Neothioviridamide, a Polythioamide Compound Produced by

Jul 14, 2017 - ABSTRACT: During genome mining for thioviridamide-like biosynthetic gene clusters that could produce polythioamide RiPP. (ribosomally s...
10 downloads 16 Views 1MB Size
Download PDF
Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Neothioviridamide, a Polythioamide Compound Produced by Heterologous Expression of a Streptomyces sp. Cryptic RiPP Biosynthetic Gene Cluster Teppei Kawahara,† Miho Izumikawa,† Ikuko Kozone,† Junko Hashimoto,† Noritaka Kagaya,‡ Hanae Koiwai,§ Mamoru Komatsu,§ Manabu Fujie,⊥ Noriyuki Sato,⊥ Haruo Ikeda,§ and Kazuo Shin-ya*,‡,∥ †

Japan Biological Informatics Consortium (JBIC), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan § Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato Sagamihara, Kanagawa 228-8555, Japan ⊥ Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son, Kunigami-gun, Okinawa 904-0495, Japan ∥ The Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan ‡

S Supporting Information *

ABSTRACT: During genome mining for thioviridamide-like biosynthetic gene clusters that could produce polythioamide RiPP (ribosomally synthesized and post-translationally modified peptides), we discovered a novel cryptic biosynthetic gene cluster. During efforts to express this biosynthetic gene using heterologous expression of this biosynthetic gene cluster, a novel compound designated as neothioviridamide was produced. We report herein the cloning and heterologous expression of the neothioviridamide biosynthetic gene cluster and the isolation, structure determination, and cytotoxic activity of neothioviridamide.

S

peptide (RiPP) containing a VMAAAASIALHC sequence in the precursor peptide.5 Since compounds that possess a thioamide functional group are rare and, moreover, 1 is the only known compound consisting of multi-thioamide functional groups, we have investigated biosynthetic genes that involve similar precursor sequences to that of 1 from our inhouse genome sequence data bank derived from more than 100 actinomycete strains. As a result of genome mining for compounds with repeated Ala residues, a gene encoding a precursor peptide containing a VMAAAATVAFHC motif in the genome of Streptomyces sp. MSB090213SC12 strain that was isolated from mangrove soil collected in Ishigaki island, Okinawa, Japan, was found. To express genes encoding

creening for bioactive microbial secondary metabolites has been performed for more than 70 years. The search for bioactive natural metabolites has resulted in great benefits to humankind, although the rate of discovery of skeletally novel compounds from bacteria has significantly decreased over time.1,2 New technologies to express cryptic biosynthetic gene clusters for secondary metabolites are needed to access novel compounds. Thioviridamide (1, Figure 1) is an N-acylated undecapeptide antibiotic that induces apoptosis selectively in EIA-transformed cells.3 The most unique structural feature of thioviridamide is the presence of five thioamide bonds formed between the amino acids.4 The gene cluster for the biosynthesis of thioviridamide in Streptomyces olivoriridis NA05001 was identified, and heterologous production of thioviridamide in Streptomyces lividans TK23 demonstrated that thioviridamide is a ribosomally synthesized and post-translationally modified © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 14, 2017

A

DOI: 10.1021/acs.jnatprod.7b00607 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Structures of thioviridamide (1) and neothioviridamide (2).

Figure 2. Comparison of biosynthetic gene clusters of thioviridamide (1) and neothioviridamide (2).

biosynthesis of this RiPP, we used various fermentation conditions employing a variety of media including the production medium for thioviridamide, but were unable to induce production. A strategy employing heterologous expression in the host SUKA strain,6−8 which is derived from Streptomyces avermitilis, was undertaken. Heterologous expression of the BAC clone prepared from Streptomyces sp. MSB090213SC12 that contains the biosynthetic gene cluster for the RiPP resulted in the production of a novel thioviridamide derivative, neothioviridamide (2). Herein, we report the heterologous expression, isolation, structure determination, and preliminary investigation of the biological activity of 2.

One of the BAC clones, pKU503J143P1-J10 (insert size: 83 095 bp), containing the gene cluster for the biosynthesis of 2 (ntv cluster), which involved almost the same biosynthetic gene cluster as that of 1, was obtained. The cloned biosynthetic gene cluster and comparison of the biosynthetic gene clusters of 1 and 2 are shown in Figure 2 and Table 1. Heterologous expression by the clone pKU503J143P1-J10 in SUKA17 resulted in the production of a small amount of 2. For gene manipulation to enhance the production of 2 and to prepare derivatives by exchanging amino acids in the precursor peptide, minimization of the gene cluster is favorable. Therefore, the excess genome region in the BAC clone was excised to establish the minimized biosynthetic gene cluster of 2. The minimized biosynthetic gene cluster of 2 was cloned by in vivo cloning using the λRED system.6 The resulting DNA fragment involving the entire ntv cluster was inserted into the integrating vector pKU592Aaac(3)IV. Unmethylated plasmid

■

RESULTS AND DISCUSSION A BAC library of the Streptomyces sp. MSB090213SC12 genome was prepared using a previously reported method.6 B

DOI: 10.1021/acs.jnatprod.7b00607 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

N1,N3-dimethylhistidinium (hdmHis) amino acid residue, and an ethenamine residue by the partial 1H−1H and 1H−13C correlations together with the comparison of the NMR data of thioviridamide as shown in Figure 3. An N-terminal 2oxopropanoic acid moiety was also established by 1H−13C long-range couplings from an end terminal methyl proton (δH 2.34) to two carbonyl carbons (δC 197.3 and δC 160.4), indicating the presence of an N-terminal pyruvic acid moiety (Figure 3). The partial amino acid sequence of 2 and their assignments were determined based on HMBC correlations from amide protons of Ala′-3, Ala′-2, Ala-1, Met′, and Val′-1 to a thioamide carbon of Ala′-2, an amide carbonyl carbon of Ala-1, thioamide carbons of Met′ and Val′-1, and a carbonyl carbon of pyruvic acid, respectively. A methyl proton (δH 1.34) of Ala-1 and an αmethine proton (δH 4.82) of Ala-2 were long-range coupled to an amide carbonyl carbon (δC 170.38) of Ala-1, which is the typical 13C chemical shift of the amide carbonyl carbon. Contrary to the 13C chemical shift of Ala-1, those of Ala′-2 and Ala′-3 were δC 203.9 and δC 203.1, respectively. These results suggested that the thioamide function in 1 is replaced by an amide function in 2. As described vide supra, 2 is derived from a RiPP gene cluster, and the order of amino acid sequence in the cyclopeptidic moiety in 2 was naturally determined as shown in Figure 3. A sulfide linkage between the two partial structures was elucidated by 1H−13C long-range coupling from a βmethine proton (δH 3.44) in the 3-Me-avCys moiety to an olefinic methine carbon (δC 101.8), of which a proton (δH 5.42) was 1H−1H coupled to an olefinic proton (δH 6.97, J = 8.4 Hz). No proton spin coupling between the olefin proton and the β-methine proton supported a 2-amino-3-[(2aminovinyl)thio]butanoic acid moiety, which originates from a Thr and a Cys residue. The assignments of Ala-4 and Ala-5 were interchangeable. The sequence and the positions of thioamide functional groups were also confirmed by MS-MS analysis (Supplementary Figure S2). The absolute configurations of the standard amino acid residues in 2 were determined using Marfey’s method.9 All Nα(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (FDAA) derivatives were identified based on comparison of their retention times, molecular formula, and UV spectra with those of standard amino acids derived from FDAA-conjugated compounds. We were unable to determine the absolute configuration of the amino acid residues except for Phe (established as L-Phe) due to racemization by hydrolysis of 1. Compound 2 exhibited cytotoxic activities against SKOV-3 (human ovarian adenocarcinoma), Meso-1 (malignant pleural mesothelioma), and Jurkat (immortalized human T lympho-

Table 1. Comparison and Functional Analyses of ORF in Thioviridamide and Neothioviridamide Biosynthetic Gene Clusters ID% SI %b

ORF

aaa

60/73

NtvA

87

41 378

37/52

NtvB

319

TvaD TvaE

329 308

30/46 33/46

NtvC NtvD

334 313

TvaF TvaG TvaH TvaI TvaJ TvaK

197 407 452 218 280 235

42/61 48/62 54/70 55/69 34/48 32/40

NtvE NtvF NtvG NtvH NtvI NtvJ

200 405 450 229 292 275

TvaL

282

33/50

NtvK

280

ORF

aaa

TvaA TvaR

75 309

TvaB TvaC

predicted function precursor peptide LuxR family transcriptional regulator hypothetical protein phosphotransferase family protein hypothetical protein phosphotransferase family protein decarboxylase methyltransferase YcaO TfuA-like protein phytanoyl-CoA dioxigenase peptidase (processing leader peptide) hypothetical protein

a

Numbers of amino acids. bID and SI indicate identity and similarity, respectively.

pKU592Aaac(3)IV::ntv cluster prepared in E. coli GM2929 hsdS::Tn10 was introduced into S. avermitilis SUKA22 by PEGassisted protoplast transformation. The SUKA22 transformant carrying the pKU592Aaac(3)IV::ntv cluster was cultured, and compound 2 was obtained from the ethyl acetate extract of the fermentation broth. Compound 2 was obtained as a colorless, amorphous powder. The molecular formula of compound 2 was determined by HR-ESIMS to be C56H85N14O10S6+. By taking into consideration this molecular formula, one thioamide bond is predicted to be exchanged by a simple amide bond compared to 1. The 1H and 13C NMR data of 2 were similar to those of 1. Since 2 was biosynthesized through the known RiPP pathway, the corresponding amino acid residues and their sequence were assumed to be VMAAAATVAFHC. The analysis of DQF-COSY and gHMBCAD data established the linear peptide moiety consisting of a thioamide Val (Val′-1), a thioamide Met (Met′), two thioamide Ala (Ala′2 and Ala′-3), and two Ala (Ala-1 and Ala-4) amino acid residues. Although many 1H and 13C signals from the cyclopeptidic substructure were not observed, the amino acidlike residues in this cyclopeptidic moiety were established to be a 3-Me-avCy, Val (Val-2), an Ala (Ala-5), a Phe, a β-hydroxy-

Figure 3. Correlations in DQF-COSY (bold lines) and gHMBCAD (arrows) spectra of 2. C

DOI: 10.1021/acs.jnatprod.7b00607 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 2. 13C and 1H NMR Spectroscopic Data of Neothioviridamide (2) δC

δH, multiplicity (J in Hz)

δC 3-Me-avCys

pyruvic acid

Val′-1

Met′

Ala-1

Ala′-2

Ala′-3

Ala-4

1 2 3

160.4 197.3 25.1

1 2 3 4 5 NH

202.3 63.1 33.6 20.1 18.3

1 2 3 4 6 NH

201.4 63.4 34.8 29.7 15.1

1 2 3 NH

170.38 54.1 17.6

1 2 3 NH

203.9 54.8a 21.3b

1 2 3 NH

203.1 58.7 21.1

1 2 3 NH

171.4 54.8a 18.1 n.a.

2.34, s

4.57, m 2.17, ovl Val-2 8.21, d (9.0)

5.28, ovl 2.04, ovl 2.46, ovl 2.02, s 10.27, d (6.6)

Ala-5

4.82, m 1.34, d (7.2) 10.16, d (5.4)

Phe

4.61, ovl 1.27, d (7.2)

hdmHisc

5.27, ovl 1.40, d (6.6) 9.91, d (5.4)

5.10, m 1.42, d (7.2)

1 2 3 5 6 NH 3-Me

n.a.e n.a. n.a. 101.8 127.6

1 2 3 4 5 NH

δH, multiplicity (J in Hz) 4.62, 3.44, 5.42, 6.97, 9.31, 1.22,

ovld m br d (8.4) dd (8.4, 8.4) br s ovl

n.a. 60.0 29.7 19.6 n.a. n.a.

3.78, 2.16, 0.98, 0.88,

ovl ovl ovl ovl

1 2 3 NH

177.1 66.5 21.3b n.a.

3.87, ovl 1.15, d (6.6)

1 2 3

n.a. 56.2 35.6

4 5, 9 6, 8 7 NH

138.3 129.5 128.2 126.5 n.a.

CO α β 2 4 5 N1-Me N3-Me NH

166.5 57.8 63.2 138.0 133.8 121.6 36.0 34.6

n.a.

n.a. 3.12, m 2.94, m 7.12, ovl 7.18, ovl 7.13, ovl

4.64, ovl 5.55, ovl 9.00, s 7.30, 3.74, 3.94, 8.56,

s s s br s

a,b Interchangeable. cavCys, S-(2-aminovinyl)cysteine moiety. dovl, overlapped with other signals. en.a., not assigned because the signals were not detected or ambiguous in the 2D NMR spectra.

cyte) cells with IC50 values of 2.1, 0.7, and 0.4 μM, respectively. Further studies on the absolute configuration and biological activities of 2 are underway. Neothioviridamde (2) consists of the unique skeletal structure involving thioamide functional groups. Since 1 and 2 are biosynthesized through the RiPP pathway, it is expected to produce a series of derivatives by exchanging amino acid residues in precursor peptides. The difference between 1 and 2 is not only in the amino acid components but also in the number of thioamide bonds.

■

selected heteronuclear multiple bond correlation using adiabatic pulses (gHMBCAD) were collected using a Varian NMR System 600 NB CL with DMSO-d6 as the solvent (δC 39.7 ppm. δH 2.49 ppm). The coupling constants (J) are given in hertz. HR-ESIMS data were recorded using a Waters LCT-Premier XE mass spectrometer. Normal- and reversed-phase MPLC were carried out using PurifPack Si-30 and Purif-Pack ODS-30 columns (Shoko Scientific Co., Ltd.), respectively. Analytical RP-UPLC was performed using a Waters ACQUITY UPLC System in conjunction with a BEH ODS column (2.1 i.d. × 100 mm, Waters), a Waters ACQUITY UPLC photodiode array eλ detector, and an LCT-Premier XE mass spectrometer. Preparative RP-HPLC was conducted using an X bridge C18 column (19 i.d. × 150 mm, Waters). MS/MS data were recorded on a Waters SYNAPT G2 mass spectrometer. Preparation of BAC Library and Minimization of BAC Clone. After the genome, embedded in 0.6% agarose gel, was partially digested with Sau3AI, the resulting fragments were separated by CHEF electrophoresis in 1% agarose gel, and approximately 100 kbp fragments were harvested from the gel. The purified DNA fragments were ligated with BamHI-cut pKU503, and the ligation products

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-2100 polarimeter using a sodium lamp at a wavelength of 589 nm. UV and IR spectra were measured with a Beckman Coulter DU730 UV/vis spectrophotometer. 1H and 13C NMR and 2D NMR data such as double-quantum-filtered correlation spectroscopy (DQF-COSY), gradient-enhanced heteronuclear single quantum coherence with adiabatic pulses (HSQCAD), and gradientD

DOI: 10.1021/acs.jnatprod.7b00607 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

introduced into E. coli NEB 10-beta (New England Biolabs, Inc., MA, USA) by electroporation.6,7 Each BAC clone was stored in five 384well plates containing Plusgrow II (100 μg/mL ampicillin and 20% glycerol) at −80 °C. Clones containing the biosynthetic gene cluster for 2 were screened by PCR amplification using two pairs of primers corresponding to upstream (1 137 848 to 1 138 452 nt of the genome) and downstream (1 149 691 to 1 150 311 nt of the genome) regions of the gene cluster (upstream primer pair, forward: 5′GCGTTACGTGGCTAGTGAACTTGA-3′ and reverse: 5′CTGCTGGTAGGCGGTGCGTTC-3′, and downstream primer pair, forward: 5′-GACTCGTTCCTCGAAAAGATCGAA-3′ and reverse: 5′-GGTTAACAGGGCTCCGTCACAGT-3′). The amplification was done as follows: after denaturation at 95 °C for 300 s, 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 60 s, and incubated at 72 °C for 5 min, then soaked at 12 °C. The minimized biosynthetic gene cluster for 2 was cloned by in vivo cloning using the λRED system as follows.6 Both upstream (1 137 998 to 1 138 256 nt of the genome) and downstream (1 150 757 to 1 151 005 nt) regions flanking the biosynthetic gene cluster for 2 were amplified by PCR using two pairs of primers (upstream forward: 5′CTCGAGactagtCAGTTCGACGCTGGACAGGAAGG-3′, upstream reverse: 5′-GAGGAtgtacaTCGAATAGATTGGGGGCAGGGACGAG-3′ and downstream forward: 5′-AGGAtgtacaTCGGAGCCCTTGGATGCTCTCTCCTC-3′, downstream reverse: 5′CTCGAGaagcttCCCAGCAAGACCATCGTGTACCAA-3′, lowercase characters indicate restriction sites, SpeI, BsrGI, BsrGI, and HindIII, respectively) with BAC clone pKU503J143P1-J10 as a template. These amplicons were cloned into pRED6 at SpeI/HindIII sites. The recombinant plasmid carrying both upstream and downstream regions was digested with BsaBI to generate linear molecules. The linearized molecules and XhoI-digested pKU503 J143P1-J10 were cotransformed into L-arabinose-induced E. coli BW25141 carrying pKD119.10 Transformants carrying the pRED::ntv cluster were selected by resistance to chloramphenicol, and the recombinant plasmid was confirmed by restriction digestion. The resulting clone was digested with SpeI/HindIII, and the 13 020 bp segment carrying the entire ntv cluster was cloned into the XbaI/HindIII site of integrating vector pKU592Aaac(3)IV. Fermentation, Extraction, and Isolation. The producing strain was cultivated in 50 mL test tubes, each containing 15 mL of a seed medium consisting of 0.5% glucose, 1.5% soybean meal, and 0.5% yeast extract at pH 7.5 (adjusted before sterilization). The test tubes were shaken on a reciprocal shaker (320 rpm) at 27 °C for 2 days. Aliquots (2.5 mL) of the culture were transferred into 500 mL baffled Erlenmeyer flasks containing 100 mL of a production medium consisting of 4.0% β-cyclodextrin, 0.5% glycerin, 2.0% pharmamedia, CuSO4·5H2O 5 mg/L, MnCl2·4H2O 5 mg/L, and ZnSO4·7H2O 5 mg/ L, pH 7.2 (adjusted before sterilization), and were cultured on a rotary shaker (180 rpm) at 27 °C for 5 days. The fermentation broth (2 L) was centrifuged, and the collected mycelial cake was extracted with acetone (400 mL × 2). After concentration in vacuo, the residual aqueous concentrate was partitioned between EtOAc and water (100 mL × 3). The EtOAc-soluble material (1.95 g) was subjected to silica gel MPLC eluted with a gradient system of n-hexane−EtOAc (0−25% EtOAc) followed by a stepwise solvent system of CHCl3−MeOH (0%, 2%, 5%, 10%, 20%, 30%, and 100% MeOH). Fractions were monitored using the UPLC-DAD-ELS-MS system. The 30% MeOH eluate (70.7 mg) was then subjected to ODS RP-MPLC by using a H2O−MeOH stepwise solvent system (20%, 40%, 60%, 80%, and 100% MeOH). The 100% MeOH fraction was purified by reserved-phase HPLC on a C18 column, using an isocratic mode with 65% CH3CN−50 mM NH4OAc to afford 2.1 mg of 2. Neothioviridamide (2): colorless amorphous powder; [α]24D −74 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 269 (4.47) nm; IR νmax (ATR) 3400 and 1640 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz), see Table 2; HRESIMS m/z at 1305.4863 [M+] (calcd for C56H85N14O10S6+, 1305.4897). Cytotoxicity Assay. The cytotoxic activities of 2 against human ovarian adenocarcinoma SKOV-3 cells, malignant pleural mesothelioma Meso-1 cells, and immortalized human T lymphocyte Jurkat cells

were examined. SKOV-3 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 μg/mL). MESO-1 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, penicillin (50 U/ mL), and streptomycin (50 μg/mL). Jurkat cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, penicillin (50 U/mL), streptomycin (50 μg/mL), and Glutamax. All cell lines were seeded in a 384-well plate at a density of 1000 cells/well in 20 μL of media and incubated at 37 °C in a humidified incubator with 5% CO2. After 4 h, 2-fold dilution samples were added to the cell culture at the concentration of 0.5% and incubated for 72 h. Cell viabilities were measured using a CellTiter-Glo luminescent cell viability assay and EnVision multilabel plate reader. Marfey’s Analysis. Compound 2 was subjected to complete acid hydrolysis (6 N HCl, 110 °C for 16 h) followed by a reaction with FDAA to obtain the respective FDAA derivatives. The retention times of these FDAA amino acid derivatives were established by UPLC monitoring with UV absorption at 340 nm and negative ion mode ESIMS.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00607. Amino acid sequences of precursor peptide in thioviridamide and neothioviridamide biosynthesis gene clusters, HR-MS and MS/MS experiments of 2, and NMR spectra of 2 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Phone/fax: +81-3-3599-8305. E-mail: [email protected] (K. Shin-ya). ORCID

Teppei Kawahara: 0000-0001-5140-4724 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported in part by a grant for “Project Focused on Developing Key Technology for Discovering and Manufacturing Drugs for Next-Generation Treatment and Diagnosis” from Japan Agency for Medical Research and Development (AMED), Japan, and from Ministry of Economy, Trade and Industry (METI), Japan.

■

REFERENCES

(1) Palazzolo, A. M. E.; Simons, C. L. W.; Burke, M. D. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 5564−5566. (2) Pye, C. R.; Bertin, M. J.; Lokey, R. S.; Gerwick, W. H.; Linington, R. G. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 5601−5606. (3) Hayakawa, Y.; Sasaki, K.; Adachi, H.; Furihata, K.; Nagai, K.; Shin-ya, K. J. Antibiot. 2006, 59, 1−5. (4) Hayakawa, Y.; Sasaki, K.; Nagai, K.; Shin-ya, K.; Furihata, K. J. Antibiot. 2006, 59, 6−10. (5) Izawa, M.; Kawasaki, T.; Hayakawa, Y. Appl. Environ. Microbiol. 2013, 79, 7110−7113. (6) Komatsu, M.; Komatsu, K.; Koiwai, H.; Yamada, Y.; Kozone, I.; Izumikawa, M.; Hashimoto, J.; Takagi, M.; Omura, S.; Shin-ya, K.; Cane, D. E.; Ikeda, H. ACS Synth. Biol. 2013, 2, 384−396. (7) Komatsu, M.; Uchiyama, T.; Omura, S.; Cane, D. E.; Ikeda, H. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 2646−2651. E

DOI: 10.1021/acs.jnatprod.7b00607 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(8) Izumikawa, M.; Kozone, I.; Hashimoto, J.; Kagaya, N.; Takagi, M.; Koiwai, H.; Komatsu, M.; Fujie, M.; Satoh, N.; Ikeda, H.; Shin-ya, K. J. Antibiot. 2015, 68, 533−536. (9) Bhushan, R.; Brückner, H. Amino Acids 2004, 27, 231−247. (10) Miyamoto, K. T.; Komatsu, M.; Ikeda, H. Appl. Environ. Microbiol. 2014, 80, 5028−5036.

F

DOI: 10.1021/acs.jnatprod.7b00607 J. Nat. Prod. XXXX, XXX, XXX−XXX