Induced production of zinniol analogues by co-cultivation of two endophytic fungi in the same ecological niche

Article abstract of DOI:10.1016/j.phytol.2019.12.007

Three zinniol analogues, pleoniols A–C (1–3), together with a known compound (4), were isolated from a mixed fermentation of two endophytic fungi, Pleosporales sp. F46 and Acremonium pilosum F47, both of which are found the pedicel of the medicinal plant Mahonia fortunei. High-performance liquid chromatography analysis showed that none of compounds 1–4 were detected when either one of the two fungi was cultured alone. The structures of compounds 1–4, including the absolute configurations, were deduced based on the interpretation of HRMS, NMR, and optical rotation data. Compounds 1–3 exhibited no antimicrobial efficacies against four Gram-positive or Gram-negative bacteria and two plant pathogenic fungi. Moreover, they demonstrated no obvious cytotoxic activities against four cancer cell lines, A549, MDA-MB-231, CT-26 and MCF-7. Our work indicates that the co-culture of endophytes in the same ecological niche is an effective approach for promoting interspecies interaction and enriching chemical diversity.

Full text of DOI:10.1016/j.phytol.2019.12.007

PhytochemistryLetters35(2020)206–210
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Induced production of zinniol analogues by co-cultivation of two endophytic
fungi in the same ecological niche
Zi-Ru Wanga, Hong-Xia Yanga, Xiao-Ping Penga, Gang Lia,*, Hong-Xiang Loua,b,
*
a Department of Natural Medicinal Chemistry and Pharmacognosy, School of Pharmacy, Qingdao University, Qingdao, 266021, People’s Republic of China
b Department of Natural Product Chemistry, Key Laboratory of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan,
250012, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three zinniol analogues, pleoniols A–C (1–3), together with a known compound (4), were isolated from a mixed
fermentation of two endophytic fungi, Pleosporales sp. F46 and Acremonium pilosum F47, both of which are found
the pedicel of the medicinal plant Mahonia fortunei. High-performance liquid chromatography analysis showed
that none of compounds 1–4 were detected when either one of the two fungi was cultured alone. The structures
of compounds 1–4, including the absolute configurations, were deduced based on the interpretation of HRMS,
NMR, and optical rotation data. Compounds 1–3 exhibited no antimicrobial efficacies against four Gram-positive
or Gram-negative bacteria and two plant pathogenic fungi. Moreover, they demonstrated no obvious cytotoxic
activities against four cancer cell lines, A549, MDA-MB-231, CT-26 and MCF-7. Our work indicates that the co-
culture of endophytes in the same ecological niche is an effective approach for promoting interspecies inter-
action and enriching chemical diversity.
Endophytes
Co-cultivation
Secondary metabolites
Zinniol analogues
Bioactivity
1. Introduction
the endophytic bacterium Bacillus wiedmannii Com1 was carried out,
and a new antibacterial ergosterol was discovered (Wang et al., 2019).
Endophytic fungi have been proven to be an important source of
natural products with unique structures and significant biological ac-
tivities (Aly et al., 2013; Gao et al., 2018). However, under the standard
laboratory culture conditions, most of their biosynthetic gene clusters
(BGCs) are silent, and many cryptic secondary metabolites have not
been explored (Rutledge and Challis, 2015; Scherlach and Hertweck,
2009). To date, some strategies have been developed to activate silent
BGCs to obtain novel natural products (Li and Lou, 2018). Among them,
co-cultivation is well-known as an effective approach to induce the
expression of BGCs through promoting interspecies interaction
(Marmann et al., 2014; Chen et al., 2015). A plethora of traits, such as
bioactive product formation (Wang et al., 2019), are typically involved
in the interspecies interaction and improve the richness of microbial
metabolites.
By further considering the interspecies interaction between endophytes
in the same ecological niche, the endophytic fungus Acremonium pi-
losum F47, also isolated from the pedicel of M. fortunei, was selected for
co-cultivation with Pleosporales sp. F46 in our current work. Herein, we
report the isolation, characterization, and antibacterial evaluation of
three new zinniol analogues, pleoniols A–C (1–3), via the co-cultivation
of Pleosporales sp. F46 with A. pilosum F47.
2. Results and discussion
In the pre-screening period, the EtOAc extracts from the co-culture
and axenic fermentations of endophytes in PDB media were analysed.
HPLC-based metabolic profiling was performed to compare their me-
tabolomes (Fig. 1). Several compounds were only detected when the
endophytic fungus Pleosporales sp F46 was co-cultivated with A. pilosum
F47 (Fig. 1), implying that several BGCs were activated. Further feeding
experiments performed by adding sterilized fungal materials to culture
media suggested that the formation of the abovementioned compounds
was very likely due to the fungal interspecies interaction (Fig. 1). To
obtain these potential bioactive compounds formed in the interspecies
Recently, an endophytic fungus Pleosporales sp. F46, was isolated
from the pedicel of the medicinal plant Mahonia fortunei by our research
group (Li et al., 2019). Its rice culture fermentation afforded seven new
heptaketides with antifungal or cytotoxic activities (Li et al., 2019). To
maximize the chemical diversity of this fungal endophyte by promoting
interspecies interaction, the co-cultivation of Pleosporales sp. F46 with
⁎ Corresponding authors at: Department of Natural Medicinal Chemistry and Pharmacognosy, School of Pharmacy, Qingdao University, Qingdao, 266021, People’s
Republic of China.
E-mail addresses: gang.li@qdu.edu.cn (G. Li), louhongxiang@sdu.edu.cn (H.-X. Lou).
https://doi.org/10.1016/j.phytol.2019.12.007
Received 16 October 2019; Received in revised form 22 November 2019; Accepted 16 December 2019
1874-3900/©2019PhytochemicalSocietyofEurope.PublishedbyElsevierLtd.Allrightsreserved.

Z.-R. Wang, et al.
PhytochemistryLetters35(2020)206–210
was revealed and was connected to C-3, which was supported by the
HMBC correlations of H3-4′/C-2′, H3-4′/C-3′, H3-4′/C-5′, H3-5′/C-2′, H3-
5′/C-3′, and H3-5′/C-4′, as well as the key HMBC correlation of H2-1′/C-
3 (Fig. 3). The strong HMBC correlation between H-1″ and C-5 was used
to place the sugar moiety at C-5 through an oxygen bridge (Fig. 3).
Thus, the planar structure of compound 1 (Fig. 2) was verified as a
zinniol analogue (Zhu et al., 2017), which was also in accordance with
the MS data.
The absolute configuration of the sugar motif was determined by the
measurement of the optical rotation (He et al., 2019; Wu et al., 2019).
Acid hydrolysis of 1 provided D-glucose, which was identified by direct
comparison with an authentic sample (Experimental Section and Fig.
S25). Therefore, the structure of compound 1 was established accord-
ingly. Compound 1 was assigned the name pleoniol A.
Compound 2 (Fig. 2) was also isolated as a colourless powder. It had
a molecular formula of C15H20O3 according to ESI-HRMS. The 1H NMR
spectrum of 2 (Fig. S10) indicated the presence of five singlet methyl
groups (δH 1.71, 1.81, 2.16, 2.17, and 2.47), one oxygenated methylene
(δH 4.29, d, J =7.0 Hz), one olefinic/aromatic proton (δH 5.58, t, J
=7.0 Hz), and two remaining singlet protons at δH 10.3 and 12.5. The
1H NMR data with the aid of 13C NMR and HSQC spectra of 2 (Figs. S11
and S12) identified five singlet methyl groups, one methylene group
(oxygenated), and an aldehyde group (δH 10.3; δC 194.7), together with
eight aromatic/olefinic carbons including one methine (δC 119.8), and
two oxygenated quaternary carbons (δC 161.7, and 163.4). The above
data for compound 2 suggested highly similar structural features to
those of compound 1, except for the absence of one oxygenated me-
thylene and a sugar moiety. Considering the key HMBC correlations
between H-10 and C-5 and C-6 and between H3-7 and C-6, as well as the
chemical shift of C-10, the aldehyde group in 2 instead of the oxyge-
nated methylene in 1 was placed at C-6 (Fig. 3). A free hydroxyl group
was placed at C-5 through the analysis of the key HMBC correlations
between 5-OH and C-4, C-5, and C-6 (Fig. 3). Accordingly, the structure
of compound 2 was determined. Compound 2 was named pleoniol B.
Compound 3 was obtained as a colourless powder and named
pleoniol C. It was assigned the molecular formula C15H20O4, showing it
had one additional oxygen atom compared with 2. Moreover, the 1H
and 13C NMR spectra of compound 3 (Figs. S17 and S18) showed that
its structure was similar to that of 2, except for the absence of H-10 and
5-OH in the 1H NMR spectrum of 3, and the upfield shift of C-10 in the
13C NMR spectrum of 3. The analysis of the above difference indicated
that the carboxyl group in 3, instead of the aldehyde moiety in 2, was
located at C-6, which was also supported by the 2D NMR data (Fig. 3
and Figs. S19–S21). The structure of the known compound 4 was elu-
cidated as (3R,4R)-3,4-dihydro-3,4,8-trihydroxy-1(2H)-naphthalenone
based on comparison of its NMR and CD data (Fig. S27) with those
reported in the literature (Borgschulte et al., 1992; Wu et al., 2019).
Zinniol and its analogues are tetraketide-derived compounds and
are frequently found in Alternaria species (Gamboa-Angulo et al., 2002;
Lou et al., 2013). In addition to their well-known phytotoxic effects
(Stierle et al., 1993), they have also been reported to have antifungal
activity (Yang et al., 2011), and anti-virus efficacy (Ai et al., 2012).
New compounds 1–3, as zinniol analogues (Zhu et al., 2017; Bitchi
et al., 2019), were screened for their antibacterial activities against the
Gram-positive bacteria Staphylococcus aureus (ATCC 6538) and Bacillus
subtilis (ATCC 9372) along with the Gram-negative bacteria Pseudo-
monas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922), and
their antifungal activities against two plant pathogens, Colletotrichum
musae [ACCC (Agricultural Culture Collection of China) 31244] and
Colletotrichum coccodes (ACCC 36067), were determined. Furthermore,
their cytotoxic activities against the A549, MDA-MB-231, CT-26, and
MCF-7 cancer cell lines were also evaluated. Unfortunately, in contrast
to the positive controls, none of the compounds were effective at the
given concentrations against the tested microorganisms or cancer cells.
Fig. 1. HPLC-DAD profiles (210 nm) of ethyl acetate extracts of the culture of
Pleosporales sp. F46, the culture of A. pilosum F47, the co-culture of Pleosporales
sp. F46 and A. pilosum F47, the culture of Pleosporales sp. F46 by feeding
sterilized fungal material from A. pilosum F47, and the culture of A. pilosum F47
by feeding sterilized fungal material from Pleosporales sp. F46.
interaction process, the EtOAc extract of the co-culture was fractionated
by flash chromatography, gel permeation chromatography (GPC) on
Sephadex LH-20, and semi-preparative HPLC to afford compounds 1–4,
which were not detected when the endophyte was cultured alone or
cultured with autoclaved fungal materials (Fig. 1).
Compound 1 (Fig. 2) was isolated as a colourless powder, and
possessed a molecular formula of C21H32O8 as determined by ESI-
HRMS. The 1H NMR spectrum of 1 (Fig. S1) showed the presence of five
singlet methyl groups (δH 1.66, 1.75, 2.11, 2.22, and 2.22), one olefinic
proton (δH 5.52), and the remaining oxygenated methylenes or me-
thines. Its 13C NMR spectrum (Fig. S2) exhibited 21 carbon signals in-
cluding two oxygenated aromatic/olefinic carbons (δC 151.5, and
155.2). The 1D NMR data (Table 1) in combination with the HSQC
spectrum (Fig. S4) established five singlet methyl groups, three oxy-
genated methylenes, and five oxygenated methines, together with eight
olefinic/aromatic carbons including one methine.
Interpretation of the 1H-1H COSY NMR spectrum (Fig. S3) identified
connections between C-1′ and C-2′, and C-1″ to C-6″, indicating the
presence of a sugar motif in the structure. The structure of compound 1
was further constructed according to the HMBC NMR data (Fig. S5).
Key HMBC correlations of H2-10 with C-1, C-5, and C-6, H3-7 with C-1,
C-2, and C-6, H3-8 with C-1, C-2, and C-3, and H3-9 with C-3, C-4, and
C-5 identified a benzene ring substituted with three methyls at C-1, C-2,
and C-4 (Fig. 3). In addition, an oxygenated methylene (C-10) was at-
tached to C-6 of the aromatic ring. An oxygenated dimethylallyl group
Fig. 2. Chemical structures of compounds 1–4.
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Z.-R. Wang, et al.
PhytochemistryLetters35(2020)206–210
Table 1
1H and 13C NMR Data for Compounds 1–3.
Position
1
2
3
δC, mult.a
δH mult.b (J in Hz)
Key HMBC
δC, mult.c
δH mult.d (J in Hz)
Key HMBC
δC, mult.c
δH mult.d (J in Hz)
Key HMBC
1
2
135.2, Cq
126.0, Cq
155.2, Cq
121.2, Cq
151.5, Cq
129.6, Cq
15.5, CH3
12.6, CH3
10.3, CH3
55.6, CH2
138.7, Cq
121.9, Cq
163.4, Cq
117.1, Cq
161.7, Cq
115.4, Cq
13.9, CH3
12.2, CH3
8.7, CH3
138.5, Cq
122.1, Cq
160.8, Cq
116.6, Cq
160.5, Cq
108.6, Cq
18.8, CH3
12.9, CH3
9.4, CH3
3
4
5
6
7
2.22, s
1, 2, 6
1, 2, 3
3, 4, 5
1, 5, 6
2.47, s
2.17, s
2.16, s
10.3, s
1, 2, 6
1, 2, 3
3, 4, 5
5, 6
2.49, s
2.18, se
2.18, se
1, 2, 6
1, 2, 3
3, 4, 5
8
2.11, s
9
2.22, s
10
4.36, d (11.0)
4.73, d (11.0)
4.15, dd (11.0, 7.0)
4.21, dd (11.0, 7.0)
5.52, t (7.0)
194.7, CH
174.7, Cq
1′
68.7, CH2
3, 2′
69.6, CH2
4.29, d (7.0)
5.58, t (7.0)
3, 2′
69.5, CH2
4.25, d (7.0)
5.58, t (7.0)
3, 2′
2′
120.6, CH
136.6, Cq
25.5, CH3
17.8, CH3
104.1, CH
74.0, CH
76.3, CH
70.6, CH
76.4, CH
61.5, CH2
1′, 3′, 4′, 5′
119.8, CH
138.6, Cq
25.9, CH3
18.0, CH3
1′, 3′, 4′, 5′
120.0, CH
138.3, Cq
25.9, CH3
18.0, CH3
1′, 3′, 4′, 5′
3′
4′
1.75, s
2′, 3′, 5′
2′, 3′, 4′
5, 2″, 5″
1″, 3″
2″, 4″
3″, 5″
4″, 6″
5″
1.81, s
1.71, s
2′, 3′, 5′
2′, 3′, 4′
1.80, s
1.71, s
2′, 3′, 5′
2′, 3′, 4′
5′
1.66, s
1″
2″
3″
4″
5″
6″
4.42, d (8.0)
3.29, t (8.0)
3.23, t (8.0)
3.05, me
3.04, me
3.67, d (10.5)
3.36, me
5-OH
a
b
c
d
e
12.5, s
4, 5
Recorded in DMSO-d6 at 125 MHz.
Recorded in DMSO-d6 at 500 MHz.
Recorded in CDCl3 at 125 MHz.
Recorded in CDCl3 at 500 MHz.
Overlapped.
250 mm). Silica gel (200–300 mesh; Qingdao Haiyang Chemical Co.,
Ltd., China) and Sephadex LH-20 (25−100 μm; Pharmacia, Uppsala,
Sweden) were used for column chromatography (CC). Thin-layer
chromatography (TLC) was performed with silica gel GF254 plates
(Qingdao Haiyang Chemical Co., Ltd., China).
3.2. Fungal material
The fungal endophytes Pleosporales sp. F46 and A. pilosum F47 were
both isolated from the pedicel of the medicinal plant M. fortunei, which
was collected from Qingdao, People’s Republic of China. The identifi-
cation of the endophytic fungus Pleosporales sp F46 has been described
previously (Li et al., 2019). The endophytic fungus A. pilosum F47 was
characterized by internal transcribed spacer (ITS) sequencing and its
ITS sequence has been deposited at GenBank (accession number
MN294707). The above fungi were deposited at the School of Phar-
macy, Qingdao University, People’s Republic of China. For large-scale
fermentation, the fungus Pleosporales sp. F46 or A. pilosum F47 was first
cultured in 30 Erlenmeyer flasks (500 mL) each containing 100 mL of
potato dextrose broth (PDB), on a rotary shaker (150 rpm) at 28 °C for 5
d. Then, the above PDB cultures from Pleosporales sp. F46 and A. pi-
losum F47 were mixed, and further incubated at 28 °C for 15 d in a
shaker (150 rpm). For more details, please see Fig. S26 in the Sup-
porting Information.
Fig. 3. Key 1H-1H COSY and HMBC correlations of compounds 1–3.
3. Experimental section
3.1. General experimental procedures
NMR spectra were recorded on a Bruker Advance spectrometer
operating at 500 (1H) and 125 (13C) MHz. Mass spectra were obtained
with an LTQ-Orbitrap spectrometer equipped with an ESI source.
Optical rotation was measured on a Jasco P-1020 digital polarimeter
(JASCO Corporation, Tokyo, Japan). IR spectra were obtained using a
Cary 600 Series FTIR spectrometer (Agilent Technologies, Germany).
Flash chromatography was performed on a Teledyne ISCO CombiFlash
Rf 200 system equipped with a C18 spherical column (20−35 μm,
100 Å, 80 g). The semi-preparative HPLC system (Agilent 1260 Infinity
II, Agilent Technologies, Germany) was equipped with a ZORBAX SB-
C18 column (4.6 × 250 mm) or an Eclipse XDP-C18 column (9.4 ×
3.3. Extraction and isolation
The entire fermentation was filtered through cheese cloth to sepa-
rate the supernatant from the mycelia. The supernatant was extracted
with ethyl acetate (EtOAc) by sonication at room temperature and then
evaporated under reduced pressure to yield a crude gum (3.8 g). The
extract was separated by flash chromatography and eluted with
MeOH–H2O (5 % MeOH for 5 min, followed by a gradient of 5–100 %
208

Z.-R. Wang, et al.
PhytochemistryLetters35(2020)206–210
MeOH over 55 min and 100 % MeOH for 25 min). All fractions were
combined to provide four major fractions (A–D) based on TLC analyses.
Based on the HPLC analyses, fractions containing secondary metabo-
lites that were detected only in the co-cultivation condition were sub-
jected to isolation and purification. Fraction A (65.0 mg) was directly
purified by semi-preparative HPLC (ZORBAX SB-C18 column, MeOH-
H2O-0.1 % FA, 70/30, 1.0 mL/min) to yield compound 4 (11.2 mg, tR
=10.5 min). Fraction B (116.0 mg) was applied to the Sephadex LH-20
GPC (MeOH) to yield four subfractions (B1-B4). Subfraction B3
(36.0 mg) was further purified by semi-preparative HPLC (ZORBAX SB-
C18 column, MeOH-H2O-0.1 % FA, 57/43, 1.0 mL/min) to provide
compound 1 (10.1 mg, tR =12.5 min). Fraction C (101.3 mg) was also
applied to the Sephadex LH-20 GPC (MeOH) to yield four subfractions
(C1-C4). Subfraction C3 (13.2 mg) was purified by semi-preparative
HPLC (Eclipse XDP-C18 column, MeOH-H2O-0.1 % FA, 77/23, 2.0 mL/
min) to generate compound 3 (2.7 mg, tR =31.0 min). By following a
similar isolation procedure as that used for fractions B and C, fraction D
(51.3 mg) was fractionated by a Sephadex LH-20 column (MeOH) and
purified by semi-preparative HPLC (Eclipse XDP-C18 column, MeOH-
H2O-0.1 % FA, 86/14, 2.0 mL/min) to afford compound 2 (1.9 mg, tR
=20.0 min).
against two plant pathogens, Colletotrichum musae (ACCC 31244) and
Colletotrichum coccodes (ACCC 36067). The cytotoxicities of the isolated
metabolites were determined against four cancer cell lines, A549, MDA-
MB-231, CT-26 and MCF-7 with the MTT method (Li et al., 2019).
Streptomycin, actidione, and adriamycin were used as the positive
controls for the analysis of the antibacterial, antifungal, and cytotoxic
activities, respectively.
4. Conclusions
Herein, we report the isolation and characterization of three new
zinniol analogues, pleoniols A–C (1–3), and a known polyketide (4),
from the co-cultivation of the endophytic fungi Pleosporales sp. F46 with
A. pilosum F47. None of compounds 1–4 were detected when either one
of the two fungi was cultured alone. The novel compounds 1–3 were
evaluated for their antimicrobial and cytotoxic activities.
Unfortunately, none of them were effective at the given concentrations
against the tested microorganisms or cancer cells. Our work revealed
that the co-culture of endophytes from the same ecological niche pro-
motes interspecies interaction and is an effective approach for enriching
chemical diversity.
3.3.1. Pleoniol A (1)
Declaration of Competing Interest
There are no competing conflicts to declare.
Acknowledgements
Colourless powder; [α]20 D = -11.9 (c 0.10, MeOH); UV λmax
202 nm; IR vmax 3515, 3410, 3278, 2914, 1687, 1370 cm−1
;
1H NMR
(500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6), see Table 1;
(+)-ESI-HRMS m/z: 435.1981 [M+Na]+ (calcd. for C21H32O8Na,
435.1989, Δ −1.8818 ppm), m/z: 395.2059 [M+H – H2O]+ (calcd. for
C
21H31O7, 395.2064, Δ −1.3351 ppm), m/z: 847.4087, [2 M + Na]+
This work was supported by the National Natural Science
Foundation of China [grant numbers 81903494 and 41706077]; and
the Scientific Research Foundation of Qingdao University.
(calcd. for C42H64O16Na, 847.4087, Δ 0.0408 ppm).
3.3.2. Pleoniol B (2)
Colourless powder; UV λmax 216, 286, 354 nm; IR vmax 3733, 2917,
Appendix A. Supplementary data
1626, 1421, 1314 cm−1
;
1H NMR (500 MHz, CDCl3) and 13C NMR
(125 MHz, CDCl3), see Table 1; (-)-ESI-HRMS m/z: 247.1338 [M - H]−
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2019.12.007.
_
(calcd. for C15H19O3 247.1340, Δ 0.5729 ppm).
3.3.3. Pleoniol C (3)
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