The Discovery of Fungal Polyene Macrolides via a Postgenomic Approach Reveals a Polyketide Macrocyclization by trans-Acting Thioesterase in Fungi

Article abstract of DOI:10.1021/acs.orglett.9b01674

Heterologous expression of a unique biosynthetic gene cluster (BGC) comprising a highly reducing polyketide synthase and stand-alone thioesterase genes in Aspergillus oryzae enabled us to isolate a novel 34-membered polyene macrolide, phaeospelide A (1).

Full text of DOI:10.1021/acs.orglett.9b01674

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
The Discovery of Fungal Polyene Macrolides via a Postgenomic
Approach Reveals a Polyketide Macrocyclization by trans-Acting
Thioesterase in Fungi
Yohei Morishita,† Huiping Zhang,‡ Tohru Taniguchi,§ Keiji Mori,∥ and Teigo Asai*,†
†Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo
153-8902, Japan
‡NMR Science and Development Division, RIKEN Spring-8 Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa
230-0045, Japan
§Faculty of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Kita
21 Nishi 11, Sapporo 001-0021, Japan
∥Department of Applied Chemistry, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16
Nakacho, Koganei, Tokyo 184-8588, Japan
S
* Supporting Information
ABSTRACT: Heterologous expression of a unique biosynthetic gene cluster (BGC) comprising a highly reducing polyketide
synthase and stand-alone thioesterase genes in Aspergillus oryzae enabled us to isolate a novel 34-membered polyene macrolide,
phaeospelide A (1). This is the first isolation of a fungal polyene macrolide and the first demonstration of fungal aliphatic
macrolide biosynthetic machinery. In addition, sequence similarity network analysis demonstrated the existence of a large
number of BGCs for novel fungal macrolides.
ungi have already provided a wide array of secondary
Fmetabolites, and many of these metabolites and their
derivatives have made vital contributions to drug develop-
ment.1 Fungal secondary metabolites therefore remain an
attractive source for drug discovery. Fungi are well-known to
harbor a large number of biosynthetic gene clusters (BGCs).2
Many of them have not yet been developed or linked to their
associated natural products. In other words, fungal genomic
information contains untapped diverse biosynthetic pathways,
and this information is one of the most promising resources for
natural product discovery in the postgenomic era.3
Synthetic biology methods based on genome mining and
heterologous biosynthesis make up a promising approach for
translating genomic information into its associated natural
products.4 The vast amount of biosynthetic knowledge that has
been accumulated to date facilitates the exploration of BGCs,
not only for known pharmaceutically beneficial natural
products but also for natural products with novel structures.
On the other hand, heterologous biosynthesis allows the
production of any natural product, even if it is highly complex,
by simply introducing the corresponding biosynthetic genes
into a model host.5 In addition, even biosynthetic genes that
are silent in the host organism are available to this method.
Therefore, to rationally discover novel natural products and
biosynthetic machineries, we have used a synthetic biology
method, so-called “postgenomic natural product discovery”.
Fungal highly reducing polyketide synthase (HR-PKS) is a
multifunctional, iterative type I PKS, which is mainly
composed of typical domains, including ketosynthase (KS),
acyltransferase (AT), dehydratase (DH), enoyl reductase
(ER), ketoreductase (KR), and acyl-carrier protein (ACP)
and/or methyl transferase (MT) domains.6 However, carbon
frameworks that differ in terms of chain length and
modification pattern are encoded by each gene.7 These
genes are thereby responsible for skeleton formation via
diverse biosynthetic pathways, so they have an important role
in generating a diversity of natural products. While HR-PKSs
are widely distributed among Ascomycota fungi, most natural
products produced by HR-PKSs remain undiscovered. We
have therefore focused on HR-PKS genes as a preferred
landmark in genome mining to find putative BGCs encoding
novel natural products and unique biosynthetic machineries.
Received: May 13, 2019
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.9b01674
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Here, we demonstrate HR-PKS-guided genome mining and
heterologous biosynthesis allow us to discover for the first time
fungal polyene macrolides and to reveal a key new offloading
system of HR-PKS for fungal aliphatic macrolide formation.
We previously collected insect-associated fungal strains as a
unique resource for natural product exploration8 and obtained
their draft genomic information via next-generation sequence
analysis. We performed HR-PKS gene-guided genome mining
in these sequenced strains and found a simple but unique BGC
composed of a HR-PKS (KS-AT-DH-ER-KR-ACP) gene,
apmlA, and a putative stand-alone thioesterase (TE) gene,
apmlB, in hairy caterpillar-associated Arthrinium phaeospermum
(Figure 1A). We then conducted a sequence similarity network
To identify the compound produced by the apml cluster and
investigate fungal aliphatic macrolide biosynthesis, we
conducted heterologous expression of the apml cluster in
Aspergillus oryzae NSAR1,16 which has been shown to be an
excellent host for heterologous biosynthesis of fungal natural
products.5b−d Initially, we introduced apmlA and/or apmlB
into A. oryzae to prepare two transformants: AO-apmlAB and
AO-apmlA. These transformants were cultivated, and the
MeOH extracts of their mycelia were analyzed using reverse-
phase HPLC (Figure 2A). An A. oryzae strain that was
Figure 1. (A) apml cluster. (B) Sequence similarity network of the
fungal HR-PKS encoded with TE and their characterized
representatives with some homologues.
analysis of the HR-PKSs versus functionally characterized or
predicted HR-PKSs using the EFI-EST web tool developed by
Gerlt and colleagues.9 Visualization of this data was carried out
using Cytoscape software. As a result, HR-PKSs including
ApmlA, which are flanked by stand-alone putative TEs, formed
a large cluster, which implies that the HR-PKSs work
cooperatively with the corresponding TEs in the production
of a class of natural products. In the same manner, functionally
similar HR-PKSs make clusters like each other (e.g., LovB and
EqiS,10 which construct decalin polyketides; Rdc5, Hpm8, and
PKS4,11 which are involved in the biosynthesis of resorcylic
macrolides; and LovF, AzaB, and AfoG,12 which produce a
linear carbon chain that is transferred to another skeleton by an
acyltransferase) (Figure 1B). It is notable that an ApmlA
cluster in the network analysis contains Bref-PKS, which has
been shown to biosynthesize the corresponding polyketide
chain of brefeldin A, a 16-membered aliphatic macrolide
(Figure 1B and Figure S1), in the presence of a putative TE,
Bref-TH, although macrolactonization has not yet been
achieved.13 In the biosynthesis of fungal resorcinolic macro-
lides like radicicol and bacterial macrolide antibiotics
represented by erythromycin, the C-terminal TE domain in
each PKS is involved in macrocyclization of the elongated
polyketide chain.11a,14 Considering the biosynthetic manners,
BGCs containing an HR-PKS and a stand-alone putative TE
may be responsible for the biosynthesis of fungal aliphatic
macrolides, an important fungal natural product group, such as
cephalosporolides, brefeldin A, berkeleylactones, and rickiols,
although there have not been reports of the biosynthesis of
these products (Figure S1).15
Figure 2. (A) HPLC profiles of the mycelial extracts of the
transformants. (B) UV absorption spectra. (C) Structures of 1−4.
(D) Two-dimensional NMR correlations of 1.
transformed using an empty vector was used as the negative
control. The HPLC profile of AO-apmlAB showed two
significant peaks, 1 (major) and 2 (minor), which were absent
in the control. Both peaks showed the same UV spectra during
diode-array detector (DAD) analysis, whereas different
molecular ion peaks were observed via LC-MS analysis at m/
z 609 [M + Na]+ and m/z 565 [M + Na]+ (panels A and B,
respectively, of Figure 2). This suggested that 1 and 2
contained the same polyene structure and that the structure of
2 had a partial structural difference corresponding to 44 Da
from 1. Although no noteworthy peaks appeared for the AO-
apmlA extract, introduction of apmlB into AO-apmlA (AO-
apmlA+apmlB) initiated the production of 1 and 2, revealing
that both ApmlA and ApmlB are required for this biosynthesis.
To isolate and determine the structures of 1 and 2, we
performed a scaled-up cultivation of the AO-apmlAB trans-
formant and extracted the cultured mycelia with MeOH.
Repeated silica gel chromatography facilitated the isolation of
1 (1.1 mg/L), whereas the properties of 2 made it difficult to
purify using our equipment. Hence, the structure of 2 was
isolated and characterized on the basis of the corresponding
B
DOI: 10.1021/acs.orglett.9b01674
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Figure 3. (A) Scheme of fragmentation and derivatization of 1: (a) Ac2O, pyridine, rt, overnight; (b) (i) O3, CH2Cl2/MeOH (1:1), −78 °C, 35
min; (ii) NaBH4, 0 °C, 30 min; (c) (i) TBDPSCl, imidazole, DMF, rt, 24 h; (ii) NaOMe, MeOH, rt, 1.5 h; (iii) 2,2-dimethoxypropane, pyridinium
p-toluenesulfonate, acetone, rt, 1.5 h. (B) Key NOE correlations (green arrow) of acetonides. (C) ΔδH(S−R) values (parts per million) of the MTPA
derivatives. (D) VCD and IR spectra of 12 compared with those of (S)- and (R)-authentic samples (c = 0.06 M for 12 derived from the ozonolysis
fragment, or c = 0.15 M for both authentic 12/CDCl3 solutions, measured at ambient temperature).
acetylated derivative 4 (Figure 2C). The molecular formula of
1 (phaeospelide A) was identified as C34H50O8 on the basis of
positive HRESIMS spectra (m/z 609.3366 [M + Na]+)
coupled with 34 signals in the 13C NMR spectrum. The four
UV absorptions (log ε) at 325 (4.77), 339 (4.75), 357 (4.53),
and 377 (4.23) nm and the narrow accumulation of the olefin
proton signals (H-7−H-12) within 0.1 ppm (δH 6.21−6.31)
indicated an all-trans-hexaene structure in 1.17 The sequential
compared with that of authentic (S)- or (R)-1,2,4-butanetriol,
resulting in the identification of a 25S configuration in 1
(Figure 3D). Fragment 5 was derived to give acetonides 8 and
9 by three-step reactions (Figure 3A). The NOESY spectral
analyses of 8 and 9 showed that both took a chair form, with all
oxygen atoms oriented in the same direction (Figure 3B).
Similarly, fragment 7 was converted to 10 and 11. The NOESY
spectral analyses of 10 and 11 revealed the following.
Compound 11 took a chair form, and its oxygen atoms at C-
29 and C-31 were in the cis configuration; 10 was in a twist-
boat form, and the oxygen atom at C-33 was oriented in the
direction opposite to C-31. Finally, the advanced Mosher’s
method was applied to 8 and 10, and the absolute
stereochemistries at C-17 and C-29 were both determined to
be S, based on ΔδH(S−R) values (Figure 3C).18 All of the
absolute stereochemistries in 1 were thus identified to be
17S,19S,21S,25S,29S,31S,33S.
The planar structure of 4 was determined to be a 32-
membered macrolide with the same hexaene motif of 1 via
positive HRESIMS spectral and NMR spectral analyses (Figure
S4 and Table S3). The detailed structural elucidation is
described in the Supporting Information. Compound 2
(phaeospelide B) was thus identified as the deacetylated
structure of 4 (Figure 2C). The structural difference between 2
and 1 was only a partial CH2-CHOH (44 Da) structure: C-16/
C-17, C-18/C-19, or C-20/C-21 in 1, which corresponds to
one polyketide elongation cycle. The absolute stereochemis-
tries in 2 were determined to be the same as those in 1, based
on their biosynthetic relevance. We further investigated the
production of 1 and 2 in Ar. phaeospermum, under several
culture conditions. However, HPLC analysis detected no
production of 1, 2, or their analogues, demonstrating the
advantage of this postgenomic approach (Figure S5).
1
correlations of H−1H COSY and HSQC-TOCSY coupled
with the HMBC and HSQC spectral analyses revealed the
partial structures of C-13−C-34 and C-2−C-6. The HMBC
correlations of H-33/C-1 revealed connectivity between C-1
and C-33 through ester linkage (Figure 2D). The six remaining
sp2 carbons were set between C-6 and C-13 to form the
hexaene. The E geometries of Δ22,23 and Δ26,27 were
characterized by vicinal couplings at J22,23 and at J26,27 of 15.3
Hz and NOESY correlations of H-21/H-23, H-22/H-24, H-
25/H-27, and H-26/H-28 (Figure S2). The assignments of all
proton and carbon resonances, listed in Table S1, were
achieved on the basis of detailed two-dimensional NMR
spectral analyses. Thus, the planar structure of 1, with 34-
membered macrolactone rings and an all-trans conjugated
hexaene, was elucidated as depicted in Figure 2 (details are
provided in the Supporting Information). The locations of all
of the olefins and hydroxy groups were consistent with the
typical polyketide rule.6 The spectral analyses of its acetyl
derivative 3 supported the planar structure of 1 (Figure S3 and
Table S2).
Compound 1 possessed seven chiral centers, at C-17, C-19,
C-21, C-25, C-29, C-31, and C-33. To determine all of the
absolute configurations, we used a chemical conversion
approach. Initially, 1 was acetylated to 3 to improve solubility
and stability. Compound 3 was then decomposed via
ozonolysis to give three fragments (5−7). Fragment 6 was
derived to give triacetate 12, whose VCD spectrum was
We also screened for antibacterial and antifungal biological
activity in 1, but we have not yet found any such activity.
C
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Scheme 1. Proposed Biosynthetic Mechanism of 1
On the basis of the structure, including the absolute
configuration of the macrolides produced by the apml BGC,
we have proposed a unique biosynthetic mechanism for
generating novel macrolides (Scheme 1). The HR-PKS ApmlA
uses acetyl-CoA and malonyl-CoA as its starter and extender
units, respectively, and provides the large carbon framework in
1 via 16 cycles of polyketide chain elongation, which is the
largest number identified in fungal iterative PKSs thus far.19
The tailoring module used depends on the extension round.
The polyol motif is installed during the first half of the rounds.
The absolute configuration of 1 reveals the intriguing function
of the KR domain as follows. During round 1, the KR domain
reduces β-ketone to an L-oriented hydroxy group, while during
later rounds, it provides hydroxy groups in the D-configuration.
The biosynthetic aspect of the KR domain is interesting in that
it can reduce β-ketone in the L- and D-configurations, according
to the situation. The characteristic conjugated hexaene moiety
is built during the later rounds (10−15), when the KR and DH
domains are at work but ER is off. Phylogenetic analysis of the
DH domain of ApmlA shows the similarity with that of Fma-
PKS, which biosynthesizes the highly conjugated polyketide
side chain of fumagillin,20 suggesting that a polyene formation
is programmed in the DH domain (Figure S6). Finally, the
mature ACP-tethered carbon chain is transferred to the serine
residue of TE, ApmlB, followed by intramolecular macro-
lactonization, generating 1. When one elongation cycle during
rounds 7−9 is skipped, compound 2 is biosynthesized instead.
In conclusion, we found a unique BGC comprising an HR-
PKS, apmlA, and a TE, apmlB, in the draft genome of the hairy
caterpillar-associated fungus Ar. phaeospermum, using HR-PKS-
guided genome mining. Heterologous expression of the cluster
in A. oryzae afforded novel natural products 1 and 2, which are
the first examples of fungal polyene macrolides. We fully
elucidated the structure of 1, including its absolute
configuration, via spectral analyses and chemical means,
revealing the characteristic 34-membered ring macrolide
structure composed of hydrophobic all-trans-hexaene opposite
a hydrophilic polyol moiety. The structure of 1 also exhibits a
unique biosynthetic mechanism, programmed by ApmlA,
featuring the largest number of chain elongation cycles
among fungal HR-PKSs and the multistereo control of its
KR domain. Our analysis results for ApmlA may serve as a clue
for understanding the enigmatic “programming” that is
encoded in fungal HR-PKS. While HR-PKSs lack a releasing
domain, various polyketide chain-release mechanisms exist,
such as spontaneous cleavage with pyrone formation,21
polyketide chain transfer from HR-PKS to nonreducing PKS
in asperfuranone biosyntesis,22 and enzymatic cleavage of the
polyketide chain catalyzed by hydrolase in lovastatin biosyn-
thesis.23 This study is the first demonstration that trans-acting
TEs catalyze macrolactonization of a polyketide chain on HR-
PKS, which is the new offloading system of HR-PKS.
Phylogenetic analysis showed a new TE family that may be
responsible for fungal macrolide formation (Figure S7). In
addition, similar sequence network analysis and phylogenetic
analysis using ApmlA as a reference revealed a large family of
HR-PKSs that cluster with ApmlB homologues (Figure 1 and
Figure S8). In other words, diverse BGCs that are probably
involved in aliphatic macrolide biosynthesis are present in
various fungal species. Previously, various aliphatic macrolides
have been reported from several fungal species, such as
Beauveria bassiana (cephalosporolides),24 Pyrenophora teres
(pyrenolides),25 Diplodia sapinea (diplodialides),26 Eupenicil-
lium brefeldianum (brefeldin A),13,27 and Eupenicillium shearii
(eushearilide).28 Therefore, the BGCs in macrolide producers
may correspond to the biosynthesis of previously reported
compounds. In contrast, the other BGCs in fungi that have not
been reported upon production of macrolides could be a
fascinating source of novel fungal aliphatic macrolides.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b01674.
Experimental details, figures and additional information,
full spectroscopic data, and NMR spectra of new
compounds (PDF)
D
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Chang, S. L.; Sung, C. T.; Wang, C. C.; Oakley, B. R. J. Am. Chem.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: tasai@bio.c.u-tokyo.ac.jp.
ORCID
Tohru Taniguchi: 0000-0002-4965-7383
Keiji Mori: 0000-0002-9878-993X
Teigo Asai: 0000-0002-8903-392X
Notes
̈
Kuhnert, E.; Bohm, A.; Pendzialek, T.; Solga, D.; Wiebach, V.; Engler,
The authors declare no competing financial interest.
H.; Berkessel, A.; Stadler, M.; Kalesse, M. Chem. - Eur. J. 2018, 24,
2200.
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ACKNOWLEDGMENTS
■
The authors thank Prof. K. Gomi (Tohoku University, Sendai,
Japan) and Prof. K. Kitamoto (The University of Tokyo) for
providing the expression vectors and the fungal strain. This
work was supported by JSPS KAKENHI [Grants 17H05055,
17H05428, and 19H04642 to T.A. from the Japan Society for
the Promotion of Science (JSPS)], the Naito Foundation, and
the Astellas Foundation for Research on Metabolic Disorders.
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