Genome Mining of Streptomyces atratus SCSIO ZH16: Discovery of

Feb 12, 2019 - Genome mining of the deep sea-derived Streptomyces atratus SCSIO ZH16 enabled the activation of a cyclodepsipeptide gene cluster and ...
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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Genome Mining of Streptomyces atratus SCSIO ZH16: Discovery of Atratumycin and Identification of Its Biosynthetic Gene Cluster Changli Sun,† Zhijie Yang,†,§ Chunyan Zhang,†,§ Zhiyong Liu,‡ Jianqiao He,†,§ Qing Liu,†,∥ Tianyu Zhang,‡,§ Jianhua Ju,*,†,§ and Junying Ma*,†

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CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China ‡ Tuberculosis Research Laboratory, State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China § University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China ∥ State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China S Supporting Information *

ABSTRACT: Genome mining of the deep sea-derived Streptomyces atratus SCSIO ZH16 enabled the activation of a cyclodepsipeptide gene cluster and isolation of its cinnamic acid-bearing product, atratumycin (1). Atratumycin’s structure was elucidated on the basis of extensive spectroscopic experiments, X-ray data, and Marfey’s method; a plausible biosynthesis and tailoring modification of 1 are also proposed and investigated. Additionally, atratumycin is active against Mycobacteria tuberculosis H37Ra and H37Rv with MICs of 3.8 and 14.6 μM, respectively.

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To obtain natural compounds encoded by other gene clusters, the in-frame deletion strain S. atratus SCSIO ZH16NS was generated by deleting an 8 kb fragment (genes spanning from ilaN-ilaS); this approach failed to not only produce ilamycins but also generate a background clean strain for genome mining.5 Accordingly, 10 different types of media (Table S1) were utilized to ferment the mutant strain. HPLC analysis of an extract generated from fermentation of this mutant strain in FYG medium revealed a very small, but interesting, chromatographic signal (Figure 1i). Subsequent LC−MS analyses of this extract revealed the clear generation of peptide fragment ions suggesting, early on, that the small peak resulting from FYG fermentations was peptidic in nature; subsequent structure elucidation efforts (vide infra) would ultimately reveal this small HPLC peak to represent cyclodepsipeptide 1. Continued application of ARTP mutagenesis using different mutagenesis times (40−120 s in 20 s increments) ultimately afforded eight engineered strains (designated as S. atratus SCSIO ZH16NS-xS where x = mutagenesis time) able to generate putative 1 in titers superior to that of the S. atratus SCSIO ZH16NS strain. HPLC metabolite analyses revealed S. atratus SCSIO ZH16NS-80S to be the optimal producer of putative 1, and this strain was chosen for all subsequent studies. Further optimization and stabilization of cyclopeptide biosynthesis was sought by supplying FYG medium with 2% macroporous

xtensive genome sequencing campaigns have revealed that most Actinomycetes contain >20 biosynthetic gene clusters (BGCs) per genome, yet only a small fraction of these are expressed under normal laboratory fermentation conditions.1 These “surplus” silent BGCs may produce bioactive compounds with the potential to rejuvenate stalled drug discovery pipelines. Toward this end, a series of genome mining strategies have been applied to unveil otherwise hidden biosynthetic options for rendering novel compounds. Notably, this approach appears to lessen the chances of rediscovering known compounds and, as such, has been proposed to optimize the chances of identifying new drug leads.2 Streptomyces atratus SCSIO ZH16 is a deep sea-derived bacterium isolated from a sediment sample. Previously, a series of ilamycin congeners with antituberculosis activity were isolated from fermentations of the wild-type (WT) strain; the BGC responsible for construction of the ilamycins has been elucidated.3 Genome sequencing and annotation of the strain revealed the presence of 26 BGCs, suggesting a far greater potential to produce specialized natural products than was previously appreciated. To optimize the biosynthetic potential of the strain, a combination of atmospheric room-temperature plasma (ARTP) mutagenesis4 and medium optimization methodologies were applied. As a result, a cyclopeptide BGC, was activated. Herein, we report the activation, isolation, structure elucidation, bioactivities, and the biosynthesis of atratumycin (1), a cyclic decapeptide with a unique 3-(2methyl phenyl)-2(E)-propenoic acid unit appended to the peptide. © XXXX American Chemical Society

Received: January 17, 2019

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DOI: 10.1021/acs.orglett.9b00208 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

configurations. The X-ray diffraction analysis with Cu Kα radiation resulted in a Flack parameter of 0.04(6), allowing us to confidently assign L-configurations to Thr, Ser, Trp, and Pro and D-configurations to Asn, Val, Leu, Tyr, and (2S, 3S)-3-OHPhe.6 Moreover, these data supported concomitant efforts in which Marfey’s method was employed to determine amino acid stereochemistries (Figure S4).7 The presence of nonproteingenic amino acids including four D-amino acids (D-Asn, D-Val, D-Leu, D-Tyr), one unnatural Thr analogue, and the (2S,3S)-3-OH-Phe unit in the backbone of 1 strongly suggested that atratumycin biosynthesis is carried out predominantly by nonribosomal peptide synthetase (NRPS) machinery. Among the 26 predicted BGCs in the S. atratus SCSIO ZH16 genome, the 13th cluster in the genome of S. atratus SCSIO ZH16 (atr gene cluster) was deemed the best candidate for encoding atratumycin biosynthesis; this logic was based on its 48% similarity to the skyllamycin BGC8 and 32% similarity to the BGC for WS9326A.9 Like 1, both skyllamycin and WS9326A are characterized by a pendant cinnamoyl moiety.8,9 The atr cluster was found to contain 45 genes including three NRPSs genes, six regulatory and transport genes, 13 biosynthetic precursor genes, three transposase related genes, two MbtH genes, one P450 monooxygenase, and 17 genes of unknown function spanning a ∼78 kb contiguous DNA region (Scheme 1A, Table S4). To understand the structural characteristics of the three NRPSs embedded within the atr gene cluster, PKS/NRPS online prediction software was used.10 These analyses revealed that the NRPS termed Atr23 encodes four modules containing 13 domains (C1−A1−T1, C2−A2−T2, C3−A3−T3, and C4−A4− T4−E1), whereas another NRPS, Atr22, encodes three modules composed of 10 domains (C5−A5−T5, C6−A6−T6, C7−A7− T7−E2). The third NRPS embedded within the atr gene cluster, Atr21, also encodes three modules composed of 11 domains (C8−A8−T8, C9−A9−T9−E3, C10−A10−T10−TE) (Scheme 1B). The Atr23, Atr22, and Atr21 NRPSs are proposed to activate L-Thr, L-Ser, L-Trp, L-Asn, L-Phe, L-Pro, LVal, Gly, L-Leu, and D/L-Tyr during atratumycin assembly line (Scheme 1B). Notably, binding pocket analyses revealed that, of the 10 A domains, seven show complementarity with amino acids in atraumycin. Conversely, the D-Asn, D-Val and D-Leu required to complete 1, perfectly match predictions of E domain compatibility within the fourth, seventh and ninth modules. A noteworthy feature is the presence of a D-Tyr in atratumycin (1), but the lack of a suitable E domain in the 10th module (Scheme 1B). We propose that the third E domain in the ninth module may carry out epimerization of both 9Leu and 10Tyr. Alternatively, the 10th A domain may directly activate the D-Tyr as is known for an A domain in the biosynthesis of fusaricidins.11 This chemistry is the focus of current investigations and will be reported on in the future. Rounding out functional assignments for the machinery driving biosynthesis of 1, we posit that the module 1 C1 domain likely catalyzes the cinnamoyl acid linkage to the growing peptide scaffold and is perhaps one of the first NRPS steps (Scheme 1B). Cinnamoyl acid moieties bearing C-2 substitution of the aryl group are rarely seen in natural products; only four kinds of cyclodepsipeptide natural products8,9,12 contain such groups, and an understanding of their biosynthesis has been elusive. To investigate the construction of this rare 3-(2-methyl phenyl)-2(E)-propenoic acid, as relates to 1, the atr gene cluster was carefully analyzed. We found 10 genes encoding for proteins with high sequence identities (from 47 to 74%) to

Figure 1. HPLC analysis of fermentation broths: (i) S. atratus SCSIO ZH16NS fermented in FYG medium; (ii) S. atratus SCSIO ZH16NS fermented in FYG medium + 2% XAD-16; (iii) S. atratus SCSIO ZH16NS-80S fermented in FYG medium; (iv−vi) S. atratus SCSIO ZH16NS-80S, Δatr23 and Δatr27 fermented with FYG medium + 2% XAD-16, respectively.

XAD-16 resin during fermentation and this improved titers of putative 1 by ∼8-fold (Figure 1iv). Thus, large-scale fermentation of S. atratus SCSIO ZH16NS-80S using these conditions ultimately afforded, after chromatographic purifications, analytically pure atratumycin (1). The molecular formula of atratumycin (1) was assigned as C68H84N12O16 on the basis of the HRESIMS ion peak at m/z 1325.6193 ([M + H]+, calcd 1325.6201) (Figure S1) and 13C and DEPT NMR spectra, which indicated 33 degrees of unsaturation. Detailed analyses of 1D and 2D NMR data (Table S2) implied the presence of 10 different amino acids: threonine (Thr), serine (Ser), tryptophan (Trp), asparagine (Asn), phenylserine (β−OH-Phe), proline (Pro), valine (Val), glycine (Gly), leucine (Leu), and tyrosine (Tyr). The 10 redundant 13C signals were assigned to a 3-(2-methyl phenyl)2(E)-propenoic acid group due to HMBC correlations of H-2 with C-1, H-3 with C-2, C-4, C-5, H-10 with C-4, C-8, C-9, and COSY correlations of H-2/H-3, H-5/H-6/H-7/H-8. The 2E configuration was verified by the H-3 coupling constant (J2, 3 = 15.7 Hz) (Figure S2). The linkage order for the 10 amino acids was elucidated on the basis of extensive MS analyses. The positive MS 2 experiment from the parent ion at m/z 1325.6201 ([M + H]+) provided abundant fragmentation ions of the compound. In the MS2 spectrum, the fragmentation ions at m/z 1098.5 (y9), 1011.5 (y8), and 825.4 (y7) were proposed to be generated from the neutral loss of one 3-(2-methyl phenyl)2(E)-propenoic acid-Tyr unit, one Ser, and one Trp, respectively. The successive losses of Asn, β-OH-Phe, Pro from the fraction y7 gave rise to ions at m/z 711.4 (y6), 548.3 (y5), and 451.3 (y4). Ions y3 (m/z 352.2), y2 (m/z 295.2), and y1 (m/z 332.2) were obtained by the losses of Val, Gly, and Leu from y4 successively. The structural fragments b1−b9 could also be explained by scission of amide linkages in the MS2 spectrum. Thus, the sequence of amino acids within 1 was confidently elucidated (Figure S1). The superfluous additional degree of unsaturation was accounted for by realizing the cyclic nature of 1. The chemical shifts for H-13 and C-13 of the Thr implicated the side chain of Thr as integral to the cyclic nature of 1 and this realization was substantiated by X-diffraction studies. Solving the X-ray structure (Table S3 and Figure S3) not only confirmed structural assignments made using NMR and MS data but also enabled us to assign all absolute B

DOI: 10.1021/acs.orglett.9b00208 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters Scheme 1. Organization of atr BGC and Proposed Biosynthetic Pathway for atratumycin (1)a

a Key: (A) organization of the atratumycin biosynthetic gene cluster in S. atratus SCSIO ZH16NS and cosmids providing whole gene cluster coverage; (B) NRPS assembly process for atartumycin (1); (C) proposed biosynthetic pathway of cinnamoyl acid unit; (D) biosynthesis of β-OHPhe.

discovering (via genome mining) new drug leads. Relatedly, anti-SMASH analyses have revealed >10 Streptomyces strains that harbor cinnamate biosynthetic gene cassettes analogous to the 10 genes containing cassette found here for 1 (Figure S5). To definitively evaluate the role of the atr gene cluster in the biosynthesis of atratumycin (1), the third A domain in atr23 was inactivated by λ-Red recombination using an apramycin gene cassette.13 Following verification of the phenotype and genotype (Figure S6), the double crossover mutant Δatr23 was obtained. HPLC-based metabolite analysis of Δatr23 mutant fermentations revealed its complete inability to produce 1, thereby confirming the indispensability of atr23 to atratumycin biosynthesis (Figure 1iv). To determine the boundary of the atr cluster, another four genes were also inactivated using the aforementioned methods. Following phenotype and genotype verifications, four double-crossover mutants were obtained (Figures S7−S10). Fermentation of the mutants and subsequent extractions and metabolite analyses revealed little to no difference in atrtumycin (1) titers relative

enzymes implicated in cinnamoyl construction during skyllamycin biosynthesis.8 We speculate that this set of 10 genes constitutes a 3-(2-methyl phenyl)-2(E)-propenoic acid biosynthetic gene cassette. Moreover, we envisioned that this cassette enables a 3-(2-methyl phenyl)-2(E)-propenoic acid assembly line that carries out a repeating cycle of condensation, reduction, dehydration, and isomerization as catalyzed by ketosynthase/chain length factor (KS-CLF (Atr6−8, Atr11)), ketoreductase (KR (Atr15)), dehydratase (DH (Atr13,14)), and the cis-trans isomerase Atr16 (Table S4 and Scheme 1C). It is noteworthy that the gene encoding the oxidoreductase proposed involved in the forming of benzene ring closure between the carbon atoms 4 and 9 in the cinnamoyl residue in skyllamycin was absent from the atratumycin gene cluster.8 Notably, identification of the atratumycin, skyllamycin and WS9326A BGCs has provided outstanding opportunities to exploit both the genetic and biochemical foundations of cinnamoyl acid biosynthesis and incorporation into natural products in constructing and/or C

DOI: 10.1021/acs.orglett.9b00208 Org. Lett. XXXX, XXX, XXX−XXX

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to S. atratus SCSIO ZH16NS-80S (Figure S11). Consequently, the atr gene cluster boundary can be narrowed to a fragment spanning 62.7 kb of sequence and composed of 33 genes (Table S4 and Scheme 1A). The sequence of the gene cluster has been deposited into the GenBank under the accession number of MK370905. The putative product of atr27 shows 60% identity with Sky32, a functionally characterized β-hydroxylase for three amino acids during skyllamycin biosynthesis.8 We therefore hypothesized that the biosynthesis of the β-OH-Phe unit in atratumycin is possibly governed by the unique cytochrome P450 monooxygenase, Atr27, which is embedded within the atr gene cluster. To investigate the function of Atr27 in atratumycin assembly line, atr27 was inactivated via aforementioned strategy (Figure S12). Fermentation and metabolite analysis of the Δatr27 mutant strain, revealed these strains complete inability to produce 1, which indicated that Atr27 functioned as a pretailoring enzyme catalyzing the βhydroxylation of Phe (Figure 1vi). In order to further characterize the β-hydroxylation of Phe was prior to or during the NRPS assembly line, in vitro reconstitution of the activity of Atr27 (Figure S13A) was conducted by using the wellcharacterized redox system, seFdx/seFdR, from Synechococcus elongatus PCC7942 and spFdx/spFdR from spinach.14 The enzymatic reaction revealed that the desired product, (2S,3S)2-amino-3-hydroxy-3-phenylpropanoic acid, was not formed when L-Phe was used as a substrate in various buffers (Figure S13B,C). These data suggest Atr27 catalyzes L-Phe to β-OHPhe in a PCP-dependent manner during the skeleton structure biosynthesis of atratumycin (Scheme 1D), similar to other cytochrome P450 monooxygenases in skyllamycin and other secondary metabolites biosyntheses (Figure S14).15 The biological activities of peptidic agents often benefit from the incorporation of nonproteinogenic amino acids; NRPSs excel at achieving this within microbial natural product producers.16 Accordingly, this suggested to us that 1 might be capable of expressing potent and useful bioactivities. Consequently, a panel of Gram-positive and Gram-negative bacteria and two Mycobacteria including Mycobacterium tuberculosis H37Ra and H37Rv were used to evaluate the antibacterial activities of atratumycin (1). Atratumycin (1) was found to exert specific activities against M. tuberculosis H37Ra and H37Rv demonstrating MICs of 3.8 and 14.6 μM, respectively (Table S5). The ability of 1 to exert cytotoxic activities was also examined. A panel of six cancerous and two normal/healthy human cell lines were subjected to 1 under standard cytotoxicity assay conditions (Table S6). We found that 1 failed to display any notable toxicities against any of the cell lines tested. In summary, activation of the atratumycin biosynthetic gene cluster was achieved using a combination of ARTP mutagenesis technology and media optimization method with a genetically engineered strain S. atratus SCSIO ZH16NS-80S. As a result, a cyclodepsipeptide with a unique cinnamoyl chain, atratumycin (1), was discovered and isolated. The BGC for 1 was identified and a biosynthetic logic affording 1 is proposed. Bioassays revealed that atratumycin (1) exerts potent antituberculosis activity and appears to lack any significant toxicity against human cells in culture. Thus, we envision that atratumycin (1), the product of a normally “silent” or “orphan” gene cluster, may represent an excellent natural product drug lead in the fight against tuberculosis.

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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.orglett.9b00208. Experimental details, bioactivities, MS and NMR data, and 1D and 2D NMR spectra (PDF) Accession Codes

CCDC 1883521 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jianhua Ju: 0000-0001-7712-8027 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the China NSF (31870046, 41706169, 81425022, U1501223, and U1706206); Guangdong NSF (2016A030312014 and 2018A0303130005), the CAS (YJKYYQ20170036), and the Special Support Program for Training High-Level Talents in Guangdong (201528018). T.Z. received support from Science and Technology Innovation Leader of Guangdong Province (2016TX03R095). We thank the analytical facility center of the SCSIO for recording spectroscopic data.

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DOI: 10.1021/acs.orglett.9b00208 Org. Lett. XXXX, XXX, XXX−XXX