Wheldone: Characterization of a Unique Scaffold from the Coculture of Aspergillus fischeri and Xylaria flabelliformis.

Wheldone (1) was isolated and elucidated from a coculture of Aspergillus fischeri (NRRL 181) and Xylaria flabelliformis (G536), where secondary metabolite biosynthesis was stimulated by antagonism between these fungi. First observed via in situ analysis between these competing fungal cultures, the conditions were scaled to reproducibly generate 1, whose novel structure was elucidated by one- and two-dimensional NMR and mass spectrometry. Compound 1 displayed cytotoxic activity against breast, ovarian, and melanoma cancer cell lines.

Fungi have been explored for new compounds for nearly 100 years, and that has led to the discovery of unique chemical diversity possessing promising biological activities, ranging from antibiotic, to immunosuppressant, to cholesterol-lowering properties.[1−5] In nature, fungi grow in competition for resources, and as such, they have evolved the ability to adapt to changes in their environment. One of the ways they stave off rival organisms is through the activation of biosynthetic gene clusters, thereby stocking their arsenal for chemical warfare.[6−10] Under standard lab conditions, fungi have been shown to produce only a fraction of their potential secondary metabolites.[11,12] As such, coculturing fungi, forcing them to compete for limited resources, may present a pragmatic strategy to stimulate the biosynthesis of novel chemical diversity.[11,13−16]

To test this, fungi with antagonistic properties were chosen to participate in coculture experiments. The draft genome for Xylaria flabelliformis (strain G536; previously named Xylaria cubensis) was reported recently,[17] and this strain biosynthesizes griseofulvin, which is an FDA-approved fungistatic compound that is known to interact with a broad range of fungi.[18−20] Fungistatic denotes that it inhibits fungal growth, rather than killing competing fungi.[21] We hypothesized that griseofulvin (and cobiosynthesized analogues) would impart stress on the competing fungal culture, especially because we observed that X. flabelliformis exudes these compounds into its surroundings.[18,22]

Aspergillus fischeri (strain NRRL 181) was chosen as the challenger due to its genetic tractability[23] and the biosynthesis of metabolite weaponry in the form of mycotoxins.[22−25] Indeed, bioinformatic analysis of the genomes of both organisms predicted the presence of as many as 48 biosynthetic gene clusters for A. fischeri(23) and 86 biosynthetic gene clusters for X. flabelliformis,[17] yet only a relatively narrow range of secondary metabolites has been reported from either fungus. Our hypothesis was that the stress caused by the chemical warfare between these organisms would activate “silent” biosynthetic gene clusters and generate unprecedented chemical diversity.[13,26−29]

In a previous study, we reported the biosynthesis of several compounds that were found only in the coculture, including one putative new structure.[22] As reported herein, the isolation and characterization of a secondary metabolite (1) (Figure ) with a novel chemical scaffold supported our postulate that coculturing could generate new chemical diversity. This experiment was repeated several times in Petri dishes and in Erlenmeyer flasks (i.e., scaled up five times), demonstrating both a reproducible and scalable way to generate new fungal metabolites.

Figure 1

Wheldone (1) was isolated from the coculture of Aspergillus fischeri and Xylaria flabelliformis.

To initiate this experiment, monocultures of X. flabelliformis and A. fischeri were examined first in situ (Petri plates) by the droplet probe[30] to generate baseline profiles of the secondary metabolites. X. flabelliformis concentrates its fungistatic metabolites toward the colony edge (i.e., the youngest part of the fungal culture).[18] Alternatively, A. fischeri had an even distribution of secondary metabolites across its mycelium (i.e., the colony edge and the colony center had similar metabolites and relative abundances).[22]

Next, cocultures of X. flabelliformis and A. fischeri were examined by droplet probe once a clear “junction” was formed (Figure and larger version in Figure S8), which is the dividing area that is essentially the “battlefield” between the two competing fungi. The profile of compounds in the X. flabelliformis side of the coculture was interesting, as secondary metabolites were primarily observed in the junction. In contrast, A. fischeri was able to upregulate the biosynthesis of mycotoxins, suggesting that it was responding to the fungistatic properties imparted by the other fungus.[22]

Figure 2

(A) Wheldone (1) was first noted as a minor component in the base peak chromatogram during in situ analysis[22] of the junction that developed between A. fischeri and X. flabelliformis (shown in the box in the coculture Petri dish at the right). (B) Base peak chromatogram of the scaled-up coculture experiment (250 mL Erlenmeyer flasks), with the inset showing the mass spectrum of 1 and its adducts. Note that the chromatographic conditions were different between panels A (in situ analysis) and B (UPLC–MS), which is why the retention time of 1 varies.

There were several known metabolites identified in the junction that were not observed in the monoculture, as reported recently.[23,25,26] However, there was one minor peak that did not match with any metabolites in an in-house database of over 525 fungal metabolites,[31] that could not be characterized via mass defect filtering,[32] and whose molecular formula and spectroscopic data did not correlate to any organic compounds in the literature. Thus this metabolite was targeted for isolation and characterization. Collectively, these data suggested that biosynthetic gene clusters, which were previously silent, could be activated via coculturing experiments to generate new chemical diversity.

To isolate and characterize the compound observed in situ in the coculture experiments, solid-phase cocultures of A. fischeri and X. flabelliformis were grown on oatmeal. (See the Supporting Information.) The organic extract (CHCl3–MeOH (1:1)) of the fermentation product underwent purification using normal-phase flash chromatography to afford six fractions. Upon further purification via C18 preparative HPLC, fraction 2 yielded compound 1 (5.48 mg) (Figure ). The purity (99%) of 1 was assessed via UPLC–MS (Figure S1). This process was repeated five times to isolate larger quantities of 1 (>15 mg), showing the reproducibility and scalability of coculturing experiments.

Compound 1 was obtained as a white amorphous powder with a molecular formula of C25H34O6, as determined via HRESIMS along with 1H, 13C, and edited-HSQC NMR data (Table and Figures S2 and S3), demonstrating an index of hydrogen deficiency of 9. The 13C NMR data (Table ) indicated the presence of 25 carbons, inclusive of 2 carbonyl, 8 vinylic, 4 oxygenated, and 11 aliphatic carbons. The 1H and edited-HSQC NMR data (Table ) indicated four methyls, five olefinic protons, four methines, and three methylenes. The HMBC correlations from H-3 to C-1 and C-2 and from H-2 to C-1 as well as the COSY cross correlations between H-3 and H-2 indicated a trans (JH-3/H-2 = 15.97 Hz) α,β-unsaturated carboxylic acid (Figures S4 and S5). The COSY correlation between H-5 and H2-6, the HMBC correlations from H2-6 to C-5, H-5 to C-7 and C-4, and H3-23 to C-7 and C-5, along with the oxygenated carbons at C-6 (δC 74.8) and C-7 (δC 95.2) established the methylated 2,5-dihydrofuran ring. HMBC correlations from H-2 to C-4 and H-3 to C-5 and C-4 formed the connection between the furan ring and the α,β-unsaturated carboxylic acid.

Table 1

1H (700 MHz), 13C (175 MHz), and HMBC NMR Data for 1 in CD3OD

pos	δC, type	δH (J, Hz)	HMBC (H → C)
1	172.2, C
2	122.6, CH	5.71 (d, 15.97)	4, 1
3	137.4, CH	7.40 (d, 16.00)	6, 2, 4, 5, 1
4	137.1, C
5	142.8, CH	6.46 (t, 1.96)	6, 7, 3, 4
6	74.8, CH2	4.75 (dd, 12.01, 1.30)	4, 5
		4.81 (dd, 12.01, 1.03)
7	95.2, C
8	76.1, CH	3.54 (d, 10.41)	23, 9, 7, 19
9	43.0, CH	3.84 (dd, 10.45, 2.12)	15, 10, 8, 7, 17, 19
10	44.0, CH	1.96 (dt, 10.78, 3.49)	16
11	31.6, CH	1.19 (m)
12	36.4, CH2	0.94 (dq, 12.82, 2.63)
		1.56 (m)
13	23.5, CH2	1.23 (m)
		1.48 (m)
14	32.8, CH2	1.50 (m)
		1.74 (m)
15	34.6, CH	2.88 (br s)
16	142.9, CH	5.81 (br s)	25, 14, 10, 19
17	133.6, C
18	121.9, CH	6.57 (s)	9, 8, 17, 19, 20
19	157.3, C
20	208.7, C
21	74.2, CH	4.41 (q, 7.04)	22, 20
22	20.8, CH3	1.35 (d, 7.04)	21, 20
23	19.3, CH3	1.42 (s)	8, 7, 5
24	20.5, CH3	0.78 (d, 6.47)	11, 12, 10
25	20.0, CH3	1.91 (d, 1.11)	17, 16, 19

The COSY NMR spectrum of 1 displayed an 11-proton spin system (H-8/H-9/H-10/H-11/H3-24/H2-12/H213/H2-14/H-15/H-16/H3-25), which served to frame the bicyclic system (Figure ). The seven membered-ring was discerned via HMBC correlations from H3-25 to C-19, H-9 to C-19 and C-17, and H-18 to C-9, C-17, and C-19. The α-hydroxy-1-propanone side chain was elucidated through the COSY correlations of H3-22 (δH/δC 1.35/20.8) and H-21 (δH/δC 4.41/74.2) and the HMBC correlations to C-20 from both H3-22 and H-21; this side chain was connected to the seven-member ring via an HMBC correlation between H-18 and C-20. The bicyclic system was connected to the furan ring system via HMBC correlations from H-8 to C-7 and C-23 and H3-23 to C-8. The absolute configuration of 1 was assigned via the Mosher ester method[33] and NOESY correlations (Figures S6 and S7), establishing the configuration as (7R,8S,9R,10R,11S,15R,21S) (Figure ).

Figure 3

Key COSY and HMBC correlations for 1.

Figure 4

ΔδH values (Δδ = δ – δ) obtained for (S)- and (R)-MTPA esters of wheldone (1) (1a and 1b, respectively) in pyridine-d5.

Compound 1 was tested against a panel of tumor cell lines (Table ), MDA-MB-231 (triple-negative human breast cancer), OVCAR-3 (human ovarian cancer), and MDA-MB-435 (human melanoma cancer), using methods described previously.[34] (See the Cytotoxicity Assay section in the Supporting Information.) Although taxol was more potent, these data demonstrated that the cell lines responded to both compounds in the same rank (i.e., taxol and 1 display the highest and lowest activities in the same cell lines), with the highest response seen in MDA-MB-435, followed by OVCAR3 and then MDA-MB-231.

Table 2

Activity of 1 against Three Tumor Cell Lines

	IC50 (μM)a
compd	MDA-MB-231	OVCAR-3	MDA-MB-435
1	7.6	3.8	2.4
taxol	0.17	0.0051	0.00043

IC50 values were determined as the concentration required to inhibit growth to 50% of control with a 72 h incubation.

Using the coculturing of A. fischeri and X. flabelliformis, one novel compound (1) with cytotoxic activity was isolated and characterized. Our strategy employed the droplet probe[30] to first pilot the coculture conditions in a Petri dish, essentially scouting for changes in the secondary metabolite profile at the intersection of the fungal cultures.[22] Then, the cocultures were scaled to reproducibly generate 1 on the milligram scale, biosynthesizing enough material for further chemical and biological evaluation. Importantly, the scaled growths imparted a much higher concentration of 1 as compared with the Petri plates, likely because the antagonistic fungi were in close contact throughout the Erlenmeyer flask, as opposed to when they grow into each other in a Petri dish, which gives a visual indication of the battlefield (Figure and Figure S8) but is likely less efficient than constant interaction.

During the peer review of this manuscript, we started coculturing X. flabelliformis with another ascomycete fungus (strain MSX79272). Natural product studies on strain MSX79272 will be reported in more detail in the future. However, on the basis of DNA barcoding,[35] we know that this strain belongs to the order Hypocreales; Aspergillus spp. are in the order Eurotiales. We were encouraged when we observed a peak in the extract from a coculture experiment of these fungi that aligned with the retention time and HRMS data for 1 (Figure S9). The peak was isolated via HPLC, and a comparison between the 1H NMR data for 1 from both coculture experiments showed that they were in concordance (Figure S9). Because 1 was generated when X. flabelliformis was used in coculture experiments with two different fungal strains, we hypothesize biosynthesis by this organism. Of the limited fungal–fungal coculture experiments in the literature (∼40),[36] this is the first example of using an alternate fungus to narrow down the biosynthetic source for the new chemical entity. Given that the genomes of both fungal strains used in this study have been sequenced and putative biosynthetic gene clusters have been predicted,[17,23] future studies will take advantage of the development of improved heterologous gene expression platforms for the targeted production of fungal secondary metabolites[37] and to identify the biosynthetic gene cluster responsible for the biosynthesis of wheldone (1).