation, we set out to engineer TycAF for

N -propargyl-L-Phe with the bioorthogonal handle on the universal backbone amine. Compared to (S)-β-Phe, amino acids with substitutions at the α- amine are even more challenging substrates for TycAF [170]. Also, no natural A domain could guide library design. We therefore solely relied on the X-ray crystal structure of GrsAF [74] and the screening power of FACS.

In GrsAF, Asp235 forms a salt bridge with the α-amine of the substrate

L-Phe, which points directly at the β13β14 loop [74]. We hypothesized that N - substituents could be accommodated by shortening or elongating the β13β14 loop in TycAF. Additionally, positions 202, 206, 210, and 381 point towards the β13β14 loop and could be randomized to bolster remodeled loop conforma- tions. Accordingly, several saturation libraries of TycAF were prepared with shortened or elongated β13β14 loops and a total of four randomized positions

(NNK). Consistent with a low activity of TycAF towards N -propargyl-L-Phe, no signal above background was observed in initial FACS screens of the result- ing libraries. However, one saturation library could be significantly enriched in

N -propargyl-L-Phe-loading TycAF variants over three rounds of FACS. In this library, residues Ser329 and Ile330 were replaced with a random NNK tripep-

70 3.2. Results tide. Additionally, position Leu210 was randomized with an NNK codon. Sequencing of individual clones after round three revealed a surprising enrich- ment of variants containing β13β14 loops that were one amino acid shorter (rather than longer) than wild type — possibly the result of hairpin excision in Saccharomyces cerevisiae. Of eleven sequences, only one corresponded to the original library design (TycAN pF,4 in Fig. 27). Ten sequences encoded three

TycA variants with a one amino acid shorter β13β14 loop relative to TycaF.

The first two (called TycAN pF,1 and TycAN pF,2) contained a large-to-small Leu210Ala or Leu210Cys mutation, respectively and a bulky Phe residue in the shortened loop. By contrast, the third variant, TycAN pF,3, featured a β13β14 loop with two Gly residues and a large Trp residue at position 210.

a b TycA F sort1

L202 G381 β13 β14

329 330 G206

L210 Phe sort2 sort3 aaloading a.u.) / (R-PE

OH OH H2N HN O O 26 53 surface display (FITC / a.u.)

Figure 26. Engineering TycAF for N -propargyl-L-Phe (53). a, Active site

of GrsA-AF with bound L-Phe (26) [74] and all residues targeted to accommodate N -propargyl-L-Phe (53) highlighted as orange sticks. b, A saturation library tar- geting residues Leu210, Ser329, and Ile330 was enriched in N -propargyl-L-Phe (53)-

loading TycAF variants over three rounds of FACS.

71 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES

Asn209. Leu210. Gln211. Glu327. Thr328. Ser329. Ile330. Cys331. tycA A A T T T G C A A G A A A C G A G C A T T . . . T G C NpF1 A A T G C G C A A G A A A C G T T T ...... T G C NpF1 A A T G C G C A A G A A A C G T T T ...... T G C NpF2 A A T T G T C A A G A A A C G T T T ...... T G C NpF2 A A T T G T C A A G A A A C G T T T ...... T G C NpF3 A A T T G G C A A G A A . . . G G T G G G . . . T G C NpF3 A A T T G G C A A G A A . . . G G T G G G . . . T G C NpF3 A A T T G G C A A G A A . . . G G T G G G . . . T G C NpF3 A A T T G G C A A G A A . . . G G T G G G . . . T G C NpF3 A A T T G G C A A G A A . . . G G T G G G . . . T G C NpF3 A A T T G G C A A G A A . . . G G T G G G . . . T G C NpF4 A A T A T G C A A G A A A C G G G T C C T T G T T G C

Figure 27. Sequences of TycA variants enriched for N -propargyl-L-Phe (53)- loading. Positions targeted for saturation mutagenesis are highlighted in red.

The TycAN pF,1 and TycAN pF,2 variants were individually tested in the yeast surface display assay for N -propargyl-L-Phe (53)-loading in the pres- ence of competing L-Phe (26) (Fig. 28). Charged with a 5:1 mixture of

N -propargyl-L-Phe (53) and L-Phe (26), both variants exhibited PE-labeling indicating that N -propargyl-L-Phe (53) could outcompete L-Phe (26). How- ever, the PE signal was suppressed by equimolar amounts of L-Phe (26).

While confirming loading of N -propargyl-L-Phe (53) by TycAN pF,1 and

TycAN pF,3, this result also indicates that both variants retain a slight prefer- ence for L-Phe (26). Overall, TycAN pF,1 showed better N -propargyl-L-Phe- loading compared to TycAN pF,3 and could conceivably be further optimized by fine-tuning of the β13β14 loop and its vicinity.

TycAN pF,1 offers an ideal starting point for the engineering of diverse sub- strate specificities. Switching of the bioorthogonal handle to the amino acid backbone should enable tailoring of the A-domain side-chain pocket com- plementary to the reprogramming of substrate specificty for backbone mod- ifications using the W239S mutation. As an evolutionary stepping stone,

TycAN pF,1 does not need to load N -propargyl-L-Phe selectively and the small

72 3.2. Results

2% 42% 56%

TycA NpF1 [Phe]

TycA NpF3

2% 26% 50% aaloadingloadi a.u.) (R-PE/

surface display (FITC / a.u.)

Figure 28. N -propargyl-L-Phe (53)-loading by variants TycAN pF,1

and TycAN pF,3. Flow cytometry of monoclonal cultures of TycAN pF,1 (top)

and TycAN pF,3 (bottom) labeled with 2 mM N -propargyl-L-Phe (53) in compe- tition with 2 mM (left), 0.4 mM (middle), and 0.08 mM L-Phe (26), respectively.

number of alterations relative to TycAF might even be advantageous for sub- sequent reversion of auxiliary mutations.

Biosynthesis of N -methylated peptides. N -Methylation is a common backbone modification in NRPs that is typically introduced by dedicated M domains. We hypothesized that a TycA pocket competent in loading

N -propargyl-L-Phe (53) should also accommodate the sterically less demand- ing substrate N -methyl-Phe and therefore enable M-domain independent methylation of the peptide backbone. To test incorporation of N -methyl-Phe into NRPs, TycAN pF,1 was reconstituted in vitro with TycB1P-SrfTEP26G

(Fig. 29a). Whereas the bimodular synthetase with TycAF did not af-

73 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES

a O TycA X TycB1-SrfTE P26G N R N N N R H O OH O O b c R = H ( 54 ), Me ( 55 ) R = H ( 48 ), Me ( 56 ) 100 100

Substrate

L-Phe 50 50

N-Me- L-Phe

dipeptideformation(%) dipeptideformation(%) N-Me- D-Phe 0 0 0 0.1 0.5 1

TycAF TycANpF1 equivalents L-Phe d TycA NpF1 TycB PFpY TycC NQYVOL

DmF PF DpY NQ Y VOL

OH NH 2 O HN O O O NH 2 O O O H H H H N N N N N N N N N OH H H H H O O O O

O O NH 2 57

Figure 29. Biosynthesis of N -methylated NRPs. a, Schematic representation of the bimodular synthetase used to test TycAN pF,1. b, Dipeptide formation by TycAF or

TycAN pF,1 paired with TycB1P-SrfTEP26G in bis-Tris propane buffer, pH 9, and supple- mented with ATP, L-Pro, and L-Phe (gray), N -methyl-L-Phe (orange), or N -methyl-D-Phe (green). c, N -methyl-D-Phe-incorporation was further tested with increasing amounts of competing L-Phe. d, When paired with TycBPFpY and TycCNQYVOL in bis-Tris propane buffer, pH 8, TycAN pF,1 afforded the doubly modified decapeptide 57.

ford N -methylated dipeptides, efficient formation of linear and cyclic N - methyldipeptides 55 and 56 was observed for TycAN pF,1 supplied with

74 3.3. Discussion

N -methyl-D-Phe (Fig. 29b). Dipeptide formation was reduced significantly

(∼10%) when N -methyl-L-Phe was provided as a substrate, suggesting that the E domain does not efficiently racemize the N -methylated substrate. Im- portantly, TycAN pF,1 could still incorporate N -methyl-D-Phe under compet- itive conditions, with only a ∼30% reduction in dipeptide formation in the presence of equimolar amounts of L-Phe (26) (Fig. 29c). This result sug- gests that N -methyl-D-Phe is the better substrate for TycAN pF,1 compared to N -propargyl-L-Phe (53), as equimolar concentrations of L-Phe (26) out- competed N -propargyl-L-Phe (53) in the yeast surface display assay. The se- lectivity of TycAN pF,1 for N -methyl-D-Phe enables incorporation into NRPs in the presence of L-Phe (26), which is especially relevant for NRPSs contain- ing other Phe-specific modules such as the tyrocidine synthetase. In prelim- inary experiments, we paired TycAN pF,1 with TycBPFpY and TycCNQYVOL to produce the doubly modified decapeptide 57 containing an N -methylated N-terminus and a bioorthogonal handle at position four (Fig. 29d, Fig. 45,

Fig. 46). The methylated D-Phe N-terminus of 57 is reminiscent of natural linear NRPs, such as the antibiotic teixobactin [44]. Our results demonstrate that A-domain reprogramming enables strategic incorporation of such build- ing blocks into NRPs without the need for auxiliary M domains.

3.3 Discussion

The design and production of bioactive NRPs by swapping and engineering NRPS modules has highlighted the potential of NRPS engineering for gener- ating unnatural products [111, 113, 115, 117, 119, 120, 171]. Fermentation of such compounds represents a green, sustainable, and economically attrac- tive alternative to solid phase peptide synthesis, particularly for structurally

75 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES complex NRPs [64]. Nevertheless, expanding structural diversity in these molecules by incorporating non-native building blocks, including backbone- modified amino acids, is still limited by our incomplete understanding of structure-function relationships in these mega-enzymes and by insufficiently robust design and assay protocols. The efficient engineering of NRPS mod- ules for incorporation of β- and N -alkylated amino acids, however, shows that high-throughput assays for catalytic activity can overcome such problems.

Successful reprogramming of TycA specificity relied on a FACS-based ac- tivity screen of functional NRPS modules displayed on yeast. This system enabled analysis of complex libraries containing >106 members, a 3-4 order of magnitude increase in throughput compared to conventional plate assays [121]. Accordingly, multiple residues in the active site could be targeted simultane- ously for saturation mutagenesis, thus facilitating identification of synergistic interactions needed to accommodate substantial changes in substrate struc- ture. To that end, directly assaying both adenylation and thioesterification activity using a “clickable” amino acid proved more effective than indirect en- gineering approaches based on ligand binding [124] or monitoring adenylation by pyrophosphate exchange [122]. As a result, major switches in substrate preference for challenging substrates could be achieved without sacrificing catalytic efficiency. In principle, this approach should be quite general for tai- loring the properties of many A domains. Extension to other bioorthogonal labelling methods [172] and performing iterative rounds of mutagenesis and screening [116] could expand its utility even further.

Backbone-modified amino acids are attractive NRP building blocks be- cause of their unique conformational properties and ability to stabilize pep- tides. While no natural A domain has been reported to activate an N -

76 3.3. Discussion alkylated substrate in the absence of an additional M domain, some A do- mains are known to activate β-amino acids [165]. However, they are rare, and the molecular recognition determinants that distinguish them from conven- tional α-synthetases are obscure. Our results provide strong support for the hypothesis [166, 167] that the length and composition of the β13β14 loop, in combination with residue 236 across the pocket, are key to the α/β-switch. Structures of the evolved β-A domains show how subtle remodeling of the ac- tive site induced by these changes leads to the 40,000-fold switch in substrate specificity. Nevertheless, no matches for the specificity-determining residues in our engineered mutants were found in a compilation of 5,572 A domain sequences [173]. It is notable in this context that TycAβF differs from two natural A domains that reportedly activate β-Phe, namely HitB from the VinN family [166] and AdmJ [174], which have completely different binding sites and β13β14 loop residues (Fig. 30).

A reprogrammed NRPS module is only useful if downstream domains toler- ate the new building block that it activates. The ability of the reprogrammed modules to function with downstream domains is therefore particularly note- worthy. The efficient production of β-amino acid containing peptides, both in vitro and in vivo, demonstrates that biosynthetic incorporation of backbone- modified amino acids into NRPs can be straightforward, robust, and scalable.

77 3. BIOSYNTHESIS OF BACKBONE-MODIFIED PEPTIDES

conser. GrsA (Phe) GISNLKVFFENSLNVTEKDRIGQFASISFDASVWEMFMALLTGASLYIILKDTINDFVKF TycA Phe) GIANLQSFFQNSFGVTEQDRIGLFASMSFDASVWEMFMALLSGASLYILSKQTIHDFAAF PolA1 (Phe) GLVSLKLTFAHTLHTTEQDRVLQFASLSFDASCWEMFNALYFGATLYIPSTETILDDQLF TxoA1 (Phe) GVCNLARAQTGAFPVEAGSRVLQWASFSFDAWVFELVMALCHGASLHLTAPGTLLAGATL TycC3 (Tyr) GIVNNVLWKKAEYQMKVGDRSLLSLSFAFDAFVLSFFTPVLSGATVVLAEDEEAKDPVSL CmdD2 (bTyr) NLSRLTPALITAFDVTPRSRVLQYSSLSFDGSISEVAMALGAGAALHLAPAHELVPGPPL AdmJ (bPhe) NLNFFLEALYPRYVSEQSENYLSIGPLYFDMTILDSMVPPLYGHSVYLYKAPFI..PAIF HitB (bPhe) SVVSLYRAILEQGLITPEDRIATTSPLQFDFALFDIGLALGTGAALVPVPREELNWPRRF TycAbF (bPhe) GIANL. QSFFQNSFGV. TEQDRIGLFA. SMSFDVSVWE. MFMALLSGAS. LYILSKQTIH. DFAAF 210 220 230 240 250 260 ***

conser. GrsA (Phe) EQYINQKEITVIT.LPPTYVVHL....DPE..R.ILSIQTLITAGSATSPSLVNKWKE.. TycA Phe) EHYLSENELTIIT.LPPTYLTHL....TPE..R.ITSLRIMITAGSASSAPLVNKWKD.. PolA1 (Phe) ERFMNEHAITIAT.LPPTYAAYL....NSD..R.LPSLSRLITAGSAVSAEFVQQWKD.. TxoA1 (Phe) VRLLREREISHLTVTPSV....LEGL.DPD..AALPALRTLVVAGEACSEALVARWAPGR TycC3 (Tyr) KKLIAASRCTLMTGVPSLFQAILECS.TPA..D.IRPLQTVTLGGEKITAQLVEKCKQLN CmdD2 (bTyr) QKLLATRAITHVTLLPAA....LRWL.SP...RGLPALDVLIVTGEACPASLVRTWASGR AdmJ (bPhe) SGIIEKYRITRLSCVPSVLEILLPQLHEATGFDKLHSLNLILFGAEKPHGQSIKTLLQHI HitB (bPhe) LAFLGDTGATQVHGVPSIWRPVLRH..EPELLAGLDRVRGILFTGEDFPLPELRHLQGLL TycAbF (bPhe) EHYLS. ENELTIIT.LP. PTYLTHL....TPE. ..R.ITSLRIMIT. AGSASSAPLV. NKWKD.. 270 280 290 300 310 ***

conser. GrsA (Phe) .KVTYINAYGPTETTICATTWVATKE..TIG.HSVPIGAPIQNTQIYIVDENLQLKS.VG TycA Phe) .KLRYINAYGPTETSICATIWEAPSN..QLSVQSVPIGKPIQNTHIYIVNEDLQLLP.TG PolA1 (Phe) .KVQYYNAYGPTEASIATSVWAASTY..DTERRAIPIGRPIWNHRLYILGAQNQLAP.IG TxoA1 (Phe) ...LMVNAYGPTEATVWATYHPCR....PEEQGAPPIGRAIANARLYVLDEHRAPVP.AG TycC3 (Tyr) PDLVIVNEYGPTESSVVAT.WQRLAG..PDA..AITIGRPIANTSLYIVNQYHQLQP.IG CmdD2 (bTyr) ...RFVNAYGPTEITVAATAMECPVTMFQETEQPPPIGCPLQSTEIYILDAHLRPVP.VG AdmJ (bPhe) PDLKVINAYGPTEGTMCCFSSEITLDSLAGD...ISIGKPFDGTK.YLLETRSGFSS..S HitB (bPhe) PHARIVNGYGATESMACS..FTEVPRPIPSDLERLSIGFPLPGFDVSLLDEHGRPVEEIG TycAbF (bPhe) .KLRYI. NAYGPTECLV. .ATIWEAPSN. ..QLSVQSVPIGK. PIQNTHIYIV. NEDLQLLP.TG. 320 330 340 350 360 370 ***

Figure 30. Sequence alignments of representative A domains. Comparison of selected Phe-, β-Phe-, and β-Tyr-activating A domains. Engineered positions in TycAβF are boxed and specificity-determining binding pocket residues [75, 76] are highlighted with asterisks.

Because NRPSs are structurally homologous and modular, the ability of the downstream condensation domains of the two pathways examined here

78 3.3. Discussion to process backbone-modified amino acids may indeed prove more general. Should additional selectivity filters emerge, for example in downstream C do- mains, alternative high-throughput engineering strategies will have to be de- veloped to alter or relax the specificity of the relevant downstream domains to access a wider range of backbone-modified products. Given the limits of chemical approaches for modifying the core of NRP natural products, robust methods for incorporating backbone-modified amino acids biosynthetically promise to enable NRP tailoring beyond the semisynthetic paradigm.

79 80 4 Perspectives

Microbes produce a plethora of bioactive secondary metabolites, that have become indispensable to human medicine. Nevertheless, the known natural product portfolio only scratches the surface of natural diversity. The realiza- tion that many of these compounds are assembled by modular biosynthetic machinery has prompted efforts to expand microbial chemistry by mixing and matching existing modules. However, reduction to practice has proven chal- lenging due to the sheer complexity of these megaenzymes and our poor un- derstanding of module boundaries and dynamics. In principle, engineering of parts would offer a more surgical alternative for reprogramming biosynthetic assembly lines.

More than 25 years ago, a single Ala-to-Gly mutation in the binding pocket of E. coli PheRS, the aminoacyl-tRNA synthase responsible for charg- ing tRNAPhe with L-Phe, enabled reprogramming of the genetic code for p- halogenated amino acids [175, 176]. Directed evolution of aminoacyl-tRNA synthases has since been instrumental in expanding the ribosomal building block repertoire to more than 100 noncanonical amino acids [134, 135]. Simi- larly, reprogramming of A domain specificity promises to be a powerful strat- egy to modulate the structures and properties of therapeutically important nonribosomal peptides. Reminiscent of PheRS engineering, the W239S mu- tation provides a minimally invasive tool to expand the L-Phe-binding pocket of gatekeeper A domains and impart nonribosomal peptides with novel func-

81 4. PERSPECTIVES tionality [123]. However, efficient methods for more general reprogramming of A-domain specificity have been lacking.

This thesis establishes yeast surface display as a robust platform for rewrit- ing the nonribosomal code. Enabled by the high throughput of FACS, we have reprogrammed the L-Phe-specific initiation module TycAF for β-amino acids, harnessing the W239S mutation for the introduction of a “clickable” side-chain handle. In principle, this strategy should be quite general, as many amino acid analogs with bioorthogonal groups, not necessarily an alkyne, can be envisioned. Also, the functional handle is not restricted to the substrate side- chain, as demonstrated by engineering TycAF for N -propargyl-L-Phe. Im- portantly, accommodating the bioorthogonal handle on the backbone amine, which is present in all amino acid substrates, opens the door to remodeling of the side-chain binding pocket. Functional A-domain display, however, re- mains a prerequisite for more general application. In the course of this thesis, we have successfully displayed four different constructs from the gramicidin

S, tyrocidine, and surfactin pathways, demonstrating that TycAF was not simply a fortuitous starting point. If required, optimization of constructs with poor display should be feasible, given the high throughput of FACS. A general and robust platform for A-domain reprogramming sets the stage for future biosynthetic engineering of NRPS pathways.

Expansion of the nonribosomal code challenges the limits of promiscuity by second-line domains. In our efforts to reprogram the gramicidin S and tyroci- dine pathways, macrocyclization by the terminal TE domain proved partially limiting. While targeted mutagenesis should allow elimination of potential steric clashes, given a better understanding of the TE domain, substitutions that impact peptide conformation, such as backbone modifications, might

82 . prove harder to accommodate. Nevertheless, such TE bottlenecks can be over- come as demonstrated by a Ser-to-Cys mutation of the catalytic nucleophile in the pikromycin TE domain, which enabled efficient macrolactonization of a backbone-modified substrate analog [177]. The analogous mutation in GrsTE was not successful, resulting in a marked decrease in Cl-gramicidin S (34) for- mation when the engineered synthetase was tested with p-chloro-L-Phe (33). However, an LC-MS-based assay is available for more thorough engineering of TE bottlenecks [90]. As shown by migration of the W239S mutation to an in- ternal elongation module, TE engineering will not always be required. In fact, the specificity profiles of GrsTE and TycTE suggest that most substitutions will be tolerated, at least to some extent [90, 151].

Of the four core domains, this leaves the C domain — by far the least ex- plored. Although we observed remarkable plasticity for unnatural substrates, C domains can be a hurdle for biosynthetic engineering, especially in combina- tion with E domains. When we transplanted the mutations for β-amino acid activation to TycB3F, analogous to W239S, addition of (S)-β-Phe resulted in complete abrogation of tyrocidine A (11) formation (Appendix 5.2). This result suggests, that (S)-β-Phe is selectively activated by the reprogrammed elongation module but not processed by the flanking C and E domains. In this extreme case, stalled peptide synthesis might not come as a surprise. Not only has the nucleophilic amine for peptide bond formation been migrated to the β-position, but the acceptor and donor C domains exhibit mismatched stereospecificities requiring efficient epimerization of the β-amino acid [84]. Nevertheless, this example underscores the need for better tools to study and engineer C domains.

83 4. PERSPECTIVES

a O

yeast EBY100 HN

ATP O H2N O S N

O S HN O C A O A E TycB1 P

TycA pY

b

10 4 O

ATP O 10 3 H2N

O 0 NH 2

aaloading a.u.) / (R-PE SH HN yeast O OH 0 10 3 10 4 EBY100 surface display (FITC / a.u.) O S A E 10 4 H2N

TycA βpY 3 10 O OH C A

SrfCL 0 A aaloading a.u.) / (R-PE

3 4 0 10 10 TycA βF-A surface display (FITC / a.u.)

R c H2N

O OH H H O N R N N N OH H NH 2 O O C A

84 .

In preliminary experiments, we have expanded the concept of yeast surface display and “click” chemistry-based high-throughput screening to C-domain catalysis. To that end, display of the full TycApY module (including the E and COM domains) was attempted to enable peptide bond formation on the surface of yeast. Indeed, this large construct (130 kDa) could be displayed as shown by FITC-based immunofluorescence labeling (Appendix Fig. 50).

When cells displaying TycApY were paired with TycB1P and supplemented with amino acids and ATP, LC-MS analysis of the supernatant revealed forma- tion of O-propargyl-D-Tyr-L-Pro-DKP (49), suggesting that display of func- tional NRPS modules on yeast is possible (Fig. 31a, Appendix Fig. 49).

A high-throughput assay requires display of acceptor modules to monitor C domain-catalyzed attachment of “click” handles, rather than release. We therefore redesigned the yeast display vector to contain an acceptor module with a free N-terminus for interaction with TycApY [179]. Given that full modules can be active on yeast, we anticipated self-cyclization of the result- ing dipeptide thioesters as the biggest hurdle for a high-throughput C domain assay. Accordingly, we tested formation of a (S)-β-Phe-containing dipep-

Figure 31. C-domain catalysis on the surface of yeast. a, Schematic representation of a DKP assay with the full initiation module TycApY displayed on the surface of yeast.

When paired with TycB1P and supplemented with O-propargyl-L-Tyr, L-Pro, and ATP, the corresponding DKP could be detected by LC-MS analysis of the supernatant (Appendix

Fig. 49). b, To enable a high-throughput assay, the acceptor module SrfCL was displayed on yeast and paired with TycAβpY in the presence of O-propargyl-(S)-β-Tyr, L-Leu, and ATP. Flow cytometry revealed PE-labeling of yeast cells (top), suggesting transfer of O- propargyl-(S)-β-Tyr to a SrfCL-bound L-Leu residue. The yeast cells displayed significantly less PE-signal when TycAβpY-A domain was used instead, lacking the ability to interact with SrfCL (bottom). c, Direct loading of stable aminoacyl-ppant mimics by Sfp could provide a more robust strategy for dipeptide tethering. The necessary CoASH analogs can be readily prepared in a one-pot biosynthesis from the respective pantetheine analog [178].

85 4. PERSPECTIVES

tide with TycAβF (see above), which cyclizes slowly without the aid of a TE domain due to the disfavored formation of a seven-membered ring. Addition- ally, L-Pro was replaced with L-Leu, which can be incorporated by pairing

TycAβpY with SrfCL due to compatible COM domains [108]. When cells displaying SrfCL without the terminal TE domain were supplemented with

TycApY, O-propargyl-L-Tyr, L-Leu, and ATP, a significant cell population not only displayed FITC fluorescence, but also the PE marker for covalent loading of the “clickable” amino acid O-propargyl-(S)-β-Tyr 44 (Fig 31b). By con- trast, only few cells were PE-positive when only the TycApY-A domain was used as a donor or without addition of L-Leu.

While these experiments provide a first proof-of-principle, more robust tethering could conceivably be achieved with stable aminoacyl-ppant analogs directly loaded onto the acceptor module by Sfp (Fig. 31c). In bypassing the specificity requirements of the A domain, such strategies would allow for engineering of not just the acceptor but also the donor site to enable general C-domain engineering in high throughput.

In addition to NRPS engineering, rewiring of existing domains and mod- ules offers additional degrees of freedom for biosynthetic engineering. For ex- ample, exchange of a core subdomain within the A domain can allow combina- torial alteration of substrate specificity [180]. While these chimeric A domains are often impaired, directed evolution can restore activity [117]. However, re- pairing extensive protein interfaces requires the ability to cover large sequence space. The ability to screen for A-domain catalysis in high throughput will therefore be crucial to restore activity in chimeric NRPSs and might help to identify better guidelines for productive (sub)domain swaps.

86 .

In principle, polyketide synthases should also be amenable to high- throughput engineering by yeast surface display. Like NRPSs, PKSs employ a thiotemplate mechanism to tether substrates and intermediates via thioesters. Furthermore, gatekeeper acyltransferases have been shown to tolerate “click- able” substrate analogs [143, 144]. Success will ultimately depend on the ability to display functional PKS domains on yeast.

As demonstrated in this thesis, a high-throughput assay for A-domain catalysis provides an invaluable tool to reprogram NRPS pathways, overcom- ing many previous limitations. Expansion of this methodology to engineer downstream processing of novel amino acids and even related PKSs promises unprecedented control over natural product biosynthesis. Our results augur well for the future of biosynthetic pathway engineering as a sustainable and scalable source for designer drugs.

87 88 5 Appendix

5.1 Materials and methods

5.1.1 Chemical synthesis

All reagents were used as received, all solvents were technical grade, and all reactions were run in open flasks fitted with PFTE coated magnetic stir bars at room temperature (RT) unless otherwise noted. Analytical thin-layer chro- matography (TLC) was performed with Merck 60 F254 pre-coated glass plates (0.25 mm) and visualized using a combination of UV detection (254 nm), p- ◦ anisaldehyde, and KMnO4 stains. RT reactions were conducted at ∼23 C, reactions run cooler than RT were performed in a cold room (4 ◦C), an ice bath (0 ◦C), or a NaCl/ice bath (-10 ◦C). Flash column chromatography was performed using SiliCycle (SilaFlash R P60, 230-400 mesh particle size) silica gel. Preparative HPLC was performed on a Waters system consisting of 515 pumps in line with a 2487 dual λ absorbance detector and a fraction collector using a Reprosil-Pur 120 C18-AQ column (150 x 20 mm, 5 µm, Dr. Maisch GmbH, Basel, Switzerland). High resolution mass spectrometry (HRMS) was performed on a Bruker maXis UHR-TOF by electrospray ionization (ESI) or a Bruker solariX by matrix-assisted laser desorption/ionization (MALDI) at the Mass Spectrometry Service of the Laboratory of Organic Chemistry (LOC) at ETH Zurich. NMR spectra were recorded on a Bruker Advance-III 400 MHz spectrometer at the NMR Service at the LOC, ETH Zurich. 1H-NMR spectra were recorded relative to residual solvent peak (CDCl3 δH 7.26 ppm, D2O δH

89 5. APPENDIX

4.79, D6-DMSO δH 2.50 ppm) and reported as follows: chemical shift (ppm), multiplicity, coupling constant (Hz), and integration. Multiplicity abbrevia- tions are as follows: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, h = hextet, ovlp = overlap, br = broad signal. 13C NMR spectra were recorded relative to residual solvent peaks (CDCl3 δC 77.0 ppm, D6-DMSO

δC 39.5 ppm).

N O 1. NaCN(BH ) O O 3 N MeOH H N N N3 H N OH H 2 2. N -propylamine, 3 N DCC, DMAP 58 59 60 DCM

60: β-Ala (Sigma, 59, 1 g, 11 mmol, 1 equiv) was dissolved in MeOH (20 mL, 0.6 mM), heated to 50 ◦C and pyridine-2-carboxaldehyde (ABCR, 58, 2.7 g,

2.4 mL, 24.7 mmol, 2.2 equiv) and NaCN(BH3) (95%, ABCR, 1.7 g, 1.4 mL, 28 mmol, 2.5 equiv) were added sequentially. The reaction was stirred for 4 h at 50 ◦C and reaction progress was monitored by LC-MS. After completion,

ZnCl2 (1 M, 50 mL) was added and the solution was acidified with TFA. The resulting mixture was extracted with DCM (3x). The combined organic extracts were washed with brine and filtered through a sodium sulfate plug, which was subsequently rinsed with DCM (2x), concentrated, and purified by preparative HPLC (H2O:MeCN + 0.1% TFA, 5% to 40% MeCN over 65 min, flowrate 10 mL/min). A portion of the crude product (159 mg, 0.6 mmol, 1 equiv) was dissolved in DCM (10 mL) and DCC (Sigma, 145 mg, 0.7 mmol,

1.2 equiv), N3-propylamine (90%, Sigma, 67 mg, 66 µL, 0.67 mmol, 1.1 equiv), and catalytic amounts of DMAP were added. The reaction was stirred at RT overnight, concentrated. and purified by preparative HPLC (H2O:MeCN +

90 5.1. Materials and methods

0.1% TFA, 5% to 40% MeCN over 65 min, flowrate 10 mL/min) to yield 60 (52 mg, 0.15 mmol, 1.4% yield). 1H NMR (400 MHz; CDOD): δ 8.74-8.72 (m, 2H), 8.13 (td, J = 7.8, 1.7 Hz, 2H), 7.69 (dt, J = 7.9, 1.1 Hz, 2H), 7.65- 7.62 (m, 2H), 4.47 (s, 4H), 3.34 (t, J = 6.6 Hz, 4H), 3.26 (t, J = 6.8 Hz, 2H), 2.67 (t, J = 6.5 Hz, 2H), 1.74 (p, J = 6.7 Hz, 2H). 13C NMR (101 MHz; CDOD): δ 173.5, 153.8, 147.5, 142.5, 126.3, 126,0, 58.1, 52.4, 50.0, 37.8, 31.9, 29.6. ESI HRMS: calculated [M+H]+ 354.2037, found 354.2036.

NH 3 N O O O H H OH N N N3 CuSO / ascorbate N N H 4 N N N O H H O O NH 61 MeOH N O O H H O N N N N H H O O NH 3 O O

H2N NH O O 2 H H OH O N N N N N N O H H N 41 O O NH N O O H H O N N N N H H O O HN O O O H N 2 NH 2 O N N 39 N

41: 61 (5.5 mg, 15.5 µmol, 1.2 equiv) and crude 39 (see 5.1.2, 17 mg, 12.8 µmol, 1 equiv) were dissolved in MeOH (0.9 mL, 17.2 mM and 14.2 mM, respectively). 100 µL of a solution of CuSO4 (100 mM, Sigma, 1.6 mg, 10 µmol, 0.78 equiv) and ascorbic acid (1 M, ABCR, 19.8 mg, 100 µmol, 7.8 equiv) was added. The reaction was run for 2 h at RT, quenched with an EDTA solution (250 mM, 2 mL, Sigma, 186 mg, 500 µmol, 38.9 equiv), acidified with TFA, and extracted with DCM (3x). The combined organic extracts were washed with brine and filtered through a sodium sulfate plug, which was subsequently rinsed with DCM (2x), concentrated, and purified

91 5. APPENDIX

by preparative HPLC (H2O:MeCN + 0.1% TFA, 30% to 100% MeCN over 30 min, flowrate 10 mL/min) to yield 41 (1 mg, 0.6 µmol, 4.6% yield). ESI HRMS: calculated [M+2H]2+ 839.9396, found 839.9392.

O O propargyl bromide, K CO H 2 3 H

HO DMF O 62 63

63: 4-hydroxybenzaldehyde (62, TCI, 4.0 g, 32.7 mmol, 1.0 equiv) was dis- solved in DMF at RT (Sigma, 33 mL, 1 M) followed by the addition of K2CO3 (Fisher, 5.4 g, 39.2 mmol, 1.2 equiv) in one portion. Propargyl bromide (80% solution in PhMe, Sigma, 5.8 g, 4.4 mL, 39.2 mmol, 1.2 equiv) was added dropwise at RT to give a brown solution that was stirred for 24 h. The re- action was quenched with aqueous NaHCO3 (sat.) and the resulting solution was extracted with EtOAc (3x). The combined organic extracts were washed with brine and filtered through a sodium sulfate plug, which was subsequently rinsed with EtOAc (2x), and concentrated. The crude product was dissolved in a minimum amount of EtOAc and subsequently precipitated with hexanes. The solid was collected via vacuum filtration through a fritted glass funnel, yielding 63 (4.6 g, 28.7 mmol, 88% yield) as a light orange solid that was car- ried onto the next step without additional purification. 1H NMR (400 MHz;

CDCl3): δ 9.90 (s, 1H), 7.87-7.84 (m, 2H), 7.11-7.07 (m, 2H), 4.78 (d, J = 2.4 13 Hz, 2H), 2.57 (t, J = 2.4 Hz, 1H). C NMR (101 MHz; CDCl3): δ 190.7, 162.3, 131.9, 130.6, 115.2, 77.5, 76.3, 55.9. MALDI HRMS: calculated [M+H]+ 161.0597, found 161.0597.

92 5.1. Materials and methods

S O N O (R)- t-butyl sulfinamide B(OCH CF ) H 2 3 3 H

O THF O 63 64

64: Adapted from literature procedure [181], (R)-t-butyl sulfinamide (TCI, 1.8 g, 15 mmol, 1.2 equiv) and 63 (2.0 g, 12.5 mmol, 1.0 equiv) were charged into a flame dried flask under N2 and dissolved in THF (anhydrous, Sigma,

125 mL, 0.1 M). B(OCH2CF3)3 (7.7 g, 25 mmol, 5.3 mL, 2.0 equiv) was added dropwise at RT and the reaction was stirred for 2 h before being quenched with NaHCO3 (sat.). The resulting solution was extracted with EtOAc (3x), the combined organic extracts were washed with brine and filtered through a sodium sulfate plug, which was subsequently rinsed with EtOAc (2x), and con- centrated. Flash chromatography (EtOAc/hexanes 10:90 to 20:80) afforded 1 64 (3.1 g, 11.8 mmol, 95%) as a colorless oil. H NMR (400 MHz; CDCl3): δ 8.52 (s, 1H), 7.83-7.81 (m, 2H), 7.06-7.04 (m, 2H), 4.76 (d, J = 2.4 Hz, 13 2H), 2.56 (t, J = 2.4 Hz, 1H), 1.25 (s, 9H). C NMR (101 MHz; CDCl3): δ 161.6, 160.8, 131.2, 128.0, 115.2, 77.8, 76.1, 57.6, 55.9, 22.6. ESI HRMS: calculated [M+H]+ 264.1053, found 264.1054.

93 5. APPENDIX

S S N O HN O t- butylbromoacetate zinc, DIBAL-H COOtBu H

O THF O 64 65

65: Adapted from literature procedure [182], zinc (Sigma, powder, 11.0 g, 168.0 mmol, 10.0 equiv) was charged into a flame dried three-neck flask fit- ted with a water condenser and a thermometer under N2 and suspended in anhydrous THF (30 mL). t-Butylbromoacetate (200 µL) was added, followed by DIBAL-H (1 M solution in PhMe, Sigma, 0.8 mL, 0.8 mmol, 0.05 equiv). The solution was warmed to 40 ◦C and additional t-butylbromoacetate (Flu- orochem, 8.2 g, 6.4 mL, 42.0 mmol, 2.5 equiv) was added dropwise while keeping the internal temperature <50 ◦C. After complete addition, the solu- tion was cooled to -10 ◦C and 64 (4.4 g, 16.8 mmol, 1.0 equiv) in THF (5 mL, ∼0.5 M total) was added dropwise. After complete addition, the reaction was warmed to 4 ◦C and stirred for 14 h before being quenched with brine, where the solution solidified. EtOAc was added and the heterogeneous biphasic mix- ture was filtered through celite, followed by EtOAc (3x) rinse. The aqueous layer was separated and the organic layer was washed with citric acid (sat.). The organic extract was filtered through a sodium sulfate plug, which was subsequently rinsed with EtOAc (2x), and concentrated. Flash chromatogra- phy (EtOAc/hexanes 30:70 to 50:50) afforded 65 (5.2 g, 13.7 mmol, 81%) as 1 a colorless oil. H NMR (400 MHz; CDCl3): δ 7.27-7.25 (m, 2H), 6.94-6.92 (m, 2H), 4.71-4.70 (m, 1H), 4.68-4.67 (m, 2H), 4.56 (br d, J = 3.8 Hz, 1H), 2.74-2.72 (m, 2H), 2.51 (t, J = 2.4 Hz, 1H), 1.39 (s, 9H), 1.21 (s, 9H). 13C

NMR (101 MHz; CDCl3): δ 170.6, 157.3, 133.9, 128.7, 115.0, 81.8, 78.6, 75.7,

94 5.1. Materials and methods

56.0, 55.7, 55.2, 43.8, 28.2, 22.8. ESI HRMS: calculated [M+H]+ 380.1890, found 380.1889.

S HN O Cl NH 3 O COOtBu HCl OH

dioxane/H O O 2 O 65 44

44: A flask was charged with 65 (2.8 g, 7.4 mmol, 1.0 equiv) which was subsequently dissolved in dioxane (3.7 mL, 0.5 M). Concentrated HCl (12.1 M, Sigma, 18.4 mL, 73.7 mmol, 10.0 equiv) was added slowly and the resulting solution was stirred for 24 h at RT before being concentrated under a stream of N2. The crude product was dissolved in a minimum amount of CH2Cl2 and subsequently precipitated with Et2O. The solid was collected via vacuum filtration through a fritted glass funnel, yielding 44 (1.8 g, 7.0 mmol, 95% 1 yield) as a light yellow solid. H NMR (400 MHz; D2O): δ 7.49-7.45 (m, 2H), 7.17-7.14 (m, 2H), 4.84 (d, J = 2.4 Hz, 2H), 3.22-3.06 (m, 2H), 2.97 (t, 13 J = 2.4 Hz, 1H), 1.32 (s, 1H). C NMR (101 MHz; D2O): δ 173.6, 157.5, 128.6, 128.5, 115.7, 78.4, 76.8, 56.0, 51.0, 37.7, 24.3 ESI HRMS: calculated [M+H]+ 220.0968, found 220.0971.

95 5. APPENDIX

Cl NH O HONBoc 3 Boc anhydride, NaOH OH OH

THF/H O O 2 O 44 66

66: A flask was charged with 44 (1.0 g, 3.9 mmol, 1.0 equiv) and H2O/THF (1:1, 39 mL, 0.1 M), NaOH (0.3 g, 11.7 mmol, 3.0 equiv) was added in a single portion, and the solution was stirred at RT until homogenous. Di-t-butyl dicarbonate (Chem Impex, 0.9 g, 4.3 mmol, 1.1 equiv) was added in a single portion and the reaction was stirred for 24 h at which point the solution was concentrated by half under a stream of N2. The resulting aqueous solution was washed with Et2O:hexanes (1x, 1:1), acidified to ∼pH 2-3 with H3PO4 and the resulting solution was extracted with CH2Cl2 (3x). The combined organic extracts were filtered through a sodium sulfate plug, which was subsequently rinsed with CH2Cl2 (2x), and concentrated to yield 66 (1.2 g, 3.6 mmol, 92%). 1 H NMR (400 MHz; D6-DMSO): δ 12.15 (s, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.25-7.21 (m, 2H), 6.93-6.89 (m, 2H), 4.85-4.83 (m, 1H), 4.76 (d, J = 2.4 Hz, 2H), 3.53 (t, J = 2.4 Hz, 1H), 2.67-2.51 (m, 2H), 1.34 (br s, 9H). 13C

NMR (101 MHz; D6-DMSO): δ 171.8, 156.1, 154.6, 135.9, 127.5, 114.4, 79.3, 78.1, 77.7, 55.3, 50.5, 41.3, 28.2. ESI HRMS: calculated [M+Na]+ 342.1312, found 342.1311.

96 5.1. Materials and methods

NH 2 N O O N S N H2N O N O

67 O O

NH 2 N HONBoc TFA NH O N 1. EDC•HCl, NHS 3 O O CH Cl S OH 2 2 N O N N H O 2. 17 , Cs CO O 2 3 O CH Cl 2 2 OH OH 66 3. TFA 47

H2O

47: Adapted from literature procedure [183], a flask was charged with 66 (0.32 g, 1.0 mmol, 1.1 equiv), N -hydroxysuccinimide (Sigma, 0.14 g, 1.2 mmol, 1.32 equiv), EDC•HCl (Chem Impex, 0.22 g, 1.2 mmol, 1.32 equiv), and

CH2Cl2 (5 mL, 0.1 M). The reaction was stirred for 4 h before addition of

H2O. The organic layer was separated and the resulting solution was extracted with CH2Cl2 (2x). The combined organic extracts were filtered through a sodium sulfate plug, which was subsequently rinsed with CH2Cl2 (2x), and concentrated to yield the crude NHS ester of 66. To this flask was added

67 (0.35 g, 0.91 mmol, 1 equiv), Cs2CO3 (Chem Impex, 0.32 g, 1 mmol, 1.1 equiv), and DMF (9 mL, 0.1 M), and the reaction was stirred for 12 h before being concentrated. Flash chromatography (MeOH/EtOAc 0:100 to 10:90) afforded partially purified acylated 67, which was subsequently dissolved in

TFA/H2O (5:1, 5 mL) for 48 h before being concentrated under a stream of

N2. Crude product was dissolved in H2O and purified by preparative HPLC

(H2O:MeCN + 0.1% TFA, 5% to 25% MeCN over 30 min, flowrate of 10

97 5. APPENDIX mL/min) to yield 47 (0.09 g, 0.14 mmol, 15% yield) as a colorless solid. 1H

NMR (400 MHz; CD3OD): δ 8.52-8.26 (m, 2H), 7.39-7.33 (m, 2H), 7.04-6.96 (m, 2H), 6.06 (d, J = 4.5 Hz, 1H), 4.74-4.69 (m, 3H), 4.64 (t, J = 7.1 Hz, 1H), 4.56 (t, J = 4.8 Hz, 1H), 4.44-4.32 (ovlp m, 3H), 4.26-4.23 (m, 1H), 3.27 (dt, J = 3.2, 1.6 Hz, 1H), 3.15-2.93 (m, 2H), 2.93-2.89 (m, 1H). 13C NMR (101 MHz; MeOD): δ 169.9, 159.8, 150.0, 145.8, 143.6, 129.8, 129.7, 116.7, 116.6, 90.5, 83.3, 79.4, 77.1, 77.0, 75.8, 72.3, 71.4, 56.7, 52.0, 49.9, 40.5, 39.0. ESI HRMS: calculated [M+H]+ 548.1558, found 548.1558.

NH 2 N O O N S N H2N O N O

67 O O

NH 2 N HONBoc TFA NH O N 1. EDC•HCl, NHS 3 O O CH Cl S OH 2 2 N O N N H O 17 2. , Cs2CO 3 CH Cl 2 2 OH OH 68 3. TFA 46

H2O

46: A flask was charged with 68 (0.25 g, 0.96 mmol, 1.1 equiv), N - hydroxysuccinimide (Sigma, 0.13 g, 1.15 mmol, 1.32 equiv), EDC•HCl (Chem

Impex, 0.22 g, 1.15 mmol, 1.32 equiv) and CH2Cl2 (10 mL, 0.1 M). The re- action was stirred for 4 h before addition of H2O. The organic layer was separated and the resulting solution was extracted with CH2Cl2 (2x). The combined organic extracts were filtered through a sodium sulfate plug, which was subsequently rinsed with CH2Cl2 (2x), and concentrated to yield the

98 5.1. Materials and methods crude NHS ester of 68. To this flask was added 67 (0.31 g, 0.79 mmol, 1 equiv), Cs2CO3 (Chem Impex, 0.28 g, 0.87 mmol, 1.1 equiv), and DMF (8 mL, 0.1 M), and the reaction was stirred for 12 h before being concentrated. Flash chromatography (MeOH:acetone 0:100 to 2:98) afforded partially puri-

fied acylated 17, which was subsequently dissolved in TFA/H2O (5:1, 5 mL) for 48 h before being concentrated under a stream of N2. Crude product was dissolved in H2O and purified by preparative HPLC (H2O:MeCN + 0.1% TFA, 5% to 25% MeCN over 30 min, flowrate of 10 mL/min) to yield 5 (0.14 1 g, 0.22 mmol, 29% yield) as a colorless solid. H NMR (400 MHz; CD3OD): δ 8.52-8.25 (m, 2H), 7.47-7.41 (m, 5H), 6.11 (d, J = 4.5 Hz, 1H), 4.78-4.71 (m, 2H), 4.61 (t, J = 4.8 Hz, 1H), 4.50-4.35 (m, 3H), 4.28 (ddd, J = 4.9, 3.7, 2.9 13 Hz, 1H), 3.20-3.00 (m, 2H). C NMR (101 MHz; CD3OD): δ 170.0, 150.2, 146.0, 143.6, 137.1, 130.7, 130.5, 128.3, 120.5, 90.5, 83.3, 75.8, 72.5, 71.4, 59.7, 52.5, 40.6. ESI HRMS: calculated [M+H]+ 494.1452, found 494.1447.

1. SOCl2 MeOH

2. propargyl bromide, K2CO 3 DMF OH OH H2N N 3. LiOH H O THF/H O (3:1) O 26 2 53

53: 26 (Chem impex, 1 g, 6.1 mmol, 1 equiv) was dissolved in MeOH (15 mL).

SOCl2 (0.66 mL, 9.2 mmol, 1.5 equiv) was added dropwise at RT and the so- lution was stirred for 16 h. The reaction was concentrated under a stream of N2 and taken up in DMF (15 mL). To this flask was added K2CO3 (1 g, 7.3 mmol, 1.2 equiv) and propargyl bromide (0.58 mL, 6.7 mmol, 1.1 equiv) and the reaction was stirred at RT for 16 h before addition of H2O. The solu- tion was adjusted to pH 12 and the product was extracted with EtOAc (3x) and concentrated. The crude material was dissolved in THF/H2O (20 mL,

99 5. APPENDIX

3:1) and LiOH was added (0.18 g, 7.3 mmol, 1.5 equiv). After 10 min, the reaction was filtered and the solvent was removed under a stream of N2. The residue was purified by preparative HPLC (H2O/MeCN + 0.1% TFA, 5-40% MeCN over 45 min, flowrate of 10 mL/min) to yield 53 (12 mg, 0.06 mmol, 1 % yield) as a white solid. 1H NMR (400 MHz; MeOD): δ 7.37-7.25 (m, 5H), 4.38 (t, J = 6.7, 5.8 Hz, 1H), 3.95 (d, J = 2.6 Hz, 2H), 3.38-3.2 (m, 3H).

5.1.2 Biocatalysis

All buffers were prepared using purified H2O (Nanopure system, Barnstead). Buffer components were used as received from specified commercial suppli- ers and used without further purification. TLC, flash chromatography, NMR spectroscopy, and mass spectrometry were performed as described in Sec- tion 5.1.1.

O TFA NH 3 O TycA βpY /TycB1 P-SrfTEP26G ATP, L-Pro HN OH N H O O 2 O 44 50 O

50 (0.15 mmol scale): A 250 mL flask was charged with 44 (0.038 g, 0.15 mmol, 1.0 equiv), L-Pro (Sigma, 0.017 g, 0.15 mmol, 1.0 equiv), and ATP (Sigma, 0.41 g, 0.75 mmol, 5.0 equiv). Buffer was added [2x concentration,

75 mL, bis-Tris propane (200 mM), NaCl (200 mM), MgCl2 (20 mM), TCEP

(2 mM)], followed by H2O (62 mL), and the pH was adjusted to 9.0 with

NaOH. TycAβpY (55 µM stock, 2 µM final, 5.5 mL, 0.2 mol%) and TycB1P-

SrfTEP26G (40 µM stock, 2 µM final, 7.5 mL, 0.2 mol%) were added to give a

100 5.1. Materials and methods

final volume of 150 mL. This solution was aliquoted into falcon tubes (3x 50 mL), which were placed in a preheated 37 ◦C water bath for 20 min before transfer to a 37 ◦C incubator for 36 h. The solution was quenched with acetone (2x v/v) and filtered through a silica plug, washed with acetone (3x). Acetone was evaporated and the resulting aqueous solution was extracted with EtOAc. The combined organic extracts were filtered through a sodium sulfate plug, which was subsequently rinsed with EtOAc (2x), and concentrated. Flash chromatography (EtOAc/acetone 100:0 to 90:10) afforded 50 (0.033 g, 0.11 1 mmol, 73%) as a colourless solid. H NMR (400 MHz; CDCl3): δ 7.26-7.22 (m, 2H), 6.99-6.96 (m, 2H), 5.85 (s, 1H), 4.79 (dd, J = 13.0, 2.2 Hz, 1H), 4.69 (d, J = 2.4 Hz, 2H), 4.58 (dd, J = 8.1, 4.3 Hz, 1H), 3.59-3.56 (m, 2H), 3.18 (t, J = 13.6 Hz, 1H), 2.78-2.68 (m, 2H), 2.53 (t, J = 2.4 Hz, 1H), 2.22-2.13 13 (m, 1H), 1.91-1.84 (m, 2H). C NMR (101 MHz; CDCl3): δ 170.0, 169.0, 157.6, 135.0, 127.1, 115.6, 78.3, 75.9, 59.7, 56.3, 55.9, 46.7, 43.8, 28.7, 23.4. ESI HRMS: calculated [M+H]+ 299.1390, found 299.1395.

O NH 2 O TycA βF/TycB1 P-SrfTEP26G ATP, L-Pro OH HN N

H2O O 45 51

51 (0.25 mmol scale): A 50 mL falcon tube was charged with (S)-β-Phe (45,

Chem Impex, 0.041 g, 0.25 mmol, 1.0 equiv), L-Pro (Sigma, 0.029 g, 0.25 mmol, 1.0 equiv), and ATP (Sigma, 0.69 g, 1.25 mmol, 5.0 equiv). Buffer was added [2x concentration, 25 mL, bis-Tris propane (200 mM), NaCl (200 mM), MgCl2 (20 mM), TCEP (2 mM)], followed by H2O (21 mL), and the pH was adjusted to 9.0 with NaOH. TycAβF (61 µM stock, 2 µM final, 1.7 mL, 0.04 mol%) and TycB1P-SrfTEP26G (44 µM stock, 2 µM final, 2.3 mL,

101 5. APPENDIX

0.04 mol%) were added to give a final volume of 50 mL. The tube was placed in a preheated 37 ◦C) water bath for 36 h and extracted with EtOAc (3x, centrifuged at 4000 x g to break emulsion). The combined organic extracts were filtered through a sodium sulfate plug, which was subsequently rinsed with EtOAc (2x) and concentrated. Flash chromatography (EtOAc/acetone 100:0 to 80:20) afforded 51 (0.042 g, 0.17 mmol, 68%) as a white solid.

(1.75 mmol scale): A 500 mL flask was charged with (S)-β-Phe (45, Chem

Impex, 0.29 g, 1.75 mmol, 1.0 equiv), L-Pro (Sigma, 1.75 g, 1.75 mmol, 1.0 equiv), and ATP (Meiya pharma, 4.8 g, 8.75 mmol, 5.0 equiv). Buffer was added [2x concentration, 175 mL, bis-Tris propane (200 mM), NaCl (200 mM),

MgCl2 (20 mM), TCEP (2 mM)], followed by H2O (148 mL), and the pH was adjusted to 9.0 with NaOH. TycAβF (61 µM stock, 2 µM final, 11.5 mL, 0.04 mol%) and TycB1P-SrfTEP26G (44 µM stock, 2 µM final, 16 mL, 0.04 mol%) were added, to give a final volume of 350 mL. This solution was aliquoted into falcon tubes (7 x 50 mL), which were placed in a preheated 37 ◦C water bath for 20 min, before transfer to a 37 ◦C incubator for 36 h. The solution was quenched with acetone (2x v/v) and filtered through a silica plug, washed with acetone (3x). Acetone was evaporated, and the resulting aqueous solution was saturated with NaCl before extraction with THF (3x). The combined organic extracts were filtered through a sodium sulfate plug, which was subsequently rinsed with EtOAc (2x), and concentrated. Flash chromatography (EtOAc/acetone 100:0 to 80:20) afforded 51 (0.251 g, 1.03 1 mmol, 59%) as a white solid. H NMR (400 MHz; CDCl3): δ 7.41-7.31 (m, 5H), 5.81 (s, 1H), 4.84 (ddd, J = 12.9, 3.0, 0.9 Hz, 1H), 4.61 (dd, J = 8.1, 4.2 Hz, 1H), 3.59 (t, J = 6.8 Hz, 2H), 3.21 (t, J = 13.6 Hz, 1H), 2.80-2.73 (m, 13 2H), 2.24-2.14 (m, 1H), 1.93-1.85 (m, 2H). C NMR (101 MHz; CDCl3): δ

102 5.1. Materials and methods

169.9, 168.8, 141.9, 129.3, 128.6, 125.7, 59.6, 56.8, 46.7, 43.7, 28.6, 23.4 ESI HRMS: calculated [M+H]+ 245.1285, found 245.1287.

NH 3

O O TycA , TycB , TycC H H F PPpY NQYVOL N N OH L-Phe, L-Pro, L-Asn, L-Gln, N N O O H H L-Tyr, L-Val, L-Orn, L-Leu O O NH N O O ATP H H O N N N N H H OH H O O O 2 O H2N O H N O 2 NH 2 O 27 39

39: O-propargyl-L-Tyr (27) was provided by Dr. Hajo Kries [145]. A 500 mL

flask was charged with 27 (see [145], 88 mg, 0.4 mmol, 2 equiv), L-Phe (Sigma,

33 mg, 0.2 mmol, 1 equiv), L-Pro (Sigma, 23 mg, 0.2 mmol, 1 equiv), L-Asn

(Sigma, 26 mg, 0.2 mmol, 1 equiv), L-Gln (Sigma, 29 mg, 0.2 mmol, 1 equiv),

L-Tyr (Sigma, 36 mg, 0.2 mmol, 1 equiv), L-Val (Sigma, 23 mg, 0.2 mmol,

1 equiv), L-Orn (Sigma, 34 mg, 0.2 mmol, 1 equiv), L-Leu (Sigma, 26 mg, 0.2 mmol, 1 equiv), ATP (Meiya pharma, 11 g, 20 mmol, 100 equiv), bis-Tris propane (Appollo scientific, 5.6 g, 20 mmol, 100 equiv), NaCl (Merck, 1.2 g,

20 mmol, 100 equiv), MgCl2 (Sigma, 0.4 g, 2 mmol, 10 equiv), and TCEP

(Sigma, 114 mg, 0.4 mmol, 2 equiv) followed by H2O (to 200 mL), and the pH was adjusted to 8.0 with HCl. The reaction was started by addition of TycAF

(175 µM stock, 0.45 µM final, 0.5 mL, 0.05 mol%), TycBPFpY (57 µM stock,

0.45 µM final, 1.6 mL, 0.05 mol%), and TycCNQYVOL (30 µM stock, 0.2 µM final, 1.2 mL, 0.02 mol%). This solution was aliquoted into falcon tubes (4 x 50 mL), which were placed in a preheated 37 ◦C water bath overnight. During the reaction 39 precipitated out of solution as determined by LC-MS analysis. Liquid was decanted and the precipitate was washed with MeOH

103 5. APPENDIX

(3x). The wash was extracted with EtOAc (3x), concentrated, and purified by preparative HPLC (H2O:MeCN + 0.1% TFA, 45% to 100% MeCN over 50 min, flowrate 10 mL/min) to yield 39 (2 mg, 1.5 µmol, 0.8 % yield) as a white solid. ESI HRMS: calculated [M+Na]+ 1346.6544, found 1346.6530.

Small molecule X-ray crystallography. Single crystals of 50 and 51 were obtained by addition of hexanes to concentrated solutions of Et2O. A suitable crystal was selected and mounted on an XtaLAB Synergy, Du- alflex, Pilatus 300K diffractometer. The crystal was kept at 100.0(1) K dur- ing data collection. Using OLEX2 [184], the structure was solved with the ShelXT [185] structure solution program using Intrinsic Phasing and refined with the XL [186] refinement package using Least Squares minimization. Re- finement statistics for compounds 50 and 51 are summarized in Table 4 and Table 5, respectively.

104 5.1. Materials and methods

Table 4. Crystal data and structure refinement for compound 50.

Empirical formula C17H18N2O3 Formula weight 298.33 Temperature (K) 100.0(1) Crystal system monoclinic Space group P21 a / b / c (A)˚ 10.53110(10) / 6.79350(10) / 10.90170(10) a / b / c (◦) 90 / 112.6870(10) / 90 Volume (A˚3) 719.593(15) Z 2 3 pcalcg (cm ) 1.377 µ (mm-1) 0.778 F(000) 316 Crystal size (mm3) 0.233 x 0.121 x 0.052 Radiation CuKα (λ = 1.54184) 2Θ range for data collection (◦) 8.792 to 158.45 Index ranges -13 ≤ h ≤ 13, -7 ≤ k ≤ 8, -13 ≤ l ≤ 13 Reflections collected 56301

Independent reflections 2972 [Rint=0.0307, Rσ =0.0112] Data / restraints / parameters 2972 / 2 / 202 Goodness-of-fit on F2 1.045 Final R indexes [I≥2σ (I)] R1=0.0266, wR2=0.0705 Final R indexes [all data] R1=0.0266, wR2=0.0706 Largest diff. peak/hole (eA˚-3) 0.18/-0.19 Flack parameter -0.01(4)

105 5. APPENDIX

Table 5. Crystal data and structure refinement for compound 51.

Empirical formula C14H16N2O2 Formula weight 244.29 Temperature (K) 100.0(1) Crystal system orthorhombic

Space group P212121 a / b / c (A)˚ 7.19770(6) / 9.6523(5) / 18.11426(9) a / b / c (◦) 90 / 90 / 90 Volume (A˚3) 1258.485(14) Z 4 3 pcalcg (cm ) 1.289 µ (mm-1) 0.707 F(000) 520 Crystal size (mm3) 0.121 x 0.076 x 0.043 Radiation CuKα (λ = 1.54184) 2Θ range for data collection (◦) 9.766 to 158.08 Index ranges -8 ≤ h ≤ 9, -12 ≤ k ≤ 12, -23 ≤ l ≤ 23 Reflections collected 90375

Independent reflections 2704 [Rint=0.0414, Rσ =0.0105] Data / restraints / parameters 2704 / 1 / 167 Goodness-of-fit on F2 1.064 Final R indexes [I≥2σ (I)] R1=0.0313, wR2=0.0824 Final R indexes [all data] R1=0.0322, wR2=0.0831 Largest diff. peak/hole (eA˚-3) 0.50/-0.16 Flack parameter 0.02(4)

106 5.1. Materials and methods

5.1.3 Cloning

All expression media and buffers were prepared using purified H2O (Nanopure system, Barnstead). Media and buffer components, kits, and enzymes were used as received from specified commercial suppliers. All commercial enzymes were purchased from NEB if not stated otherwise.

All oligonucleotide primers were purchased from Microsynth AG (Switzer- land) and all PCR utilized Phusion HF polymerase (NEB) according to the manufacturer’s instructions using the supplied HF buffer (50µL total volume, 50 ng plasmid DNA, 0.5 µM primer, 0.2 mM dNTPs (Sigma), 1µL Phusion HF). DNA was either purified via agarose gels (1%) and extracted using a Zy- moclean Gel DNA Recovery Kit (Zymo Research) or via spin columns using a DNA clean and concentrator kit (Zymo Research). E. coli transformations were conducted using electrocompetent E. coli HM0079 [149] cells (100 µL, ∼100 ng DNA), followed immediately by SOC (1 mL) rescue and incuba- tion at 37 ◦C, 400 RPM for 1 h before plating onto LB agar containing the respective antibiotic. Single colonies were selected and grown in overnight cultures using LB Miller broth (Merck) containing an appropriate antibiotic. After harvesting cells by centrifugation, plasmid DNA was isolated with a ZR Plasmid Miniprep Classic kit (Zymo Research) according to manufacturer’s specifications. All cloned variants were verified by Sanger sequencing at Mi- crosynth AG (Switzerland).

Yeast transformations for single variants were conducted with EBY100 [187] cells using a Frozen-EZ Yeast Transformation II Kit (Zymo Research) according to manufacturer specifications.

107 5. APPENDIX

Gramicidin S biosynthesis. Plasmids pSU18 grsA for the production of

GrsAF with a C-terminally His6-tag was kindly provided by Dr. Hajo Kries [145].

Plasmid pTrc99a grsB for the production of C-terminally His6-tagged

GrsBPVOL was constructed by Gibson assembly [147] of 3 fragments (pTrc99a, grsB 1, and grsB 2) containing 20 bp overlaps each using a Gibson As- sembly cloning kit (NEB) according to manufacturer specifications. Frag- ment pTrc99a was PCR amplified from pTrc99a tycB1 with primer pair pTrc99a BamHI f / pTrc99a XhoI r and gel purified. Fragments grsB 1 and grsB 2 were PCR amplified from Aneurinibacillus migulanus genomic DNA (ATCC 9999, gramicidin S producer) with primer pairs grsB 1 f / grsB 1 r and grsB 2 f / grsB 2 r, respectively. A. migulanus genomic DNA was isolated and purified by isopropanol precipitation as previously de- scribed [188]. To eliminate a second start site in grsB (Met3574), two over- lapping fragments were PCR amplified from pTrc99a grsB with primer pairs grsB NheI f / grsB M3574L r and grsB M3574L f / grsB BamHI r. The am- plified fragments were gel purified, assembled by overlap PCR with primer pair grsB NheI f / grsB BamHI r, and introduced into pTrc99a grsB via re- striction sites NheI / BamHI to yield plasmid pTrc99a grsB M3574L.

Initial attempts to produce wild-type GrsBPVOL in E. coli HM0079 [149] predominantly afforded a truncated protein after NiNTA purification (SDS-

PAGE app. MW ∼100 kDa). To identify this product and improve GrsBPVOL production, the SDS-PAGE band was cut out of the gel and analyzed by Ed- man degradation and MALDI-TOF MS (Functional Genomics Center, Uni- versity of Zurich). The sequence of the N-terminus (MLEFN) and mass of the protein (MALDI-TOF: 101,331 Da) suggested that the observed product was

108 5.1. Materials and methods produced from an alternative start site at Met3574. The mutation M3574L eliminated production of the 100 kDa side product and GrsBPVOL activity was confirmed by in vitro production of gramicidin S.

Name Sequence 5’-3’

pTrc99a BamHI f A AAA GGA TCC AGA TCT CAT CAC CAT CAC C pTrc99a XhoI r A TGT ACT CAT CTC GAG TTT CCT GTG TGA AAT TGT TAT CC grsB 1 f G AAA CTC GAG ATG AGT ACA TTT AAA AAA GAA CAT GTT CAG G grsB 1 r CCA TTC GTT TTG CCA TAC AGC grsB 2 f GCT GTA TGG CAA AAC GAA TGG grsB 2 r G ATG AGA TCT GGA TCC TTT TAC TAC AAA TGT CCC TTG TAG TAT CTG grsB NheI f G CTT CAG ATG GCT AGC TTT GCC grsB M3574L r CGT ATC ATT GAA CTC AAG TAA GAT TTG TTT CTT CT grsB M3574L f AG AAG AAA CAA ATC TTA CTT GAG TTC AAT GAT ACG grsB BamHI r AGA TCT GGA TCC TTT TAC TAC AAA TGT CCC TTG TAG TAT CTG

Tyrocidine biosynthesis Plasmid pSU18 tycA and pTrc99a tycB1 encod- ing TycAF and TycB1P with C-terminal His6-tags were kindly provided by Grunewald¨ et al. [149].

Plasmid pTrc99a tycB for the production of C-terminally His6-tagged

TycBPFF was constructed by Gibson assembly [147] of 4 fragments (A, B, C, and D) containing 20 bp overlaps each using a Gibson Assembly cloning kit (NEB) according to manufacturer specifications. The individual fragments were designed to introduce a XhoI, a SacI, and a NotI restriction by silent mutations to facilitate subsequent cloning and the ligation sites were chosen accordingly. Fragment A (4,100 bp) was PCR amplified from pTrc99a with primer pair pTrc99a gibson f / pTrc99a gibson r. Fragments B (4,700 bp), C (3,000 bp), and D (3,100 bp) were PCR amplified from Brevibacillus parabrevis

109 5. APPENDIX genomic DNA (ATCC 8185, tyrocidine producer) with primer pairs tycB 1 f / tycB 1 r, tycB 2 f / tycB 2 r and tycB 3 f / tycB 3 r, respectively. B. parabrevis genomic DNA was isolated and purified by isopropanol precipita- tion as previously described [188].

To reprogram TycB3F for O-propargyl-L-Tyr, two fragments were PCR amplified from pTrc99a tycB with primer pairs tycB 3 f / tycB3 WS r and tycB3 WS f / tycB 3 r, respectively. The two fragments were assembled by PCR with primer pair tycB 3 f / tycB 3 r and introduced into pTrc99a tycB via SacI and NotI restriction sites to yield plasmid pTrc99a tycB WS. Plas- mid pTrc99a tycB VCLV for the production of C-terminally His6-tagged

TycBPPβF was cloned analogously from three fragments obtained by PCR with primer pairs tycB 3 f / tycB3 AV r, tycB3 AV f / tycB3 b13b14 r, and tycB3 b13b14 f / tycB 3 r.

For the recombinant production of TycCNQYVOL, plasmid pTrc99a tycC was constructed as described for pTrc99a tycB by Gibson assembly of four fragments. The three fragments encoding TycCNQYVOL were PCR amplified from B. parabrevis gDNA using primer pairs tycC 1 f / tycC 1 r, tycC 2 f / tycC 2 r, and tycC 3 f / tycC 3 r, respectively.

110 5.1. Materials and methods

Name Sequence 5’-3’

pTrc99a gibson f GGA TCC AGA TCT CAT CAC CAT CAC pTrc99a gibson r CAT GGT ACC TTT CCT GTG TGA AAT TG tycB 1 f CAA TTT CAC ACA GGA AAG GTA CCA TGA GTG TAT TTA GCA AAG tycB 1 r CGC CAT CTC GAG CGA TCT TTG CGC CAT GAT C tycB 2 f GCA AAG ATC GCT CGA GAT G tycB 2 r CTC TTT CAT TGA GCT CCC tycB 3 f GGG AGC TCA ATG AAA GAG tycB 3 r GTG ATG GTG ATG AGA TCT GG

tycB3 WS f ATC GGT GTC GGA TAT GTT TGG C tycB3 WS r AAC ATA TCC GAC ACC GAT GCG TC tycB3 AV f GTT TGA CGT GTC GGT GTG GGA TAT GTT TGG tycB3 AV r CAC ACC GAC ACG TCA AAC GAG ATG CTG GC tycB3 b13b14 f CAG AGT GCC TGG TGG CGA CAC TGT GGA AAG CC tycB3 b13b14 r GTC GCC ACC AGG CAC TCT GTT GGG CCG TAT CC

tycC 1 f CAA TTT CAC ACA GGA AAG GTA CCA TGA AAA AGC AGG AAA ACA TC tycC 1 r GAG TGG AAA CTA GTG CGC AAC GAT TCG tycC 2 f TTG CGC ACT AGT TTC CAC TCC GTG CAA G tycC 2 r CAT ACA AGC TAG CAA TTT CCT CGA CGA TG tycC 3 f GGA AAT TGC TAG CTT GTA TGC AGG AAA AC tycC 3 r GTG ATG GTG ATG AGA TCT GGA TCC TTT CAG GAT GAA CAG TTC TTG

Yeast surface display. Plasmid pCT [189] was used for all yeast surface display experiments. The display vector has been modified by Dr. Rebecca Blomberg to simplify cloning as described in her Thesis [190]. In short, the NheI restriction site preceding the sequence encoding the protein of interest was changed to a NdeI restriction site and the XhoI restriction site of pCT were deleted and reinstalled before the sequence encoding the c-myc tag to yield plasmid pCTRB.

111 5. APPENDIX

For the construction of yeast display plasmids with minimal modules, the tycA-AT genes (wild type and W239S) were amplified as two fragments from plasmids pSU18 tycA and pSU18 tycA W239S [123] using primer pairs tycA- AT NdeI f / tycA-AT NdeI del r and tycA-AT NdeI del f / tycA-AT XhoI r to delete an internal NdeI restriction site. The two fragments were gel puri- fied, assembled by overlap PCR with primer pair tycA-AT2 NdeI f / tycA- AT2 NdeI r, and spin column purified. Introduction into the digested pCTRB vector via NdeI and XhoI restriction sites yielded plasmids pCTRB tycA-AT and pCTRB tycA-AT W239S.

The yeast display plasmids for full-length TycApY was assembled from two fragments. Fragment A was PCR amplified from pCTRB tycA-AT W239S with primer pair tycA-AT NdeI f / tyca-AT r. Fragment B was amplified from pSU18 tycA with primer pair tyca-AT f / tyca XhoI r. The assembly product was purified over agarose and introduced into pCTRB via NdeI and XhoI restriction sites to yield pCTRB tyca W239S.

To display the acceptor module with a free N-terminus, a redesigned dis- play vector was constructed according to [179] from five fragments. Fragment A and C encoding the signal peptide and a flexible linker, respectively, were assembled by PCR with primer pairs SP f / SP r and linker f / linker r. The srfC gene without the terminal TE domain (fragment B) was cloned from genomic DNA of Bacillus subtilis (ATCC 21332) with primer pair srfC f / srfC r. Fragments D (encoding Aga2p) and E (part of the vector) were PCR amplified from pCTRB with primer pairs aga2p f / aga2p r and vector f / vector r, respectively. All fragments were purified over agarose and fragments ABCD and CDE were assembled by PCR with primer pairs SP f / aga2p r and linker f / vector r, respectively. The two assembly products were di-

112 5.1. Materials and methods gested with EcoRI / XhoI and XhoI / XbaI, respectively, and introduced into pCTRB digested with EcoRI / XbaI to yield pCTDN srfC.

Name Sequence 5’-3’

tycA-AT NdeI f A TTA CAT ATG GTA GCA AAT CAG G tycA- CGA CTT GCC CTG TGC ATA CGG AAC TAG CGC ATG CTG C AT NdeI del r tycA- TAT GCA CAG GGC AAG TCG AT NdeI del f tycA-AT XhoI r C TGA CTC GAG CGT GCT CTT GAC AAA AAG AGC

tyca-AT r CGT GCT CTT GAC AAA AAG tyca-AT f CTT TTT GTC AAG AGC ACG tyca XhoI r T CGA CTC GAG GCG CAG TGT ATT TGC AAG C

SP f GAT CGA ATT CTA CTT CAT ACA TTT TCA ATT AAG ATG CAG TTA CTT CGC TGT TTT TCA ATA SP r C TCAT ACC AGC TGC TAA AAC GCT AGC AAT AAC AGA AAA TAT TGA AAA ACA GCG AAG TAA CTG C srfC f GC GTT TTA GCA GCT GGT ATG AGT CAA TTT AGC AAG GAT CAG G srfC r CC TCG AGT ACA TCC TGC AAG CCA TCA GAG linker XhoI f GC TTG CAG GAT GTA CTC GAG GGT GGA GGA GGT AGT GAA CAA AAG CTT ATT TCT GAA GAG GAC TTG G linker r GT TGT CAG TTC CTG GCT TCC TCC ACC ACC TGA TCC ACC ACC TCC CAA GTC CTC TTC AGA AAT AAG C aga2p f GG AAG CCA GGA ACT GAC AAC TAT ATG CGA G aga2p r G TTA TCA GAT CTC GAC CTA TTA AAA AAC ATA CTG TGT GTT TAT GGG vector f G TAT GTT TTT TAA TAG GTC GAG ATC TGA TAA CAA CAG TG vector r GTG CTC TAG ATT CCG ATG CTG tycA library construction and transformation into electrocompe- tent EBY100. The tycA library for O-propargyl-(S)-β-Tyr was constructed based on plasmid pCTRB tycA-AT W239S, that was digested with NdeI and XhoI restriction endonucleases and gel purified. The library was PCR ampli- fied as three fragments from pCTRB tycA-AT W239S using primer pairs pC- TRB f / tycA A236X r, tyca A236X f / tycA b13b14 r, and tycA b13b14 f

113 5. APPENDIX

/ pCTRB r, which were individually gel purified and assembled by overlap PCR with primer pair pCTRB f / pCTRB r.

The tycA library for N -propargyl-L-Phe was constructed based on plas- mid pCTRB tycA-AT, that was digested with NdeI and XhoI restriction en- donucleases and gel purified. The library was PCR amplified as three frag- ments from pCTRB tycA-AT using primer pairs pCTRB f / tycA L210X r, tycA L210X f / tycA b13b142 r, and tycA b13b142 f / pCTRB r, which were individually gel purified and assembled by overlap PCR with primer pair pC- TRB f / pCTRB r.

Gel-purified assembly products and digested pCTRB vector were directly transformed into freshly prepared electrocompetent EBY100 [189] cells as de- scribed by Benatuil et al. [191], exploiting 100 bp overlaps of vector and insert for homologous recombination. To that end, yeast extract peptone dextrose (YPD) medium was inoculated 1:40 with fresh preculture from a single clone of S. cerevisiae strain EBY100 to an OD600 of 0.3 and grown in a rotary shaker ◦ at 30 C and 225 rpm until OD600 1.6 was reached. The cells were then col- lected via centrifugation for 2 min at 3,000 x g and 4 ◦C. Yeast cells were washed twice with 50 mL sterile H2O and once with electroporation buffer (1

M sorbitol, 1 mM CaCl2) and resuspended in 40 mL of resuspension buffer

(0.1 M CH3COOLi, 10 mM 1,4-dithiothreitol (DTT)) and incubated for 30 min at 30 ◦C and 225 rpm. The cells were collected, washed once with 50 mL electroporation buffer, and resuspended in 1.5 mL electroporation buffer.

To 400 µL of electrocompetent EBY100 cells were added ∼3 µg of the linear NNK-insert and ∼1.5µg of digested pCTRB vector. The cell suspen- sion was immediately transferred to a electroporation cuvette (0.2 cm), elec-

114 5.1. Materials and methods troshocked with 2.5 kV and 25 µF at 4 ◦C,rescued with 8 mL 1 M sorbitol : YPD (1:1), and incubated for one hour at 30 ◦C and 225 rpm. The cells were collected by centrifugation, resuspended in 250 mL minimal SD-CAA medium (Table 6) and grown in baffled flasks in a rotary shaker at 30 ◦C and 225 rpm overnight. 100 µL of 10-2 to 10-5 dilution series of the transformed cells were plated out on SD-CAA agar to determine transformation efficiency. Background by religation of empty vector was tested with a control trans- formation without insert. The dense library cultures were diluted to a final ◦ OD600 of 0.2 in a total volume of 25 mL SD-CAA and stored at 4 .

Name Sequence 5’-3’

tycA A236X r GTC GAA CGA CAT GCT GGC AAA AAG C tycA A236X f G CTT TTT GCC AGC ATG TCG TTC GAC NNK TCC GTT AGC GAA ATG TTC ATG GCT TTG C tycA b13b14 r TTC CGT CGG GCC GTA TGC ATT TAT GTA CC tycA b13b14 f GG TAC ATA AAT GCA TAC GGC CCG ACG GAA NNK NNK NNK GCG ACG ATC TGG GAA GCC CCG TCC pCTRB r GC TAA AAG TAC AGT GGG AAC AAA GTC G

tycA L210X r ATT GGC GAT GCC TTT ATG TTC tycA L210X f GAA CAT AAA GGC ATC GCC AAT NNK CAA TCC TTT TTC CAA AAT TCG tycA b13b142 r CGT TTC CGT CGG GCC tycA b13b142 f GGC CCG ACG GAA ACG NNK NNK NNK TGC GCG ACG ATC TGG

Sequencing of EBY100 colonies. Single clones of EBY100 were regrown overnight in 3 mL SD-CAA medium and harvested by centrifugation. The plasmid DNA was extracted using the standard ZR Plasmid Miniprep Classic kit (Zymo Research) according to manufacturer specifications with additional mechanical cell disruption in the beginning of the protocol using glass beads (0.5 mm dia., BioSpec). The insert was PCR amplified with primer pair pCTRB f / pCTRB r and sequenced with tycalib seq.

115 5. APPENDIX

Name Sequence 5’-3’

tycalib seq GAC TCC GCC GAT GCA CAA TTC G

Sfp 4’-phosphopantetheinyl transferase. For the production of C- terminally His6-tagged Sfp synthase, the sfp gene was cloned from genomic DNA of Bacillus subtilis (ATCC 21332) into the pMG211 [192] vector using NdeI and XhoI restriction sites. B. subtilis genomic DNA was isolated and purified by isopropanol precipitation as previously described [188] and used to PCR amplify the sfp gene with primer pair sfp NdeI f / sfp XhoI r. The PCR product was spin column purified, digested, and gel purified. Ligation into the digested and gel-purified pMG211 vector yielded plasmid pMG211 sfp.

Name Sequence 5’-3’

sfp NdeI f GAT ATA CAT ATG AAG ATT TAC GGA ATT TAT ATG GAC CG sfp XhoI r GTG CTC GAG AAG CTC TTC GTA CGA GAC CAT TGT G

TycAβpY and TycAβF. To construct plasmids encoding selected TycA variants containing mutations required for β-amino acid incorporation, the C-terminal His6-tag of pSU18 was switched to the N-terminus in order to have a free C-terminus for interaction with downstream mod- ules. To that end, NHis tycA and NHis tycA W239S were amplified with primer pair NHis tycA EcoRI f / NHis tycA BamHI r from pSU18 tycA and pSU18 tycA W239S17, respectively, and spin column purified. The fragments and plasmid pSU18 tycA were digested with EcoRI / BamHI restriction endonucleases, gel purified, and ligated to yield plasmids pSU18NHis tycA and pSU18NHis tycA W239S, respectively. To insert the relevant mutations, tycA was amplified as three fragments from pSU18NHis tycA with primer pairs NHis tycA EcoRI f / tycA A236V r, tycA A236V f / tycA CLV r

116 5.1. Materials and methods

(Trp239) or tycA A236V W239S f / tycA CLV r (Ser239), and tycA CLV f / NHis tycA BamHI r. The PCR amplified fragments were gel purified and assembled by overlap PCR with primer pair NHis tycA EcoRI f / NHis tycA BamHI r. The assembly products and plasmid pSU18NHis tycA were digested with EcoRI / BamHI restriction endonucleases, gel purified, and ligated to yield plasmids pSU18NHis tycA VWCLV (for production of

TycAβF) and pSU18 NHis tycA VSCLV (for production of TycAβpY), respec- tively.

Name Sequence 5’-3’

NHis tycA AAT GCA GAA TTC ATT AAA GAG GAG AAA TTA ACC ATG CAT EcoRI f CAC CAT CAC CAT CAC TCC GGA AGA TCT GTA GCA AAT CAG GCC AAT CTC AT NHis tycA GC AAT TGG ATC CTA GCG CAG TGT ATT TGC AAG CAA TTC BamHI r GAA GAT tycA A236V f CC AGC ATG TCG TTC GAC GTG TCC GTT TGG GAA ATG TTC ATG GCT TTG CTG TCT GG tycA A236V CC AGC ATG TCG TTC GAC GTG TCC GTT AGC GAA ATG TTC W239S f ATG GCT TTG CTG TCT GG tycA A236V r C GTC GAA CGA CAT GCT GG tycA CLV f GCA TAC GGC CCG ACG GAA TGC CTG GTG GCG ACG ATC TGG GAA GCC tycA CLV r TTC CGT CGG GCC GTA TGC

TycAβpY-AN and TycAβF-AN. Plasmids encoding the C-terminally trun- cated A domains TycAβpY-AN and TycAβF-AN for crystallization were con- structed by PCR amplification with primer pair tycA-N f / tycA-N r and the templates pSU18NHis tycA VSCLV and pSU18NHis tycA VWCLV, respec- tively. The PCR products were gel purified, phosphorylated with T4 polynu- cleotide kinase, and ligated with T4 ligase to yield plasmids pSU18NHis tycA- AN VSCLV and pSU18NHis tycA-AN VWCLV, respectively.

117 5. APPENDIX

Name Sequence 5’-3’

tycA-N f TAG GAT CCA GAT CTC ATC ACC ATC tycA-N r GAT TCT GCC GAG AAA CTC GAT C

TycB1P-SrfTEP26G. For optimization of the dipeptide formation assay, Douglas A. Hansen equipped TycB1 with the SrfC thioesterase domain con- taining point mutation P26G for peptide offloading (SrfTEP26G) [169]. the srfC gene was cloned from Bacillus subtilis genomic DNA (ATCC 21332, sur- factin producer) into the pTrc99a37 vector. Genomic DNA was isolated and purified by isopropanol precipitation as previously described [188] and served as a template to PCR amplify the srfC gene with primer pair srfC KpnI f / srfC XbaI r. The PCR product was spin column purified and digested with KpnI and XbaI restriction endonucleases. A second fragment was PCR ampli- fied from pTrc99a tycB1 with primer pair pTrc99a f / pTrc99a r, spin column purified, and digested with MluI and KpnI restriction endonucleases. Both fragments were ligated into the MluI / XbaI-digested and gel-purified pTrc99a vector to yield plasmid pTrc99a srfC with a C-terminal His6-tag.

The P26G mutation was introduced by single primer mutagenesis with primer P26G followed by addition of restriction endonuclease DpnI. Af- ter 2 h incubation at 37 ◦C, DNA was spin column purified and directly transformed into electrocompetent HM007954 cells. The resulting plasmid pTrc99a srfC P26G served as a template for PCR amplification of the TE domain with primer pair srfC TE f / srfC TE BamHI r. Simultaneously, tycB1 was PCR amplified with primer pair tycB1 TE KpnI f / tycB1 TE r and both fragments were gel purified and assembled by overlap PCR with primer pair tycB1 KpnI f / srfC TE BamHI r. The assembled fragment and

118 5.1. Materials and methods pTrc99a srfC were digested with KpnI / BamHI restriction endonucleases, gel purified, and ligated to yield plasmid pTrc99a tycB1 SrfTE P26G.

Name Sequence 5’-3’

srfC KpnI f G AAA GGT ACC ATG TCT CAA TTT AGC AAG GAT CAG G srfC XbaI r T GAC TCT AGA TTA AGC TTA GTG ATG GTG ATG GTG ATG AGA TCT GGA TCC TGA AAC CGT TAC GGT TTG TGT ATT AAG pTrc99a f CGG CGA TTA AAT CTC G pTrc99a r G TCA GGT ACC TTT CCT GTG TGA AAT TGT TAT CC P26G ATT TTC GCA TTT CCG GGG GTC TTG GGC TAT GGC CT srfC TE f GGG GGC TCT GAT GGC TTG CAG GAT GTA srfC TE BamHI r GGT GAT GAG ATC TGG ATC CTG AAA CCG TTA CGG tycB1 TE KpnI f AGG AAA GGT ACC ATG AGT GTA TTT AGC AAA GAA CAA GTT CAG G tycB1 TE r TAC ATC CTG CAA GCC ATC AGA GCC CCC TTC CAC ATA CGC TGC CAG CGC TTG AAT CGT

Display vectors for TycAN pF,1 and TycAN pF,3. Plasmids for dis- play of TycAN pF,1 or TycAN pF,3 were constructed based on plasmid pCTRB tycA-AT, that was digested with NdeI and XhoI restriction endonu- cleases and gel purified. The inserts were PCR amplified as three frag- ments from pCTRB tycA-AT using primer pairs pCTRB f / tycA L210X r, tycA L210A/W f / tycA b13b142 r, and tycA b13b14 GGC/TFC f / pC- TRB r, which were individually gel purified and assembled by overlap PCR with primer pair pCTRB f / pCTRB r. The linear DNA was transformed into chemocompetent EBY100 cells using a Frozen-EZ Yeast Transformation II Kit (Zymo Research) according to manufacturer specifications.

For the production of C-terminally His6-tagged TycAN pF,1, three frag- ments were PCR amplified from pSU18 tycA with primer pairs pSU18 tycA f / tycA L210X r, tycA L210A f / tycA b13b142 r, and tycA b13b14 TFC f / pSU18 tycA r. The gel purified DNA fragments were assembled by PCR

119 5. APPENDIX with pSU18 tycA f / pSU18 tycA r and ligated into pSU18 tycA via EcoRI and BamHI restriction sites.

Name Sequence 5’-3’

tycA L210W f GAA CAT AAA GGC ATC GCC AAT TGG CAA TCC TTT TTC CAA AAT TCG tycA L210A f GAA CAT AAA GGC ATC GCC AAT GCG CAA TCC TTT TTC CAA AAT TCG tycA b13b14 GCA TAC GGC CCG ACG GAA GGC GGC TGC GCG ACG ATC GGC f TGG GAA GC tycA b13b14 GCA TAC GGC CCG ACG GAA ACC TTC TGC GCG ACG ATC TFC f TGG GAA GC pSU18 tycA f T TAC GAA TTC ATT AAA GAG GAG AAA TTA ACC pSU18 tycA r ATC TGG ATC CTA GCG CAG TG

5.1.4 Protein production

Modified Studier medium “ZYM-G” was used for all protein production:

ZY tryptone (Merck, 1% m/m) yeast extract (Merck, 0.5% m/m)

50 x M solution (20 mL/L) Na2HPO4 (Merck, 25 mM)

KH2PO4 (Merck, 25 mM)

NH4Cl (Merck, 50 mM)

Na2SO4 (Merck, 5 mM)

50 x G solution (20 mL/L) glycerol (Sigma, 50% v/v)

500 x Mg(II) solution (2 mL/L) MgSO4 (Merck, 1 M)

The ZY component was autoclaved, while M, G, and Mg(II) components were sterile filtered (TPP, 0.22 µm) before use (note: LB Miller broth (Merck) could be substituted for ZY with no discernible difference.) Isopropyl-β-D- thiogalactopyranoside (IPTG), ampicillin (amp), and chloramphenicol (cam) were obtained from Apollo scientific.

120 5.1. Materials and methods

HM0079 [149] cells transformed with expression plasmids were taken from glycerol cell stocks stored at 80 ◦C and grown overnight at 37 ◦C in LB Miller broth (5 mL) containing the respective antibiotic. The following morning, ZYM-G (800 mL in a 2 L baffled flask) containing the respective antibiotic was inoculated with the overnight culture (1/500 v/v) and shaken at 180 rpm ◦ and 37 C until an OD600 of 5-7 was reached, at which point the cultures were cooled to 20 ◦C. After reaching 20 ◦C, cell cultures were induced with IPTG (300 µM) and shaken at 180 rpm and 20 ◦C for ∼18 h. Cells were pelleted at 5,000 x g and 4 ◦C for 15 minutes, transferred to 50 mL falcon tube(s), and frozen at -20 ◦C.

5.1.5 Protein purification

Imidazole, NaCl, HEPES, and Tris were obtained from Merck. pH of all buffers was adjusted using a WTW bench pH/mV meter (routine meter pH 526) calibrated according to manufacturer specifications. Cell pellets were thawed and suspended in lysis buffer [3 mL / g cells, Tris (50 mM), NaCl (500 mM), glycerol (10% v/v), pH 7.4] via vortex. For lysis, the cell suspension was treated by addition of lysozyme (Sigma, 2 mg/mL), polymyxin B (Apollo, 2 mg/mL), and DNase I (Sigma, 1 mg total) on ice for 30 min before sonication (Dr. Heilscher, UP 200s sonic dismembrator, total sonication time of 6 min at 100% power with care taken to keep internal temperature <15 ◦C). Cellular debris was pelleted in a precooled (4 ◦C) centrifuge at 30,000 x g for 20 min and the supernatant was applied to NiNTA resin (Qiagen, 1 mL / 2 g cells, pre-equilibrated with five column volumes of lysis buffer). Wash buffer [5 volumes, Tris (50 mM), NaCl (500 mM), imidazole (20 mM), glycerol (10% v/v), pH 7.4] was added and the column was gently pressurized with a syringe.

121 5. APPENDIX

The enzyme of interest was eluted with elution buffer [15 mL, Tris (50 mM), NaCl (500 mM), imidazole (300 mM), glycerol (10% v/v), pH 7.4] with gentle syringe pressure.

TycA variants were further purified for reduction of endogenous amino 32 acid background in the P-PPi/ATP exchange assay. To that end, buffer was exchanged to FPLC buffer [Tris (20 mM), NaCl (20 mM), glycerol (5% v/v), pH 8.0] using centrifugal filter units (Merck, Amicon Ultra-15) and the proteins were purified by anion-exchange chromatography (GE Health- care, MonoQ 10/100, linear gradient from 0.05 to 0.5 M NaCl). All other proteins were buffer exchanged to storage buffer [HEPES (50 mM), NaCl (150 mM), glycerol (10% v/v), pH 7.4] and aliquoted. Purified proteins were ◦ immediately flash frozen in liquid N2 and stored at -80 C until use. Pro- tein concentration was determined using a Nanodrop 2000 spectrophotome- ter (Thermo Fisher) corrected by the calculated extinction coefficient (Prot- Param, http://web.expasy.org/protparam/). Protein purity was assessed by SDS-PAGE using a Phast system and 7.5% gels (GE), according to manufac- turer specifications.

5.1.6 Adenylation kinetics

Adenylation kinetics were measured in a 96-well microtiter plate format using 32 the P-PPi/ATP-exchange assay described by Otten et al. [121]. A typi- cal plate contained TycAF and L-Phe in the first row for 100% conversion

(cpmmax) and H2O in the second and last row to prevent contamination and determine background exchange, which was subsequently subtracted from all values. The remaining eight rows allowed determination of Michaelis-Menten

122 5.1. Materials and methods kinetics for two combinations of enzyme and substrate (seven dilutions) at four time points each (0.5-60 min). If necessary, FPLC-purified enzymes were used in order to reduced background exchange with endogenous amino acid contaminations. The maximal substrate concentration and the dilution factor were adjusted to result in the middle row six corresponding to the estimated

K M when possible. Enzyme concentrations and maximal time points were chosen such that reactions did not exceed 10% conversion.

Reactions were started by addition of 40 µL master mix [1.5X, Tris 32 (75 mM, pH 7.4, from 10X stock) ATP (3 mM), PPi (0.15 mM), P-PPi

(∼0.02 µCi), MgCl2 (15 mM), TCEP (1.5 mM), BSA (0.15 mg/mL), and enzyme] to 20 µL amino acid dilutions, positive control, or blank in B&W Isoplate-96 (Perkin Elmer, Waltham, Massachusetts). Following incubation at RT, reactions were quenched with 60 µL quench mix [HClO4 (3.6%, char- coal (1.6%), and Na4P2O7] and diluted to 195 µL with H2O. The charcoal with trapped 32P-ATP was pelleted at 4,000 x g for 3 min, the supernatant was re- moved, and washing was repeated 2 x with 195 µLH2O. Finally, the pellet was resuspended in 150 µL scintillation mix (Optiphase Supermix, Perkin Elmer), mixed vigorously in a plate shaker for 30 min, and repelleted. Scintillation was measured for 2 min per well in a Microplate Scintillation & Luminescence Counter TopCount NXT (Perkin Elmer).

Michaelis-Menten parameters were determined by averaging initial veloci- ties from four time points at less than 10% reaction progress. After subtraction of background, counts per minute (cpm) were converted into initial velocities

(v0) according to the following equation:

123 5. APPENDIX

cpm 1 v0 =   (1) cpmmax t 0.95·[PPi]

(CPMmax: counts per minute at full exchange; t incubation time in min)

To determine steady-state parameters, v0/[E0] ([E0]: enzyme concentra- tion) was plotted against substrate concentration and fitted to the Michaelis- Menten equation in KaleidaGraph (Synergy Software).

5.1.7 High-throughput adenylation and thioesterification assay

Yeast surface display was performed according to Boder and Wittrup [187] with the media and buffers specified in Table 6. Dense cultures of EBY100 cells transformed with the display plasmids were diluted to an OD600 of 0.1 ◦ in SD-CAA medium, grown to an OD600 of 1 at 30 C and 250 rpm, and ◦ incubated at 20 C) and 250 rpm for 16 h in SG-CAA medium (to an OD600 of ∼2). Induced cells (180 µL for single variants, 900 µL for libraries) were collected, pelleted by centrifugation (2 min at 3,000 x g and 4 ◦C), and the su- pernatant was removed. After washing with PMB (180 µL), cells were pelleted by centrifugation, resuspended in PMB (50 µL), supplemented with recombi- nant Sfp (4 µM) and CoASH (Thermo Fisher, 500 µM), and incubated at RT for 15 min to load the ppant arm. For amino acid loading, cells were pelleted by centrifugation and resuspended in PMB (180 µL) containing ATP (Sigma, 100 µM) and the appropriate amino acid. The cell suspension was incubated at RT and amino acid loading was stopped by centrifugation and removal of the supernatant. Subsequently, an azide-alkyne Huisgen cycloaddition [129,

124 5.1. Materials and methods

130] was used to label cells presenting “clickable” amino acids. Cells were incubated with an azide-PEG3-biotin conjugate (Sigma, 20 µM) in PMB (50

µL) and the reaction was started by addition of freshly mixed CuSO4 (Sigma,

100 µM), bathophenanthrolinedisulfonic acid (ABCR, 200 µM) [193], and L- ascorbic acid (ABCR, 1 mM, freshly prepared) at 4 ◦C for 2 h. The reaction was stopped by centrifugation and removal of the supernatant, followed by washing with PMB (2x, 180 µL). The cells were pelleted by centrifugation, resuspended in PMB (20 µL), and incubated with monoclonal mouse anti-c- myc antibody 9E10 (Roche, 250 ng/µL). After incubation at 4 ◦C for 30 min, the cells were pelleted by centrifugation, resuspended in PMB (20 µL), and labelled with goat anti-mouse IgG-FITC antibody (Sigma, 50 ng/µL) and a streptavidin-R-phycoerythrin conjugate (Thermo Fisher, 50 ng/µL) at 4 ◦C for another 30 min. After labelling, the cells were pelleted by centrifugation and washed with PMB (3x 180 µL). Labelled cells were resuspended in PMB and analyzed on a LSRFortessa (BD) or sorted (at ∼2000-5000 events/s) us- ing a FACSAria III (BD) at the Flow Cytometry Core Facility of ETH Zurich. Data was analysed using the FlowJo software (LLC).

The total number of yeast cells displaying constructs typically varied be- tween ∼30%-50% depending on the time of induction. Cell survival was tested by plating a defined number of sorted cells on SD-CAA plates and was >50%, where cells with more display were less viable.

The TycA library for the activation of O-propargyl-(S)-β-Tyr was enriched over three consecutive FACS rounds. In a first round, the library was screened for loading of racemic O-propargyl-β-Tyr (400 µM, RT, ∼2 min) and doubly- labelled yeast cells encoding active TycA variants were sorted into SD-CAA medium supplemented with chloramphenicol (20 µg/mL). A total of 107 cells

125 5. APPENDIX

Table 6. Buffers and media for yeast cell surface display.

SD-CAA D-glucose (Sigma 20 g) pH 6 yeast nitrogen base without amino acids (Sigma, 6.7 g) casamino acids (BD, 5 g)

Na2HPO4·7H2 (Acros, 10.19 g) and NaH2PO4·H2 (ABCR, 8.56 g)

the components were dissolved in H2O (1 L) and filter sterilized

SG-CAA analogous to SD-CAA but with D-galactose (Sigma, 20 g) instead of D-glucose pH 6

PMB NaH2PO4 (ABCR, 7.2 mM)

pH 7.4 Na2HPO4 (Acros, 40 mM) NaCl (Merck, 137 mM) KCl (Sigma, 2.7 mM)

MgCl2 (Sigma, 1 mM)

the components were dissolved in H2O and filter sterilized directly before use the buffer was supplemented with BSA (Sigma, 1 mg/mL) was processed (corresponding to a ∼10-fold oversampling) and the top 0.5% of the library, which exhibited FITC and high R-PE fluorescence, was collected by FACS. Sorted cells were regrown at 30 ◦C and directly subjected to two analogous rounds of FACS and regrowth with increasingly stringent conditions for amino acid loading (second round: 12.5 µM O-propargyl-(S)-β-Tyr, RT, ∼2 min; third round: 1 µM O-propargyl-(S)-β-Tyr in the presence of 1 mM competing 4-methoxy-L-Phe (Bachem), RT, ∼2 min). In both sorting rounds the top 1% doubly-labelled cells were isolated. The enriched library obtained after three rounds of FACS was plated onto SD-CAA medium for analysis of individual variants.

Similarly, the TycA library for the activation of N -propargyl-L-Phe was enriched over three consecutive FACS rounds with constant substrate con- centration (2 mM). In the first round, 2,780 double positive cells were sorted from a total of 8.2 106 events. In rounds two and three, 10,000 and 50,000

126 5.1. Materials and methods double positive cells were collected from 8 x 106 and 7.5 x 106 total events, respectively. The enriched libraries were plated onto SD-CAA medium for analysis of individual variants.

5.1.8 Crystallization and structure determination of β-A domains

Crystallization of the full-length engineered β-A domains (Met1-Glu548) was attempted but did not yield diffracting protein crystals. Accordingly, the respective C-terminally truncated TycA variants (Met1-Ile417, TycAβpY-AN and TycAβF-AN) were produced and crystallized as previously reported [167]. In addition to normal protein purification as described in Section 5.1.5,

TycAβpY-AN and TycAβF-AN were additionally purified by FPLC. To that end, buffer was exchanged to FPLC buffer [Tris (20 mM), NaCl (20 mM), glycerol (5% v/v), pH 8.0] using centrifugal filter units (Merck, Amicon Ultra- 15) and the proteins were purified by anion-exchange chromatography (GE Healthcare, MonoQ 10/100, linear gradient from 0.05 to 0.5 M NaCl). The pooled protein fraction was buffer exchanged to gel-filtration buffer [Tris (20 mM), NaCl (150 mM), pH 8.0] using centrifugal filter units (Amicon Ultra- 15), further purified to homogeneity by gel-filtration chromatography (GE Healthcare, Superose 12), and concentrated to 27 mg/mL.

A Phoenix crystallization robot (Art Robbins Instruments) was used to set up sitting-drop vapor-diffusion experiments in Intelli-Plates R96-3 LV (Hamp- ton Research). Initial crystallization attempts were carried out at 4 ◦C with conditions identified using the JCSG+ Suite (Qiagen) and Crystal Screen 1 and 2 (Hampton Research), and were later refined by grid screens with vary- ing pH and precipitant concentrations. Well-diffracting TycAβpY-AN and

127 5. APPENDIX

Table 7. Data collection, phasing, and refinement statistics.

TycAβF-AN TycAβpY-AN PDB code 5N82 5N81

Data collection

Space group P212121 P212121 Cell dimensions a, b, c (A)˚ 59.6, 60.4, 123.8 59.6, 60.2, 247.8 Resolution (A)˚ 50.0-1.7 (1.81-1.71)a 50.0-1.6 (1.63-1.60)a

Rmerge (%) 6.5 (65.3) 6.5 (75.7) I / σI 19.3 (2.9) 16.3 (2.3) Completeness (%) 98.9 (97.2) 99.9 (100.0) Redundancy 6.8 (6.9) 6.8 (7.0)

Refinement Resolution (A)˚ 43.2-1.7 43.2-1.6 # Reflections 48726 118611

Rwork/Rfree (%) 17.1/20.0 17.6/20.0 # Atoms Protein 3112 6205 Ligand/ion 72 119 Water 328 848 B-factors Protein 24.9 20.7 Ligand/ion 24.4 21.9 Water 33.7 30.9 Root mean square deviations (r.m.s.d) Bond lengths (A)˚ 0.006 0.006 Bond angles (◦) 0.918 0.883

a One crystal was used for data collection; values in parentheses correspond to the highest resolution shell.

128 5.1. Materials and methods

◦ TycAβF-AN crystals were obtained with 27 mg/mL enzyme at 4 C in crys- tallization buffer [Bis-Tris (100 mM), (NH4)2SO4 (200 mM), PEG3350 (Sigma Aldrich, 25% (v/v)), pH 5.5] containing 2 mM ligand 46 or 47, respectively, using the sitting-drop vapor-diffusion method. The crystals were transferred into reservoir solutions with 20% (v/v) glycerol as cryoprotectant and flash ◦ cooled at -173 C in a N2 stream. X-ray diffraction data sets were collected at the X06SA macromolecular crystallography beamline of the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) using an EIGER X 16M detector and wavelengths of 1.0000 A.˚

The diffraction data for TycAβpY-AN and TycAβF-AN were processed and scaled using the XDS [194] program package. Initial phases were de- termined by molecular replacement with Phaser [195] using the structure of the GrsAF-A domain (PDB 1AMU) [74] as a search model. The struc- ture was modified manually with Coot [196] and refined with PHENIX [197]. The final crystal data and intensity statistics are summarized in Table 7.

The final model of TycAβF-AN (PDB 5N82) consists of a single chain con- taining residues 20-180 and 183-417, ligand 46, and 328 molecules of water.

The final model of TycAβpY-AN (PDB 5N81) consists of two nearly identical chains (A and B, RMSD of 0.4 A)˚ containing residues 19-179 and 183-417, ligand 47, and 848 molecules of water. A structural similarity search was performed using Dali [198]. The cavity volumes were calculated with CASTP (http://cast.engr.uic.edu/cast/). All crystallographic figures were prepared with PyMOL (DeLano Scientific, http://www.pymol.org).

129 5. APPENDIX

5.1.9 Dipeptide synthetase reactions

DKP assays were performed based on the system described by Grunewald¨ et al. [149] at 37 ◦C (water bath) in a volume of 600 µL, and initiated by the addition of ATP (5 mM). All reactions contained TycB1P (5 µM) and the respective TycA variant. Reactions were monitored at 30, 60, 120, and 300 min time points where 100 µL of the reaction where denatured for 3 min at 95 ◦C and clarified by centrifugation at maximum speed for 3 min. The supernatant was analyzed by HPLC (Ultimate 3000, Dionex) using a Kinetex XB-C18 column (100 x 4.6 mm, 2.6 µm): 10 µL injection, monitoring 220 nm, solvent A = H2O + 0.1% TFA, solvent B = MeCN + 0.1% TFA, flow rate = 0.75 mL/min, 0-4 min ramp from 5% to 50% B, 4-6 min ramp to 95% B, 6-7 min = 95% B, 7-9 min reequilibration = 5%. Product formation was quantified by depletion of the L-Phe (analog) using linear standard curves. D-Phe-L-Pro and O-propargyl-D-Tyr-L-Pro DKP co-eluted with authentic standards [123]. All DKP products were confirmed by ESI-QTOF (Bruker Daltonics, maXis) at the MS Service of the LOC.

Reaction conditions: 50 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 1 mM TCEP, 5 mM ATP, 1 mM L-Pro, 1 mM L-Phe or analog, pH = 8.

DKP formation by yeast-displayed TycA constructs was performed analo- gous but without reducing agent. After overnight incubation at 37 ◦C (incu- bator), yeast cells were removed by centrifugation and reactions were analyzed by LC-MS (Bruker Daltonics, maXis, MS Service LOC) using a Kinetex XB-

C18 column (100 x 4.6 mm, 2.6 µm): solvent A = H2O, solvent B = MeCN + 0.1% FA, flow rate = 0.6 mL/min, 0-10 min isocratic 15% B.

130 5.1. Materials and methods

To facilitate release of backbone-modified dipeptides, TycB1P was fused to the robust surfactin TE domain [199] containing the P26G [169] mutation that is known to promote hydrolysis. Surprisingly, the bimodular synthetases consisting of the β-TycA variants and TycB1P-SrfTEP26G predominantly cat- alyzed formation of the cyclic βα-dipeptide.

Dipeptide synthetase reactions with backbone-modified amino acids were performed at 37 ◦C (water bath) in a volume of 250 µL, and initiated by the addition of substrate amino acids or mixtures thereof. All reactions con- tained TycB1P-SrfTEP26G (2 µM) and the respective TycA variants (2 µM). Reactions were performed in triplicate and monitored at 15, 30, and 45 min time points where 50 µL of the reaction mixture was aliquoted into ice-cold MeOH (150 µL), vortexed, and clarified by centrifugation (21,000 x g, 5 min, 4 ◦C). Product formation was quantified by HPLC (Ultimate 3000, Dionex) using a Reprosil Gold 120 C-18 column (100 x 2.1 mm, 3 µm): 5 µL injection, monitoring 220 nm, solvent A = H2O + 0.1% TFA, solvent B = MeCN + 0.1% TFA, flow rate = 0.75 mL/min, 0-0.2 min = 5% B, 0.2-4.75 min ramp to 40% B, 4.75-5 min ramp to 100% B, 5-5.5 min = 100% B, 5.5-5.6 min ramp to 5 % B, 5.6-7 min reequilibration = 5% B. Calibration curves generated from 5 concentrations of authentic standards (51, 50, and DKPs [123], from 4% to 200% conversion) were linear and used to quantify reaction progress.

Reactions for TycAN pF,1 were only analyzed qualitatively relative to maximal

L-Phe-L-Pro-DKP formation.

Reaction conditions: 100 mM Bis-Tris propane, 100 mM NaCl, 10 mM

MgCl2, 5 mM ATP, 1 mM L-Pro, 1 mM L-Phe analog(s), pH = 9.

131 5. APPENDIX

For total turnover number (TTN) determination of dipeptide synthetases, three concentrations were examined [amino acids (5 mM, 10 mM, 15 mM) and corresponding ATP (25 mM, 50 mM, 75 mM)] under otherwise identical conditions, allowing theoretical maximum TTNs of 2500, 5000, and 7500, respectively.

5.1.10 In vitro gramicidin S biosynthesis

Enzymatic reactions for in vitro gramicidin S and backbone-modified pen- tapeptide production were performed at 37 ◦C (water bath) in a volume of 250 µL, and initiated by the addition of substrate amino acids or mixtures thereof. All reactions contained GrsBPVOL (4 µM) and GrsAF, TycAF, or

TycAβF (1 µM). Reactions were performed in triplicate and monitored at 15, 30, and 45 min time points where 50 µL of the reaction mixture was aliquoted into ice-cold MeOH (150 µL), vortexed, and clarified by centrifugation (21,000 x g, 5 min, 4 ◦C).

Product formation was quantified by HPLC (Ultimate 3000, Dionex) using a Kinetex XB-C18 column (100 x 4.6 mm, 2.6 µm): 10 µL injection, monitor- ing 220 nm, solvent A = H2O + 0.1% TFA, solvent B = MeCN + 0.1% TFA, flowrate = 1.5 mL/min, 0-1 min = 5% B, 1-4 min ramp to 40% B, 4-5.5 min ramp to 95% B, 5.5-7 min = 95% B, 7-7.1 min ramp to 5 % B, 7.1-8.5 min reequilibration = 5% or by LC-MS (Waters H-class UPLC/SQD-2) using an Acquity UPLC BEH C-18 column (50 x 2.1 mm, 1.7 µm), 1 µL injection, mon- itoring ESI+ for [M+2H]2+ = 295.5±2 m/z or 572±2 m/z, solvent A = H2O + 0.1% TFA, solvent B = MeCN + 0.1% TFA, flow rate = 1 mL/min, initial conditions = 5% B, 0-1.5 min ramp to 80% B, 1.5-2 min ramp to 100% B, 2-2.2

132 5.1. Materials and methods min = 100% B, 2.2-2.3 min ramp to 5 % B, 2.3-3 min reequilibration = 5%. Calibration curves generated from 5 concentrations of the authentic standards

[gramicidin S (32) was purchased from Sigma, (S)-β-Phe-L-Pro-L-Val-L-Orn-

L-Leu (52) was synthesized by standard FMOC SPPS] were linear and used to quantify reaction progress.

Reaction conditions: 100 mM Bis-Tris propane, 100 mM NaCl, 10 mM

MgCl2, 20 mM ATP, 1.5 mM L-Pro, 1.5 mM L-Val, 1.5 mM L-Orn, 1.5 mM

L-Leu, and 1 mM L-Phe or (S)-β-Phe, pH = 8.

Incorporation of p-substituted L-Phe analogs into gramicidin S (32) was tested with N-terminally His6-tagged TycApY and GrsBPVOL as described in Section 5.1.9. Reactions were started by addition of ATP (20 mM), run overnight at 37 ◦C (incubator), and protein was denatured for 3 min at 95 ◦C. Product formation was analyzed by HPLC (Ultimate 3000, Dionex) using a Kinetex XB-C18 column (100 x 4.6 mm, 2.6 µm): 10 µL injection, monitoring

220 nm, solvent A = H2O + 0.1% TFA, solvent B = MeCN + 0.1% TFA, flow rate = 1.5 mL/min, 0-0.5 min = 5% B, 0.5-4 min ramp to 40% B, 4-5.5 min ramp to 100% B, 5.5-6.6 min = 100% B, 6.6-7 min ramp to 5% B, 7-8 min reequilibration = 5% B. Products were identified with an authentic gramicidin S(32, Sigma) standard and by LC-QTOF (Bruker Daltonics, maXis) at the MS Service of the LOC (Fig. 32).

Reaction conditions: 100 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 20 mM ATP, 1 mM L-Pro, 1 mM L-Val, 1 mM L-Orn, 1 mM L-Leu, 0.5 mM

L-Phe or analog, pH = 8.

133 5. APPENDIX

5.1.11 In vivo NRP production

For in vivo production of gramicidin S analog 34 and β-amino acid con- taining pentapeptide 52, LB (5 mL) containing ampicillin (250 µ/mL) and chloramphenicol (37.5 µ/mL) was inoculated with HM0079 [149] cells trans- formed with plasmids pTrc99a grsB M3574L and pSU18NHis tycA W239S or pSU18NHis tycA VWCLV, respectively and grown at 37 ˇrC,250 RPM until an OD600 = 1 was reached.

Modified Studier medium“ZYM-G”(30 mL in a 300 mL flask, as described in Section 5.1.4) supplemented with L-Phe analog (1 mM), L-Pro (1 mM),

L-Val (1 mM), L-Orn (1 mM), L-Leu (1 mM), ampicillin (250 µg/mL), and chloramphenicol (37.5 µg/mL) was inoculated with the starter culture (1/250 ◦ v/v) and incubated at 37 C, 280 RPM. Upon reaching OD600 = 1.75, the cultures were cooled (20 ◦C, 280 RPM) and induced with IPTG (100 µM).

Culture medium was sampled at 24 h and 48 h (100 µL culture into 900 µL MeOH), vortexed vigorously, and clarified by centrifugation (21,000 x g, 5 min, RT). For 52, product formation was quantified by LC-MS (Waters H-class UPLC/SQD-2) using an Acquity UPLC BEH C-18 column (50 x 2.1 mm, 1.7 µm), 500 nL injection, monitoring ESI+ for [M+2H]2+ = 295.2±2 m/z, solvent A = H2O + 0.1% TFA, solvent B = MeCN + 0.1% TFA, flow rate = 0.5 mL/min, initial conditions = 5% B, 0-4 min ramp to 30% B, 4-5 min ramp to 100% B, 5-6 min = 100% B, 6-6.5 min ramp to 5 % B, 6.5-7 min reequilibration = 5%. A calibration curve generated from 5 concentrations of the authentic standard (1-50 µM) was linear and used to calculate the titer of (S)-β-Phe-L-Pro-L-Val-L-Orn-L-Leu (52). Analogoulsy, 34 formation was analyzed qualitatively by LC-MS LC-MS (Waters H-class UPLC/SQD-2)

134 5.1. Materials and methods using an Acquity UPLC BEH C-18 column (50 x 2.1 mm, 1.7 µm), 1 µL injection, monitoring ESI+ for [M+2H]2+ = 295.5±2 m/z or 572±2 m/z, solvent A = H2O + 0.1% TFA, solvent B = MeCN + 0.1% TFA, flow rate = 1 mL/min, initial conditions = 5% B, 0-1.5 min ramp to 80% B, 1.5-2 min ramp to 100% B, 2-2.2 min = 100% B, 2.2-2.3 min ramp to 5 % B, 2.3-3 min reequilibration = 5% (Fig. 33).

5.1.12 Tyrocidine biosynthesis

Enzymatic reactions for in vitro tyrocidine production were performed at 37 ◦C (water bath) in a volume of 200 µL, and initiated by the addition of sub- strate amino acids or mixtures thereof. All reactions contained TycCNQYVOL

(1 µM), TycBPFF or TycBPFpY (1 µM), and TycAF or TycApY (1 µM). Reac- tions were performed in duplicate and monitored after 2 h where 50 µL of the reaction mixture was aliquoted into ice-cold MeOH (150 µL), vortexed, and clarified by centrifugation (21,000 x g, 5 min, 4 ◦C). Product formation was analyzed by LC-MS (Waters H-class UPLC/SQD-2) using an Acquity UPLC BEH C-18 column (50 x 2.1 mm, 1.7 µm), 1 µL injection, monitoring ESI+ for [M+2H]2+ = 295.5±2 m/z or 572±2 m/z, solvent A = H2O + 0.1% TFA, solvent B = MeCN + 0.1% TFA, flow rate = 1 mL/min, initial conditions = 5% B, 0-1.5 min ramp to 80% B, 1.5-2 min ramp to 100% B, 2-2.2 min = 100% B, 2.2-2.3 min ramp to 5 % B, 2.3-3 min reequilibration = 5%.

Reaction conditions: 100 mM Bis-Tris propane, 100 mM NaCl, 10 mM

MgCl2, 0.5 mM TCEP, 10 mM ATP, 4 mM L-Phe, 2 mM L-Pro, 2 mM, 2 mM

L-Asn, 2 mM L-Gln, 2 mM L-Tyr, 2 mM L-Val, 2 mM L-Orn, 2 mM L-Leu, and 1 mM L-Phe or analog, pH = 8.

135 5. APPENDIX

5.2 LC-MS

Biosynthesis of gramicidin S and analogs

CBA gramicidin S (32 )

calculated [M+2H]2+ 571.3602

found 571.3598

C B A Cl-gramicidin S (34 )

calculated [M+2H]2+ 605.3213

found 605.3222

CB A Br-gramicidin S (35 )

calculated [M+2H]2+ 650.2697

found 650.2699

C B A I-gramicidin S (36 )

calculated [M+2H]2+ 697.2569

found 697.2546

CB A propTyr-gramicidin S (37 )

calculated [M+2H]2+ 625.3708

found 625.3705

Figure 32. LC-MS analysis of gramicidin S biosynthesis by TycApY and GrsBPVOL supplemented with ATP, necessary amino acids, and L-Phe (26), p-chloro-L-Phe (33), p-bromo-L-Phe (28), p-iodo-L-Phe (29), or O-propargyl-L-Tyr (27), respectively. A: cyclic decapeptide, B: DKP, C: linear pentapeptide and side-product from ornithine cyclization.)

136 5.2. LC-MS

DH IF170518_invivo_lsc_40h_5 Sm (Mn, 1x1) 1: Scan ES+ 1.56 100 606 1536858 3.88e7 Area

1.59 901068 %

1.65 0.47 137144 378596 0.89 2.49 89415 1.69 24213 79623 0 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170518_invivo_lsc_40h_5 Sm (Mn, 1x1) 1: Scan ES+ 0.98 279 2320477 5.74e7 Area %

0.36 292566 1.10 0.89 286040 131810 1.68 1.87 43591 26074 0 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170518_invivo_lsc_40h_5 1: Scan ES+ 0.40 TIC 3.70e9 %

0.97

0.78 1.92 2.04 1.59 1.45 9 Time 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Figure 33. Biosynthesis of gramicidin S analog 34 by E. coli HM0079 cells co-podrucing 2+ TycApY and GrsBPVOL and supplemented with p-chloro-L-Phe (calculated [M+2H] 605.3).

137 5. APPENDIX

Biosynthesis of tyrocidine A (11) and analogs

LC-MS analysis of tyrocidine biosynthesis by the reprogrammed synthetases supplemented with all amino acids and ATP after 2 h at 37 ◦C.

IF170829 in vitro ssc 2h a9 Sm (Mn, 1x1) 1: Scan ES+ 1.73 100 636 13471640 2.79e8 Area % 1.79 2199815

1.65 1.89 275481 154954 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a9 Sm (Mn, 1x1) 1: Scan ES+ 1.62 100 644 23243064 4.45e8 Area %

0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a9 Sm (Mn, 1x1) 1: Scan ES+ 1.00 100 245 606662 1.87e7 Area

1.03 116716 %

1.04 0.94 57397 71027 2.28 1.78 2.13 1.96 27131 2.44 28138 19924 28681 25319 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a9 1: Scan ES+ TIC 4.29e9 % 1.61 1.73

0.53 0.87 1.34 2.46 5 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

2+ Figure 34. Tyrocidine A (11, calculated [M+2H] 635.8) biosynthesis by TycApY/ 2+ TycBPFF/ TycCNQYVOL (Y-tyrocidine: calculated [M+2H] 643.8).

138 5.2. LC-MS

IF170829 in vitro ssc 2h a2 Sm (Mn, 1x1) 1: Scan ES+ 1.72 100 636 28207780 5.67e8 Area

% 1.77 6812456

1.86 624066 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a2 Sm (Mn, 1x1) 1: Scan ES+ 1.53 100 644 3954918 9.68e7 Area %

0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a2 Sm (Mn, 1x1) 1: Scan ES+ 1.00 100 245 1877391 5.17e7 Area %

2.24 43496 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a2 1: Scan ES+ TIC 4.09e9

1.72 %

0.50 1.53 0.78 1.00 4 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

2+ Figure 35. Tyrocidine A (11, calculated [M+2H] 635.8) biosynthesis by TycAF/ 2+ TycBPFpY/ TycCNQYVOL (Y-tyrocidine: calculated [M+2H] 643.8).

139 5. APPENDIX

IF170829 in vitro ssc 2h a12 Sm (Mn, 1x1) 1: Scan ES+ 1.72 100 636 2300320 7.06e7 Area

% 1.76 627751 1.65 1.09 26384 1.81;94792 29154 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a12 Sm (Mn, 1x1) 1: Scan ES+ 1.61 100 644 2245973 5.97e7 Area

1.64

% 494218

1.70 0.95 1.48 61286 1.95 2.28 16828 24291 13966 15571 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a12 Sm (Mn, 1x1) 1: Scan ES+ 1.77 100 653 21294482 3.92e8 Area

% 1.83 3254529

1.93;100591 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a12 Sm (Mn, 1x1) 1: Scan ES+ 1.14 100 279 3632510 1.06e8 Area % 0.99 835938 1.23 0.59 31508 1.91 2.12 2.48 139325 137420 112763 62199 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a12 1: Scan ES+ 0.85 TIC 5.03e9 % 1.76

1.14 1.61 3 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Figure 36. TycApY/ TycBPFF/ TycCNQYVOL supplemented with p-chloro-L-Phe (33) (Cl-tyrocidine: calculated [M+2H]2+ 652.8).

140 5.2. LC-MS

IF170829 in vitro ssc 2h a4 Sm (Mn, 1x1) 1: Scan ES+ 1.72 100 636 1405139 3.39e7 Area % 1.80 174098

0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a4 1: Scan ES+ 1.55 100 644 9.07e6

1.87 % 1.58 2.32 1.07 2.43 1.52 1.69 2.07 2.36 1.44 1.76 1.91 2.14 2.23 2.49 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a4 Sm (Mn, 1x1) 1: Scan ES+ 1.77 100 653 16149961 3.57e8 Area

1.83 % 3217891 1.85 2745640

0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a4 Sm (Mn, 1x1) 1: Scan ES+ 1.00 100 245 1612770 5.31e7 Area %

1.11 1.63 0.65 0.87 1.74 2.45 5710 1.38 21172 27914 49274 22381 29622 32676 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a4 1: Scan ES+ 0.86 TIC 5.38e9 % 1.77

3 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Figure 37. TycAF/ TycBPFpY/ TycCNQYVOL supplemented with p-chloro-L-Phe (33) (Cl-tyrocidine: calculated [M+2H]2+ 652.8).

141 5. APPENDIX

IF170829 in vitro ssc 2h a16 Sm (Mn, 1x1) 1: Scan ES+ 1.73 100 636 2047103 4.41e7 Area % 1.80 231012 1.20 1.44 1.56 2.32 21911 17651 31952 16128 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a16 Sm (Mn, 1x1) 1: Scan ES+ 1.61 100 644 2197485 3.95e7 Area %

1.72 47278 2.27 2.42 20598 25024 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a16 Sm (Mn, 1x1) 1: Scan ES+ 1.78 100 675 15625495 3.02e8 Area

1.83 % 3923226

1.95 155299 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a16 Sm (Mn, 1x1) 1: Scan ES+ 1.17 100 323 2737729 8.00e7 Area %

1.25 105122 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a16 1: Scan ES+ 0.89 TIC 5.36e9

% 1.77

1.17 3 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Figure 38. TycApY/ TycBPFF/ TycCNQYVOL supplemented with p-bromo-L-Phe (28) (Br-tyrocidine: calculated [M+2H]2+ 674.8).

142 5.2. LC-MS

IF170829 in vitro ssc 2h a8 Sm (Mn, 1x1) 1: Scan ES+ 1.72 100 636 803546 1.66e7 Area % 1.82 1.41 127444 1.04 92212 1.97 2.46 0.58 0.81 1.55 1.64 2.21 31566 1.37;7830 6320 14644 9158 5114 49324050 3986 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a8 1: Scan ES+ 1.52 100 644 1.60 2.62e6

2.36 % 1.62 0.94 1.28 1.78 2.26 1.00 2.21 2.46 1.96 2.13 1.70 1.12 0.61 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a8 Sm (Mn, 1x1) 1: Scan ES+ 1.79 100 675 14979969 3.15e8 Area

1.83 % 3792031

1.88 1561869 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a8 Sm (Mn, 1x1) 1: Scan ES+ 0.89 100 245 21092126 5.19e8 Area %

1.00 3372300

0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a8 1: Scan ES+ 0.89 TIC 6.23e9 % 1.78

2 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Figure 39. TycAF/ TycBPFpY/ TycCNQYVOL supplemented with p-bromo-L-Phe (28) (Br-tyrocidine: calculated [M+2H]2+ 674.8).

143 5. APPENDIX

IF170829 in vitro ssc 2h a11 Sm (Mn, 1x1) 1: Scan ES+ 1.73 100 636 7840867 1.63e8 Area

1.79 % 1399835

1.65 235733 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a11 Sm (Mn, 1x1) 1: Scan ES+ 1.61 100 644 10142158 2.22e8 Area

% 1.66 1419954 1.75 39542 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a11 Sm (Mn, 1x1) 1: Scan ES+ 1.73 100 663 16474706 3.87e8 Area

1.77 % 3516312 1.80 1.63 2516160 138190 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a11 Sm (Mn, 1x1) 1: Scan ES+ 1.09 100 299 2186287 6.32e7 Area %

1.15 1.43 2.07 1.50 1.83 2.02 2.29 85731 16558 15137 19422 24735 15691 15116 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a11 1: Scan ES+ 0.85 TIC 3.80e9

1.72 %

1.61 1.09 1.34 4 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Figure 40. TycApY/ TycBPFF/ TycCNQYVOL supplemented with O-propargyl-L-Tyr (27) (pY-tyrocidine: calculated [M+2H]2+ 662.8).

144 5.2. LC-MS

IF170829 in vitro ssc 2h a3 Sm (Mn, 1x1) 1: Scan ES+ 1.55 100 644 243751 8.45e6 Area

% 1.61 49017 1.05 1.15 2.06 2.27 1.33 1.85 24449 18518 24824 27181 2.42 10611 18466 8660 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a3 Sm (Mn, 1x1) 1: Scan ES+ 1.73 100 636 2635324 6.01e7 Area

1.77 % 436610

1.88 1.30 1.42 36616 2.41 38268 30255 25961 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a3 Sm (Mn, 1x1) 1: Scan ES+ 1.67 100 663 47949856 8.14e8 Area %

1.79 1031346 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a3 Sm (Mn, 1x1) 1: Scan ES+ 1.00 100 245 5202355 1.36e8 Area %

1.11 2.36 126008 38902 2.46 43716 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a3 1: Scan ES+ 0.85 TIC 4.21e9 1.67 %

0.52 1.00 5 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Figure 41. TycAF/ TycBPFpY/ TycCNQYVOL supplemented with O-propargyl-L-Tyr (27) (pY-tyrocidine: calculated [M+2H]2+ 662.8).

145 5. APPENDIX

IF170829 in vitro ssc 2h a18 Sm (Mn, 1x1) 1: Scan ES+ 1.73 100 636 12176337 2.69e8 Area

% 1.78 1898022 1.64 1.80 255127 786791 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a18 Sm (Mn, 1x1) 1: Scan ES+ 1.61 100 644 11016062 1.96e8 Area %

1.76 130148 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a18 Sm (Mn, 1x1) 1: Scan ES+ 1.77 100 688 2248899 4.86e7 Area % 1.85 175641

0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a18 Sm (Mn, 1x1) 1: Scan ES+ 1.00 100 245 495020 1.60e7 Area 1.03 183704 % 0.95 1.06 65426 104561 2.26 0.62 2.08 42716 25939 20103 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a18 1: Scan ES+ 1.03 TIC 5.71e9 %

1.61 1.72 0.87 1.33 2 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Figure 42. TycApY/ TycBPFF/ TycCNQYVOL supplemented with p-benzoyl-L-Phe (31) (Bz-tyrocidine: calculated [M+2H]2+ 687.8).

146 5.2. LC-MS

IF170829 in vitro ssc 2h a17 Sm (Mn, 1x1) 1: Scan ES+ 1.73 100 636 6932514 1.42e8 Area

1.79 % 1703287

1.88 170244 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a17 Sm (Mn, 1x1) 1: Scan ES+ 1.54 100 644 1107270 2.46e7 Area %

0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a17 Sm (Mn, 1x1) 1: Scan ES+ 1.72 100 688 15847810 3.65e8 Area

1.76 6348230 %

1.88 209643 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a17 1: Scan ES+ 2.48 100 349 6.51e6

1.20 1.17 0.64 0.73 0.80 % 1.15 1.41 2.46 0.86 1.01 1.77 1.38 1.55 1.62 1.85 2.05 2.12 1.96 2.26 2.32 0 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 IF170829 in vitro ssc 2h a17 1: Scan ES+ 1.02 TIC 5.26e9 % 1.72

0.95 1.54 2 Time 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Figure 43. TycAF/ TycBPFpY/ TycCNQYVOL supplemented with p-benzoyl-L-Phe (31) (Bz-tyrocidine: calculated [M+2H]2+ 687.8).

147 5. APPENDIX

Biosynthesis of Cy5-tyrocidine analog 40

IF170924 click rxn dye ptyc 1h45min1 Sm (Mn, 1x1) 1: Scan ES+ 1.47 100 663 146183 6.82e6 Area

2.46 13471 %

1.29 45490

0 -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 IF170924 click rxn dye ptyc 1h45min1 Sm (Mn, 1x1) 1: Scan ES+ 1.35 100 1080 3571811 7.96e7 Area %

1.41 277228 1.89 1.55;23421 23719 0 -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 IF170924 proptyrocidine std new 100uM Sm (Mn, 1x1) 1: Scan ES+ 1.61 100 663 33397336 6.58e8 Area %

0 Time -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

Figure 44. Cu(I)-catalyzed cycloaddition of tyrocidine analog 39 and sulfo-Cy5-azide (Jena Bioscience) to afford the fluorescent derivative 40 (calculated [M+2H]2+ 1079.5).

148 5.2. LC-MS

Biosynthesis of N -methyl-D-Phe-containing decapeptide 57.

DN-171019_tyc_test11b Sm (Mn, 1x1) 1: Scan ES+ 1.30 100 636 2.0000Da 570037 1.64e7 Area

1.16 291093 %

2.27 1.41 2.17 53304 2.47 0.96 1.69 19052 14977 34549 22271 67625

0 -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 DN-171019_tyc_test11b Sm (Mn, 1x1) 1: Scan ES+ 1.56 100 663 2.0000Da 1022906 2.64e7 Area %

1.30 246138 0.23 1.67 2.06 1.02 2.33 133347 46994 96828 86089 128913 0 -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 DN-171019_tyc_test11b Sm (Mn, 1x1) 1: Scan ES+ 1.30 100 679 2.0000Da 7061147 1.14e8 Area %

1.51 22995 1.85 2.10 15146 23491 0 -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 DN-171019_tyc_test11b 1: Scan ES+ 0.35 TIC 0.28 4.33e9 0.26

1.29 % 0.73 1.42 0.21 0.79 2.08 1.55 1.691.82 2.12 2.37

0 Time -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

Figure 45. Biosynthesis of decapeptide 57 by TycAN pF,1/ TycBPFpY/ TycCNQYVOL supplemented with N -methyl-D-Phe and O-propargyl-L-Tyr (27) (m/z [M+2H]2+ = 678.9).

149 5. APPENDIX

DN-171019_tyc_test12b Sm (Mn, 1x1) 1: Scan ES+ 1.15 100 636 2.0000Da 220941 6.50e6 Area % 1.19 2.26 57941 18071 1.61 2.46 1.08 46740 2.13 22177 21941 1.86 15022 25270 0 -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 DN-171019_tyc_test12b Sm (Mn, 1x1) 1: Scan ES+ 1.56 100 663 2.0000Da 887003 2.50e7 Area %

0.26 1.77 2.34 1.27 2.01 90571 39346 128890 60947 67507 0 -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 DN-171019_tyc_test12b Sm (Mn, 1x1) 1: Scan ES+ 1.32 100 679 2.0000Da 6880265 1.19e8 Area %

1.49 1.70 21778 29325 2.44 12933 0 -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 DN-171019_tyc_test12b 1: Scan ES+ 0.32 TIC 0.28 4.49e9 %

0.21 0.66 0.81 1.30 1.54 2.36 1.43 0.20 0 Time -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

Figure 46. Biosynthesis of decapeptide 57 by TycAN pF,1/ TycBPFpY/ TycCNQYVOL supplemented with N -methyl-D-Phe and O-propargyl-L-Tyr (27) (m/z [M+2H]2+ = 678.9).

150 5.2. LC-MS

Abrogation of tyrocidine A (11) formation by (S)-β-Phe (45).

DN-171101_betatyc2h_9 Sm (Mn, 1x1) 1: Scan ES+ 1.48 100 636 1961462 7.36e7 Area

1.49 902733 %

1.52 259143

1.57 96306

0 -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 DN-171101_betatyc2h_9 1: Scan ES+ 0.51 TIC 4.59e9

0.25

0.27 %

0.34

0.21 0.73 1.48 1.98 1.57 2.16 1.34 1.67 2.30 0.20

0 Time -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

Figure 47. Tyrocidine biosynthesis with TycAF, TycBPFβF, and TycCNQYVOL (calculated [M+2H]2+ 635.8).

151 5. APPENDIX

DN-171101_betatyc2h_13 1: Scan ES+ 1.25 100 636 2.72e6

1.27

1.20 % 1.69 2.03 2.38 1.47 0.89 1.28 1.49 2.19 1.18 1.03 1.37 1.74 2.25 1.86 0.21 2.47 2.31 0.61 0.91 2.48 0.31 0.41 1.62

0.82 0.81 0 -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 DN-171101_betatyc2h_13 1: Scan ES+ 0.24 100 TIC 3.70e9

0.49

0.33 %

0.75 0.79 0.21 1.961.99 2.21 1.27 1.38 1.45 1.65 1.73

0.20

0 Time -0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

Figure 48. Tyrocidine biosynthesis with TycAF, TycBPFβF, and TycCNQYVOL supple- mented with (S)-β-Phe (45) (calculated [M+2H]2+ 635.8).

152 5.2. LC-MS

DKP formation on yeast

a

b

c

Figure 49. LC-MS analysis of DKP formation on yeast. a, O-propargyl-D-Tyr-L-Pro DKP standard (1 µM); b, DKP reaction with not displaying yeast cells; c, DKP formation with yeast cells displaying TycApY.

153 5. APPENDIX

5.3 Yeast cell surface display

a

b

c

Figure 50. Yeast cell surface display of full modules. a, TycApY-AT; b, TycAF; c,

TycApY.

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