Sarpeptins A and B, Lipopeptides Produced by ... - ACS Publications

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Sarpeptins A and B, Lipopeptides Produced by Streptomyces sp. KO7888 Overexpressing a Specific SARP Regulator Wilaiwan Koomsiri,†,‡ Yuki Inahashi,*,§ Kantinan Leetanasaksakul,†,‡,⊗ Kazuro Shiomi,§ Yo̅ko Takahashi,§ Satoshi O̅ mura,§ Markiyan Samborskyy,⊥ Peter F. Leadlay,⊥ Pakorn Wattana-Amorn,∥,# Arinthip Thamchaipenet,*,†,‡ and Takuji Nakashima*,§ †

Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand Omics Center for Agriculture, Bioresources, Food and Health, Kasetsart University (OmiKU), Bangkok 10900, Thailand § Kitasato Institute for Life Sciences, Kitasato University, Tokyo 108-8641, Japan ⊥ Department of Biochemistry, University of Cambridge, Cambridge CB2 1TN, U.K. ∥ Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand # Special Research Unit for Advanced Magnetic Resonance and Center of Excellence for Innovation in Chemistry, Kasetsart University, Bangkok 10900, Thailand

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‡

S Supporting Information *

ABSTRACT: Whole genome analysis of Streptomyces sp. KO7888 has revealed various pathway-specific transcriptional regulatory genes associated with silent biosynthetic gene clusters. A Streptomyces antibiotic regulatory protein gene, speR, located adjacent to a novel nonribosomal peptide synthetase (NRPS) gene cluster, was overexpressed in the wild-type strain. The resulting recombinant strain of Streptomyces sp. KO-7888 produced two new lipopeptides, sarpeptins A and B. Their structures were elucidated by highresolution electrospray ionization mass spectrometry, NMR analysis, and the advanced Marfey’s method. The distinct modular sections of the corresponding NRPS biosynthetic gene cluster were characterized, and the assembly line for production of the lipopeptide chain was proposed.

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indigoidine, and venemycin from Streptomyces nanchangensis NS3226,8 Streptomyces lavendulae FRI-5,9 and Streptomyces venezuelae,10 respectively. In this study, analysis of the genome of Streptomyces sp. KO-7888, a producer of phthoxazolins,11−14 disclosed the presence of a Streptomyces antibiotic regulatory protein (SARP)15 gene in an unidentified nonribosomal peptide synthetase gene cluster. The SARP gene was overexpressed in Streptomyces sp. KO-7888, and novel lipopeptides were successfully produced.

atural products from microorganisms are a significant source of bioactive compounds as leads in drug discovery. Filamentous Gram-positive actinomycete bacteria are particularly prolific antibiotic producers. However, the number of new compounds discovered from actinomycetes by classical screening has declined. Meanwhile, genome-scale analysis of actinomycetes has revealed a large pool of “silent” or “cryptic” secondary metabolite gene clusters which are potential sources of new compounds.1−4 Mining actinomycete genomes, therefore, is a promising way to discover new antibiotics, particularly from Streptomyces; for example, 2-alkyl-4-hydroxymethylfuran3-carboxylic acid from Streptomyces coelicolor5 and avermitilol from Streptomyces avermitilis6 have been uncovered in this way. Such silent biosynthetic gene clusters (BGCs) can be activated to express the pathway-specific enzymes that synthesize the corresponding novel compounds using various strategies including heterologous expression, RNA polymerase mutation, and overexpression of pathway-specific regulators.7 Activation of transcriptional regulators located adjacent to the silent BGCs has been shown to induce such BGCs to produce potential new compounds. For example, overexpression of nanR1 and nanR2, far3, and vemR resulted in the production of nanchangmycin, © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The genome of the phthoxazolin-producing strain Streptomyces sp. KO-7888 was sequenced and analyzed by Rapid Annotation Using Subsystem Technology (RAST) version 2.0.16−18 The genome spans 8.5 Mbp and has 7545 putative protein-coding sequences. Secondary metabolite gene clusters of the genome were investigated using Antibiotic and Secondary Metabolite Analysis Shell (antiSMASH),19−21 which predicted 24 BGCs Received: January 25, 2019

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DOI: 10.1021/acs.jnatprod.9b00074 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Production of sarpeptins A (1) and B (2) of Streptomyces sp. KO-7888/pOSV556t::speR.

Figure 2. Structures of sarpeptins A (1) and B (2).

To investigate the function of SpeR, the full-length speR gene was amplified using specific primers. The gene was then ligated to pOSV556t under the control of the ermE* promoter, and the resulting plasmid was used to transform Escherichia coli ET12567/pUZ8002 before introducing it into Streptomyces sp. KO-7888 by intergeneric conjugation to obtain Streptomyces sp. KO-7888/pOSV556t::speR. The recombinant strain and Streptomyces sp. KO-7888/pOSV556t as a control were compared for production of new compounds. HPLC analysis clearly demonstrated that Streptomyces sp. KO-7888/pOSV556t::speR produced two new compounds, namely, sarpeptins A (1) and B (2), that were not produced by the control strain (Figures 1 and 2). Streptomyces sp. KO-7888/pOSV556t::speR was incubated in 6 L of YD medium at 27 °C for 7 days. The supernatant of the culture broth was extracted and subjected to column chromatography and followed by HPLC purification to obtain 6.9 and 12.9 mg of 1 and 2, respectively (Scheme S1). Compounds 1 and 2 were isolated as pale yellow powders that were determined by high-resolution electrospray ionization

encoding for the production of butyrolactones, ectoine, indole, lantibiotics, melanin, nonribosomal peptides, polyketides, siderophores, and terpenes. In addition to the BGC for phthoxazolins A−D, the genome encodes a large number of cryptic BCGs that potentially govern production of novel compounds. The number of BGCs found is consistent with the 10−30 cryptic BGCs reported in genomes of other actinomycetes.1−4 The genome annotation from RAST revealed 613 genes that showed similarity to authentic regulatory genes. Combining the results from RAST and antiSMASH analysis, over 25% of these genes (164 genes) showed similarity to regulatory genes that are involved in secondary metabolite production from actinomycetes22 including asnC, luxR, LAL, marR, merR, SARP, and tetR classes. A SARP regulatory gene, designated speR, located adjacent to an unidentified NRPS gene cluster, was chosen for further experiment. Analysis of SpeR by the Conserved Domain Database revealed a conserved helixturn-helix domain and bacterial transcriptional activation domain at the N-terminus (Figure S1). B


Journal of Natural Products Table 1. 1H and

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Article

C NMR Data of Sarpeptins A (1) and B (2) in DMSO-d6 1

no.

δC, type

OH-Asp-CONH2 1 170.9, C 2 55.8,a CH 3 71.1,a CH 4 172.6, C 1-NH2 2-NH Leu 1 2 3

172.0, C 51.7, CH 39.9, CH2

4 5 6 2-NH Thr 1 2 3 4 2-NH Tyr 1 2

24.1, CH 22.3, CH3 21.2, CH3

3

35.9, CH2

4 5, 9 6, 8 7

170.8, C 59.2, CH 66.5, CH 19.9, CH3

171.1, C 54.8, CH

127.7, C 130.1, CH 114.9, CH 155.7, C

δH (J in Hz)

4.52, m 3.98, d (4.8)

δC type 171.0, C 53.4, CH 71.1, CH 172.8, C

7.11, br 7.08, br 8.18, d (8.4)

4.22, 1.54, 1.45, 1.61, 0.85, 0.79, 7.74,

1

2

m m m m d (6.4) d (7.8) d (7.6)

4.08, dd (8.0, 4.0) 4.01, m 1.02, d (6.4) 7.89, d (8.0)

4.32, m 2.88, dd (14.0, 4.0) 2.78, dd (14.0, 9.2) 6.97, d (8.8) 6.58, d (8.8)

δH (J in Hz)

no.

4.62, dd (9.0, 4.4) 4.02, d (4.4) 7.15, br 7.10, br 8.27, d (9.0)

172.4, C 51.6, CH 40.2, CH2 24.3, CH 23.4, CH3 21.6, CH3

170.7, C 59.0, CH 66.8, CH 20.0, CH3

171.3, C 54.9, CH 36.4, CH2

4.34, m 1.46, m 1.61, 0.85, 0.81, 7.89,

4.13, 3.93, 1.00, 7.87,

m d (6.4) d (7.2) d (6.8)

dd (8.0, 4.0) m d (6.4) d (8.0)

4.44, ddd (9.2, 8.4, 4.0) 2.86, dd (14.0, 4.0)

8 9 10

2.74, dd (14.0, 9.2) 127.9, C 130.4, CH 115.1, CH 155.9, C

δC, type

2-NH 7-OH OH-Asp 1 170.7, C 2 55.8,a CH 3 71.1,a CH 4 170.8, C 2-NH Gly-1 1 168.7, C 2 42.2, CH2 2-NH Gly-2 1 169.6, C 2 42.2, CH2 2-NH fatty acid 1 165.7, C 2 124.9, CH 3 134.1, CH 4 126.5, CH 5 139.1, CH 6 25.4, CH2 7 38.1, CH2 27.0, CH 22.3, CH3 22.3, CH3

δH (J in Hz)

2 δC type

7.76, d (7.6) 9.10

4.51, m 3.97, d (4.8)

169.2, C 55.7, CH 70.1, CH 173.1, C

7.97, d (9.2)

3.77, d (5.4) 8.20, t (5.4)

3.84, d (5.6) 8.37, t (5.6)

169.1, C 42.3, CH2

169.7, C 42.4, CH2

7.33, dd (15.0, 11.7) 6.11, dd (11.7, 11.1) 5.76, dt (11.1, 8.0) 2.22, dt (8.0, 8.0) 1.24, m 1.53, m 0.85, d (6.4) 0.85, d (6.4)

33.6, CH 19.0, CH3 28.9, CH2

6.97, d (8.4) 11

4.64, dd (9.0, 2.4) 4.40, d (2.4) 7.90, d (9.0)

166.0, C 125.1, CH 134.4, CH 126.7, CH 139.5, CH 25.4, CH2 35.9, CH2

6.08, d (15.0)

δH (J in Hz) 7.80, d (8.4) 9.08

11.3, CH3

3.77, d (5.6) 8.17, t (5.6)

3.82, d (6.0) 8.34, t (6.0)

6.07, d (15.2) 7.33, ddd (15.2, 11.4, 0.8) 6.11, dd (11.4, 11.2) 5.76, dt (11.2, 8.0) 2.22, 1.33, 1.15, 1.31, 0.83, 1.30, 1.10, 0.81,

m m m m d (7.6) m m d (7.2)

6.58, d (8.4) a

Not clearly observed due to overlap.

Figure 3. COSY and HMBC correlations for sarpeptins A (1) and B (2).

cm−1) and amide carbonyl (1650 cm−1) functional groups, while the IR bands of 2 corresponded to amine or hydroxy (3282 cm−1) groups, and the same IR band for amide carbonyl was

mass spectrometry (HRESIMS) to have molecular formulas of C41H60N8O16 and C42H62N8O16, respectively (Table S1). The specific IR bands of 1 corresponded to amine or hydroxy (3309 C



Article

Figure 4. (A) Organization of the spe NRPS gene cluster of Streptomyces sp. KO-7888. (B) Schematic representation of the sarpeptin NRPS housed in SpeA, SpeB, and SpeC and proposed pathway for the biosynthesis of sarpeptins A (1) and B (2). The formation of the two HO-Asp residues is proposed to occur by β-hydroxylation of PCP-bound Asp monomers (catalyzed by candidate hydroxylases, SpeH and SpeI) before they are incorporated into the growing peptide chain. Fragmentation of the released linear peptide to release sarpeptins A and B is proposed to be triggered by α-hydroxylation of Gly-8 by putative flavin-dependent monooxygenase SpeJ. Subsequent hydrolysis of the hemiaminal may be spontaneous or assisted by putative hydrolase SpeK.

observed (Table S1). The wavelength of maximum absorbance of both 1 and 2 at 265 nm indicated that the compounds have an aromatic residue (Table S1). The structural elucidation was mainly focused on 2. The 1H and 13C NMR signals of 2 were assigned by a combination of COSY, HSQC, and HMBC spectra (Table 1 and Figures 3, S2− S6). The HSQC spectra revealed 8 sp2 methine carbons, 10 sp3 methine carbons, 7 sp3 methylene carbons, and 5 methyl carbons, while 10 carbonyl carbons and 2 sp2 nonprotonated carbons were indicated by 13C NMR. The COSY and HMBC spectra indicated the presence of a fatty acid moiety and 7 amino acids: one 3-hydroxy aspartic acid amide (OH-Asp-CONH2); one leucine (Leu); one threonine (Thr); one tyrosine (Tyr); one 3-hydroxy aspartic acid (OH-Asp); and two glycines (Gly) (Figure 3). The fatty acid moiety was deduced to be 8-methyl2,4-decadienoic acid. The coupling constants between H-2 and H-3 (J = 15.2 Hz) and between H-4 and H-5 (J = 11.2 Hz) indicated the configuration of the double bonds were E and Z, respectively (Figure 3). The HMBC correlations among the

amino acid residues from Gly-1-NH (δH 8.34) to Gly-2-CO (δC 169.7), from OH-Asp-NH (δH 7.90) to Gly-1-CO (δC 169.1), from Tyr-NH (δH 7.80) to OH-Asp-CO (δC 169.2), from ThrNH (δH 7.87) to Tyr-CO (δC 171.3), from Leu-NH (δH 7.89) to Thr-CO (δC 170.7), and from OH-Asp-CONH2-NH (δH 8.27) to Leu-CO (δC 172.4) established the sequence to be Gly-GlyOH-Asp-Tyr-Thr-Leu-OH-Asp-CONH2 (Figure 3). The presence of heptapeptide was also supported by the MS/MS fragmentation pattern of 2 (Figure S12). The stereochemistries of Tyr, Thr, and Leu residues were determined by the advanced Marfey’s method.23 The acid hydrolysates of 2 were derivatized with D-FDLA (1-fluoro-2,4-dinitrophenyl-5-D-leucine amide) and analyzed by LC-MS to compare with the authentic amino acid standards. The retention times of FDLA derivatives indicated the chiralities of Tyr, Thr, and Leu were L, L, and D, respectively (Figure S13). Finally, the fatty acid and heptapeptide moieties were connected by the HMBC correlation from Gly-2-NH (δH 8.34) to fatty acid-CO (δC 166.0) (Figure 3). Therefore, the structure of 2 was elucidated as D



Article

N-8-methyl-2E,4Z-decadienoyl Gly-Gly-OH-Asp-L-Tyr-L-ThrD-Leu-OH-Asp-CONH2. Compound 1 was 14 mass units (CH2) lighter than 2 (Table S1). The 1H and 13C NMR signals of 1 were assigned using a combination of COSY, HSQC, and HMBC spectra (Table 1 and Figures 3, S7−S11). The NMR spectra were very similar to those of 2 except for a difference in the fatty acyl moiety. The COSY and HMBC spectra indicated the fatty acid of 1 was 8methyl-2,4-nonadienoic acid (Figure 3). The coupling constants between H-2 and H-3 (J = 15.0 Hz) and between H-4 and H-5 (J = 11.1 Hz) indicated the geometry of the double bonds to be E and Z, respectively (Figure 3). From the results of amino acid analysis by the advanced Marfey’s method, the chiralities of Tyr, Thr, and Leu were determined to be L, L, and D, respectively (Figure S13). Therefore, the structure of 1 was elucidated as N8-methyl-2E,4Z-nonadienoyl Gly-Gly-OH-Asp-L-Tyr-L-Thr-DLeu-OH-Asp-CONH2. The new lipopeptides, sarpeptins A (1) and B (2), are linear. Most lipopeptides previously characterized from actinomycetes, such as taromycin A from Saccharomonospora sp. CNQ-490,24 daptomycin from Streptomyces roseosporus,25 A54145 from Streptomyces f radiae,26 laspartomycin from Streptomyces viridochromogenes var. komabensis,27 and friulimicin from Actinoplanes f riuliensis,28 are cyclic lipopeptides. However, linear lipopeptides have been reported from other bacteria, for example, gageostatins A−C from Bacillus subtilis,29 dragonamide E from Lyngbya majuscula,30 and syringafactins from Pseudomonas syringae pv. tomato DC3000.31 Those linear lipopeptides have shown various antimicrobial and antiparasitic activities.29−31 No compounds of similar structure to the sarpeptins have previously been reported. Sarpeptins A (1) and B (2) showed no antimicrobial activity against Bacillus subtilis ATCC6633, Escherichia coli NIHJ, Kocuria rhizophila ATCC9341, Mycobacterium smegmatis ATCC607, Pseudomonas aeruginosa IFO3080, Staphylococcus aureus ATCC6538p, Xanthomonas campestis pv. oryzae KB88, Aspergillus niger ATCC6275, Candida albicans ATCC64548, Mucor racemosus IFO4581, and Saccharomyces cerevisiae ATCC9763. The linear lipopeptide structures of sarpeptins A (1) and B (2) produced when the SARP regulatory gene, speR, located nearby (Figure 4) was overexpressed are almost perfectly correlated with the catalytic domains of nonribosomal peptide synthetases (NRPSs) encoded by speA, speB, and speC. The substrate specificity of each adenylation (A) domain was predicted (Table 2) based on established active site motifs for recognition of amino acid substrates.32,33 The biosynthesis of sarpeptins is proposed to start by attachment of decanoic acid to acyl carrier protein (SpeE) catalyzed by fatty acyl-AMP ligase (SpeD). Then, the fatty acyl chain is oxidized by SpeF and/or SpeG (acyl CoA dehydrogenases) to obtain the decadienoyl moiety. In the first NRPS extension module, glycine is specifically activated by the first A domain of SpeA. Since there is no Gly-A domain in the second module (Figure 4), it is possible that the A domain of the first module is required for the successive attachment of the first two (Gly) residues. The iterative use of a single A domain in this way to incorporate two successive amino acid residues was previously reported in the biosynthesis of safracin from Pseudomonas f luorescens A2-234 and saframycin A from Streptomyces lavendulae NRRL 11002.35 Thereafter, chain elongation of sarpeptins is predicted to continue using the successive A-T-C (adenylation-thiolationcondensation) core domains of the three NRPSs, SpeA, SpeB, and SpeC, in this order (Figure 4). The D-Leu of the lipopeptide

Table 2. Active-Site Residues of A-Domains of Sarpeptin NRPS (Spe) residue position according to SpeA AGly numbering A domain SpeA AGly AAsp ATyr AThr ALeu SpeB AAsp AGly APro AGly SpeC AGly AAla AGly

643

644

647

686

709

711

734

741

D D D D D

I L A F A

L T S W L

Q K T N F

L V A V A

G G A G G

M A A M A

V V V V V

D D D D

L I V I

T L Q L

K Q Y Q

V L V L

G G A G

A L H V

V V V I

D D D

I V I

L F L

Q I Q

L V L

G A G

V I L

I V V

chain is likely to arise via the action of an epimerase encoded by the E domain located at the C-terminus of extension module 5 at the end of SpeB (Figure 4). The origin of the two HO-Asp residues is strongly suggested by the presence of two standalone α-ketoglutarate-dependent oxygenases, SpeH and SpeI, encoded in the spe cluster (Figure 4). It has been shown in other NRPS systems that this modification takes place on the amino acid bound to the peptidyl carrier protein domain of the appropriate module in the NRPS assembly line.36,37 Hydroxylation of an Asp residue is a widespread modification in NRPSmediated biosynthesis of acylpeptide siderophores, where it provides an additional bifunctional ligand for Fe(III) chelation.38 Strikingly, the final linear structure of sarpeptins is a heptapeptide, significantly shorter than the tridecapeptide predicted from the organization of the NRPS. We initially considered two possible pathways for sarpeptin biosynthesis on this unexpectedly long assembly line (Figure 4). There is an interesting precedent for the premature release of a linear hexapeptide from the assembly line for the calcium-dependent antibiotic (CDA) when the A domain for the Asp-specific module 7 of CDA is mutated in its specificity motif.39 However, hydrolytic activity at this point would release sarpeptins as Ccarboxylic acids rather than the observed C-terminal amides. An attractive alternative is that the full-length spe gene cluster product is released by the C-terminal thioesterase domain, after which the Gly-8 residue immediately C-terminal of OH-Asp-7 is specifically hydroxylated at the α-carbon to form a hemiaminal by putative flavin-dependent monooxygenase SpeJ (Figure 4). This would readily hydrolyze to yield the C-terminal amide directly. In other NRPS systems such hemiaminal formation at X−Gly bonds is catalyzed by a two-component flavin-dependent monooxygenase.40,41 Subsequent hydrolysis of the hemiaminal may be spontaneous or assisted by putative alpha/beta hydrolase SpeK (Figure 4). Further experiments are in progress to explore the mechanism of sarpeptin release. Taken together, the results presented here strongly support the identification of this cryptic spe NRPS as governing the biosynthesis of sarpeptins A (1) and B (2). E



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Article

(methanol with 0.1% formic acid); B (5−100%) for 30 min at 0.5 mL/ min and detected at UV 254 nm. Fermentation and Isolation of Sarpeptins A and B from Streptomyces sp. KO-7888/pOSV556t::speR. Streptomyces sp. KO7888/pOSV556t::speR was inoculated into a 500 mL baffled flask containing 100 mL of YD medium and incubated on a rotary shaker at 27 °C, 210 rpm for 3 days. Then, 1% of the seed culture was transferred into 60 flasks containing 100 mL of YD medium each (total 6 L) and incubated further for 7 days. The supernatant was collected by centrifugation at 1000g for 10 min and was applied to HP20 column chromatography (60 mm i.d. × 100 mm) eluted by 50% methanol and 100% methanol. The 100% methanol fraction was applied to silica gel FL100D column chromatography (25 mm i.d. × 110 mm; Fuji Silysia Chemical, Aichi, Japan) eluted by a stepwise gradient of 200 mL of chloroform/methanol (100:0, 100:1, 100:2, 9:1, 1:1, and 0:100 (v/v)). The eluted fractions of 1:1 and 0:100 were subjected to ODS column chromatography (25 mm i.d. × 110 mm; Senshu Scientific, Tokyo, Japan) and eluted by a stepwise gradient of 200 mL of acetonitrile (10%, 20%, 30%, 40%, 50%, 60%, 80% and 100% each). The fractions eluted with 20−50% acetonitrile were purified by HPLC on a Pegasil ODS SP100 column (20 mm i.d. × 250 mm, Senshu Scientific) with 35% acetonitrile containing 0.05% formic acid at 8 mL/min and detected at UV 254 nm. The fractions eluting at 20.6 and 32.0 min were dried in vacuo to obtain 1 (6.9 mg) and 2 (12.9 mg). Amino Acid Analysis. Amino acid configurations were determined by the advanced Marfey’s method.23 The sample (0.1 mg) was completely hydrolyzed with 0.1 mL of 6 M HCl at 100 °C for 3 h. The hydrolysate was concentrated to dryness and dissolved in 0.05 mL of H2O. The hydrolysate and 1 mM amino acid standards (each 0.05 mL) were mixed with 0.02 mL of 1 M NaHCO3 and 0.05 mL of 1% 1-fluoro2,4-dinitrophenyl-5-D-leucinamide (D-FDLA) in acetone. The mixture was incubated at 37 °C for 1 h, and then 0.025 mL of 1 M HCl was added to stop the reaction. The mixture was dried in vacuo and dissolved in 1 mL of acetonitrile before analysis by LC-MS. LC-MS Analysis. Samples were injected into an ExionLC (AB Sciex, Framingham, MA, USA) coupled to a TripleTOF 5600 (AB Sciex). Chromatographic separation was performed on a Capcell Core C18 column (3.0 i.d. × 100 mm; Osaka Soda, Osaka, Japan) at 40 °C. Gradient elution was with solvent A (water with 0.1% formic acid) and solvent B (methanol with 0.1% formic acid): 50% B (0−2 min); 50− 100% B (2−18 min); 100% B (18−20 min); at flow rate 0.5 mL min−1. Fragmentation was recorded with the following parameters: ion spray voltage = 5500 V, source gas = 50 L min−1, curtain gas = 25 L min−1, declustering potential = 80 V, temperature = 500 °C, collision energy = 10 and 45 V, and collision energy spread = 15 V. Sarpeptin A (1): pale yellow powder; [α]26.5D +12.7 (c 0.1, MeOH); IR (ATR) νmax 3309, 2958, 1650, 1519 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 921.4192 [M + H]+ (calcd for C41H61N8O16, 921.4200). Sarpeptin B (2): pale yellow powder; [α]26.5D +10.6 (c 0.1, MeOH); IR (ATR) νmax 3282, 2958, 1650, 1519 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 935.4356 [M + H]+ (calcd for C42H63N8O16, 935.4378). Antimicrobial Activities. Antifungal and antibacterial activities of the purified compounds were analyzed by paper disc diffusion assay. Cell suspensions of B. subtilis ATCC6633 (5 × 105 cfu/mL), E. coli NIHJ (5 × 105 cfu/mL), K. rhizophila ATCC9341 (2 × 105 cfu/mL), M. smegmatis ATCC607 (5 × 105 cfu/mL), P. aeruginosa IFO3080 (5 × 105 cfu/mL), S. aureus ATCC6538p (5 × 105 cfu/mL), and X. campestis pv. oryzae KB88 (1 × 106 cfu/mL) were individually mixed into 7 medium (0.5% peptone, 0.5% meat extract, and 0.8% agar, pH 7.0); while A. niger ATCC6275 (1 × 106 spores/mL), C. albicans ATCC64548 (2 × 105 cfu/mL), M. racemosus IFO4581 (2 × 105 spore/mL), and S. cerevisiae ATCC9763 (1 × 106 cfu/mL) were individually mixed into GY medium (1.0% glucose, 0.5% yeast extract, and 0.8% agar, pH 6.0) and poured into Petri dishes. After that, the paper dishes containing the purified compounds at 30, 10, 3, and 1 μg were placed on the test agar plates and incubated at 37 °C for 24 h for antibacterial assay and at 27 °C for 24−48 h for antifungal assay.

EXPERIMENTAL SECTION

General Experimental Procedures. Maximum absorption wavelengths were observed by a Hitachi U-2810 spectrophotometer (Hitachi, Tokyo, Japan). The accurate mass and molecular formulas of the purified compounds were established by LC-ESIMS spectra (AB Sciex QSTAR Elite Hybrid LC/MS/MS Systems, AB Sciex, Framingham, MA, USA), while a Horiba FT-710 Fourier transform IR spectrometer (Horiba Ltd., Kyoto, Japan) was used to obtain IR spectra (ATR). Optical rotation was determined by a JASCO model DIP-1000 polarimeter (Jasco, Tokyo, Japan). NMR spectra were observed by an Agilent 400-MR DD2 (Agilent Technologies, Santa Clara, CA, USA) with 1H NMR at 400 MHz and 13C NMR at 100 MHz in DMSO-d6. The chemical shifts are represented in ppm and are referenced to DMSO-d6 in 1H NMR (2.48 ppm) and to DMSO-d6 in 13 C NMR (39.5 ppm) spectra. Genome Analysis of Streptomyces sp. KO-7888. The genome sequence of Streptomyces sp. KO-7888 was determined at the University of Cambridge Department of Biochemistry Sequencing Facility and was analyzed by RAST16−18 version 2.0 (http://rast.nmpdr.org) to annotate all open reading frames. The genome was next analyzed by antiSMASH 3.019−21 (http://antismash.secondarymetabolites.org) to classify the secondary metabolite gene clusters. After annotation, transcriptional regulatory genes were located and identified. Their structures were predicted by the Conserved Domain Database (CDD, http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). The sequence of the spe BGC has been deposited at DDBJ/EMBL/GenBank under the GenBank accession number MN068049. Overexpression of a SARP Transcriptional Regulatory Gene of Streptomyces sp. KO-7888. The full-length SARP transcriptional regulatory gene, speR, of Streptomyces sp. KO-7888 was amplified from genomic DNA using specific primers: forward primer, 5′agagtcgaccggcatgcaggaggggtgtcatggagttccgagt-3′, and reverse primer, 5′-ttaccagatctgcagccatcaggccgcggaccgctcg-3′. The PCR conditions using Q5 high-fidelity DNA polymerase (New England Biolabs Inc., USA) were as follows: 98 °C for 30 s; 35 cycles of 98 °C for 10 s, 60 °C for 5 s, 72 °C for 1 min; and 72 °C for 3 min. The PCR fragment was ligated using a Gibson assembly42 to an integrative overexpression plasmid, pOSV556t, containing the ermE* promoter (kindly provided by Gregory L. Challis, University of Warwick, UK). The recombinant vector was introduced into E. coli NEB10-beta (New England Biolabs Inc., USA) by a standard heat-shock procedure. Then, the recombinant vector was extracted by QIAprepMiniprep (QIAGEN, Germany) and was transferred into E. coli ET12567/pUZ800243 by electroporation to prepare for intergeneric conjugation.44 Next, E. coli ET12567/ pUZ8002 containing the recombinant vector was grown in LB medium containing ampicillin (100 μg/mL), kanamycin (50 μg/mL), and chloramphenicol (50 μg/mL) at 37 °C for 4 h and was centrifuged at 1000g for 10 min. The cells were washed twice with LB to remove antibiotics and resuspended to 106 cfu/mL. At the same time, 10 μL of spore suspension of Streptomyces sp. KO-7888 (109 spore/mL) was added to 500 μL of LB medium, and the mixture incubated at 50 °C for 10 min for spore germination. Finally, 100 μL of E. coli ET12567/ pUZ8002/pOSV556t::speR was mixed with a 500 μL spore suspension of Streptomyces sp. KO-7888 and spread onto mannitol soybean agar containing 10 mM MgCl2. After incubation at 27 °C for 24 h, the plate was overlaid with nalidixic acid (25 μg/mL) and hygromycin (50 μg/ mL) and incubated further at 27 °C for 3 days. Characterization of Secondary Metabolites of Streptomyces sp. KO-7888/pOSV556t::speR. The wild-type and the recombinant strains were inoculated into 10 mL of YD medium (1% yeast extract and 1% dextrose) and incubated on a rotary shaker (210 rpm) at 27 °C for 3 days. Then, 0.1 mL of 3-day seed culture was transferred into 10 mL of YD and incubated further for 14 days. Next, 10 mL of culture broth was extracted by adding 10 mL of ethanol and rotary shaken at 27 °C, 210 rpm for 1 h. The supernatant was collected by centrifugation at 1000g for 15 min and analyzed by LC-UV using an Inertsil ODS-4 column (3 mm i.d. × 250 mm, GL Sciences Inc., Tokyo, Japan) with gradient elution of solvent A (water with 0.1% formic acid) and solvent B F



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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.jnatprod.9b00074. Isolation scheme, physicochemical properties, 1D and 2D NMR spectra, LC-MS analyses of D-FDLA derivatives, and HRESIMS for 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (Y. Inahashi): [email protected]. Tel: (+813) 3444 6161. *E-mail (A. Thamchaipenet): [email protected]. Tel: (+662) 562 5444. *E-mail (T. Nakashima): [email protected]. Tel: (+813) 3444 6161. ORCID

Kazuro Shiomi: 0000-0003-1264-5116 Arinthip Thamchaipenet: 0000-0002-8749-0414 Present Address ⊗

(K. L.) National Omics Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS W.K. has received a Royal Golden Jubilee Ph.D. scholarship (Grant No. PHD/0205/2553), Thailand Research Fund. Y.I. was supported by a Kitasato University Research Grant for Young Researchers. This work was financially supported by JSPS-NRCT under the Japan-Thailand Research Cooperative Program; the Institute for Fermentation, Osaka (IFO), Japan; Omics Center for Agriculture, Bioresources, Food and Health, Kasetsart University (OmiKU), Bangkok, Thailand; and the Herchel Smith Chair of Biochemistry Fund, University of Cambridge, U.K.

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