Exploration of Fungal Metabolic Interactions Using Imaging Mass

Jun 19, 2018 - Application of matrix-assisted laser desorption/ionization imaging mass spectrometry to microbiology and natural product research has o...
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Article Cite This: J. Nat. Prod. 2018, 81, 1527−1533

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Exploration of Fungal Metabolic Interactions Using Imaging Mass Spectrometry on Nanostructured Silicon Pi-Yu Chen,† Chi-Ying Hsieh,† Chao-Jen Shih,†,‡ Yuan-Jing Lin,§ Chia-Wen Tsao,§ and Yu-Liang Yang*,† †

Agricultural Biotechnology Research Center, Academia Sinica, 11529 Taipei, Taiwan Bioresource Collection and Research Center, Food Industry Research and Development Institute, 30062 Hsinchu, Taiwan § Department of Mechanical Engineering, National Central University, 32001 Taoyuan, Taiwan ‡

J. Nat. Prod. 2018.81:1527-1533. Downloaded from pubs.acs.org by UNIV OF SUSSEX on 07/27/18. For personal use only.

S Supporting Information *

ABSTRACT: Application of matrix-assisted laser desorption/ionization imaging mass spectrometry to microbiology and natural product research has opened the door to the exploration of microbial interactions and the consequent discovery of new natural products and their functions in the interactions. However, several drawbacks of matrix-assisted laser desorption/ionization imaging mass spectrometry have limited its application especially to complicated and uneven microbial samples. Here, we applied nanostructured silicon as a substrate for surface-assisted laser desorption/ionization mass spectrometry for microbial imaging mass spectrometry to explore fungal metabolic interactions. We chose Phellinus noxius and Aspergillus strains to evaluate the potential of microbial imaging mass spectrometry on nanostructured silicon because both fungi produce a dense mass of aerial mycelia, which is known to complicate the collection of high-quality imaging mass spectrometry data. Our simple and straightforward sample imprinting method and low background interference resulted in an efficient analysis of small metabolites from the complex microbial interaction samples. cause flaking, as well as non- or decreased electric conductivity. In the latter case, the charge can build up on the target over time, manifesting as high-intensity gradient signals during the beginning of the spectral acquisition, and eventually resulting in little or no signal intensity. Charge buildup is common when imaging thick microbial samples, such as Actinomyces and fungi that can produce thick aerial hyphae or filaments. In MALDI IMS, the size of matrix−analyte cocrystals is a major factor affecting the spatial resolution. The stainless steel sieve used to apply solid matrices prior to sample dehydration is an efficient, low-cost, and widely used method for uniform dry-coating and provides favorable spatial resolution (ca. 100 μm or greater).1,3,9 Unfortunately, the excess solid matrices used in the sieve method can seriously contaminate instruments. The uneven coating and matrix aggregation sometimes generate artificial distribution of signals. In addition, the intense matrix-associated signals observed can suppress the intensity of the analyte signals and subsequently cause unexpected spatial distributions to be observed. After dehydrating the microbial samples prepared on agar-based media, liquid matrices can be deposited on the surface of the sample using a sprayer device.10,11 However, the rehydration, extraction, and recrystallization of matrix−analyte cocrystals on

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pplication of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) to microbes was developed in 2009. It has since proved capable of differentiating phenotypically relevant and irrelevant chemotypes in microbial colonies and communities.1 Aided by the development of ever more sophisticated mass spectrometry, IMS and intact-cell mass spectrometry techniques have facilitated various microbiology and natural product studies.2−8 IMS can be used to investigate the metabolic secretome of various microbial species and has enabled the discovery of numerous secreted and colony-associated metabolites that are produced by microbes to interact with and respond to neighboring organisms and environmental stresses, and these interactions may have meaningful ecological implications. Using IMS, a large number of metabolites can be observed in heterogeneous mixtures of microorganisms harvested from various natural sources, thus revealing the importance of metabolic exchange in a polymicrobial environment. Sample preparation is a critical process in all IMS experiments. One of the challenges of performing microbial IMS in a high-vacuum environment is sample flaking.3 Flaking is caused by factors such as bubbles under or inside the growth medium, high-salt medium components and microbial molecules, vegetative hyphae of microbes inside the growth media, insufficient matrix saturation of the samples, or the use of smooth or polished stainless steel target plates. The thickness of the microbial sample is also a factor that may © 2018 American Chemical Society and American Society of Pharmacognosy

Received: October 17, 2017 Published: June 19, 2018 1527

DOI: 10.1021/acs.jnatprod.7b00866 J. Nat. Prod. 2018, 81, 1527−1533

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Figure 1. Preparation of MALDI IMS (A) and SALDI IMS on nSi (B) of microbial samples and scanning electron microscopy images of (C) unetched wafer with gold deposition, (D) top view, and (E) transection of nSi substrate. (F) Contact angle of water droplet on fresh nSi is ∼135°.

by Tsao et al. to have better signal intensity than DIOS.28−31 The combination of nSi and dynamic electrowetting provided a limit of detection (LOD) to the order of several attomoles for various model peptides.28 In this study we decided to apply the nSi substrate for microbial IMS because (1) it is a costeffective method without a complex process, as nSi is simply produced via metal-assisted chemical etching; (2) nSi is easily chemically modified; and (3) it is easy to produce a suitable amount of nSi with high yields required to be compatible with the areas of interest for microbial IMS.

the dehydrated agar surface are not always homogeneous, which can cause challenges with interpretation. The dislocation of analytes caused by solvents used for spraying the liquid matrices can be avoided by applying solid matrices or spraying liquid matrices on the agar surface prior to dehydration.12 Developing new matrices for MALDI IMS, especially for small-molecule analysis, is crucial. Although numerous types of matrices have been introduced for use in small-molecule analysis, the application of matrices in MALDI IMS experiments, especially for microbial MALDI IMS, is still extremely limited because of the aforementioned unique properties of microbial samples. In addition, matrices produce high background signals below m/z 600 that limit the efficiency of detection of small molecules, which is another critical obstacle to MALDI IMS analysis. To overcome the aforementioned challenges associated with microbial IMS, we aim to develop matrix-free techniques for microbial IMS. So far, numerous matrix-free materials such as nanostructured surfaces,13 nanoparticles,14,15 nanowires,16 nanotubes,17 clathrate nanostructures,18 and others19 have been developed for small-molecule analysis. However, few of these applications have been successfully implemented in microbial IMS. We surveyed silicon-based substrates, which can potentially be applied to microbial IMS. Desorption and ionization on silicon (DIOS), nanostructure initiator mass spectrometry (NIMS), nanostructure-assisted laser desorption/ionization (NALDI) and nanophotonic laser desorption ionization have been used to perform tissue section IMS,19−23 microbial intact-cell profiling,6 and microbial IMS.24 Although independent study has shown that NALDI outperforms DIOS according to several parameters,25 the NALDI mass spectra collected in positive ion mode have characteristic background ions such as m/z 197, 235, 243, etc., due to the formation of Au+, [AuF2]+, [AuSi(H2O)]+, and other related cluster ions, which interfere with the analysis of small molecules.26 Singlecrystal silicon nanowires (SiNWs) are a good platform since the laser energy per pulse can be reduced and more ionized signals were detected than with MALDI and DIOS.16 Ordered silicon nanocavity arrays have been developed for smallmolecule analysis.27 Nanostructured silicon (nSi) was reported

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RESULTS AND DISCUSSION

We chose the p-type (100) silicon wafer as the substrate. Gold (Au, 3 nm thick) was deposited on the wafer by e-beam evaporation (Figure 1C) and then dipped in a 1:1:1 volume ratio of HF/H2O2/EtOH solution to create the nSi surface. The etching time was optimized to 300 s.28,29 After cleaning and drying the silicon wafer, the nanostructured silicon was characterized by scanning electron microscopy as shown in Figure 1D,E. The contact angle of fresh nSi was more than 130° (Figure 1F). The MS signal intensity was closely related to the surface wettability. Therefore, storage and environmental stability are important for the use of nSi. Nitrogen gas and vacuum storage inhibited nSi surface oxidation and maintained the high surface wettability to provide better MS desorption/ionization efficiency. A vacuum oven desiccation process can activate the nSi surface and further enhance MS detection sensitivity (up to 1000 times) for up to three months.31 We then simply imprinted the microbial sample back side onto the nSi surface and then removed the sample before collecting IMS data (Figure 1B and Supporting Video). To facilitate the analytes transferred from the microbial sample to the nSi surface without bubble interference, the nSi substrate was rinsed with methanol quickly, then dried by nitrogen gas prior to imprinting. The MS signal intensities were saturated when the period of imprinting was over 15 min, which demonstrated that imprinting for a longer time period does not improve the S/N and IMS quality. The disadvantage of collecting IMS from the back side of microbial samples is that some colony-associated signals may be missed, a 1528

DOI: 10.1021/acs.jnatprod.7b00866 J. Nat. Prod. 2018, 81, 1527−1533

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Figure 2. (A) Morphologies of Aspergillus strains 3G and 3Y. (B) Phellinus noxius cocultured with Aspergillus strains 3G and 3Y from day 1 to day 7. (C) UPLC-MS profiles (TIC) of Aspergillus strains 3G and 3Y cultured in large-scale solid potato dextrose agar fermentation.

phenomenon well-demonstrated by 3D microbial IMS.32 However, it is very difficult to collect high-quality IMS data directly from the surface of many fungal or Actinomyces samples by using any kind of IMS techniques. Previously, we have used three approaches for fungal samples: removing the aerial hyphae then collecting IMS from top or directly collecting IMS from the back side or from transection. When using MALDI IMS, it was inconvenient to collect data from the back side because of the problem of sample flaking (the fungal surface makes the sample difficult to adhere to the target plate). Even when collecting MALDI IMS from the transection, flaking occurs frequently during the dehydration step. Although the MS profiles of microbial samples can be observed by using ambient ionization mass spectrometry, such as desorption electrospray ionization mass spectrometry (DESI MS),33 nanoDESI,34 or liquid microjunction surface sampling probe,35 the collection of high-quality IMS data directly from microbial samples8,36 or imprinted membranes37,38 remains challenging because the spatial resolution is relatively poor and many physical parameters and solvent systems need to be optimized. The spatial resolution of MALDI IMS is highly associated with the size and homogeneity of matrix−analyte cocrystals. These two factors are difficult to optimize especially for microbial samples because of the matrix aggregation on the microbial surface. By using the nSi substrate with the imprinting approach, we could skip the matrix deposition to

improve the spatial resolution because the surface of nSi consists of relatively homogeneous Si nanowires (the pore diameters were around 500 nm, Figures 1D,E). In addition, the sample flaking during the dehydration step in the MALDI IMS sample preparation was avoided. Here we chose Phellinus noxius and Aspergillus strains to evaluate microbial IMS on nSi because they produce a dense mass of aerial hyphae and it is difficult to collect high-quality IMS data using traditional sample preparation methods. P. noxius is an aggressive fungal pathogen that is the causative agent of brown root rot disease in woody plants.39,40 The disease generally spreads through root-to-root contact or through infested wood debris in soil. It is prevalent in tropical and subtropical regions and has a wide host range covering over 200 woody plant species. In order to discover antifungal agents from microbial sources, we isolated Aspergillus strains 3G and 3Y from the soil. They share the same internal transcribed spacer region sequence of nuclear rDNA together with several housekeeping gene sequences but can be easily differentiated since their morphologies are obviously different (Figures 2A, S1, and S2). Both Aspergillus extracts, prepared through large-scale solid fermentation on potato dextrose agar (Figure S2A and B), displayed an inhibitory effect on P. noxius. Following the bioactivity-guided fractionation, LC-MS and HPLC-ELSD profiling analysis revealed that extracts of strains 3G and 3Y contain nearly equal amounts of the active 1529

DOI: 10.1021/acs.jnatprod.7b00866 J. Nat. Prod. 2018, 81, 1527−1533

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Figure 3. Microbial IMS on nSi of Aspergillus strains cocultured with Phellinus noxius. (A) Aspergillus strains 3Y and 3G single culture and coculture with P. noxius for 3 days. (B) Aspergillus 3G cocultured with P. noxius for 5 days. (C) Structures of Aspergillus metabolites observed in microbial IMS on nSi. 325 [M + H]+: sterigmatocystin (1); 245 [M + H]+: L-Leu-L-Leu, L-Ile-L-Leu, L-Leu-L-Ile, and L-Ile-L-Ile (2); 261 [M + H]+: cyclo(Phe-Ile) or cyclo(Phe-Leu) (3); 578 [M + Na]+: fellutamide B (4); 580 [M + Na]+: fellutamide C (5); 594 [M + Na]+: new fellutamide (6).

polyketide, sterigmatocystin (1, yield = 4.9% versus 3.3%, minimal inhibitory dose (MID) = 125 μg) (Figures 2C and S3). However, this result conflicted with the antagonistic assay, which indicated only strain 3G had an inhibitory effect on P. noxius in coculture (Figure 2B). We then applied microbial IMS on the nSi substrate to explore the metabolic interactions between Aspergillus strains 3G and 3Y and P. noxius. Aspergillus strains 3Y and 3G were cocultured with P. noxius for 3 days and then monitored by microbial IMS on an nSi substrate (Figure 3). The three metabolites 1−3 were produced only by strain 3G in coculture conditions. The metabolite 1 was the active polyketide sterigmatocystin secreted by strain 3G into the agar media to contribute to the inhibitory effect. In contrast, strain 3Y was unable to produce sterigmatocystin (1) during coculture, which explained why only strain 3G showed an inhibitory effect in the antagonistic assay. In addition to sterigmatocystin (1), Aspergillus can produce other antifungal agents under coculture conditions. We further cocultured strain 3G with P. noxius for 5 days (Figure 3B). The microbial IMS results showed that strain 3G produced metabolite 2, and the distribution overlapped the entire inhibition zone. Compound 2 was identified as dipeptides L-Leu-L-Leu, L-Ile-L-Leu, L-Leu-L-Ile, and L-Ile-L-Ile by comparison with tandem mass fragmentation and UPLC-MS retention time of authentic samples (Figure S12). However, these four dipeptides were unable to inhibit the growth of P. noxius. Compound 3 localized under strain 3G was identified as cyclo(Phe-Ile) or cyclo(Phe-Leu) based on the tandem mass fragmentation (Figure S13). One other interesting observation from the five-day coculture sample was that sterigmatocystin (1) was distributed only in strain 3G growth regions rather than in the inhibition zone (Figure 3B). This implied that sterigmatocystin (1) was not the only antifungal agent produced by strain 3G. In addition, a cluster of lipopeptides distributed specifically in the boundary between strain 3G and P. noxius, which suggests the production of

lipopeptides by strain 3G, is induced in coculture. Through fragmentation-based molecular networking analysis,41 they were identified as fellutamides [fellutamides B (4) and C (5)42 and a new analogue (6)], which were first isolated from Penicillium (Table 1 and Figure S11).43 A. nidulans was Table 1. High-Resolution Tandem Mass Fragmentations of Fellutamide B (4), Fellutamide C (5), and New Fellutamide (6)

4 b2

b3

y2

y2-H2O

1530

5

6

R

CHO

CH2OH

COOH

calcd found Δ (ppm) calcd found Δ (ppm) calcd found Δ (ppm) calcd found Δ (ppm)

313.2127 313.2121 −1.9 441.2713 441.2707 −1.4 244.1661 244.1655 −2.5 226.1556 226.1550 −2.7

313.2127 313.2116 −3.5 441.2713 441.2701 −2.7 246.1818 246.1808 −4.1 228.1712 228.1703 −3.9

313.2127 313.2122 −1.6 441.2713 441.2710 −0.7 260.1610 260.1606 −1.5 242.1505 242.1501 −1.7

DOI: 10.1021/acs.jnatprod.7b00866 J. Nat. Prod. 2018, 81, 1527−1533

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products in microbial interactions. This will facilitate more effective exploration of natural products.

reported to be capable of producing fellutamide B (4) by inhibiting histone deacetylase activity or replacing the promoters of the biosynthetic gene cluster.44,45 Fellutamides are cytotoxic against several cancer cell lines; in addition, they are reported as antifungal agents through targeting the proteasome.45−47 Interestingly, even though we did not observe significant inhibitory phenotype between strain 3Y and P. noxius, strain 3Y produced more fellutamides than 3G under single and coculture conditions (Figure 3A). The MID of fellutamide B (4) was 125 μg, and it also induced P. noxius to produce yellow pigments at the sub-MID level (Figure S14). Phellinus species have been reported to produce various yellow polyphenol pigments, styrylpyrones, which showed significant biological activity. Antioxidant activity is an important protective mechanism for Phellinus, and styrylpyrones, the main contributors, are reported to be more potent than vitamin E.48 On the basis of these results, we can conclude that the pure polyketide sterigmatocystin (1) was active against P. noxius. Sterigmatocystin (1) was not consistently produced during coculture and small-scale single culture, and it may contribute a partial inhibitory effect against P. noxius. Other metabolites such as fellutamides may result in a synergistic inhibitory effect on P. noxius. In contrast, P. noxius produced yellow pigments in response to antifungal agent fellutamide B (4). Purified and synthetic metabolites of fungal interactions were used to evaluate the LOD of the nSi substrate and MALDI MS (Table S1). Although the LODs of tested metabolites using an nSi substrate were competitive with those using MALDI MS (picomoles), the metabolites were difficult to observe by MALDI IMS in fungal samples because of the dense mass of fungal aerial mycelia (Figure S4). We then applied the same imprinting sample preparation approach to collect microbial IMS on a commercial NALDI plate. Surprisingly, the metabolic interactions observed on nSi were not observed in NALDI IMS (Figure S5). The most important goal of this research is the development and application of a new IMS technique to explore natural products from microbial samples. The development of modern spectroscopic techniques over the last three decades is considered a remarkable breakthrough in dereplication and de novo structural elucidation. However, the true biological functions of the natural products in the microbial world are still poorly understood.49 Two factors limiting the understanding of the biological functions of natural products are insufficient techniques that can be applied in situ and lack of methods for real-time monitoring of natural products in biological samples. Microbes are able to produce various metabolites based on different culture conditions. For example, sterigmatocystin (1) (large-scale versus small-scale solid culture conditions) and fellutamides (4−6) (single versus coculture conditions) in this study are difficult to explore using traditional analytic approaches. IMS is one of the few techniques capable of overcoming these limitations. MALDI IMS has been applied in microbial natural products research over the past decade, and there have been many reports about its strengths and weaknesses.2,3,5 Microbial IMS on nSi is a simple and straightforward sample preparation approach with a clean signal background (versus MALDI IMS) and provides more informative spatial distribution of small metabolites (versus NALDI IMS). The development of microbial IMS on nSi reported herein has the potential to improve the performance of IMS in the study of the true biological functions of natural

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EXPERIMENTAL SECTION

General Experimental Procedures. IMS data and LOD were collected using a Bruker Autoflex Speed MALDI-TOF/TOF MS spectrometer. A laser frequency of 1000 Hz and positive reflectron mode were applied. The high- and low-resolution MS and tandem MS data were collected by a Thermo Orbitrap Elite MS and a Waters Xevo TQ-S triple quadrupole MS spectrometer equipped with a Waters ACQUITY UPLC system, respectively. The NMR data were collected by Bruker Avance II 500 and 600 MHz NMR. RP-HPLCPDA (Hitachi 2130, 2455) and ELSD (Alltech 3300) were used for purification and quantification of target metabolites. Fabrication of Nanostructured Silicon. The method was modified from Tsao’s reports.28−31 P-type (100) six-inch silicon wafers with 1−100 Ω cm resistivity that were pretreated with e-beam deposition of 3 nm of Au (Figure 1C) were purchased from Advanced Furnace Systems Corp. (Tainan County, Taiwan). Hydrofluoric acid (HF, 49%) was purchased from Sigma. Ethanol (EtOH, electronic grade) was from Echo. Hydrogen peroxide (H2O2, 31%) and methanol (MeOH, 99.9%, electronic grade) were purchased from J. T. Baker. The Au-coated silicon wafer was immersed in an HF/H2O2/ EtOH mixture (1:1:1, v/v/v), in which etching proceeded for 300 s at 25 °C to create silicon nanowires on the wafer. After etching, the wafer was fully rinsed with methanol, and the wafer surface was dried under a nitrogen stream. Teflon containers were used for all HF solutions. The etched wafer was bathed in methanol for 5 h, dried with nitrogen gas, and stored in a desiccator. The surface of the nSi substrate was characterized by scanning electron microscopy (NOVA600, FEI, USA) (Figure 1D,E). Safety Considerations. The etching of a silicon wafer with HF and H2O2 should only be conducted in a well-ventilated fume hood while wearing a chemical smock, face shield, and double-layered nitrile gloves. Care must be taken not to breathe in HF and H2O2 fumes. Calcium gluconate gel must be available for burn treatment when etching with HF. If contact with HF is suspected, remove all contaminated clothing while flushing vigorously with cold water and apply calcium gluconate gel on the affected area. Gently massage the exposed area for at least 15 min until pain is relieved. Immediately seek medical treatment in a hospital and repeat application of calcium gluconate gel as necessary during transit. Characterization of Phellinus noxius and Aspergillus Strains 3G and 3Y. P. noxius (accession number: ITS MH071443) was provided by Dr. Feng-Chia Hsieh, Biopesticides Division, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Council of Agriculture, Taiwan. Aspergillus strains 3G and 3Y were isolated from soil from Yuli, Taiwan, in June 2012. 3G and 3Y were cultured on potato dextrose agar (PDA) for 5 days. A Viogene DNA extraction kit was used to obtain the genomic DNA. The internal transcribed spacer (ITS), 18S, calmodulin gene (CF), DNA replication licensing factor (Mcm7), RNA polymerase 2 (RPB2), and pre-rRNA processing protein (Tsr1) gene sequences were amplified using PCR. The primer sequences of each gene are listed in Table S2. The CF, Mcm7, RPB2, and Tsr1 gene sequences were further combined for phylogenetic analysis. Multiple sequence alignments were analyzed using ClustalW of MEGA5 (http://www. megasoftware.net/),50 and phylogenetic trees were created using the neighbor-joining method of MEGA5 (Figure S1). Accession numbers of genes from Aspergillus strain 3G: ITS MH071386; 18S MH071383; CF MH109154; Mcm7 MH109155; RPB2 MH109156; Tsr1 MH109157. Accession numbers of genes from Aspergillus strain 3Y: ITS MH071387; 18S MH071384; CF MH109158; Mcm7 MH109159; RPB2 MH109160; Tsr1 MH109161. Preparation of Microbial IMS Using MALDI, nSi, and NALDI. Aspergillus 3G and 3Y and P. noxius were cultured individually on PDA for 5 days at 30 °C. The spores of 3G and 3Y were collected and then transferred to a new PDA plate. After 4 days of incubation, P. noxius was inoculated close to 3G and 3Y for 3 or 5 days of coculture 1531

DOI: 10.1021/acs.jnatprod.7b00866 J. Nat. Prod. 2018, 81, 1527−1533

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at 30 °C. For MALDI IMS analysis, the targeted area was cut and placed on the top of a MALDI stainless steel target plate. Universal matrix (CHCA/DHB, 1:1, Sigma) was sprinkled on top of the sample surface using a 20 μm sieve followed by dehydration in a 37 °C oven for 3 h. For nSi and NALDI IMS analysis, the target area was cut out and the back side was placed on the top of a NALDI plate (Bruker) or a methanol-rinsed nSi substrate for 15 min of imprinting. After removing the target sample, nitrogen gas was used to clean the NALDI or nSi surface prior to IMS analysis (Supporting Video). The imprinted nSi substrate was fixed on a stainless steel slide by threeway conductive tape and subjected to MS spectrometry using a Bruker MTP slide adapter II. MALDI and NALDI target plates were directly subjected to MS spectrometry. The mass spectra were collected using the following settings: 250 shots/raster, 19.0 and 16.6 kV for the ion source 1 and 2, 8.7 kV for lens, 21 and 9.4 kV for reflector 1 and 2, respectively. We targeted the samples from m/z 100−2000 with 20% laser power, and the raster was set at 1000 μm. The universal matrix and peptide standard I (Bruker) were used for calibration. All image data were processed with TIC normalization and analyzed using Fleximaging 3.0. Isolation and Identification of Target Metabolites from Aspergillus strains 3G and 3Y. Aspergillus strains 3G and 3Y were cultured on 100 PDA plates (as shown in Figure S2A and B) for 4 days at 30 °C. The cultured agar was extracted by ethyl acetate. The ethyl acetate extracts were further purified by silica gel chromatography eluted with n-hexane and ethyl acetate (8:2) and monitored by MALDI TOF/TOF MS to give pure sterigmatocystin. The 10 mg/ mL of 3G and 3Y extracts were subjected to HPLC-ELSD analysis for the quantification of sterigmatocystin (1) (0−30 min 5% to 100% ACN, 30−40 min 100% ACN; Discovery HS C18, 4.6 mm × 25 cm, SUPELCO; flow rate: 0.8 mL/min) (Figure S3). Peak area percentage was used to present the yield of sterigmatocystin. For the purification of fellutamides, strain 3G was cultured on 1000 PDA plates at 30 °C for 4 days and then cocultured with P. noxius for another 3 days. The cocultured agar was extracted using ethyl acetate. The extract was purified by Sephadex LH-20 chromatography eluted with methanol. The target fractions containing lipopeptides were collected for further purification by RP-HPLC-PDA eluted with a water and acetonitrile gradient (0−30 min 5% to 100% ACN, Discovery HS C18, 10 mm × 25 cm, SUPELCO; flow rate: 2.4 mL/ min) to give fellutamide B (4, tR = 22.2 min). The structures of fellutamide C (5) and new fellutamide (6) were proposed based on the high-resolution tandem mass fragmentations deduced from UPLC-MS analysis of coculture extract (0−6 min 5% to 99.5% ACN, 6−8 min 99.5% ACN, 8−8.2 min 99.5% to 5% ACN, 8.2−10 min 5% ACN, both ACN and H2O contain 0.1% formic acid; ACQUITY UPLC BEH C18, 1.7 μm, 2.1 × 100 mm; flow rate: 0.4 mL/min) (Table 1 and Figure S11). The dipeptides 245, including LLeu-L-Leu, L-Ile-L-Leu, L-Leu-L-Ile, and L-Ile-L-Ile, were synthesized by the Peptide Synthesis Laboratory, Institute of Biological Chemistry, Academia Sinica, using a general solid phase peptide synthesis method. The natural dipeptides 245 (2) were identified by UPLC-MS analysis of coculture extract and synthetic dipeptides 245 (0−3 min 5% to 7% ACN, 3−4 min 7% to 10% ACN, 4−5 min 10% to 12.5% ACN, 5−5.5 min 12.5−15% ACN, 5.5−6 min 15% to 25% ACN, 6− 6.2 min 25% to 30% ACN, 6.2−6.5 min 30% to 99.5% ACN, 6.5−8 min 99.5% ACN, 8−8.2 min 99.5% to 5% ACN, 8.2−10 min 5% ACN, both ACN and H2O contain 0.1% formic acid; ACQUITY UPLC BEH C18, 1.7 μm, 2.1 × 100 mm; flow rate: 0.4 mL/min) (Figure S12). The purified and synthetic metabolites were subjected to structural elucidation, LOD evaluation, and antifungal assay. Detailed identification data of target metabolites (UPLC-MS analysis, tandem mass fragmentation, and NMR data) are summarized in the Supporting Information.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00866. Figures S1 to S14, Tables S1 to S2, identification of targeted metabolites, LOD, and antifungal assay; the MS2 data set of Apsergillus strain 3G cocultured with P. noxius (MSV000082223) is publically available via MassIVE (PDF) Supporting video of the step-by-step imprinting method (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yu-Liang Yang: 0000-0002-3533-5148 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the Ministry of Science and Technology, Taiwan (MOST 104-2320-B-001019-MY2). LC-MS data were collected in the Metabolomics Core Facility, Agriculture Biotechnology Research Center, Academia Sinica, and NMR data were collected in the High Field Nuclear Magnetic Resonance Center, Academia Sinica. The synthetic dipeptides 245 (2) were provided by the Peptide Synthesis Laboratory, Institute of Biological Chemistry, Academia Sinica. We thank Chia-Chi Peng for producing the Supporting Video displaying the imprinting method.

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REFERENCES

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