Antibacterial Radicicol Analogs from Pochonia chlamydosporia and

Jun 6, 2019 - Chemical investigation of fungus Pochonia chlamydosporia strain 170, derived from rice fermentation sediment samples, afforded seven ...
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Cite This: J. Agric. Food Chem. 2019, 67, 7266−7273

Antibacterial Radicicol Analogues from Pochonia chlamydosporia and Their Biosynthetic Gene Cluster Feifei Qin,†,‡,§ Yan Li,†,§ Runmao Lin,‡,§ Xi Zhang,‡,§ Zhenchuan Mao,§ Jian Ling,§ Yuhong Yang,§ Xia Zhuang,§ Shushan Du,‡ Xinyue Cheng,*,‡ and Bingyan Xie*,§ ‡

College of Life Sciences, Beijing Normal University, Beijing 100875, People’s Republic of China Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China

§

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S Supporting Information *

ABSTRACT: Chemical investigation of fungus Pochonia chlamydosporia strain 170, derived from rice fermentation sediment samples, afforded seven radicicol analogues, including two new compounds, monocillin VI (1) and monocillin VII (2), and five known compounds, monocillin II (3), monorden D (4), monocillin IV (5), monocillin V (6), and pochonin M (7). The structures of compounds 1−7 were established primarily by analysis of nuclear magnetic resonance data, and the absolute configurations of the secondary alcohol in compounds 1 and 2 were assigned by the modified Mosher method. All seven compounds have modest antibacterial activities, with a minimal inhibitory concentration (MIC) of 25.6 μg/mL for compounds 1 and 3−7 and 51.2 μg/mL for compound 2, on inhibition of the growth of the plant pathogen Xanthomonas campestris (the positive control ampicillin showed a MIC value of 12.8 μg/mL), indicating that the fungus has the potential to control bacterial disease. The biosynthetic gene cluster and putative biosynthetic pathways of these radicicol analogues in the P. chlamydosporia genome were proposed. These findings increase our knowledge of the chemical potential of P. chlamydosporia and may allow us to better utilize the fungus as a biological control agent. KEYWORDS: Pochonia chlamydosporia, radicicol analogues, antibacterial activity, Xanthomonas campestris pv. campestris, biosynthetic gene cluster

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INTRODUCTION Pochonia chlamydosporia (Goddard) Zare & W. Gams (Hypocreales, Clavicipitaceae), which is a facultative parasite and rhizosphere-colonizing fungus, has become one of the most promising biocontrol agents thus far.1 Until now, a lot of secondary metabolites from P. chlamydosporia and other Pochonia spp. have been identified.2,3 These compounds are mainly classified as three groups, resorcylic acid lactones (RALs), pyranones, and alkaloids, with broad biological activities, including antibacterial, antifungal, antimalarial, antinematicidal, antiviral, antitumor, antioxidative, and other activities.2,3 Among these groups, RALs are a member of macrocyclic polyketides containing a 2,4-dihydroxybenzoate residue attached to a 12−14-membered lactone ring system, which are the dominant type of secondary metabolites in P. chlamydosporia and mainly composed of radicicols (monorden and analogues), pochonins, and monocillins.3−5 It was reported that radicicol and analogues have significant antifungal, antiviral, antiparasitic, anticancer, and antimicrobial activities6−9 and are also potent heat-shock protein 90 (Hsp90) inhibitors.10 Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc) is a pathogen of black rot disease, which is the most destructive disease of crops belonging to the Brassicaceae family, with no effective existing method to control.11−13 During the course of our ongoing screening for new natural products from biocontrol fungi Pochonia spp. with antibacterial activity against X. campestris, an ethyl acetate (EtOAc) extract of the rice fermentation of P. chlamydosporia strain 170 © 2019 American Chemical Society

(PC170) showed modest inhibitory activity against X. campestris. Bioassay-guided fractionation of this extract led to the identification of seven radicicol analogues, including two new compounds, monocillin VI (1) and VII (2), and five known compounds, monocillin II (3), monorden D (4), monocillin IV (5), monocillin V (6), and pochonin M (7).3,4 In this paper, we report on the isolation and structure elucidation of the radicicol analogues and their antibacterial activities toward Xcc. Moreover, we also predicted the radicicol biosynthetic gene cluster in the P. chlamydosporia genome, which would contribute to our understanding of the biosynthetic pathway of this class of compounds.

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MATERIALS AND METHODS

Experimental Procedures. Optical rotations were measured on an Anton-Paar MCP 200 polarometer. Circular dichroism (CD) spectra were recorded on a Chirascan spectropolarimeter. Ultraviolet (UV) spectroscopy data were recorded on a Shimadzu Biospec-1601 spectrophotometer. 1H and 13C nuclear magnetic resonance (NMR) data were acquired with a Bruker AVANCE 500 MHz spectrometer with a 5 mm triple resonance cryoprobe at 298 K. The referenced NMR solvent signals were acetone-d6, δH 2.05/δC 29.8 and 206.1, and pyridine-d5, δH 7.21, 7.58, and 8.73. Heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments were optimized for 145 and 8 Hz, respectively. Received: Revised: Accepted: Published: 7266

March 29, 2019 June 4, 2019 June 6, 2019 June 6, 2019 DOI: 10.1021/acs.jafc.9b01977 J. Agric. Food Chem. 2019, 67, 7266−7273

Article

Journal of Agricultural and Food Chemistry Table 1. NMR Data of Compounds 1 and 2 in Acetone-d6 compound 1 position

δCa, mult.

1 2 3 4 5 6 7 8

171.1, 105.7, 164.7, 101.8, 162.6, 112.2, 139.3, 49.4,

qC qC qC CH qC CH qC CH2

9 10

207.7, qC 39.1, CH2

11

21.9, CH2

12 13 14 15 16 17 18

δHb (J in Hz)

compound 2 δCa, mult.

HMBCc

170.8, 104.1, 164.2, 101.5, 162.8, 108.5, 144.0, 133.2,

6.31, d (2.5)

1, 2, 3, 5, 6

6.22, d (2.5)

1, 2, 4, 5, 8

4.20, d (17.2) 3.97, d (17.2)

2, 6, 7, 9 2, 6, 7, 9

2.60, dt (17.5, 5.0) 2.45, ddd (17.5, 8.5, 5.0) 1.57, m

9, 11, 12 9, 11, 12 9, 10, 12, 13

24.6, CH2 30.6, CH2 133.3, CH

1.69, m 2.17, m 5.74, dt (15.5, 7.0)

9, 10, 11, 13, 14 11, 12, 14, 15 12, 13, 15, 16

130.0, 73.9, 75.9, 16.6,

5.56, 4.13, 5.29, 1.36,

13, 14, 16 14, 15, 18 1, 15, 16, 18 16, 17

CH CH CH CH3

dd (15.5, 7.0) t (5.0) dq (6.5, 5.0) d (6.5)

qC qC qC CH qC CH qC CH

132.8, CH 69.8, CH 39.6, CH2 130.1, CH 127.4, CH 38.4, CH2 71.4, CH 18.8, CH3

δHb (J in Hz)

HMBCc

6.14, d (2.4)

2, 3, 6

6.21, d (2.4)

2, 5, 8

6.65, d (16.0)

2, 6, 7, 9, 10

5.62, dd (16.0, 4.8) 4.31, m

7, 8, 10, 11 9, 12

2.32, 2.24, 5.37, 5.42, 2.40, 2.20, 5.06, 1.25,

8, 9, 12 8, 9, 12 11, 13 11 12, 15, 16 12, 15, 16 1, 13, 14, 16 14, 15

m m m m m m dd (11.3, 6.3) d (6.3)

a

Recorded at 125 MHz. bRecorded at 500 MHz. cHMBCs are from proton(s) stated to the indicated carbons. Chemical Extraction, Isolation, and Purification. The rice cultures of P. chlamydosporia strain 170 were extracted twice for 12 h with ethyl acetate (200 mL each flask). After filtering, the organic solvent was evaporated to dryness under vacuum to afford a 15 g crude extract, which was further fractionated by silica gel vacuum liquid chromatography (VLC) using petroleum ether (PE)−EtOAc gradient elution. The fraction that eluted with 25% EtOAc (142 mg) was purified by reversed-phase high-performance liquid chromatography (RP-HPLC, Agilent Zorbax SB-C18 column, 5 μm, 9.4 × 250 mm, 75% MeOH in H2O over 20 min, 2 mL/min) to afford compounds 3 (22.06 mg, tR of 12.54 min), 4 (3.32 mg, tR of 15.45 min), and 5 (4.01 mg, tR of 18.20 min). The fraction that eluted with 35% EtOAc (243 mg) was also purified by the same semi-preparative RP-HPLC (60% MeOH in H2O over 20 min, 60−100% MeOH in H2O over 7 min and 40 s, 2 mL/min) to afford compounds 1 (72.07 mg, tR of 13.26 min), 2 (7.67 mg, tR of 22.76 min), and the mixture containing compounds 6 and 7, and then the mixture was further purified by RP-HPLC (Agilent Zorbax SB-C18 column, 5 μm, 4.6 × 150 mm, 65% MeCN in H2O over 20 min, 1 mL/min) to afford compounds 6 (1.17 mg, tR of 10.38 min) and 7 (3.30 mg, tR of 12.13 min). Monocillin VI (1): white powder; [α]25 D , +28.68 (c 0.1, MeOH); UV (MeOH) λmax (log ε), 215 (2.67), 264 (2.38), 302 (2.07) nm; CD (c 3.0 μM, MeOH) λmax (Δε), 207 (+7.85), 215 (−12.82), 225 (+8.89), 298 (+21.40) nm; 1H, 13C, and HMBC NMR data for compound 1 are listed in Table 1; HRESIMS, m/z 357.1309 [M + Na]+ (calculated for C18H22O6Na+, 357.1309). Monocillin VII (2): white powder; [α]25 D , −66.90 (c 0.1, MeOH); UV (MeOH) λmax (log ε), 234 (2.62), 270 (2.23), 310 (1.93) nm; 1 H, 13C, and HMBC NMR data of compound 2 are listed in Table 1; HRESIMS, m/z 291.1226 [M + H]+ (calculated for C16H19O5+, 291.1227). Monocillin IV (5): [α]25 D , +45.15 (c 0.1, MeOH); CD (c 3.1 μM, MeOH) λmax (Δε), 206 (+11.71), 216 (−21.81), 230 (+7.58), 297 (+47.12) nm. Preparation of (R)-MTPA Ester (1a) and (S)-MTPA Ester (1b). (R)-(−)- and (S)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl (MTPA) esters were prepared as described in a previous study.14 A sample of compound 1 (1.0 mg, 0.003 mmol) was dissolved in

High-resolution electrospray ionization mass spectrometry (HRESIMS) data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. Fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas (300 °C) with a flow rate at 10 L/min, and the pressure of the nebulizer was 10 psi. The mass spectrometry (MS) experiments for compounds 1 and 2 were performed in positive ion mode, while compounds 3−7 were performed in negative ion mode. Full-scan spectra were acquired over a scan range of m/z 100−1000 at 1.03 spectra/s. All solvents used were of analytical grade. Column chromatography was performed with the 100−200 or 200−300 mesh silica gel (Qingdao Marine Chemical, Inc., China). Semipreparative high-performance liquid chromatography (HPLC) was performed on an Agilent 1260 G7111A Quaternary Pump equipped with a G7117C diode array detector (DAD). Origin of Strains. P. chlamydosporia strain 170 was originally isolated from Meloidogyne incognita eggs and stored in the −80 °C refrigerator as a conidial culture. The strain was also deposited in the Chinese General Microbiological Culture Collection Center under CGMCC 8860. This strain was grown on potato dextrose agar (PDA) medium (200 g of potato, 20 g of glucose, and 10 g of agar in 1 L of distilled water) for routine culturing. X. campestris pv. campestris was isolated from Brassica oleracea L. and stored at −80 °C. The strain was maintained at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This strain was grown in Luria-Bertani (LB) broth culture (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 1 L of distilled water). Fermentation. Five agar plugs from PDA cultures were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of SMYA seed medium (1 g of Bacto neopeptone, 40 g of maltose, 10 g of yeast extract, and 4 g of agar in 1 L of distilled water). Seed media were incubated for 5 days of shaking with 220 rpm. A scaled-up fermentation was then carried out in ten 500 mL Erlenmeyer flasks containing 40 g of rice and 60 mL of distilled water in each flask, and then the contents were autoclaved at 121 °C for 20 min. After cooling to 25 °C, 1.0 mL of the seed culture was inoculated in each flask, mixed with the rice, and incubated at 28 °C for 30 days. 7267

DOI: 10.1021/acs.jafc.9b01977 J. Agric. Food Chem. 2019, 67, 7266−7273

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Journal of Agricultural and Food Chemistry

Figure 1. Structures of compounds 1−7: (A) chemical structures of compounds 1−7, (B) Δδ values (ppm) = δS − δR obtained for (S)- and (R)MTPA esters 1b and 1a, and (C) Δδ values (ppm) = δS − δR obtained for (S)- and (R)-MTPA esters 2b and 2a. pyridine (1.0 mL) in a 10 mL round-bottomed flask. After (S)-MTPA Cl (4.0 μL, 0.02 mmol) was quickly added and the flask was sealed, all contents were stirred at 25 °C for 24 h. Then, reaction mixture was purified by semi-preparative RP-HPLC (Agilent Zorbax SB-C18 column, 5 μm, 9.4 × 250 mm, 90% CH3OH in H2O for 20 min, 2 mL/min) to afford R-MTPA ester (1a) (0.3 mg, tR of 13.3 min), with the relevant 1H NMR data (CDCl3, 500 MHz; Figure S8 of the Supporting Information): δ 6.36 (d, J = 2.6 Hz, 1H, H-4), 6.10 (d, J = 2.5 Hz, 1H, H-6), 5.96 (dt, J = 14.9, 6.3 Hz, 1H, H-14), 5.55 (m, 1H, H-15), 5.45 (m, 1H, H-17), 5.33 (s, 1H, H-16), 3.89 (d, J = 17.1 Hz, 2H, H2-8), 2.48 (dt, J = 17.9 and 5.4 Hz, 2H, H2-10), 1.68 (dd, J = 12.6 and 5.8 Hz, 2H, H2-11), 1.34 (d, J = 6.4 Hz, 3H, CH3-18). Similarly, another sample of compound 1 (1.0 mg, 0.003 mmol), (R)MTPA Cl (4.0 μL, 0.02 mmol), and pyridine (1.0 mL) were put into a 10 mL round-bottomed flask to react at 25 °C for 24 h and purified the same as described for compound 1a to afford S-MTPA ester (1b) (0.3 mg, tR of 13.8 min), with the relevant 1H NMR data (CDCl3, 500 MHz; Figure S8 of the Supporting Information): δ 6.37 (d, J = 2.5 Hz, 1H, H-4), 6.11 (d, J = 2.5 Hz, 1H, H-6), 5.78 (s, 1H, H-14), 5.51 (m, 1H, H-15), 5.47 (m, 1H, H-17), 5.33 (s, 1H, H-16), 3.90 (d, J = 17.2 Hz, 2H, H2-8), 2.42 (dt, J = 18.4 and 5.9 Hz, 2H, H2-10), 1.42 (d, J = 6.2 Hz, 3H, CH3-18). Preparation of (R)-MTPA Ester (2a) and (S)-MTPA Ester (2b). A MeOH solution of compound 2 (1.0 mg, 0.003 mmol) was transferred to a NMR tube, and the sample was then evaporated to dryness under vacuum. Pyridine-d5 (0.5 mL) and (S)-MTPA Cl (4.5 μL, 0.025 mmol) were quickly added to the NMR tube; the tube was sealed; all contents were mixed thoroughly by shaking the NMR tube; and then the solution was allowed to stand at 25 °C for 24 h. 1H NMR data for the resulting R-MTPA ester (2a) were obtained without purification, with the key 1H NMR signals (pyridine-d5, 500 MHz; Figure S9 of the Supporting Information): δ 6.48 (d, J = 16.0 Hz, 1H, H-8), 5.94 (m, 2H, H-9, H-10), 5.50 (m, 1H, H-13), 5.38 (m, 1H, H-12), 5.15 (dd, J = 9.7 and 6.3 Hz, 1H, H-15), 2.67 (m, 1H, H11a), 2.53 (m, 1H, H-11b), 2.32 (m, 1H, H-14a), 2.15 (m, 1H, H14b), 1.16 (d, J = 6.3 Hz, 3H, CH3-16). Similarly, another sample of compound 2 (1.0 mg, 0.003 mmol), (R)-MTPA Cl (4.5 μL, 0.025 mmol), and pyridine-d5 (0.5 mL) were put into another NMR tube to

react at 25 °C for 24 h and processed as described above for compound 2a to afford S-MTPA ester (2b), with the key 1H NMR signals (pyridine-d5, 500 MHz; Figure S9 of the Supporting Information): δ 6.66 (d, J = 16.0 Hz, 1H, H-8), 6.10 (dd, J = 16.0 and 5.4 Hz, 1H, H-9), 5.95 (dd, J = 9.8 and 5.4 Hz, 1H, H-10), 5.45 (m, 1H, H-13), 5.25 (m, 2H, H-12, H-15), 2.67 (m, 1H, H-11a), 2.46 (m, 1H, H-11b), 2.30 (m, 1H, H-14a), 2.10 (m, 1H, H-14b), 1.22 (d, J = 6.2 Hz, 3H, CH3-16). Bioactivity Assay. Antibacterial bioassays were performed with three replicates by following the National Center for Clinical Laboratory Standards (NCCLS) recommendations.15 The test bacterial strain, X. campestris pv. campestris, was grown on LB agar. Targeted microbes (3−4 colonies) were incubated in the 25 mL glass tube in LB medium at 28 °C for 24 h, and sterile water were added to adjust the density of bacteria at 106 cells/mL. Test compounds [with 10 mg/mL as mother solution in dimethyl sulfoxide (DMSO) and serial dilutions] were transferred to a 96-well clear plate in triplicate, and the bacteria suspension was added to each well to a final volume of 200 μL. Ampicillin was used as the positive control. After incubation, viability was determined by direct inspection of the wells under a microscope with the aid of PrestoBlue resazurin dye (Life Technologies) as the viability indicator. The minimal inhibitory concentration (MIC) value was defined as the lowest concentration of the test compound that resulted in the cultured bacteria with 100% inhibition or no detectable growth as observed by microscopy compared to the growth of the untreated control. Analysis of the Gene Cluster. To find differences in the radicicol biosynthetic gene cluster among published fungi, we first identified secondary metabolite genes in the genome of P. chlamydosporia strain 17016 using the tools antiSMASH17 (version 4.0.2) and SMURF.18 PKS and nrPKS domain structures and gene cluster organizations were determined by antiSMASH. Then, homologous genes were obtained by BLAST analyses on the website of the National Center for Biotechnology Information (NCBI). DNAMAN version 7 and Easyfig version 2.2.3 were used for sequence analysis. 7268

DOI: 10.1021/acs.jafc.9b01977 J. Agric. Food Chem. 2019, 67, 7266−7273

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Journal of Agricultural and Food Chemistry

Figure 2. CD spectra of compounds 1 and 5: (A) CD spectrum of monocillin VI (1) and (B) CD spectrum of monocillin IV (5).

Figure 3. MIC (μg/mL) of radicicol analogues 1−7 against X. campestris pv. campestris in a 2-fold liquid dilution assay. The 2-fold serial dilution of radicicol analogues from 204.8 to 0.4 μg/mL was tested in 96-well plates. The positive control was ampicillin at a 2-fold serial dilution from 200 to 0.4 μg/mL. The negative controls were medium (CK−) and inoculum (CK+).

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RESULTS AND DISCUSSION Structure Elucidation of Radicicol Analogues. The known compounds 3−7 (Figure 1A) isolated from the crude extracts were identified as monocillin II (3),19 monorden D (also named as pochonin D) (4),6 monocillin IV (5),19 monocillin V (6),19 and pochonin M (7),20 by comparison of their NMR (Figures S1−S5 of the Supporting Information) and MS data to those previously reported. The new compound monocillin VI (1) obtained in this study is a white powder, with a molecular formula of C18H22O6 (eight degrees of unsaturation) established by HRESIMS (m/z 357.1309 [M + Na]+). Analysis of its 1H, 13C, and HMQC NMR spectroscopic data (Table 1) revealed one methyl group, five methylenes, two oxymethines, eight aromatic/olefinic carbons (four of which were protonated), one carboxyl carbon (δC 171.1), and one ketone carbon (δC 207.7). Interpretation of the 1H−1H correlation spectroscopy (COSY) and HMBC NMR data for compound 1 established its structure as a 14membered resorcylic acid lactone, which was a C-16 hydroxylated analogue of the known compound monocillin IV (5), a co-isolated metabolite that was originally identified from Monocillium nordinii.19 Therefore, the gross structure of monocillin VI (1) was proposed as 16-hydroxymonocillins IV (5). The disubstituted double bond (C-14/C-15) geometry in

the side chain was deduced to be trans from the large coupling constant (J14,15 = 15.5 Hz) of the olefinic protons. The absolute configuration of the C-16 secondary alcohol in compound 1 was assigned by application of the modified Mosher method.21 Treatment of compound 1 with (S)-MTPA Cl afforded the R-MTPA ester (1a), and treatment of compound 1 with (R)-MTPA Cl afforded the S-MTPA ester (1b). To assign the absolute configuration at C-16, the difference in chemical shift values (Δδ = δS − δR) for the diastereomeric esters 1b and 1a was calculated. On the basis of the Δδ results summarized in Figure 1B, the 16S absolute configuration of compound 1 was proposed. Because the absolute configuration of C-17 in compound 5 was determined to be a R configuration through its single-crystal X-ray diffraction and total synthetic studies,22,23 the C-17 absolute configuration in compound 1 was assigned to be the same as that of compound 5 because of the identical CD curves between compounds 1 and 5 (Figure 2) and the positive specific rotation values of compounds 1 and 5 (1, [α]25 D = +28.68; 5, [α]25 D = +45.15). The molecular formula of monocillin VII (2) was established as C16H18O5 (eight degrees of unsaturation) by analysis of its HRESIMS (m/z 291.1226 [M + H]+) and NMR data (Table 1). A comparison of the 1H and 13C NMR spectra of compound 2 to those of compounds 1 and 3−7 revealed 7269

DOI: 10.1021/acs.jafc.9b01977 J. Agric. Food Chem. 2019, 67, 7266−7273

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Journal of Agricultural and Food Chemistry

Figure 4. Structure and arrangement of genes involved in radicicol biosynthesis in the P. chlamydosporia genome: (A) radicicol biosynthetic gene cluster in the genome of P. chlamydosporia strain 170, (B) synteny analysis of DNA sequences of the gene cluster among the three strains, and (C) structures of two polyketide synthases (hrPKS and nrPKS).

rotation value ([α]25 D = −66.90) of compound 2 were different from those of compound 1. However, the C-15 absolute configuration in compound 2 was assumed to be the same as that in known compounds 3−7 based on the biogenetic analogy. Bioactivities of the Radicicol Analogues in Inhibiting Growth of the Bacterium X. campestris. The antibacterial activity of the seven compounds was measured in a 2-fold liquid dilution series (Figure 3). All compounds showed inhibitory effects against X. campestris pv. campestris. The compounds 1 and 3−7 showed modest activity against Xcc, with MIC values of 25.6 μg/mL, and compound 2 showed a slightly weaker antibacterial activity, with a MIC value of 51.2 μg/mL. The positive control ampicillin showed a MIC value of 12.8 μg/mL. Identification of the Radicicol Biosynthetic Gene Cluster in P. chlamydosporia. Core enzymes of radicicol biosynthesis have been reported in previous studies,24−28 including in the P. chlamydosporia strain ATCC 16683.25 Taking advantage of the two published genomic data sets for P. chlamydosporia strain 170 and strain 123,16,28 genes involved in radicicol biosynthesis in P. chlamydosporia were identified and compared. In the PC170 genome, with antiSMASH analysis,

that compound 2 displayed similar signals characteristic of those RALs, while there was a difference in that the 2,4dihydroxybenzoate group was fused to a 12-membered lactone ring in compound 2, unlike a 14-membered lactone ring in compounds 1 and 3−7. Interpretation of the HMBC data for compound 2 established the same 2,4-dihydroxybenzoate partial structure as that found in compounds 1 and 3−7, with one carboxyl group attached to C-1. Analysis of its 1 H−1H COSY NMR data established an isolated spin system of C-8−C-16 (including OH-10). HMBC cross peaks from H8 to C-2, C-6, and C-7 and from H-6 to C-8 indicated that C-8 is attached to the 2,4-dihydroxybenzoate group at C-7, while another key HMBC correlation from H-15 to C-1 connected both C-15 and carboxyl C-1 (δC 170.9) to the same oxygen atom to form a 12-membered lactone ring fused to the 2,4dihydroxybenzoate unit at C-1 and C-7, respectively. On the basis of these data, the planar structure of compound 2 was established. The 10S absolute configuration of the secondary alcohol in compound 2 was deduced using the modified Mosher method,21 as illustrated in Figure 1C. Because the structure of the lactone ring in compound 2 was significantly different from that in compound 1, the CD spectrum (Figure S10 of the Supporting Information and Figure 2) and specific 7270

DOI: 10.1021/acs.jafc.9b01977 J. Agric. Food Chem. 2019, 67, 7266−7273

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Journal of Agricultural and Food Chemistry

Figure 5. Putative biosynthetic pathways of compounds 1−7.

X. campestris pv. campestris (MIC = 25.6 μg/mL). Compound 2 (monocillin VII) has a lower activity (MIC = 51.6 μg/mL) than the other compounds, which have a different structure of 2,4-dihydroxybenzoate residues attached to a lactone ring system; perhaps, the lactone ring is critical for the antibacterial activity. Our results indicate that the fungus P. chlamydosporia has application potential in the control of bacterial disease. Moreover, through antiSMASH, the radicicol biosynthetic gene cluster was identified in the P. chlamydosporia genome, which includes 18 gene modules. Among them, the five core members (hrPKS, nrPKS, halogenase, CYP450, and MFS) are conserved among radicicol biosynthetic gene clusters known in fungi (P. chlamydosporia, Colletotrichum sublineola, Glomerella graminicola, and Chaetomium chiversii) (Figure S8 of the Supporting Information). However, the other 13 related members involved in the cluster of P. chlamydosporia strain 170 are not conserved. On the basis of our current knowledge, the biosynthetic pathways of the seven radicicol analogues are suggested as follows (Figure 5): Monocillin II (3) is an early intermediate in the biosynthetic pathway, which has a typical of RAL biosynthesis documented in previous studies.5,25,27 The other five compounds (1, 2, and 4−6) are perhaps produced from compound 3 by redox, epoxidation, and halogenation reactions. In contrast to the above six compounds with the 5 + 4 mechanism to maintain the chemical modularity of the two PKSs, compound 2 is suggested to have the 4 + 4 mechanism, which caused a salient difference in structure with other compounds. To fully understand the radicicol biosynthetic pathway in P. chlamydosporia, more experimental evidence is needed.

we identified a gene cluster with 18 gene modules (Figure 4A), which included the five core enzymes of radicicol biosynthesis, i.e., two polyketide synthases [a highly reducing PKS (hrPKS) and a non-reducing PKS (nrPKS)], a halogenase, a cytochrome P450, and a major facilitator superfamily (MFS) transporter. In addition to the above five core members, 13 other members are involved in the biosynthetic gene cluster, including a meiotically upregulated protein, a hemolysin-III family protein, an importin 11, a threonyl-tRNA synthetase, a max protein, an acetylserotonin methyltransferase-like protein, a polybromo-1, and six hypothetical proteins. We also identified homologous genes in the PC123 genome28 and compared them to genes of the reported gene cluster of P. chlamydosporia strain ATCC 16683.25 Synteny analysis among the three strains of P. chlamydosporia showed that the sequence identity was greater than 90% (Figure 4B). Hence, we suggest that this biosynthetic gene cluster is the most likely candidate for radicicol biosynthesis. The structures and domains of the two polyketide synthases in P. chlamydosporia are shown in Figure 4C. RALs are the dominant type of secondary metabolites in P. chlamydosporia, and a number of radicicol derivatives have been isolated from P. chlamydosporia var. catenulate P0297 and P. chlamydosporia var. chlamydosporia strain TF-0480.6,20 In this study, chemical investigation of the fungus P. chlamydosporia strain 170 derived from rice fermentation sediment samples provided seven RALs, including two new radicicol analogues (monocillin VI and monocillin VII) and five known compounds (i.e., monocillin II, monorden D, monocillin IV, monocillin V, and pochonin M). Bioassays showed that most of these radicicol derivatives have antiviral, antiparasitic, and antifungal activities2,3 but seldom report on antibacterial activity. In our study, we found that these radicicol analogues have modest antibacterial activities against the plant pathogen 7271

DOI: 10.1021/acs.jafc.9b01977 J. Agric. Food Chem. 2019, 67, 7266−7273

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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.jafc.9b01977. NMR and HRESI mass spectra of monocillin VI (1) (Figure S1), NMR and HRESI mass spectra of monocillin VII (2) (Figure S2), NMR and ESI mass spectra of monocillin II (3) (Figure S3), NMR and ESI mass spectra of monorden D (4) (Figure S4), NMR and ESI mass spectra of monocillin IV (5) (Figure S5), NMR and ESI mass spectra of monocillin V (6) (Figure S6), NMR and ESI mass spectra of pochonin M (7) (Figure S7), NMR spectra of compounds 1a and 1b (Figure S8), NMR spectra of compounds 2a and 2b (Figure S9), CD spectrum of monocillin VII (2) (Figure S10), comparison of radicicol biosynthetic gene clusters among five fungal strains (Figure S11), and information on genes involved in the radicicol biosynthetic gene clusters of P. chlamydosporia strain 170, strain 123, and strain ATCC 16683 (Table S1) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +86-10-58809696. E-mail: [email protected]. *Telephone: +86-10-82109546. E-mail: [email protected]. ORCID

Bingyan Xie: 0000-0001-9640-8956 Author Contributions †

Feifei Qin and Yan Li contributed equally to this work.

Funding

This work was supported by the National Key Research and Development (R&D) Plan of China (2016YFC1201100). Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.jafc.9b01977 J. Agric. Food Chem. 2019, 67, 7266−7273