CrocadepsinsâDepsipeptides from the Myxobacterium

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Crocadepsins – Depsipeptides from the Myxobacterium Chondromyces crocatus found by a Genome Mining Approach Frank Surup, Konrad Viehrig, Shwan Rachid, Alberto Plaza, Christine K. Maurer, Rolf W. Hartmann, and Rolf Müller ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00900 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 10, 2017

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ACS Chemical Biology

Crocadepsins – Depsipeptides from the Myxobacterium Chondromyces crocatus found by a Genome Mining Approach Frank Surup,†,# Konrad Viehrig,‡ Shwan Rachid,‡,φ Alberto Plaza,‡,ξ Christine K. Maurer, §,#; Rolf W. Hartmann §,# and Rolf Müller†,‡,#,* †

Helmholtz Center for Infection Research (HZI), Department Microbial Drugs, Inhoffenstraße 7, 38124 Braunschweig (Germany), Email: [email protected] ‡

Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research and Pharmaceutical Biotechnology, Saarland University, Campus, Building C2.3, 66123 Saarbrücken (Germany). § Department of Drug Design & Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Saarland University, Campus E8.1, 66123 Saarbrücken (Germany). # German Centre for Infection Research Association (DZIF), partner site Hannover-Braunschweig, Inhoffenstraße 7, 38124 Braunschweig (Germany) ABSTRACT: Analysis of the genome sequence of the myxobacterium Chondromyces crocatus Cm c5 revealed the presence of numerous cryptic megasynthetase gene clusters, one of which we here assign to two previously unknown chlorinated metabolites by a comparative gene inactivation and secondary metabolomics approach. Structure elucidation of these compounds revealed a unique cyclic depsipeptide skeleton featuring β- and δ-amide bonds of aspartic acid and 3-methyl ornithine moieties, respectively. Insights into their biosynthesis were obtained by targeted gene inactivation and feeding experiments employing isotope-labeled precursors. The compounds were produced ubiquitously by the species Chondromyces crocatus and were found to inhibit the carbon storage regulator-RNA interaction.

Nonribosomal peptides (NRPs), polyketides (PKs) and hybrids thereof have a vast medical importance in drug development as exemplified by prominent representatives such as 1-3 tacrolimus, daptomycin and cyclosporin. Their diverse biological activities are based on a virtually endless structural variety, which mainly results from the abundance of poten4 tial building blocks. With the ajudazoles, chondramides, crocacins, chondrochlorens, thuggacins, crocapeptins and crocagins, already six classes of NRP and NRP/PK products and one postranslationally modified ribosomal peptide class are known from the myxobacterial strain Chondromyces crocatus Cm c5, elevating this strain to an exceptional sec5-11 ondary metabolite producer. However, whole genome sequencing of strain Cm c5 and subsequent annotation indicated the presence of additional 13 megasynthetases for generating NRP and PK natural products on top of the 6 previ12 ously known. In order to correlate these biosynthetic gene clusters to chemical products, we deployed a genome mining approach in which the crocadepsins presented herein were discovered.

RESULTS AND DISCUSSION Genome mining Using targeted gene inactivation via single-crossing-over mutagenesis, 11 non ribosomal peptide synthetase (NRPS) genes, each representing one hypothetical gene cluster were inactivated. After cultivation of the resulting mutants, their

crude extracts were investigated by comparative LC-HRMS 13 analysis as described previously. Inactivation of two NRPS genes, which were later annotated as cdpA and cdpL led to abolition of production of metabolites (1) and (2) with molecular ions at m/z 872.3 and 858.3 in the respective chromatograms. 39 31

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Figure 1A Crocadepsin gene cluster from Chondromyces crocatus Cm c5. NRPS and PKS genes are schown in dark blue, other ORFs are shown as light blue arrows. Figure 1B Annotated ORFs in the cdp gene cluster, with proposed function according to closest homologue found via BLAST against NRD. *BLAST hits from Chondromyces apiculatus DSM1436 were ignored, in that case the 2nd closest hit was selected. Figure 1C Effect of gene inactivation on production of crocadepsin derivatives in Cm c5 extracts. Production levels are shown relative to chondramide B production levels. Masses were normalized to crocadepsin A levels in the wild type (w. t.), which was set to “1”. tn5Crc: Cm c5 control mutant. 1

Structure elucidation To obtain sufficient material for structure elucidation and bioactivity tests, 11.2 mg of 1 and 9.4 mg of 2 were isolated from extracts of a 70 L scale Cm c5 wild type fermentation. The structures were subsequently determined by extensive 2D NMR spectroscopy, derivatization and degradation experiments and named crocadepsins. HRESIMS provided the molecular formula C40H51N7O13Cl for crocadepsin A (1) and C39H49N7O13Cl for crocadepsin B (2); the presence of a chlorine atom was indicated by the characteristic isotopic pat-

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tern. The H NMR and H, C HSQC spectra of 1 in DMSO-d6 revealed characteristic peptide resonances with several exchangeable amide NH protons in the region between 6.5 and 13 10.06 ppm. The analysis of the C NMR spectrum supported the peptidic nature of 1 by 8 carbonyl carbon signals between δC 167 and 179 ppm. The interpretation of 1H,1H COSY and 1 13 TOCSY, H, C HSQC and HMBC spectra identified aspartic acid and the unusual amino acid derivatives β-methyl ornithine (βMeOrn), 3-hydroxy-asparagine (Has), dehydrobutyrine (Dhb), and cinnamic acid (Cin). The presence of a βhydroxy-O-methyl-chlorotyrosine (βHmct) moiety was es-


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CdpI

Figure 2 Proposed biosynthesis of crocadepsin A (1). Trans cimmanic acid-CoA serves as free starter molecule that condenses with the threonine attached to the first PCP of CpdA. This intermediate is extended by CdpA, CdpB, CdpL and CdpF. Modifications of the amino acid residues by CdpC and CdpH are presumably introduced during assembly. β−methyl-ornithine is generated by CdpI, CdpJ and CdpK from arginine prior to incorporation by CdpL, as shown in the box on the bottom left. Methylation at the beta-carbon of ornithine is introduced during a transient alpha-keto intermediate, which is shown in brackets. The assembly line releases a deschloro derivative, which is halogenated by CdpE to yield crocadepsin A (1). Crocadepsin B (2) is produced, when CdpB incorporates malonyl-CoA instead of methylmalonyl-CoA (not shown). tablished by HMBC correlations. The sequence of entities 1 13 was assigned by inter-residue H, C HMBC correlations (Figure S1, Supporting Information). The high field shift of 19–H (δH 5.91) indicated an ester linkage at this position, which 1 13 was confirmed by the H, C HMBC correlation of H19 to the carboxyl carbon at δC 169.1 of Asp. Because the HMBC exper2 3 iment cannot differentiate J and J correlations, it remained ambiguous whether Asp was connected via C-1 or C-4. This issue was addressed by an 1,1 ADEQUATE experiment. Correlations from 2-H2 to C-1 and 3-H to C-4 in this spectrum indicated the connection via the C-terminal carboxyl atom, and thereby established the unique planar depsipeptide structure of 1. Crocadepsin B (2) differs from 1 by the absence of methyl C26. 1 1 A H, H ROESY correlation between 28-NH and 29-H 28,29 demonstrated the cis-configuration of the ∆ double bond, 32,33 while the trans-configuration of the ∆ double bond was 3 established by the large coupling constant JH32,H33=15.8 Hz. 28,29 The ∆ Z-configuration was further confirmed by the ra14 ther high field chemical shift (δ 5.65) of H-29. Crocadepsin A (1) features nine chiral centers. Their stereochemistry was determined by a combination of NMR spectroscopy, chemical degradation, and derivatization. First, the relative configuration of all substituents in βHmct, βMeOrn, and Has moieties was deduced by the J-based method devel15 oped by Murata and Tachibana (Figure S2). Subsequently, the absolute L-configuration of Asp and β-hydroxy asparagine was established by hydrolysis and derivatization with 16 FDAA, respectively FDLA. Derivatization with Mosher’s acid (MTPA, α-methoxy-α-(trifluoromethyl)phenylacetic acid) revealed S configuration of C6 and R-configuration of C17 (Figure S3); thus finalizing the elucidation of the absolute stereochemistry of 1 and 2. Gene cluster analysis and mutagenesis results

In our initial mutagenesis effort, production of 1 and 2 was correlated to the hybrid NRPS/PKS gene cluster shown in figure 1A. Consequently, the identified ORFs were annotated and analyzed for homology to known proteins with the aim to assign their possible function in biosynthesis of this novel metabolite class. The best matching sequences of most of the ORFs in the crocadepsin gene cluster were derived from ORFs of Chondromyces apiculatus strain DSM436 (Cm a2), suggesting the presence of another crocadepsin gene cluster in this strain. We investigated the published sequence of Cm 18 a2 and found an almost identical biosynthetic gene cluster, which differs from the Cm c5 crocadepsin cluster only by the absence of the putative MFS transporter gene, which is replaced by an aminomutase gene, as shown in table S4. The ORFs up- and downstream of the cluster are different in Cm c5 and Cm a2, confirming the cluster's boundaries. A detailed comparison of both sequences is presented in the supplemental information. We then aimed to inactivate the remaining ORFs found between cdpA and cdpL in Cm c5 to study their function on crocadepsin production. Mutants with disrupted genes cdpC, cdpD, cdpE, cdpF, cdpG, cdpJ, cdpK were obtained, genetically verified by PCR, and their extracts analyzed (see Figure 1C and S15). Inactivation of the third NRPS gene cdpF led to complete loss of crocadepsin production in the mutants, which was also the case for mutants of cdpC, encoding a cytochrome P450 monooxygenase. Inactivation of the halogenase gene cdpE strongly reduced crocadepsin signals in the respective extract, but instead a peak with m/z of 838.3 was observed. This mass corresponded to non-chlorinated crocadepsin A; re-examination of the wild-type confirmed that this non-chlorinated derivative was indeed produced as a minor metabolite. Since the activity of another additional halogenase is improbable, residual activity for the disrupted gene product or am impure mutant are more likely reasons for minor production. Mutants of the arginase gene cdpK and the methyl transferase gene cdpJ



both showed strongly reduced crocadepsin production, but no alternative derivatives were observed. Inactivation of the two putative transporter genes cdpD and cdpG led to reduced crocadepsin signals as well. Biosynthesis proposal: The findings from the chemical structure, sequence analysis and mutagenesis were combined with feeding experiments to provide a crocadepsins biosynthesis proposal. Crocadepsin assembly starts with activation and incorporation of threonine by CdpA, which is condensed to phenlyalanine-derived trans-cinnamic acid-CoA by the initial C-domain. Acylation of the free amino-terminus by the first C-domain of an NRPS is a common feature in myxobacterial NRPS pathways, and the crocacins from Cm c5 have been shown to be N-capped with trans-cinnamic acid-CoA, 7 too. The free hydroxy-group of threonine is eliminated in a yet unresolved step, resulting in the dehydrobutyrine moiety found in 1 and 2. Dehydrobutyrine residues have been found 14,19 in microcystins, cyanobacterial NRP-PK hybrids, and 20 nisins, ribosomally assembled peptides. The biosynthesis of these residues is currently unclear, but expected to result from phosphorylation of the free OH of threonine and subsequent elimination. Genes for putative phosphothreonine lyases, e.g. homologues to NisB, were however not found in 21 the crocadepsin gene cluster. Crocadepsin assembly then continues with the incorporation of tyrosine, which is Omethylated during assembly by the methyl transferase domain in the same NRPS module and chlorinated in orthoposition by the flavin-dependent halogenase CdpE. Halogenation by CdpE was verified by mutagenesis and probably occurs on the released depsipeptide. The tyrosine residue is further hydroxylated in the β-position (C19). The reaction is carried out by the cytochrome P450 monooxygenase CpdC on the PCP-bound intermediate, as observed in novobiocin 22 biosynthesis . This free β-hydroxy-group is later required for product release via lactonization with the aspartatic acid residue, and the complete loss of crocadepsin production in cdpC mutants supports this hypothesis. Although insertional mutagenesis frequently results in polar effects, absence of 1 and 2 in cdpC mutants is not expected to result from inhibition of gene expression of downstream genes, because the following downstream gene cdpD is encoded on the opposite strand. Depsipeptide assembly is then continued by extension with either malonyl-CoA or methylmalonyl-CoA by the PKS CdpB, thereby forming either crocadepsin A or B (1, 2), which was 13 indirectly confirmed by feeding with of C-labeled methio2 nine. MS fragmentation showed that SAM-derived methyl is not incorporated in methyl C26 of 1 (Table S3). The peptide is then extended with asparagine at the NRPS CdpL. Hydroxylation to ß-hydroxy-asparagine is assumed to be catalyzed by CdpH, a hydroxylase with high similarity to SyrP from Pseudomonas syringae, which catalyzes a similar 23 step in syringomycin biosynthesis. Mutants of cdpL were not obtained. The next extender unit, ß-methyl-ornithine (βMeOrn), is expected to be formed from arginine and a SAM-derived methyl group, since the biosynthesis gene cluster contains the arginase gene cdpK and a methyltransferase gene cdpJ colocalized with the aminotransferase cdpI to form βMeOrn. The origin of this extender unit was corroborated by labeling 13 13 2 with C6-arginine and C-methionine and MS experiments

(see table S3). The A-domain CdpL-A4, responsible for recognition and activation of the building block at that position, is not predicted to incorporate arginine. Therefore, ornithine or βMeOrn are expected to be formed prior to activation and loading onto the NRPS. A proposed biosynthesis for βMeOrn, resulting from the concerted action of CdpI, CdpJ and CdpK, is shown in the box at the bottom of figure 2. CdpK catalyzes hydrolysis of urea from arginine, then a transient α-keto-acid is formed by the transaminase CdpJ, which can be ß-methylated by CdpI. An analogous mechanism was described for the 3-methyl glutamate moiety 24 in daptomycin biosynthesis. After activation, ßMeOrn is condensed with asparagine via the distal δ-amino group. Since a δ-connection of a modified ornithin was reported for 25 scabichelin , we submitted the sequences of both C-domains 26 for an alignment using the napdos platform, to test whether these C-domains are phylogentically related. They do, however, group into different clades, thus there are no indications for a common clade of C-domains catalyzing a nucleophilic attack from a terminal amino group. The final building block aspartate is incorporated by the onemodular NRPS CdpF. Crocadepsin is then released by the TE domain of CdpF via lactonisation between the ß-hydroxygroup of tyrosine and the proximal carboxy-group of aspartate. This requires adenylation of Asp and subsequent tethering to the PCP-domain via its β-carboxy group, as shown in fig. 2 (CdpF). From the sequence information, the substrate specificity of the responsible adenlyation domain CdpF-A5 does not predict aspartic acid as a substrate, but points towards 2-aminoadipic acid (Aad), see table S5. The identified unusual non-ribosomal code, which is similar in the Adomains of the terminal module of the crocadepsin clusters from Cm c5 and Cm a2, might be explained by a different coordination of Asp in the binding pocket, resulting in its adenylation at the β-carboxylic acid instead of the standard α-carboxy activation. A β−connection of an Asp residue has 27 also been observed in microcystin and interestingly, the corresponding A-domain in the microcystin NRPS is also predicted to incorporate Aad instead of Asp. We therefore hypothesize that this type of connection is at least in part determined by the substrate specificity of the A-domain of the respective module. Biological activty Crocadepsins A and B (1 and 2) contain a β-hydroxy-γ-amino acid residue known as component of many protease inhibi28 tors. We could however not detect any inhibitory activity against the proteases chymotrypsin, pepsin or papain, and neither did we observe any antibacterial, antifungal or cyto29 toxic effects in our standard screening panel. Interestingly, we observed a moderate inhibition of the interaction between the carbon storage regulator A (CsrA) of Yersinia 30 pseudotuberculosis and RNA (IC50 33 µM for 1, 29 µM for 2). CsrA as a global bacterial post-transcriptional regulator is affecting mRNA translation and/or stability and occurs widespread among bacteria. As CsrA is known to be essential for full virulence of several pathogens, inhibition of the respective interaction represents a promising anti-infective drug target. Whether crocadepsins are of further use in this application is currently under investigation. In addition, crocadepsins might possess a currently unknown but important


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ACS Chemical Biology ecological function: While production levels varied greatly, the production of 1 and/or 2 could be demonstrated for all (total of 11) Chondromyces crocatus strains investigated so far in the course of our screening program for bioactive molecules (see table S6), but only occasionally for other Chon31 dromyces species.

D

Crocadepsin A (1): colorless oil; [α]25 +2.1 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 225 (sh), 285 (4.00) nm; IR (KBr) 3374, 2943, 1650, 1627, 1534, 1385, 1621, 1222, 1094, 1026, 767, 563 cm 1 1 13 ; H and C NMR see Table S1; HRESIMS m/z 872.3220 + ([M+H] , calcd for C40H51N7O13Cl, 872.3228), 870.3075 ([MH] , calcd for C40H49N7O13Cl, 870.3082). D

Conclusions We discovered two unique cyclic depsipeptides named crocadepsin A (1) and B (2) by a genome mining approach that was based on targeted knock out of megasynthetases in the myxobacterium Chondromyces crocatus Cm c5. The metabolites contain inter alia rare isopeptid connections of β-methyl ornithine and Asp. Analysis of the gene cluster revealed insights into the biosynthesis of the metabolites and their amino acid building blocks. Our study underlines the great potential of genome based screening approaches for finding novel secondary metabolites exhibiting exciting chemical scaffolds. We emphasize that the screening for new natural products even from intensively studied organisms such as C. crocatus Cm c5 exhibits great potential for finding new types of chemistry and unusual biosynthetic pathways.

Crocadepsin B (2): colorless oil; [α]25 +2.0 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 226 (sh), 285 (4.07) nm; IR (KBr) -1 1 13 3394, 2946, 1655, 1534, 1384, 1262, 1024 cm ; H and C NMR + see Table S2; HRESIMS m/z 858.3071 ([M+H] , calcd for C39H49N7O13Cl, 858.3071), 856.2906 ([M-H] , calcd for C39H47N7O13Cl, 856.2926).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ORCID Frank Surup: 0000-0001-5234-8525 Present Addresses φ Faculty of Science and Health, University of Koya, Koya, Kurdistan KOY45 (Iraq) ξ Sanofi-Fraunhofer Natural Product Center, Industriepark Hoechst, 65926 Frankfurt, (Germany)

METHODS

Author Contributions

Bacterial strains and growth conditions

The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

C. crocatus Cm c5 was cultivated in 70 L of Pol 0.3 medium (Probion 3 g/L, soluble starch 3 g/L, MgSO4 × 7 H2O 2 g/L, CaCl2 × 2 H2O 0.5 g/L) in a biofermenter Biostat UE100 (Braun Melsungen, Melsungen, D) at 30 °C for 11 days. After 3 days, 1% Amberlite XAD-16 was added to the fermentation broth.

Funding Sources The authors gratefully acknowledge funding by the German Ministry of Education and Research (BMBF). Notes The authors declare no competing financial interest.

Fermentation and isolation Amberlite XAD-16 and cell mass of C. crocatus Cm c5 were harvested by centrifugation. Cells were separated from XAD16 by flotation and discarded. The XAD was washed with water and subsequently eluted with 50% aqueous methanol, methanol and acetone. All fractions were analyzed by HPLC−UV−MS for the presence of target compounds. Consequently, the methanol extract was subjected to solvent partition using 85% aqueous methanol, which was extracted twice with heptane. Subsequently, the aqueous methanol was adjusted to 70% methanol and extracted twice with dichloromethane. The dichloromethane fraction containing the main amount of 1 and 2 was fractionated by RP MPLC [column 480 × 30 mm, ODS/AQ C18 (Kronlab), gradient 30−100% methanol in 60 min, ﬂow 30 mL/min, UV peak detection at 210 nm]. Fractions containing 1 and 2 were combined and further purified by preparative RP HPLC (column 250 × 21 mm, VP Nucleodur C18 Gravity 5 μm, gradient 10−30% acetonitrile in 25 min, 0.2% acetic acid, ﬂow 20 mL/min). Finally, preparative RP HPLC (column 150 × 40 mm, VP Nucleodur C18 Gravity 7 μm, gradient 10−30% acetonitrile in 25 min, 0.05% TFA, flow 50 mL/min) provided 11.7 mg of 1 (RT 18-19min) and 4.9 mg of 2 (RT 17-18min).

ACKNOWLEDGMENT We thank C. Kakoschke for recording NMR spectra, A. Gollasch and T. Hoffmann for ESI-MS measurements, D. Telkemeyer for strain maintenance, W. Collisi for conducting bioassays, B. Trunkwalter for help with labeling experiments, the fermentation team for large scale cultivation, V. Wray for helpful discussions, M. Fruth for the heterologous expression and purification of CsrA and R. Jansen for proofreading the manuscript.

ASSOCIATED CONTENT Supporting Information. Supporting Information, containing experimental details, further analysis, and NMR/HRMS spectra, is available free of charge via the Internet at http://pubs.acs.org.

ABBREVIATIONS FDAA, 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide; FDLA, 1-fluoro-2-4-dinitrophenyl-5-L-leucine amide; MTPA, αmethoxy-α-(trifluoromethyl)phenylacetic acid; βMeOrn, βmethyl ornithine; Has, 3-hydroxy-asparagine; Dhb, dehydrobutyrine; Cin, cinnamic acid; βHmct, β-hydroxy-O-methylchlorotyrosine.



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