Genome mining approach for harnessing the cryptic gene cluster in Alternaria solani: Production of PKS-NRPS hybrid metabolite, didymellamide B

Article abstract of DOI:10.1016/j.tetlet.2016.05.043

Using a heterologous expression system in Aspergillus oryzae we characterized cryptic biosynthetic genes found in Alternaria solani. This system enabled the isolation of an antifungal metabolite, didymellamide B, which was previously not detected in the extract of the A. solani. The co-production and chemical transformation of didymellamide congeners enabled us to decipher the biosynthetic pathway of didymellamide B as follows: (1) formation of a linear polyketide chain with 3-acyltetramate, (2) oxidative ring expansion to yield a 2-pyridone skeleton, and (3) non-enzymatic endo-selective [4+2] cycloaddition to afford a trans-decalin. These results demonstrate the robustness and reliability of the A. oryzae expression system.

Full text of DOI:10.1016/j.tetlet.2016.05.043

Accepted Manuscript
Genome mining approach for harnessing the cryptic gene cluster in Alternaria
solani: Production of PKS-NRPS hybrid metabolite, didymellamide B
Takahiro Ugai, Atsushi Minami, Katsuya Gomi, Hideaki Oikawa
PII:
S0040-4039(16)30566-4
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.05.043
TETL 47662
Reference:
Tetrahedron Letters
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Accepted Date:
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8 May 2016
12 May 2016
Please cite this article as: Ugai, T., Minami, A., Gomi, K., Oikawa, H., Genome mining approach for harnessing
the cryptic gene cluster in Alternaria solani: Production of PKS-NRPS hybrid metabolite, didymellamide B,
Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.05.043
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Genome mining approach for harnessing the
cryptic gene cluster in Alternaria solani:
Production of PKS-NRPS hybrid metabolite,
didymellamide B
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Takahiro Ugai, Atsushi Minami, Katsuya Gomi and Hideaki Oikawa*

1
Tetrahedron Letters
Genome mining approach for harnessing the cryptic gene cluster in Alternaria solani:
Production of PKS-NRPS hybrid metabolite, didymellamide B
Takahiro Ugai a , Atsushi Minami a*, Katsuya Gomib and Hideaki Oikawa a*
aDivision of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
b Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Using a heterologous expression system in Aspergillus oryzae we characterized cryptic
biosynthetic genes found in Alternaria solani. This system enabled the isolation of an antifungal
metabolite, didymellamide B, which was previously not detected in the extract of the A. solani.
The co-production and chemical transformation of didymellamide congeners enabled us to
decipher the biosynthetic pathway of didymellamide B as follows: 1) formation of a linear
polyketide chain with 3-acyltetramate, 2) oxidative ring expansion to yield a 2-pyridone
skeleton, and 3) non-enzymatic endo-selective [4+2] cycloaddition to afford a trans-decalin.
These results demonstrate the robustness and reliability of the A. oryzae expression system.
Available online
Keywords:
polyketide synthase
didymellamide
natural product biosynthesis
heterologous expression
fungi
2009 Elsevier Ltd. All rights reserved.
Introduction
A recent bioinformatics analysis of the various fungal genome
has shown that 20–100 biosynthetic gene clusters of secondary
metabolites are found in a single strain, and nearly half of these
are polyketide synthase (PKS) genes, suggesting that polyketides
are the most abundant metabolites in fungi.1 Functions of these
gene clusters have however not been examined. Genome mining
using heterologous production are one of the most versatile
methods used for deciphering cryptic gene clusters. Recently, we
have successfully synthesized meroterpenoids, terpenes and
polyketide metabolites using the Aspergillus oryzae heterologous
expression system.2 As part of our ongoing project on the total
biosynthesis of bioactive fungal polyketides such as cholesterol-
lowering lovastatin and antifungal agent griseofulvin, we focus
on the heterologous expression of gene clusters, including the
PKS or PKS-non-ribosomal peptide synthetase (PKS-NRPS).3
Alternaria solani is a phytopathogenic fungus, which causes
potato early blight.4 This fungus produces various types of
phytotoxic metabolites, including polyketides alternariol,
altersolanol A, altertoxin, macrosporin, and solanapyrone.4 The
biosynthesis of solanapyrone that involves an intriguing Diels-
Alderase (DAase) Sol5 has been extensively studied.5
Heterologous expression of several A. solani PKS successfully
yielded polyketide pyrones.6 The polyketide metabolite,
alternaric acid was isolated as an antifungal agent in 1949 and
was shown to contribute the disease development caused by A.
solani.7 Based on conventional feeding experiments with isotope
labeled acetate, we previously proposed a unique strategy for the
construction of the polyketide scaffold, including a condensation
of two different polyketide chains.8
Figure 1. Fungal PKS-NRPs hybrids harboring 2-pyridone and decalin
moieties.
During the biosynthetic study of alternaric acid, we faced an
interesting phenomenon on a single gene cluster consisting of a
functionally unknown PKS-NRPS. Although the relevant genes
were apparently expressed, no corresponding metabolite was
detected on several analytical methods. We became interested in
this unusual phenomenon on the PKS and decided to determine
the structure of the relevant polyketide. In this communication,
we describe the successful production of didymellamide B (1) 9
polyketide metabolites with pyridone and decalin moieties by the
heterologous expressions of three genes using A. oryzae host

2
Tetrahedron
Scheme 1. Proposed biosynthetic pathway of didymellamide B.
(Figure 1). Co-production of didymellamide congeners enabled
us to propose the biosynthetic pathway of 1 involving a non-
enzymatic [4+2] cycloaddition.
asolB) and other modification genes (asolDEH) encoding
flavoprotein, methyltransferase, short-chain dehydrogenase, and
auxiliary proteins (asolFG) (Figure S2). Three asolSCA showed
high similarity (49-60% identity) to the biosynthetic genes of
tenellin,10 a PKS-NRPS product with a pyridone moiety. Next,
we attempted to isolate the relevant polyketide metabolites in the
extracts from several fermentation media but failed to detect any
metabolite with TLC, HPLC and LC-MS, indicating its low
productivity.
To determine structure of the polyketide metabolite, we
prepared a transformant AO-asolSC with the plasmid (pUARA2-
asolSC) by using the A. oryzae expression system. The
metabolite profiles showed that AO-asolSC produced a new
metabolite 2 (123 mg/kg of rice) (Figure 2). HR-MS data
revealed the molecular formula to be C24H29NO4 (unsaturation
degree, 11) and the characteristic absorption at 283 nm in the UV
spectrum suggested the presence of a 3-acyltetramate moiety
derived from the tyrosine (Figure S3).11 This finding was further
supported by 1H- and 13C-NMR, and HSQC data of 2 (p-
hydroxyphenyl group:C 155.1, 130.3, 127.8, 115.7; tricarbonyl
methane moiety of 3-acyltetramate: C 194.0, 192.0, 175.3 and
101.9). Extensive analysis of the COSY correlations revealed that
2 possessed a decalin moiety. Detailed analysis of the NMR data
including NOESY data (Figure S4) enabled us to determine the
structure of 2 (except C5-stereochemistry), which is named as
protodidymellamide , as shown in Figure 1.
Results and discussion
In the draft genome sequence of the alternaric acid producer
A. solani A-17, we found 11 highly reducing PKS (HR-PKS)
genes by a BLAST search. RT-PCR of those genes in the mycelia
of A. solani under alternaric acid producing conditions showed
apparent expression of the PKS-NRPS gene asolS (Figure S1).
The gene cluster comprising the asolS gene contained the trans-
acting ER (asolC), two cytochrome P450 genes (asolA and
In the biosynthesis of tenellin, TenA, a chytochrome P450,
catalyzed the characteristic oxidative ring expansion from
tetramate to 2-pyridone.12 To characterize the function of the
homologous gene asolA (60% identity), a triple transformant
AO-asolSCA was then prepared. This transformant produced two
new metabolites 1 and 3 (1: 22 mg/kg, 3: 84 mg/kg), and the UV
spectra were closely related to the structurally related 2-pyridone
polyketide metabolites (Figure 2, Figures S3 and S4).13 The
molecular formula (C24H27NO4) was deduced from HR-MS and
comparison of the NMR spectrum of 1 with that of natural
product confirmed that 1 was identical with the antifungal
metabolite didymellamide B isolated from a marine-derived
fungus, Stagonosporopsis cucurbitacearum (previous name:
Didymella bryoniae).9
Figure 2. HPLC profiles of the metabolites from (A) wild type strain
283 nm, (B) transformant with asolSC 283 nm, and (C) transformant
with asolSCA 347 nm.
1
In H- and 13C-NMR spectra of the second metabolite 3, a
part of signals appeared as a duplicated pair, suggesting that 3

3
was a 1:1 mixture of diastereomers. A comparison of 1H- and 13C-
NMR data of 3 with those of 1 revealed that 3 possessed the
same 3-acyl-2-pyridone scaffold (H; 7.48; C 188.1, 170.6,
158.8, 139.4, 111.5, 106.8), and that difference was located at a
decalin moiety of 1. Further detailed NMR analysis showed the
presence of two partial structures, a conjugated diene connected
with terminal methyl group and a -hydroxycarbonyl moiety
(Table S1, Figure S5), suggesting that 3 had a linear chain
instead of trans-decalin via [4+2] cycloaddition as shown in
Scheme 1. A treatment of 3 with acetic anhydride in the presence
of DMAP at 85°C followed by methanolysis afforded 1 which
was identical to the natural didymellamide B9 in all respects. The
conversion of 1 from 3 indicated that under the reaction
conditions, C9-acetylation of 3 and the subsequent elimination
took place to give triene 5a, which underwent stereoselective
cycloaddition to furnish acetate of 1 as a single isomer. This
chemical transformation provided the further support on the
structure of 3.
These experimental results enabled us to discuss the
biosynthetic pathway of didymellamide as shown in Scheme 1. In
biosynthesis of tenellin, TenS, close homolog of PKS-NRPS
AsolS, in collaboration with TenC generates a linear tetramate
intermediate and the subsequent P450 TenA catalyzes a ring
expansion to afford a linear 2-pyridone.12 Together with [4+2]
cycloaddition by PKS-NRPS hybrid LNKS,14 we initially
speculated that AsolS/AsolC catalyzed formation of a putative
linear tetramate 4 and the subsequent cycloaddition gave 2 in the
AO-asolSC. However, the conversion of 2 into 1 (route B) was
not observed in the incubation of the single gene transformant
AO-asolA (Figure S6). Isolation of -hydroxyketone 3 is another
key issue in the discussion of the didymellamide biosynthesis.
Isolation of 3 as a 1:1 epimeric mixture suggested that 3 was not
derived by the AsolS catalyzed reaction because ketoreduction of
PKS was known to proceed in a highly stereoselective manner15
and -hydroxyketone 3 was obtained only as a tetramate form but
not as a 2-pyridone form. Recently, we reported several unusual
side reactions caused by the expression host A. oryzae.16 Co-
production of 3 with 1 suggested that the unusual product may be
attributed to the action of the expression host A. oryzae hydratase
which trapped linear intermediate 5a to convert into 3 although
nonenzymatic hydration can not be excluded. Considering all
data shown here, we propose that the biosynthesis of 1 proceeds
via route A (4-5a-1) and the [4+2] cycloaddition of 5a proceed
nonenzymatically. Non-enzymatic cycloaddtions were reported
during the studies of the bifunctional DAases Sol5 (oxidase)17
and LNKS (PKS)14 as side reactions in which endo-adducts were
preferentially obtained in the aqueous medium. In this case, the
endo-selectivities observed in the aqueous medium were
explained by a hydrophobic effect.
oxidative transformations. These modification reactions are
under investigation.
Conclusions
In summary, we have succeeded in the heterologous
expression of the cryptic gene cluster found in A. solani to obtain
a marine-derived antifungal agent didymellamide B from the A.
oryzae transformant introducing PKS-NRPS, trans-ER and P450
genes asolSCA. The co-production of didymellamide shunt
products enabled us to propose the exact sequence of oxidative
ring expansion and non-enzymatic cycloaddition. It should be
pointed out that 1 was not produced at a detectable level in the A.
solani, even though the expression of the relevant genes was
confirmed by RT-PCR of the host mRNA. Temporary or low
expression of the gene clusters was often observed in the
infection process of the phytopathogenic fungi.1b The successful
production of didymellamide B in high yield proves that the
heterologous expression system of A oryzae is a powerful tool to
address the structural determination of a metabolite from a
cryptic gene cluster.
Acknowledgments
This work was supported by Grant-in-Aid for Scientific
Research (A)15H01835 to H.O. We are grateful to Prof.
Kiyotaka Koyama for giving us a didymellamide B producer,
Stagonosporopsis cucurbitacearum.
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5
HIGHLIGHT
• Heterologus production of fungal metabolite with
3 genes
• biosynthetic gene dependent production of fungal
polyketide
• non-enzymatic [4+2] cycloaddition of highly
reactive linear triene
• endo-selective Diels-Alder reaction generating
trans-decalin with 2-pyridone