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org/OrgLett Letter

Discovery of a Cyclic Depsipeptide from Chaetomium mollipilium by

the Genome Mining Approach
Yuto Homma, Akihiro Sugawara, Yohei Morishita, Kento Tsukada, Taro Ozaki, and Teigo Asai*
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ABSTRACT: Genome mining and bioinformatics analyses allowed us to rationally find a candidate biosynthetic gene cluster for a
new cyclic depsipeptide of Chaetomium mollipilium. A heterologous reconstitution of the identified biosynthetic pathway predictably
afforded a new cyclic depsipeptide composed of L-leucine, L-tryptophan, and a polyketide moiety. Interestingly, the 10-membered
macrocycle structure generated equilibrium to an unprecedented cyclol structure. This study demonstrates the advantage of a
synthetic biology method in achieving rational access to new natural products.

T he discovery of natural products is one of the most

fundamental research areas of natural product chemistry,
prompting the growth of various research fields. In the
postgenomic era, genetic information has emerged as an
attractive source for new natural products, which has the
potential to replace conventional bioresources.1 Bioinformatic
analyses suggest that a large number of biosynthetic gene
clusters (BGCs) that encode cryptic natural products remain
uncharacterized.1,2 A combination of genome mining and
heterologous expression is one of the most powerful ways to
convert genomic information to compounds.
In addition, this methodology enables rational access to new
natural products hidden in genomic information. Genome
mining combined with bioinformatic analyses, such as
phylogenetic and comparative analysis, can estimate the
possibility of whether a target cluster is linked to new natural
products. Reconstructing the biosynthetic pathway in a
heterologous host affords all the natural products programmed
in the identified BGCs.3
Filamentous fungi are one of the most famous producers of
biologically important natural products. In addition, they
possess many untapped BGCs.1 Furthermore, excellent
heterologous expression systems using Aspergillus oryzae or
A. nidulans have been established,4 making fungi appropriate
sources for synthetic biology-based natural product discovery.
Thus, far, our research group has discovered various natural
products from fungal genomic information through this
approach.5 Here, we describe a discovery of a new fungal
cyclic depsipeptide from Chaetomium mollipilium.
Focused on fungal depsipeptides and lipopeptides because of
their attractive structure and biological activity,6 we mined
clusters that contain both a highly reducing polyketide
synthase (HR-PKS) gene and a nonribosomal peptide
synthetase (NRPS) gene using in-house draft genomic
sequences, and we found a candidate gene cluster (cmlp
cluster) in one fungus, Chaetomium mollipilium. The cluster
was composed of an HR-PKS gene (cmlpA), an AMP-binding
domain-containing enzyme gene (cmlpB), an acyltransferase
(AT)-like enzyme gene (cmlpC), and an NRPS gene (cmlpD),
and the cluster showed high similarity with that of
emericellamide A (Figure 1A),7 suggesting that the cmlp
cluster is responsible for the biosynthesis of a cyclic
depsipeptide. We carried out a homology search in the
National Center for Biotechnology Information (NCBI)
database and found a dozen of BGCs containing cmlpD
homologues (Figure S1). We also classified their NRPSs on the
basis of the number of Adenylation domains (A domains),
Received: April 4, 2022
Published: May 11, 2022

© 2022 American Chemical Society https://doi.org/10.1021/acs.orglett.2c01172

3504 Org. Lett. 2022, 24, 3504−3509
Organic Letters
Figure 1. (A) cmlp cluster from Chaetomium mollipilium compared with the eas cluster from Aspergillus nidulans. T, thiolation; C, condensation; A,
adenylation; E, epimerization; KS, ketosynthase; AT, acyltransferase; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; MT, methyl
transferase; ACP, acyl carrier protein. (B) Homologous BGCs (cm3, BllRfg, and f rb clusters) of the eas cluster. (C) Cyclic depsipeptides linked to
homologous BGCs of the eas cluster.
because the number of A domains in an NRPS generally relates
to the number of incorporated amino acids in a product
synthesized by an NRPS (Figure 1C).7,8 For example,
emericellamide A contains five amino acids, and EasA, an
NRPS responsible for the biosynthesis of emericellamide A,
possesses five A domains.7
The number of A domains in the NRPSs in this type of BGC
was bioinfomatically annotated and categorized into two, three,
five, six, eight, and twelve.7,8 Among them, Cm3A (three A
domains),8a EasA (five A domains),7 BllRfg_NRPS (eight A
domains),8b and FrbA (12 A domains)8c have been connected
to the corresponding cyclic depsipeptides, beauveriolide I
(three amino acids),8a emericellamides (five amino acids),7 Bll-
rafflesfungin (eight amino acids),8b and FR901469 (12 amino
acids),8c respectively (Figure 1B,C). In contrast, NRPSs with
two and six A domains have not been related to any natural
products. In addition, no cyclic depsipeptides with two amino
acids have ever been reported from Chaetomium fungi. The
above bioinformatics analysis suggested a cmlp cluster to
produce a new cyclic depsipeptide; therefore, we conducted a
heterologous expression study of the cmlp cluster.
pubs.acs.org/OrgLett Letter
In previous studies on emericellamides, four genes,
easABCD, were shown to be essential for biosynthesis by
gene knockout experiments.7 The same set of the biosynthetic
genes are conserved in the cmlp cluster (Figure 1A), suggesting
that cmlpABCD are also indispensable for biosynthesis. In the
hypothetical biosynthetic pathway of emericellamides,7 EasB
(HR-PKS) produces a polyketide carboxylic acid, which is then
activated and transferred to the T domain of EasA (NRPS).
EasA extends two amino acids and cyclizes the extended chain
to produce cyclic depsipeptides as final products. Although
EasD (AMP-binding domain-containing enzyme) and EasC
(AT-like enzyme) were proposed to be involved in the
activation and loading of the EasA product, the enzyme
responsible for the hydrolytic release of the PKS product was
not identified. However, EcdI, a fatty-acyl-AMP ligase that
shares 46% sequence identity with EasD, was shown to activate
and transfer linoleic acid to the T domain of EcdA (NRPS) in
the biosynthesis of echinocandin B.9 This finding suggests that
EasD and orthologous CmlpB are solely responsible for the
lipoinitiation of NRPS. Therefore, we hypothesized that
CmlpC would catalyze hydrolytic cleavage of the polyketide
chain from the ACP of HR-PKS CmlpA.
3505 https://doi.org/10.1021/acs.orglett.2c01172
Org. Lett. 2022, 24, 3504−3509
Organic Letters pubs.acs.org/OrgLett
Figure 2. (A) HPLC profiles of extracts of A. oryzae transformants
harboring the genes of the cmlp cluster. (B) Structure of
chaetodepsipeptin A (2a) and chaetodepsipeptin B (2b) and chemical
conversions of 2a and 2b to 1, 3, and 4.
On the basis of our hypothesis, we heterologously expressed
cmlpA, cmlpAC, and cmlpABCD in A. oryzae to construct AO-
cmlpA, AO-cmlpAC, and AO-cmlpABCD, respectively. AO-
cmlpA did not produce any products derived from the
introduced gene (cmlpA), whereas AO-cmlpAC produced
polyketide 1 (Figure 2A).
The structure of 1 was determined to be (2R,3R,4S)-3-
hydroxy-2,4-dimethylhexanoic acid by NMR spectral analyses,
derivatization to a reported methyl ester,10 and applying
advanced Mosher’s method (Figure 2A Figure S8; Table S7).11
LC−MS and HPLC analysis of the mycelial extracts of AO-
cmlpABCD showed two products, 2a (chaetodepsipeptin A)
and 2b (chaetodepsipeptin B) (Figure 2A and Figure S9).
Both compounds showed the same pseudo molecular ion peak
at m/z 442 [M + H]+, whereas the peak at m/z 424 [M − H2O
+ H]+, which was assumed to be the dehydrated pseudo
molecular ion peak, was only observed in the mass spectrum of
2b (Figures S10). We separated 2a and 2b by preparative
HPLC; subsequently, the separated fractions were reanalyzed
by HPLC. Both fractions showed 2a and 2b, indicating that 2a
and 2b were in equilibrium (Figure S11). Because it was
impossible to isolate 2a and 2b, we elucidated their structure as
a mixture. The ratios of 2a and 2b were altered depending on
the NMR solvent. The ratio of 2a to 2b was 2:3 in CDCl3
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Letter
(Figure S23), and the NMR analyses including 2D NMR
revealed the structure of cyclic depsipeptide 2a and its
derivative 2b with a quite unique cyclol system (6-hydroxy-
5-oxa-1,8-diazabicyclo[4.4.0]decane) (Figure 2B and Figure S9
and Tables S8 and S9).12 We assigned 2b, which showed an
ion peak of dehydration to the cyclol form because of the
existence of a hydroxy group (δH 3.96 for 1-OH; δC 99.8 for C-
1). During investigation of the ratio of 2a/2b in various
solvents, we found that the abundance of 2b increased in
pyridine-d5 (Figure S9). The NMR spectral analyses measured
in pyridine-d5 clearly supported the unique cyclol structure. In
addition, the NOESY spectra revealed the relative config-
uration of 2b except for C-22 in the polyketide moiety (Figure
S9). Subsequently, we conducted acidic hydrolysis of the 2a
and 2b mixture for applying advanced Marfey’s method.13 The
ethyl acetate extraction of the hydrolysis solution afforded
polyketide carboxylic acid 1. 1H and 13C NMR data of 1 from
the acidic hydrolysis was completely consistent with those of 1
obtained from AO-cmlpAC. Applying Marfey’s method to the
hydrolysis solution demonstrated the configuration of leucine
to be L (Figure S2). The configuration of tryptophan could not
be determined by Marfey’s method because of the epimeriza-
tion at the α position under acidic conditions (Figure S2).
Combining the information on the NOESY analysis, the
structure of the polyketide moiety, and the absolute stereo-
chemistry of L-leucine, the absolute configurations of 2a and
2b were fully determined (Figure 2B). To confirm the
structure of cyclic depsipeptide form 2a, our effort focused
on obtaining derivatives that do not produce an equilibrium
form. Treatment of a mixture of 2a and 2b with LiAlH4 was
converted to the acyclic diol compound 3, as expected, further
supporting the existence of 10-membered macrocycle form 2a
(Figure 2B and Figure S12; Table S10). We also applied
Marfey’s method to 3 and confirmed the absolute config-
urations of both leucine and tryptophanol residues to be L
(Figure S3). The L-configuration of the tryptophanol residue is
consistent with the above NOESY analysis of 2a/2b, further
supporting our conclusion. We named 2a and 2b chaetodep-
sipeptin A and chaetodepsipeptin B, respectively.
Next, we aimed to convert a derivative, maintaining its
bicyclic structure to confirm the cyclol structure. A dehydrated
form 4 was obtained by a dehydration reaction of 2a and 2b
(DMSO at 100 °C) (Figure 2B and Figure S13; Table S11). As
4 retained a 6/6 bicyclic scaffold of 2b, we reasonably
concluded that 4 was derived from the cyclol form 2b through
dehydration at C-1. The above chemical conversions of the
equilibrium mixture into 3 and 4 clearly confirmed that both
forms 2a and 2b were present in the solvent.
On the basis of the heterologous expression and metabolite
identifications, we proposed the biosynthetic pathway of the
depsipeptide 2a/2b (Figure 3). Remarkably, CmlpC is the first
example of an AT-like enzyme responsible for hydrolytic chain
release from HR-PKS.14 Although an AT-like enzyme, which
can hydrolyze stalled acyl units, has been known in the trans-
AT PKS system,15 CmlpC is classified into a protein family
different from such a proofreading AT (PedC [proofreading
AT in pederin biosynthesis]: PF00698 in Pfam; CmlpC:
PF02458), suggesting that Cmlp machinery employs a distinct
strategy for the release of polyketide chains. CmlpD
incorporates polyketide 1, which is probably adenylated by
CmlpB and catalyzes the condensation of L-leucine and L-
tryptophan. After the linear N-acyldipeptide scaffold is
constructed, the terminal C domian (CT domain)16 of
https://doi.org/10.1021/acs.orglett.2c01172
Org. Lett. 2022, 24, 3504−3509
Organic Letters pubs.acs.org/OrgLett
Figure 3. Putative biosynthetic pathways of 2a and 2b.
CmlpD catalyzes cyclization to generate cyclic depsipeptide
form 2a. Although CT domain-mediated diketopiperazine
formation, followed by a nucleophilic attack of the hydroxy
group to the amide carbonyl of L-tryptophan and subsequent
isomerization to 2a, could not be ruled out (Figure S4),12,17
the biosynthetic relationship with that of emericellamide made
us assume that this cyclol formation via diketopiperazine is less
likely than that via 10-membered macrocycle 2a. This NRPS
assembly system requires two A domains, three T domains,
two C domains, and one CT domain. However, CmlpD has a
T1C1T2C2A1T3C3A2T4CT domain system; therefore, one TC
unit is extra. Among the three C domains, the active site
HHXXXDG motif, is conserved in C1 and C3 domains,
whereas the corresponding motif in C2 is mutated to
SPXXXDG (Figure S5),18 indicating the C2 domain to be
inactive. We further demonstrated that C1, C3, and CT were
indispensable for the activity of CmlpD by site-directed
mutagenesis of histidine residues in the conserved motif of
each C domain (Figure S5). According to the general NRPS
biosynthetic manner, the A1 domain of CmlpD selectively
activates L-leucine and transfers it to the downstream T3
domain. In the same manner, the A2 domain likely activates
and transfers L-tryptophan to the T3 domain. To examine
which of the remaining T domains, T1 or T2, is required for
CmlpD activity, we prepared two CmlpD mutants, CmlpDΔT1
and CmlpDΔT2, by point mutation of the conserved serine
residue, the 4′-phosphopantetheine attachment site, to alanine.
Each cmlpD mutant gene was heterologously expressed with
cmlpABC in A. oryzae to construct AO-cmlpABCDΔT1 and AO-
cmlpABCDΔT 2 . HPLC analysis showed that AO-
cmlpABCDΔT1 abolished the production of 2a and 2b,
whereas AO-cmlpABCDΔT2 still produced 2a and 2b (Figure
2A). This result demonstrated that the T1 domain is essential
for the activity of CmlpD. Therefore, we concluded that the T2
and C2 domains are not involved in the biosynthesis of 2a.
Genome mining and bioinformatics analyses allowed us to
find a candidate biosynthetic cluster (cmlp cluster) for a new
natural product, and the heterologous expression of the cluster
predictably gave us a new cyclic depsipeptide, 2a. The 10-
membered ring in the depsipeptide 2a generated an
equilibrium to unprecedented cyclol structures 2b (6-
hydroxy-5-oxa-1,8-diazabicyclo[4.4.0]decane), which was
clearly supported by chemical derivatization. Although
ergometrin and lentopeptin are known to possess a 6/5 cyclol
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Letter
structure, which is quite rare in natural products, the 6/6 cyclol
structure disclosed herein is the first case as a natural product,
to the best of our knowledge. The heterologous expression
study revealed not only the biosynthesis of this type of natural
product but also a new AT-mediated hydrolytic chain-releasing
system of HR-PKS. Through this study, we demonstrated
rational access to new natural products by a combination of
genome mining and heterologous expression.
■ ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.2c01172.
Experimental procedures and characterization data;
figures of BGCs, HPLC profiles, cyclol formation routes,
structures, and mass, NMR, COSY, HMBC, HMQC,
and NOESY spectra; tables of oligonuceotides, con-
struction of expression plasmids, A. oryzae trans-
formants, and NMR data (PDF)
■ AUTHOR INFORMATION
Corresponding Author
Teigo Asai − Graduate School of Pharmaceutical Sciences,
Tohoku University, Sendai 980-8578, Japan; orcid.org/
0000-0002-8903-392X; Email: teigo.asai.c8@tohoku.ac.jp
Authors
Yuto Homma − Graduate School of Pharmaceutical Sciences,
Tohoku University, Sendai 980-8578, Japan
Akihiro Sugawara − Graduate School of Pharmaceutical
Sciences, Tohoku University, Sendai 980-8578, Japan;
orcid.org/0000-0003-1266-7497
Yohei Morishita − Graduate School of Pharmaceutical
Sciences, Tohoku University, Sendai 980-8578, Japan
Kento Tsukada − Graduate School of Pharmaceutical
Sciences, Tohoku University, Sendai 980-8578, Japan
Taro Ozaki − Graduate School of Pharmaceutical Sciences,
Tohoku University, Sendai 980-8578, Japan; orcid.org/
0000-0002-4673-4478
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.2c01172
https://doi.org/10.1021/acs.orglett.2c01172
Org. Lett. 2022, 24, 3504−3509
Organic Letters pubs.acs.org/OrgLett
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Professor Katsuya Gomi (Tohoku University) and
Professor Katsuhiko Kitamoto (The University of Tokyo) for
providing the expression vectors and the fungal strain. This
work was supported by JSPS KAKENHI (grant no. 19H04642
to T. Asai from Japan Society for the Promotion of Science
(JSPS)), AMED under grant no. JP21wm0325003, JST
FOREST Program (grant no. JPMJFR205W, Japan), the
Naito Foundation and the Uehara Memorial Foundation.
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