Inducing Secondary Metabolite Production by Co-culture of the

The results of this work indicated that the production of 1–5 involved a ... NMR and DEPT data (Table 1) of 1 indicated one methyl, three methylene,...
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Inducing Secondary Metabolite Production by Co-culture of the Endophytic Fungus Phoma sp. and the Symbiotic Fungus Armillaria sp. Hong-Tao Li,# Hao Zhou,# Rong-Ting Duan, Hong-Yu Li, Lin-Huan Tang, Xue-Qiong Yang, Ya-Bin Yang, and Zhong-Tao Ding* Key Laboratory of Functional Molecules Analysis and Biotransformation, Yunnan Provincial Department of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, People’s Republic of China J. Nat. Prod. Downloaded from pubs.acs.org by MIDWESTERN UNIV on 02/20/19. For personal use only.

S Supporting Information *

ABSTRACT: Co-culturing the endophytic fungus Phoma sp. YUD17001 from Gastrodia elata with Armillaria sp. in liquid nutrient medium resulted in the production of five new secondary metabolites, including two phenolic compounds, phexandiols A and B (1 and 2), three aliphatic ester derivatives, phomesters A−C (3−5), and a known fatty acid (6). The structures and absolute configurations of these compounds were elucidated by the interpretation of data from detailed spectroscopic analysis, Mosher’s method, and electronic circular dichroism spectra, together with consideration of the biogenetic origins. None of the five new compounds were detected in single-strain cultures under identical fermentation conditions. The results of this work indicated that the production of 1−5 involved a complicated interaction process. None of the new compounds possessed significant cytotoxicity or antimicrobial activities.

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the known examples is the fungal community associated with Gastrodia elata (Orchidaceae) habitats. G. elata, a prominent traditional Chinese herbal medicine, is a rootless and leafless achlorophyllous orchid. It requires a symbiotic relationship with the mycorrhizal fungi Armillaria sp. (Tricholomataceae) during its vegetative growth.17,18 We were attracted by this special symbiotic community. The secondary metabolites induced by fungal co-culture of the endophytic fungus Phoma sp. YUD17001 and the symbiont Armillaria sp. were investigated. The new components produced in this system were initially detected by TLC (Figure S1) and then confirmed by analytical HPLC (Figure S2) and were not present in cultures of pure strains. This led to the discovery of five new secondary metabolites. Herein, the detailed isolation and structure elucidation process of 1−5 are presented.

icroorganisms with novel skeleton structures and pronounced biological activities have historically served as significant sources for drug leads.1,2 The vast majority of previous studies have involved the isolation of secondary metabolites from microbes grown in pure culture.3,4 In some cases, biosynthetic pathways are not active in single-strain culture, resulting in unrealized metabolic potential.5 Cocultivation of microorganisms has been described as a promising strategy to induce the production of novel metabolites.6−9 In the co-culture system, two strains grow together either symbiotically or in competition for nutrients and space, establishing an equilibrium population and lifestyle state.9,10 The resulting interactions may lead to induced production of new secondary metabolites not generated by individual strains.11−14 Until now, most co-cultivation research has focused on strains from completely unrelated environments,2 whereas the co-cultivation of microorganisms from the same or similar habitats remains an intriguing topic with unforeseen implications.15,16 Indeed, microorganism co-culture is inspired by the natural microbe communities that are omnipresent in nature.15 One of © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 14, 2018

A

DOI: 10.1021/acs.jnatprod.8b00685 J. Nat. Prod. XXXX, XXX, XXX−XXX

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methine, and two nonprotonated carbons. The planar structure of 1 was further elucidated by 2D NMR spectroscopic analysis (Figure 1). The presence of the ortho-disubstituted phenyl and

Figure 1. Key 1H−1H COSY, HMBC, and ROESY correlations of 1 and 2.

alkyl chain was confirmed by the 1H−1H COSY correlations of H-2/H-3/H-4/H-5 and H-7/H-8/H2-9/H2-10/H2-11/H3-12. In the HMBC spectrum, the long-range correlations from H-7 at δH 4.77 (d, 1H, J = 6.0 Hz) to C-6 (δC 129.2), C-1 (δC 156.2), and C-5 (δC 169.2) were observed, indicating the phenyl and alkyl chains were connected by C-6 (δC 129.2) and C-7 (δC 74.8) (Figure 1). Furthermore, considering the molecular formula and NMR data, the remaining three protons should be present in the form of hydroxy protons assigned to C-1 (δC 156.2), C-7 (δC 74.8), and C-8 (δC 76.1). Consequently, the planar structure of 1 was established as in Figure 1. The relative stereochemistry of 1 was deduced from the ROESY experiment (Figure 1). The significant ROESY correlations of H-7 with H-8 and of H-5 with H-7 and H-8

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RESULTS AND DISCUSION Phexandiol A (1) was isolated as a pale yellow oil, and the molecular formula C12H18O3 was determined by HR-ESIMS, implying the presence of four degrees of unsaturation. The IR spectrum displayed absorption peaks at 3385, 1623, 1509, 1220, and 1099 cm−1, indicating the presence of hydroxy groups and double bonds. The 1H NMR data of 1 (Table 1) showed four adjacent aromatic protons at δH 6.76 (d, 1H, J = 8.1 Hz), 6.81 (t, 1H, J = 7.2 Hz), 7.07 (t, 1H, J = 7.8 Hz), and 7.25 (d, 1H, J = 7.8 Hz). The 13C NMR and DEPT data (Table 1) of 1 indicated one methyl, three methylene, six Table 1. 1H and

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C NMR Spectroscopic Data for Compounds 1−5 (Methanol-d4)

1a no.

δC, type

1 2

156.2, C 116.5, CH

3 4 5

129.3, CH 120.4, CH 129.2, CH

6

129.2, C

7

74.8, CH

8 9 10

76.1, CH 33.6, CH2 29.2, CH2

11 12 13

δH (J in Hz) 6.76, d (8.1) 7.07, t (7.8) 6.81, t (7.2) 7.25, d (7.8)

2b δC, type 161.5, C 126.6, CH 121.6, CH 131.0, CH 110.9, CH

δH (J in Hz) 7.35, d (7.6) 6.90, t (7.2) 7.20, t (7.6) 6.79, d (8.0)

130.2, C

94.6, CH 71.9, CH 35.8, CH2

23.6, CH2

4.77, d (6.0) 3.75, m 1.39, m 1.46, m 1.26, m 1.25, m

73.9, CH

14.3, CH3

0.85, t (7.2)

14.4, CH3

19.9, CH2

5.32, d (3.6) 4.30, t (4.0) 3.68, m 1.60, m 1.52, m 1.60, m 1.42, m 0.96, t (7.2)

3b δC, type 175.9, C 34.6, CH2

4b

δH (J in Hz)

δC, type

2.35, t (7.2)

175.3, C 34.7, CH2

22.1, CH2 37.6, CH2 73.2, CH

1.66, m 1.51, m 4.00, q (6.4)

136.4, CH 128.9, CH 41.3, CH2 72.0, CH 40.0, CH2 19.9, CH2 14.4, CH3 52.0, OCH3

δH (J in Hz)

5b δC, type

δH (J in Hz)

2.38, t (7.2)

175.4, C 34.9, CH2

2.34, t (7.2)

22.1, CH2 37.6, CH2 73.2, CH

1.68, m 1.52, m 4.02, m

22.2, CH2 37.6, CH2 73.2, CH

1.67, m 1.51, m 4.00, m

5.49, dd (15.6, 6.8) 5.68, m

136.4, CH

5.49, dd (15.6, 6.8) 5.68, m

136.4, CH

5.49, dd (15.2, 6.8) 5.68, m

2.19, 3.60, 1.46, 1.39, 1.48, 1.36, 0.93, 3.66,

41.3, CH2 71.9, CH 40.0, CH2

2.19, m 3.59, m 1.45, m 1.38, m 1.47, m 1.35, m 0.93, t (6.8) 4.15, dd (11.2, 4.4) 4.06, dd (11.2, 6.0) 3.82, m 3.56, d (0.8) 3.54, d (1.6)

41.3, CH2 72.0, CH 39.9, CH2

14 15

m m m m m m t (6.4) s

128.9, CH

19.8, CH2 14.4, CH3 66.8, CH2

71.1, CH 64.0, CH2

16 17 18 19

128.9, CH

14.4, CH3 65.4, CH2

2.19, 3.58, 1.45, 1.37, 1.46, 1.34, 0.93, 4.07,

29.4, CH2 24.4, CH2

1.65, m 1.38, m

30.0, CH2 40.3, CH2 173.2, C 22.5, CH3

1.51, m 3.16, t (6.8)

19.9, CH2

m m m m m m t (6.0) t (6.8)

1.93, s

a

Measured at 600 or 150 MHz for 1H and 13C NMR, respectively. bMeasured at 400 or 100 MHz for 1H and 13C NMR, respectively. B

DOI: 10.1021/acs.jnatprod.8b00685 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. Experimental and calculated ECD spectra of compounds 1 and 2.

data of 3 were similar to those of (E)-5,9-dihydroxydodec-6enoic acid (6),19 which was also isolated from both the coculture system extracts and the Phoma sp. YUD17001. The methoxy group (δC 52.0, δH 3.66) in 3, together with the chemical shift of C-1 (δC 175.9), indicated the presence of a methyl ester. This was confirmed by the correlation of H3-13 (δH 3.66) with C-1 in the HMBC spectrum (Figure 3).

suggested that there were two possible isomers [(7R,8R)-1 and (7S,8S)-1] of compound 1. To resolve the stereochemical dilemma, the absolute configuration of 1 was elucidated by comparison of the experimental electronic circular dichroism (ECD) data and the quantum chemical time-dependent density functional theory (TDDFT)-calculated ECD. Each isomer was optimized using DFT at the B3LYP/6-311+G(d,p) level in the Gaussian 09 program. The optimized isomer was calculated using TDDFT/GIAOs at the B3LYP/6-311+G(d,p) level in the Gaussian 09 program to generate its specific rotation. The agreement of the calculated ECD spectrum of the (7R,8R)-1 isomer with the experimental spectrum confirmed the absolute configuration of 1 as (7R,8R)-1 (Figure 2a). Phexandiol B (2) was obtained as a colorless oil. The 13C NMR spectrum of 2 displayed 12 signals including two nonprotonated carbons, four possible aromatic methines (δC 131.0, 126.6, 121.6, and 110.9), three oxygenated methines (δC 94.6, 73.9, and 71.9), two methylenes, and one methyl moiety. The 1H NMR data of 2 revealed the presence of four adjacent aromatic protons at δH 6.79 (d, 1H, J = 8.0 Hz), 6.90 (t, 1H, J = 7.2 Hz), 7.20 (t, 1H, J = 7.6 Hz), and 7.35 (d, 1H, J = 7.6 Hz). The signal systems of the NMR of 2 resembled those of compound 1, suggesting structural similarity. The most evident difference was that one of the methylene signals for compound 1 was replaced by an oxygenated methine (C-9, δC 71.9) in 2. The molecular formula of 2 was deduced as C12H16O3 on the basis of its HR-ESIMS, requiring one more degree of unsaturation than compound 1. Further aided by 1H−1H COSY and HMBC experiments (Figure 1), the planar structure of 2 was established as dihydrobenzofuran with the C-9 hydroxylation as in 1. Its relative configuration was proposed on the basis of key ROESY correlations (Figure 1). The observation of ROESY correlations between H-7 and H-8 combined with no significant correlations of H-9 with H-7 and H-8 suggested that there were two possible isomers, (7S,8S,9R)-2 and (7R,8R,9S)-2. The absolute configuration of 2 was elucidated as (7S,8S,9R)-2 by comparing its experimental ECD spectrum to those calculated (Figure 2b). The structure of 2 was assigned as shown. Phomester A (3) was isolated as a colorless oil with the molecular formula C13H24O4, as determined by HR-ESIMS, requiring two degrees of unsaturation. The 1H and 13C NMR

Figure 3. Key 1H−1H COSY and HMBC correlations of 3−5.

Ultimately, taking into account the 2D NMR spectral data, the planar structure of 3 was identified as depicted in Figure 3. To determine the absolute configuration of compound 3, the theoretical ECD was compared to the experimental spectrum; this was not conclusive. Accordingly, 3 was converted to the Mosher esters 3a and 3b with (S)- and (R)-MTPA-Cl.20 The chemical shift differences ΔδSR were significant (Figure 4) and showed that compound 3 should have S configurations at both the C-5 and C-9 positions. The structure of 3 was defined as shown. Phomester B (4) was obtained as a colorless oil with a molecular formula of C15H28O6 deduced from its HR-ESIMS. Comparison of the NMR data (Table 1) for 4 with those of 3

Figure 4. Reactions of compound 3 with Mosher esters. C

DOI: 10.1021/acs.jnatprod.8b00685 J. Nat. Prod. XXXX, XXX, XXX−XXX

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internal transcribed spacer (ITS) sequencing, and basic local alignment search tool (BLAST) searching followed.21 The sequence is available at GenBank under the accession number MH665638. The result from the BLAST search indicated that the sequence was highly similar (100%) to that of Phoma sp. QC-2014a (GenBank accession no. KP330435). The strain was identified as Phoma sp. YUD17001. The rhizomorphs of Armillaria sp. were obtained from the epidermis of G. elata collected from the same location and at the same time as YUD17001. The rhizomorphs were surface sterilized with 96% ethanol for 3 min. Small segments of the white inner parts of the fresh rhizomorphs were excised and placed on malt extract agar (MEA: 20 g L−1 malt extract, 2 g L−1 yeast extract, 16 g L−1 agar). The isolates were then incubated at 24 °C for 2 weeks. Rhizomorph tips that developed from the primary cultures were transferred to PDA medium and further incubated under the same conditions. This procedure was repeated until the pure cultures were obtained. The strain was identified by 18S rRNA amplification and sequence analysis of the ITS genes. NCBI-BLAST analysis of these sequences identified Armillaria sp. 2TG-2016 (accession no. KT822279) among the top matches with 99% identity,21 and it was identified as Armillaria sp. (GenBank accession no. MK079569). Both strains were deposited at the School of Chemical Science and Technology, Yunnan University, China. Co-culture Conditions. Liquid co-cultures were established by adding precultures of Armillaria sp. and Phoma sp. YUD17001 to fermentation media. The spores of both Armillaria sp. and Phoma sp. YUD17001 were inoculated into 250 mL Erlenmeyer flasks each containing 50 mL of potato dextrose broth medium (PDB) and incubated at 28 °C on a rotary shaker at 180 rpm. Armillaria sp. was cultured for 7 days and Phoma sp. YUD17001 for 3 days, to generate precultures. The precultures (25 mL) of both Armillaria sp. and Phoma sp. YUD17001 fungal strains were simultaneously transferred to 500 mL Erlenmeyer flasks containing 150 mL of PDB medium and incubated at 28 °C and 200 rpm for 14 days to establish mature cocultures (5 L total). Extraction and Isolation. The mycelia were removed from the co-cultures (5 L) by filtration. The filtrate was extracted with ethyl acetate (EtOAc) three times, and the solvent was removed under vacuum to obtain the EtOAc extract (2.8 g). The EtOAc extract was separated into five fractions (Fr. 1 to Fr. 5) by column chromatograph on silica gel, eluting stepwise with a CHCl3−MeOH gradient (CHCl3, CHCl3−MeOH = 30:1 v/v, CHCl3−MeOH = 10:1 v/v, CHCl3− MeOH = 1:1 v/v, MeOH). Fr. 1 was isolated by a silica gel column with a petroleum ether−ethyl acetate mixture (10:1 v/v) to afford 2 (3.8 mg). Fr. 2 was divided into three parts (Fr. 2-1 to Fr. 2-3) by a Sephadex LH-20 column with MeOH; then Fr. 2-1 was isolated by a Lichroprep RP-18 column with MeOH−H2O (70:30 v/v) to obtain 3 (9.0 mg). Fr. 2-3 was fractionated by a silica gel column with CHCl3− acetone (20:1 v/v) to afford 1 (3.5 mg). Fr. 3 was divided into two parts (Fr. 3-1 and Fr. 3-2) by a Lichroprep RP-18 column with MeOH−H2O (25:75−80:20 v/v), and Fr. 3-1 was separated by semipreparative HPLC with MeOH−H2O (45:55 v/v) to afford 4 (4.8 mg). Fr. 3-2 was fractionated by a Sephadex LH-20 column with MeOH to obtain 5 (2.0 mg). Fr. 4 was fractionated by a silica gel column with CHCl3−acetone (2:1 v/v) to afford 6 (4.0 mg). Phexandiol A (1): pale yellow oil; [α]23D +4.0 (c 0.14, MeOH); UV−vis (MeOH) λmax (log ε) 223 (3.75), 273 (1.22) nm; IR (KBr) νmax 3385, 1623, 1509, 1220, and 1099 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 233.1147 [M + Na]+ (calcd for C12H18O3Na, 233.1148). Phexandiol B (2): colorless oil; [α]23D +70.0 (c 0.11, MeOH); UV−vis (MeOH) λmax (log ε) 225 (4.01), 280 (1.59) nm; IR (KBr) νmax 3352, 1629, 1503, 1478, and 1172 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 231.0990 [M + Na]+ (calcd for C12H16O3Na, 231.0992). Phomester A (3): colorless oil; [α]23D +1.1 (c 0.18, MeOH); UV− vis (MeOH) λmax (log ε) 229 (1.04), 258 (1.10) nm; IR (KBr) νmax 3426, 2923, and 1729 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 267.1563 [M + Na]+ (calcd for C13H24O4 Na, 267.1567).

suggested that both compounds contain compound 6 as a moiety. The remaining spectral data indicated the presence of a glycerol moiety. Further analysis of the 2D NMR spectra (Figure 3) indicated 4 is the glyceride of (E)-5,9-dihydroxydodec-6-enoic acid. Meanwhile, the absolute configuration of 4 was elucidated as 5S, 9S, and 14R by the Mosher method (details in Supporting Information, Figure S3). Phomester C (5), isolated as a colorless oil, had the molecular formula C19H35NO5 as determined by HR-ESIMS. Comparison of the 1D NMR data with those of 3 and 4 (Table 1) confirmed the presence of 6 as a moiety in 5. In the 1H−1H COSY spectrum of 5, vicinal homonuclear coupling correlations from H2-13 to H2-17 suggested the alkyl chain extended from C-13 to C-17, and the HMBC correlations from H2-13 to C-1 revealed the connection of C-1 and C-13 via an ester bond. The HMBC correlation from H3-19 to C-18 indicated the presence of an acetyl group, linked to C-17 through an amide bond, as shown by the HMBC correlation from H2-17 to C-18, together with the chemical shift of C-17 (δC 40.3) and the molecular formula. Consequently, the planar structure of 5 was identified as illustrated in Figure 3. The Mosher reactions for 5 could not be performed due to lack of material, but, based on the biological relationship, the absolute configuration of 5 was inferred to be 5S and 9S. Although phexandiols A and B (1 and 2) and phomesters AC (3−5) displayed different structures, their primary skeletons contain 12 carbons. To explain the possible biogenetic relationship of 1−5, their plausible biosynthetic pathways were proposed in Scheme S1. These new compounds were evaluated for their cytotoxic and antimicrobial activities, but proved to be inactive (details in Supporting Information). To illuminate the interaction mechanisms of the co-culture system for the production of 1−5, crude extract feeding experiments were conducted. None of the five compounds were detected in either strain individually fed with crude extracts of the other strain. We could infer that the production of 1−5 necessitated a complicated interaction process.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV−vis spectra were recorded using a Shimadzu UV-2550 PC spectrometer (Shimadzu Co., Ltd., Tokyo, Japan). IR (KBr) spectra and optical rotations were obtained on a Thermo Nicolet AVATAR 360 FTIR spectrometer (Thermo Nicolet Co., Madison, WI, USA) and Horiba SEPA-300 polarimeter (Horiba, Kyoto, Japan), respectively. ECD spectra were recorded using a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., UK). NMR spectra including 1D and 2D spectra were acquired at room temperature on a Bruker Avance-400 and 600 MHz instrument (Bruker, Karlsruhe, Germany). HRESIMS data were obtained on an Agilent G3250AA (Agilent, Santa Clara, CA, USA). Reversed-phase HPLC was performed on an Agilent Eclipse XDB-C18 (5 μm) column. Silica gel (200−300 mesh, Qingdao Marine Chemical Group Co., Qingdao, China), Lichroprep RP-18 gel (40− 63 mm, Merck, Darmstadt, Germany), and Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden) were used for column chromatography (CC). Analytical thin-layer chromatography (TLC) was carried out using HSGF 254 plates (Qingdao Marine Chemical Group Co., Qingdao, China) and visualized by spraying with anisaldehyde−H2SO4 reagent. Fungal Strain. The strain YUD17001 was isolated from the rhizomes in the underground portion of G. elata collected from a plantation field at Xiaocaoba, Zhaotong, Yunnan Province, China, in December 2016. The rhizomes were treated by the direct inoculation method on potato dextrose agar medium (PDA). The purified strain was incubated on PDA medium at 28 °C; 18S rRNA extraction, PCR, D

DOI: 10.1021/acs.jnatprod.8b00685 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Phomester B (4): colorless oil; [α]23D −6.4 (c 0.27, MeOH); UV− vis (MeOH) λmax (log ε) 221 (1.16), 266 (1.05) nm; IR (KBr) νmax 3391, 2933, and 1716 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 327.1779 [M + Na]+ (calcd for C15H28O6Na, 327.1778). Phomester C (5): colorless oil; [α]23D +12.4 (c 0.22, MeOH); UV− vis (MeOH) λmax (log ε) 233 (1.72), 266 (1.88) nm; IR (KBr) νmax 3423, 2917, 1740, and 1689 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 380.2407 [M + Na]+ (calcd for C19H35NO5Na, 380.2407). Cytotoxicity Assay. The in vitro cytotoxicity of compounds 1−5 against human promyelocytic leukemia (HL-60), human hepatoma (SMMC-7721), non-small-cell lung cancer (A-549), breast cancer (MCF-7), and human colorectal carcinoma (SW480) cell lines was determined by the MTS method.22 Cisplatin (DDP) was used as the positive control. All assays were performed in triplicates in three independent experiments. Antimicrobial Assay. Antimicrobial assays were performed in 96well sterilized microplates using a microdilution method.23 Compounds 1−5 were tested for antimicrobial activities against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Candida albicans. As positive controls, nystatin (Taicheng Pharmaceutical Co., Ltd., Guangdong, China) exhibited antifungal activity against C. albicans with an MIC of 4 μg/mL, and kanamycin (Yunke Biotechnology, Kunming, China) exhibited antibacterial activities against B. subtilis, E. coli, and S. aureus with MICs of 4, 8, and 4 μg/mL, respectively. All experiments were repeated three times.

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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.8b00685.

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Spectral data (1D and 2D NMR, HRESMS) and bioactivity assay results for 1−5, along with the plausible biosynthetic pathways of 1−5, together with other details (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Z.-T. Ding). ORCID

Hong-Tao Li: 0000-0002-1350-094X Hao Zhou: 0000-0002-9256-6022 Zhong-Tao Ding: 0000-0002-7860-060X Author Contributions #

H.-T. Li and H. Zhou contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from the Natural Science Foundation of China (Nos. 81603005 and 81860623), a project of Yunling Scholars of Yunnan Province, a grant from the Science and Technology Project of Yunnan Provincial Department of Science and Technology (No. 2017FD059), and a grant from the Scientific Research Foundation of Yunnan Provincial Department of Education (No. 2016zzx003).

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