Phylogenomic Analysis of the Microviridin Biosynthetic Pathway

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Phylogenomic analysis of the microviridin biosynthetic pathway coupled with targeted chemoenzymatic synthesis yields potent protease inhibitors Muhammad N. Ahmed, Emmanuel Reyna González, Bianca Schmid, Vincent Wiebach, Roderich Suessmuth, Elke Dittmann, and David P Fewer ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00124 • Publication Date (Web): 13 Apr 2017 Downloaded from http://pubs.acs.org on April 14, 2017

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ACS Chemical Biology

Phylogenomic analysis of the microviridin biosynthetic pathway coupled with targeted chemo-enzymatic synthesis yields potent protease inhibitors Muhammad N. Ahmed1, †, Emmanuel Reyna-González2, †, Bianca Schmid3, Vincent Wiebach3, Roderich D Süssmuth3, Elke Dittmann2*, David P. Fewer1,* 1 Microbiology and Biotechnology Division, Department of Food and Environmental Sciences P.O.Box 56, Viikki Biocenter, Viikinkaari 9, FIN-00014 University of Helsinki, Finland. 2 Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Straße 2425, 14476 Potsdam-Golm, Germany 3 Institute of Chemistry, Technical University Berlin, Straße des 17. Juni 124, 10623 Berlin, Germany Abstract Natural products and their semi-synthetic derivatives are an important source of drugs for the pharmaceutical industry. Bacteria are prolific producers of natural products and encode a vast diversity of natural product biosynthetic gene clusters. However, much of this diversity is inaccessible to natural product discovery. Here we use a combination of phylogenomic analysis of the microviridin biosynthetic pathway and chemo-enzymatic synthesis of bioinformatically predicted microviridins to yield new protease inhibitors. Phylogenomic analysis demonstrated that microviridin biosynthetic gene clusters occur across the bacterial domain and encode three distinct subtypes of precursor peptides. Our analysis shed light on the evolution of microviridin biosynthesis and enabled prioritization of their chemoenzymatic production. Targeted one-pot synthesis of four microviridin types encoded by the cyanobacterium Cyanothece sp. PCC 7822 identified a set of novel and potent serine protease inhibitors the most active of which had an IC50 value of 21.5 nM. This study advances the genome mining techniques available for natural product discovery and obviates the need to culture bacteria.

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Introduction Natural products are low molecular weight metabolites with diverse chemical structures and potent biological activities1. They are the source and/or inspiration for many drugs in use today.1,2 Natural product biosynthetic gene clusters (BGCs) are prevalent in microbial genomes.3-5 Genome mining studies demonstrate that bacteria encode far more natural products than are typically expressed under normal growth conditions.4-8 Consequently traditional bioactivity-guided fractionation approaches to natural product discovery harnesses just a small percentage of the potential microbial natural product repertoire.2,9 Accessing this pool of silent secondary metabolites has proven arduous.6,9 Numerous methodologies combine bioinformatic approaches for structural prediction and prioritization with the activation of cryptic natural product BGCs in their natural hosts or expression in heterologous hosts.2,9,10 However, only a small fraction of strains harboring natural product BGCs can be cultivated and the heterologous production of complex natural products is often challenging.6, 9 A recent alternative genome mining method employs solid phase peptide synthesis (SPSS) to produce synthetic natural products predicted through genome mining obviating the need for culturing or heterologous expression.11 Ribosomally synthesized and post-translationally modified peptides (RiPPs), produced through the post-translational modification of short precursor peptides, are a rapidly growing class of natural products.12 RiPP biosynthetic pathways often encode an array of tailoring enzymes, which release these peptides from the structural constraints imposed on proteins.1215 The tailoring enzymes recognize a leader sequence in the precursor peptide and modify residues in the core region of the precursor.12 This allows RiPP tailoring enzymes to coordinate a concerted targeted biosynthetic effort on a specific precursor peptide.13 A number of recent structural studies have revealed the conformational impact of leader peptide and tailoring enzyme interactions on the maturation of RiPPs.14,15 Precursor peptides provided in trans or fused to their maturation enzymes allow the in vitro reconstitution of a variety of RiPP types using conformationally-activated tailoring enzymes.16-21 RiPP precursor peptides can be synthesized by an economical SPPS approach and be processed by their natural tailoring enzymes.22 Microviridins are tricyclic N-acetylated members of the RiPP family of natural products that are crosslinked through intramolecular ω-ester and/or ω-amide bonds thereby yielding a unique unprecedented cage-like structure.23-25 The formation of the lactone and lactam rings is catalyzed by two dedicated ATP-grasp ligases.24,25 The maturation of microviridins also requires the activity of a GNAT-type N-acetyltransferase and cleavage of the microviridin precursor peptide to produce mature microviridin.24,25 The ATP-grasp ligases interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism.15,26 The cyclization reactions have a strict order with the large lactone ring being formed first, followed by the smaller lactone ring and the lactam ring.27 Microviridins are potent and selective inhibitors of a variety of serine proteases including chymotrypsin, trypsin and elastase.23, 28 The full in vitro reconstitution of the microviridin pathway has recently been achieved using conformationally-activated ATP-grasp ligases and an Nacetyltransferase.16 Here we develop a chemo-enzymatic genome mining approach to exploit the chemical diversity encoded in the microviridin biosynthetic pathways. We carried out a comprehensive phylogenomic mining study to catalog the structural diversity of microviridins and to reconstruct the evolutionary history microviridin BGCs. We used this data as the basis for a chemo-enzymatic approach to natural product discovery by synthesizing a cocktail of 2 ACS Paragon Plus Environment

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microviridins predicted from a cryptic microviridin BGC encoded in the genome of the cyanobacterium Cyanothece sp. PCC 7822. The results of this study expands the genetic and chemical diversity of the microviridin family, provides new insights into the evolution of the microviridin BGC and led to the discovery of potent serine protease inhibitors.

Results and discussion Bioinformatic analysis identified 174 microviridin BGCs from the genomes of a variety of bacterial genera belonging to the cyanobacteria, bacteroidetes and proteobacteria phyla (Supporting Information Table S1). Microviridin BGCs were widely distributed in cyanobacteria phylum (69 BGCs) while the microviridin BGCs were largely restricted to the genus Chryseobacterium in the bacteroidetes phylum (88 BGCs). Microviridin BGCs were scattered among the beta, gamma and delta classes of the proteobacteria phylum (17 BGCs). Microviridin BGCs ranged from 1-12 kb in length and encoded between 2 and 17 genes (Figure 1). The vast majority of the microviridin BGCs encoded MdnB and MdnC, lactone and lactam ring-forming ATP-grasp ligases. The mdnB and mdnC genes had a strictly conserved order in all microviridin BGCs and were translationally coupled in many strains (Figure 1). By contrast, only a subset of microviridin BGCs encode the MdnD Nacetyltransferase that catalyzes the acetylation of the tricyclic core peptide at the Nterminus.24 The microviridin BGCs frequently encoded either the MdnE ABC transporter that is proposed function as scaffold protein29 or membrane proteins comprising a C39 peptidase domain that are known to cleave double-glycine motifs in a number of bacteriocin leader peptides.30,31 The latter proteins were often accompanied by HlyD3 homologs, which help in the transportation of the proteases across membranes.32 Many of microviridin BGCs encoded further proteins with possible roles in the post-translational modification of the core sequence during the maturation of microviridin (Figure 1). The 174 microviridin BGCs encoded 308 precursor peptides that differed considerably in their length and composition (Figure 2). The precursor peptides ranged in length from 44-126 amino acids and shared 38-100% sequence homology. All microviridin precursor peptides encoded the highly conserved PFFARFL α-helix motif at the N-terminus that plays an essential role in the activation of both ATP-grasp ligases.15,26 The C-terminal region of the precursor peptides encoded 1-5 core sequences containing the TxKxPSD motifs that is cyclized during the maturation of microviridins.24 Many of the precursor peptides encoded the double-glycine motif (LSX2ELX2IXGG) found in the leader sequence of bacteriocins, lantibiotics and signal peptides.30,31 The microviridin core peptides were divergent but rich in Asp, Thr, Ser and Lys residues that could form additional lactone and lactam rings via their side chains, respectively. Three principal classes of precursor peptides were observed based on sequence conservation and the arrangement of the core peptides. Class I precursor peptides contain a fused leader peptide for each single microviridin core peptide. The majority of strains encoded a single precursor peptide in featuring a class I precursor, Cyanothece sp. PCC 7822 exceptionally encoded 10 precursor peptides (Figure 1). Class I precursors mostly lacked double-glycine motifs and were often associated with an MdnE type of membrane transporter. All known microviridins characterized by bioactivity-guided purification or heterologous expression are derived from class I precursors. The marinostatin precursor encoded an authentic doubleglycine motif between the leader and core peptides and short additional sequence stretches Nterminal and C-terminal to the core peptide, which do not appear in the final marinostatin 3 ACS Paragon Plus Environment


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product (Figure 2). The maturation of marinostatin is poorly understood and neither maturation proteases nor transporters are encoded in the vicinity of the marinostatin BGC (Figure 1). Class II precursor peptides contained a single leader peptide and up to 5 consecutive core peptides (Figure 2). The consecutive core peptides were either separated by Gly-rich sequence spacers or lacked a visible spacer and/or a double-glycine cleavage site (Figure 2). Correspondingly, the class II microviridin BGCs regularly encode membrane proteins with C39 cleavage domain. It is not clear whether these microviridin precursor peptides form multiple separate microviridins or whether class II precursors generate a single product with microviridin cage repeats. The class II and III precursor proteins were of similar length. However, only the C-terminus clearly resembles a microviridin precursor peptide core sequence. The sequence diversity of precursor peptides was reflected in a sequence similarity network that showed three distinct clusters (Figure 2). Cluster I and II are dominated by cyanobacterial precursor peptides, while cluster III contained mainly precursor peptides from bacteriodetes with a lower sequence diversity (Figure 2). The proteobacterial precursor peptides were all scattered amongst all three clusters suggesting a complex pattern of diversification. The evolutionary history of the microviridin and marinostatin BGCs was reconstructed using PhyML analysis of the MdnC ATP-grasp ligase, the only enzyme common to all pathways (Figure 3). The MdnC enzymes were diverse with sequence similarities ranging from 40100%. The MdnC enzymes from bacteriodetes and cyanobacteria were placed in separate monophyletic groups while the MdnC homologs from proteobacteria are placed in the base of cyanobacteria and bacteriodetes in a paraphyletic assemblage (Figure 3). MdnC homologs from Planktothrix, Microcystis and Chryseobacterium species form monophyletic subclusters (Figure 3). Marinostatin is a depsipeptide protease inhibitor consisting of 11 amino acids and two internal ester linkages.33-36 Marinostatins lack the internal ω-amide bond found in microviridins and the marinostatin gene cluster lacks MdnB.33 The phylogenetic analysis demonstrates that the marinostatin BGC does not cluster separately to the microviridin gene cluster but instead is located on an internal branch (Figure 3). The phylogenetic analysis presented here suggests that marinostatins are derived microviridin resulting from the deletion of the MdnB ATP-grasp ligase resulting in the presence of lactone bonds and the absence of lactam rings found in microviridins (Figure 3). Eight strains encoding microviridin/marinostatin biosynthetic pathways lacking the MdnB ATP-grasp ligase were found and none of these formed a separate group to the microviridin MdnCs, suggesting that the loss of the MdnB ATP-grasp ligase is a recurrent phenomenon. Microviridins are known from strains of the cyanobacterial genera Microcystis, Planktothrix and Nostoc.28 However, cryptic microviridin gene clusters have been reported from a range of bacteria in multiple studies.24,25,28,37 Our results demonstrate that the microviridin pathway is more widely distributed throughout the bacterial domain than anticipated and found in genomes of the cyanobacterial, proteobacterial and bacteriodetes phyla. We uncovered a far greater diversity and a wider distribution of microviridins than expected. The precursor peptides are rich in Asp, Thr, Ser and Lys residues that could form additional lactone and lactam rings via their side chains, respectively. Our results suggest that the known chemical diversity of microviridins and marinostatins is just a small fraction of the potential chemical diversity encoded in the microviridin and marinostatin BGCs. We anticipate further variation on the unique tricyclic and bicyclic cages of microviridin and marinostatin.


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The microviridin BGC from Cyanothece sp. PCC 7822 encodes 10 precursor peptides and was selected for a chemo-enzymatic approach to natural product discovery (Supporting Information Figure S1). A closer inspection of the core peptide sequences MdnA1-A10 revealed length variation from 12-14 amino acids (Figure 4). Moreover, the core peptides contained either a Leu, Arg or Lys residue at the crucial position 5 that interacts with the active site of serine proteases and determines the specificity of microviridins.26,16 This suggested that the Cyanothece sp. PCC 7822 strain produces a cocktail of different microviridins with protease inhibition bioactivity. In order to test the hypothesis five peptide sequences, MdnA3, MdnA6, MdnA7, MdnA8, MdnA9, representing the different core peptide lengths and either containing Leu, Arg or Lys at position 5 were synthesized using a SPSS approach (Supporting Information Tables S2S3). The five peptides were subsequently incubated with the lactone ring-forming ATP-grasp ligase MvdD from Planktothrix agardhii CYA 126/8 that was constitutively activated by the N-terminally fused MvdE leader peptide (LP-MvdD), as described recently.16 While four of the peptides could be successfully bicyclized (Supporting Information Figure S2), the MdnA9 core peptide could not be cyclized under the conditions tested (data not shown). Each of the four bicyclized peptides were further tricyclized using the lactam ring-forming MvdC enzyme of P. agardhii CYA 126/8 (LP-MvdC), and of these four peptides three could be further acetylated by MvdB, rendering fully maturated microviridins. It is worth mentioning that only MdnA3 and MdnA6 could be fully maturated with a yield above 80%. MdnA8 could not be acetylated in vitro (Supporting Information Table S2). The bicyclic MdnA7 and MdnA8 were used in the bioactivity assays because a recent study has revealed little difference in activity against elastase between bicyclic and tricyclic microviridins.26 The bicyclic MdnA7 and MdnA8 variants, with Leu at position 5, were active against elastase as expected from previous studies26, with an IC50 of 1.05 ± 0.20 µM for MdnA7 and IC50 of 21.85 ± 3.35 µM for MdnA8 (Table 1). The tricyclic MdnA3 and MdnA6 variants, with positively charged Arg or Lys at position 5, were anticipated to be trypsin inhibitors.16,26 The IC50 values obtained were 28.4 ± 4.0 nM for MdnA3 and 21.5 ± 0.6 nM for MdnA6 (Table 1). The fully maturated microviridin derived from MdnA6 is the most potent microviridin type inhibitor against trypsin that has been characterized to date. Cyanothece sp. PCC 7822 was obtained from the Pasteur Culture Collection of Cyanobacteria in order to test if the strain produces any of the predicted microviridins. Four of the ten predicted microviridins (MdnA3, MdnA4, MdnA6 and MdnA7) could be detected in a methanol extract of the strain using MALDI-TOF measurements, although in tiny amounts (Supporting Information Figure S4a). Furthermore, MALDI-TOF/TOF revealed fragment ions confirming the correct tricyclization of synthetic MdnA6 (Supporting Information Figure S4b). Notably, only microviridins relating to core peptides with a length of 13 or 14 amino acids could be detected from the cyanobacterium. These data confirm that microviridins obtained by the chemo-enzymatic approach indeed resemble authentic microviridins produced by the cyanobacteria. The amounts produced by the cyanobacteria are too low for a detailed characterization or would require laborious large-scale cultivation. However, the in vitro chemo-enzymatic synthesis of microviridins provides a fast avenue to obtain a sufficient amount of these peptides. This systematic phylogenomic survey revealed three distinct classes of microviridin precursors. We successfully demonstrated that our recently developed chemo-enzymatic synthesis approach16 can be expanded to the synthesis of bioinformatically predicted class I precursors encoded in cryptic microviridin BGCs. Cyanothece sp. PCC 7822 encodes an 5 ACS Paragon Plus Environment


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unusual class I microviridin BGC with 10 consecutive microviridin precursor genes. The in vitro synthesis and characterization of the predicted microviridins demonstrates the potential as well as the current limitations of the chemo-enzymatic approach. The lactone-forming ATP-grasp ligase of Planktothrix agardhii CYA 126/8 was capable of cyclizing microviridin core peptides of Cyanothece sp. PCC 7822 ranging from 12-14 amino acids in length thereby demonstrating the promiscuity of the enzymes regarding core peptide length. However, the precursor MdnA9 could not be cyclized. The MdnA9 precursor peptide shows deviations in the α-helix region of the leader peptide as well as the core peptide that comprises an additional Glu residue at the C-terminus that could potentially be involved in an alternative cyclization (Fig. 4). There was no acetylation by the MvdB acetyltransferase in the case of the precursor peptide MdnA8, which is only 12 amino acids long. This suggests that the activity of the enzyme is compromised when the precursor peptide core is shorter than 13 amino acids even though this enzyme accepts variations in the peptide core sequence. The in vitro synthesis of divergent microviridins may thus require the utilization of ATP-grasp ligase enzymes with their cognate leader peptide sequences. It should also be possible to develop synthetic protocols for class II and class III microviridin precursor peptides through the use of cognate enzyme and leader peptide pairs. Conclusions Microbes are widely held to be a near inexhaustible source of natural products.38 Analysis of the ever-increasing number of genome sequences has demonstrated that natural products BGCs are exceptionally common in bacteria.3-5,7 However, the bulk of this natural product chemical diversity remains inaccessible to natural product discovery because gene clusters are silent or are encoded in slow growing microbes.6,9 Numerous procedures for the heterologous expression, refactoring and activation of natural product BGCs have been developed to improve natural product discovery methods.6,9,38-42 Recently the synthesis of bioinformatically predicted natural products using SPSS has also been developed as an alternative solution to this problem.11 Here we used chemo-enzymatic synthesis of synthetic microviridins to obviate the need to culture or express the microviridin BGC in a heterologous host. This approach employed the use of two ATP-grasp ligase enzymes to install lactone and lactam bonds in a synthetic precursor peptide.16 The use of this one-pot method on synthetic sequences encoded in the genome of Cyanothece sp. PCC 7822 led to the discovery of new microviridin peptides some of which are active in low nanomolar concentrations. Numerous in vitro reconstitution methods have been developed for a diversity of natural product classes.16,17,18,19,43 RiPP biosynthetic pathways are particularly amenable to reconstitution experiments given the lower complexity of the biosynthetic pathways encoding these natural products.13 Reconstitution of the microviridin,16 cyanobactin,17,19 lantibiotic,18,20 thiopeptide,21 lassopeptide,22 and LAP43 RiPP biosynthetic pathways has provided detailed insights into biosynthesis of these natural products as well as suggesting engineering strategies for the production of new natural products. It is conceivable that such chemoenzymatic methods for reconstituting natural product pathways could be applied more widely in genome mining studies targeting the discovery of new natural products.

Methods Microviridin genome mining The MdnC protein sequence from Planktothrix agardhii NIVA-CYA 126/8 (WP_042156020.1) was used to query the non-redundant database at NCBI using a BLASTp search (E < 1e-50). This allowed the identification of candidate microviridin BGCs encoding MdnA, MdnB and MdnC homologs in members of the cyanobacteria, proteobacteria and 6 ACS Paragon Plus Environment

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bacteriodetes phyla with sequence identities of 40-100%. A representative MdnC homolog from each phylum was used in reciprocal BLASTp searches to ensure all microviridin BGC had been identified (E < 1e-50). The precursor peptides of RiPP BGCs are short and often overlooked in genome annotations.8 Genes encoding microviridin precursor peptides were identified in Artemis, if not already annotated, by searching for short ORFs containing the conserved PFFAxFL α-helix motif at the precursor peptide leader sequence and/or the presence of a microviridin core containing the TxKYPSD motif. The microviridin precursor peptides were aligned using the ClustalW and sorted into three classes based on sequence similarity and the presence or conserved elements involved in the maturation of microviridins. WebLogo was used to generate graphical representations of sequence conservation patterns within a multiple sequence alignment of the microviridin precursor peptides. A sequence similarity network of the 308 microviridin precursor sequences was generated using the EFI-Enzyme Similarity Tool (EFI-EST).44 The sequence similarity network contained 308 nodes with 22,368 edges constructed by pairwise BLAST alignments better than an E value of 10−5. Cytoscape 3.2.1 was used to visualize the sequence similarity network using the organic layout.45 Phylogenetic analysis A phylogeny of the MdnC ATP-grasp ligase was constructed in order to illustrate the diversity of the microviridin BGCs and to resolve the relationship between microviridin and marinostatin BGCs. Taxon selection was based on sequence conservation and the overrepresented sequences from the Microcystis, Planktothrix and Chryseobacterium genera were reduced to a single representative. The resulting selection of 69 MdnC protein sequences were aligned using MUSCLE to produce an alignment of 380 positions.46 Ambiguous and poorly aligned regions of the aligned sequences were stripped from the alignment using GBLOCKS.47 The following parameters used were used to extract the conserved blocks from the alignment using GBLOCKS: minimum number of sequences for a conserved position=35, minimum number of sequences for a flanking position=35, maximum number of contiguous non-conserved positions=68, minimum length of block=5. The remaining 291 positions were retained and used for the construction of a phylogenetic tree using PHYML.48 Phylogenetic trees were constructed using the JTT amino acid substitution model, four substitution rate categories, an estimated proportion of invariable sites, an estimated gammadistribution shape parameter, and using a BIONJ starting tree. The stability of the in-group relations was assessed with 1000 bootstrap replicates. The resulting phylogenetic tree was rooted using midpoint rooting using the RETREE and visualized using TREEVIEW. Solid-phase peptide synthesis Automated solid-phase peptide synthesis was performed in 50 µmol scale. Resin loading: 1 g of trityl chloride polystyrene (TCP) resin (0.9 mmol/g) was pre-swollen in 10 ml dry dichloromethane (DCM). After removal of the solvent a mixture of the amino acids (0.6 mmol) and 3 equivalents of N,N-diisopropylamine (DIPEA) dissolved in 5 ml dry DCM was added to the resin and was mixed for 30 min at RT. The resin was washed (2x 5 ml N,Ndimethylformamide (DMF), 2x 5 ml DCM). Capping of non-reacted functional groups of the resin was performed with DCM, MeOH and DIPEA 80:15:5 (2x 10 ml, 10 min). After washing (5x 5 ml DMF), Fmoc-removal was achieved with DMF/piperidine (4:1, 5 ml, 1x 2 min, 1x 20 min). After final washing (2x 5 ml DMF, 1x 5 ml MeOH, 3x 5 ml DCM), the resin was dried in vacuo. Coupling of Fmoc/tBu-protected amino acids: To 200 mg of the resin (~0.5 mmol/g), a 0.25 M solution of the amino acid in DMF (2.5 eq relative to resin loading) was added. After 7 ACS Paragon Plus Environment


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addition of a 0.5 M solution of DIPEA in DMF (2.5 eq) and a 0.25 M solution of O(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU) in DMF (2.5 eq), the reaction solution was shaken for 2 x 15 min. For couplings subsequent to the fifth amino acid, double couplings with 30 min coupling times were performed. For couplings subsequent to the tenth amino acid, a third coupling with 45 min was performed. Fmocremoval was achieved with DMF/piperidine (4:1, 2.5mL, 4x 2.5 min). The resin was washed with DMF (6x 2.5 ml). Global deprotection: The resin was transferred to a 5 ml syringe with frit and cap. After addition of the cleavage cocktail (trifluoroacetic acid (TFA), H2O, triethylsilane (TES), DODT (3,6-dioxa-1,8-octane-dithiole) 92.5:2.5:2.5:2.5), the syringe was shaken for 3 h. The peptide was precipitated in cold diethyl ether and centrifuged. The supernatant was removed and the precipitate was washed with diethyl ether twice. The peptide was resolved in acetonitrile (MeCN)/H2O (2:1) and lyophilized. Preparative HPLC purification of peptides: Crude precursor variants were dissolved in H2O/MeCN (1:1; v:v) and purified by C18 reversed-phase HPLC (Agilent Technologies 1260 Series HPLC system equipped with an UV/Vis detector operating at λ = 210 nm using a reverse phase Agilent C18 column (212 x 250 mm, particle size 10 µm). The following gradient system was used (flow-rate: 20 mL · min-1; UV-detection at λ = 210 nm) for the different precursor variants: Elution started with a linear gradient from X% to Y% buffer B (Supporting Information Table S2) for 15 min, followed by 100% buffer B for 5 min (Buffer A, 0.1% TFA in H2O; buffer B, 0.1% TFA in methanol). The retention times for each peptide are displayed in Supporting Information Table S2. General information on devices and chemicals: N-Fmoc protected amino acids were purchased from Orpegen. TCP resin was bought from Intavis. TBTU, DiPEA and DMF (99.8%) were purchased from Iris Biotech. TFA, TIS and 3,6-dioxa-1,8-octanedithiol were purchased from Sigma-Aldrich. MeOH and MeCN were purchased from Fischer Scientific. Peptide synthesis was carried out on a Prelude parallel synthesizer from Protein Technologies. All loading and amino acid coupling reactions were carried out under N2 atmosphere. Enzyme expression and purification The enzymes were expressed in E. coli BL21 cells as follows: 400 mL cultures were grown at 37ºC with agitation of 220 rpm to an OD600= 0.5. Expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The cultures were further incubated over 3 days at 22ºC with agitation and the cells were harvested by centrifugation. Cell pellets were resuspended in 15 mL of Lysis Buffer (300 mM NaCl and 50 mM NaH2PO4 pH 8.0) + 10 mM imidazole and lysed with a sonicator (HD 3100, Bandelin) for 10 min (3 min on/ 3 min off) with an amplitude of 70%. Cell debris was removed by centrifugation and the supernatant was incubated with 1 mL of Ni-NTA agarose slurry (PureCube Ni-NTA agarose, Cube Biotech) at 4ºC. Afterwards the resin was washed once with 5 mL Lysis Buffer + 20 mM imidazole and once with 5 mL Lysis Buffer + 50 mM imidazole. Finally the bound enzyme was eluted with 2.5 mL of Lysis Buffer + 250 mM imidazole. The eluate was further purified by gel filtration chromatography (HiLoad 16/600 SuperDex 200 column, Äkta Prime Plus, GE Healthcare) with buffer 150 mM NaCl, and 20 mM Tris-HCl, pH 7.5. The enzymes were then concentrated by centrifugation (Amicon Ultra-4 centrifugal filter units, Millipore) to use them in the cyclization assays.


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Cyclization assays and MALDI MS analysis of synthetic microviridins The reactions were set up as follows: 100 mM Tris-HCl pH 8.0, 2.5 mM ATP, 5 mM MgCl2, 25 mM KCl, 25 µg MvdD (in the case of tricyclization also 25 µg MvdC), and 80 µg of precursor peptide (in the case of trans activation, also 80 µg of the leader peptide). After 16 hours of incubation at 37ºC the reactions were quenched with 2.0 µl 0.5 M EDTA pH 8.0 and 200 µl methanol 100%. In the case of one-pot reactions, instead of stopping the reaction, acetyl coenzyme A (1 mM) and 10 µg of MvdB were added to the mix and further incubated for 6 hours, followed by the addition of EDTA and methanol. The cyclization results (Supporting Information Table S3) were analyzed via HPLC with the following program with fractionation: 20% acetonitrile for 1 min, and 20 to 38% acetonitrile in 20 min (Prominence UFLC XR HPLC, Shimadzu; column: SymmetrieShield RP18, 4.6 x 100 mm, particle size 3.5 µM). Candidate fractions were analyzed via MALDI-TOF MS (Samples dissolved in 10 µL trifluoroacetic acid 0.1%; 0.3 µL of the sample solution were spotted with 0.3 µL of αCyano-4-hydroxycinnamic acid matrix 10 mg/mL, and analyzed with a Bruker microflex LRF equipped with a nitrogen laser (λ= 337 nm). The accelerating voltage used was 19 kV. The analysis was done in the positive-ion reflectron mode, and in general 5-10 laser shots were averaged to generate a spectrum. The MALDI spectra of the processed precursor peptides can be seen in Supporting Information Figure S2. After cyclization and fractionation in the HPLC, the samples were lyophilized and weighed on an analytical balance (MX5 Microbalance, Mettler Toledo). HPLC-MS/MS analysis of synthetic microviridins To confirm the position of the rings in the microviridins a bicyclic MdnA3 and a tricyclic MdnA6 were analyzed via HPLC-MS/MS. All MS/MS measurements were conducted using an LTQ-Orbitrap XL system (Thermo Fisher Scientific, Bremen, Germany) coupled to an analytical Agilent 1200 HPLC system (Agilent, Waldbronn, Germany) equipped with a Grom-Sil-120-ODS-4-He column (50 x 2 mm; Grace, Deerfield, IL, USA). Chromatographic separation was achieved as described previously (Reyna-Gonzalez et al., 2016). For mass spectrometric analysis, the following ESI settings were applied: a capillary temperature: 330°C; sheath gas flow: 7L/min, aux gas flow: 25L/min; source voltage: 4.8 kV; and 45 V was used for the capillary voltage, whereas the tube lens voltage was set to 125. The FT micro scans were set to 1 with 400 ms maximum C-trap fill time. MS scans were recorded using the Orbitrap analyzer with a resolution of R = 60,000. First a MS scan was recorded and present ions were analyzed and chosen for tandem MS fragmentation with normalized collision energies (CID) ranging from 35% - 80% (Supporting Information Figure S3). Isolation width was set to 1 m/z. Following data acquisition, multiple charged spectra were deconvoluted using Xtract, implemented in the Xcalibur 2.0 software (Thermo Fisher Scientific). MALDI-TOF analysis of microviridins produced by Cyanothece sp. PCC 7822 To determine if Cyanothece sp. PCC 7822 produce microviridins, MALDI-TOF MS measurements of intact cells were conducted as previously reported.49 Briefly, 1 mL of the cyanobacterial culture was harvested by centrifugation, frozen at -80°C and dried by lyophilization. Lyophilized cells were resuspended in 20 µl of a 1:1:1 (v/v/v) mixture of ethanol, water and acetonitrile. The cell suspension was mixed with an equal volume of a 20 mg/ml dehydroxybenzoic acid (DHB) solution and 0.5 µl were spotted on a ground steel target plate (Bruker Daltonics, Bremen, Germany) using the dried droplet method. Spectra were recorded on an ultrafleXtreme MALDI-TOF mass spectrometer. The accelerating voltage was set to 20 kV, while the analysis was performed in positive reflector mode in a mass range of 1000 – 3500 Da with a laser power of 70 % and detector gain set to 12x. 9 ACS Paragon Plus Environment


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Between 3000 and 5000 shots were accumulated to record the spectra. External calibration was performed using the peptide calibration mix II (Bruker). To further confirm the results obtained, cells were harvested as described above and lysed using bead beating (0.5 mm mesh) as described before.50 The samples were then centrifuged and the supernatant was removed (Water extract). Afterwards 200 µl of MeOH were added to the lysed cells and subjected to an ultrasonic bath for 15 min. After centrifugation, the supernatant was collected (MeOH extract) and both samples were measured using MALDITOF/TOF (Supporting Information Figure S4) and HPLC-MS/MS as described above. MALDI-TOF/TOF spectra were recorded using the post source decay (PSD) method with a laser power increase of 50 % and detector gain boost of 100 %. Recorded spectra were analyzed using the flexanalysis software (Bruker). Protease inhibition assays The inhibition assays were performed in 96-well microtiter plates as follows: 30 µL of the enzyme solution (trypsin [100 µg/mL] or elastase [1.5 U/mL]) and 60 µL of the buffer (50 mM Tris-HCl, 20 mM CaCl2 pH 7.5) were pipetted into a series of 6 wells. 10 µL of a 2000 µM peptide solution (microviridin variant) were then added to well 1 and serially diluted until well 5; well 6 was used as control. After completing the serial dilution of the sample, 10 µL were taken from well 5 and discarded to maintain the same volume in all wells. The plates were then incubated for 5 min at 37 ºC, and subsequently 90 µL of the substrate solution (trypsin assay: N-benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPNA) [2.17 mg/mL]; elastase assay: Succinyl-Ala-Ala-Ala-p-nitroanilide (Suc-ALA3-pNA) [1 mg/mL]) were added to each well. The enzymatic activity was measured spectrophotometrically (Varioscan Flash, Thermo Scientific) at 410 nm after incubation at 37ºC for 15 min for the elastase assay, and 30 min for the trypsin assay. The assays were done twice for each sample. Obtained values were plotted in Origin to determine the IC50 of each sample (Table 1). All the substrates and enzymes were purchased from Sigma Aldrich. Associated content Supporting Information Available: This material is available free of charge via the Internet. Supporting Information Table S1 (XLSX) Supporting Information Tables S2-S3 and Supporting Information Figures S1-S4 (PDF) Author information Corresponding Authors: Email: [email protected] Email: [email protected] Author Contributions: †These authors contributed equally to this work.

Acknowledgements This work was supported by funding of the Academy of Finland (259505) to D.P. Fewer, a grant of the German Research Foundation (Di910/7-1) to E. Dittmann and funding by the Cluster of Excellence, Unifying Concepts in Catalysis (UniCAT) granted by the German Research Foundation (DFG) and coordinated by TU Berlin to E. Dittmann and R.D. 10 ACS Paragon Plus Environment

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Süssmuth. We would like to thank Muriel Gugger from the Institut Pasteur for kindly providing us with a culture of Cyanothece sp. PCC 7822.

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Tables and Figures

Table 1 Elastase and trypsin inhibition using synthetic microviridins inspired by the cryptic microviridin biosynthetic gene cluster encoded in the Cyanothece sp. PCC 7822 genome. Peptide

Maturation

Elastase IC50 (µM)

Trypsin IC50 (nM)

MdnA3 MdnA6 MdnA7 MdnA8

Ac-tricyclic Ac-tricyclic Bicyclic Bicyclic

ND ND 1.05 ± 0.20 21.85 ± 3.35

28.4 ± 4.0 21.5 ± 0.6 ND ND

(ND; not determined)

Figure 1. A selection of microviridin and marinostatin biosynthetic gene clusters from bacteria belonging to the proteobacteria, bacteriodetes and cyanobacteria phyla. The biosynthetic gene clusters encode 1-10 precursor peptides and enzymes involved in tailoring and maturation of microviridins and marinostatins.

Figure 2. The diversity of 308 precursor peptides encoded in the microviridin and marinostatin biosynthetic gene clusters. (A) A sequence similarity network showing three classes of precursor peptides encoded in microviridin and marinostatin biosynthetic gene clusters of members of the bacteriodetes, proteobacteria, and cyanobacteria phyla. (B) The precursor peptides could be divided into three classes based on the presence of processing signals and the number of core peptides. MdnA leader peptides, core peptides and additional sequence stretches of unknown function (SS) are marked.

Figure 3. A maximum-likelihood phylogeny based on the MdnC ATP-grasp ligase showing the relationship between known microviridin and marinostatin producers as well as cryptic microviridin biosynthetic gene clusters. The only strains included in this analysis that produce a known microviridin are found in the Microcystis and Planktothrix (boxed) and marinostatin are found in Altermonas sp. B-10-31 (boxed). Microviridin biosynthetic gene clusters encoding the MdnD acetyltransferase are marked with a solid circle. The Cyanothece sp. PCC 7822 strain is marked in bold. The strains encoding marinostatin or putative marinostatin homologs are marked in yellow. The phylogeny is rooted at the midpoint and the values are the node indicate bootstrap values above 50%. Branch length is proportional to sequence change. 15 ACS Paragon Plus Environment


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Figure 4. A schematic representation of the chemo-enzymatic synthesis of synthetic microviridins from Cyanothece sp. PCC 7822. (A) The 11.9 kb microviridin biosynthetic gene cluster from Cyanothece sp. PCC 7822 encodes 10 consecutive MdnA precursor peptides. (B) An alignment of the predicted MdnA core sequences showing the ester and amide bonds. The presence of positively charged amino acids in the fifth position (marked in red) suggested that this strain might encode a cocktail of serine protease inhibitors. (C) The selected core peptide variants were incubated with the lactone ring-forming ATP-grasp ligase MvdD and the lactam ring-forming ATP-grasp ligase MvdC of Planktothrix agardhii NIVACYA 126 both containing the covalently attached microviridin leader peptide at the Nterminus. N-acetylation was achieved using the MvdB acetyltransferase of P. agardhii.16 (D) The acetylated tricyclic structure of MdnA3 and MdnA6 and the bicyclic structure of MdnA7 and MdnA8.


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Figure 1. A selection of microviridin and marinostatin biosynthetic gene clusters from bacteria belonging to the proteobacteria, bacteriodetes and cyanobacteria phyla. The biosynthetic gene clusters encode 1-10 precursor peptides and enzymes involved in tailoring and maturation of microviridins and marinostatins. 170x62mm (300 x 300 DPI)

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Figure 2. The diversity of 308 precursor peptides encoded in the microviridin and marinostatin biosynthetic gene clusters. (A) A sequence similarity network showing three classes of precursor peptides encoded in microviridin and marinostatin biosynthetic gene clusters of members of the bacteriodetes, proteobacteria, and cyanobacteria phyla. (B) The precursor peptides could be divided into three classes based on the presence of processing signals and the number of core peptides. MdnA leader peptides, core peptides and additional sequence stretches of unknown function (SS) are marked. 83x82mm (300 x 300 DPI)


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Figure 3. A maximum-likelihood phylogeny based on the MdnC ATP-grasp ligase showing the relationship between known microviridin and marinostatin producers as well as cryptic microviridin biosynthetic gene clusters. The only strains included in this analysis that produce a known microviridin are found in the Microcystis and Planktothrix (boxed) and marinostatin are found in Altermonas sp. B-10-31 (boxed). Microviridin biosynthetic gene clusters encoding the MdnD acetyltransferase are marked with a solid circle. The Cyanothece sp. PCC 7822 strain is marked in bold. The strains encoding marinostatin or putative marinostatin homologs are marked in yellow. The phylogeny is rooted at the midpoint and the values are the node indicate bootstrap values above 50%. Branch length is proportional to sequence change. 83x152mm (300 x 300 DPI)



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Figure 4. A schematic representation of the chemo-enzymatic synthesis of synthetic microviridins from Cyanothece sp. PCC 7822. (A) The 11.9 kb microviridin biosynthetic gene cluster from Cyanothece sp. PCC 7822 encodes 10 consecutive MdnA precursor peptides. (B) An alignment of the predicted MdnA core sequences showing the ester and amide bonds. The presence of positively charged amino acids in the fifth position (marked in red) suggested that this strain might encode a cocktail of serine protease inhibitors. (C) The selected core peptide variants were incubated with the lactone ring-forming ATP-grasp ligase MvdD and the lactam ring-forming ATP-grasp ligase MvdC of Planktothrix agardhii NIVA-CYA 126 both containing the covalently attached microviridin leader peptide at the N-terminus. N-acetylation was achieved using the MvdB acetyltransferase of P. agardhii.16 (D) The acetylated tricyclic structure of MdnA3 and MdnA6 and the bicyclic structure of MdnA7 and MdnA8. 170x234mm (300 x 300 DPI)


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Phylogenomic Analysis of the Microviridin Biosynthetic Pathway

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