Discovery, Biosynthesis, and Heterologous Production of

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Discovery, Biosynthesis, and Heterologous Production of Streptoseomycin, an Anti-Microaerophilic Bacteria Macrodilactone Bo Zhang,†,∥ Kai Biao Wang,†,∥ Wen Wang,† Shu Feng Bi,† Ya Ning Mei,§ Xin Zhao Deng,† Rui Hua Jiao,† Ren Xiang Tan,*,†,‡ and Hui Ming Ge*,† †

State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, School of Life Sciences, Nanjing University, Nanjing 210023, China ‡ State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210023, China § Department of Clinical Laboratory, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China S Supporting Information *

ABSTRACT: Streptoseomycin (1), which is a rare macrodilactone with potent activities against microaerophilic bacteria, featuring a pentacyclic 5/14/10/6/6 ring system together with an ether bridge, was characterized by a combination of spectroscopic method and Xray analysis from a marine Streptomyces seoulensis. Sequencing and characterization of a ∼76-kb biosynthetic gene cluster led to the proposition of the biosynthetic pathway of 1. Heterologous expression of the gene cluster using a BAC vector in Streptomyces chartreusis 1018 led to the successful production of 1.

H

[C31H37NO11Na]+ 622.2259). 13C NMR (Table S1 in the Supporting Information) and HSQC spectral analyses confirmed the presence of 31 carbon signals, including 8 quaternary, 14 methine, 7 methylene, and 2 methyl carbons. The 1H−1H COSY spectrum displayed four spin systems, H-2 to H-12, H-15 to H-19 and H-21, H-30 to H-31, and H-24 to H-25 (Figure 1). The four distinctive spin systems were

elicobacter pylori is a Gram-negative microaerophilic pathogenic bacterium that infects over two-thirds of the human population in the world.1 And its colonization in the stomach has been addressed to play key roles in a series of secondary gastroduodenal diseases, such as superficial chronic gastritis, gastric ulceration, and even gastric cancer.2 Meanwhile, more and more drug-resistant H. pylori strains are being isolated from infected individuals, solidifying the challenge to the established treatment protocols such as triple therapy.3 Therefore, the discovery of novel antibiotics stands as an urgent need in the treatment of the H. pylori infection and its related diseases. Actinomycetes represent one of the most prolific sources for novel bioactive natural products.4 In order to explore the huge genetic potential of yet unexplored metabolites, we are focusing our efforts on actinomycetes lived in special niche, such as those living in the bodies of insects or marine environments.5 During this screening, one strain, Streptomyces seoulensis A01, which was isolated from a marine prawn obtained in the Yellow Sea in China, showed that its metabolic extract possessed promising bioactivity against H. pylori in broth dilution assay. The subsequent large-scale fermentation and bioactivity-guided fractionation led to the isolation of an unusual macrodilactone, streptoseomycin (1) with potent bioactivity against H. pylori and a panel of microaerophilic bacteria. Compound 1 was genetically linked to its biosynthetic gene cluster in S. seoulensis A01 through genome sequencing and bioinformatics analysis, as well as heterologous production in other Streptomyces hosts. Compound 1 was isolated as colorless needles. The molecular formula of 1 was determined to be C31H37NO11 by its HRESIMS data ([M + Na]+ at m/z 622.2256, calculated for © XXXX American Chemical Society

Figure 1. Key 2D correlations and X-ray crystal structure of 1.

connected through HMBC and 13C chemical shift analysis. HMBC correlations from H-3, H-5, H-7, and H-11 to C-13, and from H-6, H-12 to C-4 indicated the presence of a decalin subunit (rings D and E) (see Figure 1). In addition, an ether bridge between C-9 and C-13 was deduced on the basis of HMBC correlation of H-9 with C-13, as well as the typical oxygen-bearing chemical shifts for C-9 (δ 64.9) and C-13 (δ 76.6). HMBC correlations were observed from H-17 and H-3 to an ester carbonyl carbon C-1 (δC 173.6), from an olefinic Received: March 29, 2018

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DOI: 10.1021/acs.orglett.8b01006 Org. Lett. XXXX, XXX, XXX−XXX

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Figure S1 in the Supporting Information) previously isolated from Nocardia argentinensis ATCC 31306, Nocardioides sp. OMR-59, Actinoplanes coloradoensis AB 921J-26, and Saccharothrix xinjiangensis G60/1571, respectively.6 All of these compounds shared the similar tricyclic ring system (rings C, D, and E) with an unusual ether bridge as highlighted in red in compound 1 (Scheme 1). Feeding experiments of C-13 labeling

proton H-15 to C-13, revealing the presence of a 10-membered lactone ring (ring D) connected with ring B at C-4 and C-13. A methyl and methoxyl group were attached to C-14 and C-2, respectively, through HMBC correlations of H-20 to C-13, C14, and C-15, and H-22 to C-2, respectively. The HMBC correlations from H-19 to C-31, H-21, and H-25 to a carbonyl carbon C-23 (δC 172.9) as well as the typical chemical shifts for oxygenated carbons for C-19 (δC 71.5) and C-31 (δC 153.7) indicated C-19 and C-31, and C-21 and C-23 were connected through an ether and an ester bond linkage, respectively. The magnitude of the 3JH,H vicinal coupling constant (13.5 Hz) between H-30 and H-31 suggested an Egeometry for the C-30 and C-31 olefinic bond. The HMBC correlations from H-31 and H-25 to C-29, and H-30 and H-24 to C-26 established a 14-membered macrolactone ring (ring B). Thus, far, only two carbonyl carbons C-27 (δC 172.1) and C28 (δC 172.0) and one exchangeable proton (δH 10.77) were not assigned. In the HMBC spectrum, the exchangeable proton signal showed discernible correlations with C-26 and C-29, suggesting that this molecule might contain a maleimide group (ring A), completing the planar structure elucidation of 1. Compound 1 possessed ten chiral carbons, which were difficult to fully assign by NMR interpretation. Luckily, a high quality single crystal of 1 was obtained from MeOH−CH2Cl2 (2:1). The X-ray crystallography experiment measured in Cu Kα radiation at low temperature confirmed the elucidated planar structure and further determined the absolute configurations of the stereocenters in 1 to be 2S, 4R, 7R, 9R, 11R, 12S, 13S, 16S, 17S, 18R (Figure 1). Next, compound 1 was tested for antibacterial activities against eight bacteria. Notably, it is highly active against Helicobacter pylori, which is a leading pathogenic bacterium that infects the human stomach, with the MIC value of 2 μg mL−1, as well as a panel of microaerophilic bacteria, including Lactobacillus acidophilus, Bif idobacterium bifidum, Eubacterium brachy, Propionibacterium acnes with their MICs in the range of 4−8 μg mL−1 (see Table 1). In contrast, compound 1 only

Scheme 1. Structure of Streptoseomycin (1)

precursors have been conducted, and it is found that C1, C3, C5, C7, C9, and C11 can be labeled by feeding C-13 labeling sodium acetate and C13, C15, and C17 can be labeled by feeding C-13 labeling sodium propionate, indicating that this tricyclic ring follows the typical polyketide origin.7 Meanwhile, the feed of C-13 labeling sodium acetate and succinate confirmed that the five-carbon component of the maleic anhydride is directly derived from α-KG. However, the fourcarbon part was not labeled by C-13 labeling succinate raising the question of whether this part derived from TCA. Moreover, to date, no genetic and biochemical information has been reported for this class of compounds. To gain the first insight into the biosynthetic machinery of this class of compounds, the producing strain S. seoulensis A01 was fully sequenced, resulting in the assembly of one contiguous linear genome data. On the basis of the structure of 1 and AntiSMASH results,8 a candidate type-I PKS gene cluster spanning ∼76 kb (GenBank Accession No. MG891745) was identified, which includes 32 open reading frames (Figure 2A). The functions of gene products were analyzed by BLAST and are summarized in Table S2 in the Supporting Information. The cluster harbors three giant genes encoding three PKS megaenzymes, designated stmA, stmB, and stmC, in which all domains are functional, because they contained all essential catalytic residues (Figure S10 in the Supporting Information).9 From the canonical rule of type I PKS assembly line, we reasoned that biosynthesis starts from the incorporation of one methylmalonyl-CoA unit, followed by condensation of two methylmalonyl-CoA and six malonyl CoA units. This hypothesis is supported by bioinformatics analysis of substrate specificity of individual AT domains (Figure S10). After the appropriate modifications by KRs and DHs, the ACP bound full-length polyketide chain will be cyclized by TE domain to release a nascent macrolactone (2), whose scaffold agrees perfectly with the tricyclic ring in 1. One of the common structural features among this class of compounds (Figure S1) is the presence a decalin ring system, which is envisioned to be biosynthesized through an intramolecular Diels−Alder (IMDA) reaction. In accordance with this hypothesis, the newly generated PKS product (2) indeed has the diene and dienophile moieties essential for IMDA reaction as highlighted in red in 2 (Figure 2C). To date, only a few Diels−Alderases have been identified from the producing strains of lovastatin, spinosyn, pyrroindomycin, versipelostatin, and solanopyranone.10 Importantly, all the Diels−Alderases discovered so far showed extremely low sequence homology, as

Table 1. Antibacterial Activities of Compound 1 MIC (μg mL−1) strain

1

positive control

Helicobacter pylori Lactobacillus acidophilus Bifidobacterium bif idum Eubacterium brachy Propionibacterium acnes Staphylococcus aureus Micrococcus luteus Bacillus subtilis

2 4 4 8 8 32 32 64

1a 0.25b 0.25b 0.25b 0.5b 0.25c 0.5c 0.12c

a Gentamicin was used as the positive control. bOrnidazole was used as the positive control. cTetracycline was used as the positive control.

exhibited weak activities against three aerobic bacteria Staphylococcus aureus, Micrococcus luteus, and Bacillus subtilis with their MIC values of 32, 32, and 64 μg mL−1, respectively. It is noteworthy that no cytotoxicity was found in 1 toward African green monkey kidney VERO cells, rat pheochromocytoma PC12 cells, and human monocytic THP-1 cells at concentrations up to 20 μg mL−1. Compound 1 belongs to a small tricyclic−macrolactone family with only four members discovered to date, including nargenicin, nodusimicin, coloradocin, and branimycin (see B

DOI: 10.1021/acs.orglett.8b01006 Org. Lett. XXXX, XXX, XXX−XXX

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Figure 2. Biosynthesis of 1. (A) Gene cluster of 1 in S. seoulensis A01, (B) relative location of positive BACs, and (C) proposed biosynthetic pathway for 1.

exemplified by LovB, SpnF, PyrE3, VstJ, Sal5, etc.,10 making it difficult to predict and discover a novel Diels−Alderase. Following IMDA cyclization, the tricyclic compound (3) will be further oxidized by some of P450 monooxygenases, StmP1P4, or redox enzymes, StmO1-O4, to introduce four hydroxyl groups at C-2, C-18, C-19, and C-21. Presumably similar to PtmO5, which is a P450 monooxygenase catalyzing the 11S,16S-ether ring formation found in platecimycin,11 one of the P450s could play a role in the formation of the 9R,13Rether ring, which is another key structural feature among these tricyclic-type compounds (Figure S1 in the Supporting Information). Moreover, compound 1 possessed a unique maleimide moiety. To our knowledge, the biosynthesis for maleimide has not been reported. However, it could share a similar biosynthetic pathway to dialkylmaleic anhydride, as seven genes stmGHINQRS showed high homologies to the genes in dialkylmaleic anhydride pathway of tautomycetin and tautomycin (Figure S11 and Table S2 in the Supporting Information).12 Thus, a putative route is proposed through an intramolecular aldol condensation between α-ketoglutarate and an unknown four-carbon unit. The subsequent CoA transfer and dehydration steps could be catalyzed by CoA transferase StmH and dehydratase StmS, respectively. The final step involves coupling between tricyclic ring and maleimide part, presumably through the actions of one P450 to generate the 19,31-ether bond linkage as well as an esterase StmI,

showing 49% identity to TtnK in tautomycetin biosynthesis, for forming the 21,23-ester bond. Taken together, a plausible biosynthetic pathway for 1 was deduced, as shown in Figure 2C. To verify the involvement of stm gene cluster in 1 biosynthesis, we intended to use bacterial artificial chromosomes (BACs), which has the ability to harbor up to a 600 Kb DNA fragment,13 to clone the stm gene cluster for heterologous expression. pMSBBAC2, which is a BAC vector designed for heterologous expression of large gene cluster in Streptomyces host, was chosen for this work. pMSBBAC2 features a set of essential elements consisting of the origin of replication (ori), which allows maintenance of the BAC DNA in E. coli, the φC31 integrase gene (int) and its attachment site (attP) for integration of DNA fragment into the chromosome of heterologous Streptomyces species host, origin of DNA transfer (oriT) for efficient conjugation from E. coli to Streptomyces, and aac3(IV) gene for resistant selection in both E. coli and Streptomyces. Based on the established protocol,13 a BAC library of S. seoulensis A01 strain was constructed. The average insert length was ∼130 kb, as determined by pulsed-field gel electrophoresis (PFGE) analysis with HindIII digestion from 30 randomly selected clones (Figure S12 in the Supporting Information). The BAC library was screened using three pairs of primers, which are considered to be located upstream, midstream, and downstream of the stm gene cluster, respectively. One positive C

DOI: 10.1021/acs.orglett.8b01006 Org. Lett. XXXX, XXX, XXX−XXX

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cytotoxic activities, which could be a good starting point for antibiotic development. The stm gene cluster reported here represents the first genetic evidence for biosynthesis of the tricyclic−macrolactone class of compounds (Figure S1). Future identification of other gene clusters from this class and comparative biosynthetic study will enable us to reveal the nature’s inventory for creating these similar, but distinct structural features. Moreover, the producing strains for other tricyclic compounds in this family all belong to rare actinobacteria,6 whose genetics are notoriously difficult to be manipulated. The successful expression of the stm gene cluster in heterologous host has set the stage for future biosynthetic study, as well as to utilize combinatorial biosynthetic strategy for the production of new derivatives.

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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.8b01006. Experimental procedures and NMR spectroscopic data for 1 (PDF) Accession Codes

CCDC 1821748 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 data_ [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: +44 1223 336033.

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Figure 3. Expression of stm gene cluster using BAC vector in heterologous host. (A) Physical map of pHG0101 harboring the intact stm gene cluster. (B) Pulsed-field gel electrophoresis of pHG0101 BAC. [Legend: M1, λ-Hind III digest; 1, BAC pHG0101; 2, BAC pHG0101 digested by NdeI; 3, BAC pHG0101 digested by EcoRV; 4, BAC pHG0101 digested by XhoI; and M2, Lambda PFG Ladder.] (C) LC-MS analysis of 1 produced by nascent and heterologous hosts. [Legend: (i) S. seoulensis A01 wild-type, (ii) S. charteius 1018/ pHG0101, (iii) S. charteius 1018/pHG0102, and (iv) S. charteius 1018/ pHG0103.]

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (R. X. Tan). *E-mail: [email protected] (H. M. Ge). ORCID

Ren Xiang Tan: 0000-0001-6532-6261 Hui Ming Ge: 0000-0002-0468-808X Author Contributions ∥

These authors contributed equally.

Notes

We thus introduced pHG0101 into four different Streptomyces hosts, including S. coelicor M1154, S. lividans SBT18, S. albus J1074, and S. charteius 1018 via conjugation. Fermentation of these four recombinant strains was thus carried out, utilizing the original production medium along with S. seoulensis A01 wild-type strain as the positive control. LC/MS analysis of the metabolic extracts showed S. charteius 1018/pHG0101 was able to produce 1 (Figure 3C, trace (ii), while, no products were observed in other three hosts. As negative controls, two BAC plasmids harboring partial stm gene cluster, pHG0102 (lacking stmE, D, Y, and G) and pHG0103 (lacking partial stmC, stmX and stmO4) were also introduced into S. charteius 1018. As expected, no 1 production was observed in either S. charteius 1018/pHG0102 or S. charteius 1018/pHG0103 (Figure 3C, traces (iii) and (iv)), confirming the necessity of these enzymes in the biosynthesis of 1. In summary, here, we have presented the isolation and characterization of a rare macrodilactone known as streptoseomycin (1) from a marine S. seoulensis A01. Compound 1 showed potent antimicroaerophilic bacteria but very weak

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China (Nos. 21572100, 81522042, 81773591, 81421091, 81500059, 81673333, 21672101, and 21661140001) and the China Postdoctoral Science Foundation.

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DOI: 10.1021/acs.orglett.8b01006 Org. Lett. XXXX, XXX, XXX−XXX