Suppression with N

Article pubs.acs.org/jnp

Actinomycete Metabolome Induction/Suppression with N‑Acetylglucosamine Yousef Dashti,† Tanja Grkovic,† Usama Ramadan Abdelmohsen,‡,§ Ute Hentschel,‡ and Ronald J. Quinn*,† †

Eskitis Institute for Drug Discovery, Griffith University, Brisbane, QLD 4111 Australia Department of Botany II, Julius-von-Sachs Institute for Biological Sciences, University of Würzburg, Julius-von-Sachs-Platz 3, D-97082 Würzburg, Germany

‡

S Supporting Information *

ABSTRACT: The metabolite profiles of three sponge-derived actinomycetes, namely, Micromonospora sp. RV43, Rhodococcus sp. RV157, and Actinokineospora sp. EG49 were investigated after elicitation with N-acetyl-D-glucosamine. 1H NMR fingerprint methodology was utilized to study the differences in the metabolic profiles of the bacterial extracts before and after elicitation. Our study found that the addition of N-acetyl-Dglucosamine modified the secondary metabolite profiles of the three investigated actinomycete isolates. N-Acetyl-D-glucosamine induced the production of 3-formylindole (11) and guaymasol (12) in Micromonospora sp. RV43, the siderophore bacillibactin 16, and surfactin antibiotic 17 in Rhodococcus sp. RV157 and increased the production of minor metabolites actinosporins E−H (21−24) in Actinokineospora sp. EG49. These results highlight the use of NMR fingerprinting to detect changes in metabolism following addition of N-acetyl-D-glucosamine. N-Acetyl-D-glucosamine was shown to have multiple effects including suppression of metabolites, induction of new metabolites, and increased production of minor compounds.

M

walls, as well as the extracellular matrix of animal cells.26,28,29 When the cell wall is remodeled during cell growth, a significant amount of GlcNAc is released into the environment and becomes available for cell signaling.26,27 A number of studies have demonstrated that the addition of GlcNAc to the culture medium of fungi30−32 and bacteria33−35 activates cellular signaling in these microorganisms. GlcNAc induced the production of undecylprodigiosin and actinorhodin antibiotics in Streptomyces coelicolor under poor nutritional conditions.35 Furthermore, the addition of GlcNAc to the culture medium of Pseudomonas aeruginosa enhanced the production of the phenazine-type antibiotic pyocyanin.36 Herein, we investigated the effect of GlcNAc on metabolic profiles of nine sponge-derived actinomycetes, including Nocardiopsis sp. SBT366, Streptomyces sp. SBT343, Geodermatophilus sp. SBT350, Streptomyces sp. SBT345, Streptomyces sp. SBT346, Micromonospora sp. SBT373, Micromonospora sp. RV43, Rhodococcus sp. RV157, and Actinokineospora sp. EG49. The three latter strains, which revealed changes in metabolic profile upon addition of GlcNAc to the culture media, were investigated in more detail. LC-UV-MS as well as 1H NMR fingerprint methodology were used as the main tools for comparison and detailed analysis of the chemical

any of the commercially available nature-derived drugs have been isolated from bacteria and fungi.1,2 In particular, actinomycetes are well-known for their ability to produce natural products with structural complexity and diverse biological activities.3−7 However, genome sequencing data have shown that those microorganisms are capable of producing still a far greater number of metabolites and potential drugs.8−15 Actinomycetes produce secondary metabolites to communicate between individual bacteria or between species as well as to defend themselves in various habitats.16−18 However, when actinomycetes are grown under standard laboratory culture conditions, many of the biosynthetic gene clusters remain silent or are poorly expressed.19−21 Accordingly, significant research effort has been dedicated to desilence the underlying gene clusters with the aim to identify new natural products and molecular scaffolds, which may eventually yield novel drug candidates.22−24 In this context, different strategies have been proposed to activate the cryptic genes including mimicking the natural environment and coculturing of microorganisms.19−21,25 Chemical signals produced from one microorganism in coculture can activate the expression of metabolites in the other. One compound that acts as a signaling molecule and induces a variety of responses in microbes is N-acetyl-D-glucosamine (GlcNAc).26,27 The amino sugar GlcNAc is a component of peptidoglycan in the bacterial cell walls and chitin in fungal cell © 2017 American Chemical Society and American Society of Pharmacognosy

Received: July 20, 2016 Published: March 29, 2017 828

DOI: 10.1021/acs.jnatprod.6b00673 J. Nat. Prod. 2017, 80, 828−836

Journal of Natural Products

Article

amounts of the pentamer 9 and hexamer 10 precluded use of specific rotations to determine their absolute configurations. Investigation of the extract of Micromonospora sp. RV43 treated with GlcNAc identified 3-formylindole (11)45 and 2R,3R -guaymasol (12).46 The absence of compounds 11 and 12 by NMR and LC-MS in the untreated bacterial extract suggested that these are induced metabolites. Activating Cryptic Biosynthetic Gene Clusters of Surfactin Lipopeptides and the Siderophore Bacillibactin in Rhodococcus sp. RV157. The 1H NMR fingerprint study of the fractions from analytical reversed-phase HPLC of the extracts of Rhodococcus sp. RV157 revealed some distinctive resonances unique to the untreated sample, which after an NMR-directed isolation were assigned to N-acetyltyramine (13)47 and N-propionyltyramine (14) (Figure 2).48 The LCUV chromatogram indicated the presence of an additional compound in the extract of untreated Rhodococcus sp. RV157; however, no signal corresponding to this metabolite was detected in the 1H NMR spectrum of the related fraction from analytical HPLC. UV-guided large-scale isolation identified the compound to be avenalumic acid (15).49 The coupling constants of the olefinic protons H-7 and H-8 (J7,8 = 15.3 Hz) as well as H-9 and H-10 (J9,10 = 15.1 Hz) enabled the assignment of the double-bond configurations to be 2-E,4-E. It is known that compound 15 is unstable upon exposure to air or daylight,49 and this may be the reason that the compound was not detected in the proton NMR spectrum of the untreated sample collected from analytical HPLC, or it may be due to the large extinction coefficient of the compound, which allows detection of a small amount by UV while the concentration is not within the NMR detection limit. Comparative investigation of the 1H NMR spectra revealed that Rhodococcus sp. RV157 not only stopped production of metabolites 13−15 upon GlcNAc addition to the culture medium, but also showed evidence of induced secondary metabolites. NMR-directed isolation identified the first induced metabolite as the catecholic siderophore bacillibactin (16).50 Siderophores play an essential role in achieving a successful infection of the host by pathogens;51 however, the main role of siderophores, including bacillibactin, is to supply iron from the environment to the microorganisms.52 Here, the addition of GlcNAc to Rhodococcus sp. RV157 induced production of bacillibactin. Although the gene cluster responsible for the biosynthesis of bacillibactin has been reported from Rhodococcus imtechensis RKJ300 and Rhodococcus equi,53,54 the metabolite has never been previously isolated from this genus. 16S rDNA analysis revealed that our strain Rhodococcus sp. RV157 is closely related to Rhodococcus equi. This work is the first report of the isolation of bacillibactin from the genus Rhodococcus. Another induced metabolite identified in Rhodococcus sp. RV157 was the surfactin lipopeptide 17.55,56 NMR data and the size and magnitude of the specific rotations of surfactin 17 matched published values for the Glu-Leu-D-Leu-Val-Asp-DLeu-Leu amino acid sequence of the peptide.57,58 The length and substitution type of the branching methyl in the β-hydroxy fatty acid chain of the surfactin were identified based on a combination of mass spectrometry evidence and 13C NMR chemical shifts of the terminal methyl groups.58 HRESIMS of surfactin 17 exhibited a peak of the sodium adduct ion at m/z 1030.6412 [C51H89N7O13Na]+, and the 13C NMR spectrum in DMSO-d6 showed characteristic carbon resonances of the methyl groups at δC 11.1 and 19.0 ppm, indicating a fatty acid

profile of the bacteria before and after treatment with GlcNAc. Our results show that the addition of the amino sugar GlcNAc to the liquid cultures of Micromonospora sp. RV43, Rhodococcus sp. RV157, and Actinokineospora sp. EG49 altered the production of natural products expressed under normal culture conditions and induced or suppressed the production of a number of other small molecules.

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RESULTS AND DISCUSSION To monitor the changes in the metabolic profile of the bacteria after exposure to GlcNAc, the EtOAc extracts (6 mg) of untreated and treated actinomycetes were fractionated by analytical HPLC using an established literature procedure,37 and the fractions were then analyzed by 1H NMR spectroscopy (Supporting Information, Figure S1). Fractionation reduces the complexity of the metabolites present in the crude extract, and therefore analysis of the metabolites by 1H NMR becomes less complicated.38 The 1H NMR spectra of the corresponding fractions of treated and untreated samples were compared to obtain more detailed information about changes in chemical profiles of the bacteria after elicitation with GlcNAc.25 NMR was then used to guide isolation of compounds using semipreparative HPLC. The effects of GlcNAc on the metabolic production of the three investigated actinomycetes are presented in this study. Alterations in the Expression of the Secondary Metabolites of Micromonospora sp. RV43 by GlcNAc. Using NMR fingerprinting methodology to investigate fractions from analytical reversed-phase HPLC of Micromonospora sp. RV43 following an NMR-guided isolation, glutaric acid (1),39 three phenolic compounds, namely, 4-hydroxyphenyl acetic acid (2),40 4-hydroxybenzoic acid (3),41 and 4-hydroxybenzaldehyde (4),42 and different analogues of 3-hydroxybutyrate from mono- to hexamers (6−10)43 were identified only in the extract of bacteria not treated with GlcNAc (Figure 1). The

Figure 1. Metabolites 1−10 were identified only in the extract of untreated Micromonospora sp. RV43. Addition of GlcNAc induced the production of compounds 11 and 12.

stereogenic carbon of 3-hydroxybutyric acid and the 3hydroxybutyrate unit of poly-3-hydroxybutyrate identified in bacterial extracts has been reported to be in the Rconfiguration.44 Comparison of the specific rotation of the dimer 6, trimer 7, and tetramer 8 with the specific rotation values reported for the synthesized compounds revealed that the configurations of all of the stereogenic centers were R.43 However, the low purity of the monomer 5 and the low 829



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Figure 2. Metabolites 13−15 identified in the EtOAc extract of untreated Rhodococcus sp. RV157 and metabolites 16 and 17, induced metabolites, identified in the EtOAc extract of Rhodococcus sp. RV157 treated with N-acetylglucosamine.

Figure 3. Addition of GlcNAc to the culture medium of Actinokineospora sp. EG49 enhanced production of secondary metabolites, which resulted in the isolation of known compounds 18−20 and new metabolites 21−24.

chain length of 13 carbons with anteiso structure.58 In addition to surfactin 17, several other analogues with methylene differences in their structures, as revealed by HRESIMS data, were also identified only in Rhodococcus sp. RV157 treated with GlcNAc.

Surfactins have been reported to display a wide range of bioactivities including antimicrobial,59 antiviral,60 antitumor,61 blood anticoagulant,62 and immunosuppressive.63 These lipopeptides, due to their surfactant properties in conjunction with some favorable characteristics including low toxicity, biodegradability, and biocompatibility, gained considerable 830



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Table 1. 1H (900 MHz) and 13C (225 MHz) NMR Data of Actinosporins E and F Recorded in DMSO-d6 actinosporin E (21) position

δC

1 2

196.7, C 53.0, CH2

3 4

71.7, C 43.3, CH2

4a 5 6 6a 7 7a 8 9 10 11 11a 12 12a 12b 13 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″

148.1, 134.0, 128.8, 134.2, 180.1, 120.9, 155.6, 121.7, 135.3, 119.6, 137.1, 183.9, 134.7, 134.2, 29.7, 98.3, 70.0, 70.0, 71.7, 70.0, 17.8,

C CH CH C C C C CH CH CH C C C C CH3 CH CH CH CH CH CH3

δH (J in Hz)

actinosporin F (22) HMBC (δH to δC)

2.70, d (13.8) 3.05, d (13.8)

1, 3, 4, 13 1, 3

3.07, d (16.6) 3.22, d (16.6)

2, 3, 4a, 12b 3, 4a, 12b

7.68, d (8.1) 8.14, d (8.1)

4, 6a, 12b 4a, 7, 12b

7.64, d (7.9) 7.81, t (7.9) 7.61, d (7.9)

7a, 11 8, 11a 7a, 9, 12

1.34, 5.64, 3.96, 4.00, 3.34, 3.52, 1.08,

s d (1.8) (3.5, 1.8) dd (9.4, 3.5) under H2O peak m d (6.1)

2, 3, 4 8, 2′ 4′ 4′ 5′, 6′ 4′, 5′

δC

δH (J in Hz)

HMBC (δH to δC)

155.3, C 113.1, CH

7.30, s

1, 4, 12b, 13

138.7, C 121.5, CH

7.43, s

2, 5, 12b, 13

137.4, 128.3, 123.1, 131.0, 183.0, 121.1, 155.5, 117.1, 133.9, 123.9, 146.4, 64.5, 139.4, 120.1, 21.4, 100.3, 69.9, 71.4, 72.0, 69.9, 18.0, 99.5, 70.2, 70.2, 71.7, 69.8, 17.8,

C CH CH C C C C CH CH CH C CH C C CH3 CH CH CH CH CH CH3 CH CH CH CH CH CH3

7.86, d (8.6) 8.04, d (8.6)

4, 6a, 12b 4a, 7, 12b

7.23, d (8.0) 7.63, t (8.0) 7.37, d (8.0)

7a, 11 8, 11a 7a, 9, 12

6.87, s

2.47, 5.59, 4.40, 3.96, 3.40, 3.60, 1.21, 5.50, 4.09, 3.86, 3.32, 3.49, 1.08,

s d (1.8) dd (3.6, 1.8) dd (9.2, 3.6) t (9.2) m d (6.1) d (1.8) dd (3.4, 1.8) dd (9.4, 3.4) under H2O peak m d (6.2)

6a, 7a, 11, 12a, 11a

2, 3, 4 1, 2′, 3′

4′, 5′ 8, 2″

4″, 5″

HPLC, and the collected fractions were analyzed by 1H NMR. Comparison of the 1H NMR spectra of the fractions from both samples revealed similar proton resonances; however, the intensity of the resonances corresponding to the minor metabolites increased in the sample treated with GlcNAc. Actinosporins A (18) and C (19) were obtained by NMR-directed isolation of untreated Actinokineospora sp. EG49. Although evidence of more actinosporin-like compounds was observed in the proton spectrum of the untreated sample, the yields were not sufficient for purification and structure elucidation. Addition of GlcNAc to the culture media boosted production of secondary metabolites, and in addition to the known compounds 18−20, new metabolites actinosporins E− H (21−24) were identified (Figure 3). The molecular formula of actinosporin E (21) was determined to be C25H24O9, based on HRESIMS and NMR data (Table 1). The 1H NMR spectrum of 21 in DMSO-d6 showed 14 resonances comprising five sp2-hybridized methines, five sp3-hybridized methines, two sp3-hybridized methylenes, and two sp3-hybridized methyls. Analysis of gCOSY correlations and 1H−1H coupling constants revealed 1,2,3-trisubstituted and 1,2,3,4-tetrasubstituted D and B rings, similar to those of compound 20. The lack of aromatic singlet proton resonances of ring A and the presence of sp3-hybridized methylenes H-2 (δH 2.70 and 3.03) and H2-4 (δH 3.06 and

attention for applications in environmental industries as biosurfactants.64 There are increasing efforts to enhance the yield and reduce the cost of surfactin production from various strains of Bacillus spp.65 However, the effect of GlcNAc on production of surfactins has never been investigated, and the result of this study indicates the potential application of GlcNAc on surfactin production for biotechnology purposes. Furthermore, surfactins have been reported only from Bacillus spp., and therefore Rhodococcus species can be considered as a new source of surfactin lipopeptides. Increasing Production of Poorly Expressed New Metabolites Actinosporins E−H in Actinokineospora sp. EG49 by GlcNAc. Actinokineospora sp. EG49 is a source of angucycline-type compounds. Glycosylated actinosporins A−D had been isolated from the species.66,67 Furthermore, mixed fermentation of Actinokineospora sp. EG49 and Nocardiopsis sp. RV163 resulted in production of induced metabolites that were not detected in individual cultures. However, it was determined that these induced metabolites were most likely produced by Nocardiopsis sp. RV163, as one of them showed antibacterial activity against Actinokineospora sp. EG49.25 Here, in an attempt to induce the production of secondary metabolites in Actinokineospora sp. EG49, GlcNAc was added to the culture medium. The EtOAc extracts of untreated and treated Actinokineospora sp. EG49 were fractionated by analytical 831



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Figure 4. Comparison of the experimental and calculated ECD spectra of (a) actinosporin E (21) and (b) actinosporin F (22).

actinosporin compounds and was similar in structure to compound 20. Two characteristic anomeric signals at δH 5.50 and 5.59, which had HSQC correlations to δC 99.5 and 100.3, respectively, as well as doublets at δH 1.08 and 1.21 indicated the presence of two rhamnose moieties in the structure. The connections of the sugars to the aglycone were identified through a 3JCH correlation of H-1′ (δH 5.59) to C-1 (δC 155.3) and H-1″ (δH 5.50) to C-8 (δC 155.5). The coupling constant of the anomeric protons revealed that both are α sugars, and the absolute configurations were determined to be L based on GC-MS analysis. While compound 20 showed two carbon resonances typical of a quinone at δC 180.3 and 185.5, compound 22 showed only one resonance for a quinone carbonyl at δC 183.0 and an oxygenated methine signal at δH 6.87 and δC 64.5. An HMBC experiment revealed a 3JCH correlation of this methine proton to C-6a (δC 131.0), C-7a (δC 121.1), C-11 (δC 123.9), and C-12b (120.1). The NMR data in conjunction with a 2 Da difference in mass of 22 compared to the quinone 20 indicated that C-12 was reduced in 22. Actinosporin F is very similar to pseudonocardone A identified from a Pseudonocardia sp. derived from a fungusgrowing ant.70 They have the same aglycone apart from an OMe group at position 8 in pseudonocardone A and a hydroxy group at position 8 in actinosporin F. ECD calculations were used to assign the absolute configuration of C-12. Both rhamnose moieties were omitted for calculation of the ECD spectra. Conformational analysis of both R and S stereoisomers was performed using the OPLS2005 force field followed by DFT/B3LYP/6-31G(d,p) optimization. Three conformers were obtained for each stereoisomer within a 2 kcal/mol energy window from the global minimum in the gas phase. Calculations of ECD spectra were carried out using TDDFT/CAM-B3LYP/6-31G(d,p) in MeOH. The calculated ECD spectrum for the S enantiomer was in good agreement with the experimental spectrum (Figure 1), and therefore the absolute configuration of C-12 was assigned as S. Actinosporin G (23) gave a peak in the positive HRESIMS spectrum at m/z 473.1207 [M + Na]+, consistent with a molecular formula of C25H22O8. The NMR data of 23 were very similar to 20, with the difference in the resonances related to the rhamnose moiety. One anomeric signal at δH 5.64 (δC 98.6) and one methyl resonance at δH 1.29 (δC 17.8) indicated

3.20) indicated that ring A was nonaromatic. Ring A was further confirmed based on correlations of the methyl protons H3-13 (δH 1.34) to the carbons C-2 (δC 53.0), C-3 (δC 71.7), and C-4 (δC 43.3); correlations of methylene protons H-2 to C1 (δC 196.7) and C-4 (δC 43.3); and correlations of the methylene protons H-4 to C-2 (δC 53.0) and C-12b (δC 134.2). The position of the hydroxy group on C-3 was deduced through the tertiary carbinol resonance at δC 71.7. The presence of an anomeric proton at δH 5.64 along with a methyl doublet in the aliphatic region at δH 1.08 (J = 6.1 Hz) indicated the presence of a rhamnose moiety in the structure, which was further confirmed by the characteristic chemical shift for the anomeric carbon at 98.3 ppm. An HMBC experiment revealed that the sugar was O-linked to the aglycone at C-8 (3JCH correlation between H-1′ at δH 5.48 to C-8 at δC 155.6). On the basis of the magnitude of the coupling constant of the anomeric proton (J = 1.8 Hz) as well as 1JCH of 172.9 Hz between H-1′ and C-1′, the configuration of the anomeric proton of the rhamnose residue was established to be α. The absolute configuration of the sugar was determined to be L (Figure S27) by GC-MS analysis after hydrolysis of compound 21, followed by reaction of the sugar with L-cysteine methyl ester and subsequent acetylation.68 The absolute configuration of C-3 was established upon comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. The experimental ECD spectrum of actinosporin E showed two positive Cotton effects (CEs) at 380 and 220 nm as well as two negative CEs at 330 and 270 nm. Because the sugar moiety has little influence on the ECD spectra69 and also to reduce calculation time, the rhamnose moiety was omitted for ECD calculations. Conformational analysis of both R and S enantiomers using the OPLS-2005 force field within a 2 kcal/ mol energy window followed by DFT/B3LYP/6-31G(d,p) optimization showed the presence of 12 conformers for each enantiomer. Calculation of the ECD spectra was carried out using TDDFT/CAM-B3LYP/6-31G(d,p) in MeOH. Comparison of the calculated and the experimental ECD spectra (Figure 4) showed agreement with the R enantiomer; thus the absolute configuration of C-3 in compound 21 was assigned to be R. The molecular formula of C31H34O12 was established for actinosporin F (22) based on HRESIMS. The NMR data (Table 1) indicated that 22 was related to the known 832



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experiments were performed with a MeOH−H2O gradient solvent system. Milipore Milli-Q PF filtered H2O and HPLC grade solvents were used for chromatography. Microbial Fermentation and Extract Preparation. Nocardiopsis sp. SBT366, Streptomyces sp. SBT343, Geodermatophilus sp. SBT350, Streptomyces sp. SBT345, Streptomyces sp. SBT346, and Micromonospora sp. SBT373 were cultivated from the marine sponges Chondrilla nucula, Petrosia f iciformis, Chondrilla nucula, Agelas oroides, Petrosia ficiformis, and Chondrilla nucula, respectively. Rhodococcus sp. RV157 and Micromonospora sp. RV43 were isolated from the Mediterranean sponges Dysidea avara and Aplysina aerophoba, while Actinokineospora sp. EG49 was cultivated from the Red Sea sponge Spheciospongia vagabunda.71 Each strain was fermented in 10 Erlenmeyer flasks (2 L), each containing 1 L of ISP 2 medium in artificial seawater (Sigma-Aldrich), and incubated at 30 °C for 7 days with shaking at 150 rpm. For the elicitation experiments, N-acetyl-Dglucosamine (50 μM, Sigma-Aldrich) was added immediately after inoculation. After fermentation, the broth was filtered, and the supernatant was extracted with EtOAc (2 × 5 L) to yield the EtOAc extract. The cells and mycelia were macerated in a double volume of MeOH with shaking overnight and then filtered (methanolic extract). Chromatographic Procedures for Small-Scale Fractionation. The EtOAc extracts (6 mg) were dissolved in DMSO (600 μL), then divided into five increments and analyzed by analytical HPLC using a Phenomenex Onyx Monolithic C18 column (4.6 × 100 mm). The HPLC gradient was as follows: a linear gradient was performed from 90% H2O−10% MeOH to 50% H2O−50% MeOH in 3 min at a flow rate of 4 mL/min followed by a convex gradient to 100% MeOH in 3.5 min at a flow rate of 3 mL/min; held at 100% MeOH for 0.5 min at a flow rate of 3 mL/min; held at 100% MeOH for 1 min at a flow rate of 4 mL/min; then a linear gradient to 90% H2O−10% MeOH in 1 min at a flow rate of 4 mL/min, then held in the same condition for 2 min. Fractions collected from analytical HPLC were then dried for analysis by 1H NMR. Chromatographic Procedures for Large-Scale Fractionation and Isolation. EtOAc extracts (60 mg) were preadsorbed to C18bonded silica and then packed into a stainless steel HPLC guard cartridge (10 × 30 mm) that was subsequently attached to a semipreparative Phenomenex Onyx Monolithic reversed-phase C18 HPLC column (10 × 100 mm). Initially isocratic conditions of 10% MeOH were used for 10 min; then a linear gradient from 10% to 100% MeOH was performed over 40 min and continued isocratic for 10 min at a flow rate of 9 mL/min. Sixty fractions collected in 1 min increments over 60 min were dried for NMR and mass spectrometry studies. Metabolites Identified from Untreated Micromonospora sp. RV43. Compound 1 (1.8 mg) eluted in fraction 4; the phenolic compounds 2 (0.3 mg), 3 (1.1 mg), and 4 (1.1 mg) were eluted in fractions 9, 6, and 8, respectively. The metabolite 5 was eluted from fraction 2, and the polyesters 6 [1.7 mg, [α]31D −38.4 (c 0.045, CHCl3)], 7 [2.4 mg, [α]31D −30 (c 0.05, CHCl3)], 8 [1.6 mg, [α]31D −16.2 (c 0.025, CHCl3)], 9 (0.8 mg), and 10 (0.6 mg) were obtained from fractions 11, 23, 33, 42, and 48, respectively. Metabolites Identified from Micromonospora sp. RV43 Treated with GlcNAc. Compound 11 (0.3 mg) eluted in fraction 15, and compound 12 (1.4 mg) was obtained from fraction 24. Metabolites Identified from Untreated Rhodococcus sp. RV157. Compounds 13 (1.7 mg) and 14 (0.6 mg) eluted in fractions 6 and 9, respectively, and different isomers of avenalumic acid (15) were eluted in fractions 20 (0.2 mg) and 23 (0.1 mg). Metabolites Identified from Rhodococcus sp. RV157 Treated with GlcNAc. Bacillibactin (16) (1.2 mg) eluted in fraction 26, and surfactin lipopeptides eluted as a mixture in fractions 45 to 48. In order to purify surfactin 17, fraction 46 (8.5 mg) was subjected to HPLC using a semipreparative Phenomenex Onyx Monolithic reversed-phase C18 column (10 × 100 mm). A linear gradient at a flow rate of 9 mL/ min from 75% to 90% MeOH was performed over 50 min followed by a gradient to 100% MeOH over 5 min, then stayed isocratic for 5 min. Sixty fractions were collected in 1 min increments and were dried for MS and NMR analyses. Compound 17 eluted in subfractions 29 to 31.

the presence of only one rhamnose moiety in 23. The position of the sugar moiety on the structure was determined on the basis of the HMBC correlation of the anomeric proton resonance at δH 5.64 to the C-8 at δC 155.4. The coupling constant value of the anomeric proton was consistent with an α configuration, and GC-MS analysis confirmed the absolute configuration to be L. A molecular formula of C25H22O9 was established for actinosporin H (24) by positive HRESIMS. Similar NMR data to those of compound 19 were observed with the exception of the sugar signals. Signals of only one rhamnose moiety were detected in the NMR spectra of 24. Connection of the rhamnose to C-1 was established based on an HMBC correlation from the anomeric proton at δH 5.49 (H-1′) to C-1 at δC 155.1. The coupling constant of the anomeric proton allowed identification of an α configuration, and the absolute configuration of the sugar was also identified as L by GC-MS.

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CONCLUSION In the present study, 1H NMR fingerprinting was used to investigate GlcNAc-mediated elicitation of the metabolome of three actinomycetes, Micromonospora sp. RV43, Rhodococcus sp. RV157, and Actinokineospora sp. EG49. The metabolic profiles of all three investigated bacteria changed upon addition of GlcNAc to their culture media. Upon elicitation with GlcNAc, Micromonospora sp. RV43 stopped the production of secondary metabolites 1−10 and initiated production of compounds 11 and 12. Upon exposure to GlcNAc, Rhodococcus sp. RV157 produced bacillibactin 16 and the surfactin lipopeptide 17. The compounds 16 and 17 were not identified in the extract of the untreated sample. Here we report, for the first time, identification of bacillibactin and surfactin lipopeptides from a Rhodococcus species. Elicitation of Actinokineospora sp. EG49 with GlcNAc boosted the production of low-yielding compounds 20−24. These results highlight that the elicitation with GlcNAc represents an effective strategy for the induction of silent biosynthetic pathways and thus increases the chemical diversity of microbial natural products. NMR fingerprinting was shown to be effective to follow metabolism of the three actinomycetes. GlcNAc was shown to have multiple effects including suppression of metabolites, induction of new metabolites, and increased production of minor compounds.

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GENERAL EXPERIMENTAL PROCEDURES

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter with a 10 cm cell. UV spectra were acquired on a Jasco V650 UV/vis spectrophotometer. A JASCO J-715 spectropolarimeter was used to record circular dichroism spectra. IR spectra were recorded on a Bruker Tensor 27 spectrophotometer (Billerica, MA, USA). NMR spectra were recorded on Varian Inova 500, Varian Inova 600, or Bruker 900 MHz. The 1H and 13C NMR chemical shifts were referenced to the solvent peaks at δH 2.50 and δC 39.5 for DMSO-d6 or δH 3.31 and δC 49.0 for CD3OD. For NMR fingerprint experiments, fractions were dissolved in 230 μL of DMSO-d6 and run in a 3 mm NMR tube. The standard VnmrJ 3.2 Proton pulse sequence was run on a Varian Inova 600 MHz spectrometer with the following parameters: pw = 45°, p1 = 0 μs, d2 = 0 s, d1 = 1 s, at = 1.7 s, sw = 9615 Hz, nt = 512 scans. A Waters ZQ electrospray mass spectrometer with a Phenomenex Luna C18 column (4.6 × 50 mm, 3 μm) was used for LC-MS analyses. A Phenomenex Onyx Monolithic C18 column (4.6 × 100 mm) and a Phenomenex Onyx Monolithic C18 column (10 × 100 mm) were used for analytical and semipreparative HPLC, respectively. All HPLC and LC-MS 833



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Surfactin 17: amorphous, white solid (3.2 mg); 1H and 13C NMR data were in good agreement with those reported in the literature;58 [α]25D −26 (c 0.1, MeOH); HRESIMS m/z 1030.6412 [M + Na]+ (calcd for C51H89N7O13Na, 1030.6416). Metabolites Identified from Actinokineospora sp. EG49 Treated with GlcNAc. The known metabolites 18−20 eluted in fractions 21, 26, and 23, respectively. Compound 21 eluted in fraction 17 as a mixture with diketopiperazines. The metabolite was then purified by solvent/solvent extraction using H2O−CHCl3, and the compound partitioned in the H2O extract. Compound 22 eluted as a mixture in fraction 24. In order to purify the compound, fraction 24 was subjected to HPLC using a semipreparative Phenomenex Onyx Monolithic reversed-phase C18 column (10 × 100 mm). A linear gradient at a flow rate of 9 mL/min from 30% to 50% MeOH was performed over 50 min followed by a gradient to 100% MeOH over 5 min, then stayed isocratic for 5 min. Sixty fractions were collected in 1 min increments, and compound 22 eluted in fraction 29. Compound 23 eluted in fraction 34 as an impure metabolite, and the fraction therefore was subjected to semipreparative HPLC using a Phenomenex Onyx Monolithic reversed-phase C18 column (10 × 100 mm). A linear gradient at a flow rate of 9 mL/min from 50% to 70% MeOH was performed over 50 min followed by a gradient to 100% MeOH over 5 min, then stayed isocratic for 5 min. Sixty fractions were collected in 1 min increments, and compound 23 eluted in fractions 22 and 23. Compound 24 was also obtained as an impure metabolite from fraction 36. In order to purify the compound, the same HPLC condition as used for compound 23 was utilized and compound 24 eluted in fractions 27 and 28. Actinosporin E, 21: amorphous, yellow solid (0.8 mg); [α]24D −54 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 220 (4.03), 260 (3.95), 311 (3.56) nm; ECD (0.05 mg/mL, MeOH), λmax (Δε) 383 (0.5), 323 (−0.6), 268 (−1.7), and 220 (1) nm; IR νmax 3664, 3392, 2972, 2901, 1673, 1591, 1407, 1395, 1383, 1252, 1233, 1066, 893, 881 cm−1; 1H NMR (900 MHz, DMSO-d6) and 13C NMR (225 MHz, DMSO-d6), Table 1; HRESIMS m/z 491.1314 [M + Na]+ (calcd for C25H24NaO9, 491.1312). Actinosporin F, 22: amorphous, yellow solid (1.5 mg); [α]25D −144 (c 0.025, MeOH); UV (MeOH) λmax (log ε) 223 (4.37), 279 (4.15) nm; ECD (0.1 mg/mL, MeOH), λmax (Δε) 373 (−2.8), 310 (1.2), 281 (6.7), 264 (−1), 237 (−1.2), 226 (2.8), and 210 (−2.2) nm; IR νmax 3664, 3404, 2972, 2903, 1651, 1597, 1455, 1407, 1395, 1384, 1269, 1232, 1076, 1065, 956, 893, 765 cm−1; 1H NMR (900 MHz, DMSOd6) and 13C NMR (225 MHz, DMSO-d6), Table 1; HRESIMS m/z 621.1943 [M + Na]+ (calcd for C31H34NaO12, 621.1942). Actinosporin G, 23: amorphous, red solid (1.3 mg); [α]25D +130 (c 0.003, MeOH); UV (MeOH) λmax (log ε) 225 (4.23), 310 (3.88) nm; 1 H NMR (600 MHz, DMSO-d6) δH 8.18 (1H, d, J = 8.6 Hz, H-5), 8.09 (1H, d, J = 8.6 Hz, H-6), 7.80 (1H, t, J = 8.1 Hz, H-10), 7.73 (1H, d, J = 8.1 Hz, H-11), 7.63 (1H, d, J = 8.1 Hz, H-9), 7.35 (1H, s, H-4), 6.97 (1H, s, H-2), 5.64 (1H, brs, H-1′), 4.00 (1H, m, H-2′), 3.98 (1H, m, H-3′), 3.53 (1H, m, H-5′), 3.35 (1H, m, H-4′), 2.44 (1H, s, H-13), 1.09 (1H, d, J = 6.3 Hz, H-6′); 13C NMR (125 MHz, DMSO-d6) δC 187.2 (C-12), 180.7 (C-7), 155.4 (C-8), 154.7 (C-1), 140.5 (C-3), 137.6 (C-4a), 137.6 (C-11a), 135.2 (C-10),134.8 (C-5), 134.4 (C-6a), 132.8 (C-12a), 121.9 (C-9), 121.8 (C-6), 120.4 (C-7a), 120.3 (C11),119.5 (C-4), 117.9 (C-12b), 116.7 (C-2), 98.6 (C-1′), 71.7 (C-4′), 70.1 (C-2′), 70.1 (C-3′), 70.1 (C-5′), 21.0 (C-13), 17.8 (C-6′); HRESIMS m/z 473.1207 [M + Na]+ (calcd for C25H22NaO8, 473.1206). Actinosporin H, 24: amorphous, orange solid (0.8 mg); [α]25D +140 (c 0.004, MeOH); UV (MeOH) λmax (log ε) 232 (3.86), 308 (3.50), 430 (3.21) nm; 1H NMR (600 MHz, CD3OD) δH 7.75 (1H, t, J = 8.0 Hz, H-10), 7.59 (1H, d, J = 8.0 Hz, H-11), 7.42 (1H, s, H-5), 7.26 (1H, d, J = 8.0 Hz, H-9), 7.22 (1H, s, H-4), 7.13 (1H, s, H-2), 5.49 (1H, brs, H-1′), 4.03 (1H, m, H-2′), 3.81 (1H, dd, J = 9.4, 3.5 Hz, H-3′), 3.77 (1H, m, H-5′), 3.49 (1H, t, J = 9.4 Hz, H-4′), 2.47 (1H, s, H-13), 1.29 (1H, d, J = 6.2 Hz, H-6′); 13C NMR (150 MHz, CD3OD) δC 186.8 (C-12), 162.8 (C-8), 157.4 (C-6), 155.1 (C-1), 143.1 (C-3), 138.5 (C-10), 137.8 (C-4a), 138.0 (C-11a), 141.7 (C-12a), 123.9 (C9), 120.2 (C-4), 120.1 (C-6a), 118.9 (C-11), 117.8 (C-5), 117.0 (C-

12b), 116.2 (C-7a), 112.4 (C-2), 100.5 (C-1′), 73.7 (C-4′), 72.1 (C3′), 71.6 (C-2′), 70.9 (C-5′), 21.9 (C-13), 17.8 (C-6′); HRESIMS m/z 489.1154 [M + Na]+ (calcd for C25H22NaO9, 489.1158). The structures of all known compounds were confirmed upon comparison of spectrometric (low- and high-resolution MS) and spectroscopic (1H, 13C, and 2D, where necessary) data with published literature values. Determination of the Absolute Configurations of the Sugars. Compounds 21−24 (0.5 mg each) were hydrolyzed with 250 μL of 2 N trifluoroacetic acid at 120 °C for 1 h. The hydrolyzed products were then dried under a nitrogen flow, and the residue was dissolved in 100 μL of dry pyridine. The pyridine solutions of the hydrolyzed compounds 21−24 as well as pure D- and L-rhamnose (100 μL of a 0.04 mol/L solution) were reacted with 100 μL of a pyridine solution of L-cysteine methyl ester hydrochloride (0.06 mol/L) at 60 °C for 1 h. Acetic anhydride (150 μL) was then added to the reaction mixtures and kept at 120 °C for 20 min. The mixtures were dried under a nitrogen flow and redissolved in 1 mL of CH2Cl2, and the polar material (underivatized or partially derivatized sugars) was extracted with H2O (1 mL). The organic phase was then dried under a nitrogen flow and dissolved in 200 μL of MeOH for GC-MS analysis. The GC-MS conditions were as follows: injection temperature 250 °C; initial oven temperature 45 °C, then increased linearly to 300 °C at 20 °C/min, then held for 17.25 min. Analyses were performed on a 30 m length Restek (Rxi-5 ms) diphenyl dimethyl polysiloxane column with an internal diameter of 0.25 mm. Conformational Analysis, Geometrical Optimization, and ECD Calculations. Conformational analyses of compounds 21 and 22 were performed with Schrödinger MacroModel 9.1 software using the OPLS 2005 (Optimized Potential for Liquid Simulations) force field in H2O. Conformers occurring within a 2 kcal/mol energy window from the global minimum were chosen for geometrical optimization and energy calculation using density functional theory (DFT) with the B3LYP functional and the 6-31G(d,p) basis set in the gas phase with the Gaussian 09 program.72 Vibrational analysis was done at the same level to confirm minima. The ECD spectra calculations were performed using TD-DFT/CAM-B3LYP/6-31G(d,p) in MeOH using the SCRF method, with the CPCM model. ECD curves were reconstructed on the basis of rotator strengths with a halfband of 0.25 eV using SpecDis v1.61.73 The spectra were combined after Boltzmann weighting according to their population contribution.

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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.6b00673. NMR spectra, low-energy conformations and their Boltzman population, Cartesian coordinates of the optimized geometry (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61-7-3735-6000. Fax: +61-7-3735-6001. E-mail: r. quinn@griffith.edu.au. ORCID

Tanja Grkovic: 0000-0002-6537-3997 Ronald J. Quinn: 0000-0002-4022-2623 Present Address §

Department of Pharmacognosy, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt. Notes

The authors declare no competing financial interest. 834



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ACKNOWLEDGMENTS This research was supported under the Australian Research Council’s Discovery Projects funding scheme (project number DP160101429). Y.D. acknowledges Griffith University for the provision of the Griffith University International Postgraduate Research Scholarship (GUIPRS) and Griffith University Postgraduate Research Scholarship (GUPRS). U.H. received financial support from DFG (SFB 630 TP A5). The authors wish to acknowledge funding from the Australian Research Council for support toward NMR and MS equipment (LE0668477 and LE0237908).

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