Identification of the Biosynthetic Gene Cluster for the Anti-infective

Mar 6, 2015 - Deep Sea Actinomycetes and Their Secondary Metabolites ... Genome mining reveals the biosynthetic potential of the marine-derived strain...
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Identification of the Biosynthetic Gene Cluster for the Anti-infective Desotamides and Production of a New Analogue in a Heterologous Host Qinglian Li, Yongxiang Song, Xiangjing Qin, Xing Zhang, Aijun Sun, and Jianhua Ju* 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, 164 West Xingang Road, Guangzhou 510301, People’s Republic of China S Supporting Information *

ABSTRACT: The desotamides (DSAs) are potent antibacterial cyclohexapeptides produced by Streptomyces scopuliridis SCSIO ZJ46. We have identified the 39-kb dsa biosynthetic gene cluster by whole-genome scanning. Composed of 17 open reading frames, the cluster codes for four nonribosomal peptide synthetases and associated resistance, transport, regulatory, and precursor biosynthesis proteins. Heterologous expression of the dsa gene cluster in S. coelicolor M1152 afforded desotamides A and B and the new desotamide G. Cluster identification and its demonstrated amenability to heterologous expression provide the foundation for future mechanistic studies as well as the generation of new and potentially clinically significant DSA analogues.

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combination of 454 and Hiseq2000 technologies, and the acquired sequence reads were then assembled into five contigs covering 8.1 Mb. Bioinformatics analysis of the S. scopuliridis SCSIO ZJ46 genome data using online antiSMASH software4 revealed the presence of three putative NRPS gene clusters. Among these, an NRPS gene cluster, designated the dsa cluster, containing 17 open reading frames (ORFs) and spanning 39 kb of contiguous genomic DNA was found to possess three consecutive NRPS genes (dsaGHI) proposed to constitute the putative desotamide gene cluster. The three NRPS genes encode a total of six modules in accordance with expectation based on the peptidic scaffold of the desotamides. Additionally, genes associated with resistance, transport, regulation, and precursor (L-allo-Ile) biosynthesis were found both upstream and downstream of dsaGHI (Table S1). The genetic organization of the putative desotamide gene cluster is depicted in Figure 1A, and the nucleotide sequence of the gene cluster has been deposited in GenBank under accession number KP769807. The desotamide peptide backbone is composed of six amino acids: L-Trp or L-N-formyl-kynurenine (NFK), L-Leu, D-Leu, LVal or L-allo-Ile, L-Asn, and L-Gly. Consistent with this structural composition, three NRPS genes (dsaI, dsaH, and dsaG), encoding a total of six modules, are apparent within the cluster. The ORF dsaI encodes two modules composed of six domains (A1-PCP1-C1-A2-PCP2-C2), and dasH codes for two

he cyclohexapeptide desotamide A (1) and desotamides B−D (2−4) have recently been isolated from the deepsea-derived Streptomyces scopuliridis SCSIO ZJ46.1 Importantly, desotamides A and B show notable antibacterial activities against pathogenic Gram-positive strains of Streptococcus pneumoniae NCTC 7466, Staphylococcus aureus ATCC 29213, and methicillin-resistant clinical isolate Staphylococcus epidermidis (MRSE) shhs-E1.1 Structure−activity relationship (SAR) studies have revealed the Trp moiety to be crucial to this antibacterial scaffold.1 Desotamide A (1) was originally isolated from fermentation cultures of a soil-derived Streptomyces in 1997, although, at the time, no biological activity was reported.2a Another two analogues, desotamides E and F, were recently reported from a soil Streptomyces nov. sp. (MST115088) exhibiting growth inhibitory activity against Grampositive bacteria.2b Furthermore, the biosynthetic pathway for the desotamides has, to date, eluded characterization. Herein, we report the identification and elucidation of the gene cluster (dsa) governing desotamide biosynthesis; heterologous expression of the dsa gene cluster in Streptomyces lividans TK64 and Streptomyces coelicolor M1152 has also been achieved, enabling access to a new congener, desotamide G (5). The desotamides contain nonproteinogenic amino acids DLeu and L-allo-Ile, inspiring the hypothesis that modular nonribosomal peptide synthetases (NRPSs) generate the peptidic backbone of these natural products.3 On this basis we sought to identify the gene cluster by application of whole genome scanning and annotation methods. A sequence scan of the S. scopuliridis SCSIO ZJ46 genome was carried out using a © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 6, 2015

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DOI: 10.1021/acs.jnatprod.5b00009 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. (A) Genetic organization of the desotamide (dsa) gene cluster; cosmids harboring the entire candidate gene cluster are indicated by solid lines. (B) Proposed biosynthetic pathway for the desotamides.

standard PCR-targeting methods6 in the natural S. scopuliridis SCSIO ZJ46 producer. These efforts, not surprisingly on the basis of earlier studies, revealed S. scopuliridis SCSIO ZJ46 to be recalcitrant to genetic engineering; NRPS knockout experiments failed. We therefore sought to express the dsa cluster in a more genetically tractable heterologous host. A genomic cosmid library of S. scopuliridis SCSIO ZJ46 was constructed using the SuperCos 1 vector, and ∼3000 clones were picked and placed into 96-well plates. Primers for dsaH, which is centrally located within the cluster, were used to screen the S. scopuliridis SCSIO ZJ46 cosmid library using PCR methods, and a panel of 10 positive cosmids was identified. To select the cosmid harboring all of the biosynthetic genes, another two primer pairs targeting dsaA and orf(+1), genes constituting the putative left and right borders of the gene cluster, were used to further screen the 10 dsaH-positive cosmids. Cosmids 07-6H and 07-9A tested positive for all three PCR probes and were selected for end-sequencing. Sequencing results and bioinformatics analyses revealed that cosmid 07-6H contained 17 ORFs spanning from dsaA to dsaQ, whereas cosmid 07-9A contained 21 ORFs spanning from orf(−3) to orf(+1) (Figure

modules with six domains (A3-PCP3-E-C3-A4-PCP4). The corresponding enzymes DsaI and DsaH are proposed to activate L-Trp, L-Leu, D-Leu, and L-Val during desotamide assembly because the presence of the DsaH E domain is fully consistent with the presence of a D-Leu residue, although the substrate-specific sequence (or binding pockets)5 of A1, A2, and A3 (Table S2) does not match the predicted substrates. The E domain is also proposed to epimerize the L-Leu of the growing Dsa-PCP3-tethered desotamide precursor. Furthermore, the substrate-specific sequence DALWMGGV of the DsaH-A4 domain is fully consistent with the L-Val activation step necessary for desotamide B (2) construction. DsaG encodes two modules with seven domains (C4-A5-PCP5-C5-A6-PCP6TE); the substrate-specific sequences DLTKVGEV and DILQVGLI are fully consistent with established activation domains for L-Asn and Gly, respectively (Figure 1B). Thus, the overall domain organization of NRPS proteins within the dsa cluster suggests that this cluster likely drives the biosynthesis of 1. To confirm involvement of the candidate dsa gene cluster in desotamide biosynthesis, we first sought to inactivate dsaI using B

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then performed the same fermentations in the presence of 3% crude sea salt, and the same analysis is implied. S. lividans TK64/07-6H and S. lividans TK64/07-9A still produced only trace quantities of 1. However, fermentations of S. coelicolor M1152/07-6H and S. coelicolor M1152/07-9A both afforded significantly higher amounts of 1; 2 was also observed, although neither 3 nor 4 was produced (Figure 2). Moreover, a previously unrecognized new metabolite, 5, having a slightly longer retention time than 1 and exhibiting UV absorption signals characteristic of 1 and 2 at 275 nm, was observed (Figure 2). HRESIMS analyses revealed that 5 has a molecular formula of C35H52N7O8, only one mass unit greater than that of 1. We named this new compound desotamide G (5). To obtain quantities of 5 sufficient for NMR characterization studies, a large-scale (7 L) fermentation of S. coelicolor M1152/07-6H was carried out. Following extraction and purification, a full set of 1D (1H and 13C) and 2D (COSY, HSQC, and HMBC) NMR spectra of 5 was acquired, enabling full assignment of all

1A), suggesting that both cosmids harbor the entire candidate gene cluster. To confirm that the genes contained within cosmids 07-6H and 07-9A code for desotamide construction, their heterologous expression in S. lividans TK64 and a genome-minimized S. coelicolor strain M11527 was investigated. SuperCos 1-based cosmids 07-6H and 07-9A were first modified by replacing each kanamycin resistance gene within the SuperCos 1 vector with a pSET152AB-derived fragment; the pSET152AB vector constructed in our laboratory8 using RED recombination approaches contains an apramycin resistance gene and elements for conjugation and site-specific recombination (oriT, integrase gene, and φC-31 site). Second, the resulting cosmids, designated 07-6H-AB and 07-9A-AB, were transferred into S. lividans TK64 and S. coelicolor M1152, respectively, by conjugation and stable integration into their chromosomes via attB/attp-site-specific recombination, to yield S. coelicolor M1152/07-6H, S. coelicolor M1152/07-9A, S. lividans TK64/076H, and S. lividans TK64/07-9A strains, respectively. The engineered strains were then fermented using the same medium previously used for wild-type S. scopuliridis SCSIO ZJ46 fermentations omitting the addition of 3% crude sea salt. Extracts of the resulting fermentations were then prepared and analyzed by HPLC-UV and HPLC-MS. All four strains produced only trace quantities of 1 (Figure 2). On the basis of the marine nature of the native Streptomycete producer, we

Figure 3. HMBC correlations identified for desotamide G (5). 1

H and 13C signals (Table S3 and Figure 3). Importantly, the H and 13C NMR spectroscopic data of 5 and 1 are extremely similar. However, analysis of the HRMS2 pattern of 5 reveals that, although 5 is structurally similar to 1, it contains an Asp residue, whereas 1 contains an Asn at the same position within the peptide backbone. The application of Marfey’s analysis (Figure S3) and chiral-phase HPLC (Figure S4) of the acid hydrolysates of 5 and 1 afforded the same HPLC profiles, demonstrating that 5 and 1 share the same absolute configurations. Unlike compound 1, which displays significant antibacterial activities against Streptococcus pnuemoniae, Staphylococcus aureus ATCC 29213, and MRSE shhs-E1, compound 5 showed no notable antibacterial activities against these pathogens (MIC > 113 μg/mL). The Asn residue of 1 is, on the basis of these findings, vital to desotamide antibacterial activity against the bacteria evaluated here. To examine whether compound 5 was generated through biotransformation from compound 1 by S. coelicolor M1152, an incubation experiment with the addition of compound 1 to an S. coelicolor M1152 culture was performed. HPLC analysis (Figure S5) of the biotransformation products revealed that approximate 50% of the exogenously added 1 could be converted into 5 during the S. coelicolor M1152 fermentation. This result indicates that desotamide G (5) is a product of asparagine hydrolysis and S. coelicolor M1152 may produce an amidase to detoxify desotamide A (1) under the heterologous expression conditions. The heterologous expression of cosmids 07-6H and 07-9A revealed that these cosmids do indeed harbor all of the genes 1

Figure 2. HPLC analyses of the fermentation extracts. (I) S. scopuliridis SCSIO ZJ46 with supplement of 3% crude sea salt; (II and III) negative controls of the host strain S. lividans TK64 without or with supplement of 3% crude sea salt, respectively; (IV and V) S. lividans TK64/07-6H without or with supplement of 3% crude sea salt, respectively; (VI and VII) S. lividans TK64/07-9A without or with supplement of 3% crude sea salt, respectively; (VIII and IX) negative controls of the host strain S. coelicolior M1152 without or with supplement of 3% crude sea salt, respectively ; (X and XI) S. coelicolior M1152/07-6H without or with supplement of 3% crude sea salt, respectively; (XII and XIII) S. coelicolior M1152/07-9A without or with supplement of 3% crude sea salt, respectively. C

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BLAST program (http://blast.ncbi.nlm.nih.gov/). The NRPS domains were determined using an NRPS-PKS online Web site (http://nrps. igs.umaryland.edu/nrps/).12 Genomic Library Construction and Screening. The S. scopuliridis SCSIO ZJ46 genomic cosmid library was constructed using SuperCos 1 according to the manufacturer’s protocol included with the vector kit. About 3000 clones were picked and placed into 96well plates and stored at −80 °C. Three pairs of primers associated with the orfs dsaH, dsaA, and orf(+1) (Table S4) were designed and used to screen the genomic cosmid library using PCR methods. Construction of the Desotamide Gene Cluster Heterologous Expression Strains. To heterologously express the dsa cluster, cosmids 07-6H and 07-9A harboring the desotamide gene cluster were modified following our established method.8 Briefly, the aac(3)IVoriT-intφC31 cassette was first excised from plasmid pSET152AB8 by BamHI/EcoRI digestion and then used to replace the kanamycin resistance gene on the vector backbone of SuperCos 1-based cosmids 07-6H and 07-9A via Red/ET-mediated recombination in E. coli BW25113/pIJ790. Recombinant cosmids 07-6H-AB and 07-9A-AB were introduced into E. coli ET12567/pUZ8002 and then transferred into S. lividans TK64 and S. coelicolor M1152 by conjugation to generate the heterologous expression strains S. coelicolor M1152/076H, S. coelicolor M1152/07-9A, S. lividans TK64/07-6H, and S. lividans TK64/07-9A. Metabolite Analyses of Wild-Type and Recombinant Heterologous dsa Expression Strains. The fermentation of wildtype S. scopuliridis SCSIO ZJ46 was carried out following the reported precedure1 using modified Am2ab medium supplemented with 3.0% crude sea salt (Guangdong Salt Industry Group Corporation, Guangzhou, China). For heterologous expression strains, cells were first grown on mannitol soya medium11 agar at 28 °C for 5 to 7 days to achieve sporulation and then used to inoculate 250 mL flasks containing 50 mL of modified Am2ab medium with or without supplemental 3.0% crude sea salt. Fermentations were then carried out at 28 °C with an agitation speed of 200 rpm for 8 days. Following fermentations, each culture broth was extracted with butanone (1 × 100 mL), and solvents were then removed under reduced pressure. The extracts were dissolved into 500 μL of MeOH and centrifuged at 13000g for 10 min; the supernatant was subjected to HPLC analysis, which was performed on an Agilent Technologies 1260 Infinity system using a Phenomenex ODS column (150 × 4.6 mm, 5 μm) with a linear gradient of 0% to 80% solvent B (solvent B: 0.1% HOAc−85% CH3CN in H2O; solvent A: 0.1% HOAc−15% CH3CN in H2O) over 25 min at a flow rate of 1 mL/min and with UV detection at 275 nm. Large-Scale Fermentation and Isolation of Desotamide G (5). To isolate the new desotamide analogue, large-scale fermentation of the recombinant strain S. coelicolor M1152/07-6H was carried out using a two-stage fermentation process. Briefly, a spore suspension (50 μL) of S. coelicolor M1152/07-6H was inoculated into a 250 mL flask containing 50 mL of the modified Am2ab medium with supplemental 3.0% crude sea salt. After incubation for 30 h at 28 °C on a rotary shaker at 200 rpm, each of the seed cultures was trans-inoculated into 1 L flasks containing 200 mL of modified Am2ab medium with supplemental 3.0% crude sea salt and grown at 28 °C for 9 days at 200 rpm. Subsequently, the fermentation cultures (7 L) were centrifuged (3800g, 10 min) to yield the supernatant and mycelium. The supernatant was extracted with equal volume of butanone three times; the extract solutions were combined, and the solvent was removed under reduced pressure to give extract A. Similarly, the mycelium was repeatedly extracted with a total volume of 1 L of acetone three times to give extract B. Extracts A and B were combined after HPLC analysis and subjected to silica gel CC using gradient elution with a CHCl3−MeOH mixture (150:0, 147:3, 144:6, 141:9, 138:12, 135:15, 120:30, and 75:75), supplemented with 0.1% HOAC, to give eight fractions (Afr.1−Afr.8). Afr.3−Afr.8 were combined and purified by MPLC with an ODS column, eluted with a linear gradient from 10% to 40% solvent B (A: H2O−HOAC, 100:0.1, B: CH3CN− HOAC, 100:0.1) over the course of 20 min and from 40% to 80% solvent B over the course of 50 min to give the refined fraction Bfr.5 containing 5. Fraction Bfr.5 was further purified by preparative HPLC

required for biosynthesis of 1 and 2. Post assembly line oxidation of the Trp C-2′/C-3′ olefin in desotamide A by a tryptophan 2,3-dioxygenase may generate 3. Deformylation of the N-formyl-kynurenine residue in 3 may then give rise to 4. However, bioinformatics analysis of all the biosynthetic genes and another 10 ORFs upstream or downstream of the gene cluster failed to reveal any solid candidates for the oxidase associated with Trp C-2′/C-3′ olefin modification. The absence of a clear oxidase candidate suggests that this oxidation may be catalyzed by a nonspecific tryptophan 2,3-dioxygenase or a counterpart coded for elsewhere in the S. scopuliridis SCSIO ZJ46 genome. Tryptophan 2,3-dioxygenase has been reported to be widespread in Streptomyces.9 This hypothesis is further supported by the absence of 3 and 4 in the extracts of the recombinant heterologous expression strains. In conclusion, we have identified and characterized the dsa gene cluster by genome sequencing, bioinformatics analysis, and heterologous expression methods, laying a foundation for future mechanistic studies of desotamide biosynthesis. In addition, heterologous expression of the dsa gene cluster in S. coelicolor M1152 enabled the unexpected production of a new desotamide analogue for SAR studies. This heterologous expression system for products of the dsa cluster provides a new strategy for desotamide analogue production through the application of combinatorial biosynthetic methods.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation was obtained with an MCP-500 polarimeter (Anton Paar). The UV spectrum was recorded with a UV-2600 spectrometer (Shimadzu). The IR spectrum was measured using an IRAffinity-1 spectrophotometer (Shimadzu). NMR spectra were acquired with an Avance 500 spectrometer (Bruker) at 500 MHz for the 1H nucleus and 125 MHz for the 13C nucleus. Coupling constants (J) were given in Hz. Highresolution mass data were determined using a Maxis quadrupole-timeof-flight mass spectrometer (Bruker). Column chromatography (CC) was performed using silica gel (100−200 mesh, Yantai Jiangyou Silica Gel Development Co., Ltd.). Reversed-phase medium-pressure preparative liquid chromatography (RP-MPLC) was carried out using a Cheetah MP200 (Agela Technologies) column filled with ODS (40−63 μm, YMC). RP-HPLC was performed using an LC3000 solvent delivery module equipped with a Smartline UV detector 2550 (Knauer) and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Bacterial Strains and Plasmids. The desotamide producer S. scopuliridis SCSIO ZJ46 has been previously described.1 S. coeliecolor M1152 and S. lividans TK64 was used as the host strains for heterologous expression of the dsa gene cluster. Escherichia coli DH5α was used as the host for cloning purposes.10 E. coli XL 1-Blue MR (Stratagene) was used as the host strain for construction of the S. scopuliridis SCSIO ZJ46 genomic cosmid library. E. coli ET12567/ pUZ8002 was used to transfer DNA into the Streptomyces from E. coli by conjugation. E. coli BW25113/pIJ790 was used as the host for Red/ ET-mediated recombination6 during construction of the heterologous expression plasmids 07-6H-AB and 07-9A-AB. All E. coli strains were grown in Luria−Bertani medium at 37 or 30 °C. SuperCos1 (Stratagene) was used for the construction of the S. scopuliridis SCSIO ZJ46 genomic cosmid library. Whole Genome Scanning and Bioinformatics Analysis. The S. scopuliridis SCSIO ZJ46 genomic DNA used for scanning was isolated according to a slightly modified protocol.11 Whole genome scanning was accomplished using a combination of 454 and Hiseq2000 sequencers at Shenzhen BGI Diagnosis Technology Co., Ltd. Secondary metabolite biosynthetic gene clusters were detected and analyzed using online antiSMASH software (http://antismash. secondarymetabolites.org/).4 ORFs were analyzed using online FramePlot 4.0beta software (http://nocardia.nih.go.jp/fp4/), and their functional predictions were accomplished with an online D

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(6) Gust, B.; Challis, G. L.; Fowler, K.; Kieser, T.; Chater, K. F. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1541−1546. (7) Gomez-Escribano, J. P.; Bibb, M. J. Microb. Biotechnol. 2011, 4, 207−215. (8) Zhang, Y.; Huang, H.; Chen, Q.; Luo, M.; Sun, A.; Song, Y.; Ma, J.; Ju, J. Org. Lett. 2013, 15, 3254−3257. (9) Kurnasov, O.; Jablonski, L.; Polanuyer, B.; Dorrestein, P.; Begley, T.; Osterman, A. FEMS Microbiol. Lett. 2003, 227, 219−227. (10) Sambrook, J. E.; Russell, D. W. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989. (11) Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hopwood, D. A. Practical Streptomyces Genetics; John Innes Foundation: Norwich, UK, 2000. (12) Bachmann, B. O.; Ravel, J. Methods Enzymol. 2009, 458, 181− 217. (13) CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, Approved Standard-8th ed.; Clinical and Laboratory Standards Institute: Villanova, PA, 2009; M07-A8.

with an ODS column, eluted with a linear gradient from 45% to 80% solvent B (A: H2O−HAc, 100:0.1; B: CH3CN−HAc, 100:0.1) over 23 min at a flow rate of 10.0 mL/min (using detection at 275 nm), to afford desotamides A (54.1 mg) and G (68.2 mg) with retention times of 20.4 and 21.9 min, respectively. Desotamide G (5): white, amorphous powder; [α]25D −31.1 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 220 (4.40), 281 (3.66) nm; IR (ATR) νmax 3260, 1630, 1539 cm−1; 1H and 13C NMR spectroscopic data, Supporting Information Table S3; HRESIMS m/z 698.3848 [M + H]+ (calcd for C35H52N7O8, 698.3872); m/z 720.3678 [M + Na]+ (calcd for C35H51N7NaO8, 720.3691). Biotransformation of Desotamide A (1) to Desotamide G (5) Using S. coelicolor M1152. A spore suspension of S. coelicolor M1152 was inoculated into 50 mL of modified Am2ab medium with supplemental 3.0% crude sea salt in a 250 mL flask. After incubation at 28 °C for 30 h at 200 rpm, desotamide A (3 mg) dissolved in 20 μL of dimethyl sulfoxide (DMSO) was added to the culture. The culture was grown at 28 °C for 7 days at 200 rpm. Negative controls without supplemental desotamide A or strain S. coelicolor M1152 were set. Subsequent extraction and HPLC analysis (Figure S5) were conducted using the same methods as those for the wild-type strain. Antibacterial Activities Assay. Antibacterial activities of compounds 1 and 5 were assessed using sequential 2-fold serial dilutions in MH broth according to previously reported methods provided by the Clinical and Laboratory Standards Institute (CLSI).1,13 Vancomycin was used as an antibacterial agent control.

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ASSOCIATED CONTENT

S Supporting Information *

Spectra of ESIMS/MS, 1D and 2D NMR, Marfey’s method analyses, and chiral-phase HPLC analysis for compound 5 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-20-89023028. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported, in part, by the National Natural Science Foundation of China for Young Scientists (31400072, 41206135), the National High Technology Research and Development Program of China (2012AA092104), the Programs of Chinese Academy of Sciences (XDA11030403, KGZD-EW-606), and a special financial fund for innovative developments of the Marine Economic Demonstration Project (GD2012-D01-001). Additionally, we thank the analytical facility center (Ms. Xiao and Mr. Li) of the South China Sea Institute of Oceanology for recording NMR data.

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REFERENCES

(1) Song, Y.; Li, Q.; Liu, X.; Chen, Y.; Zhang, Y.; Sun, A.; Zhang, W.; Zhang, J.; Ju, J. J. Nat. Prod. 2014, 77, 1937−1941. (2) (a) Miao, S.; Anstee, M. R.; LaMarco, K.; Matthew, J.; Huang, L. H. T.; Brasseur, M. M. J. Nat. Prod. 1997, 60, 858−861. (b) Khalil, Z. G.; Salim, A. A.; Lacey, E.; Blumenthal, A.; Capon, R. J. Org. Lett. 2014, 16, 5120−5123. (3) Mootz, H. D.; Schwarzer, D.; Marahiel, M. A. ChemBioChem 2002, 3, 490−504. (4) Medema, M. H.; Blin, K.; Cimermancic, P.; de Jager, V.; Zakrzewski, P.; Fischbach, M. A.; Weber, T.; Takano, E.; Breitling, R. Nucleic Acids Res. 2011, 39, 339−346. (5) Challis, G. L.; Ravel, J.; Townsend, C. A. Chem. Biol. 2000, 7, 211−224. E

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