Electrocatalytic hydrogen evolution with gallium hydride and ligand-centered reduction

Article abstract of DOI:10.1039/c8sc05247f

Ga^III chloride tetrakis(pentafluorophenyl)porphyrin (1) was synthesized and shown to be a highly active and stable post-transition metal-based electrocatalyst for the hydrogen evolution reaction (HER). Electrochemical and spectroscopic studies

Full text of DOI:10.1039/c8sc05247f

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RESEARCH ARTICLE
Genome mining for fungal polyketide-diterpenoid
hybrids: discovery of key terpene cyclases and
multifunctional P450s for structural
diversification†
Cite this: DOI: 10.1039/c8qo01124a
Wei-Guang Wang, *a Lian-Qiong Du,a Shan-Ling Sheng,a Ao Li,b Yan-Ping Li,c
Gui-Guang Cheng,d Gan-Peng Li,a Guiling Sun,e Qiu-Fen Hua and
f
Yudai Matsuda
*
A biosynthetic gene cluster for chevalone E (1) and its oxidized derivatives have been identified within the
genome of the endophytic fungus Aspergillus versicolor 0312, by a mining strategy targeting a polyke-
tide-diterpenoid hybrid molecule. The biosynthetic pathway has been successfully reconstituted in the
heterologous fungus Aspergillus oryzae. Interestingly, two P450 monooxygenases, Cle2 and Cle4, were
found to transform 1 into seven new analogues including 7 and 8 that possess a unique five-membered
lactone ring. Furthermore, the replacement of the terpene cyclase gene with that from another fungus
led to the production of sartorypyrone D (11), which has a monocyclic terpenoid moiety. Finally, some of
the compounds obtained in this study synergistically enhanced the cytotoxicity of doxorubicin (DOX) in
breast cancer cells.
Received 17th October 2018,
Accepted 29th November 2018
DOI: 10.1039/c8qo01124a
rsc.li/frontiers-organic
terpenoids have been intensively studied in recent years.2,3
The majority of the fungal meroterpenoids have a polyketide
Introduction
Natural products are one of the essential resources for drug motif as a non-terpenoid moiety,1 but biosynthetic studies on
discovery. Not only do they directly provide many bioactive these hybrid natural products at genetic/enzymatic levels have
small molecules, but they also provide inspiration for synthetic mainly focused on those harboring a relatively small terpenoid
drug development. Meroterpenoids are naturally occurring moiety (C15 or less), and only a few biosynthetic gene clusters
organic compounds that in part originate from the terpenoid involved in the biosynthesis of fungal polyketide-diterpenoids
pathway, among which those found in filamentous fungi (PK-DTs) have been identified to date.4,5 Therefore, mining
exhibit significant diversity in their biological activities and and utilization of unexploited fungal biosynthetic gene clus-
molecular architectures.1,2 The fungal meroterpenoids have ters (BCGs) for PK-DTs should accelerate the discovery of new
thus received broad attention from natural product research- molecules and provide fascinating insight into the biosynthetic
ers, and the biosynthesis and total syntheses of fungal mero- logic governing the occurrence of fungal meroterpenoids.
As an experimental strategy, heterologous expression of bio-
synthetic genes offers an efficient way to enable genome
mining, especially when the target genes either are silent in
the host organism or are difficult to be activated. The fungus
Aspergillus oryzae is a particularly robust platform developed
for heterologous expression,6–8 since the metabolite(s) derived
from the expression of the introduced genes can generally be
obtained in a good yield and are easily purifiable due to a low
background. Thus, the A. oryzae system has been utilized for
successful genome mining of several fungal biosynthetic
genes, and this led to the discovery of several natural products
with new scaffolds.9–12 However, only pathways involving a few
biosynthetic genes have been so far reconstituted in A. oryzae
for the sake of genome mining, although it is known that
>10 genes can be readily expressed in the fungus.8,13 Thus, it
aKey Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs
Commission and Ministry of Education, Yunnan Minzu University, Kunming 650031,
China. E-mail: wwg@live.cn
bCollege of Pharmacy and Bioengineering, Chongqing University of Technology,
Chongqing 400054, People’s Republic of China
cSchool of Chinese Pharmacy, Yunnan University of Traditional Chinese Medicine,
Kunming 650500, China
dYunnan Institute of Food Safety, Kunming University of Science and Technology,
Kunming, 650500, China
eKey Laboratory of Plant Stress Biology, State Key Laboratory of Cotton Biology,
School of Life Sciences, Henan University, Kaifeng 475004, China
fDepartment of Chemistry, City University of Hong Kong, 83 Tat Chee Avenue,
Kowloon, Hong Kong SAR, China. E-mail: ymatsuda@cityu.edu.hk
†Electronic supplementary information (ESI) available: Experimental details,
supplementary figures, and spectral data. See DOI: 10.1039/c8qo01124a
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would be interesting to see whether A. oryzae can serve as a therefore, it was expected that the gene cluster would be
useful tool to investigate the function of a biosynthetic gene responsible for the biosynthesis of a new natural product.
cluster comprising a relatively large number of genes found by
genome mining.
On the basis of the predicted functions of each proteins
encoded by the cle cluster, the biosynthetic pathway for the
In this report, we describe the discovery and characteriz- metabolite synthesized by the cluster can be proposed by
ation of the gene cluster responsible for the biosynthesis of analogy to the known pathways for the biosynthesis of other
the fungal PK-DT chevalone E (1)14 and its oxidized derivatives fungal meroterpenoids.2 Initially, the PKS Cle1 yields a polyke-
(2–8) (Fig. 1A), and demonstrate the successful reconstitution tide product, which is then geranylgeranylated by the prenyl-
of the biosynthetic pathway in A. oryzae. In addition, we discov- transferase Cle5 utilizing GGPP synthesized by the GGPS Cle6.
ered another gene cluster similar to that for chevalones, and The next step would involve the epoxidation of one of the
the terpene cyclase encoded by this cluster interestingly double bonds in the geranylgeranyl moiety, catalyzed by the
accepts the same substrate as that for chevalone E synthesis FAD-dependent monooxygenase (FMO) Cle3. Subsequently,
but produces a differently cyclized product sartorypyrone D the terpene cyclase Cle7 would catalyze the cyclization of the
(11) (Fig. 1A). Finally, we report that compounds 1, 2, and 5–8 prenyl group initiated by protonation and ring-opening of the
and doxorubicin (DOX) have a synergistic effect on breast epoxide. The two cytochrome P450 monooxygenases (Cle2 and
cancer cell growth inhibition.
Cle4) would decorate the backbone structure generated by the
above-mentioned five enzymes to provide the pathway end
product.
To analyze the function of each gene in the cle cluster and
to obtain the metabolite produced by the cluster, the cle genes
were heterologously expressed in the A. oryzae NSAR1 strain16
according to the proposed pathway. Initially, the PKS gene cle1
was solely expressed in A. oryzae, but unfortunately, no new
product was detected as compared with the negative control
strain without any cle genes (Fig. 2A, lanes i and ii).
Nevertheless, further introduction of the prenyltransferase and
GGPS genes, cle5 and cle6, resulted in the production of a
hydrophobic molecule 9 (Fig. 2A, lane iii), whose molecular
formula was determined to be C26H38O3 by the HR-MS ana-
lysis. Following large-scale cultivation, 9 was isolated in
sufficient amounts for structural characterization, with NMR
analysis revealing that 9 is a geranylgeranylated form of triace-
tic acid lactone (TAL) at the C-3 position (Fig. 2B). Although it
is unclear why the single expression of the PKS gene did not
generate TAL, the observation strongly suggests that the PKS
Cle1 is a TAL synthase, which is consistent with the domain
organization of Cle1 (SAT-KS-AT-PT-ACP-ACP). Coexpression of
the FMO gene cle3 in the transformant producing 9 then gen-
erated a new compound 10 (Fig. 2A, lane iv), which is much
more hydrophilic than 9. Compound 10 possesses a similar
structure to that of 9, but it has two additional hydroxyl groups
at the terminus of the geranylgeranyl chain (Fig. 2B). The diol
moiety should be the consequence of the hydrolysis of the
epoxide ring synthesized by the FMO Cle3. Finally, the five-
gene expression system including the terpene cyclase gene cle7
afforded the cyclized product 1 (Fig. 2A, lane v), which was
Results and discussion
To find a BGC suitable for our genome mining study, we
initially searched for a genomic region that encodes both a
polyketide synthase (PKS) and a geranylgeranyl pyrophosphate
(GGPP) synthase (GGPS) in the several fungal genome
sequences available to us, since it is well known that BGCs for
fungal meroditerpenoids, including indole-diterpenoids,
encode a GGPS to provide the substrate for a prenyltransfer-
ase.2,15 Consequently, we focused on a gene cluster of seven
genes, derived from the endophytic fungus Aspergillus versico-
lor 0312 isolated from Paris polyphylla var. yunnanensis. The
gene cluster, designated as the cle cluster, encodes a non-redu-
cing PKS (Cle1), a prenyltransferase (Cle5), a GGPS (Cle6), and
a terpene cyclase (Cle7) (Fig. 1B and Table S1;† DDBJ/EMBL/
GenBank accession number: LC422695), and therefore implies
that the cle cluster is involved in the biosynthesis of a PK-DT
meroterpenoid. Importantly, the cle cluster is significantly
different from the known clusters for fungal PK-DTs,4,5 and
identified as the known natural product chevalone
(Fig. 2B).14
E
Given the production of chevalone E (1) by the five enzymes
for the backbone structure synthesis, the two P450s Cle2 and
Cle4 would decorate the chevalone E scaffold into further oxi-
dized metabolites. To investigate the order of involvement of
the P450s, cle2 and cle4 were individually introduced into the
transformant that synthesizes 1, and both transformants inter-
estingly produced new compounds. The transformant with
cle2 yielded two new metabolites 2 and 3 (Fig. 2A, lane vi),
Fig. 1 (A) The structures of chevalone E (1) and sartorypyrone D (11). (B)
Schematic representations of the cle and sre clusters. PKS: polyketide
synthase; GGPS: geranylgeranyl pyrophosphate synthase; FMO: FAD-
dependent monooxygenase.
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experiment to verify the metabolites resulting from the actions
of the P450s as true pathway intermediates. To this end, 4, 5,
and 6 were fed to the transformant only lacking cle4, while 2,
3, 6, and 7 were incubated with the strain without cle2
(Fig. S2†). The result revealed that 4, 5, and 6 are all further
oxidized into downstream metabolites by Cle2 to eventually
yield the products with the five-membered lactone, 7 or 8
(Fig. 3A), indicating that Cle2 has a broad substrate specificity
and oxidizes the C-20 position of all the tested compounds
into carboxylate. On the other hand, the substrate specificity
of Cle4 appears to be stricter than that of Cle2 as evidenced by
the observation that 3 and 6 remained unchanged in the pres-
ence of Cle4 (Fig. 3B, lanes iii and vi). Meanwhile, 2 and 7
underwent a single round of oxidation by Cle4 to afford 6 and
8, respectively (Fig. 3B, lanes ii and v).
On the basis of the structures obtained from the heter-
ologous reconstitution and substrate feeding experiments,
plausible pathways leading to the predicted end product 8 can
be proposed as follows (Fig. 4). First, the non-reducing PKS
Cle1 synthesizes the α-pyrone TAL from one molecule of acetyl-
CoA and two molecules of malonyl-CoA. The membrane-bound
Fig. 2 (A) HPLC profiles of culture supernatant extracts from A. oryzae
NSAR1 transformants. Transformants (i) with only empty vectors; (ii) with
cle1; (iii) with cle1 + 5 + 6; (iv) with cle1 + 3 + 5 + 6; (v) with cle1 + 3 +
5 + 6 + 7; (vi) with cle1 + 2 + 3 + 5 + 6 + 7; (vii) with cle1 + 3 + 4 + 5 +
6 + 7; (viii) with cle1 + 2 + 3 + 4 + 5 + 6 + 7; and (ix) with cle1 + 3 + 5 +
6 + 7 + sre3. The chromatograms were monitored at 230 nm. (B)
Structures of the metabolites isolated in this study.
both of which are oxidized at the C-20 methyl group; com-
pounds 2 and 3 have hydroxyl and carboxyl groups at this posi-
tion, respectively (Fig. 2B). Thus, Cle2 could accept 1 as a sub-
strate to perform three consecutive oxidations at C-20 to trans-
form the methyl group into carboxylate. On the other hand,
the separate expression of the other P450 gene cle4 generated a
single new product 5 (Fig. 2A, lane vii) with two β-hydroxyl
groups at C-11 and C-12 (Fig. 2B), suggesting that Cle4 can
oxidize the two adjacent carbon atoms. Finally, coexpression
of cle2 and cle4 with the other five genes provided a much
more complicated metabolite profile with four additional new
molecules (4 and 6–8). Among these compounds, 4 was found
to be a less oxidized analogue of 5, containing only the C-11
alcohol, while 6 is an isomer of 5 and harbors two hydroxyl
groups at C-11 and C-20 (Fig. 2B). Interestingly, 7 has a unique Fig. 3 Functional analyses of Cle2 and Cle4. (A) HPLC profiles of
culture supernatant extracts from the A. oryzae transformant harboring
cle1 + 2 + 3 + 5 + 6 + 7 incubated with (i) only culture media; (ii) 4;
(iii) 5; and (iv) 6. (B) HPLC profiles of culture supernatant extracts from
the A. oryzae transformant harboring cle1 + 3 + 4 + 5 + 6 + 7 incubated
five-membered lactone ring, and 8 is an oxidized analogue of 7
with one additional hydroxyl group at C-12 and seems to be
the end product of the pathway (Fig. 2B).
In order to more deeply explore the functions of the two
with (i) only culture media; (ii) 2; (iii) 3; (iv) 6; and (v) 7. The chromato-
P450s Cle2 and Cle4, we then performed a substrate-feeding grams were monitored at 254 nm.
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Fig. 4 Proposed biosynthetic pathway leading to 8.
prenyltransferase Cle5 then accepts TAL as its substrate to pathway intermediate for 8. It should be noted that the precy-
perform a C-3 geranylgeranylation reaction to afford 9, in which clized intermediate 9 appears to be widely involved in the bio-
the pathway-dedicated GGPS Cle6 is required to provide GGPP, synthesis of many fungal meroterpenoids. For example, the
the other substrate of Cle5. Subsequently, the FMO Cle3 forms fungus Eurotium chevalieri produces chevalone A, a close ana-
an (S)-epoxide ring at the terminal olefin of the geranylgeranyl logue of 1 with the α-pyrone, as well as the acetylated derivative
group to provide 10′, which is then protonated by the terpene of 1 with the γ-pyrone moiety.17 The fact that no such cyclized
cyclase Cle7 to undergo the cyclization reaction that yields the metabolite carrying the α-pyrone was obtained in our study
pentacyclic pathway intermediate chevalone E (1). Importantly, suggests that the terpene cyclase Cle7 has a stricter product
the α-pyrone seen in 9 is transformed into γ-pyrone after the selectivity. Furthermore, partially cyclized analogues of 1 with
cyclization event, which could be attributed to the occurrence of mono- or tricyclic terpenoid moieties, such as aszonapyr-
the tautomerization of the TAL-derived moiety during the ones17,18 and sartorypyrones,19–21 have been discovered in
terpene cyclization. After the generation of the cyclized product other fungi. These observations indicate that there are several
1, 1 could follow several possible pathways to be converted into different terpene cyclases that can accept the same substrate
oxidized derivatives. In the absence of the action of one of the 10′ but then produce differently cyclized products, as seen in
P450s Cle4, 1 is subjected to oxidation at C-20 by Cle2 to give the biosynthesis of fungal meroterpenoids derived from 3,5-di-
the alcohol 2 and the carboxylate 3. On the other hand, if Cle2 methylorsellinic acid.22,23 To investigate whether our A. oryzae
is missing from the pathway, 1 is initially oxidized at C-11 by expression system can be readily utilized to obtain analogous
Cle4 to yield 4, which is further oxidized at C-12 by the same natural products of chevalones with a different terpenoid
enzyme to afford 5.
moiety, we searched for a gene cluster that is similar to the cle
In the coexistence of Cle2 and Cle4, the end product 8 cluster but is involved in the biosynthesis of a different natural
could be achieved by several routes. In this process, the charac- product. As a result, a gene cluster found in Aspergillus felis
teristic lactone ring seems to be spontaneously formed after 0260 was selected and hereby named the sre cluster (Fig. 1B
the placement of the carboxyl group at C-20 and the β-hydroxyl and Table S1;† DDBJ/EMBL/GenBank accession number:
group at C-11, or alternatively, it could also be generated by LC428283). The terpene cyclase gene in the sre cluster, sre3,
the oxidation of lactol that would occur after alcohol dehydro- was coexpressed with cle1, cle5, cle6, and cle3, which yielded
genation to aldehyde. Collectively, all of the chevalone E 11, an isomer of chevalone E (1) (Fig. 2A, lane ix). The cyclized
derivatives isolated in this study except for 3 serve as the product 11 was identified to be sartorypyrone D,21 demonstrat-
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ing that Sre3 (38% protein sequence identity with Cle7) also (half maximal inhibitory concentrations) from 2.9 to
accepts 10′ as Cle7 but performs a distinct cyclization reaction. 100 μM.17,24 Therefore, we evaluated the cytotoxicity of selected
Given that the sre cluster encodes the acetyltransferase Sre5, compounds (1, 2, and 5–8) against two breast cancer lines
the end product of the sre pathway would be sartorypyrone A, (MDA-MB-231 and MCF-7) but found that all of the tested com-
which is the acetylated form of 11 (Fig. 5).
pounds did not show inhibitory activity even at a concentration
It is reported that chevalone analogues inhibit the growth of 40 μM. Although DOX (1.25 μM) treatment alone for 48 h
of a variety of malignant solid cancer cells with IC50 values caused an inhibition of about 50% decrease of cell viability in
MDA-MB-231 (Fig. 6) and MCF-7 cells (Fig. S2†), co-treatment
of DOX with the indicated compounds resulted in an
enhanced effect on the reduction of cell viability in both
MDA-MB-231 and MCF-7 cells. To characterize the interactions
between the DOX and the indicated compounds, the drug
combination index (CI) was further calculated using the
CompuSyn software.25 We observed that all of the tested com-
pounds synergistically enhanced the cytotoxicity of DOX in
both MDA-MB-231 (Fig. 6G) and MCF-7 cells (Fig. S2G†), as all
Fig. 5 Proposed biosynthetic pathway leading to sartorypyrone A.
of the CI values calculated were less than 1.
Conclusions
In summary, we have discovered a gene cluster responsible for
the biosynthesis of novel chevalone E analogues, by a genome
mining approach that targeted a PK-DT hybrid, and we unam-
biguously characterized the functions of each enzyme encoded
by the cle cluster. Our result provides the first example in
which the complete biosynthesis of a fungal PK-DT hybrid has
been successfully reconstituted in a heterologous host and
indicates the usefulness of the A. oryzae system for obtaining
new natural products. Furthermore, we have obtained another
PK-DT, sartorypyrone D (11), which is synthesized by a cycliza-
tion mode distinct from that for 1. Since geranylgeranyl-TAL-
derived meroterpenoids are widespread in fungi, our finding
further facilitates future biosynthetic studies on compounds
that are structurally related to chevalone E (1) and sartorypyr-
one A. Importantly, we have found two P450s, Cle2 and Cle4,
which can derivatize 1 into seven new analogues (2–8).
Interestingly, two of the new analogues, 7 and 8, possess a
characteristic five-membered lactone ring, which has never
been found in the structures of fungal meroterpenoids and are
only found in a few natural products.26,27 We have thus
demonstrated that the genome mining study focusing on
fungal PK-DTs is a promising way to discover new bioactive
natural products. Future studies will involve the identification
of gene clusters similar to the cle cluster, and the combinator-
ial biosynthesis utilizing biosynthetic genes obtained from
more different TAL-derived meroterpenoid pathways to create
new molecules with desired functions.
Fig. 6 Compounds 1, 2, and 5–8 synergistically enhance the cyto-
toxicity of DOX in MDA-MB-231 breast cancer cells. (A–F) MDA-MB-231
cells were treated with DOX, compounds 1, 2, 5–8, or the combination
of both agents at a 1 : 8 M ratio. After 48 h, cell viability was determined
Experimental section
using the colorimetric MTT assay. (G) Combination index values were General experimental procedures
calculated using CompuSyn software to determine drug interaction.
Solvents and chemicals were purchased from Merck KGaA Ltd
(Darmstadt, Germany). Oligonucleotide primers were pur-
chased from Tsingke (Beijing, China) and are listed in
CI < 1 indicates synergism, CI = 1 indicates additive effects, and CI > 1
indicates antagonism. The data are presented as mean standard devi-
ation of three independent experiments.
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Table S2.† PCR was performed using an Applied Biosystems®
For the genome sequence of A. felis 0260, the 6.7 Gb of
SimpliAmp™ Thermal Cycler (Thermo Fisher) with Phanta HiSeq raw reads was treated with NGSQCToolkit32 (v2.3.3), fas-
Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd). tuniq33 (v1.1), and BLESS34 (v1.02) software. Sequence assem-
Analytical and preparative HPLC were performed on an Agilent bly was performed with platanus35 (v1.2.4) to yield 54 scaffolds
1260 system, using a ZORBAX SB-C18 column (4.6 mm i.d. × with N50 of 1.62 Mb covering approximately 32.6 Mb.
250 mm, Agilent Co. Ltd) and a ZORBAX SB-C18 column
Gene structure annotation was obtained by using
(9.4 mm i.d. × 250 mm, Agilent Co. Ltd), respectively. Silica gel AUGUSTUS (http://bioinf.uni-greifswald.de/webaugustus) with
column chromatography was performed using Synthware the Aspergillus fumigatus gene model used as the training set.
C189463. NMR spectra were obtained at 800 MHz (1H) and All of the predicted gene products were used to construct the
200 MHz (13C) with a Bruker Avance III HD 800 MHz NMR database to perform the local BLAST search.
spectrometer at Kunming Institute of Botany, Chinese
Academy of Sciences. Samples for LC-HRMS analysis were
injected into a Thermo Scientific Dionex Ultimate 3000
UHPLC system equipped with a Thermo high resolution Q
Exactive focus mass spectrometer; chromatographic column:
Thermo Scientific Hypersil GOLD C18 (2.1 × 100 mm, 1.9 μm);
flow rate: 0.2 mL min−1; column temperature: 25 °C.
Construction of fungal expression plasmids
To construct one-gene containing fungal transformation plas-
mids, each gene in the cle cluster or the sre cluster was first
amplified from the A. versicolor 0312 and A. felis 0260 genomic
DNA, with the primers listed in Tables S2 and S3.† The full-
length cle and sre genes were purified and then introduced
into the SmaI-digested pTAex336 vector using an In-Fusion®
HD Cloning Kit (Clontech Laboratories, Inc.) according to the
manufacturer’s protocol (Table S3†). For the introduction of
cle2, cle3, or cle4 into pBARI,8 a DNA fragment with the amyB
promoter (PamyB) and the amyB terminator (TamyB) was
amplified from the pTAex3-based plasmids constructed above
and ligated into the HindIII-digested vector (Table S3†). To
construct the co-expression plasmid of cle2 and cle4, cle5 and
cle6, cle3 and cle7, and cle3 and sre3 two different gene frag-
ments containing PamyB and/or TamyB with cle2, cle3, cle4,
cle5, cle6, cle7, or sre3 were amplified from the pTAex3-based
plasmids and ligated into HindIII-digested pBARI, XbaI-
digested pAdeA,37 or SmaI-digested pUSA38 using the In-
Fusion® HD Cloning Kit, to generate pBarI-cle2 + 4, pAdeA-
cle5 + 6, pUSA-cle3 + 7, and pUSA-cle3 + sre3, respectively
(Table S3†).
Strains and media
Aspergillus versicolor 0312 and Aspergillus felis 0260 were iso-
lated from the healthy tissue of Paris polyphylla var. yunnanen-
sis collected from Kunming, Yunnan province. These two
strains were cultivated at 30 °C, 200 rpm, in DPY medium (2%
dextrin, 1% hipolypepton (Nihon Pharmaceutical Co., Ltd),
0.5% yeast extract (Oxoid), 0.5% KH2PO4, and 0.05%
MgSO4·7H2O) for 3 days, and used as a source for whole
genome sequencing and the cloning of each gene in the cle
and sre clusters, respectively.
Aspergillus oryzae NSAR1 (niaD−, sC−, ΔargB, adeA−)16 was
used as the heterologous fungal host. The A. oryzae transfor-
mants were grown in shaking cultures in DPY medium for
three days at 30 °C and at 200 rpm for the production of the
metabolites derived from the introduced genes.
Standard DNA engineering experiments were carried out
using Escherichia coli DH5α (Takara Bio Inc.). E. coli cells har-
boring each plasmid were cultivated in Luria–Bertani (LB)
medium and were selected with ampicillin.
Transformation of Aspergillus oryzae NSAR1
Transformation of A. oryzae NSAR1 was performed by the pre-
viously reported protoplast–polyethylene glycol method.39 To
co-express cle1, cle5, and cle6, two plasmids, pTAex3-cle1 and
Whole genome sequencing and analysis
Genome sequencing of A. versicolor 0312 and A. felis 0260 was pAdeA-cle5 + 6, were used for the transformation. To co-
performed by Novogene Co., Ltd (Beijing, China) with an express cle1, cle3, cle5, and cle6, three plasmids, pTAex3-cle1,
Illumina Novaseq system.
pBarI-cle3, and pAdeA-cle5 + 6, were used for the transform-
For the genome sequence of A. versicolor 0312, we generated ation. To co-express cle1, cle3, cle5, cle6, and cle7, three plas-
6.3 Gb of HiSeq reads to estimate the genome features with mids, pTAex3-cle1, pUSA-cle3 + 7, and pAdeA-cle5 + 6, were
the GCE28 (v1.0.0) software. Given the unique k-mer depth of used for the transformation. To co-express cle1, cle3, cle5, cle6,
74 under 15-mers, the genome size was calculated to be and sre3, three plasmids, pTAex3-cle1, pUSA-cle3 + sre3, and
36.1 Mb. A total of 5.1 Gb of SMRT reads were produced on the pAdeA-cle5 + 6, were used for the transformation. To co-
PacBio Sequel system, and the subreads were corrected, express cle1, cle2, cle3, cle5, cle6, and cle7, four plasmids,
trimmed, and assembled using CANU29 (v1.6) with the para- pTAex3-cle1, pBarI-cle2, pUSA-cle3 + 7, and pAdeA-cle5 + 6,
meters “genome size = 36 m”. The consensus sequences were were used for the transformation. To co-express cle1, cle3, cle4,
corrected by Arrow(v2.2.1) with SMRT subreads and polished cle5, cle6, and cle7, four plasmids, pTAex3-cle1, pBarI-cle4,
by Pilon30 (v1.22) with the Illumina short reads. Lastly, we pUSA-cle3 + 7, and pAdeA-cle5 + 6, were used for the transform-
obtained 17 contigs with N50 of 3.96 Mb that constitute 95.6% ation. To co-express cle1, cle2, cle3, cle4, cle5, cle6, and cle7,
(34.5 Mb) of the genome. The BUSCO31 (v3.0.2) analysis indi- four plasmids, pTAex3-cle1, pBarI-cle2 + 4, pUSA-cle3 + 7, and
cated that 98.9% of the 290 fungal conserved genes were pAdeA-cle5 + 6, were used for the transformation. To construct
present.
negative control strains without one or more genes, the corres-
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ponding empty vectors or plasmids harboring a single gene at 570 nm was measured using a microplate reader (Tecan
were used for the transformation.
Infinite 200 Pro, Hombrechtikon, Switzerland) and presented
as relative cell viability.
HPLC analysis of each product
Metabolites from each A. oryzae transformant were analyzed by
HPLC, with a solvent system of H2O (solvent A) and acetonitrile Conflicts of interest
(solvent B), at a flow rate of 1.0 ml min−1 and a column temp-
The authors declare no competing financial interests.
erature of 35 °C. Separation was carried out with solvent
B/solvent A using a linear gradient from 10 : 90 to 100 : 0
within 30 min, 100 : 0 for 5 additional min, and a linear gradi-
ent from 100 : 0 to 10 : 90 within the following 5 min. Products
from the feeding experiments were analyzed with the same
method described above except for the addition of 0.1% tri-
fluoroacetic acid in solvent A.
Acknowledgements
We are grateful to Prof. Katsuya Gomi (Tohoku University) and
Prof. Jun-ichi Maruyama (The University of Tokyo) for kindly
providing the expression vectors and the fungal strain. This
project was financially supported by the National Natural
Science Foundation of China (No. 31760096 and 81660717),
the Yunnan Innovative Research Team for Discovery and
Comprehensive Utilization of Functional Small Molecules in
Medicinal Plants, and the Science Foundation of Yunnan, the
National Natural Science Foundation of Yunnan provinces (No.
2017FB133), and the Union Special Fund of Yunnan Provincial
Science and Technology Department and Yunnan University of
Traditional Chinese Medicine [No. 2015FB205(-025)]. This
work is dedicated to Professor Han-Dong Sun, Kunming
Institute of Botany, Chinese Academy of Sciences, on the
occasion of his 80th birthday.
Feeding experiment
The A. oryzae transformant harboring cle1 + 2 + 3 + 5 + 6 + 7
was cultivated in the presence of 1 mg of either 4, 5, or 6,
while the A. oryzae transformant harboring cle1 + 3 + 4 + 5 +
6 + 7 was cultivated in the presence of 1 mg of either 2, 3, 6, or
7. Both transformants were cultivated in 20 mL of DPY media
at 30 °C, 200 rpm, for 72 h. After the incubation, each culture
broth was extracted with ethyl acetate, and the mycelia were
extracted with acetone at room temperature, concentrated, and
reextracted with ethyl acetate. Both extracts were combined
and further analyzed by HPLC using the same method
described above.
Isolation and purification of each metabolite
Notes and references
To isolate each metabolite, the broth from one liter of the
culture was extracted with ethyl acetate. Mycelia were extracted
with acetone at ambient temperature overnight, concentrated
in vacuo, and reextracted with ethyl acetate. Both extracts were
then combined and subjected to silica-gel column chromato-
graphy and further purification by semi-preparative HPLC. The
detailed purification methods for all of the isolated com-
pounds are provided in the ESI.†
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