EXPANDING THE STRUCTURAL DIVERSITY OF THE RiPP CLASS OF NATURAL PRODUCTS
BY
NIDHI KAKKAR
DISSERTATION
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2018
Urbana, Illinois
Doctoral Committee:
Professor Wilfred A. van der Donk, Chair Professor John A. Gerlt Professor Yi Lu Associate Professor Douglas A. Mitchell
Abstract
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a diverse and rapidly expanding class of natural products. Despite starting out as linear chains of amino acids, RiPP natural products acquire structural diversity ranging from small molecules like microcin C7 to 48-mer polytheonamides with varying post-translational modifications installed by biosynthetic enzymes. These conformational restrictions allow these natural products to exhibit a wide range of therapeutic activities like antifungal, antibacterial, allelopathic, and antiviral.
Despite the structural and functional disparities, they all share a common feature: the presence of amino acid sequence in the precursor peptide called the ‘leader peptide’ that helps guide the biosynthetic enzymes towards the remaining precursor peptide termed the ‘core peptide’ to act on specific residues and install different PTMs. This leader-peptide guided biosynthetic route provides tremendous opportunity to bioengineer libraries of analogues of RiPPs. Exploiting this feature, in Chapter 2, I report my investigations on a strategy wherein by swapping the leader peptides from unrelated RiPP biosynthetic pathways and modifying the core peptide sequences, different biosynthetic enzymes can perform their characteristic chemistries on the core peptide residues.
In Chapter 3, I report the design and usage of chimeric leader peptides that can be bound by multiple enzymes from unrelated RiPP pathways to create new-to-nature hybrid RiPPs. This strategy involves the combination of a cyclodehydratase with modification enzymes from different lanthipeptide RiPP classes. These hybrids provide insight into enzyme promiscuity and compatibility, and the biosynthetic order of events, and establish a general platform for the engineering additional hybrid RiPPs.
ii In Chapter 4, I develop two different strategies for non-proteinogenic amino acid incorporation in the class I lantibiotic nisin and the class II lantibiotic lacticin 481 using amber stop codon technology.
iii
To my family and my teachers
iv Acknowledgements
"If I have seen further, it is by standing upon the shoulders of giants" -Sir Issac Newton
First of all, I want to thank my mom, Raj, and my sister, Swati, for their unconditional love and support throughout my life. Their belief in me led me on the course that brought me seven thousand miles away from home, but despite the physical distance, I have always felt their presence with me. Mom, it is because of your love, hard work and prayers that I have accomplished anything and everything in my life, and try as I will, would not be able to pay for it. I love you, and am forever grateful to you.
Apart from my family, the one person that I have been privileged to observe up close is my advisor Professor Wilfred van der Donk. Anyone who has heard of him in the scientific community knows how brilliant a scientist he is but not many would know that he is such a kind- hearted, respectful and humble person. I was fortunate enough to attend his graduate class twice, first as the student and then as the TA, and watching him teach with ever-lasting patience is truly remarkable. I really want to thank him for his constructive guidance, patience and honest advice in projects and life in general. He is not just a great scientific advisor but also a fabulous mentor.
His support encouraged me immensely through the good and specially the bad times. I owe him my deepest gratitude and not just for the PhD training!
I would also like to thank my committee members, Professor Douglas A. Mitchell,
Professor Yi Lu and Professor John A. Gerlt for their suggestions and advice during the graduate studies. Special thanks to Prof. Satish K. Nair for providing his valuable input on my Original
Research Paper. I want to convey my thanks to our collaborators, Professor Wenshe Liu, Professor
v Michael C. Jewett and Professor Douglas A. Mitchell for generously providing several bacterial strains and plasmids and for sharing their expertise in different techniques with me.
I am eternally grateful to so many of the past and present members of van der Donk laboratory for their friendship and wisdom. Right from my first year till this day, I found a great friend and mentor in Dr. Ayse Okesli. Her pleasant personality always brightened up the bay. I want to thank Dr. Yanxiang Shi, Dr. Noah Bindman, Dr. Subha Mukherjee and other senior students for their generous help. I would also like to express my sincere gratitude towards the post- doctoral fellows, Dr. Mark C. Walker, Dr. Lindsay Repka, Dr. Ian Bothwell, Dr. Liujie Huo, Dr.
Debapriya Dutta, Dr. Tania Caetano, Dr. Qi Zhang, Dr. Rebecca Splain, Dr. Julian Hegemann and
Dr. Chi Pan Ting for all the useful discussions and the friendly banter in lab. Mark, you are a blessing that Wilfred brought into the lab in my second year and I want to specially thank you for your kind and helpful attitude, and your encyclopedic knowledge about cloning techniques that has saved my life so many times. Thank you, Marc R. Gancayco and Subhanip Biswas, for making this journey entertaining. Your witty remarks, pranks and unintentional humor kept the fun alive at work. I also want to thank Nan Holda for our interesting conversations and her ever uplifting attitude. I also want to thank my friends Rohini Singh, Tejas Ghirnikar and Abhishek Sheshadri for their love, compassion and often unsolicited sarcasm.
Lastly, I want to thank all the teachers and professors that taught me in school, and during bachelors and masters for their love, support and unwavering encouragement. This day would not have been possible without them. Beloved Ma’ams and Sirs, I respect you and I applaud you for all your efforts. ‘A teacher takes a hand, opens a mind and touches a heart’ and you have done all three for me. Dhanyawad.
vi Table of Contents
Chapter 1: Gifts from Microorganisms...... 1
1.1 Introduction...... 1
1.2 Ribosomally synthesized and post-translationally modified peptides (RiPPs)...... 4
1.3 Lanthipeptides...... 7
1.4 Linear azole-containing peptides (LAPs)...... 13
1.5 Discussion...... 16
1.6 References ...... 18
Chapter 2: Leader-Peptide Guided Generation of Non-Natural Hybrid RiPP Products –
Swapping the Leader Peptide Strategy...... 28
2.1 Introduction...... 28
2.2 Sortase-mediated swapping of leader peptides...... 30
2.3 Design strategy...... 31
2.4 Results and discussion...... 37
2.5 Methods...... 48
2.6 References...... 60
Chapter 3: Leader-Peptide Guided Generation of Non-Natural Hybrid RiPP Products –
Chimeric Leader Peptide Strategy...... 67
3.1 Introduction...... 67
3.2 Results...... 70
vii 3.3 Discussion...... 87
3.4 Methods...... 89
3.5 References...... 99
Chapter 4: Incorporation of Non-Proteinogenic Amino Acids in Class I and II Lantibiotics.....107
4.1 Introduction...... 107
4.2 Results and discussion...... 108
4.3 Conclusion...... 118
4.4 Methods...... 118
4.5 References...... 145
viii Chapter 1: Gifts from Microorganisms
1.1 Introduction
Natural products (NPs) possess splendid structural, functional and chemical diversity that is yet to be matched by any synthetic libraries of compounds and continue to be the source of inspiration for novel discoveries and breakthroughs in chemistry, biology, and medicine. Their structures have evolved over millions of years to be extremely efficient in their tasks, be it housekeeping or combat against environmental agents. The history of medicine is filled with examples where a natural product saved thousands of lives through its medicinal properties (e.g. penicillin in World War II) or led to the discovery of an even more potent small molecule. And there is more to be discovered, uncovered and explored in this new age of natural product drug discovery.
Besides the four well-established and well-researched classes of NPs, terpenoids, alkaloids, polyketides, and non-ribosomal peptides (NRPs), a relatively new class of NPs that has come to light and garnered tremendous scientific interest is the ribosomally synthesized and post- translationally modified peptides (RiPPs).1-5 Unlike their non-ribosomal counterparts, these peptides do not require large, modular, multifunctional enzymes known as non-ribosomal peptide synthetases for installing non-proteinogenic amino acids into the peptide backbone.3, 4, 6 RiPPs acquire a similar extent of structural diversity as NRPs through exhaustive post-translational modifications (PTMs) of a ribosomally synthesized precursor peptide (see Figure 1.1).1 This allows RiPPs to possess a wide variety of structural features that endow them with desirable medicinal properties like resistance to proteolysis and a lower entropic cost of target binding.3, 5
Therefore, not surprisingly, a plethora of RiPPs exhibit a range of activities such as antifungal,
1 O H O N NH O O NH O NH2 S HN O O N H HN O HN S O S
N O NH HN H N O NH O HN O O O S HN O NH O HN HN HN S N H O O H N HN O HN O HN 2 O NH S O N HN O O NH2 NH S NH HN O 2 N HN H O O HO O HN O Me H O HN HO N H2N O nisinA N H NH HN lanthipeptide O O O
NH N NH 2 O O N N S OH HO H N N N 2 O H NH2 H N N O N P O N NH2 H2N O H O S N O OH OH N N H2N O O HN O HN S O HN microcin C7 NH S NH HO O O O NH O O O microcin H H H HN S N N N N S N N OH H O H H N O O O N HO O O O S HO O streptide NH2 S N N nosiheptide H O N O thiopeptide
NH HN O S H O H N HN O S S S HN N O H H H H trunkamide N N N N O O O N N N O O NH H H H cyanobactin O S S O S O H N NH thioviridamide thioamide O sporulation killing factor OH NH sactipeptide HN 2 N N NH HN
O O S O H HO N NH microcin J25 NH N S H HN O O N lasso peptide HN H H O HN O N O O HN NH O HN Ile Gly Thr H2N O O O Gly Pro S O S NH HN OH Val NH O H O His Gly Ala Ile HO N HN S O Val Phe O H N SerGly O N S O O NH N Pro Tyr H H HN O O N O Phe O N H HN NH N O N NH H N O Tyr O O Gly HN NH
NH2
Figure 1.1 Chemical structures of selected RiPP subfamily members. The second line mentions the RiPPs subfamily the natural product belongs to.
2 antibacterial, allelopathic, and antiviral (see Table 1.1).1, 3 Compared to ~3000 nucleotides required to add/change a single amino acid in the NRP system, RiPPs only need three nucleotides, and it is due to this enormous advantage that the ribosomal world has an edge over the non-ribosomal one with respect to chemical research and drug discovery.4 Additionally, through the cost reduction of genome sequencing techniques and the advances made in bioinformatics for more accurate protein function annotation,7 more and more RiPP gene clusters with potentially new PTMs are being uncovered,8 and new connections are being established between classes of enzymes previously thought to be unrelated, thereby paving the way for a better understanding of the evolution of biology in the microbial world.
Table 1.1 Selected RiPPs with proven or potential applications as drugs.3
Compound Organism Year of Bioactivity Reference discovery Actagardine Actinoplanes 1976 Antibacterial (Gram- 9 (Gardimycin) garbadinensis positive) α-Amanitin Amanita phalloides 1964 Antitumor 10 Duramycin Streptomyces 1958 Antibacterial (Gram- 11 (Moli1901, cinnamomeus forma positive), lancuvotide) azacoluta antiviral Labyrinthopeptin Actinomadura 2010 Antiallodynic, 12, 13 namibiensis antiviral Microbisporicin Microbispora 2008 Antibacterial (Gram- 14 (NAI-107) corallina positive), Microviridin Microcystis viridis 1990 Serine protease 15 inhibitor Nisin Lactococcus lactis 1928 Antibacterial (Gram- 16 positive)
3 1.2 Ribosomally synthesized and post-translationally modified peptides (RiPPs)
To date, more than twenty RiPP families (MW < 10 kDa) are known, which are subdivided into members according to their PTMs.1 Typically, RiPP biosynthesis starts with the translation of a precursor peptide on the ribosome (Figure 1.2). The precursor peptide contains two distinct regions: a leader peptide and a core peptide. In most RiPPs, the leader peptide is attached to the
N-terminus of the core peptide and is usually important for recognition by most of the biosynthetic enzymes for installing primary PTMs, and for export.17 However, in certain cases, the leader peptide is found to be appended to the C-terminus of the core peptide and is called a “follower” peptide.18-20 After translation, the biosynthetic enzymes recognize and bind to the leader peptide, and install different class-defining PTMs (summarized in Table 1.2) on specific residues of the core peptide. The modified peptide then undergoes proteolytic cleavage to remove the leader peptide and the resulting mature RiPP is transported outside the host cell. In certain gene clusters, besides the leader peptide-dependent biosynthetic enzymes, certain secondary enzymes are present that can act on the core peptide independent of the leader peptide and install chemical functionalities that are crucial for the bioactivites.21 Despite playing a crucial role in biosynthesis as a recognition element, the term ‘leader peptide’ is loosely defined for classification purposes since we cannot negate that the recognition motif can extend well into the core peptide as well. So, for all purposes, ‘leader peptide’ is the portion of precursor peptide that is cleaved off to form a mature RiPP natural product, and the part that is retained in the mature product constitutes the
‘core peptide’.
This leader-peptide guided strategy for the biosynthesis of such a diverse class of RiPP natural products sheds light on the observation of several precursor peptides with practically similar leader peptides but hypervariable core sequences that can be present in the same
4
Figure 1.2 General biosynthetic pathway for RiPPs. The precursor peptide contains a core region that is transformed into the mature product. Many of the posttranslational modifications are guided by the leader peptide and the recognition elements in the biosynthetic enzymes.
biosynthetic gene clusters.22 This finding speaks volumes about the inherent promiscuity of RiPP biosynthetic enzymes and the overall ease of evolution in nature or genetic engineering in a laboratory setting, for creating structural analogs at a low genetic cost.
The remainder of this chapter will focus on two subfamilies of RiPPs: lanthipeptides and linear azol(in)e-containing peptides that are the topics of this thesis.
5 Table 1.2 Summary of all known RiPPs subfamilies.5, 23-25
RiPPs subfamily Defining PTM
Lanthipeptides Lan and/or MeLan thioether bis-amino acids Linaridins dehydroamino acids but lacking Lan/MeLan Proteusins linear peptides containing D-amino acids and C-methylations Linear azol(in)e-containing linear peptides containing (methyl)oxazol(in)e or/and thiazol(in)e peptides heterocycles Cyanobactins N-to-C macrocyclic peptides produced by cyanobacteria sometimes further decorated with azole(in)es and/or prenylations Thiopeptides a central six-membered nitrogen-containing ring additional PTMs include dehydrations and cyclodehydrations Bottromycins an N-terminal macrocyclic amidine a C-terminal follower peptide instead of N-terminal leader peptide Microcins produced by members of the Enterobacteriaceae family include some members of the lasso peptide and LAP families Lasso peptides an N-terminal macrolactam with the C-terminal tail threaded through the ring Microviridins lactones made from Glu/Asp and Ser/Thr side chains and lactams made from Lys and Glu/Asp residues Sactipeptides intramolecular thioether linkages between Cys side chains and α-carbons of other amino acids Bacterial head-to-tail cyclized N-to-C cyclized peptides differing from cyanobactins in the biosynthetic peptides machinery employed for macrocyclization Amatoxins and Phallotoxins N-to-C cyclized peptides produced by fungi Cyclotides N-to-C cyclized peptides produced by plants containing a cysteine knot composed of three disulfides Orbitides N-to-C cyclized peptides produced by plants lacking disulfides Conopeptides venom peptides produced by snails; the degree and type of PTM varies Glycocins glycosylated antimicrobial peptides Autoinducing peptides peptides containing a cyclic ester or a thioester Methanobactin peptidic chelators used by methanotrophic bacteria Streptide a Trp-to-Lys carbon-carbon crosslink Thioamides peptides containing thioamide linkages installed post-translationally Pyrroloquinoline quinone (PQQ), small molecules generated from the post-translational modification of a pantocin, and thyroid hormones precursor peptide or protein
6 Table 1.2 (cont.)
Phomopsins 13-member macrocyclic ring formed by an ether bridge linking an Ile residue to the phenyl ring of a Tyr and contain dehydroamino acids and no proteinogenic amino acids Ustiloxins 13-member macrocyclic ring formed by an ether bridge linking an Ile residue to the phenyl ring of a Tyr and contain proteinogenic amino acids N-methylated peptides N-methylation of the peptide backbone by a methyltransferase domain encoded within the leader sequence of the precursor protein
1.3 Lanthipeptides
Lanthipeptides, for example nisin (Figure 1.1), are characterized by the presence of the thioether containing amino acids called lanthionine (Lan) or 3-methyllanthionine (MeLan) (Figure
1.3). These characteristic PTMs are installed in two steps biosynthetically. In the first step, selected
Ser/Thr residues in the core region of a precursor peptide, called LanA, are dehydrated to 2,3- didehydroalanine (Dha) and (Z)-2,3-didehydrobutyrine (Dhb), respectively (Figure 1.3).1, 2 In the second step, specific Cys thiols attack the unsaturated amino acids in a Michael-type addition reaction to form (Me)Lan.1, 2 Originating from an anti-addition of Cys on Dha or Dhb, the stereochemistry depends on whether the thiol attacks from above or below the plane of the double bond to generate: (2S,6R)/(2R,6R)-Lan or (2S,3S,6R)/(2R,3R,6R)-MeLan. The (2S,6R)-Lan and
(2S,3S,6R)-MeLan are more commonly present (also known as the DL isomers; D configuration at the former Ser/Thr and L configuration at the former Cys).1, 2, 5 The (2R,6R)-Lan and
(2R,3R,6R)-MeLan rings (also known as the LL isomers) are formed only when the core peptide has a Dhx-Dhx-Xxx-Xxx-Cys motif (Dhx represents Dha or Dhb and Xxx represents amino acids other than Ser, Thr and Cys) since two consecutive dehydroamino acids impose conformational restrictions that disfavor the Si face attack of Cys.26, 27 This observation was of special interest
7 since it showed that the substrate, and not the enzyme, determines the outcome of the reaction which happens rarely in enzymatic reactions.
Figure 1.3 Biosynthetic route for the formation of the most common post-translational modifications found in lanthipeptides. Depending upon the face from which Michael-type attack of Cys thiols takes place, two stereochemistries for the thioether rings are possible as shown.
Lanthipeptides are classified into four categories based on the mechanism of installation of the thioether rings and the number of enzymes involved in each step. Class I lanthipeptides are produced by the action of two enzymes: a lanthipeptide dehydratase (LanB) and a lanthipeptide cyclase (LanC).1, 2 The mechanism for dehydration of Ser/Thr by LanB involves an unanticipated
8 co-substrate: glutamyl-tRNAGlu. Studies carried out on the lanthipeptide dehydratase NisB involved in nisin biosynthesis revealed that dehydration proceeds through glutamylation of Ser/Thr residues within the precursor peptide NisA (Figure 1.4).28, 29 In the proposed mechanism,28, 29 upon deprotonation, the Ser/Thr side chain attacks the α-carbon of glutamate bound to tRNAGlu, thereby eliminating tRNAGlu. The intermediate then collapses with the abstraction of the α-H of the glutamylated Ser/Thr resulting in Dha or Dhb, and glutamate. LanB does not aminoacylate tRNA itself but instead uses the cellular pool of glutamyl-tRNAGlu for the activation of Ser/Thr. Structural characterization of NisB demonstrated that the protein consists of two domains: one at the N- terminus that catalyzes glutamylation and the other at the C-terminus that facilitates glutamate elimination. The core peptide moves between the two domains during dehydration.28 The identification of glutamyl-tRNAGlu as the co-substrate took more than 25 years since the nisB gene was sequenced primarily owing to the fact that NisB shares no significant structural homology with tRNA synthetases or other enzymes involved in secondary metabolism that use aminoacylated-tRNAs (aa-tRNAs) like cyclodipeptide synthetases.30-32 The lack of such homology however hints towards a new mode of interaction between class I lanthipeptide dehydratases and aa-tRNAs.
In class II lanthipeptides, the thioether rings are installed by a bifunctional lanthionine synthetase, LanM, containing an N-terminal dehydratase domain and a C-terminal cyclase domain.1 The dehydrations in class II lanthipeptides are carried out in two steps as well: the ATP- dependent phosphorylation of Ser/Thr residues followed by phosphate elimination (Figure 1.4).33
The N-terminal domains share homology with PoyF dehydratase involved in the biosynthesis of polytheonamides,34 and the C-terminal domains are homologous with the class I LanC cyclases.
Most LanC proteins and cyclase domains of LanM are zinc metalloproteins and have one His and
9
Figure 1.4 Proposed dehydration mechanism employed by class I lanthipeptide dehydratases and class II–IV lanthionine synthetases.
two Cys ligands at the active site.35-38 However, the cyclase domain of the class II lantibiotic synthetase ProcM that modifies twenty nine different precursor peptides has three Cys ligands at its active site and forms a distinct clade in the phylogenetic tree of class II LanM synthetases.38
This difference is speculated to cause two main rifts from the general trend of cyclase activity.
Firstly, a His ligand at the active site can act as the base that deprotonates the α-proton of the thioether rings causing ring opening.39 Therefore, NisC and class II lanthipeptide synthetase
HalM2 both have been shown to be capable of opening up multiple rings under the conditions of
10 catalysis, even though only the ring-closed peptide is observed at equilibrium (Figure 1.5).39 In contrast, the Michael-type cyclizations catalyzed by ProcM are irreversible.39
Route 1: Exchange of α-D with solvent
Xn Xn Xn
O R NH H2O protonation O R NH O R NH D H B HN S O HN S O + HN S O BH B α-D-labeled (Me)Lan a-H-labeled (Me)Lan
conjugate addition H2O to Dhx
conjugate Xn retro- Xn Xn conjugate addition NEM to NEM O R NH addition O S NH O NEM S NH
HN S O HN R O HN R O BH+
Route 2: Opening of thioether ring
Figure 1.5 Cyclizations catalyzed by both NisC and HalM2 are reversible, as shown with α- D-labeled modified NisA and modified HalA2. D/H exchange in α-D-labeled substrate could occur either by a deprotonation/reprotonation mechanism (route 1) or by a reversible cyclization mechanism (route 2). The reversibility of cyclization was confirmed for α-D-labeled mHalA2 and mNisA by trapping of the ring-opened species by conjugate addition to NEM.39
Secondly, this difference in the identity of the active site ligands aids in the promiscuous activity of ProcM since the presence of three thiolate ligands increases the positive charge density on the zinc ion, thereby, increasing the enzyme reactivity.40 Surprisingly, this does not come at a cost of low substrate selectivity since ProcM essentially makes single products of a specific ring topology. This observation is indebted to the fact that ProcM-catalyzed product formation is under
11 kinetic control, and therefore, high regioselectivity of product formation is governed by the selectivity of the initially formed ring.40
The recently reported crystal structure of the class II lanthionine synthetase CylM, involved in cytolysin biosynthesis, revealed for the first time that structurally the N-terminal domain of
LanM resembles the catalytic unit of eukaryotic lipid kinases despite having no sequence homology with it.41 Co-crystallization experiments with AMP and mutagenesis data revealed that the kinase active site and the phosphate-elimination active site are overlapping, which suggests that these enzymes carry out phosphorylation and phosphate elimination in a processive manner.41
This hypothesis is further supported by the dependence of the phosphate elimination on ADP, and the absence of detectable phosphorylated precursor peptide intermediates.41, 42
So far we have seen two different mechanisms and two different routes of evolution for enzymes involved in the formation of (Me)Lan rings. A third route is found to be adopted by class
III and IV synthetases. These are trifunctional enzymes composed of a central Ser/Thr kinase domain, an N-terminal phosphoSer/phosphoThr lyase domain, and a C-terminal cyclization domain.13, 43-45 The dehydration occurs through a phosphate intermediate like in class II, however, the activation of the Ser/Thr –OH group with ATP, and the elimination of phosphate group from the intermediate occurs in two separate domains, resembling class I dehydratase. Although C- terminal cyclization domains display sequence homology to each other as well as to the LanC proteins, three metal binding residues that are fully conserved in class I, II, and IV cyclases are absent in the class III cyclization domains. This abnormality is suspected to be responsible for the formation of carbacyclic structures termed labionin by some of the class III enzymes.12 Labionins are formed by a second Michael-type addition of the enolate intermediate formed upon initial thioether ring formation into a second Dha.12
12 Some RiPPs comprise of D-amino acids, such as the two component lanthipeptides carnolysin and bicereucin that contain D-aminobutyrate (D-Abu) while the latter also contains D- alanine (D-Ala).46, 47 The mechanism involves the reduction of Dha to D-Ala and Dhb to D-Abu,46,
47 and the enzymes involved can be used to make more structurally diverse analogs.
1.4 Linear azole-containing peptides (LAPs)
The LAPs are a heterogeneous subclass of RiPPs with the characteristic feature being the presence of azole or azoline heterocycles within a linear peptide, unlike macrocyclized RiPPs like cyanobactins and thiopeptides.1, 8 As for all RiPPs, their biosynthesis initiates with the translation of a precursor peptide with a N-terminal leader region and C-terminal core peptide. The biosynthetic machinery involved are classified as protein B, a flavin-dependent dehydrogenase, protein C which is a member of the E1-ubiquitin activating (E1-like) superfamily, and a protein D
(cyclodehydratase from the YcaO superfamily).1, 8 In certain gene clusters, genes for protein C and
D are fused together.8 Upon binding to the leader peptide, the CD enzyme complex carries out
ATP-dependent cyclodehydration of specific Ser, Thr or Cys residues to form azolines with the removal of water from the carbonyl group of the preceding amide, giving rise to an azoline heterocycle.1, 8, 48 In the next step, protein D carries out the oxidation of azolines to azoles.1, 8
The first conclusive proof for these previously unknown PTMs was obtained in 1996 when the in vitro reconstitution of the biosynthetic enzymes of microcin B17 was reported by Walsh and coworkers.49 However, at that time only the dehydrogenase was correctly assigned owing to the yellow color from binding to its co-factor, flavin mononucleotide.50 It has been proposed to perform the dehydrogenation via an E-2 mechanism involving a conserved Lys-Tyr motif,49, 51
13 which likely deprotonates the Cα of the azoline followed by a hydride transfer resulting in azole formation (Figure 1.6).8
Figure 1.6 Plausible mechanism for the dehydrogenation of methyloxazoline catalyzed by B enzymes. The conserved Lys-Tyr motif is likely involved in deprotonating the Cα of the azoline, which is followed by hydride transfer.
For more than a decade, the specific roles that proteins C and D were playing could not be easily distinguished primarily since very few LAPs gene clusters had been reported then, and most of the cyclodehydratases available were dependent on other proteins in the complex for activity.
In 2012, with the in vitro reconstitution of the Balh cyclodehydratase from Bacillus sp. Al Hakam, the Ycao protein, BalhD, and not BalhC was shown to carry out ATP hydrolysis.48 The exact role played by the protein C still remains elusive although it has been proposed to act as a scaffolding or docking protein.30 The last and perhaps the most important piece of the puzzle was to determine the mechanism of azoline formation thereby determining the role of ATP in the reaction. Two
14 different mechanisms had been proposed48, 52: 1) direct activation wherein the phosphate group of
ATP is directly added to the heterocyclized intermediate, so as to act as a leaving group (Pi) for elimination (Figure 1.7), or 2) ATP acts as an allosteric regulator for the enzyme, and the active conformation of the enzyme is adopted upon ATP hydrolysis. Through isotope labelling studies in
18O labelled water, it was shown that no 18O was incorporated in the free phosphate released upon
ATP hydrolysis.14 This result supported the direct activation since the second mechanism proposed would involve the attack of bulk water, labelled water in this case, and result in the γ-phosphate being labelled upon release. The direct activation mechanism garners special interest since the
YcaO protein hydrolyzes the stable and relatively inert amide bond in the peptide backbone for heterocyclization. More insights into this mechanism and protein structure can open doors for the evolution of new biological catalysts performing new chemistries.
15
Figure 1.7 Proposed mechanisms for azoline biosynthesis by YcaO domains. Direct activation suggests the incorporation of phosphate(s) directly into the heterocyclized intermediate, providing a leaving group (Pi) for elimination.
1.5 Discussion
The 1945 Nobel Prize in Physiology or Medicine to Sir Alexander Fleming, Ernst B. Chain, and Sir Howard Florey for the discovery of penicillin and the 1952 award to Selman A. Waksman for the discovery of streptomycin marked the advent of the Golden Age of natural products drug discovery. In these celebrated years, several breakthrough discoveries were made, the most prominent ones resulting in the 2015 Nobel Prize to William C. Campbell and Satoshi Omura, for the discovery of the microbial natural product avermectin and to Youyou Tu for the discovery of the plant natural product artemisinin. Derivatives of avermectin have high activity against onchocerciasis and lymphatic filariasis,53, 54 and artemisinin is used for the treatment of malaria.55
16 However, it has been ~35 years since the avermectin and artemisinin families of drugs were first introduced as human therapeutics and unfortunately resistant strains have started to slowly evolve against them.53, 56 While developing microbial drug resistance is inevitable,57 this fast-rising tide can be curbed with a combination drug therapy, new use of known NP-based drugs (since NPs are not designed as mechanism based drugs and often interact with multiple biological targets),58-61 and/or with discovery of new drugs.
The RiPP class of natural products with their novel chemical scaffolds and diverse functionalities provide a unique platform to leap further into discovery and development of paradigm-changing drug molecules. Total synthesis of these NP and their analogs for structure- activity relationship (SAR) studies is not profitable due to their complex structures. However,
RiPPs biosynthetic systems do a phenomenal job in reaching this goal. Additionally, from a bioengineering perspective, the combinatorial nature of RiPP biosynthesis makes them more amenable for engineering vast libraries of derivatives and scanning for new bioactive molecules.
There are multiple ways to approach this task. First, directed evolution through the use of a randomized library of core peptides in phage or yeast display can help identify mutants with desired activities.62, 63 Second, incorporation of unnatural amino acids using amber suppression strategies can not only aid in making a more potent derivative but also in mode of action studies through labeling with fluorescent or cross-linking tags.64-68 Third, the leader-peptide guided feature of RiPPs biosynthesis can be put to task in combining different enzymes to create novel scaffolds.17
Fourth, alternative to the in vivo approaches, in vitro biosynthesis with reconstituted enzymes has resulted in promising leads in certain examples.13, 69, 70 Moreover, studies where the leader peptide can be used in trans to the core peptide,71, 72 or is fused to the biosynthetic enzyme,73, 74 has vast potential since it eliminates the need for difficult-to-synthesize long precursor peptides. Last, new
17 compounds can be discovered by characterizing the vast number of RiPP gene clusters that are being reported every day in the databases.7, 75 Two of these strategies have been explored in the following chapters. Chapter 2 and 3 describe leader peptide-guided bioengineering strategies that enable recognition and processing by multiple enzymes from unrelated RiPP pathways to create new-to-nature hybrid RiPPs. And, Chapter 4 presents methodologies for the production of analogues of two lantibiotics, belonging to different classes, that contain non-proteinogenic amino acids using amber stop codon suppression technology.
1.6 References
1. Arnison, P. G. B., M. J.; Bierbaum, G.; Bowers, A. A.; Bulaj, G.; Camarero, J. A.;
Campopiano, D. J.; Clardy, J.; Cotter, P. D.; Craik, D. J.; Dawson, M.; Dittmann, E.; Donadio, S.;
Dorrestein, P. C.; Entian, K.-D.; Fischbach, M. A.; Garavelli, J. S.; Göransson, U.; Gruber, C. W.;
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27 Chapter 2: Leader-Peptide Guided Generation of Non-Natural Hybrid RiPP Products -
Swapping the Leader Peptide Strategy
2.1 Introduction
As mentioned in Chapter 1, since the RiPP class of natural products is synthesized on the ribosome as a precursor peptide, it provides a relatively easy way to bioengineer new natural product analogs endowed with new or an improved range of activities such as antifungal, antibacterial, allelopathic, and antiviral.1-4 There have been several reports wherein through site- directed mutagenesis, or stop codon suppression strategies, the core peptide sequence was engineered to biosynthesize new analogs.5-8 However, in this and the next chapter, we focused on the role of leader peptide during biosynthesis. A decade ago, the role of the leader peptide during biosynthesis was poorly understood, but through the elucidation of the mechanism of installation of key post-translational modifications (PTMs) in RiPP natural products and the availability of crystal structures of precursor peptides bound to biosynthetic enzymes, the community has made spectacular progress.2, 9-17 Among the many functional roles proposed for the leader peptide, the most prominent ones suggest they serve 1) as a means of protection for the native producer since it greatly reduces or abolishes antimicrobial activity of the mature RiPPs as long as it is bound to it, 2) as a recognition element for the secretion of secondary metabolites, 3) as an allosteric agent in facilitating the biosynthetic enzyme to adopt an active conformation for catalysis, and most importantly for this work, 4) as a guiding tool for these enzymes to act on specific residues following the leader peptide.1, 3 It is this last feature, the leader peptide-directed installation of a myriad of PTMs of the RiPPs class, that we sought to better understand and exploit for
28 bioengineering of a new class of hybrid RiPP natural products with a mix-and-match of different biosynthetic enzymes and their corresponding PTMs.
There have been reports showcasing the ability of leader peptides to direct its respective enzymes to modify non-natural cargos attached. For instance, the nisin biomachinery has been widely used to modify core peptides derived from other lanthipeptide and non-lanthipeptide related sequences attached to the NisA leader peptide in Lactococcus lactis.18-23 The versatility of the biomachinery was exploited to improve the properties of pharmaceutically relevant peptides in vivo.20, 21 However, our goals were to establish methodology that would allow installation of not just PTMs belonging to one class, but to combine PTMs from different classes, and therefore, truly make a new, hybrid, non-natural class of RiPP.
Because of the impressive progress made in the field of genome mining, tens of thousands of bacterial genomes have been sequenced, and this flood of genetic information is waiting to be tapped for its full potential to be realized. Besides academic curiosity, the ultimate goal for studying these genomes and their constituent gene clusters is for synthetic biology to be viewed with equal, if not higher, applicability than synthetic organic chemistry in the sheer ease and potential to create complicated, chemically diverse molecules for the pharmaceutical, food, cosmetics, or electronic industries. Realization of this goal would require, at its foundation level, a platform to perform combinatorial biosynthesis and a better understanding of the very basal requirements of enzymes to be able to efficiently work with each other in a plug-and-play fashion.
In most well-studied RiPP gene clusters, the recognition motifs in the leader peptide responsible for binding to the biosynthetic enzymes have been identified,2 however this is not true for the majority of cases reported in GenBank. So, through our efforts, we aimed to establish a general strategy to achieve this hefty task of combinatorial biosynthesis of new, conformationally
29 restrained and pharmacophore-rich products that expand the current proteinogenic repertoire using known examples of RiPP gene clusters as model systems.
2.2 Sortase-mediated swapping of leader peptides
Given the promiscuity of RiPP modifying enzymes and their clearly defined recognition sequences, it would seem straightforward to engineer modifying enzymes from unrelated biosynthetic pathways into the same pathway, but at the start of these studies, this strategy had yet to be achieved in the laboratory. Since the leader peptide holds the key in the recognition by modification enzymes and the subsequent modification of the core peptide, we envisioned a platform wherein different leader peptides from different biosynthetic pathways could be chemically attached to the same core peptide, and then the latter could be modified by respective biosynthetic pathways, thereby creating a new class of hybrid RiPP products. From a practical standpoint, there are three main considerations to be made: 1) a facile method is required to cleave the first leader off the core peptide modified by the first set of biosynthetic enzymes, and then a method is needed to attach a new leader peptide onto the modified core, 2) the sequence of the core peptide should include specific native motifs (derived from the native sequence) that would tolerate catalysis by not just one but all enzymes used in the strategy, and 3) the promiscuity of the biosynthetic enzymes in the later stages should allow a partially modified core to be accepted as a substrate despite having never-seen-before modifications. With their native leader peptides as well as short recognition sequences in the core peptides, the modifying enzymes were anticipated to bind to their respective recognition regions in the core peptides and sequentially install posttranslational modifications. Importantly, these modifications, in principle, can be rationally chosen and do not need to occur together in nature.
30 2.3 Design strategy
To test this strategy, we chose to create a hybrid of the lanthipeptide lacticin 481 and the microcin B17. Lacticin 481 is a class II lantibiotic that is acted upon by one bifunctional lanthipeptide synthetase enzyme, LctM.3 During the biosynthesis of lacticin 481, LctM performs both dehydrations and cyclizations on its precursor peptide LctA (Figure 2.1).3 The enzyme first phosphorylates the Ser/Thr targeted for dehydration, and then eliminates the phosphate to produce
Figure 2.1 Biosynthesis of the class II lantibiotic lacticin 481. LctM dehydrates Ser and Thr residues in the core peptide of LctA, and catalyzes Michael type addition of Cys to Dha and Dhb. Next, LctT removes the leader peptide. Although it is drawn that cyclizations commence after all dehydrations are complete, the two can proceed alternately. The chemical structures for Dha, Dhb and lanthionine and methyllanthionine are shown as well as short-hand notations. Abu stands for 2-aminobutyric acid.
31 the dehydro-amino acids (dehydroalanine, Dha and dehydrobutyrine, Dhb). A separate active site catalyzes the Michael-type addition of Cys residues to the dehydro amino acids to form lanthionine and methyllanthionine.3
From previous studies investigating the biosynthesis of lacticin 481, we gathered the sequence and position dependence of dehydration activity of LctM: the presence of flanking Gly residues around a Ser prevents its dehydration by LctM, and LctM-catalysed dehydration is dependent on the distance to the leader peptide (Table 2.1), as has been reported for several other
RiPP biosynthetic enzymes.24, 25 Utilizing this information, we hypothesized that the first few residues of the LctA core peptide act as a spacer, and can therefore be substituted with a recognition motif derived from the core peptide for a different modification enzyme, specially one having modifiable residues surrounded by Gly residues. This requirement led us to microcin B17, which has a spacer region between leader and core peptides that is rich in Gly, and the common recognition motif in the core is a Ser or Cys preceded by a Gly (Figure 2.2).12, 26
32 Table 2.1 Sequence and position dependence of dehydration by LctM. A concise summary of findings about the extent of dehydration of different LctA mutants upon being modified by LctM.24, 25
LctA (–24to14) –24 –14 –1 1 4 MKEQNSFNLLQEVTESELDLILGA - KGGSGVIHTISHECNMNSWQFVFTCCS
His6-substrate Number of Maximum Observations Conclusions dehydrations number of in major dehydrations product possible wt-LctA 4 5 Ser4 escapes dehydration Truncants of wt-LctA
LctA(–24to14)- 2 3 Ser17 escapes Two Gly VGSGEA dehydration residues LctA(–24to14)- 3 3 Ser17 flanking a Ser VISHEA undergoes deactivate dehydration LctM Mutations/Insertions in wt-LctA
LctA(G3I/G5H) 4 5 Ser4 escapes A minimum dehydration distance LctA(K1- 4 5 Ser7 escapes between G2insAAA) dehydration Ser/Thr to be LctA(K1- 5 5 Ser7 undergoes modified and G2insAAA)- dehydration the leader (G3I/G5H) peptide is LctA(K1- 5 (with three 5 Thr9 undergoes required for G2insAAAALLKT) copies of lctM) dehydration efficient dehydration by LctM
Microcin B17 is a potent inhibitor of DNA gyrase and was the first post-translationally modified peptide antibiotic for which the biosynthesis was reconstituted in vitro.27, 28 Its biosynthesis involves modification of the precursor peptide McbA by the ternary complex formed by its biosynthetic enzymes. The complex includes a cyclodehydratase McbD that along with a scaffolding/regulatory protein McbC generates oxazoline and thiazoline structures from Gly-Ser
33 and Gly-Cys sequences in an ATP-dependent fashion, respectively. The complex also contains a flavin-dependent dehydrogenase that oxidizes these intermediates to oxazoles and thiazoles.28, 29
Through photocrosslinking experiments, it has been concluded that McbD is crucial for binding to the leader peptide of McbA.30 Recently, using substrate analogs and isotopic labeling studies,
Mitchell and co-workers showed that the D protein is involved in directly phosphorylating peptide amide backbone using ATP resulting in the formation of a phosphorylated hemiorthoamide intermediate which upon collapse results in the formation of an azoline moiety.29
Studies carried out by Walsh and co-workers in understanding the biosynthetic pathway of microcin B17 in the late 1990s showed that 1) the directionality of microcin B17 biosynthetic enzymes is from N- to C- terminus, 2) a ten-Gly residue long region between the leader peptide and the first modified Ser in the core peptide is a spacer since it helped maintain appropriate spatial distance between the binding and catalytic sites of the enzyme complex, and 3) the spacer region can be truncated from ten Gly residues to three Gly residues without affecting the binding affinity.26, 28, 30 Based on these observations, we chose a seven-residue motif ‘GGGGSCG’ from the beginning of the core peptide of McbA to be included in our hybrid core. Additionally, since lacticin 481 has its post-translational modifications concentrated in the C-terminus, we hypothesized that there would be little perturbation in binding of the McbBCD enzymes to the
McbA core motif introduced near the N-terminus of the core peptide of the hybrid substrate.
34
Figure 2.2 Biosynthesis of linear azol(in)e containing peptide, microcin B17. (A) McbCD complex acts on Gly-Ser and Gly-Cys motifs in the precursor peptide McbA in the first step, which is followed by dehydrogenation performed by flavin-dependent McbB to form oxazole and thiazole functionalities. (B) Reactions for generation of oxazol(in)e and thiazol(in)e motifs.
The last task in this design strategy was to find a suitable means to swap the two leader peptides. In most of the class II lantibiotic precursor peptides, the C-terminus of the leader peptide is Gly rich1 and there is only one well-studied promiscuous enzyme that can perform ligation of
Gly rich peptides: sortase A from Staphylococcus aureus.31-34 Sortases are membrane-associated transpeptidases that anchor proteins on the cell wall of Gram-positive bacteria as shown in Figure
2.3.34 The proteins to be displayed on the bacterial surface have a conserved recognition motif for
35
Figure 2.3 Schematic representation of how proteins are anchored to the cell wall via sortase in Gram-positive bacteria.34 Sortase A is a calcium-assisted transpeptidase that binds surface proteins to the peptidoglycan cell wall of Gram-positive bacteria. In the first step, sortase A identifies its recognition motif, LPXTG, in the incoming protein and cleaves the peptide bond between Thr and Gly residues through the attack of active site Cys and forms an enzyme-bound intermediate. This is followed by the amino group of N-terminus Gly of peptidoglycan precursor attacking the carbonyl group of thioester intermediate, thereby releasing the enzyme and anchoring the protein onto the cell surface.
sortase: Leu-Pro-Xxx-Thr-Gly (LPXTG, where X is any amino acid and Gly cannot be a free carboxylate), at or near their C-terminus. Upon recognition, the bond between the Thr and Gly residues is cleaved by active site Cys of sortase A thereby forming an acyl-enzyme intermediate.
Then, this intermediate is resolved by nucleophilic attack by the free amino group of the cell wall precursor lipid II. This lipid II-linked protein conjugate is incorporated during cell wall synthesis and consequently the protein is displayed at the surface.
36 N-terminal labeling via sortase A requires: 1) a display of 1-5 Gly residues at the N- terminus of a peptide, 2) the recognition motif LPXTG at the C-terminus of the other peptide
(Figure 2.4).33, 34 Therefore, in our strategy, we envisioned to ligate the second leader peptide with an LPETG motif at the C-terminus to the core peptide with Gly residues at the N-terminus as shown in Figure 2.4 and Figure 2.5.
Figure 2.4 N-terminal labeling via sortase A. Specific to our system, the sortase-mediated ligation requires a) McbA leader with C-terminal LPXTG motif provided it is followed by at least one amino acid and b) a ligating partner with two-to-five Gly residues at the N-terminus, i.e., LctM-modified hybrid core peptide with leader peptide cleaved.
2.4 Results and discussion
2.4.1 Modification of LctA leader-hybrid core with LctM and subsequent proteolytic removal of the leader peptide
As per the design strategy (Figure 2.5), the McbA core motif GGGGSCG was substituted in place of residues from positions 1-5 of the LctA core to obtain a hybrid core peptide with
37 sequences favorable for both LctM and McbBCD to act upon. In order to cleave off the LctA leader after LctM-catalyzed modifications had been installed, an Ala–1Glu mutation was introduced since previous reports had shown the –1 position of LctA to be highly amenable for substitution without affecting LctM catalysis.35 As diagrammed in Figure 2.5, the hybrid peptide was tested by expressing His-tagged hybrid peptide and LctM in Escherichia coli (see Table 2.3 for plasmids).
The modified peptide was affinity purified, and analyzed via matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). A mixture of products was observed, with the most processed one being three-fold dehydrated. Even upon increasing the number of copies of the gene encoding LctM from one to three, the extent of dehydration did not improve (Figure 2.6).
38
Figure 2.5 Schematic depicting the strategy for sortase-mediated swapping of leader peptides to create a library of hybrid RiPPs. The figure details the three-step approach starting with LctM-catalyzed modification of the hybrid core attached to the LctA leader peptide. After the modification, the leader peptide is cleaved off. In the second step, sortase A ligates the hybrid core with Gly residues at the N-terminus with the McbA leader peptide terminating with the LPETGG motif. The final step involves the installation of oxazole and thiazole onto the hybrid core attached to the McbA leader peptide in a McbBCD assay.
39 Figure 2.6 MALDI-TOF MS spectrum of His-tagged LctA/A-1E/GGGGSCG/N15R modified by two/three copies of lctM in E. coli. The N15R mutation was introduced to improve solubility.36 Despite increasing the number of copies of lctM expressed in E. coli, the 4-fold dehydrated peptide was not obtained as the major product. m/z calculated for [M+H]+ peak for 3-fold dehydrated peptide = 7496.29, m/z observed for [M+H]+ peak for 3-fold dehydrated peptide = 7496.
Taking inspiration from a previously reported LctA mutant,25 wherein residues were inserted in between the first half of the LctA core, the GGGGSCG motif was inserted between
Lys1 and Gly2. This His-tagged hybrid peptide upon co-expressing with a single copy of lctM generated a fully modified peptide with the expected loss of four water molecules (Figure 2.7).
40 Proteolytic cleavage with LysC followed by HPLC purification afforded the LctM-modified core peptide with four Gly residues at the N-terminus.
Figure 2.7 MALDI-TOF MS spectrum of His-tagged LctA/K1G2insGGGGSCG/N22R/F28H peptide modified by expressing a single copy of LctM in E. coli. The N22R and F28H mutations were introduced to improve solubility.36 By inserting the McbA core-derived ‘GGGGSCG’ motif between K1 and G2 residues of LctA core peptide instead of subsitutuing the first seven residues of LctA core with that motif, a single copy of lctM efficiently dehydrated the four dehydratable residues in the hybrid core. m/z calculated for [M+H]+ peak for 4-fold dehydrated peptide = 7796.57, m/z observed for [M+H]+ peak for the labeled 4-fold dehydrated peptide = 7796.
41 2.4.2 Sortase-Tag Expressed Protein Ligation (STEPL)33 for attaching the McbA leader peptide onto the LctM-modified core peptide
For sortase-mediated ligation to work, the STEP ligation strategy33 involved attaching the
His-tagged catalytic unit of sortase A onto the McbA leader peptide separated by the recognition motif of sortase LPETGG, and a linker as shown in Figure 2.8. During the affinity purification, the His-tagged fused protein in the cell lysate would stay bound to the Ni beads. After washing and incubating with a peptide with Gly residues at the N-terminus and calcium ions, the sortase- tagged McbA leader would form a binary complex with the ligating partner. After the ligation, the ligated product would not be bound to the Ni beads and elute.
Figure 2.8 Scheme for STEPL. An additional motif from spacer region of McbA, VGIGG was introduced after the McbA leader peptide to maintain the distance between the McbBCD recognition site on McbA leader peptide and the first residue in the core peptide that is modified. The Histagged-catalytic unit of sortase A is fused to this McbA leader with C-terminal LPETG motif. The fused protein is expressed and loaded on a nickel column. Upon addition of calcium and hybrid core peptide with N-terminal glycine residues, the sortase enzyme catalyzes peptide ligation, resulting in its elution from the column.
42 To optimize the ligation conditions, a short three-residue test peptide, GGG, was used at different concentrations while varying the concentration of CaCl2 and the incubation time and temperature for ligation. Maximum yields for the ligated product were obtained when 100 µM of short peptide and 100 µM of CaCl2 were used at 37 °C for 5 hours (Figure 2.9).
Using these optimized ligation conditions and stirring the 100 µM of the LctM-modified core with the loaded Ni beads, the ligated peptide was observed in the solution upon MALDI-TOF
MS analysis as shown in Figure 2.10. HPLC purification afforded the clean ligated peptide wherein
Figure 2.9 MALDI-TOF MS spectrum of STEPL of the McbA leader with the test peptide, GGG (100 µM) in the presence of 100 µM CaCl2 at 37 °C. McbA leader shown contains an additional motif from McbA spacer region, VGIGG at the C-terminus and TwinStrep tag at the N- terminus. m/z calculated for [M+H]+ peak for ligated product, McbA leader-LPET-GGG = 6956.66, m/z observed for [M+H]+ peak for the ligated product = 6956.
43 the McbA leader with LPET insert in the linker region was attached to the LctM-modified hybrid core.
Figure 2.10 MALDI-TOF MS spectrum of the product of STEPL of McbA leader with LctM- modified hybrid core (100 µM) in the presence of 100 µM CaCl2 at 37 °C. In this case, McbA leader and the core are not separated by the additional spacer region, VGIGG (see Figure 2.8). The peaks observed in the absence of core peptide were due to non-specific binding of E. coli proteins to the column. m/z calculated for [M+H]+ peak for ligated peptide = 6479.45, m/z observed for [M+H]+ peak for the ligated peptide = 6478.
44 2.4.3 McbBCD in vitro assay29
The last step in the strategy was to feed the purified hybrid peptide to the MbcBCD complex for the installation of thiazoles and oxazoles. McbB, McbC and McbD were purified as reported earlier.29 To test the activity of the ternary complex, and to test the feasibility of the in vitro reaction in our hands, a control experiment was performed using the wild type precursor peptide McbA as the substrate. After the in vitro reaction followed by thrombin cleavage to remove the Maltose Binding Protein (MBP) tag, MALDI-TOF MS analysis showed that the precursor peptide underwent nine-fold modifications each accompanied with a loss of 20 Da after 12 h incubation at RT (Figure 2.11). However, when the ligated peptide was incubated with the
McbBCD enzymes (10 µM), no modifications were observed even after 24 h.
Figure 2.11 McbBCD in vitro assay with MBP-tagged native precursor peptide, wt-McbA followed by thrombin cleavage. MALDI-MS analysis shows loss of 180 Da accounting for nine modifications introduced in the core peptide. m/z calculated for [M+H]+ peak for 9-fold modified peptide = 6116.12, m/z observed for [M+H]+ peak for the same = 6117.
45 2.4.4 Optimizations for the in vitro reaction with McbBCD enzymes
To optimize the final step, we looked closely at all possibilities that might cause interference with the binding and modifications by McbBCD enzymes. The first possibility was that the recognition motif of sortase, LPET, present in the linker region might hinder the binding of the peptide to the active sites of the McbBCD complex. The second possibility was that the
LctM-installed thioether rings at the C-terminus of the peptide caused too large a perturbation from the linear peptide sequence for the enzyme complex to bind to the peptide and/or act on the modifiable residues in the core peptide.
To test the first possibility, a control experiment was designed wherein the LPET motif was introduced into wt-McbA in place of Gly residues at the same position as in the ligated peptide.
Upon incubating this control peptide with McbBCD enzymes followed by thrombin cleavage to remove the MBP-tag, the peptide was observed to undergo the desired nine modifications (Figure
2.12), which ruled out the possibility that replacement of the GGGG motif with LPET in the linker region of the precursor peptide was causing the observed absence of oxazole/thiazole installation in the hybrid peptide.
46 Figure 2.12 McbBCD in vitro assay with MBP-tagged McbA mutant with LPET motif replacing GGGG residues from position 6 to 9, followed by thrombin cleavage. MALDI-MS analysis shows loss of 180 Da accounting for nine modifications introduced in the core peptide. m/z calculated for [M+H]+ peak for 9-fold modified peptide after thrombin cleavage= 6326.37, m/z observed for [M+H]+ peak for the same = 6328.
This result, while disappointing, emphasized how important the order of installation of these post-translational modifications (PTMs) is for creating hybrid RiPP products with any combination and variety of PTMs. In order to address the problem at hand, we envisioned three approaches. First, introduce a spacer region between the motif derived from McbA, and the
Ser/Thr/Cys in the LctA core peptide that can be acted upon by LctM. Since no crystal structure has been reported for the McbBCD complex, the length of the spacer region would have to be
47 optimized by trial-and-error. Second, to use a cyclization-impaired mutant of LctM37 which would only carry out dehydrations of Ser and Thr but no cyclizations. Alternatively, we could use the nisin biosynthetic machinery as opposed to lacticin 481 since the nisin gene cluster has two separate enzymes, one for dehydration (NisB) and one for cyclization (NisC) that can act independent of each other.10, 38 So, the NisC-installed thioether rings could be introduced after the
McbBCD assay. Finally, we considered changing the order in which we installed the PTMs, starting with thiazoles and oxazoles first since they resemble a natural amino acid Pro chemically and would not drastically disrupt the flexibility of the peptide. But with that strategy, we might run into the problem of regioselectivity by having no control over McbBCD enzymes potentially modifying Ser/Cys in the motif derived from LctA.
Despite having these future directions in mind, we realized that we needed to refine our strategy so as to not have to define the order of installations of PTMs by trial-and-error method (to be tested in the final step of the strategy) but to let the kinetic and thermodynamic parameters carve out the map for the formation of one or many products, as the case maybe, and then through optimizations, guide the pathway towards the desired species, just as nature has been doing for millions of years in evolving the enzymes and optimizing the structure of natural products. Charles
Darwin said the underlying principle for all events is the survival of the fittest, how you define fit varies from physically/chemically endowed to energetically favorable in the grand scheme of events.39 The new and improved strategy is discussed in the next chapter.
2.5 Methods
All oligonucleotides, restriction endonucleases, and DNA polymerases were purchased from Integrated DNA Technologies, New England Biolabs or Invitrogen. Media components for
48 bacterial cultures were purchased from Difco laboratories. Chemicals were purchased from Fisher
Scientific or from Aldrich unless noted otherwise. Non-canonical amino acids were purchased from Chem Impex International. Solvents commonly used in peptide purification, including trifluoroacetic acid (TFA) and acetonitrile,40 were obtained in RP-HPLC grade or better and used directly without further purification. Endoproteinase GluC and LysC were purchased from Roche
Biosciences or Worthington Biosciences. E. coli DH5α was used as host for cloning and plasmid isolation, and E. coli BL21 (DE3) was used as a host for co-expression.
2.5.1 General methods
All polymerase chain reactions (PCR) were carried out on a C1000™ thermal cycler (Bio-
Rad). DNA sequencing was carried out by ACGT Inc. using appropriate primers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was carried out on a UltrafleXtreme TOF/TOF mass spectrometer (Bruker Daltonics) at the Mass Spectrometry
Facility (UIUC).
2.5.2 Construction of histagged-lctA/A-1E/GGGGSCG/N17R/lctM/pRSFDuet-1
The mutations were introduced in four steps by the QuickChange method using Histagged- lctA(wt)/lctM/pRSFDuet-1 as template.41 The primers are listed in Table 2.2. In the first step, A-
1E/K1G mutations were introduced followed by S4G/G5S mutations. In the third step, CG was inserted between S5 and V6. And, finally N17 was mutated to R. For all PCR amplifications, the following conditions were used: denaturation (98 °C for 20 s), annealing (appropriate temp for 45 s), and extension (70 °C for 5 min) over 20 cycles. The PCR reaction included 25 µL of KOD
HotStart master mix, 50-100 ng of template DNA and primers (1 µM each) in a total volume of 50
49 µL. The amplification products were run on a 1% agarose gel. The 50 µL PCR reaction mixture was incubated with 2 µL DpnI and 5 µL of NE Buffer 4 at 37 °C for 1 h. After incubation, the reaction mixture was purified using a QIAquick PCR Purification Kit (QIAGEN) and 5 µL of the final solution was used for transforming E. coli DH5a cells via heat shock and were plated on
Luria Bertani (LB)-kanamycin agar plates and grown at 37 °C for 18 h. Four colonies were picked and incubated in 10 mL of LB-kanamycin medium at 37 °C for 14 h, followed by isolation of the plasmids using a QIAprep Spin Miniprep Kit (QIAGEN). The sequences of the resulting plasmids were confirmed by DNA sequencing.
2.5.3 Construction of histagged-lctA/K1insGGGGSCG/N22R/F28H/lctM/pRSFDuet-1
The gblock Histag-EcoRI-LctA_K1G2instGGGGSCG_N22R_F28H_NotI_pRSFDuet shown in Table 2.2 was ordered from Integrated DNA Technologies and digested with EcoRI and
NotI overnight. The histagged-lctA(wt)/lctM/pRSFDuet-1 was digested with the same restriction enzymes overnight and purified by QIAquick PCR Purification Kit. The resulting DNA inserts (50 ng) were ligated with the respective digested vector in the ratios 1:3 and 1:10 at 50 °C for 2 h using
10 µL of Gibson ligation master mix (NEB) in a 20 µL reaction. After incubation, 10 µL of reaction mixture was used for transformation of E. coli DH5a cells, and the cells were plated on LB- kanamycin agar plates and grown at 37 °C for 18 h. Colonies were picked up and incubated in 10 mL of LB-kanamycin medium at 37 °C for 14 h, followed by isolation of the plasmids using a
QIAprep Spin Miniprep Kit (QIAGEN). The sequences of the resulting plasmids were confirmed by DNA sequencing.
50 2.5.4 Construction of McbAleader-VGIG-LPET-linker-SrtA/pRSFDuet-1
The plasmid was prepared by digesting gblock TwinStrep_McbAld_VGIGG_LPETG shown in Table 2.2 and empty pRSFDuet-1 separately with NdeI and XhoI restriction enzymes overnight, and then ligating the digested DNA fragments through Gibson ligation as described above. The TwinStrep tag was removed using restriction enzymes, NdeI and NheI, and the plasmid was ligated back together via Gibson ligation protocol.
2.5.5 Construction of MBP-McbA/GGGG6to9LPET/pET15b
The GGGG6to9LPET mutations were introduced via QuikChange as described above using MBP-McbA/pET15b as template.
51 Table 2.2 Oligonucleotide sequences of primers used for preparing plasmids.
Primer Name Primer Sequence (5'-3')
LctA/A-1E/K1G/FP GAA TTG GAC CTT ATT TTA GGT GAA GGC GGC GGC
AGT GGA GTT ATT CAT AC
LctA/A-1E/K1G/RP GTA TGA ATA ACT CCA CTG CCG CCG CCT TCA CCT AAA
ATA AGG T CC AAT T C
LctA/S4G/G5S/FP CTT ATT TTA GGT GAA GGC GGC GGC GGC AGC GTT ATT
CAT ACA ATT TCT CAT GAA TG
LctA/S4G/G5S/RP CAT TCA TGA GAA ATT GTA TGA ATA ACG CTG CCG CCG
CCG CCT TCA CCT AAA ATA AG
LctA/CGinstbeforeV6/FP CTT ATT TTA GGT GAA GGC GGC GGC GGC AGC TGC
GGC GTT ATT CAT ACA ATT TCT CAT G
LctA/CGinstbeforeV6/RP CAT GAG AAA TTG TAT GAA TAA CGC CGC AGC TGC CGC
CGC CGC CTT CAC CTA AAA TAA G
LctA/N15R/F21H/FP CAT ACA ATT TCT CAT GAA TGT CGT ATG AAT AGC TGG
CAA CAT GTA TTT AC
LctA/N15R/F21H/RP GTA AAT ACA TGT TGC CAG CTA TTC ATA CGA CAT TCA
TGA GAA ATT GTA TG
52 Table 2.2 (cont.)
Histag-EcoRI- AT G GGC AGC AGC CAT CAC C AT CAT CAC
LctA_K1G2instGGGGSCG_N22R_ CAC AGC CAG GAT CCG AAT TCG ATG AAA
F28H_NotI_pRSFDuet GAA CAG AAC TCC TTC AAC CTG CTG CAG
GAA GTG ACC GAA AGC GAA CTG GAT CTG
AT T CTG GGT GCA AAG GGC GGT GGC GGT
TCT TGT GGC GGC GGC AGC GGC GTA AT C
CAC ACC AT C AGC CAC GAG TGT CGT AT G
AAC TCT TGG CAG CAT GTA TTC ACC TGC
TGC AGC TAA GCG GCC GCA TAA TGC TTA
AGT CGA ACA GAA AGT AAT CGT ATT GTA
CAC GGC
SrtAend/HisTag/XhoI/RP AAT AAT CTC GAG TTA ATG ATG ATG ATG ATG
ATG TTT GAC TTC TGT AGC TAC AAA GAT TTT
ACG TTT TTC
NdeI/(GGS)5/SrtA-59/FP AT T AT T CAT AT G GGC GGC AGC GGC GGC
AGC GGC GGC AGC GGC GGC AGC GGC GGC
AGC CAA GCT AAA CCT CAA ATT CCG AAA
GAT AAA TC
TwinStrep_McbAld_VGIGG_LPETG GAT AAC AAT TCC CCA TCT TAG TAT AT T AGT
TAA GTA TAA GAA GGA GAT ATA CAT AT G TCA
GCT TGG TCA CAC CCA CAA TTC GAA AAG
53 Table 2.2 (cont.)
GGT GGT GGA TCA GGA GGA GGT TCA GGA
GGT TCT GCA TGG TCA CAT CCA CAG
TTCGAGAAGGCTAGC ATGGAACTGAAAG
CTTCCGAATT CGGTGTGGTA CTGAGCGTTG
ACGCTCTGAA GCTGAGCCGC CAGTCTCCTC
TGGGT GTG GGC ATT GGC GGT CTGCC
GGAAACCGGC GGC GGC AGC GGC GGC AGC
GGC GGC AGC GGC GGC AGC GGC GGC AGC
CAAGCTAAACCTCAAATTCCG
McbA_6LPET9_newFqc CA TTA GGT GTT GGC ATT GGT GGT CTG
CCG GAA ACC GGC GGC GGC GGT AG
McbA_6LPET9_Rqc CTA CCG CCG CCG CCG GTT TCC GGC AGA
CCA CCA ATG CCA ACA CCT AAT G
54 Table 2.3 List of plasmids used.
Plasmid Marker Insert/Mutation Reference
histagged-lctA(wt)/ Kanamycin Histagged-lctA(wt) inserted 41
lctM/pRSFDuet-1 between EcoRI and NotI
sites; lctM inserted between
BglII and XhoI sites
histagged-lctA/A-1E/ Kanamycin Histagged-lctA/A- This work
GGGGSCG/N17R/ 1E/K1G/S4G/G5C/CG
lctM/pRSFDuet-1 inserted before V6 in wt-
lctA; N17R mutation
introduced
histagged- Kanamycin GGGGSCG inserted between This work
lctA/K1insGGGGSCG/ K1 and G2 of lctA(wt); N22R
N22R/F28H/ and F28H mutations
lctM/pRSFDuet-1 introduced
lctM/pACYCDuet-1 Chloramphenicol lctM gene inserted between 42
BglII and XhoI sites
lctM/pCDFDuet-1 Spectinomycin lctM gene inserted between 42
BglII and XhoI sites
55 Table 2.3 (cont.)
pGBMCS-SortA Ampicillin srtA gene from Addgene,
Staphylococcus aureus (strain Plasmid
N315) with 1-59 residues #21931
deleted, GB1 tag on N
terminus and His-tag on C
terminus
McbAleader-VGIGG-LPET- Kanamycin TwinStrep_McbAld_VGIGG_ This
linker-SrtA/del1-59/ LPETG work
pRSFDuet-1 inserted as gblock, TwinStrep
tag removed later
MBP-McbA/pET15b Ampicillin MBP-tagged mcbA gene 29
MBP- Ampicillin GGGG(6 to 9) mutated to This
McbA/GGGG6to9LPET/ LPET using MBP- work
pET15b McbA/pET15b as template
MBP-McbB/pET15b Ampicillin MBP-tagged mcbB gene 29
MBP-McbC/pET15b Ampicillin MBP-tagged mcbC gene 29
MBP-McbD/pET28b Kanamycin MBP-tagged mcbD gene 29
56 2.5.6 Protein expression and purification
E. coli BL21(DE3) cells were transformed with plasmids encoding the tagged proteins and selected using appropriate antibiotics. Starter cultures were grown in 10 mL of LB with antibiotics and used to inoculate 1 L of similarly prepared media. After reaching an OD600 of ~0.8, cells were induced with IPTG. For the sortase-fused proteins, the overexpression was carried out at 37 °C for
4 h with 1 M IPTG, and the other overexpressions were performed at 18 °C with 250-400 mM
IPTG for 16-20 h.
Next, cells were harvested by centrifugation at 4000 ´ g for 30 min at 4 °C. Cell pellets were stored at –20 °C. Proteins used for in vitro assays were purified using affinity chromatography via the MBP tag. MBP-tagged proteins were purified from E. coli cells using amylose resin (NEB). The resin was pre-equilibrated with 5 bed volumes of Buffer A [50 mM Tris pH 7.5, 500 mM NaCl, 2.5% (v/v) glycerol, 0.1% (v/v) Triton X-100]. Cell pellets were suspended in Buffer A and sonicated three times for 30 s at 10-13 W using a Misonix MICROSON XL2000
Cell Disruptor with 10 min incubations at 4 °C between each 30 s sonication period to prevent sample heating. The lysate was clarified by centrifugation at 34000 ´ g for 1 h, and applied to the equilibrated resin, and washed with 10 bed volumes of Buffer B [50 mM Tris pH 7.5, 400 mM
NaCl, 2.5% (v/v) glycerol]. Protein was then eluted using Elution Buffer [50 mM Tris pH 7.5, 300 mM NaCl, 2.5% (v/v) glycerol, 10 mM maltose] and collected into Amicon Ultra 15 mL Filter centrifugal filters [30kDa NMWL (Nominal Molecular Weight Limit)]. The eluted protein was concentrated and a buffer exchange was performed into Storage Buffer thrice [50 mM HEPES,
300 mM NaCl, 2.5% (v/v) glycerol] through centrifugation at 4000 ´ g. The final protein concentration was calculated from the absorbance at 280 nm (NanoDrop 2000 UV-Vis
Spectrophotometer) with the predicted protein extinction coefficient
57 (http://web.expasy.org/protparam/). All proteins were stored at –80 °C. Note that after initial column loading, all wash and storage buffers were supplemented with 1 mM tris-(2- carboxylethyl)-phosphine (TCEP).
2.5.7 Purification of MBP-tagged or His-tagged peptides
All peptides that were MBP-tagged were purified as described above. Typically, >20 mg of MBP-tagged peptide (corresponding to >1 mg of substrate after affinity tag removal) was obtained from 1 L of culture.
The His6-tagged peptides were purified by the following procedure. The harvested cells were resuspended in 25 mL of LanA Buffer 1 [6 M guanidine hydrochloride, 20 mM NaH2PO4, pH 7.5 at 25 °C, 500 mM NaCl, 0.5 mM imidazole], and lysed by sonication (50% amplitude, 1.0 s pulse, 2.0 s pause, 5 min). The sample was subjected to centrifugation (23,700 × g for 30 min at
4 °C) and the supernatant containing the His6-tagged peptide was retained for further purification.
The His6-tagged peptide was then purified by immobilized metal affinity chromatography (IMAC) using 2-4 mL of His60 Ni Superflow Resin (Clontech). The supernatant was applied to the column and the column was washed with two column volumes of LanA Buffer 1 followed by two column volumes of LanA Buffer 2 [4 M guanidine hydrochloride, 20 mM NaH2PO4, pH 7.5 at 25 °C, 300 mM NaCl, 30 mM imidazole], and then eluted with three column volumes of LanA Elution Buffer
[4 M guanidine hydrochloride, 20 mM Tris, pH 7.5 at 25 °C, 100 mM NaCl, 1 M imidazole]. The elution fractions containing the peptide were desalted by using a Vydac C4 SPE column and lyophilized and stored at –80 °C.
58 2.5.8 Proteolysis of full-length His-tagged peptides, and HPLC purification
After solid phase extraction, the full-length peptides were purified further on a C5-
Phenomenex column using a Shimadzu Prep-HPLC Instrument and were observed to elute in a time range of 22-24 min when the gradient was set from 2% solvent B to 100%A (solvent A =
0.1% TFA in water, solvent B = 0.0866% TFA in 80% ACN/20% water) over a time period of
45.0 min at a flow rate of 7.0 mL /min. The full-length hybrid peptides were proteolytically cleaved by LysC or GluC at a concentration of 1:100 (w/w) in 50 mM Tris buffer (pH 8.0) overnight at room temperature. The extent of cleavage was monitored using MALDI-TOF MS. The peptides were then purified using a C18-Phenomenex column on an Agilent Analytical HPLC Instrument and were observed to elute in a time range of 21-23 min when the gradient was set from 2% solvent
B to 100%A (solvent A = 0.1% TFA in water, solvent B = 0.0866% TFA in 80% ACN/20% water) over a time period of 45.0 min at a flow rate of 1.0 mL /min.
2.5.9 STEPL ligation33
A 500 mL culture of cells expressing McbAleader-VGIGG-LPET-linker-SrtA/del1-
59/pRSFDuet-1 were harvested by centrifugation and resuspended in 10 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 1 mg/mL lysozyme, 1 EDTA-free Complete Mini protease inhibitor tablet (Roche), pH 7.5). Lysates were incubated at room temperature for 30 min while gently shaking, and then sonicated for 4 min and then centrifuged at 7000 x g for 15 min. The supernatant was then incubated with 0.3 mL of His60 Ni Superflow Resin (Clontech, pre-equilibrated with lysis buffer) for an hour. Then, the resin was added to a column and washed with 10 mL of STEPL buffer (20 mM Tris-base, 50 mM NaCl, pH 7.5). The beads were resuspended in 1.5 mL of STEPL
59 buffer with 100 µM of CaCl2 and the ligating partner, and incubated at 37 °C while gentle rocking.
The progress of the reaction was monitored by MALDI-TOF.
2.5.10 McbBCD in vitro assay29
The MBP-tagged McbB, McbC and McbD proteins were pooled at a 1:1:1 molar ratio such that the overall concentration of each was 100 µM. To this sample, 0.2 µg of thrombin (from bovine plasma) was added, and the sample was proteolytically digested for 4 h at 22 °C to remove the
MBP tags. A 300-µL sample of 20 µM MBP-McbA and low-salt synthetase buffer (50 mM Tris pH 8.5, 25 mM NaCl, 5 mM MgCl2, 10 mM dithiothreitol, 5 mM ATP, pH 8.0) and the above mixture of McbB, McbC and McbD was added to a final concentration of 10 µM to initiate the reaction. The reaction was monitored after 5-, 10- and 24 h time periods via MALDI-TOF MS after thrombin cleavage.
2.6 References:
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J. D., Wenzel, S. C., Willey, J. M., and van der Donk, W. A. Ribosomally synthesized and post- translationally modified peptide natural products: overview and recommendations for a universal nomenclature, Nat. Prod. Rep. 2013, 30, 108-160.
5. Inokoshi, J., Koyama, N., Miyake, M., Shimizu, Y., and Tomoda, H. Structure-Activity
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61 9. Garg, N., Salazar-Ocampo, L. M., and van der Donk, W. A. In vitro activity of the nisin dehydratase NisB, Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 7258-7263.
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16. Latham, J. A., Iavarone, A. T., Barr, I., Juthani, P. V., and Klinman, J. P. PqqD is a novel peptide chaperone that forms a ternary complex with the radical S-adenosylmethionine protein
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62 17. Dong, S. H., Tang, W., Lukk, T., Yu, Y., Nair, S. K., and van der Donk, W. A. The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold, Elife 2015, 4, e07607.
18. Rink, R., Wierenga, J., Kuipers, A., Kluskens, L. D., Driessen, A. J. M., Kuipers, O. P., and Moll, G. N. Production of dehydroamino acid-containing peptides by Lactococcus lactis, Appl.
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P., and Rink, R. Production of a class II two-component lantibiotic of Streptococcus pneumoniae using the class I nisin synthetic machinery and leader sequence, Antimicrob. Agents Chemother.
2010, 54, 1498-1505.
20. Kluskens, L. D., Kuipers, A., Rink, R., de Boef, E., Fekken, S., Driessen, A. J., Kuipers,
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21. Rink, R., Kuipers, A., de Boef, E., Leenhouts, K. J., Driessen, A. J., Moll, G. N., and
Kuipers, O. P. Lantibiotic structures as guidelines for the design of peptides that can be modified by lantibiotic enzymes, Biochemistry 2005, 44, 8873-8882.
22. Rink, R., Kluskens, L. D., Kuipers, A., Driessen, A. J., Kuipers, O. P., and Moll, G. N.
NisC, the Cyclase of the Lantibiotic Nisin, Can Catalyze Cyclization of Designed Nonlantibiotic
Peptides, Biochemistry 2007, 46, 13179-13189.
23. van Heel, A. J., Mu, D., Montalban-Lopez, M., Hendriks, D., and Kuipers, O. P. Designing and producing modified, new-to-nature peptides with antimicrobial activity by use of a combination of various lantibiotic modification enzymes, ACS Synth. Biol. 2013, 2, 397-404.
63 24. Chatterjee, C., Patton, G. C., Cooper, L., Paul, M., and van der Donk, W. A. Engineering dehydro amino acids and thioethers into peptides using lacticin 481 synthetase, Chem. Biol. 2006,
13, 1109-1117.
25. Bindman, N. A., and van der Donk, W. A. A General Method for Fluorescent Labeling of the N-Termini of Lanthipeptides and Its Application to Visualize their Cellular Localization, J.
Am. Chem. Soc. 2013, 135, 10362-10371.
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29. Dunbar, K. L., Melby, J. O., and Mitchell, D. A. YcaO domains use ATP to activate amide backbones during peptide cyclodehydrations, Nat. Chem. Biol. 2012, 8, 569-575.
30. Milne, J. C., Roy, R. S., Eliot, A. C., Kelleher, N. L., Wokhlu, A., Nickels, B., and Walsh,
C. T. Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17,
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64 functionalization of PEGylated capsules for targeting applications, Angew. Chem. Int. Ed. 2012,
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66 Chapter 3: Leader-Peptide Guided Generation of Non-Natural Hybrid RiPP Products –
Chimeric Leader Peptide Strategy*
3.1 Introduction
Bryce Dallas Howard said in Jurassic World “our advances in gene splicing have opened up a whole new frontier. We have learned more from genetics in the past decade than a century of digging up bones’’. Indeed, genetic advancements have uncovered an ability to glimpse at our evolutionary timeline: what events might have happened in the past, how we arrived at our present state of being, and what we can potentially achieve in the future.1-6 Of course, humankind would like to understand and achieve all three, but most important may be the last category. Genome sequencing projects can help us in advancing synthetic biology to not just engineer analogues of natural products that we have come to know as highly potent drugs, but to also create new molecules that have never been produced/isolated under natural stimuli from their host strains.4-6
As mentioned in the previous chapter, one approach to accessing new structural and functional space is through bioengineering of new-to-nature hybrid molecules that may have desired chemical and biological properties and applications in the pharmaceutical and food industry.
Looking at the evolution of biosynthetic pathways in nature, several factors play a role in determining the product: enzymes kinetics, substrate specificity and enzyme promiscuity, environmental stresses and the subsequent changes in the desired concentration and functional role of the natural product, the metabolic rate of production, and positive or negative feedback loops resulting in rate enhancement or inhibition, respectively.7, 8 Therefore, designing a biosynthetic
* Reproduced in part with permission from Burkhart, B.J.; Kakkar, N.; Hudson, G.A.; van der Donk, W.A.; Mitchell, D.A. Chimeric leader peptides for the generation of non-natural hybrid RiPP products. ACS Cent. Sci. 2017, 6, 629-638. Copyright 2017 American Chemical Society. Dr. Brandon Burkhart contributed towards section 3.2.2 and 3.3.
67 route to create a new molecule needs an iterative process with high turnaround so that different permutations and combinations of biosynthetic enzymes can be tested: their number, their compatibility, and their order of action in the scheme. Since our attempts described in chapter 2 using the ‘swapping leader peptides’ approach was not very high-throughput, we needed a new approach for combinatorial biosynthesis.
A different way to understand the recognition of precursor peptides and their subsequent modification by RiPP biosynthetic enzymes is by identifying the conserved recognition residues in the leader peptide (called recognition sequences, RS) and enzymes (called RiPP recognition element, RRE), either found together as a motif in their 1D structure or brought together spatially through coevolution of amino acids to adopt a specific conformation in the 3D structure for/upon binding to the enzyme.9-16 Recently, it was reported that the majority of known prokaryotic RiPP enzymes contain a conserved ∼90-residue PqqD-like domain as RRE, which allows them to bind to the RSs in the leader peptides in most reported cases.15 Since the leader peptide-binding domain and the catalytic domain of RiPP modification enzymes are spatially apart, they are able to perform with high specificity (at the leader peptide-binding site) as well as high promiscuity (at the catalytic site).15, 17
Taking advantage of the RSs on the leader peptides and the corresponding RREs on modification enzymes (not considering exceptions wherein the leader peptide-dependent enzymes lack this domain or do not use it),18-20 we envisioned a “chimeric leader peptide” strategy that would enable the rational combination of RiPP modifications. In this method, RSs from two leader peptides are concatenated to form a new bifunctional leader peptide within a single precursor peptide. With their native RSs present, we hypothesized that two primary modifying enzymes would bind to the respective regions on the chimeric leader peptide and sequentially install
68 posttranslational modifications, resulting in a hybrid RiPP product. In the following sections, it will be demonstrated that, after optimizing the core sequence and properly designing the coexpression plasmids, this approach successfully combines a thiazoline forming cyclodehydratase (HcaD/F) with primary modifying enzymes from several different RiPP classes
Figure 3.1 Overview of RiPP biosynthesis.† (a) A generic RiPP gene cluster and the key maturation steps. (b) Chimeric leader peptide strategy for combining modifying enzymes from unrelated pathways. Examples of primary RiPP modification enzymes combined in this work include (c) azoline-containing RiPPs, and (d) Cβ-cross-linked lanthipeptides. (e) Examples of secondary RiPP modification enzymes used in this work to install D-Ala residues and decarboxylate C-terminal Cys residues.
† Edited version of figure made by Dr. Brandon Burkhart from the Mitchell laboratory at UIUC.
69 including class I and II lanthipeptides (NisB/C and ProcM, respectively; Figure 3.1c and 3.1d).
We also demonstrate how leader peptide-independent tailoring enzymes such as NpnJA and MibD
(Figure 3.1e) can be included. Therefore, this chapter details a broad foundation for the generation of designer RiPP hybrids. The design principles for each example are explained on a case-by-case basis.
3.2 Results
3.2.1 Thiazoline-lanthipeptide (class I) hybrid
In order to test our underlying hypothesis of chimeric leader peptides being able to direct different RiPP biosynthetic enzymes to act on the same core peptide, we first sought to combine an azoline forming cyclodehydratase (HcaD/F) with a lanthipeptide synthetase (NisB/C). HcaD/F is a thiazoline-forming cyclodehydratase (Figure 3.1d) and was chosen because of two main reasons: 1) the RS of the precursor peptide (HcaA) was identified to be towards the N-terminus of the leader (Figure 3.2a ), and 2) because of the long HcaA leader, we had an opportunity to replace the second half that is not important for enzyme binding to make a hybrid leader.21, 22 NisB
(dehydratase) and NisC (cyclase) are involved in biosynthesis of the class I lantibiotic nisin. In a first-of-its-kind mechanism, NisB/C forms the characteristic lanthionine (Lan) or methyllanthonine (MeLan) rings found in lanthipeptides through a two-step process involving glutamyl-tRNA.17, 23 First, NisB catalyzes dehydration of Ser/Thr residues to form the corresponding dehydroalanine (Dha) or dehydrobutyrine (Dhb) in a tRNA-dependent manner.17, 23
Next, NisC catalyzes a 1,4-nucleophilic addition of a Cys thiol to the Cβ of the dehydro-amino acid to form a thioether ring.24 The vast number of reports investigating how and where NisB/C bind to and modify the precursor peptide NisA and its mutants made it an ideal candidate to be used in this strategy (Table 3.1).25-28
70 Table 3.1 Sequence and position dependence of dehydration by NisB. A concise summary of findings about the extent of dehydration of different NisA mutants by NisB. The notation XXXX(−ato−b)YYYY implies XXXX motif from position −a to −b in the peptide was replaced by YYYY motif. The entries shown in red are for the positions in the C-terminal region of the NisA leader which were being investigated in this study.25-28
NisA −20 −10 −1 MSTKDFNLDLVSVSKKDSGASPR – ITSISLLCTPGCKTGALMGCNMKTATCHCSIHVSK
Positions of mutations in NisA Extent of Role played in nisin biosynthesis leader dehydration STKD(−22to−19)AAAA Complete Important for binding to NisB and forming complex with NisT, but not for dehydrations FNLD(−18to−15)AAAA Up to 4 Important for interactions with NisB and NisC, and for dehydrations VSVSKK(−13to−8)HHHHHH 8 The residues towards the N-terminus of LVSV(−14to−11)AAAA Up to 3 NisA leader are more important for binding SKKD(−10to−7)AAAA Up to 4 to NisB and their substitution to Ala reduces SGAS(−6to−3)AAAA Between 4-8 the number of dehydrations GASPR(−5to−1)DDDDK 8 Yields go down ASP(−4to−2)VSL 8 ASP(−4to−2)IDG 8 ASP(−4to−2)IEG 8 ASPR(−4to−1)DDDK 8 PR(−2to−1)AA Between 3-8 Pro is important for binding to NisB and causing dehydrations
A chimeric precursor peptide was constructed from the HcaA and NisA precursor peptides to serve as a potential substrate for both HcaD/F and NisB/C (Figure 3.1a). To incorporate both the RS from HcaA and NisA, we replaced the non-essential C-terminal region of the HcaA leader peptide with the essential region of the NisA leader peptide.22 We next engineered the core region
71 of the chimeric precursor peptide. Since the C-terminus of the NisA leader peptide is amenable to substitutions without significant impairing catalysis (see Table 3.1), we inserted a “GGRCG” motif derived from the HcaA core peptide in the C-terminal region of the NisA leader as shown in Figure
3.2a at a position that would be appropriately distanced from the RS of HcaA to facilitate HcaD/F processing but too close to the NisA RS to allow for NisB/C processing. This motif was then followed by the native NisA core sequence so that it would be spaced approximately at a similar distance from the NisA RS on the chimeric leader peptide as it is in NisA itself. Through this example, we set ourselves to test three main challenges: 1) whether the presence of two potentially competing RREs would cause interference in creating a hybrid 2) whether a chimeric leader peptide (LP) can be designed when both the RREs are near the N-terminus of their respective LPs,
−8 to −4 of HcaA LP, and −18 to −15 of NisA LP, which makes it more challenging for the design of a chimeric leader peptide, and 3) whether we can combine four RiPP primary modification enzymes, namely NisB, NisC, HcaD and HcaF.
The designed hybrid peptide, named Hyp 1.1, was tested by expressing HcaD/F, NisB/C, and His6-Hyp1.1 in Escherichia coli (see Table 3.3 for plasmids). Co-expressions of the peptide with HcaD/F or NisB/C separately acted as controls. The modified peptide was affinity purified, and analyzed via MALDI-TOF mass spectrometry (Figure 3.2b). As can be seen from Figure 3.2c, only when all four enzymes were expressed, did the peptide undergo close to the maximum extent of modifications (the maximum being ten dehydrations). We do not at present know why co- expression of all four enzymes with the hybrid peptide leads to much better conversion than when the peptide is co-expressed with only two enzymes. One possible explanation is that co-expression of all four proteins and the His6-tagged peptide results in less precursor peptide because of the additional protein expression burden and therefore the peptide that is produced is more completely
72
Figure 3.2 Production of a thiazoline−lanthipeptide (class I) hybrid Hyp1.1. (a) Design of the Hyp1.1 sequence from HcaA and NisA precursor peptides. The portions of the leader peptides that were combined are colored. The recognition sequences (RSs) bound by the RREs of the cognate enzymes are indicated with a pink box. The orange underlined regions were combined to generate the chimeric core region. (b) Overview of experimental procedure. Numbering is based on the core position, not peptide length after digestion. (c) The peptide was most modified when co-expressed with HcaD/F and NisB/C, and had up to nine losses of water molecules as analyzed via MALDI TOF-MS. m/z calculated for [M+H]+ peak for 9-fold dehydrated peptide = 10659, m/z observed for [M+H]+ peak for the same = 10706. The peaks for intermediates were separated by 18 Da.
73 modified. Since both NisB-catalyzed dehydrations and HcaD/F-catalyzed cyclodehydrations involve a 18 Da change, analysis of the products was challenging. Thus, we needed a way to reduce the number the products we observed and then carefully ascertain the type of modifications that were associated with a given product. Therefore, we decided to truncate the NisA part of the core peptide from the C-terminus at Met 24 (Figure 3.3a). We assumed that would not be a problem as long as NisB/C would still modify residues from the N-terminus to the C-terminus of NisA as observed for the wild type system.29
Upon co-expression with HcaD/F and NisB/C, the hybrid peptide Hyp1.2 was affinity purified and digested with AspN and analyzed by MALDI-TOF MS (Figure 3.3b). A mixture of products was observed with the most modified species having lost six water molecules (Hyp1.2a in Figure 3.3c), one more than what we expected from dehydration of the NisA core segment, which implied that one Cys residue was cyclodehydrated. To probe the structure of the most processed species, we leveraged the fact that thiazolines are readily hydrolyzed to Cys under mild acid treatment with a concomitant gain of 18 Da and we did indeed observe a gain of 18 Da upon mild acid treatment (Figure 3.3c). Treatment with iodoacetamide under reducing conditions, which would alkylate free Cys, yielded very little product suggesting that the thioether rings were formed in high yield (Figure 3.3c).
74
Figure 3.3 Production of a thiazoline−lanthipeptide (class I) hybrid peptide. (a) Design of the Hyp1.2 sequence from HcaA and NisA precursor peptides. Portions of the leader peptides that were combined are colored. The sequences of Hyp1.1 and Hyp1.2 peptides are shown to highlight the optimizations made. (b) Overview of experimental procedure. (c) The mass of Hyp1.2a (MALDI-TOF-MS) is consistent with one thiazoline, three dehydrations, and two (Me)Lan. These modifications are supported by acid hydrolysis (blue arrow, +18 Da) and by lack of significant labeling by iodoacetamide (green arrow, +57 Da). m/z calculated for [M+H]+ peak for 6-fold dehydrated peptide = 3484.37, m/z observed for [M+H]+ peak for the 6-fold dehydrated peptide = 3484.
75 Since the most processed species was not the major product, we further truncated the NisA core region to Thr20 and mutated Thr20 to Ala. Our hypothesis was that Thr20, being towards the
C-terminus, was the last residue to be dehydrated by NisB,29 and was poorly modified (Figure
3.3a). Upon co-expression of the new hybrid peptide, Hyp1.3, with four enzymes, followed by
IMAC purification and AspN digestion, we again observed a mixture of products but this time the most extensively processed peptide, dubbed Hyp1.3a, was 5-fold dehydrated (Figure 3.4d). If fully modified, it would contain one thiazoline, one Dhb, one Dha, one MeLan, and one Lan (Figure
3.4c and 3.4d). To confirm the structure, we performed mild acid hydrolysis, which resulted in a concomitant gain of 18 Da and no significant adduct formation upon treatment with iodoacetamide under reducing conditions, consistent with the proposed structure.
While previous nisin biosynthetic manipulation introduced new tailoring enzymes from related pathways30, 31 and hybrids have been synthetically prepared,32, 33 the enzymatic incorporation of a non-native modification (thiazoline) has not been reported. Our data show the feasibility of combining disparate RiPP pathways to generate new-to-nature hybrids by rationally designing a chimeric leader peptide. These results also demonstrate that two RiPP enzymes that compete for modification of the same residue (Cys) can still be rendered compatible. In this first example, discrete regions in Hyp1.3 were used to mimic the native peptides and a similar approach could be used to append different modified structures to the N-terminus of other lanthipeptides.
76
Figure 3.4 Production of a thiazoline−lanthipeptide (class I) hybrid. (a) Design of the Hyp1.3 sequence from HcaA and NisA precursor peptides. Portions of the leader peptides that were combined are colored. (b) Overview of experimental procedure. (c) Structure of thiazoline−lanthipeptide (class I) hybrid Hyp1.3a upon AspN digestion. Thiazolines are blue while dehydrations and (Me)Lan are red. Numbering is based on the core position, not peptide length after digestion. (d) The mass of Hyp1.3a (MALDI-TOF-MS) is consistent with one thiazoline, two dehydrations, and two (Me)Lan. These modifications are supported by acid hydrolysis (blue arrow, +18 Da) and by lack of labeling by iodoacetamide (green arrow, +57 Da). m/z calculated for [M+H]+ peak for 5-fold dehydrated peptide = 3234.76, m/z observed for [M+H]+ peak for the 5- fold dehydrated peptide = 3233.
77 3.2.2 Hybrids made with a secondary tailoring enzyme, the reductase NpnJA
In addition to showcasing the use of chimeric leader peptides for primary biosynthetic enzymes, we investigated its utility for general use since some natural biosynthetic gene clusters incorporate secondary tailoring enzymes that install functional groups critical for bioactivity of the finished products.34 We chose to investigate a combination of the same cyclodehydratase, HcaD/F and the class II lanthipeptide synthetase ProcM, a highly promiscuous enzyme that acts on 30 different native precursor peptides (ProcAs) and installs rings of different size and topology.35-38
Therefore, we envisioned it would make a good candidate to explore other designs of the hybrid core peptides. Furthermore, with ProcM the diversity of examples would be expanded to include a case wherein the leader peptide does not have a RRE.18
Regarding the design of the chimeric leader peptide, since only the C-terminal residues of
ProcA leader are essential for binding to ProcM and the majority of the N-terminus is dispensable,35 our collaborators, Professor Douglas A. Mitchell and Dr. Brandon Burkhart, introduced the last nineteen residues of the ProcA3.3 leader peptide after the first 25 residues of the HcaA leader region. For the core region, the entire core peptide of Proc3.3 with a Thr18Ala mutation was used so as to prevent Cys14 from forming the native MeLan ring with Thr18, and a
Met20Asp mutation was introduced to discourage HcaD/F from cyclodehydrating Cys21. With this design, they showed the formation of Hyp2.1a peptide with a thiazoline moiety present within a MeLan ring (Figure 3.5).
78 Figure 3.5 Deduced structure of Hyp2.1a containing a thiazoline within a MeLan.
To include a tailoring enzyme for this hybrid system based on ProcA3.3 and HcaA, we chose the D-Ala-forming Dha reductase NpnJA from Nostoc punctiforme PCC 73102 since the presence of D-amino acids in a RiPP natural product can improve the proteolytic stability of
39-43 peptides in vivo, and can help in stabilizing beneficial turns in peptides/proteins. NpnJA reduces
39 Dha to D-Ala using NAPDH as co-substrate. While being highly substrate tolerant, NpnJA has been shown to prefer hydrophobic flanking residues (Leu, Val, Ile, Lys, Arg, Phe, Ala, Trp, Asn, and His),39 and therefore a D2V/T3S double substitution was introduced into the core peptide to create Hyp2.2 (Figure 3.6a).
Upon co-expressing Hyp2.2, HcaD/F, ProcM and NpnJA in E. coli, a minor +2 Da product was observed while the majority of the peptides still retained the Dha residue at position 3 in the
39 core peptide. However, when we carried out the in vitro NpnJA assay on Hyp2.2a with NADPH as co-substrate overnight, we observed that the major species gained 2 Da (labeled Hyp2.2b), indicating that the NpnJA was able to reduce Dha3 into D-alanine (Figure 3.6d).
79
Figure 3.6 Combination of two primary and one secondary RiPP modifications. (a) Design of hybrid peptide. Orange underlining highlights changed residues. The dashed line indicates the leader peptide of the Proc3.3. (b) Experimental overview for combining HcaF/D, ProcM, and NpnJA. (c) Deduced structure of Hyp2.2b. (d) MALDI-TOF-MS analysis of Hyp2.2 products. The initially formed hybrid has a Dha, thiazoline (arrow indicates hydrolysis), and MeLan. The formation of D-Ala from Dha is indicated by the +2 Da shift (triangle) of the Hyp4.4a species upon addition of NpnJA. The weak ion labeled as M – 4H2O is likely a peptide that underwent two cyclodehydrations and two dehydrations. m/z calculated for [M+H]+ peak for 3-fold dehydrated peptide = 3154.46, m/z observed for [M+H]+ peak for 3-fold dehydrated peptide = 3154.
80 3.2.3 Further exploration of thiazoline-lanthipeptide (class II) hybrids
The general tolerability of HcaD/F and ProcM with ProcA3.3 derived core sequences encouraged us to design a new hybrid sequence based on a peptide with a completely different core sequence, ProcA1.1 so that we could form MeLan rings of different ring size and topology.
Like Hyp2.2, this hybrid (Hyp3.1) appended the HcaA minimal leader sequence to the C-terminal portion of the ProcA1.1 leader peptide (Figure 3.7a). To make Cys3 a better substrate for the cyclodehydratase HcaD/F (Cys16 was not expected to be acted on by HcaD/F as it cannot modify terminal Cys residues), we modified the sequence context around it to mimic the repeat sequence from the HcaA core peptide, “RCG” as was done in case of Hyp1.1. Additionally, we mutated,
Thr12 to Ser since Cys thiols react faster with Dha than with Dhb residues.44 Co-expression of
Hyp3.1 with ProcM and HcaD/F resulted in mainly unmodified peptide as the major product, and two partially modified minor products: a one-fold dehydrated species containing one MeLan between Ser12 and Cys16 and no thiazoline, and a two-fold dehydrated species containing two
MeLan rings, exactly what we observe in prochlorosin 1.1 (Figure 3.8). So, under these conditions,
Cys3 was either not modified at all or was forming a MeLan ring with a dehydrated Thr7.
Therefore, to eliminate the possibility of Cys3 forming a thioether ring, Thr7 was mutated to Ala in Hyp3.2. Unfortunately, co-expression of MBP-tagged Hyp3.2, ProcM, and HcaD/F resulted in mostly unmodified peptide and a minor species with one dehydration (Figure 3.7c). Hence,
ProcA1.1 does not appear to be as amenable for hybrid product generation as ProcA3.3.
81
Figure 3.7 Production of hybrid thiazoline−lanthionine (class II) hybrid, Hyp3.2. (a) Design of hybrid peptides. Orange underlining indicates edited positions of the core peptide. The dashed line indicates the leader peptide of the ProcA1.1. (b) Experimental overview for combination of HcaD/F and ProcM. (c) MALDI-TOF-MS analysis of Hyp3.2 products. Numbering is based on the core position, not peptide length after GluC digestion. m/z calculated for [M+H]+ peak for unmodified peptide = 2083.97, m/z observed for [M+H]+ peak for unmodified peptide = 2082.
82
Figure 3.8 Structure of prochlorosin1.1 observed after ProcM installs modifications on its precursor peptide, ProcA1.1.
3.2.4 Hybrids with the oxidative decarboxylase MibD as secondary tailoring enzyme
We also successfully combined HcaD/F with MibD, a flavin-dependent enzyme from
Microbispora sp. 107891 involved in the biosynthesis of the lanthipeptide microbisporicin, to produce a C-terminally decarboxylated, linear azoline-containing peptide (Figure 3.9). MibD is a
LanD enzyme that carries out oxidative decarboxylation of a C-terminal Cys to the corresponding aminoenethiolate, and its preferred sequence for activity is S-(F/W/N)-(N/C)-S-(F/Y/W/N)-C-C.45
This motif was mimicked by changing the C-terminus of the peptide Hyp4.1, derived from Proc2.8 instead of Proc1.1 as discussed in the previous section, to SAFAC to create test peptide Hyp4.2
(Figure 3.9a).
83
Figure 3.9 Design and production of decarboxylated linear azol(in)e-containing peptide, Hyp4.2. (a) A hybrid peptide was designed that would enable modification by HcaD/F and the leader-independent tailoring enzyme MibD from Microbispora sp. 107891. Although part of the ProcM leader region was present, ProcM was not used in this experiment. (b) Anticipated hybrid biosynthesis. As both thiazoline and decarboxylation are smaller modifications occurring on different ends of the core peptide, the enzymes can presumably act in any order. (c) Overview of experiment. Hyp4.2 was co-expressed with HcaD/F in pET-3.4-FD and MibD in pCDFDuet (see plasmid list in Table 3.3). The peptide was purified, digested with AspN, and analyzed by MALDI TOF-MS. (d) The mass spectrum shows several ions, but the most intense peak (labeled Hyp4.2a) corresponded to the desired hybrid product. (e) Deduced structure of the hybrid Hyp4.2a product after AspN digestion. m/z calculated for [M+H]+ peak for Hyp4.2a = 2725.16, m/z observed for [M+H]+ peak for Hyp4.2a = 2725.
84
Figure 3.10 Chemical structure of post-translation modification, AviCys. Flavin-dependent LanD enzymes first oxidizes the terminal Cys to a thioaldehyde, followed by spontaneous decarboxylation to form the thioenolate. Attack of this enethiol onto the Dha/Dhb residue, yields the AviCys product.45-48
The peptide was used to attempt the production of a hybrid peptide containing an AviCys ring as found in microbisporicin.45 Chemically, AviCys (S-[(Z)-2-aminovinyl]-D-cysteine) is formed by the attack of the aminoenethiolate onto a nearby Dha or Dhb (Figure 3.10).45-48
Therefore, to provide a dehydrated residue, we added ProcM to our system. In addition to adding another kind of functionality to our growing repertoire of PTMs for hybrid RiPP natural products, this setup would also have allowed to further probe the mechanism of formation of AviCys. Till date, it is not known whether these rings are formed enzymatically or non-enzymatically, and whether its formation occurs with the help of an enzyme. In other words, it is not known whether the decarboxylase (LanD), the cyclase (LanC or LanM), or an unknown enzyme is involved.45-48
Co-expression of MBP-tagged Hyp4.2 with ProcM, HcaD/F and MibD resulted in a major species that underwent a loss of 82 Da, a combination of 46 Da due to MibD-catalyzed oxidative decarboxylation and 36 Da due to the loss of two water molecules (Figure 3.11b). However, acid hydrolysis of the product showed the absence of a thiazoline ring, which was essential for the peptide to be considered a hybrid species. The lack of significant changes in the mass spectra upon
IAA treatment under reducing conditions hints towards the formation of an AviCys ring at the C- terminus of the peptide, however because at present it is not known if an aminoenethiolate reacts with IAA, this conclusion needs further verification in future work.
85
Figure 3.11 Combination of two primary and one secondary RiPP modification. (a) Overview of experiment. Hyp4.2 was co-expressed with HcaD/F in pET-3.4-FD, MibD in pCDFDuet and ProcM in pETDuet (see plasmid list in Table 3.3). The peptide was purified, digested with AspN, and analyzed by MALDI TOF-MS. (b) The mass spectrum shows several ions, but the most modified species on the left was acted upon by ProcM and MibD only as seen by lack of addition of 18 Da upon mild acid hydrolysis. m/z calculated for [M+H]+ peak for peptide labeled M - 36 - 46 Da = 2707.15, m/z observed for [M+H]+ peak for the same = 2708.
86 3.3 Discussion
Rational design of biosynthetic pathways to produce custom molecules has long been a goal of combinatorial biosynthesis and natural product synthetic biology. Despite progress in understanding natural pathways, the chemical space that can be explored by current engineering techniques is limited compared to what is theoretically possible.49, 50 In this work, we have demonstrated how a chimeric leader peptide has the potential to unlock the vast chemical space afforded by RiPPs. This concept was inspired by how natural pathways can combine different enzymes (Figure 3.11) and represents a major step forward in RiPP engineering. Although prior work has demonstrated enzyme swapping within related pathways, such as for nisin and the cyanobactins,30, 31, 51-55 this study is to our knowledge the first demonstration of leveraging the recognition sequences and programmability of RiPP enzymes to mix-and-match the primary, class-defining structural features from unrelated classes. In total, four mechanistically unique enzymes were shown to be tolerant to manipulation of their leader peptides and insertion of non- native sequences. The primary difficulty we encountered was ensuring that the enzymes acted at the desired locations and in the correct order. Being from unrelated pathways, there were no natural biosynthetic checkpoints to guide processing toward a single hybrid product, meaning the enzymes could compete for the same residue or act in a fashion that blocked the other enzyme. However, modest editing of the core sequence was sufficient to exploit the innate selectivity of an enzyme to yield the desired product. Future work with other enzymes that exhibit different substrate preferences may lead to a toolbox of enzymes for use in specific applications. Optimizing expression levels of each enzyme through plasmid design also adds another layer of control.
Despite the successful use of these design principles in this work, there are likely some RiPP modifications that are inherently incompatible or too difficult to generally combine given their
87
Figure 3.12 Natural “hybrid” RiPP biosynthetic gene clusters.‡ (a) Linear azol(in)e-containing peptides such as plantazolicin have only the cyclodehydratase as its primary, class-defining modifying enzyme. A tailoring enzyme also dimethylates the N-terminus upon leader peptide proteolysis. (b) Cyanobactins are defined by the presence of a PatG-like protease which recognizes a C-terminal motif to cyclize peptides after removal of the N-terminal leader region (by a PatA- like N-terminal protease). However, most cyanobactin gene clusters have acquired a cyclodehydratase and thus install two primary modifications (azolines and N-to-C macrocyclization). (c) Thiopeptides have three major modifications: azol(in)es, dehydrated amino acids, and a 6-membered nitrogen-containing heterocycle. Because the cyclodehydratase and dehydratase can be found in other RiPP subclasses, the cycloaddition enzyme is the class-defining feature of thiopeptides. Unexpectedly the dehydratase, which is normally leader-peptide dependent
‡ Figure was made by Dr. Brandon Burkhart from the Mitchell laboratory at UIUC.
88 Figure 3.12 (cont.) in lanthipeptide pathways,56 acts in a leader independent fashion in characterized thiopeptide pathways.57
different structural requirements or limited promiscuity of their enzymes. Nonetheless, based on the enzymes combined here, we predict that many other hybrid RiPP combinations will be feasible.
In summary, chimeric leader peptides appear to be a broadly applicable and effective platform for creating hybrid RiPPs. In the cases shown here, the method afforded significant product yields (∼1 mg/L of culture for the core peptide without any optimization) and employed a standard coexpression plasmid system. Moreover, RiPP enzymes can be chosen even if the enzymes act on the same residue. Our approach was successful regardless of native leader peptide length (8 vs 60+), its binding affinity (∼5 µ M for ProcA/ProcM vs ∼50 nM for HcaA/HcaF),22, 38 how it was bound (RRE or non-RRE binding site), or whether the substrate binding site was previously known. Further, our approach is amenable to the inclusion of leader-independent enzymes. Accordingly, the chimeric leader peptide strategy holds much potential for combinatorial
RiPP biosynthesis and opens the door for the generation of additional hybrid RiPP compounds.
3.4 Methods
3.4.1 General methods
Materials were purchased from Fisher Scientific, Gold Biotechnology, or Sigma-Aldrich unless otherwise noted. Tryptone and yeast extract were purchased from Dot Scientific. Restriction enzymes (REs), AspN endoprotease, KOD HotStart polymerase, T4 DNA ligase, and deoxynucleotides (dNTPs) were purchased from New England Biolabs (NEB). Oligonucleotide primers were synthesized by Integrated DNA Technologies (IDT) and are listed in Table 3.2. DNA
89 spin columns were purchased from QIAGEN, and DNA sequencing was performed by ACGT Inc.
All polymerase chain reactions (PCRs) were conducted on an S1000 thermal cycler (Bio-Rad).
3.4.2 Cloning
Plasmids were constructed using restriction enzymes and DNA ligase or by Gibson assembly (GA). For RE cloning, the target gene was amplified with KOD HotStart polymerase, and after PCR cleanup using the QIAquick PCR Purification Kit Protocol, the amplicon was digested with restriction enzymes for ligation into a similarly digested and purified vector. For amplifications which gave more than one product, a gel extraction was performed. Ligations were performed with T4 DNA ligase at 23 °C for 1 h (100 ng vector and 3-fold excess insert). For GA, the standard protocol was followed with guidance from the NEBuilder Assembly tool
(nebuilder.neb.com). Inserts were generated using KOD HotStart polymerase and purified as above. Vector backbones were linearized using PCR or restriction enzymes and similarly purified.
Genes for the cyclodehydratase (HcaF and HcaD),22 lanthipeptide synthetase (ProcM and
58 39 45 NisB/C), dehydroalanine reductase (NpnJA), and flavoenzyme decarboxylase (MibD) were cloned previously from their native organisms. All new plasmids used in this study are listed in
Table 3.3 and were constructed using primers listed in Table 3.2.
The hybrid peptides were built from HcaA or ProcA precursor peptides through overlap extension PCR and initially cloned into a modified pET28 vector with an N-terminal maltose- binding protein (MBP) affinity tag (pET28-MBP). This intermediate was then used to clone the gene encoding MBP-tagged hybrid peptides into Duet vectors or to express and purify the peptide for in vitro activity assays. The gene encoding the NisA-derived hybrid peptide was synthesized as a gblock (IDT) and was cloned directly into a Duet vector in frame with a His6 tag. Duet vectors
90 pACYCDuet, pCDFDuet, pETDuet, and pRSFDuet were used for coexpression in E. coli (see
Table 3.3), as has been well established,59 and genes were sequentially cloned into each multiple cloning site. To insert the cyclodehydratase HcaF and HcaD genes into a single multiple cloning site, the genes were taken together as a single amplicon from pETDuet-HcaF-HcaD.
3.4.3 Site-directed mutagenesis
Changes to the sequence of hybrid peptides were made with cloned PFU or KOD polymerase (a proofreading DNA polymerase isolated from Thermococcus kodakaraensis) and the site-directed mutagenesis (SDM) primers in Table 3.2 following the Agilent QuikChange protocol.
3.4.4 In vivo production of modified hybrid peptides
E. coli BL21(DE3) chemically competent cells were cotransformed with pairs of Duet vectors (Table 3.3) and selected with 100 µg/mL ampicillin for pETDuet, 50 µg/mL kanamycin for pRSFDuet or pET28, and 34 µg/mL chloramphenicol for pACYCDuet. Initial cultures were started from single colonies and grown in 10 mL of Luria−Bertani (LB) broth at 37 °C supplemented with appropriate antibiotics for several hours, and then used to inoculate 0.5 or 1 L of the same media. Cells were grown to OD600 of ~0.8, put on ice for 10 min, and induced with
0.6 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) at 22 °C. After 18 h induction, cells were harvested at 4000 x g for 15 min at 4 °C, washed with TBS (Tris-buffered saline: 25 mM Tris pH
8.0, 150 mM NaCl), and centrifuged again at 4000 x g for 10 min at 4 °C. Cell pellets were stored at −20 °C.
91 3.4.5 Protein expression and purification
E. coli BL21(DE3) cells were transformed with plasmids containing the genes encoding the tagged proteins and selected using appropriate antibiotics. Starter cultures were grown in 10 mL LB with antibiotics and used to inoculate 1 L of similarly prepared media. After reaching an
OD600 of ~0.8, cells were induced overnight with IPTG. Next, cells were harvested by centrifugation at 4000 x g for 15 min at 4 °C, washed with TBS, and then subjected to centrifugation at 4000 x g for 10 min at 4 °C. Cell pellets were stored at −20 °C. Proteins used for in vitro assays were purified using affinity chromatography via the MBP or His6 tag.
MBP-tagged proteins were purified from E. coli cells using amylose resin (NEB). The resin was preequilibrated with 5 bed volumes of Buffer A [50 mM Tris pH 7.5, 500 mM NaCl, 2.5%
(v/v) glycerol, 0.1% (v/v) Triton X-100]. Cell pellets were suspended in Buffer A and sonicated 3 times for 30 s at 10-13 W using a Misonix MICROSON XL2000 Cell Disruptor with 10 min incubations at 4 °C between each 30 s sonication period to prevent sample heating. The lysate was clarified by centrifugation at 34000 x g for 1 h on a Sorvall RC 6 plus centrifuge, and the supernatant applied to the equilibrated resin, and washed with 10 bed volumes of Buffer B [50 mM Tris pH 7.5, 400 mM NaCl, 2.5% (v/v) glycerol]. Protein was then eluted using Elution Buffer
[50 mM Tris pH 7.5, 300 mM NaCl, 2.5% (v/v) glycerol, 10 mM maltose] and collected into
Amicon Ultra 15 mL Filter centrifugal filters [30 kDa NMWL (Nominal Molecular Weight
Limit)]. The eluted protein was concentrated and a 10-fold buffer exchange was performed into
Storage Buffer [50 mM HEPES, 300 mM NaCl, 2.5% (v/v) glycerol] through centrifugation at
4000 x g. The final protein concentration was calculated from the absorbance at 280 nm
(NanoDrop 2000 UV-Vis Spectrophotometer) with the predicted protein extinction coefficient
(http://web.expasy.org/protparam/) and the Bradford assay (Pierce™ Coomassie Protein Assay
92 Kit). All proteins were stored at −80 °C. Note that after initial column loading, all wash and storage buffers were supplemented with 1 mM tris-(2-carboxylethyl)-phosphine (TCEP).
His6-tagged proteins were purified identically to the above protocol except the buffer composition differed as follows: Buffer A [50 mM Tris pH 7.5, 500 mM NaCl, 2.5% (v/v) glycerol,
15 mM imidazole], Buffer B [50 mM Tris pH 7.5, 400 mM NaCl, 2.5% (v/v) glycerol, 30 mM imidazole], and Elution Buffer [50 mM Tris pH 7.5, 300 mM NaCl, 2.5% (v/v) glycerol, 250 mM imidazole].
3.4.6 Purification of modified hybrid peptides
All hybrid peptides except Hyp1.x were MBP-tagged and purified as described above.
Typically, >20 mg of MBP-tagged peptide (corresponding to >1 mg of core peptide after affinity tag removal) was obtained from 1 L of culture. Hyp1.x was expressed as a His6-tagged peptide and purified by the following procedure. The harvested cells were resuspended in 25 mL of LanA
Buffer 1 [6 M guanidine hydrochloride, 20 mM NaH2PO4, pH 7.5 at 25 °C, 500 mM NaCl, 0.5 mM imidazole], and lysed by sonication (50% amplitude, 1.0 s pulse, 2.0 s pause, 5 min). The sample was subjected to centrifugation (23,700 x g for 30 min at 4 °C) and the supernatant containing the His6-tagged peptide was retained for further purification. The His6-tagged peptide was then purified by immobilized metal affinity chromatography (IMAC) using 2-4 mL of His60
Ni Superflow Resin (Clontech). The supernatant was applied to the column and the column was washed with 2 column volumes of LanA Buffer 1 followed by 2 column volumes of LanA Buffer
2 [4 M guanidine hydrochloride, 20 mM NaH2PO4, pH 7.5 at 25 °C, 300 mM NaCl, 30 mM imidazole], and then eluted with 3 column volumes of LanA Elution Buffer [4 M guanidine hydrochloride, 20 mM Tris, pH 7.5 at 25 °C, 100 mM NaCl, 1 M imidazole]. The elution fractions
93 containing the peptide were desalted by Vydac C4 SPE column and lyophilized and stored at −80
°C.
3.4.7 In vitro modification assays
In vitro assays were performed with purified proteins and peptides as described previously.22, 39, 45, 60 In general, peptides were supplied at 50 µM. Proteins were present at 5 to 20
µM and allowed to react for 16 h. Reactions were carried out in synthetase buffer [50 mM Tris pH
7.5, 125 mM NaCl, 20 mM MgCl2, 1 mM TCEP]. However, assays with the dehydroalanine reductase (NpnJA) were performed in 50 mM HEPES pH 7.4 with 1.5 mM NADPH.
3.4.8 MALDI-TOF-MS
Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI
TOF-MS) was used to analyze posttranslational modifications based on their characteristic mass changes and reactivity towards mild acid or iodoacetamide treatment. Upon in vitro or in vivo modification, peptides were digested with a commercial protease (AspN or GluC) following the manufacturer’s protocol. Hydrochloric acid (200 mM) or iodoacetamide (20 mM at 8 pH) were individually added to an aliquot of the digestion and allowed to react for 1 h in the dark at 22 °C except in case if acid hydrolysis of Hyp1.x peptides, the incubation time was 5-15 min. Next, the samples were desalted with a ZipTip (Millipore) according to the manufacturer’s instructions and eluted into 3.5 µL of 70 % MeCN. Then, 0.5 µL was applied to a MALDI target plate with 1 µL of matrix and subsequently mixed until crystallized. The samples were analyzed using a Bruker
Daltonics UltrafleXtreme MALDI-TOF mass spectrometer in reflector/positive mode.
94 Table 3.2 Primer sequences. Restriction enzyme sites are underlined. F, forward; R, reverse.
Primer Name Primer sequence (5’ to 3’) Purpose
ACA CCC GGT TGT AAA ACA GGA
Hyp1.2 F GCT CTG TAA GGT TGT AAC ATG SDM
AAA ACA GCA ACT TGT C
GAC AAG TTG CTG TTT TCA TGT
Hyp1.2 R TAC AAC CTT ACA GAG CTC CTG SDM
TTT TAC AAC CGG GTG T
GT ATT TCG CTA TGT ACA CCC
Hyp1.3 F GGT TGT AAA GCG TAA GGA GCT SDM
CTG TAA GGT TGT AAC ATG A
TCA TGT TAC AAC CTT ACA GAG
Hyp1.3 R CTC CTT ACG CTT TAC AAC CGG SDM
GTG TAC ATA GCG AAA TAC
CGG CGG AGG CGT TTC CGG CAT Hyp2.2 F SDM CCA AGC GGT GCT GCA C
CTT GGA TGC CGG AAA CGC CTC Hyp2.2 R SDM CGC CGC TAG CGG CTT CC
Amplifying vector backbone
GCG GCC GCA TAA TGC TTA AGT containing MBP-tagged part- Hyp_backbone F CGA ACA GAA AGT AAT CG HcaA leader, HcaD and
HcaF for gibson ligation
95 Table 3.2 (cont.)
Amplifying vector backbone CCA ATA CAC AAC ATT ACC AGT containing MBP-tagged Hyp_backbone R TTG ATA ATC ATT AAG GTT TAA part-HcaA leader, HcaD and TGA TTG TAG TTC TTG TTG HcaF for gibson ligation
GAT TAT CAA ACT GGT AAT GTT
MBP-HcaProcA1.1 GTG TAT TGG GAC TTA GAA AAA Amplifying ProcA1.1 for
F GAG CAT CGC CAA ACC CTG gibson ligation
GTT CGA CTT AAG CAT TAT GCG Amplifying ProcA1.1 for MBP-HcaProcA1.1 GCC GCT TAT CAG CAC ACA TTG gibson ligation Gibson R ATA GTA AAA CGG TTA GCA ligation
GTG CAG GGT ACT GCT AAC CGT
Hyp3.1_T12S F TTT AGC ATC AAT GTG TGC TGA SDM
TAA AAG
CTT TTA TCA GCA CAC ATT GAT
Hyp3.1_T12S R GCT AAA ACG GTT AGC AGT ACC SDM
CTG CAC
G GGT TTT CGC TGT GGT CAG
Hyp3.1_RCG_T12 GGT A CTG CTA ACC GTT TTA Gibson ligation S F1 GCA TCA ATG TGT GCT GAT AAA
AGC TTG
96 Table 3.2 (cont.)
Hyp3.1_RCG_T12 CCA GCC ACA CCT TCC AGA TCA Gibson ligation S R1 TCA TCA GAC A
Hyp3.1_RCG_T12 CTG CTA ACC GTT TTA GCA TCA Gibson ligation S F2 ATG TGT GCT GAT AAA AGC TTG
TAC CCT GAC CAC AGC GAA AAC Hyp3.1_RCG_T12 CCC CAG CCA CAC CTT CCA GAT Gibson ligation S R2 CAT CAT CAG ACA
GT TTT CGC TGT GGT CAG GGT
Hyp3.2 F GCT GCT AAC CGT TTT AGC ATC SDM
AAT G
CAT TGA TGC TAA AAC GGT TAG
Hyp3.2 R CAG CAC CCT GAC CAC AGC GAA SDM
AAC
CT CCA TCC TAT AGC GCA TTT
Hyp4.2 F GCA TGC TAA GCG GCC GCA TAA SDM
T
ATT ATG CGG CCG CTT AGC ATG Hyp4.2 R SDM CAA ATG CGC TAT AGG ATG GAG
97 gblock sequence synthesized (IDT) for Hyp1.1 (vector backbone, gray; HcaA leader peptide, blue;
NisA leader peptide; mutation for HcaD/F thiazoline; NisA core peptide, black):
CACAGCCAGGATCCGAATTCGAGCTCGATGAATCAGTTTCAACAAGAACTACAATC ATTAAACCTTAATGATTATCAAACTGGTAATGTTGTGTATTGGGATATGAGTACAAA AGATTTTAACTTGGATTTGGTATCTGTTTCGAAGAAAGGCGGCCGTTGCGGTCCACG CATTACAAGTATTTCGCTATGTACACCCGGTTGTAAAACAGGAGCTCTGATGGGTTG TAACATGAAAACAGCAACTTGTCATTGTAGTATTCACGTAAGCAAATAAAAGCTTGC GGCCGCATAATGCTTAAGTC
Translated peptide from gblock sequence (coloring as above): MNQFQQELQSLNLNDYQTGNVVYWDMSTKDFNLDLVSVSKKGGRCGPRITSISLCTPG CKTGALMGCNMKTATCHCSIHVSK
Table 3.3 New plasmids generated for this study. HcaF-HcaD are the thiazoline cyclodehydratase, NisB and NisC are a class I lanthipeptide synthetase, and ProcM is the class II lanthipeptide synthetase. Constructs for the tailoring enzymes MibD and NpnJA are reported elsewhere.39, 45 Abbreviations: MCS, multiple cloning site; MBP, maltose binding protein. The entries in italics were made by Dr. Brandon Burkhart from Mitchell laboratory at UIUC.
Name Plasmid backbone Gene(s)
(copy number61) MCS1; MCS2
pCDF-1.1 pCDFDuet (~20-40) His6-Hyp1.1
pCDF-1.2 pCDFDuet (~20-40) His6-Hyp1.2
pCDF-1.3 pCDFDuet (~20-40) His6-Hyp1.3
pET-2.1-FD pETDuet (~40) MBP-Hyp2.1; HcaF-HcaD
pET-2.2-FD pETDuet (~40) MBP-Hyp2.2; HcaF-HcaD
98 Table 3.3 (cont.)
pET-3.1-FD pETDuet (~40) MBP-Hyp3.1; HcaF-HcaD
pET-3.2-FD pETDuet (~40) MBP-Hyp3.2; HcaF-HcaD
pET-4.1-FD pETDuet (~40) MBP-Hyp4.1; HcaF-HcaD
pET-4.2-FD pETDuet (~40) MBP-Hyp4.2; HcaF-HcaD
pACYC-M pACYCDuet (~10) (empty); ProcM
pRSF-F-D pRSFDuet (>100) HcaF; HcaD
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106 Chapter 4: Incorporation of Non-Proteinogenic Amino Acids in class I and II Lantibiotics§
4.1 Introduction
The extensive genome sequencing efforts of the past decade have provided unparalleled information regarding the potential capacity of organisms to produce highly diverse biologically active natural products.1 As mentioned in Chapter 1, RiPPs are a large class of such molecules and the lanthipeptides form the largest group among RiPP natural products.2-5 Many lanthipeptides possess antimicrobial activity and are called lantibiotics. These cyclic peptides have garnered increasing attention with respect to engineering for three main reasons. First, they are ribosomally synthesized and hence variants of the precursor peptide can be made with relative ease using mutagenesis. Second, the biosynthetic enzymes have been shown to be inherently promiscuous facilitating analogue formation. And third, the post-translational modifications, by restricting the conformational flexibility, allow for higher chemical stability and tighter and/or more specific binding to the natural target, thereby improving the bioavailability and bioactivity.4, 6, 7
Many efforts have been made to increase the diversity of lantibiotics by site-directed mutagenesis to afford variants with substitutions by proteinogenic amino acids.8-17 More recently, several studies have also shown the potential of expanding the repertoire of substitutions to non- proteinogenic amino acids (or unnatural amino acids, UAAs) for a subset of lanthipeptides.18-23
Prompted by these recent reports, we investigated incorporation of UAAs into the class I lanthipeptide nisin and the class II lanthipeptide lacticin 481. The methods described in this chapter are complementary to previous attempts and illustrate approaches to ensure both incorporation of
§Reproduced from the manuscript under review titled ‘Incorporation of non-proteinogenic amino acid incorporation in class I and II lantibiotics’.
107 the UAAs and full post-translational modification. Like the chimeric leader approach described in
Chapter 3, this methodology provides a versatile approach for creating new structural scaffolds for
RiPPs.
4.2 Results and discussion
4.2.1 Lacticin 481 as model system
Lacticin 481 was first isolated in the early 1990s from Lactococcus lactis subsp. lactis
CNRZ 481 and exhibits inhibitory activity against a range of indicator strains.24 It is the prototypical member of a large group of natural variants with similar ring topology that includes nukacin ISK-1 and members of the salivaricin family.25 These compounds exhibit diverse antimicrobial potency, but they all contain the mersacidin-like lipid II binding motif within their
A-ring (Figure 4.1a).8, 10, 26-28 As mentioned in Chapter 2, during the biosynthesis of lacticin 481, the lanthipeptide synthetase LctM performs both dehydrations and cyclizations on its precursor peptide LctA (Figure 4.1).29 The enzyme first phosphorylates the Ser/Thr targeted for dehydration, and then eliminates the phosphate to produce the dehydro amino acids.30 A separate active site catalyzes the addition of Cys residues to the dehydro amino acids.31 In a previous study, our laboratory showed that substituting aromatic residues in the C-ring with UAAs using substrates containing core peptides prepared by solid-phase peptide synthesis (SPPS) could improve the bioactivity of lacticin 481.32 These findings were a good starting point for biological incorporation of UAAs at those positions. In this study, we aimed to investigate the factors required for successful use of stop codon suppression technology for incorporation on UAAs into lantibiotics.
In a recent report, our group showed the genetic incorporation of hydroxy-amino acids into lantibiotics using the pyrrolysine tRNA synthetase and its cognate tRNA.20 Using that platform,
108
Figure 4.1 a) Biosynthesis of lacticin 481, b) Commonly found PTMs in lanthipeptides, color code is as per the residues shown in (a).
109 we herein demonstrate the production of several analogs of lacticin 481 and compare their bioactivities with the parent compound. Pyrrolysine tRNA synthetase (PylRS) and its mutants are robust and versatile enzymes that have been employed to charge pyl-tRNA with several lysine and phenylalanine derivatives.33 We chose the N346A/C346A mutant of the Methanosarcina mazei
PylRS and its cognate pyl-tRNA to incorporate o-Br-Phe, o-NO2-Phe, m-Br-Phe, and m-CF3-Phe at three positions (Trp19, Phe21 and Phe23) of lacticin 481.34-36 pRSFDuet plasmids were prepared encoding N-terminally His-tagged LctA mutants containing the amber stop codon at each of these positions (W19/F21/F23), LctM, and pyltRNA. These plasmids were used together with the previously reported pEVOL plasmid carrying orthogonal translation machinery.36 Since the mutant PylRS has low selectivity with respect to Phe and its derivatives, and therefore results in background incorporation of Phe in the growing peptide chain, we carried out the co-expressions in synthetic media37 lacking Phe. Additionally, to improve the solubility of these analogs Asn15 in lacticin 481 was mutated to Arg.32 Co-expression of His-tagged-LctA/N15R/TAG mutants,
LctM, PylRS and pyltRNA was carried out in E. coli BL21(DE3) in synthetic media supplemented with the desired non-proteinogenic amino acid. After immobilized metal affinity chromatography
(IMAC) purification of the peptide, analysis by matrix-assisted laser desorption ionization time- of-flight mass spectrometry (MALDI-TOF MS) showed the formation of full-length LctA with four dehydrations and containing the UAA.
The incorporation was complete and well-tolerated at all positions for all four Phe derivatives tested (for a representative example, see Figure 4.2, all other examples are shown in
Figures 4.8-4.18 in the Methods section 4.4). Thus, the lacticin 481 production system in E. coli was not affected by the expression of the additional machinery required for UAA incorporation nor by the need to grow in synthetic media. The yields of the peptides were in the range of 0.5-0.8
110
Figure 4.2 MALDI-TOF mass spectrum of lacticin 481/N15R/Trp19m-CF3Phe produced by LctM in E. coli BL21(DE3). The dashed line shows the position of the expected peptide if stop codon supression did not occur.
mg/L without optimization.
Since the first residue of the core peptide is Lys, digestion with endoprotease LysN yielded the lacticin 481 analogs, which were tested for bioactivity against L. lactis HP. All mutants were active, with five of the mutants slightly more active than lacticin 481: Trp19o-ClPhe, Trp19m-
BrPhe, Trp19o-NO2Phe, Phe21o-NO2Phe and Phe23o-ClPhe (Figure 4.3), similar to previous observations with aromatic substitutions at these positions prepared with SPPS.28 The MIC values determined were 195 nM for Trp19o-NO2Phe, and 390 nM for Trp19o-ClPhe, Trp19m-BrPhe,
Phe21o-NO2Phe and Phe23o-ClPhe as was the case for the wt-lacticin 481. The improved agar activity for the four analogs could be due to the different diffusion rates.
111
Figure 4.3 Agar diffusion assay of wt-lacticin 481 and its mutants against L. lactis HP.
4.2.2 Nisin as model system
Next, we turned to the class I lantibiotic nisin, which is the best-characterized member of the family. It has been employed in the food industry as a food preservative for the last five decades and significant bacterial resistance is yet to develop.38 As mentioned in Chapter 3, nisin is biosynthesized by two proteins, the dehydratase NisB and cyclase NisC (Figure 4.4). Nisin exerts its antimicrobial activity through a highly potent dual mode of action. The A- and B-rings recognize and sequester the pyrophosphate moiety of lipid II,39 and the C-, D- and E-rings insert into the membrane creating pores.40-42 Despite the high antimicrobial potency and non-toxicity to humans, nisin is not employed as a human therapeutic, in part because of its low solubility in water, and degradation at acidic and alkaline pH.43-47
112
Figure 4.4 Biosynthesis of the class I lantibiotic nisin A. The process is shown as first completion of dehydration and then cyclization, but this is not necessarily the case. The cyclization catalyzed by NisC is reversible as shown by experiments involving resubjection of modified NisA to the cyclization conditions.48
We previously reported the heterologous expression of nisin in E. coli, which opened up the possibility to investigate generation of nisin analogs using amber stop codon suppression.
Taking into account the positions that improved the bioactivity of nisin in previous site-directed mutagenesis studies,49 as well as the positions at which degradation has been reported,44 we chose
113 to place amber stop codons at positions encoding Thr2, Ser5 and Met21. Since a previously reported nisin mutant (Ile4Val/Ser5Phe/Leu6Gly) is a known inhibitor of Lactococcus lactis that overcomes the naturally occurring self- protection mechanism,12 we prepared the same variant but with an amber stop codon for mutations at Ser5. Plasmids were constructed encoding the His- tagged NisA mutants and NisB (in pRSFDuet), NisC and pyltRNA (in pCDFDuet), and the pylRS mutant and another copy of pyltRNA (in pEVOL). These plasmids were used for co-expression in
E. coli BL21(DE3) using synthetic media lacking Phe and supplemented with m-Br-Phe and m-
CF3-Phe. Unfortunately, we observed that the full-length peptide with the unnatural amino acid was only partially dehydrated by NisB, and that truncants of the peptide were observed in sizable amounts due to incomplete amber codon suppression. As a control, we also expressed wild type
(wt) NisA under these conditions and observed that it also was incompletely dehydrated by NisB
(see Figure 4.19 in the Methods section 4.4), indicating that the incomplete dehydration is not the result of the UAA substitutions. We investigated whether the level of NisB expression was insufficient and switched the media to LB, which previously led to full dehydration,18 but this resulted in substantial decline in stop codon suppression (Figure 4.5). Thus, unlike the observations for lacticin 481, where the requirements for stop codon suppression did not affect the lantibiotic processing machinery, for nisin the additional burden placed upon the cell requires additional optimization of the post-translational modification process. Similar findings were also recently reported in another study on incorporation of UAAs into nisin.22
To improve the activity of NisB in E. coli grown in synthetic media, we expressed multiple copies of NisB (up to three) which afforded a mixture of peptides with improved dehydration
114
Figure 4.5 MALDI-TOF mass spectrum of His6-NisA-Thr2m-BrPhe modified by NisB and NisC in E. coli BL21(DE3) grown in LB.
(see Figure 4.20 in the Methods section 4.4), but the problem of incomplete stop codon suppression persisted. While amber stop codons have been widely used for genetic incorporation of UAAs, the method in some cases leads to poor incorporation efficiencies and thus lower yield.50-52 This inherent problem is thought to arise in part because of competition between Release Factor 1 (RF1) and the orthogonal tRNA charged with the UAA during peptide synthesis.53, 54 In prokaryotes, RF1 is responsible for recognizing the amber stop codon UAG and terminating peptide synthesis.53, 55
Thus, for an unrestrictive reassignment of the amber codon from a stop signal to an UAA incorporating process, deletion of the prfA gene encoding RF1 has been reported by generation of an E. coli strain named genomically recoded organism (GRO).56 In GRO, 321 UAG codons were replaced by synonymous UAA codons, thereby eliminating the basal need for RF1, and the prfA gene could be deleted. The GRO provides significant advantages for incorporating UAAs into
115 proteins by amber suppression.57 To address the problem of truncation of NisA mutants in our studies, we decided to use an even more evolved GRO developed by Jewett and coworkers.58 This
E. coli strain (C321.ΔprfA-T7RNAPΔrneΔompTΔlon) has the T7 polymerase gene inserted and the genes for endonuclease (rne) and proteases (ompT and lon) deleted from the genome. These mutations are used commonly in recombinant protein expression to increase mRNA stability and reduce degradation of heterologous proteins, respectively.59-62 Given cytosolic expression of the lanthipeptides in this work, we suspected that the rne and lon mutations could provide benefits.
Transformation of this strain with the nisin plasmids overcame the problem of NisA truncation.
Moreover, we could switch to LB media and observed fully-modified full-length nisin analogues as the product (Figure 4.6), although the growth rate as well as the cell yields were reduced, presumably because of the high antibiotic pressure during cell culture (kanamycin, chloramphenicol and spectinomycin were required to maintain plasmids), the number of proteins being expressed in this GRO, and the lower temperature at which the strain was employed (30 oC).
The UAA-containing nisin variants were produced in the range of 0.2-0.5 mg/L prior to leader peptide removal.
116
Figure 4.6 MALDI-TOF mass spectrum of NisA-Thr2m-BrPhe modified by NisB and NisC in GRO expressing a single copy of nisB grown in LB medium.
Because of these low yields, we only tested the Ser5m-BrPhe mutant for bioactivity after trypsin digestion, and it was found to have lower activity than wt nisin (Figure 4.7). Very recently,
Schultz and coworkers reported a different strategy to improve the dehydration of nisin mutants containing para-substituted-Phe and Lys derivatives.22 In that study, additional copies of E. coli tRNAGlu/GluRS were expressed, and Methanocaldococcus jannaschii tRNATyr/TyrRS or
Methanosarcina barkeri pyrrolysine tRNAPyl/PylRS were used for incorporation of the non- proteinogenic amino acid. Our strategy explored a different approach to address the problem of truncation and incomplete dehydration, offering alternative strategies for future studies.
117
Figure 4.7 Agar diffusion assay of wt nisin A (left) and nisin-I4V/Ser5m-BrPhe/L6G (right) against L. lactis HP.
4.3 Conclusion
In summary, we report two different approaches for the production of lantibiotic variants containing UAAs depending on the demands of the biosynthetic machinery. This study provides a platform for further exploration of bioactivities and stabilities of different lantibiotics including nisin. This work also adds to the growing list of examples of incorporation of UAAs into RiPPs via stop codon repression methods.18, 21, 22, 63-65
4.4 Methods
4.4.1 Materials
All oligonucleotides, restriction endonucleases, and DNA polymerases were purchased from Integrated DNA Technologies, New England Biolabs or Invitrogen. Media components for bacterial cultures were purchased from Difco laboratories. Chemicals were purchased from Fisher
Scientific or from Aldrich unless noted otherwise. Non-canonical amino acids were purchased from Chem Impex International. Solvents commonly used in peptide purification, including trifluoroacetic acid (TFA) and acetonitrile, were obtained in RP-HPLC grade or better and used directly without further purification. Endoproteinase LysN and trypsin were purchased from Roche
118 Biosciences or Worthington Biosciences. E. coli DH5α was used as host for cloning and plasmid isolation, and E. coli BL21 (DE3) or genomically recoded organism (GRO) C321.ΔprfA-
T7RNAPΔrneΔompTΔlon66 were used as a host for co-expression. GRO was obtained from the
Jewett laboratory at Northwestern University.
4.4.2 General methods
All polymerase chain reactions (PCR) were carried out on a C1000™ thermal cycler (Bio-
Rad). DNA sequencing was carried out by ACGT Inc. using appropriate primers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) was carried out on an UltrafleXtreme TOF/TOF mass spectrometer (Bruker Daltonics) at Mass Spectrometry
Facility (UIUC).
4.4.3 Construction of pRSFDuet-1/nisA mutants+nisB and pCDFDuet-1/nisC/pylT
The amber codon TAG was introduced into nisA/nisB/pRSFDuet-1 at different positions of nisA, generating co-expression vectors encoding NisA-T2B, NisA-I4V/S5B/L6G, and NisA-
M21B, where B stands for the amber stop codon. The mutants were generated using QuikChange
PCR with the nisA/nisB/pRSFDuet-1 vector as the template.18 The primer sequences are listed in
Table 4.1. The PCR mixture included 1× Phusion HF buffer 67, dNTP mixture (2 mM each), primers (stock solution 100 µM each, final concentration 1 µM each), DMSO (2%, v/v), template plasmid nisA/nisB/pRSFDuet-1 (~ 40 ng) and Phusion High-Fidelity DNA Polymerase (0.02
U/µL). The PCR amplification was performed by twenty five cycles of denaturing (98 °C for 10 s), annealing (57.5 °C for 30 s), and extending (72 °C for 150 s). The product pNisA1.x (Table
4.3) was purified by QIA quick PCR Purification Kit (QIAGEN). The purified PCR product was
119 mixed with 2 µL of 10x NEBuffer 4 and 2 µL of DpnI (stock solution 20,000 U/mL, New England
Biolabs), followed by incubation at 37 °C for 1 h. DpnI was heat-inactivated by incubation at 80
°C for 20 min. A 5 µL aliquot of the resulting solution was used for heat shock transformation of
E. coli DH5a.
One copy of nisC was inserted between BglII and XhoI restriction sites into the MCS2 of pCDFDuet-1 via PCR amplification, digestion, and ligation using nisC/pACYCDuet-118 as the
PCR template. In order to raise the copy number of MmPylT expressed in the host cells, one copy of Mmpyrrolysyl-tRNA (pylT) was introduced between BamHI and NotI restriction sites into the
MCS1 of the constructed nisC/pCDFDuet-1 using the the pEVOL-pylT-mmPylRS(2X)-
N346A/C348A vector as the PCR template, resulting in pNisTC (Table 4.3). The primer sequences are listed in Table 4.1.
A copy of nisB with E. coli-optimized codons was inserted into MCS2 of pNisTC after the nisC gene separated by ribosome binding site (rbs) via PCR amplification using appropriate primers and then Gibson ligated using the vendor’s protocol to arrive at pNisTCB plasmid. An additional copy of nisB was inserted in a similar fashion after the first copy of nisB gene separated by a rbs to produce pNisTCBB.
Table 4.1 Sequences of oligonucleotide primers used to prepare analogs of nisin.
Primer Name Primer Sequence (5'-3') proK_BamHI_FP ATA ATA T GG GAT CCG T GT GCT T CT CAA AT G CCT
GAG GCC tRNA_NotI_RP ATA ATA TGG CGG CCG CCA TGC AAA AAA GCC
TGC TCG TTG
120 Table 4.1 (cont.)
NisA_T2B_FP CAG GTG CAT CAC CAC GCA TTT AGA GTA TTT
CGC TAT G
NisA_T2B_QRP GCG TGG TGA TGC ACC TGA AT C T T T CT T CG
NisA_M21B_FP GAG CTC TGA TGG GTT GTA ACT AGA AAA CAG
CAA CTT G
NisA_M21B_QRP GTT ACA ACC CAT CAG AGC TCC TGT TTT ACA
ACC
NisC_T7_PacI_FP TCC AGG CAT TAA TTA ACG AAA TTA ATA CGA CTC
ACT ATA GGG G
NisC_AvrII_RP ATA ATA T GC CTA GGT CAT TTC CTC TTC CCT CCT
TTC
NisC/RBS/optNisB/Fgib GAT TTT TTG AAA GGA GGG AAG AGG AAA TGA
AGG AGA TAT ACC ATG ATC AAA AGC TCC TTT
AAA GCA CAG optNisB/PacI/pCDFDuet/Rgi GCG GTG GCA GCA GCC TAG GTT AAT TAA TTA b TTT CAT ATA TTC TTC GCT CAC AAA CAG ACG
NisBend/RBS/NisB/Fgib TTC GGA AGA ATA CAT GAA ATG AAG GAG ATA
TAC CAT GAT AAA AAG TTC ATT TAA AGC TCA
ACC G pCDF/PacI/NisB/Rgib GCG GTG GCA GCA GCC TAG GTT AAT TAA TCA
TTT CAT GTA TTC TTC CGA AAC AAA C
121 4.4.4 Construction of pRSFDuet-1/lctA mutants+lctM+pylT
The amber codon TAG was introduced into histagged-lctA(wt)/lctM/pylT/pRSFDuet-1 at different positions of lctA, generating co-expression vectors encoding LctA-W19B, LctA-F21B, and LctA-F23B, where B stands for the amino acid to be introduced via the amber stop codon. The mutants were generated using QuikChange PCR with the histagged- lctA(wt)/lctM/pylT/pRSFDuet-120 vector as the template. The primer sequences are listed in Table
4.2. To improve solubility of these peptides,32, 68 the N15R mutation was introduced via
QuikChange PCR.
Table 4.2 Oligonucleotide sequences of primers used for preparing analogs of lacticin 481.
Primer Name Primer Sequence (5'-3')
LctA_Trp19Stop_N15R_FP CA ATT TCT CAT GAA TGT CGC ATG AAT AGC TAG CAA
TTT G
LctA_Trp19Stop_N15R_RP CAA ATT GCT AGC TAT TCA TGC GAC ATT CAT GAG
AAA TTG
LctA_F21Stop_FP* ATG AAT AGC TGG CAA TAG GTA TTT ACT TGC TGC
LctA_F21Stop_RP* GCA GCA AGT AAA TAC CTA TTG CCA GCT ATT C
LctA_Phe23Stop_FP* AGC TGG CAA TTT GTA TAG ACT TGC TGC TC
LctA_Phe23Stop_RP* GAG CAG CAA GTC TAT ACA AAT TGC CAG C
LctA_N15R_FP_new CA ATT TCT CAT GAA TGT CGC ATG AAT AGC TGG C
LctA_N15R_RP_new GCC AGC TAT TCA TGC GAC ATT CAT GAG AAA TTG
122 4.4.5 Heterologous production, purification, and characterization of lacticin 481 analogs
The lacticin 481 analogs were expressed using E. coli BL21 (DE3) cells transformed with pLctAMx, and pMmPyl (Table 4.3). An overnight culture of these cells was added to a culture flask containing synthetic media37 lacking Phe, and containing kanamycin (25 mg/mL) and chloramphenicol (12.5 mg/mL). The culture was then incubated in a 37 ºC shaker (220 rpm) until the optical density reached 1.0. After cooling at 4 ºC for 15 min, overexpression was induced with arabinose (0.2% final concentration) and isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.25 mM final concentration) in the presence of non-proteinogenic acid (2 mM final concentration; dissolved using appropriate amount of 1 M NaOH right before use), and the flask was incubated for 12 h in a shaker at 37 ºC and 220 rpm. The following morning the cells were harvested by centrifugation (11,867 ×g, 4 ºC, 20 min), the supernatant was discarded and the cells were lysed using a cell homogenizer (Avestin Emulsiflex-C3; 5,000 PSI) in LanA start buffer (2 mL per 100 mL culture; 20 mM NaH2PO4, 500 mM NaCl, 0.5 mM imidazole, 20% glycerol, pH 7.5). The soluble and insoluble fractions were then separated by centrifugation (22,789 ×g, 4 °C, 20 min) and the soluble fraction was saved for purification. The insoluble fraction was suspended in LanA lysis buffer (1 mL per 100 mL culture; 6 M guanidine-HCl, 20 mM NaH2PO4, 500 mM NaCl, 0.5 mM imidazole, pH 7.5) and the suspension was sonicated. The soluble and insoluble fractions were again separated by centrifugation (22,789 ×g, 4 °C, 20 min) and the modified His6-LanA peptide was purified from the combined soluble layers using a Ni HisTrap HP column (5 mL, GE
Healthcare). The column was washed with LanA wash buffer (4 M guanidine-HCl, 20 mM
NaH2PO4, 500 mM NaCl, 30 mM imidazole, pH 7.5) to remove nonspecific binding proteins. The
His6-LanA peptide was eluted with LanA elute buffer (3 × 5 mL, 4 M guanidine-HCl, 20 mM
NaH2PO4, 100 mM NaCl, 500 mM imidazole, pH 7.5). Finally, the peptide solution was desalted
123 by C4 SPE column (Grace Vydac).
4.4.6 Proteolysis of full-length His-tagged modified LctA mutants, and purification of lacticin
481 analogs
After solid phase extraction, the full-length peptides were purified further on a C5
Phenomenex column in a Shimadzu Prep-HPLC Instrument and were observed to elute in a time range of 22-25 min when the gradient was set from 2% solvent B to 100%A (solvent A = 0.1%
TFA in water, solvent B = 0.0866% TFA in 80% ACN/20% water) over a time period of 45.0 min at a flow rate of 7.0 mL /min. Lacticin 481 analogues were obtained via the proteolytic removal of the leader peptide using the endoproteinase LysN. The full-length peptides were dissolved in water, and incubated in 50 mM Tris buffer (pH 8.0), followed by the addition of LysN at a concentration of 1:100 (w/w) with respect to the peptide and incubated overnight at room temperature. Extent of cleavage was monitored using MALDI-TOF MS. The peptides were then purified using a C18-Phenomenex column in a Agilent Analytical HPLC Instrument and were observed to elute in a time range of 23-26 min when the gradient was set from 2% solvent B to
100%A (solvent A = 0.1% TFA in water, solvent B = 0.0866% TFA in 80% ACN/20% water) over a time period of 45.0 min at a flow rate of 1.0 mL /min.
4.4.7 Heterologous production, purification, and characterization of nisin analogues using E. coli BL21 (DE3)
The nisin analogs were expressed using E. coli BL21 (DE3) cells transformed with pNisABx, pMmPyl, and either pNisTC or pNisTCB or pNisTCBB (Table 4.3).
124 An overnight culture of E. coli BL21 (DE3) cells was added to a culture flask containing synthetic media37 lacking Phe, and containing kanamycin (17 mg/mL), chloramphenicol (10 mg/mL) and spectinomycin (17 mg/mL). The culture was then incubated in a 37 ºC shaker (220 rpm) until the optical density reached 1.0. After cooling at 4 ºC for 15 min, overexpression was induced with arabinose (0.2% final concentration) and isopropyl β-D-1-thiogalactopyranoside
(IPTG, 0.25 mM final concentration) and the flask was incubated for 16 h in a shaker at 18 ºC and
220 rpm. The following morning the cells were harvested by centrifugation (11,867 ×g, 4 ºC, 20 min), the supernatant was discarded, and the cells were lysed using a cell homogenizer (Avestin
Emulsiflex-C3; 5,000 PSI) in LanA start buffer (2 mL per 100 mL culture; 20 mM NaH2PO4, 500 mM NaCl, 0.5 mM imidazole, 20% glycerol, pH 7.5). The soluble and insoluble layers were then separated by centrifugation (22,789 ×g, 4 °C, 20 min) and the soluble layer was saved for purification. The insoluble layer was suspended in LanA lysis buffer (1 mL per 100 mL culture; 6
M guanidine-HCl, 20 mM NaH2PO4, 500 mM NaCl, 0.5 mM imidazole, pH 7.5) and the suspension was sonicated. The soluble and insoluble layers were again separated by centrifugation
(22,789 ×g, 4 °C, 20 min) and the modified His6-LanA peptide was purified from the combined soluble layers using a Ni HisTrap HP column (5 mL, GE Healthcare). The column was washed with LanA wash buffer (4 M guanidine-HCl, 20 mM NaH2PO4, 500 mM NaCl, 30 mM imidazole, pH 7.5) to remove nonspecific binding proteins. The His6-LanA peptide was eluted with LanA elute buffer (3 × 5 mL, 4 M guanidine-HCl, 20 mM NaH2PO4, 100 mM NaCl, 500 mM imidazole, pH 7.5). Finally, the peptide solution was desalted with a C4 SPE column (Grace Vydac).
125 Table 4.3 List of plasmids used.
Plasmid Marker Insert/Mutati Site Reference
on phistagged- Kanamycin His-tagged- nisA inserted 2 nisA/nisB/pRSFDuet-1 nisA (wt) and between
nisB genes BamHI and
HindIII; nisB
inserted
between NdeI
and XhoI pNisAB1 Kanamycin His-tagged- This work derivative of phistagged- nisA-T2B (B = nisA/nisB/pRSFDuet-1 amber stop
codon) and
nisB genes pNisAB2 Kanamycin His-tagged- This work derivative of phistagged- nisA- nisA/nisB/pRSFDuet-1 I4V/S5B/L6G
(B = amber
stop codon)
and nisB genes
126 Table 4.3 (cont.) pNisAB3 Kanamycin Histagged- This work derivative of phistagged- nisA-M21B (B nisA/nisB/pRSFDuet-1 = amber stop
codon) and
nisB genes pNisC/pCDFDuet-1 Spectinomycin nisC BglII and XhoI This work
(MCS2)
pNisTC Spectinomycin nisC and BamHI and This work
mmPylT tRNA NotI (MCS1)
in pCDFDuet-1 in
pNisC/pCDFD
uet-1 pNisTCB Spectinomycin nisB, nisC and rbs-nisB gene This work
mmPylT tRNA inserted right
in pCDFDuet-1 after the nisC
gene in
pNisTC
127 Table 4.3 (cont.) pNisTCBB Spectinomycin nisB (2X), nisC Additional This work
and mmPylT copy of rbs-
tRNA in nisB inserted
pCDFDuet-1 right after the
nisB gene in
pNisTCB pMmPyl Chloramphenic pylT- Sequential 36
-ol mmPylRS(2X)- insertion of
N346A/C348A two copies of
in pEVOL N346A/C348A
pylT between
SpeI and SalI;
pylT gene
inserted
between
ApaLI and
XhoI
128 Table 4.3 (cont.) phistagged- Kanamycin Histagged- lctA(wt) 20 lctA(wt)/lctM/pylT/pRSFD lctA(wt), lctM inserted uet-1 and pylT between
EcoRI and
NotI; lctM
inserted
between BglII
and XhoI; pylT
inserted
between AgeI
and DrdI pLctAM1 Kanamycin Histagged- This work derivative of phistagged- lctA- lctA(wt)/lctM/pylT/pRSFD N15R/W19B (B uet-1 = amber stop
codon), lctM
and pylT
129 Table 4.3 (cont.) pLctAM2 Kanamycin Histagged- This work derivative of phistagged- lctA- lctA(wt)/lctM/pylT/pRSFD N15R/F21B (B uet-1 = amber stop
codon), lctM
and pylT pLctAM3 Kanamycin Histagged- This work derivative of phistagged- lctA- lctA(wt)/lctM/pylT/pRSFD N15R/F23B (B uet-1 = amber stop
codon), lctM
and pylT
4.4.8 Heterologous production, purification, and characterization of nisin analogues using
GRO
The nisin analogues were expressed using GRO transformed with pNisABx, pMmPyl and pNisTC (Table 4.3). This strain was always maintained at temperatures below 32 ºC (mostly 30
ºC) to avoid triggering lambda-red mediated mutagenesis.66, 69
An overnight culture of E. coli BL21 (DE3) cells was added to a culture flask containing
LB media containing kanamycin (17 mg/mL), chloramphenicol (10 mg/mL) and spectinomycin
(17 mg/mL). The culture was then incubated in a 30 ºC shaker (220 rpm) until the optical density
130 reached 0.8. After cooling at 4 ºC for 15 min, overexpression was induced with arabinose (0.2% final concentration) and isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.4 mM final concentration) in the presence of non-proteinogenic acid (4 mM final concentration; dissolved using an appropriate amount of 1 M NaOH right before use) and the flask was incubated for 20 h in a shaker at 18 ºC and 220 rpm. The following morning the cells were harvested by centrifugation
(11,867 ×g, 4 ºC, 20 min), the supernatant was discarded and the cells were lysed using a cell homogenizer (Avestin Emulsiflex-C3; 5,000 PSI) in LanA start buffer (2 mL per 100 mL culture;
20 mM NaH2PO4, 500 mM NaCl, 0.5 mM imidazole, 20% glycerol, pH 7.5). The soluble and insoluble fractions were then separated by centrifugation (22,789 ×g, 4 °C, 20 min) and the soluble layer was saved for purification. The insoluble fraction was suspended in LanA lysis buffer (1 mL per 100 mL culture; 6 M guanidine-HCl, 20 mM NaH2PO4, 500 mM NaCl, 0.5 mM imidazole, pH
7.5) and the suspension was sonicated. The soluble and insoluble layers were again separated by centrifugation (22,789 ×g, 4 °C, 20 min) and the modified His6-LanA peptide was purified from the combined soluble layers using a Ni HisTrap HP column (5 mL, GE Healthcare). The column was washed with LanA wash buffer (4 M guanidine-HCl, 20 mM NaH2PO4, 500 mM NaCl, 30 mM imidazole, pH 7.5) to remove nonspecific binding proteins. The His6-LanA peptide was eluted with LanA elute buffer (3 × 5 mL, 4 M guanidine-HCl, 20 mM NaH2PO4, 100 mM NaCl, 500 mM imidazole, pH 7.5). Finally, the peptide solution was desalted by using a C4 SPE column
(Grace Vydac).
4.4.9 Proteolysis of full-length His-tagged NisA mutants, and purification of nisin analogues
After solid phase extraction, the full-length peptides were purified further on a C5-
Phenomenex column using a Shimadzu Prep-HPLC Instrument and were observed to elute in a
131 time range of 23-25 min when the gradient was set from 2% solvent B to 100% A (solvent A =
0.1% TFA in water, solvent B = 0.0866% TFA in 80% ACN/20% water) over a time period of
45.0 min at a flow rate of 7.0 mL /min. Nisin analogues were obtained via the proteolytic removal of the leader peptide using the endoproteinase trypsin. The full-length peptides were dissolved in water, and incubated in 50 mM Tris buffer (pH 8.0), followed by the addition of trypsin at a concentration of 1:100 (w/w) with respect to the peptide and incubated for 3 h at room temperature.
The extent of cleavage was monitored using MALDI-TOF MS. The peptides were then purified using a C18-Phenomenex column in an Agilent Analytical HPLC Instrument and were observed to elute in a time range of 22-24 min when the gradient was set from 2% solvent B to 100% A
(solvent A = 0.1% TFA in water, solvent B = 0.0866% TFA in 80% ACN/20% water) over a time period of 45.0 min at a flow rate of 1.0 mL /min.
4.4.10 Agar diffusion growth inhibition assay of lacticin 481, nisin and analogues
HPLC-purified peptides were dissolved in water, lyophilized and redissolved in water.
Isolation of authentic lacticin 481 has been described previously.20 For lacticin 481 bioactivity assays, overnight cultures of L. lactis HP were diluted in M-17 agar containing 0.5 % glucose to an OD600 of 0.05. The concentrations of the peptide solutions were determined on Agilent
Analytical HPLC Instrument by measuring their absorbance at 220 nm and then comparing that to absorbance of solution of wt-lacticin 481 of known concentration. A total of 10 µL of 12.5 µM solution was spotted. The plates were incubated at 30 ºC for 20 h.
For nisin bioactivity assays, overnight cultures of L. lactis HP were diluted in M-17 agar containing 0.5% glucose to an OD600 of 0.05. A total of 10 µL of 5 µM commercial, HPLC- purified nisin and analogues was spotted. The plates were incubated at 30 ºC for 18 h.
132 4.4.11 Determination of MIC and IC50 values against liquid cultures of L. lactis HP
Ninety-six well microtiter plates were used to determine MIC and IC50 values. A total volume of culture in each well was 100 µL; the experimental wells contained 15 µL of diluted peptide at defined concentrations and 85 µL of a culture of L. lactis HP indicator strain (OD600 =
0.9 – 1.0) in GM17 medium (M17 media with 2% glucose) diluted 1-to-10 in fresh liquid GM17 medium. The peptide concentrations ranged from 25 µM to 11.9 nM. Each plate contained two lanes for controls: 85 µL fresh GM17 medium and 15 µL sterile Millipore water, and 85 µL of untreated 1-in-10 diluted culture and 15 µL sterile Millipore water. The optical density at 600 nm
(OD600) was recorded at hourly intervals from 0 to 5 h with an additional measurement at 18 h using a BioTek Synergy 2 plate reader. Plates were incubated at 30 ºC between readings. The MIC values reported are the concentrations where no growth was observed after 18 h.
Blanks (growth medium and sterile Millipore water only) were averaged and then subtracted from the averages of triplicate experimental readings. Growth curves vs. peptide concentration were developed, and curve fits for IC50 determination were produced by fitting data with OriginPro 8 software using the dose-response curve. IC50 values were calculated from this fit. The fitted curves are shown in Figure 4.24.
133 Mass spectra for the eleven lacticin 481 analogs are shown below. For calculated and observed masses, see Table 4.4.
Figure 4.8 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/W19o-ClPhe modified by LctM in E. coli BL21(DE3).
Figure 4.9 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/W19m-BrPhe, modified by LctM in E. coli BL21(DE3).
134
Figure 4.10 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/W19o-NO2Phe, modified by LctM in E. coli BL21(DE3).
Figure 4.11 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/F21m-BrPhe, modified by LctM in E. coli BL21(DE3).
135
Figure 4.12 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/F21m-CF3Phe, modified by LctM in E. coli BL21(DE3).
Figure 4.13 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/F21o-ClPhe, modified by LctM in E. coli BL21(DE3).
136
Figure 4.14 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/F21o-NO2Phe, modified by LctM in E. coli BL21(DE3).
Figure 4.15 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/F23m-BrPhe, modified by LctM in E. coli BL21(DE3).
137
Figure 4.16 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/F23m-CF3Phe, modified by LctM in E. coli BL21(DE3).
Figure 4.17 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/F23o-ClPhe, modified by LctM in E. coli BL21(DE3).
138
Figure 4.18 MALDI-TOF mass spectrum of His-tagged-LctA-N15R/F23o-NO2Phe, modified by LctM in E. coli BL21(DE3).
Table 4.4 Masses observed in MALDI-TOF mass spectra of LctA mutants after purification of fully modified full-length peptides.
Mutant Mass calculated for Mass observed for [M+H]+ peak [M+H]+ peak
His-tagged-LctA-N15R/W19o- 7326.53 7326 ClPhe
His-tagged-LctA-N15R/W19m- 7370.99 7371 BrPhe
His-tagged-LctA-N15R/W19m- 7360.09 7360 CF3Phe (Fig. in the main text)
His-tagged-LctA-N15R/W19o- 7337.07 7337 NO2Phe His-tagged-LctA-N15R/F21m- 7410.03 7411 BrPhe
139 Table 4.4 (cont.)
His-tagged-LctA-N15R/F21m- 7399.13 7399 CF3Phe
His-tagged-LctA-N15R/F21o- 7365.57 7366 ClPhe
His-tagged-LctA-N15R/F21o- 7376.13 7375 NO2Phe
His-tagged-LctA-N15R/F23m- 7410.03 7411 BrPhe
His-tagged-LctA-N15R/F23m- 7399.13 7400 CF3Phe
His-tagged-LctA-N15R/F23o- 7365.57 7366 ClPhe
His-tagged-LctA-N15R/F23o- 7376.13 7376 NO2Phe
140
Figure 4.19 MALDI-TOF mass spectrum of wild type NisA modified by NisB and NisC in E. coli BL21(DE3) in synthetic media at 18 °C. Although the desired 7- and 8-fold dehydrated peptides are present, peptides that are dehydrated to a lesser extent are prevalent. m/z calculated for [M+H]+ peak for 7-fold dehydrated His-tagged-NisA = 7319.45, m/z observed for [M+H]+ peak for the labeled 7-fold dehydrated His-tagged-NisA = 7319.
141 Figure 4.20 MALDI-TOF mass spectra of NisA-Thr2m-BrPhe modified by NisB and NisC in E. coli BL21(DE3) in synthetic media with multiple copies of nisB gene expressed.
Mass spectra for the three modified NisA analogs are shown below.
Figure 4.21 MALDI-TOF mass spectrum of NisA-I4V/S5B/L6G with S5 substituted by m- BrPhe, modified by NisB and NisC in GRO.
142
Figure 4.22 MALDI-TOF mass spectrum of NisA-I4V/S5B/L6G with S5 substituted by m- CF3Phe, modified by NisB and NisC in GRO.
Figure 4.23 MALDI-TOF mass spectrum of NisA-M21/m-BrPhe, modified by NisB and NisC in GRO. The peptide was expresed in very low yields.
143 Table 4.5. Masses observed in MALDI-TOF mass spectra of NisA mutants after purification of full-length peptides expressed in GRO.
Mutant Mass calculated for the Mass observed for the major peak, [M+H]+ major peak, [M+H]+
His-tagged-NisA-T2m-BrPhe (Fig. 7444.42 7445 in the main text)
His-tagged-NisA-I4V/S5m- 7388.31 7389 BrPhe/L6G
His-tagged-NisA-I4V/S5m- 7377.41 7378 CF3Phe/L6G
His-tagged-NisA-M21m-BrPhe 7396.32 7396
0.6 0.6
wt-lacticin 481 Lacticin 481/N15R/W19mBrPhe 0.5 0.5
0.4 0.4
0.3 0.3
600 600
OD OD 0.2 0.2
0.1 0.1
0.0 0.0
-8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 log(conc. in M) log(conc. in M)
MIC = 390 nM MIC = 390 nM IC50 = 185 ± 10 nM. IC50 = 192 ± 9 nM
Figure 4.24 IC50 fitting curves for wt-lacticin 481 and five analogs that were found to be more active against L. lactis HP from the agar diffusion assays.
144
0.6 0.6 Lacticin 481/N15R/W19oClPhe 0.5 Lacticin 481/N15R/W19oNO2Phe 0.5
0.4 0.4
0.3 600
600 0.3
OD OD 0.2 0.2
0.1 0.1
0.0 0.0
-8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 log(conc. in M) log(conc. in M)
MIC = 390 nM MIC = 195 nM IC50 = 138 ± 8 nM. IC50 = 98 ± 8 nM
0.6
Lacticin 481/N15R/F23oClPhe 0.5 0.5 Lacticin 481/N15R/F21oNO2Phe
0.4 0.4
0.3 0.3
600
600
OD OD 0.2 0.2
0.1 0.1
0.0 0.0 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 log(conc. in M) log(conc. in M)
MIC = 390 nM MIC = 390 nM IC50 = 123 ± 21 nM. IC50 = 190 ± 9 nM
Figure 4.24 (cont.)
4.5 References
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