Characterization of the Biosynthetic Gene Cluster for the Antibiotic

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Characterization of the Biosynthetic Gene Cluster for the Antibiotic Armeniaspirols in Streptomyces armeniacus Yongjian Qiao,† Jiayan Yan,† Jia Jia,‡ Jiao Xue,† Xudong Qu,† Yunfeng Hu,§ Zixin Deng,† Hongkai Bi,*,‡ and Dongqing Zhu*,†

J. Nat. Prod. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 02/12/19. For personal use only.

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Key Laboratory of Combinatorial Biosynthesis and Drug Discovery Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan, Hubei Province 430071, People’s Republic of China ‡ Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu Province 210029, People’s Republic of China § CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, Guangdong Province 510301, People’s Republic of China S Supporting Information *

ABSTRACT: Armeniaspirols (1−3) are potent antibiotics against Gram-positive pathogens. Through a biosynthetic investigation, we identified four enzymes involved in the structural modification of 1−3. Manipulation of their activity led to the generation of 4−6 and nine novel analogues, 7−15. Bioactivity assessments revealed that the pyrrole chloro group and the methyl group are important for the antimicrobial activities of armeniaspirols, which lays the foundation for future structure optimization and mechanism of action studies of armeniaspirols.

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biosynthetic manipulations is an alternative reasonable method. With this latter aim, we herein performed a biosynthetic investigation of the armeniaspirols to explore their active chemical space. Through demethylation and dechlorination steps, a total of nine novel analogues (7−15) and 4−6 were obtained, resulting in the identification of the important role of the 9-chloro group and the methyl group on the pyrrole in the antimicrobial activities of armeniaspirols. S. armeniacus DSM 43125 was cultured in ISP-2 medium, and four secondary metabolites were identified from the fermentation broth (Figure S1 A1). Three peaks, 1−3, were characterized to be armeniaspirols A−C (1−3) based upon the LC-ESI-HRMS, UV, and 1H NMR analysis (Figure S11−S13, Table S5), consistent with previous reports.6

alogenated pyrroles (halopyrroles) are common chemical moieties found in bacterial secondary metabolites with remarkable biological activities, including pyoluteorin from Pseudomonas fluorescens,1 pyralomicin from Nonmuraea spiralis,2 and pyrrolomycin,3 marinopyrrole,4 and chlorizidine5 from Streptomyces. Armeniaspirols A−C (1−3), with a unique chlorinated spiro[4.4]non-8-ene scaffold, and compounds 4−6 were isolated from Streptomyces armeniacus DSM 19369 (Figure 1).6 Compounds 1−3 displayed high activities against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE).6,7 The complex structures of natural products present challenges for optimization through chemical approaches, but the generation of chemical derivatives through

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RESULTS AND DISCUSSION In order to obtain the biosynthetic gene cluster of armeniaspirols and novel chemical analogues through genetic methods, the whole genome of S. armeniacus DSM 43125 was sequenced and scanned. A 48-kb DNA region containing 27 ORFs designated the arm gene cluster was identified to be responsible for the biosynthesis of armeniaspirols (Figure 2A, Table S3, accession no. MF401191). Four conserved genes Figure 1. Structures of previously reported armeniaspirols (1−6) and the new congeners (7−15) produced in this study © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 3, 2018

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DOI: 10.1021/acs.jnatprod.8b00753 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. Products of S. armeniacus Wild-Type Strain, Gene Deletion Mutant Strains, and Complementation Strainsa name (deleted gene) A WT QY1 (Δarm3-8) QY8 (Δarm21) B QY6 (Δarm16) QY6::pWHU3069 C JY1 (Δarm5) QY3 (Δarm8) QY3::pWHU3080 QY4 (Δarm9) QY4::pWHU3067 QY14 (Δarm9Δarm16) D QY5 (Δarm15) QY5::pWHU3068 QY12 (Δarm8Δarm15) QY13 (Δarm9Δarm15)

new compound

1−3 + − − − + + − + − + − − + − −

involved in the formation of dichloropyrrolyl-S-PCP, including arm20 encoding proline carrier protein, arm4 encoding amino acid adenyltransferase, arm3 encoding acyl-CoA dehydrogenase, and arm21 encoding FADH2-dependent halogenase, were identified in the arm gene cluster (Figure 2B).8 The genes arm6, arm7, and arm18 were proposed to encode type I polyketide synthases. According to the protein sequence analysis and biosynthetic analysis (Table S4), Arm6 and Arm8, but not Arm18, were proposed to catalyze the conversions of dichloropyrrolyl-S-PCP to 4−6 (Figure 2B). An alternative possibility is that 4−6 are formed through a biomolecular condensation of two polyketide chains as described previously.6 Δarm3-8 mutant strain QY1 and Δarm21 mutant strain QY8 were generated through gene deletion (Figures S2, S3). Through HPLC and LC-ESI-HRMS analysis, neither QY1 nor QY8 produced any detectable armeniaspirols (Figure S1 A2, A3). These results together confirmed that the arm gene cluster was responsible for the biosynthesis of armeniaspirols. The final step of armeniaspirol biosynthesis may be the methylation of the pyrrole nitrogen. As the only methyltransferase in the gene cluster, Arm16 was proposed to be involved in the conversion of N-des-methyl armeniaspirols A− C to 1−3 (Figure 2B). To prove our hypothesis and obtain the novel N-des-methyl armeniaspirols, Δarm16 mutant strain QY6 was constructed (Figure S4). Based on HPLC and LCESI-HRMS analysis, QY6 accumulated three new compounds (7−9), but not 1−3 produced by the wild-type strain (Figure S1 B1). Complementation of QY6 with arm16 restored the production of 1−3 (Figure S1 B2). After isolation and

− − 7−9

4−6, 10−12, 13−15 4−6, 10−12, 13−15 4−6, 10−12 4−6, others 4−6 4−6

a

(A) Wild-type strain, large DNA fragment (arm gene cluster) deletion mutant, and conserved FADH2-dependent halogenase gene arm21 deletion mutant; (B) predicted N-methyltransferase gene arm16 deletion mutant and complementation strain; (C) predicted FADH2-dependent halogenase gene arm5, arm8, and arm9 deletion mutants and complementation strains; (D) predicted FAD-dependent monoxygenase gene arm15 deletion mutants and complementation strain.

Figure 2. Armeniaspirol biosynthetic gene cluster (A), proposed biosynthetic pathway of armeniaspirols (B), and antimicrobial activities (minimum inhibitory concentration, MIC) of compounds 1−15 (C). KS, ketosynthase domain; AT, acyltransferase domain; DH, dehydratase domain; KR, β-ketoreductase domain; ACP, acyl carrier protein domain; R, thioester reductase domain. B

DOI: 10.1021/acs.jnatprod.8b00753 J. Nat. Prod. XXXX, XXX, XXX−XXX

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chemical analysis using MS, UV, and NMR, 7−9 were characterized to be N-des-methyl armeniaspirols A−C (Figures S17−S19, Table S6). The methyltransferase Arm16 was further expressed and purified from E. coli BL21 (DE3) with a yield of 8.7 mg/L (Figure S9). The recombinant methyltransferase Arm16 was incubated with the substrates 7−9, respectively. The LC-MS analysis showed that 7−9 were consumed to form 1−3, respectively (Figure 3A, Figure S10A,C), which further confirmed that the Arm16 was a new methyltransferase responsible for the methylation of the pyrrole nitrogen.

Initially, our hypothesis was that Arm8 and Arm9 catalyzed the chlorination of 10−12 to form 7−9, followed by the methylation catalyzed by Arm16 to generate 1−3. However, the discovery of 13−15 suggested another possible pathway: Arm16 catalyzed the methylation of 10−12 to form 13−15, followed by the chlorination catalyzed by Arm8 and Arm9 to generate 1−3. The former pathway is preferred based on the following reasons: first, 10−12 were not accumulated in Δarm16 mutant strain QY6; second, 10−12 were not fully converted to 13−15 in Δarm8 mutant strain QY3 and Δarm9 mutant strain QY4, which suggests that 10−12 might not be the native substrates of the methyltransferase Arm16; finally, methyltransferases generally exhibited relatively low substrate specificity, compared with the high specificity for halogenases. To further confirm our proposal, the recombinant Arm16 was incubated with the mixture of 7 and 10, and samples were analyzed at different time by LC-MS, which showed that only trace amounts of 10 were consumed to form 13 in 16 h, while the conversion from 7 to 1 was almost quantitative in 30 min (Figure 3B). These results are supported by analysis of the incubations of the recombinant Arm16 with the mixture of 8 and 11 and the mixture of 9 and 12, respectively (Figure S10B,D). The above results together support our proposal of the final two biosynthetic steps: Arm8 and Arm9 perform the chlorination of 9-des-chloro-N-des-methyl armeniaspirols A− C (10−12) to form N-des-methyl armeniaspirols A−C (7−9) and then Arm16 methylates 7−9 to form the final products 1− 3. The other three compounds produced by QY3 and QY4 were proposed to be compounds 4−6 according to their exact masses measured by ESI-MS. To obtain compounds 4−6, the enzyme responsible for the cyclization of 4−6 needed to be identified. The only predicted FAD-dependent monoxygenase gene arm15 in the gene cluster seemed to be a good candidate. The gene arm15 was deleted in the wild-type strain to generate the Δarm15 mutant strain QY5 (Figure S8). QY5 was cultured, and the resulting culture extracted was analyzed by HPLC and LC-ESI-HRMS. QY5 did not produce compounds 1−3, but 4−6 and many other intermediates accumulated (Figure S1 D1). Complementation of QY5 with arm15 restored the production of 1−3 (Figure S1 D2). To increase the productions of 4−6 and avoid the interference of other compounds, arm15 was deleted in QY3 and QY4 to generate Δarm8Δarm15 mutant strain QY12 and Δarm9Δarm15 mutant strain QY13, respectively. As expected, all interferential peaks disappeared in the extracts of QY12 and QY13 and the production of the compounds 4−6 increased significantly (Figure S1 D3, D4). By scale-up fermentation of QY13, 4−6 were isolated and characterized (Figures S14−S16, Table S5). This result revealed that Arm15 is involved in the cyclization of 4−6 to form 10−12. In order to explore the active chemical space of armeniaspirols, the bioactivities of the nine new analogues (7−15) were analyzed. According to the MIC (minimal inhibition concentration) assessments (Figure 2C), N-desmethyl armeniaspirols A−C (7−9) and 9-des-chloro armeniaspirols A−C (13−15) displayed lower antibacterial activities compared with 1−3, revealing that the methyl group and 9-chloro group on the pyrrole are important for the antimicrobial activities. The conclusion is corroborated by the bioactivities of 9-des-chloro-N-des-methyl armeniaspirols A−C (10−12), which are only weakly active against Gram-positive

Figure 3. LC-MS analysis of incubations of recombinant Arm16 with 7 (A) and with the mixture of 7 and 10 (B). (a) Pure compound 7; (b) pure compound 1; (c) control reaction using heat-inactivated Arm16 with 7; (d) 2 h reaction using Arm16 with 7; (e) pure compound 13; (f) pure compound 1; (g) 0 h reaction using Arm16 with the mixture of 7 and 10 as control; (h) 0.5 h reaction using Arm16 with the mixture of 7 and 10; (i) 16 h reaction using Arm16 with the mixture of 7 and 10.

The formation of 7−9 from 4−6 is obscure and may include three steps: dechlorination, cyclization, and chlorination. We proposed that some of the three extra predicted FADH2dependent halogenases encoded by arm5, arm8, and arm9 are involved in these conversions. To prove this hypothesis and obtain novel des-chloro armeniaspirols, Δarm5 mutant strain JY1, Δarm8 mutant strain QY3, and Δarm9 mutant strain QY4 were constructed (Figures S5−S7). HPLC and LC-ESI-HRMS analysis showed that JY1 produced 1−3 (Figure S1 C1) and that both QY3 and QY4 abolished the production of 1−3 and accumulated the same intermediates containing nine new chemicals (Figure S1 C2, C4). Complementations of QY3 with arm8 and QY4 with arm9 restored production of 1−3 (Figure S1 C3, C5). From scale-up fermentation of QY4, six compounds were isolated and characterized. The ESI-MS, UV absorbance bands, and NMR spectra revealed that 10−12 are 9-des-chloro-N-des-methyl armeniaspirols A−C (Figures S20−S22, Table S7) and 13−15 are 9-des-chloro armeniaspirols A−C (Figures S23−S25, Table S8). We proposed that 13− 15 in QY3 and QY4 were biosynthesized by the methylation of 10−12 catalyzed by Arm16. To confirm the hypothesis, arm16 was deleted in Δarm9 mutant strain QY4 to generate Δarm9Δarm16 mutant strain QY14. As expected, 13−15 biosynthesized by QY3 and QY4 disappeared in the extract of QY14 (Figure S1 C6). C

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from nt 522 of arm3 to nt 728 of arm8 had been replaced by the 1465-bp fragment harboring aac(3)IV (Figure S2). As described above, arm21 mutant QY8 were also obtained (Figure S3). To avoid the polarity effect caused by aac(3)IV in mutant strains, the DNA fragment harboring aac(3)IV was not used to replace arm16. The internal 802-bp fragment of tsr from 4 nt to 806 nt on pJTU127818 was replaced by 1465-bp aac(3)IV (amplified from the DNA of pIB139 by using primer pair target-F and target-R) using a PCR targeting system to generate the vector pYJ49. A 2099-bp DNA fragment harboring partial arm13, arm14, arm15, and partial arm16 was amplified with primer pair YJ17F-2/YJ17R and digested with SacI and XbaI, while a 2155-bp DNA fragment harboring partial arm16, arm17, and partial arm18 was amplified with primer pair YJ18F/ YJ18R-1 and digested with XbaI and HindIII. The two DNA fragments were inserted into the SacI and HindIII sites of pYJ49 to generate plasmid pWHU3048. Plasmid pWHU3048 was conjugated into S. armeniacus DSM 43125. Apramycin (50 μg/mL) was used to select exconjugants. The single crossover strains were inoculated on SFM plates without any antibiotic under relaxed conditions in order to get double-crossover strains that had lost apramycin resistance. The primer pair YJ56F and YJ56R was used to select the arm16 mutant QY6, which gave the PCR product of 286 bp compared to 1038 bp with wild-type strain DSM 43125. The in-frame deletion mutant QY6 has a 6-bp XbaI scar that has replaced an internal 758 bp of arm16 from nt 37 to nt 794 (Figure S4). As described above, arm5 mutant JY1, arm8 mutant QY3, arm9 mutant QY4, and arm15 mutant QY5 were also obtained (Figures S5−S8). The construction of plasmids used to complement mutants of S. armeniacus is listed in Table S1, illustrated by the examples of the complementation of arm16 mutant QY6. The recombinant pIB139derived plasmid pWHU3069 harboring arm16 (Table S1) recovered from E. coli DH10B was transformed into E. coli ET12567/pUZ8002, and the unmethylated plasmid was conjugated into S. armeniacus Δarm16 mutant QY6, using apramycin to select the respective exconjugants. The complementation strain QY6::pWHU3069 was confirmed by PCR using the primer pair M13F and M13R to give the 1182-bp PCR product. As described above, the complementation strains QY3::pWHU3080, QY4::pWHU3067, and QY5::pWHU3068 were also obtained. Analysis of Products from S. armeniacus Strains. The cultivation of S. armeniacus strains and the analysis of the resulting products were as previously described.6 The S. armeniacus strains (the wild-type strain DSM 43125, mutant strains, and complementation strains) were cultured in the liquid ISP-2 medium (0.4% yeast extract, 1% malt extract, 0.4% glucose, pH 7.0) containing 2 g/L CaCO3 at 30 °C and 200 rpm for 3 days, respectively. Then 10% (v/v) of the culture was transferred in 50 mL of fresh ISP-2 medium and fermented at 30 °C and 200 rpm for 6 days. The culture was separated from the biomass fraction and the supernatant fraction by centrifugation. The supernatant fraction was extracted with an equal volume of ethyl acetate twice. The biomass fraction was extracted with methanol, disrupted by sonication, and centrifuged to remove the cell debris. The retained supernatant was concentrated on a rotovap, resuspended in water, and twice extracted with an equal volume of ethyl acetate. The combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated on a rotovap. The residue was resuspended in methanol and filtered with a microporous membrane (0.22 μm, nylon). HPLC (Shimadzu, SPD-M20A/LC-20AT) and LC-ESI-HRMS (Thermo Scientific LTQ Orbitrap XL, negative ion mode) were used to analyze each sample. The HPLC conditions (mobile phase A: water; mobile phase B: acetonitrile; UV detection λ: 300 nm) were as follows: elution gradient I (for Thermo Scientific C18 reversed-phase HPLC column, 250 × 4.6 mm, 5 μm, used in HPLC and LC-ESIHRMS): 5−95% B for 25 min, 95% B for 10 min, 95−5% B for 5 min, 5% B for 5 min at a flow rate of 1 mL/min; elution gradient II (for Thermo Scientific C18 reversed-phase HPLC column, 250 × 10 mm, 8 μm, used in HPLC): 20−85% B for 25 min, 85% B for 10 min, 85− 20% B for 3 min, 20% B for 7 min at a flow rate of 3 mL/min.

pathogens. Interestingly, compounds 4−6(compared with 1− 3) showed comparable activities against Staphylococcus aureus and displayed higher activities against Enterococcus faecium, Enterococcus faecalis, and Bacillus subtilis, suggesting that the spiro[4.4]non-8-ene scaffold may not be necessary for antimicrobial activity. Thus, further structure optimization based on 4−6 is promising. Compounds 1−15 did not display activities against Gram-negative bacteria, including Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumonia, Salmonella typhimurium, Shigella dysenteriae, and E. coli (Table S9).

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EXPERIMENTAL SECTION

General Experimental Procedures. The bacterial strains and plasmids used in this work are listed in Table S1. Primer sequences are listed in Table S2. Reagents and solvents purchased from SigmaAldrich were of the highest quality available and were used without further purification. Restriction enzymes, T4 DNA ligase, and DNA polymerase were purchased from New England Biolabs and used according to the manufacturer’s specifications. Ni-NTA affinity columns were purchased from GE Healthcare. Amicon Ultra centrifugal filter units were purchased from Millipore. DNA primers were synthesized by TsingKe Inc., Wuhan, China. Growth media and conditions used for E. coli and Streptomyces strains and standard methods for handling E. coli and Streptomyces in vivo and in vitro were as described previously,9 unless otherwise noted. All DNA manipulations were performed following standard procedures.10 DNA sequencing was carried out at TsingKe Co. Ltd. (Wuhan, China). Genome sequencing of DSM 43125 was performed by BGI Co. Ltd. (Wuhan, China) using the Illumina HiSeq 2000 System. ORFs of the secondary metabolite biosynthetic gene clusters w e r e i d e n t i fi ed u si n g an ti S M A SH (h t t p : / / a n t i s m a s h . secondarymetabolites.org),11,12 FramePlot (http://nocardia.nih.go. jp/fp4/),13 and secondFind (http://biosyn.nih.go.jp/2ndfind/). The NRPS-PKS online tools (http://www.nii.ac.in/~pksdb/sbspks/ master.html, and http://nrps.igs.umaryland.edu/) were used to analyze PKSs.14,15 All proteins were handled at 4 °C unless otherwise stated. Protein concentrations were determined according to the method of Bradford, using a PerkinElmer Lambda 25 UV/vis spectrophotometer with bovine serum albumin as the standard.16 Protein purity was estimated using SDS-PAGE and visualized using Coomassie Brilliant Blue stain. Gene Inactivation and Complementation. The construction of plasmids used to generate mutants of S. armeniacus is listed in Table S1, illustrated by the examples of arm3-8 mutant QY1 and arm16 mutant QY6. A 2385-bp DNA fragment harboring partial arm1, arm2, and partial arm3 was amplified with primer pair YJ10F and YJ10R and digested with BamHI and EcoRI, while a 2440-bp DNA fragment harboring partial arm8 and partial arm9 was amplified with primer pair YJ11F and YJ11R and digested with EcoRI and HindIII. The two DNA fragments were inserted into the BamHI and HindIII sites of pDQ4417 to generate plasmid pYJ35. A 1465-bp DNA fragment harboring aac(3)IV amplified with primer pair XJ28F and XJ28R and digested with EcoRI was inserted into the EcoRI site of pYJ35 to generate plasmid pWHU3041. Plasmid pWHU3041 was conjugated into S. armeniacus DSM 43125. Apramycin (50 μg/mL) was used to select exconjugants. The double-crossover strains that lost thiostrepton resistance were confirmed by PCR using three primer pairs, YJ25F and YJ25R, YJ27F and YJ27R, and YJ35F and YJ35R, located in the lost part of arm3-8. The PCR products of the wild-type S. armeniacus DSM 43125 were 2060 bp, 2264 bp, and 1842 bp, while no PCR product of arm3-8 mutant QY1 was observed (Figure S2). The primer pair YJ34F located in 5′-partial arm3 and YJ34R-1 located in 3′-partial arm8 were also used to confirm mutant QY1. No PCR product of wild-type strain was observed because there is an 18 003bp interval between the primer pair YJ34F and YJ34R-1, while the PCR product of QY1 was 1534 bp, in which a 17 991-bp fragment D

DOI: 10.1021/acs.jnatprod.8b00753 J. Nat. Prod. XXXX, XXX, XXX−XXX

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N-Des-methyl armeniaspirol B (8): (C17H17Cl2NO4) UV/vis λmax 210, 232, 297 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.70 (d), 7.44 (d), 2.53 (m), 1.48 (m), 1.16 (m), 1.48 (m), 0.80 (d), 0.80 (d), 9.81 (s) ppm; 13C NMR (100 MHz, 303 K in DMSO-d6) δ 167.4, 113.3, 124.6, 110.2, 171.4, 113.1, 189.0, 94.7, 140.6, 128.1, 164.3, 22.4, 26.4, 38.4, 27.7, 22.9, 22.9 ppm; HR-ESI-MS m/z 368.04861 ([M − H]−, calcd m/z 368.04564). N-Des-methyl armeniaspirol C (9): (C18H19Cl2NO4) UV/vis λmax 210, 232, 296 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.70 (d), 7.44 (d), 2.54 (d), 1.47 (m), 1.27/1.08 (m), 1.27 (m), 1.27/1.08 (m), 0.77 (m), 0.77 (m), 9.81 (s) ppm; 13C NMR (100 MHz, 303 K in DMSO-d6) δ 167.0, 113.2, 124.6, 110.4, 171.4, 113.1, 189.2, 94.7, 140.6, 128.2, 164.3, 22.5, 26.1, 36.1, 34.0, 29.3, 11.6, 19.4 ppm; HRESI-MS m/z 382.06473 ([M − H]−, calcd m/z 382.06129). 9-Des-chloro-N-des-methyl armeniaspirol A (10): (C17 H18 ClNO4) UV/vis λmax 212, 293 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.73 (d), 7.45 (d), 7.41 (s), 2.52 (m), 1.50 (m), 1.27 (m), 1.25−1.32 (m), 1.25−1.32 (m), 1.25−1.32 (m), 0.85 (t), 9.49 (s) ppm; 13C NMR (100 MHz, 303 K in DMSO-d6) δ 166.6, 113.0, 124.3, 110.8, 170.8, 112.5, 191.0, 95.1, 137.8, 132.0, 166.4, 22.3, 28.6, 29.0, 31.5, 22.5, 14.4 ppm; HR-ESI-MS m/z 334.09045 ([M − H]−, calcd m/z 334.08461). 9-Des-chloro-N-des-methyl armeniaspirol B (11): (C17H18ClNO4): UV/vis λmax 211, 293 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.72 (d), 7.45 (d), 7.41 (s), 2.53 (m), 1.46− 1.59 (m), 1.20 (m), 1.46−1.59 (m), 0.85 (d), 0.85 (d), 9.49 (s) ppm; 13 C NMR (100 MHz, 303 K in DMSO-d6) δ 166.6, 113.0, 124.3, 110.8, 170.8, 112.5, 191.0, 95.1, 137.8, 132.0, 166.5, 22.5, 26.5, 38.6, 27.6, 22.9, 22.9 ppm; HR-ESI-MS m/z 334.09055 ([M − H]−, calcd m/z 334.08461). 9-Des-chloro-N-des-methyl armeniaspirol C (12): (C18H20ClNO4) UV/vis λmax 212, 292 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.71 (d), 7.44 (d), 7.41 (s), 2.51 (m), 1.51 (m), 1.05−1.37 (m), 1.05−1.37 (m), 1.05−1.37 (m), 0.83 (t), 0.83 (d), 9.49 (s) ppm; 13C NMR (100 MHz, 303 K in DMSO-d6) δ 166.9, 113.0, 124.2, 110.5, 170.8, 112.7, 190.8, 95.1, 137.8, 132.0, 166.6, 22.6, 26.2, 29.0, 36.3, 34.0, 29.4, 11.7, 19.4 ppm; HR-ESI-MS m/z 348.10620 ([M − H]−, calcd m/z 348.10026). 9-Des-chloro armeniaspirol A (13): (C18H20ClNO4) UV/vis λmax 212, 227, 295 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.76 (d), 7.49 (d), 7.53 (s), 2.61 (s), 2.55 (m), 1.50 (m), 1.23−1.31 (m), 1.23−1.31 (m), 1.23−1.31 (m), 0.83 (t) ppm; 13C NMR (100 MHz, 303 K in DMSO-d6) δ 167.1, 113.1, 124.4, 110.9, 171.3, 113.0, 190.1, 97.3, 136.5, 132.1, 164.5, 25.4, 22.3, 28.5, 29.0, 31.5, 22.5, 14.4 ppm; HR-ESI-MS m/z 348.10635 ([M − H]−, calcd m/z 348.10026). 9-Des-chloro armeniaspirol A (14): (C18H20ClNO4) UV/vis λmax 212, 227, 295 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.77 (d), 7.49 (d), 7.53 (s), 2.61 (s), 2.55 (m), 1.48−1.57 (m), 1.19 (m), 1.48−1.57 (m), 0.83 (d), 0.83 (d) ppm; 13C NMR (100 MHz, 303 K in DMSO-d6) δ 166.9, 113.1, 124.4, 111.0, 171.3, 113.0, 190.1, 97.3, 136.4, 132.1, 164.5, 25.4, 22.4, 26.4, 38.5, 27.7, 22.9, 22.8 ppm; HRESI-MS m/z 348.10638 ([M − H]−, calcd m/z 348.10026). 9-Des-chloro armeniaspirol A (15): (C19H22ClNO4) UV/vis λmax 213, 227, 295 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.76 (d), 7.49 (d), 7.52 (s), 2.61 (s), 2.55 (m), 1.51 (m), 1.05−1.37 (m), 1.05−1.37 (m), 1.05−1.37 (m), 0.81 (t), 0.81 (d) ppm; 13C NMR (100 MHz, 303 K in DMSO-d6) δ 166.9, 113.1, 124.4, 111.0, 171.3, 113.0, 190.1, 97.3, 136.4, 132.1, 164.5, 25.3, 22.5, 26.1, 36.2, 33.9, 29.4, 11.6, 19.3 ppm; HR-ESI-MS m/z 362.12192 ([M − H]−, calcd m/z 362.11591). Expression and Purification of Recombinant Arm16 Protein. A 906-bp DNA fragment harboring arm16 amplified with primer pair YJ42F-1/YJ42R and digested with EcoRI and HindIII was inserted into the corresponding site of pET28a to generate plasmid pWHU3050. E. coli BL21(DE3) harboring the plasmid pWHU3050 was grown in Luria−Bertani (LB) medium supplemented with 50 μg/ mL kanamycin at 37 °C until the OD600 reached 0.6−0.8. IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 0.1 mM, and the culture was further incubated at 18 °C overnight. The cells were then harvested by centrifugation at

Production, Isolation, Purification, and NMR Analysis of Armeniaspirols A−C and the Intermediates. S. armeniacus wildtype strain DSM 43125, Δarm9Δarm15 mutant strain QY13, Δarm16 mutant strain QY6, and Δarm9 mutant strain QY4 were cultured using the liquid ISP-2 medium as previously described. The compounds were extracted from the culture as previously described. The compounds were purified first by using HPLC (Beijing QingBoHua Technologies Co. Ltd.) with a Daisogel C18 reversedphase HPLC column (250 × 20 mm, 10 μm). The HPLC conditions (mobile phase A: water; mobile phase B: methanol; UV detection λ: 300 nm) were as follows: 10−90% B for 35 min, 90% B for 15 min, 90−10% B for 5 min, 10% B for 10 min at a flow rate of 10 mL/min. The obtained compounds were concentrated on a rotovap and further purified by using HPLC (Shimadzu, SPD-M20A/LC-20AT) with a Thermo Scientific C18 reversed phase HPLC column (250 × 10 mm, 8 μm). The HPLC conditions (mobile phase A: water; mobile phase B: acetonitrile; UV detection λ: 300 nm) were as follows: 70% B for 30 min. The purified compounds were concentrated on a rotovap and dried on a freeze-dryer. Armeniaspirols A−C (1−3, 11.7 mg, 9.5 mg, and 9.1 mg) were purified from the culture of the wild-type strain DSM 43125 (ca. 20 L). Compounds 4−6 (10.4, 13.2, and 9.7 mg) were purified form the culture of Δarm9Δarm15 mutant strain QY13 (ca. 20 L). Compounds 7−9 (12.1, 10.6, and 9.4 mg) were purified form the culture of Δarm16 mutant strain QY6 (ca. 20 L). Compounds 10−15 (12.2,11.4,10.8,14.3, 13.6, and 15.8 mg) were purified form the culture of Δarm9 mutant strain QY4 (ca. 40 L). Carbon-13 and proton spectra were recorded on an Agilent 400 MHz NMR spectrometer operating at 400 and 100 MHz for 1H and 13 C respectively, equipped with a 5 mm PFG One NMR Probe. All experiments were carried out at 303 K in DMSO-d6. The chemical shifts were reported in ppm and referenced to the solvent resonance signal (DMSO-d6: 1H δppm 2.50, 13C δppm 39.9). Characterization of Compounds 1−15. Spectra of the compounds 1−15 are shown in Figures S11−S25 and Tables S5−S8. Armeniaspirol A (1): (C18H19Cl2NO4) UV/vis λmax 208, 232, 299 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.79 (d), 7.54 (d), 2.69 (s), 2.59 (t), 1.53 (m), 1.26 (m), 1.26 (m), 1.26 (m), 0.83 (m) ppm; HR-ESI-MS m/z 382.06818 ([M − H]−, calcd m/z 382.06129). Armeniaspirol B (2): (C18H19Cl2NO4) UV/vis λmax 208, 232, 299 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.77 (d), 7.53 (d), 2.69 (s), 2.58 (t), 1.53 (m), 1.18 (m), 1.53 (m), 0.83 (d), 0.83 (d) ppm; HR-ESI-MS m/z 382.06808 ([M − H]−, calcd m/z 382.06129). Armeniaspirol C (3): (C19H21Cl2NO4) UV/vis λmax 209, 232, 298 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.76 (d), 7.52 (d), 2.69 (s), 2.57 (td), 1.51 (m), 1.28/1.09 (m), 1.30 (m), 1.28/1.09 (m), 0.79 (t), 0.81 (d) ppm; HR-ESI-MS m/z 396.08386 ([M − H]−, calcd m/z 396.07694). Compound 4: (C17H19Cl2NO3) UV/vis λmax 245, 351 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.50 (d), 7.80 (d), 7.09 (s), 2.55 (m), 1.44 (m), 1.25−1.34 (m), 1.25−1.34 (m), 1.25−1.34 (m), 0.87 (t), 10.50 (s), 12.81 or 13.22 (s), 12.81 or 13.22 (s) ppm; HRESI-MS m/z 354.07129 ([M − H]−, calcd m/z 354.06637). Compound 5: (C17H19Cl2NO3) UV/vis λmax 245, 351 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.50 (d), 7.79 (d), 7.09 (s), 2.54 (m), 1.46 (m), 1.21 (m), 1.55 (m), 0.85 (d), 0.85 (d), 10.52 (s), 12.78 or 13.22 (s), 12.78 or 13.22 (s) ppm; HR-ESI-MS m/z 354.07156 ([M − H]−, calcd m/z 354.06637). Compound 6: (C18H21Cl2NO3) UV/vis λmax 245, 351 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.49 (d), 7.84 (d), 7.07 (s), 2.54 (m), 1.06−1.53 (m), 1.06−1.53 (m), 1.06−1.53 (m), 1.06−1.53 (m), 0.83 (dt), 0.83 (dt), 10.46 (s), 12.94 or 13.22 (s), 12.94 or 13.22 (s) ppm; HR-ESI-MS m/z 368.08725 ([M − H]−, calcd m/z 368.08202). N-Des-methyl armeniaspirol A (7): (C17H17Cl2NO4) UV/vis λmax 210, 232, 296 nm; 1H NMR (400 MHz, 303 K in DMSO-d6) δ 6.65 (d), 7.41 (d), 2.51 (m), 1.46 (m), 1.22 (m), 1.22 (m), 1.22 (m), 0.80 (m), 9.79 (s) ppm; 13C NMR (100 MHz, 303 K in DMSO-d6) δ 167.1, 113.2, 124.6, 110.4, 171.4, 113.1, 189.2, 94.7, 140.6, 128.2, 164.3, 22.2, 28.5, 28.9, 31.6, 22.5, 14.4 ppm; HR-ESI-MS m/z 368.04868 ([M − H]−, calcd m/z 368.04564). E

DOI: 10.1021/acs.jnatprod.8b00753 J. Nat. Prod. XXXX, XXX, XXX−XXX

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5000g for 15 min and resuspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 7.4). After cell disruption by sonication, the cell debris was removed by centrifugation at 20000g for 60 min, and the supernatant was loaded into a Ni-NTA column pre-equilibrated with lysis buffer. After collecting the flow-through and washing with 20 mM imidazole in lysis buffer at a flow rate of 2 mL/min, the protein was eluted with elution buffer (50 mM TrisHCl, 300 mM NaCl, 500 mM imidazole, pH 7.4) at a flow rate of 1 mL/min. The fractions were analyzed by SDS-PAGE. The pooled Arm16 protein was concentrated and buffer-exchanged into storage buffer (50 mM NaH2 PO4, 100 mM NaCl, pH 7.4, containing 10% glycerol) using an Amicon Ultra centrifugal filter (Ultracel-10K). The yield of purified recombinant Arm16 was typically ca. 8.7 mg/L of culture. Incubation of the Recombinant Arm16 with N-Des-methyl Armeniaspirols A−C (7−9) and 9-Des-chloro-N-des-methyl Armeniaspirols A−C (10−12). Compound 7 (50 μM) was incubated at 30 °C with recombinant Arm16 (10 μM) and Sadenosylmethionine (SAM) (0.5 mM) in 100 μL of Tris buffer (50 mM Tris-HCl, 200 μM MgCl2, pH 8.0). After 2 h, the reaction mixture was quenched with an equal volume of methanol, followed by centrifugation (12 000 r min−1, 1 min) to remove the precipitated protein. The supernatant was analyzed by LC-MS: 20−85% B for 15 min, 85% B for 5 min, 85−20% B for 3 min, 20% B for 7 min at a flow rate of 1 mL/min. The incubation of the recombinant Arm16 with 8 and 9 (respectively) was performed with the same method. The mixture of 7 and 10 was incubated at 30 °C with the recombinant Arm16 and SAM (0.5 mM) in 100 μL of Tris buffer (50 mM Tris-HCl, 200 μM MgCl2, pH 8.0). Samples were withdrawn at 0, 0.5, and 16 h. The samples were quenched with an equal volume of methanol, followed by centrifugation (12 000 r min−1, 1 min) to remove the precipitated protein. The supernatant was analyzed by the above LC-MS method. The incubation of the recombinant Arm16 with the mixture of 8 and 11 and the mixture of 9 and 12 (respectively) was performed with the same method. Antibacterial Activity Assay. Staphylococcus aureus Newman, methicillin-resistant Staphylococcus aureus USA300, Enterococcus faecium ATCC19434, Enterococcus faecalis FA2-2, Bacillus subtilis 168, Acinetobacter baumannii ATCC19606, Pseudomonas aeruginosa PAO1, Klebsiella pneumonia ATCC35657, Salmonella typhimurium ATCC14028, Shigella dysenteriae ATCC13313, and E. coli MG1655 were cultured in LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.0) at 37 °C aerobically. Antibacterial activities were evaluated by examining MIC, which was determined by the broth microdilution assay, as follows. Twofold serial dilutions of the test compounds were prepared in a 96-well microtiter plate containing 100 μL of LB broth and were inoculated into each well to give a final concentration of 1 × 105 to 3 × 105 CFU/mL. The plates were incubated at 37 °C for 24 h. After incubation, the plates were examined visually, and MIC was determined to be the lowest concentration that resulted in no turbidity.

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Xudong Qu: 0000-0002-3301-8536 Dongqing Zhu: 0000-0002-6298-512X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (NSFC Nos. 81373305 and 31401057), CAS Key Laboratory of Tropical Marine Bioresources and Ecology, Chinese Academy of Sciences (LMB141008), and Hubei Provincial Natural Science Foundation of China (No. 2017CFB618).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00753.

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REFERENCES

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Table of strains, plasmids, primers, predicted proteins of the gene cluster, and predicted domains of PKS genes, HPLC analysis, construction of deletion mutants, SDSPAGE analysis of protein purified, ESI-MS and NMR spectra of the compounds (PDF)

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DOI: 10.1021/acs.jnatprod.8b00753 J. Nat. Prod. XXXX, XXX, XXX−XXX