Genomics-driven discovery of a new cyclodepsipeptide from the guanophilic fungusAmphichorda guana

Article abstract of DOI:10.1039/d1ob00100k

Two potential non-ribosomal peptide synthetases (NRPSs) were identified in the genome of a guanophilic fungusAmphichorda guanaby bioinformatics analysis and gene knockout experiments. Liquid chromatography coupled with mass spectrometry (LC-MS) guided isolation led to the discovery of a new cyclodepsipeptide isaridin H (1) and seven known analogs, desmethylisaridin E (2), isaridin E (3), isariin A (4), iso-isariin B (5), iso-isariin D (6), isariin E (7), and nodupetide (8). The absolute configuration of isaridin H (1) was achieved by Marfey's method. Isaridin H (1) showed significant antifungal activity againstBotrytis cinereaandAlternaria solani.

Full text of DOI:10.1039/d1ob00100k

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Genomics-driven discovery of a new
cyclodepsipeptide from the guanophilic
fungus Amphichorda guana†
Cite this: Org. Biomol. Chem., 2021,
19, 1960
Received 20th January 2021,
Accepted 12th February 2021
Min Liang,‡a,b Hai-Ning Lyu,
‡
a,c Zi-Ying Ma,a,b Er-Wei Li,a Lei Cai*a and
a,b
DOI: 10.1039/d1ob00100k
Wen-Bing Yin
*
rsc.li/obc
Two potential non-ribosomal peptide synthetases (NRPSs) were been widely applied as pharmaceuticals and agrochemicals,6–9
identified in the genome of a guanophilic fungus Amphichorda and some are known as mycotoxins.10–13 They are mainly dis-
tributed in the genera Acremonium, Aspergillus, Beauveria,
Fusarium, Isaria and Amphichorda.10,14–17 Amphichorda, a rare
genus with long white synnemata, is isolated from humid
habitats.18–20 Amphichorda was first established by Fries in
1825, and currently comprises only two species, A. felina and
A. guana, among which, A. guana is a novel bat guano-loving
fungus recently found in a Karst cave in Guizhou province,
China.21 A number of bioactive secondary metabolites (SMs)
have been isolated from A. felina, such as cyclosporin C, isar-
iins and isaridins.17,20,22–24 However, the secondary metabolic
profile of A. guana was fully unknown. In this study, we
sequenced the genomic DNA of A. guana and found that it con-
tains two NRPSs which were predicted to assemble CDPs.
Guided by LC-MS, a novel CDP isaridin H (1), along with
seven known analogs, desmethylisaridin E (2), isaridin E (3),
isariin A (4), iso-isariin B (5), iso-isariin D (6), isariin E (7) and
guana by bioinformatics analysis and gene knockout experiments.
Liquid chromatography coupled with mass spectrometry (LC-MS)
guided isolation led to the discovery of a new cyclodepsipeptide
isaridin H (1) and seven known analogs, desmethylisaridin E (2),
isaridin E (3), isariin A (4), iso-isariin B (5), iso-isariin D (6), isariin E
(7), and nodupetide (8). The absolute configuration of isaridin H (1)
was achieved by Marfey’s method. Isaridin H (1) showed significant
antifungal activity against Botrytis cinerea and Alternaria solani.
Introduction
Natural products such as peptides, polyketides, and terpenoids
are important drug resources due to their highly diverse struc-
tures.1 The skeletons of these compounds are constructed by
core biosynthetic enzymes for each class of natural products,
namely non-ribosomal peptide synthetases (NRPSs) for nodupetide (8), were isolated. The antifungal and antibacterial
peptides,2 polyketide synthases (PKSs)3 for polyketides, and activities of the isolates were also described.
terpene cyclases4 for cyclic terpenoids. Among these, non-ribo-
somal peptides (NRPs) represent a broad range of important
medicinal agents, such as the immunosuppressant cyclospor-
Results and discussion
ine, the antifungal caspofungin, the antibacterial daptomycin
and so on.5
To mine novel CDPs, the genomic DNA of A. guana LC5815
was subjected to PacBio sequencing. In silico analysis of the
draft genome of A. guana LC5815 using antiSMASH 4.0.1
revealed 54 secondary metabolite biosynthesis gene clusters
(BGCs) including 21 PKSs, 8 NRPSs (Table S3), and 6 hybrid
PKS-NRPSs. NRPs are biosynthesized by large, modular, multi-
Cyclodepsipeptides (CDPs) are a class of NRPs featuring
molecular ring formation involving an ester group. Most CDPs
(i.e., PF1022A, enniatins, destruxins and beauvericins) have
enzyme NRPS machinery. Each module of an NRPS is respon-
sible for the selection, loading, and condensation of a single
amino acid building block. The general NRPS module com-
prises three enzyme domains: adenylation (A), peptidyl carrier
protein (PCP; also known as the thiolation domain), and con-
densation (C). Proteinogenic and non-proteinogenic amino
acid building blocks are selected and activated by A domains,
and then other domains incorporate these amino acid sub-
aState Key Laboratory of Mycology and CAS Key Laboratory of Microbial
Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy
of Sciences, Beijing 100101, P.R. China. E-mail: cail@im.ac.cn, yinwb@im.ac.cn
bUniversity of Chinese Academy of Sciences, Beijing, 100049, China
cArtemisinin Research Center, and Institute of Chinese Materia Medica, China
Academy of Chinese Medical Sciences, Beijing 100700, China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1ob00100k
‡These authors contributed equally.
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strates into the final NRP products.25 Importantly, modifying
enzymes increases the diversity of products. To identify the
BGCs of CDPs, we retrieved the sequenced genome data of
A. guana LC5815 using a local Basic Local Alignment Search
Tool Protein (BLASTP) program. According to the structures of
CDPs which have been reported in A. felina and bioinformatics
tool analysis (antiSMASH, NRPSsp, CDD, interPro), two candi-
date BGCs in our novel species A. guana (cluster 20 and cluster
31) were expected to be involved in the biosynthesis of CDPs.
Cluster 20 includes a 22 906 bp core gene (AG06652) encoding
a putative NRPS with five A-domains. Meanwhile, cluster 31
includes a 24 262 bp core gene (AG03264) encoding a putative
NRPS with six A-domains. The candidate gene clusters are
illustrated by using the native BLAST (Fig. S1, S2 and Tables
S4, S5†) and the A domain specificities are shown in Table S6†
Fig. 1 HPLC analysis of fermentation products from WT A. guana and
mutants (ΔAG06652, ΔAG03264). The control strain and mutants were
grown in rice medium at 28 °C for 7 days; their fermentation products
were extracted using ethyl acetate (for detailed information see the
ESI†). Detection was carried out at 210 nm.
by
bioinformatics
software
analysis
(NRPSsp
and
NRPSpredictor3). We used the amino acid sequences of
AG06652 and AG03264 as a search query for the BLASTP
program. The results indicated that AG06652 had 58% identity
and 97% similarity to NRPS of Thermothelomyces thermophilus
ATCC 42464 (GenBank accession number XP_003664570.1),
and 33% identity and 92% similarity to destruxin synthetase
in Metarhizium robertsii ARSEF 23 (GenBank accession number
XP_007826232.2). Meanwhile, AG03264 had 62% identity and
99% similarity to destruxin synthetase of M. robertsii ARSEF 23
(GenBank accession number XP_007826232.2), and 39% iden-
tity and 96% similarity to cyclosporin
C synthetase of
Beauveria felina (GenBank accession number ATQ39428.1)
respectively. Similarly, AG03265 had 74% identity and 93%
similarity to aldo-keto reductase of M. robertsii ARSEF 23
(GenBank accession number XP_007826234.2) (Fig. S3†). The
biosynthetic pathway for potential CDPs can also be proposed
based on a mode for destruxin biosynthesis.26
To prove the two BGCs related to CDP production, we per-
formed gene deletion experiments. The AG06652 (NRPS) and
AG03264 (NRPS) genes were disrupted by homologous replace-
ments in A. guana, respectively.9 Correct transformants were
verified by diagnostic PCR (Fig. S4†). Subsequently, mutants
were cultured in rice medium for 7 days and subjected to
metabolite analysis. HPLC analysis of fermentation extracts of
wild-type A. guana and mutants (ΔAG06652 and ΔAG03264)
showed that most of those compounds are absent in the
mutants compared to the wild-type (WT) (Fig. 1). Interestingly,
the phenotypes of the mutants showed significant differences
in comparison with the WT because mutants cannot produce
synnemata (Fig. S5†). It is reported that synnemata play impor-
tant roles in ecological significance and are related to CDP pro-
duction (Fig. S5†).27,28,29–32
Fig. 2 (A) Total ion chromatography (TIC) from the positive ionization
mode of LC-MS of the A. guana LC5815 chemical extract in the range of
500–800 Da. A. guana LC5815 was grown in rice medium at 28 °C for 7
days, and then extracted using ethyl acetate. (B) The putative novel CDP
sequence indicated by the pronounced fragment ions in its LC-MS/MS
spectrum. (C) HPLC analysis of the target fraction isolated from the
crude extract of the wild type.
To identify these compounds missing from the mutant the protonated molecular ion at m/z 642.3978 [M + H]+
strains, we performed comparative metabolite analysis (Fig. 2B). Meanwhile, two know CDPs, isariin A (m/z 638.4315)
between deletion strains and the WT by LC-MS analysis of and iso-isariin B (m/z 596.3779), were deduced (Fig. S6†).
their extracts. The results showed that many high molecular
Combined with the results of knockout experiments and
weight compounds exist in the WT, but not in the mutants mass spectrometry, it was speculated that the missing com-
(Fig. 2A), implying the possible novel CDPs. The results were pounds were CDPs. To mine more CDPs in the bat guano-
further confirmed by LC-ESI-MS/MS profiling which pin- loving fungus A. guana, scale-up fermentation was carried out.
pointed the successive cleavage of amino acid moieties from The ethyl acetate extracts were separated by silica gel column
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Table 1 1H and 13C NMR spectroscopic data for isaridin H (1) (DMSO-
chromatography using different organic solvents to give four
fractions, and the targeted fraction was selected for further
purification using Sephadex LH-20 and semipreparative HPLC
(Fig. 2C) (for detailed information see the ESI†). Fortunately, a
novel CDP isaridin H (1), along with seven known analogs, des-
methylisaridin E (2), isaridin E (3), isariin A (4), iso-isariin B
(5), iso-isariin D (6), isariin E (7), and nodupetide (8), were iso-
lated from A. guana (Fig. 3). The known compounds were
determined by the comparison to the reported data (Tables
d6, 500 MHz for 1H NMR, 125 MHz for 13C NMR)
Position
δH (J Hz)
δC
1
2
3
171.3
33.8
34.2
2.65 m, 2.37 dd (15.2, 11.8)
3.71 m, 3.08 m
4
5
170.3
57.9
4.16 m
6
7
2.04 ddd (16.6, 11.6, 4.9)
0.64 d (6.7)
30.4
17.9
S9–S15†).24,28,30,33–35 Even the known isariin
E (7) was
8
0.81 d (6.7)
19.6
9
168.3
65.3
25.9
19.0
19.5
29.0
172.7
49.8
reported; however, no detailed NMR spectra were provided. In
this study, we report the NMR spectra of isariin E (7) for the
first time (for detailed information see the ESI†).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
4.62 d (10.4)
2.24 m
0.82 d (6.4)
0.94 d (6.4)
2.62 m
Isaridin H (1) was obtained as a white powder with [α]D25
−27 (c 0.1, MeOH), which was further proved to have a mole-
cular formula of C34H51N5O7 from the protonated molecular
ion at m/z 642.3859 (calculated for C34H52N5O7N, 642.3890) in
its high-resolution electrospray ionization mass spectrometry
(HR-ESI-MS). The UV spectrum showed absorption at 207 nm
(Fig. S16†). The IR spectrum indicated the presence of amine
(3274 cm−1), ester carbonyl (1740 cm−1), amide carbonyl
4.95 m
3.04 m, 2.84 m
37.2
137.4
129.4
128.3
126.7
128.3
129.4
173.7
58.6
27.5
25.1
47.3
167.4
75.0
7.38 d (7.5)
7.32 d (7.5)
7.25 m
7.32 m
7.38 m
(1647 cm−1), and aromatic (1545 cm−1
) functionalities
1
4.50 t (7.8)
2.19 m, 1.43 m
1.78 m
(Fig. S17†). The H NMR spectrum of 1 revealed the presence
of three NH protons (δH 9.11, 8.28, and 7.33), five aromatic
protons (δH 7.38–7.25, for H-19 to H-23), five CH protons [δH
4.95 (H-16), 4.91 (H-30), 4.62 (H-10), 4.50 (H-25), and 4.16
(H-5)], six methyl groups (δH 0.94–0.64, for H3-7, H3-8, H3-12,
H3-13, H3-33, and H3-34), and one N-methyl group (δH 2.62, for
H3-14) (Fig. S10†). Analysis of the 13C NMR spectrum of 1 indi-
cated the presence of six carbonyls, one quaternary aromatic
carbon, five tertiary aromatic carbons, one tertiary oxymethine,
four tertiary nitrogenated methines, seven methylenes, three
methines, six methyls, and an N-methyl, suggesting a CDP
structure (Fig. S11† and Table 1).33 With this information in
mind, the assignment of proton and carbon signals to the
amino acid residues was subsequently accomplished by inter-
preting their 1H–1H COSY, HSQC, and HMBC spectra
(Fig. S12–S14†), indicating that 1 was composed of Ala, HMPA
(2-hydroxy-4-methylpentanoic acid), Pro, Phe, N-Me-Val, and
Val residues. The sequence of amino acids in 1 was also con-
firmed on the basis of MS analysis (Fig. 2B). The absolute con-
figurations of the four alpha amino acids in 1 were determined
3.78 m, 3.22 m
4.91 dd (10.1, 4.9)
1.78 m, 1.52 m
1.65 m
0.85 d (6.6)
0.91 d (6.6)
37.6
24.4
22.6
21.5
through a combination of Marfey’s method and the use of
reversed-phase HPLC,34,36 and thus, the four amino acid units
(Val, N-Me-Val, Phe, and Pro) in
1 were assigned as
L-configured (Fig. S7†). The absolute configuration of C-30 in
HMPA was deduced as S from the ROESY correlation between
H-25 and H-30 (Fig. 4 and Fig. S15†).33 Hence, the structure of
1 was assigned as a new CDP and named isaridin H (Fig. 4).
Based on the CDPs isolated, we further confirmed that two
gene clusters were responsible for the biosynthesis of CDPs.
The new CDP isaridin H (1) and two analogs (2 and 3)
were also subjected to antimicrobial bioassays. These com-
pounds showed weak inhibitions against Escherichia coli and
Staphylococcus aureus. However, the novel CDP isaridin H (1)
Fig. 3 Structures of the isolated compounds 1–8.
1962 | Org. Biomol. Chem., 2021, 19, 1960–1964
Fig. 4 1H–1H COSY, key HMBC and ROESY correlations of 1.
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showed inhibition against Bacillus subtilis at 12.5 μM
(Fig. S8†). Most notably, 1 exhibits significant antifungal activi-
ties against Botrytis cinerea and Alternaria solani (Fig. S8 and
Table S8†). The IC50 value of antifungal activity was 10.0 µg
mL−1 (15.6 µM) against A. solani (Fig. S8†).
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Conclusions
To summarize, we identified two NRPS BGCs responsible for
the biosynthesis of CDPs by bioinformatics analysis and gene
deletion experiments in A. guana. LC-MS analysis showed that
A. guana could produce many high molecular weight com-
pounds which contain many CDPs. In this study, we have
identified eight CDPs, including one new CDP isaridin H (1)
and seven known analogs. The structure of new CDP was
identified by NMR, Mafey’s method and LC-ESI-MS/MS. In
addition, the new CDP isaridin H (1) was found to have anti-
fungal activity against several phytopathogens. Furthermore,
we proposed a biosynthetic model for the new CDP isaridin H
(1) according to the domain organisations of AG06652 and
AG03264 (Fig. S9†). Since neither of the two non-ribosomal
peptide synthases individually recognizes all assembled amino
acids according to A domain specificities, it is not clear
whether the two enzymes perform assembly functions
together.37 These isolated CDPs indicate that A. guana has
great potential to mine novel SMs. Our data also provide
insights into the biosynthetic origin of CDPs (isaridins and
isariins) in Amphichorda. It is worth noting that the discovery
of new active CDPs could accelerate the development of these
important molecules for agricultural and pharmacological
applications.
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
We would like to thank Drs Jinwei Ren and Wenzhao Wang
(Institute of Microbiology, CAS) for NMR, and MS data collec-
tion. This work was supported by the National Key Research
and Development Program of China (2018YFA0901901), the
National Natural Science Foundation of China (81872771),
the Key Research Program of Frontier Sciences, CAS
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