Investigation of the Biosynthesis of the Lasso Peptide Chaxapeptin

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Investigation of the Biosynthesis of the Lasso Peptide Chaxapeptin Using an E. coli-Based Production System ́ ez,†,‡ Uwe Linne,‡ Fernando Albericio,§,⊥,∥ Judit Tulla-Puche,*,§,# Helena Martin-Gom and Julian D. Hegemann*,‡,¶ †

Institute for Research in Biomedicine, Baldiri Reixac 10, 08028 Barcelona, Spain Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg, Germany § Department of Inorganic and Organic Chemistry−Organic Chemistry Section, University of Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain ⊥ CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Baldiri Reixac 10, 08028 Barcelona, Spain ∥ School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa # Institut de Biomedicina de la Universitat de Barcelona (IBUB), 08028 Barcelona, Spain ¶ Department of Chemistry, University of Illinois at Urbana−Champaign, 600 S. Mathews Avenue, Urbana, Illinois 61801, United States

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S Supporting Information *

ABSTRACT: Lasso peptides are natural products belonging to the family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and are defined by their unique topology. Even though lasso peptide biosynthetic gene clusters are found in many different kinds of bacteria, most of the hitherto studied lasso peptides were of proteobacterial or actinobacterial origin. Despite this, no E. coli-based production system has been reported for actinobacterial lasso peptides, while there are numerous examples of this for proteobacterial lasso peptides. Here, a heterologous production system of the lasso peptide chaxapeptin was established in E. coli. Chaxapeptin, originally isolated from Streptomyces leeuwenhoekii strain C58, is closely related to the lasso peptide sungsanpin (produced by a marine Streptomyces sp.) and shares its inhibitory activity against cell invasion by the human lung cancer cell line A549. Our production system not only allowed isolation of the mature lasso peptide outside of the native producer with a yield of 0.1 mg/L (compared to 0.7 mg/L from S. leeuwenhoekii) but also was used for a mutational study to identify residues in the precursor peptide that are important for biosynthesis. In addition to these experiments, the stability of chaxapeptin against thermal denaturation and proteases was assessed.

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an aspartate or glutamate residue at positions 7−9 of the peptide. This ring is threaded by the linear C-terminal tail, which in turn is held in position by sterically demanding residues placed above and below the ring. Such a lasso fold can be extremely stable despite relying only on steric interactions, sometimes withstanding prolonged incubation at 95 °C6,14−26 or even autoclaving at 120 °C26 without loss of the structure. There are also examples of heat-sensitive lasso peptides (i.e., they unthread into a branched-cyclic peptide at elevated temperatures) described in the literature.6,14−25 The compact and rigid lasso fold often confers resistance against proteolytic degradation.6,14−21,23−25,27

asso peptides are natural products that exhibit many interesting biological properties, including antimicrobial, antiviral, enzyme inhibitory, and receptor antagonistic activities.1−12 These compounds belong to the superfamily of RiPPs, which share as a common feature in their biosynthesis that a genetically encoded precursor peptide is matured to the respective natural product through processing by other enzymes.1 The precursor peptide itself can be subdivided into an N-terminal leader sequence, which is needed for enzymatic recognition and is removed during the maturation process, and a C-terminal core peptide, where modifications are introduced.1 Lasso peptides are defined by their unique three-dimensional structures that are reminiscent of the knot in a lariat, which is also the origin of their name.2−4,13 The lasso fold consists of a macrolactam that is formed between the Nterminal α-amino group and the carboxylic acid side chain of © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 16, 2018

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DOI: 10.1021/acs.jnatprod.8b00392 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Until now, there is no known example of a functional E. colibased production system for lasso peptides from actinobacteria, which is surprising considering that many of the initially discovered lasso peptides were isolated from actinobacteria and that genome mining showed a plethora of putative lasso peptide biosynthetic gene clusters in this phylum.2−4,12,33,35 This suggests that previous attempts to establish actinobacterial lasso peptide production in E. coli have failed, although there are only a few examples in the literature where this is actually stated.28 There are however examples where actinobacterial lasso peptides were successfully heterologously produced in actinobacterial host organisms.18,28,36,37 The efficiency of production in these reports ranges from barely detectable amounts28 to 15 mg/L of culture.18 In all of these cases, lasso peptide production was accomplished only after considerable effort.18,28,36,37 Additionally, use of such actinobacterial host organisms is associated with long cultivation times. An E. coli-based production platform for lasso peptides from actinobacteria should permit much easier genetic manipulation of the biosynthetic gene cluster and significantly reduce cultivation times. In this study, we investigate which residues in the CptA precursor peptide are essential for lasso peptide production. For this, we established a heterologous production system in E. coli that allowed isolation of chaxapeptin and enabled us to probe corresponding residues by mutational analysis. By performing high-resolution liquid chromatography (LC)− mass spectrometry (MS), tandem MS, and ion mobility MS (IMS) we were able to validate that the heterologously produced chaxapeptin is identical to the one isolated from a native producer. In addition to these experiments, we also further investigated the stability of chaxapeptin by carrying out thermal stability and proteolysis assays with proteases not tested against this lasso peptide before.

For the closely related lasso peptides sungsanpin and chaxapeptin (both isolated from Streptomyces spp.), an interesting inhibitory activity was observed in cell invasion assays employing the human lung cancer cell line A549.5,7,10 Despite their similarity (Figure 1), these compounds originate

Figure 1. (a) Three-dimensional structure of chaxapeptin (PDB code 2N5C).7 The macrolactam is shown in yellow, the ring-forming Asp8 in red, and the tail region in blue. (b) Schematic representation of the chaxapeptin sequence using the same color code. (c) Biosynthetic gene cluster of chaxapeptin from S. leeuwenhoekii C34. (d) Amino acid sequence of the CptA precursor and the sungsanpin lasso peptide.7,10 Residues investigated in this study are highlighted.

from very different habitats, with a marine Streptomyces sp. producing sungsanpin,10 while chaxapeptin-producing strains (Streptomyces leeuwenhoekii strains C34 and C58) were isolated from the Atacama Desert.5,7 As the genome of the latter has been sequenced and published, closer inspection of the corresponding biosynthetic gene cluster is possible (Figure 1). The genes essential for chaxapeptin production are cptA, cptC, cptB1, and cptB2. During the lasso peptide biosynthesis, the precursor peptide CptA is first recognized by the RiPP recognition element (RRE) protein CptB1, which then mediates interaction with the protease CptB2.3,4,7,9,11,12,15,18,28−31 After cleavage of the precursor peptide into the N-terminal leader peptide (originally needed for RRE recognition) and the C-terminal core peptide (which consists only of the residues found in the final lasso peptide) by CptB2, the macrocyclase CptC activates the Asp8 side chain in an ATP-dependent manner.3,4,18,32 CptC then catalyzes the macrolactam formation, which yields the mature chaxapeptin.3,4,18,32 The exact mechanism of how the machinery accomplishes the complex lasso fold instead of making a branched-cyclic peptide has not been elucidated. The majority of studied lasso peptide biosynthetic gene clusters are from proteobacteria and feature a fused B protein, which means their gene clusters contain a single ORF encoding a protein with an N-terminal RRE and a C-terminal cysteine protease domain.6,12,14,19−24,33,34 The only non-proteobacterial lasso peptide for which an E. coli production system has been established is paeninodin, whose biosynthetic gene cluster stems from Paenibacillus dendritiformis C454.15 This cluster also contains separate B1 and B2 proteins, similar to the one producing chaxapeptin.

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RESULTS AND DISCUSSION Generation of an E. coli-Based Chaxapeptin Production System. For generation of a chaxapeptin production system, the complete gene cluster was amplified from gDNA and cloned into a pET41a vector. Expression of this plasmid (cptACB1B2 pET41) was carried out in E. coli BL21(DE3) under established conditions19−21,23,24 using M9 minimal medium. MeOH extraction of the cell pellet did not yield any detectable amounts of chaxapeptin. Following the procedures previously reported for optimization of heterologous production of several proteobacterial lasso peptides,17,20,24,38 we replaced the intergenic region between cptA and cptC with a λt0 terminator, followed by the mcjBDC promoter sequence that was originally found in the lasso peptide biosynthetic gene cluster of microcin J25 from E. coli AY25.26,38,39 As expression of the resulting plasmid (cptA_Termλt0_PrommcjBCD_cptCB1B2 pET41) still did not yield any detectable amounts of chaxapeptin, we reasoned that the native ribosomal binding sites (RBS) in front of cptB1 and cptB2 might not be recognized well in E. coli. To address this, E. colioptimized RBS sequences were added in front of cptB1 and cptB2. The resulting construct (cptA_Termλt0_PrommcjBCD_cptC_RBS_cptB1_RBS_cptB2 pET41) produced enough lasso peptide to enable isolation of 0.7 mg of chaxapeptin from 6 L of culture after expression for 3 d at 20 °C. To possibly improve yields, M9 minimal medium was compared to LB and TB medium when expressing for either 1 d at 37 °C or for 3 d at 20 °C (Supporting Information Figure B

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Figure 2. Comparison of (a) Streptomyces-isolated and (b) E. coli-produced chaxapeptin. Shown are the tandem MS spectra with the monoisotopic peaks of identified b fragments colored in red. The UV peaks of the lasso peptides in LC and the IMS drift times of the triply charged lasso peptide ions are shown in inlets.

Mutational Analysis of cptA. While the yield obtained with the E. coli production system (∼0.1 mg/L) is less than that of the native producer (∼0.7 mg/L), production time is cut from 10 d (3 d seed culture + 7 d for the actual production culture) to 4 d (1 d for the overnight culture + 3 d for the expression culture) and the E. coli plasmid allows quick and easy mutagenesis for exchanging specific residues in CptA. Thus, we could investigate the importance of conserved residues in the CptA precursor for lasso peptide maturation. To our knowledge, only clusters from Proteobacteria and a single cluster from a Firmicutes have been used for these kinds of experiments in heterologous E. coli-production systems,15,19−21,24,25,44 and because of this there is a need to validate if conserved residues shown to be important for lasso peptide processing in known systems are also important in a cluster of actinobacterial origin. A set of plasmids with mutations in the cptA gene were generated. The D8E variant was generated to test if exchange of the ring-forming residue is possible, a feat that has failed in every system tested so far.15,20,21,24,25 Previous studies have also shown that lasso

S1 and Table S1). These experiments showed the highest production level was accomplished in M9 minimal medium after expression for 3 d at 20 °C and that yields in rich media were significantly lower. To verify that the heterologously produced compound is identical to native chaxapeptin, both compounds were analyzed side by side via LC-MS, tandem MS, and IMS. The LC retention time indicates the similarity of the compounds, while tandem MS data can confirm the primary structure of a peptide and should show different peak intensity patterns for a branched-cyclic versus a lasso peptide with the same amino acid sequence.24,40,41 IMS was recently shown to be a powerful tool that also allows discrimination of lasso and branchedcyclic topologies at high charge states.15,42,43 Data from these experiments showed that LC retention time, tandem MS spectra, and IMS drift times were identical for both compounds and thus proved the identity of our E. coliproduced lasso peptide (Figure 2). The observed fragmentation spectra of chaxapeptin are furthermore in agreement with a previous study of this compound.7 C

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peptide residues that were previously shown to be important for lasso peptide maturation in proteobacterial clusters15,19−21,24,25,44 are of comparable importance in actinobacterial systems. It also confirms that the YxxP motif in the leader peptide, which was previously shown to be highly conserved in systems with split B-proteins,12 plays an important role in lasso peptide biosynthesis. Extended Stability Tests of Chaxapeptin. After investigating the biosynthetic machinery of chaxapeptin via mutational analysis, we wanted to assess how stable this particular lasso peptide is. Previously it was shown that chaxapeptin was resistant to degradation by the proteases trypsin, chymotrypsin, pepsin, and papain.7 We tested this lasso peptide against thermolysin, which could potentially cleave for example at the Phe10 or Leu12 residues in the loop region, and carboxypeptidase Y, which has the ability remove residues from the C-terminal tail that are far enough away from the shielding ring in lasso peptides. These proteases had no effect on chaxapeptin (Supporting Information Figures S2 and S3), which may be explained by its compact fold and short Cterminal tail section below the ring. Chaxapeptin heated to 95 °C for extended periods of time shows no unthreading (Supporting Information Figure S4), highlighting that the lasso fold of chaxapeptin is heat stable. In summary, this study is the first example of the successful heterologous production of an actinobacterial lasso peptide in E. coli. While this E. coli-based chaxapeptin production platform allows isolation of the lasso peptide in moderate yields (∼0.1 mg/L), it is not efficient enough to be used as a basis for a structure−activity relationship study of the inhibitory properties of chaxapeptin on invasion by A549 cells,7,10 and further optimization will be necessary. Potential routes to accomplish this could be using an E. coli codon optimized gene cluster, use of different promoters, and/or optimization of the production conditions. The E. coli-based production system described allowed a mutational analysis study of a lasso peptide cluster originally from a Streptomyces strain to be performed. By these means, it was demonstrated that previously gathered knowledge about residues important for lasso peptide biosynthesis in proteobacterial systems15,19−21,24,44 appears to be transferable to lasso peptide biosynthetic machineries originating from actinobacteria. In addition it was shown that chaxapeptin is a heat-stable lasso peptide that is resistant against a broader panel of proteases than reported previously,7 both useful properties for further optimizing its inhibitory activity against cell invasion by A549 lung cancer cells in light of a possible medical application that needs a stable peptide scaffold.7,10

peptide biosynthetic machineries have a high affinity for the residue at position 1 of the core peptide and that exchanges greatly diminish or completely abolish lasso peptide production.15,19,21,24,25 Therefore, a G1A variant was investigated. The penultimate threonine of the leader peptide has been present in every previously investigated lasso peptide precursor and was shown to be crucial for efficient lasso peptide processing. Some biosynthetic machineries tolerate exchange of this residue to other amino acids close in size and shape to Thr (e.g., Cys, Val, Ile for microcin J25 and capistruin), whereas substitution with Ala showed a significant impact in all systems investigated so far.15,19−21,24,25,44 Therefore, a T-2A variant was also evaluated. Finally, a conserved glycine at position 8 was proven to be important for production of the proteobacterial caulonodin lasso peptides,19 and such a residue is also found in CptA. While Gly-8 is present in many of the published proteobacterial clusters, it is absent in the precursor of the lasso peptide paeninodin found in a Firmicutes strain.15 To determine if this residue is needed in actinobacterial clusters, the G-8A variant was generated. In addition to these four residues that were shown to be important in various mutational analysis studies, a recent lasso peptide genome mining study identified a highly conserved YxxP motif in the leader peptide regions of clusters encoding a split B protein such as found in the chaxapeptin operon.12 Hence, substitution of Y-17A/P-14A was also tested. All of these variants were expressed alongside a WT reference in M9 minimal medium for 3 d at 20 °C, and the cell pellet extracts were analyzed by high-resolution LC-MS. Resulting data were analyzed for the predicted masses of the respective lasso peptides, and corresponding UV peaks were integrated for quantification. The results of these experiments are shown in Figure 3.

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EXPERIMENTAL SECTION

General Experimental Procedures, Bacterial Strains, and Materials. Streptomyces leeuwenhoekii C34 (DSM no. 42122) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). E. coli Top10 cells were used for cloning and mutagenesis, while E. coli BL21(DE3) was employed for expressions. All PCRs were carried out using Phusion DNA polymerase, and all Gibson assembly reactions were performed with Gibson assembly master mix from New England Biolabs. Carboxypeptidase Y and thermolysin were purchased from Alfa Aesar and Sigma-Aldrich, respectively. Cloning of the cpt Gene Cluster into pET41a and Optimization of the Plasmid. The complete cpt gene cluster was amplified from S. leeuwenhoekii C34 gDNA using Phusion polymerase with GC-buffer, 10% DMSO, and the primers FP_cptACBB and

Figure 3. Mutational analysis of cptA in the production plasmid. Experiments were performed in triplicates. A plus sign indicates that only the mass of the lasso peptide could be detected, while a minus sign shows that no production was observed.

As can be seen, all of the exchanges had a significant impact on overall lasso peptide yield. Exchanges of residues involved in the macrolactam formation (G1A and D8E) abolished production, while exchanges of conserved residues in the leader region reduced production to a fraction of the WT level (G-8A) or so low that they could only be detected by MS but not UV (Y-17A/P-14A and T-2A). This shows that precursor D

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RP_cptACBB (Supporting Information Table S2), which also introduced 5′ overhang regions utilized for Gibson assembly. pET41a was linearized for Gibson assembly by PCR using Phusion polymerase with HF buffer and the primers FP_pET41a and RP_pET41a (Supporting Information Table S2), which allowed cloning of cptACB1B2 behind the T7 promoter in a way that the T7 promoter RBS was placed in front of cptA (primers in Supporting Information Table S2). For the Gibson assembly reaction, insert and vector backbone DNA were mixed with the Gibson assembly master mix and incubated for 1 h at 50 °C. Gibson assembly was also used to incorporate the Termλt0-PrommcjBCD sequence in between cptA and cptC, thereby placing the RBS of the mcjBCD promoter in front of cptC. For amplification of the Termλt0-PrommcjBCD sequence a previously generated lasso peptide production plasmid24 was used as PCR template, and amplification was accomplished by using Phusion polymerase with HF buffer and the primers FP_TermProm and RP_TermProm (Supporting Information Table S2). Linearization of cptACB1B2 pET41a was also achieved by PCR employing Phusion polymerase, GC-buffer, 10% DMSO, and the primers FP_cpt-TermProm and RP_cpt-TermProm (Supporting Information Table S2). The Gibson assembly reaction was carried out as described above. Finally, E. coli-optimized RBS sequences (taken from the T5 promoter sequence in the pQE60 plasmid) were introduced in front of cptB1 and cptB2 in a stepwise manner by site-directed ligaseindependent mutagenesis (SLIM) using established protocols.45,46 In short, plasmid DNA was amplified with two sets of primers, each set consisting of a base primer (FP or RP) complementary to the flanking region of the RBS insertion and an overhang primer that not only binds to the flanking region but also has a nonbinding overhang with the sequence to be introduced on its 5′ end (FPTail or RPTail). After checking if the PCRs worked on an agarose gel, 10 μL of the PCR with FP and RPTail primers was mixed with 10 μL of the PCR with FPTail and RP primers, 20 μL of H2O, and 10 μL of SLIM hybridization buffer (750 mM NaCl, 125 mM Tris, 100 mM EDTA, pH 8.0). DNA is then denatured and rehybridized by incubation at 99 °C for 3 min, followed by three cycles of first incubating at 65 °C for 5 min and then for 40 min at 30 °C. In this way, double-stranded hybrid DNA consisting of DNA strands from each PCR was generated, where the respective complementary overhang regions hybridized with each other and in this way yielded circular DNA that could be transformed and proliferated in E. coli. For RBS incorporation between cptB1 and cptB2, the primer pairs FP_cptB1_RBS_cptB2/RPTail_cptB1_RBS_cptB2 and FPTail_cptB1_RBS_cptB2/RP_cptB1_RBS_cptB2 were used, while FP_cptC_RBS_cptB1/RPTail_cptC_RBS_cptB1 and FPTail_cptC_RBS_cptB1/RP_cptC_RBS_cptB1 were utilized for RBS introduction between cptC and cptB1 (Supporting Information Tabe S2). All SLIM-PCRs were carried out using Phusion DNA polyermase with GC buffer and 10% DMSO. The DNA sequence of the resulting cptA_Termλt0_PrommcjBCD_cptC_RBS_cptB1_RBS_ cptB2 pET41 plasmid is listed in the Supporting Information. Chaxapeptin Isolation from S. leeuwenhoekii C34. A 100 mL aliquot of GYM medium (10 g/L malt extract, 4 g/L glucose, 4 g/L yeast extract, pH 7.0) was inoculated by addition of 10 μL of S. leeuwenhoekii C34 spore suspension and incubated for 3 d at 30 °C. Then, 5 mL of the seed culture was used to inoculate 1 L of GYM medium, which in turn was shaken for 7 d at 30 °C. After that time, cells were harvested by centrifugation. Culture supernatants were extracted by addition of 4 g of XAD-16 resin and slowly stirred for 1 h at room temperature (RT). The supernatant was removed by filtration, and the resin was first washed with water and then eluted with 100 mL of MeOH. For extraction of the cell pellet, cells were resuspended in 150 mL of MeOH and shaken overnight at 4 °C. Afterward, the extract was centrifuged and the clear supernatant was collected. Solvent from both extracts was removed in vacuo. Dried pellet and supernatant extracts were resuspended in 8 mL of 50% of MeOH, cleared by centrifugation and pressing through a syringe filter (0.45 μm), and finally applied to preparative HPLC

using a microbore 1100 HPLC system (Agilent) with a VP 250/21 Nucleodur C18 Htec 5 μm column (Macherey-Nagel) at RT and a flow rate of 18 mL/min. Absorbance was measured at 215 nm. For the first round of purification, linear gradients of solvent A (water−0.045% formic acid) and solvent B (MeOH−0.05% formic acid) were used: 20% to 35% B in 5 min, followed by a linear increase of 35% to 95% B in 30 min. For the second round of purification, the solvent system was switched to solvent C (water−0.1% trifluoroacetic acid) and solvent D (MeCN−0.1% trifluoroacetic acid) using a linear gradient from 10% to 60% D in 30 min. In this way, a total of 0.7 mg/ L of chaxapeptin could be obtained from the cell pellet extract, while no lasso peptide was detected in the supernatant extract. Heterologous Production and Isolation of Chaxapeptin. All E. coli culture media used in this study were supplemented with 50 μg/mL kanamycin as resistance marker. Heterologous production screens were carried out in M9 minimal medium [17.1 g/L Na2HPO4· 12 H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 1 mL/L MgSO4 solution (2 M), 0.2 mL/L CaCl2 solution (0.5 M), pH 7.0; after autoclaving, 10 mL/L sterilized glucose solution (40% w/v) and 2 mL/L vitamin mix (see Supporting Information Table S3) were added] under the following conditions: 500 mL of M9 minimal medium in a 2 L baffled flask was inoculated by addition of 5 mL of a 37 °C lysogeny broth (LB) overnight culture. Cells were grown at 37 °C until reaching an optical density at 600 nm (OD600) of 0.4−0.45, and then the temperature was reduced to 20 °C. Cultures were induced 1 h later (OD600 of 0.5−0.7) by addition of 100 μL of a 0.5 M stock solution of isopropyl β-D-1-thiogalactopyranoside (IPTG), yielding a final IPTG concentration of 0.1 mM. Expressions were carried out for 3 d at 20 °C and then harvested by centrifugation. Cell pellets were extracted by resuspending them in 50 mL of MeOH and shaking them overnight at 4 °C. Afterward, extracts were spun down, and the clear supernatant was collected and evaporated to dryness under reduced pressure. The dry residue was resuspended in 1 mL of 50% methanol, cleared by centrifugation, and analyzed by highresolution LC-MS (see below). As the heterologously expressed lasso peptide biosynthetic gene cluster did not contain a dedicated ABC transporter and as unaided export of peptidic natural products of this size is unlikely, the culture supernatant was not extracted. After establishing general chaxapeptin production, a media screen was performed to investigate if yields could be optimized. For this screen, M9 minimal medium, LB medium (10 g/L casein peptone, 5 g/L yeast extract, 10g/L NaCl, pH 7.0), and terrific broth (TB) medium (12 g/L casein peptone, 24 g/L yeast extract, 2.2 g/L KH2PO4, 9.4 g/L K2HPO4, 4 mL/L glycerol, pH 7.0) were used. Expressions were carried out either for 1 d at 37 °C or 3 d at 20 °C. For continuous expression at 37 °C, 500 mL of medium in a 2 L baffled flask was inoculated by addition of 5 mL of a 37 °C LB overnight culture, and cells were grown until reaching an OD600 of 0.5−0.7 in the case of M9 and LB medium or 1.4−1.6 in the case of TB medium. At this point, cells were induced by addition of 100 μL of a 0.5 M IPTG stock solution and grown overnight at 37 °C. Cultures were harvested and worked up as described above. Expressions for 3 d at 20 °C were generally carried out as described above, with minor changes when using LB and TB medium. For LB, temperature was shifted down to 20 °C when the OD600 reached 0.2− 0.25, but induction would still take place 1 h later at an OD600 of 0.5− 0.7. For TB medium, the temperature was shifted down to 20 °C when the OD600 reached 0.9−1.0 and induction would take place 1 h later at an OD600 of 1.4−1.6. These experiments showed (Supporting Information Figure S1 and Table S1) that the highest production level of chaxapeptin is accomplished in M9 minimal medium when the optimized production plasmid is expressed for 3 d at 20 °C. Large-scale heterologous production of chaxapeptin was carried out with 6 L of M9 minimal medium over 3 d at 20 °C as described above and worked up in a similar manner. The resulting dried pellet extract was resuspended in 8 mL of 50% MeOH, cleared by centrifugation, pressed through a syringe filter (0.45 μm), and purified by two rounds of preparative HPLC as described above. In this way, 0.7 mg of chaxapeptin was isolated from a 6 L E. coli expression culture. E

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Thermal Stability Assay. A 100 μL solution of purified chaxapeptin (100 μg/mL) in 50% MeCN was incubated at 95 °C for 1, 2, 3, or 4 h. Samples were cooled to 4 °C and analyzed via analytical HPLC (Waters Alliance 2695) with an automatic injector and a photodiode array detector (Waters 2998) using a Phenomenex Luna C18 5 μm (4.6 mm × 250 mm) reversed-phase analytical column run with a linear gradient from 35% to 40% MeCN (with 0.045% trifluoroacetic acid) over 40 min at 25 °C. UV signals were recorded at 220 nm, and the system was run at a flow rate of 1 mL/min.

LC-MS, Tandem MS, and IMS. Cell pellet extracts were analyzed with a microbore 1100 HPLC system (Agilent) that was connected to a high-resolution LTQ-FT Ultra instrument (Thermo Fisher Scientific). LC separation was accomplished by using an EC 125/2 Nucleodur 300-5 C18 ec column (Macherey-Nagel) at a flow rate of 0.2 mL/min employing the following gradient of solvent A (water− 0.1% trifluoroacetic acid) and solvent B (MeCN−0.1% trifluoroacetic acid): Holding 2% B for 2 min, followed by a linear increase from 2% to 30% B in 18 min, and then a linear increase from 30% to 95% B in 15 min and holding 95% B for 2 min. Fragmentation was accomplished by selecting the singly charged ions for collisioninduced fragmentation within the linear ion trap using online LC-MS. The energy for fragmentation was set to 35%. For IMS analysis, samples were prepared to contain a final concentration of 4% formic acid and 0.5% sulfolane to establish high charge states that would allow the discrimination between lasso peptides and analogous branched-cyclic peptides.15,43 For measurements, a Synapt G2-Si (Waters) hybrid quadrupole ion-mobility timeof-flight mass spectrometer with an ESI source was used in positive mode at the settings shown in Supporting Information Table S5. Mutational Analysis. SLIM45,46 was used to mutate cptA in the chaxapeptin production plasmid using the primers listed in Supporting Information Table S4. SLIM-PCRs were accomplished by using Phusion polymerase with GC buffer and 10% DMSO. The Y17A/P-14A mutation was introduced with the primer pairs FP_cptA_-17to-11/RPTail_cptA_Y-17A-P-14A and FPTail_cptA_Y-17A-P-14A/RP_cptA_-17to-11. For G-8A, the primer pairs FP_cptA_-8to-2/RPTail_cptA_G-8A and FPTail_cptA_G-8A/ RP_cptA_-8to-2 were used. For T-2A, the primer pairs FP_cptA_8to-2/RPTail_cptA_T-2A and FPTail_cptA_T-2A/RP_cptA_-8to-2 were employed. For G1A, the primer pairs FP_cptA_1to8/ RPTail_cptA_G1A and FPTail_cptA_G1A/RP_cptA_1to8 were utilized. For D8E, the primer pairs FP_cptA_1to8/RPTail_cptA_D8E and FPTail_cptA_D8E/RP_cptA_1to8 were used. All mutant plasmids and a WT control were expressed in triplicate in 500 mL of M9 minimal medium as described above and worked up under the aforementioned conditions. Of the resulting 1 mL of concentrated extracts, 100 μL was applied to high-resolution LC-MS (see above). When the mass of a predicted lasso peptide was detected, the UV chromatogram was checked for peaks with the same retention time. If found, the peak was integrated to estimate the production level of the lasso peptide (see Supporting Information Table S6). Protease Stability Assays. For the carboxypeptidase Y assay, 10 μL of a carboxypeptidase Y stock solution (1 mg/mL) in MES buffer (50 mM MES, 1 mM CaCl2 at pH 6.75) was added to 25 μL of a solution of purified chaxapeptin (20 μg/mL) in the same buffer, and the mixture was incubated for 4 h at 25 °C. The reaction was quenched by the addition of 75 μL of MeCN, filtered, and analyzed via analytical HPLC (Waters Alliance 2695) with an automatic injector and a photodiode array detector (Waters 2998) employing a Phenomenex Luna C18 5 μm (4.6 mm × 250 mm) reversed-phase analytical column run with a linear gradient from 35% to 40% MeCN (with 0.045% trifluoroacetic acid) over 40 min at 25 °C. UV absorption was detected at 220 nm, and the system was run at a flow rate of 1 mL/min. For the thermolysin assays, 50 μL of a chaxapeptin stock solution (100 μg/mL in buffer) was mixed with 15 μL of a thermolysin stock solution (1 mg/mL in the same buffer) and incubated under different conditions. A buffer consisting of 50 mM Tris and 0.5 mM CaCl2 at pH 8 was used when incubating for 2 h at 70 °C, while 10 mM Tris at pH 8 was used as buffer when incubating overnight at 37 °C. In both cases, the reaction was quenched by the addition of 4 M HCl until reaching pH 2. Samples were then filtered and analyzed via analytical HPLC (Waters Alliance 2695) with an automatic injector and a photodiode array detector (Waters 2998) utilizing an Xbridge C18 2.5 μm (4.6 mm × 75 mm) reversed-phase analytical column with a linear gradient from 30% to 60% MeCN (with 0.045% trifluoroacetic acid) over 8 min at 25 °C. UV detection was performed at 220 nm, and the system was run at a flow rate of 1 mL/min.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00392.

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Chromatograms of the media screens, thermal and protease stability assays, tables with oligonucleotide primer sequences, IMS settings, observed UV integrals, and the recipe for the M9 vitamin mix (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail (J. Tulla-Puche): [email protected]. *E-mail (J. D. Hegemann): [email protected]. ORCID

Julian D. Hegemann: 0000-0002-3859-8744 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Prof. M. A. Marahiel for general support with this project and J. Bamberger for help with the IMS data evaluation. J.D.H. thanks the Deutsche Forschungsgemeinschaft for financial support (DFG Research Fellowship 309199717). This work was partially funded by MINECO (CTQ2015-68677R) and the Generalitat de Catalunya (2017 SGR 1498). H.M.-G. and J.T.-P. thank MINECO for a Severo Ochoa predoctoral fellowship and a Ramon y Cajal contract, respectively.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.8b00392 J. Nat. Prod. XXXX, XXX, XXX−XXX