Natural products are an extremely useful source of chemicals for development into drugs, pesticides and reagents because they generally demonstrate a diversity of unique biological activities or chemical structures [1]. Secondary metabolites of microorganisms, such as penicillin, streptomycin and amphotericin B, have been used as highly successful medications in human health. However, it is not easy to discover potentially useful drug candidates from natural products. Thus, a new screening system is often required to promote exploratory research to unearth novel chemicals.

We have screened microbial culture broths to identify secondary metabolites that specifically inhibit the drug-sensitive recombinant Saccharomyces cerevisiae. The budding yeast, S. cerevisiae, is a model organism of eukaryotes and it is often used in screening and target identification of bioactive compounds from natural product sources [2, 3]. However, the high level of drug resistance already reported in S. cerevisiae has complicated the screening process. Consequently, a multidrug-sensitive budding yeast, S. cerevisiae 12geneΔ0HSR-iERG6, has been produced which is a very useful tool for antifungal screening because the resistant strain is devoid of a drug efflux system, and its expression of the gene related to the permeability barrier is a down-regulated system in the presence of glucose [4, 5]. Using the S. cerevisiae 12geneΔ0HSR-iERG6 screening system, we isolated a new compound, named pestynol [6], from a culture broth of the filamentous fungus, Pestalotiopsis humus FKI-7473. Here, we report another new antibiotic, named pestiocandin (1), isolated from a culture broth of the same fungus.

One loopful of strain FKI-7473, grown on an LcA slant (0.1% glycerol, 0.08% KH2PO4, 0.02% K2HPO4, 0.02% MgSO4·7H2O, 0.02% KCl, 0.2% NaNO3 and 1.5% agar, pH 6.0), was inoculated into a 500-mL Erlenmeyer flask containing 100 mL of a seed medium (2% glucose, 0.2% yeast extract, 0.05% MgSO4·7H2O, 0.5% polypepton (FUJIFILM Wako Pure Chemical Co., Osaka, Japan), 0.1% KH2PO4 and 0.1% agar, pH 6.0) and incubated on a rotary shaker at 27 °C for 4 days. Twenty-five millilitres of the seed culture was inoculated into each of 20 Ulpack 47 (culture bags) (HOKKEN Co. Ltd., Tochigi, Japan) containing a production medium (500 g of water-sodden rice). Static fermentation was carried out at 27 °C for 14 days. The stationary culture was extracted with acetone (18 L). After centrifugation, the supernatant was collected and concentrated in vacuo to remove acetone. The aqueous solution (4 L) was extracted three times with an equal volume of EtOAc. The organic layer was concentrated to dryness to afford a crude extract (50 g). The extract was chromatographed on a silica gel (Silica gel 60 (0.063–0.200 mm), Merck KGaA, Darmstadt, Germany) column and eluted stepwise with a mixture of CHCl3–MeOH (100:0, 100:1, 100:5, 100:10, 1:1 and 0:100). The 1:1 fraction (5.7 g) was applied to an ODS silica gel (YMC*GEL ODS-A, YMC Co., Ltd., Kyoto, Japan) column (60 i.d. × 140 mm) and eluted stepwise with a mixture of MeOH–H2O (20:80, 30:70, 50:50, 70:30, 80:20, 90:10 and 100:0). The 80:20 fraction was concentrated in vacuo to remove solvents.

The dried material (197 mg) was dissolved in a small amount of MeOH and applied to preparative HPLC (column, Pegasil ODS SP100: size, 20 i.d. × 250 mm; Senshu Scientific Co. Ltd., Tokyo, Japan) with an isocratic solvent system of 45% CH3CN at a flow rate of 10 mL/min. The eluted peak, from 80 to 84 min, was collected to give 21.5 mg of 1 (Fig. 1a) as a yellow oil. It was soluble in MeOH, acetonitrile, CHCl3 and DMSO but not in H2O. The compound showed [α]D23 + 21 (c = 0.1, CH3OH), IR (ATR) νmax 3362, 2925, 2850, 2359, 2335, 1716, 1699, 1653, 1558, 1541, 1457 1264, 1150, 1043 cm−1 and UV (MeOH) λmax nm(ε) 206 (65,600), 223 (43,200), 263 (30,500). The molecular formula of 1 was identified as C43H62O16Na (found m/z 857.3962 [M+Na]+, calculated m/z 857.3936 [M+Na]+) by HR-ESIMS. All connections for 1H and 13C in 1 were revealed by an HSQC study. The NMR data showed the presence of two methyl, 12 methylene, 11 sp3 methine, 12 sp2 methine, four fully substituted sp2 and two carbonyl carbons (Table 1). 1H–1H correlations in COSY from H-1 (δH 4.80) to H-6 (δH 3.89 and 4.04) showed the existence of a sugar moiety. The glycoside was elucidated to be the glucose type through analysis of the coupling patterns of proton signals, ROESY correlations (Fig. S7) and HOHAHA spectra (Fig. S8). Furthermore, the JH1,2 value (J = 10.0 Hz) indicated that it is a β-glucoside. The HMBC correlations from H-9 (δH 6.28) to C-7 (δC 114.4), C-8 (δC 158.7), C-10 (δC 159.3) and C-11 (δC 109.4), from H-11 (δH 6.41) to C-7, C-9 (δC 104.5), C-10, C-12 (δC 143.1) and C-13 (δC 63.7) and from H-13 (δH 4.55 and 4.64) to C-7, C-11 and C-12 proved the existence of a 3,5-dihydroxybenzyl alcohol unit. HMBC correlations from H-1 to C-7, C-8 and C-12 showed that this unit was combined with the glucose unit by a C-glycosidic bond. 1H–1H COSY between H-1′ (δH 4.35) and H-2′ (δH 3.48), H-3′ (δH 3.46) and H-4′ (δH 3.75) and H-5′ (δH 3.67) and H-6′ (δH 4.13 and 4.20) and HMBC correlations from H-1′ to C-3′ (δC 74.7), from H-2′ to C-1′ (δC 105.4) and C-3′, from H-3′ to C-1′ and C-2′ (δC 72.6), from H-4′ to C-2′ and C-3′, from H-5′ to C-1′, C-3′, C-4′ (δC 70.2) and C-6′ (δC 64.5) and from H-6′ to C-4′ and C-5′ (δC 73.9) indicated the presence of another sugar moiety containing an anomeric proton. The glycoside was elucidated to be the galactose type through analysis of the coupling patterns of proton signals, ROESY (Fig. S7) and HOHAHA spectra (Fig. S9). Moreover, the JH1′,2′ value (J = 7.1 Hz) showed that it is a β-galactoside. HMBC correlations from H-4 (δH 3.97) to C-1′ and from H-1′ to C-4 indicated that the galactose connected to the glucose unit through an O-glycosidic bond. Connections from C-2′′ (δC 121.6) to C-12′′ (δC 18.2) were elucidated by 1H–1H COSY. In addition, the presence of the oxygenated hydrocarbon unit was proved by HOHAHA spectra observed from H-12′′ (δH 1.74) to H-7′′ (δH 4.14) (Fig. S10) and from H-3′′ (δH 7.25) to H-7′′ (Fig. S11) and decoupling of proton signals of H-6′′ (δH 2.38), H-8′′ (δH 5.53), H-11′′ (δH 5.69) and H-3′′ (Figs. S12–14). HMBC correlations from H-2′′ (δH 5.87), H-3′′ (δH 7.25) and H-3 (δH 5.18) to C-1′′ (δC 168.9) indicated that this unit was connected to the glucose unit through the carbonyl carbon of C-1′′. The remaining 12 carbons were suggested to be an unsaturated fatty acid by 1D and 2D NMR data including HSQC-TOCSY (Fig. S15). The alignment from C-2′′′ (δC 121.9) to C-5′′′ (δC 29.2) was elucidated by 1H–1H COSY between H-2′′′ (δH 5.91) and H-3′′′ (δH 7.05), H-3′′′ and H-4′′′ (δH 2.25) and H-4′′′ and H-5′′′ (δH 1.49), and the alignment from C-10′′′ (δC 33.0) to C-12′′′ (δC 14.5) was elucidated by HMBC correlations from H-12′′′ (δH 0.90) to C-10′′′ and C-11′′′ (δC 23.7). The remaining four carbons were assigned as methylene carbons by HSQC, and their alignment was shown by HSQC-TOCSY correlations from H-4′′′ to C-6′′′ (δC 30.4) and C-8′′′ (δC 30.7), from H-5′′′ to C-7′′′ (δC 30.5) and C-9′′′ (δC 30.4) and from H-12′′′ to C-10′′′ (δC 33.0). HMBC correlations from H-2′′′, H-3′′′ and H-6′ to C-1′′′ (δC 168.0) indicated that it was connected to the galactose unit through the carbonyl carbon of C-1′′′. Thus, the planar structure of 1 was concluded to be as shown in Fig. 1b. All geometric isomerisms of 1 were elucidated to be the E-form by large coupling constants of JH2′′,3′′, JH4′′,5′′, JH8′′,9′′, JH10′′,11′′ and JH2′′′,3′′′. Compound 1 belongs to the papulacandin class of antibiotics [7, 8] and, to our knowledge, this represents the first report of the isolation of 1 from Pestalotiopsis sp.

Fig. 1
figure 1

a Structure of pestiocandin (1). b Key correlations of 1H–1H COSY and HMBC in pestiocandin (1)

Full size image
Table 1 NMR spectroscopic data for pestiocandin (1) in CD3OD
Full size table

There are two types of connection between glucose unit and the benzyl alcohol in papulacandin class antibiotics. The papulacandin type has a spirocyclic moiety, whereas the chaetiacandin type, including 1, does not [9, 10]. Two types of galactose units have been reported for chaetiacandin-type antibiotics, a β-galactofuranoside type (like furanocandin [10]) and β-galactopyranoside type (like the fusacandins A and B [11]). Compound 1 contains β-galactopyranoside as shown above and is confirmed by its carbon chemical shifts [12].

Minimum inhibitory concentration (MIC) values of 1 were evaluated against Gram-positive and Gram-negative bacteria, yeasts and a filamentous fungus by a liquid dilution method using 96-well microplates. Compound 1 showed moderate or weak growth inhibition against all Gram-positive bacteria tested as well as inhibition in some Gram-negative bacteria, yeasts and a filamentous fungus (Table 2).

Table 2 MIC values of pestiocandin (1)
Full size table

In yeast, our tests showed that 12 genes related to drug sensitivity were disrupted in S. cerevisiae 12geneΔ0HSR-iERG, while four genes related to drug sensitivity were disrupted in S. cerevisiae BY25929. Compound 1 showed moderate growth inhibition against S. cerevisiae 12geneΔ0HSR-iERG6, and weak growth inhibition against S. cerevisiae BY25929. Conversely, it did not inhibit growth in wild-type yeasts such as S. cerevisiae BY4741. Papulacandin class antibiotics, such as the papulacandins [13], L-687,781 [14], fusacandins A and B [15] and F-10748 C1 and C2 [16], have been reported to inhibit fungal β(1,3)-D-glucan synthase. However, 1 did not show potent antifungal activity.

In conclusion, a new papulacandin class antibiotic was isolated from the fungus, Pestalotiopsis humus FKI-7473. Our research has also confirmed that multidrug-sensitive S. cerevisiae organisms have become useful tools to facilitate isolation and identification of potentially promising compounds for development into much-needed new antibiotics.