Heterologous production of asperipin-2a: Proposal for sequential oxidative macrocyclization by a fungi-specific DUF3328 oxidase

Article abstract of DOI:10.1039/c8ob02824a

Asperipin-2a is a ribosomally synthesized and post-translationally modified peptide isolated from Asperigillus flavus. Herein, we report the heterologous production of asperipin-2a and determination of its absolute structure. Notably, the characteristic bicyclic structure was likely constructed by a single oxidase containing the DUF3328 domain.

Full text of DOI:10.1039/c8ob02824a

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Heterologous production of asperipin-2a:
proposal for sequential oxidative macrocyclization
by a fungi-specific DUF3328 oxidase†
Cite this: Org. Biomol. Chem., 2019,
7, 39
1
Received 13th November 2018,
Accepted 28th November 2018
a
a
b,c
a
a
Ying Ye, Taro Ozaki, * Myco Umemura, Chengwei Liu, Atsushi Minami and
a
DOI: 10.1039/c8ob02824a
Hideaki Oikawa
*
rsc.li/obc
Asperipin-2a is a ribosomally synthesized and post-translationally involved in the oxidation and macrocyclization of the precur-
modified peptide isolated from Asperigillus flavus. Herein, we sor peptide that furnishes the 13-membered ring of ustilox-
1
1
report the heterologous production of asperipin-2a and determi- ins. The details of the macrocyclization process as well as the
nation of its absolute structure. Notably, the characteristic bicyclic function of DUF3328 proteins in the other gene clusters are yet
structure was likely constructed by a single oxidase containing the to be elucidated.
DUF3328 domain.
1 is a bicyclic peptide that possesses two macrocyclic ether
rings consisting of 14- and 17-membered paracyclophans. The
putative biosynthetic gene cluster for 1 is composed of four
genes encoding the precursor peptide (AFLA_041400, referred
to aprA in this study), UstYa/Yb homolog (AFLA_041390, aprY),
a transporter (AFLA_041380, aprT), and isoflavone reductase-
related enzyme (AFLA_041370, aprR) (Fig. S1†). As proposed in
the biosynthesis of ustiloxin B, AprY containing the function-
ally unknown domain DUF3328 likely catalyzes oxidative
macrocyclization during the biosynthesis of 1. However, in the
previous study, only aprA and aprY gene deletions were investi-
RiPPs (ribosomally synthesized and post-translationally modi-
fied peptides) are a diverse and rapidly expanding class of
natural products with potent biological activities as well as
1
unique structures. Although RiPPs can be found in all three
domains of life (archaea, bacteria, and eukaryotes), they have
rarely been identified in fungi. In Basidiomycetes, α-amanitin,
phallacidin, and omphalotin were demonstrated to be
2
–4
RiPPs.
In contrast, in Ascomycetes, there were no such
reports until the biosynthetic gene cluster for ustiloxin B was
discovered (Fig. 1).
mopsin A from Phomopsis leptostromformis and epichloëcyclins
from the genus Epichloë belong to the RiPP class. Led by the
identification and characterization of the biosynthetic genes
for ustiloxin B, Nagano and co-workers extensively searched
similar gene clusters in fungal genomes and classified them
5
–7
Recently, it was also reported that pho-
10
gated and the biosynthetic pathway of 1 remains unclear. In
the present study, we report the heterologous expression of the
four genes involved in the production of 1, showing that
DUF3328 oxidase is involved in the biosynthesis of this com-
pound. The improved production yield of 1 also allowed us to
8
,9
1
0
into more than 40 types. They also identified asperipin-2a (1)
as a product derived from one of the gene clusters. The co-
occurrence of genes encoding precursor peptides and enzymes
with the DUF3328 domain is a unique feature of these gene
clusters. By employing an Aspergillus oryzae expression system,
we revealed that two oxidases UstYa and UstYb, both of which
contain the DUF3328 domain, and tyrosinase UstQ were
a
Department of Chemistry, Faculty of Science, Hokkaido University,
Sapporo 060-0810, Hokkaido, Japan. E-mail: ozaki@sci.hokudai.ac.jp,
hoik@sci.hokudai.ac.jp
b
Bioproduction Research Institute, National Institute of Advanced Industrial Science
and Technology (AIST), Tsukuba 305-8566, Ibaraki, Japan
c
Computational Bio Big Data Open Innovation Laboratory (CBBD-OIL), AIST,
Tsukuba 305-8566, Ibaraki, Japan
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob02824a
Fig. 1 Asperipin-2a (1) and related cyclic peptides.
Org. Biomol. Chem., 2019, 17, 39–43 | 39
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determine its absolute configuration by chemical degradation,
chiral HPLC, and NMR analyses.
To reconstitute the biosynthetic pathway, four genes, aprA,
aprY, aprR, and aprT, were amplified from genomic DNA of
Aspergillus flavus. The aprA gene was cloned into the plasmid
1
2
pUSA2 to construct pUSA2-aprA, while the aprY gene was
1
2
inserted into pUARA2 to generate pUARA2-aprY (Fig. S2†).
The two other genes, aprR and aprT, were cloned into
1
3
pAdeA2 to construct pAdeA2-aprRT (Fig. S2†). Sequencing
the aprA gene revealed that the precursor peptide contains
eight repeats of the core sequence “FYYTGY” while the AprA
sequence deposited in NCBI contains eleven repeats (see ESI,
page S17†). The two plasmids, pUARA2-aprY and pAdeA2-
aprRT, were introduced into A. oryzae NSAR1 (AO-WT) to gene-
rate AO-aprYRT. The resulting transformant did not produce 1
as the precursor peptide was absent in this strain. We then
introduced pUSA2-aprA into this transformant to generate
AO-aprAYRT. As we expected, the resulting transformant suc-
cessfully produced 1 (Fig. 2). The 1D- and 2D-NMR spectra of
the isolated compound as well as its HR-ESI-MS were in good
agreement with previously reported spectra, confirming that
the above four genes were sufficient for the biosynthesis of 1
(Table S3†). We also prepared the transformants AO-aprAYR
and AO-aprAYT to test whether AprR and/or AprT were essen-
tial for biosynthesis. Although these showed significantly
decreased levels of production, both transformants still pro-
duced trace amounts of 1, suggesting that AprR and AprT were
required for biosynthesis and that adventitious proteins in the
host strain complement their function. Considering that del-
etion of aprY in A. flavus abolished the production of 1 in the
Scheme 1 Putative biosynthetic pathway for 1.
previous study, this observation suggests that AprY is involved generated the expected products, thus suggesting the impor-
in the formation of the bicyclic structure (Scheme 1).
tance of both the third and the sixth tyrosine for cyclization
Using AO-aprYRT as a host strain, we also investigated the (Fig. S3†). As no related metabolites such as monocyclic com-
conversion of two AprA analogs, AprA/Y3F and AprA/Y6F. As pounds, were observed, the cyclization might be a sequential
either the third or the sixth tyrosine of the core peptide is process. However, further experimental verification is necess-
substituted with phenylalanine, we expected that these ary to address this hypothesis.
mutants could not undergo cyclization and that monocyclic
A BLAST search using AprA as a query retrieved orthologous
products might be obtained. We constructed the plasmids precursor proteins from at least three strains including
pUSA2-aprA/Y3F and pUSA2-aprA/Y6F and introduced them Aspergillus oryzae, Aspergillus parasiticus, and Aspergillus arachi-
into AO-aprYRT. Neither AO-aprAYRT/Y3F nor AO-aprAYRT/Y6F dicola (Table S4 and Fig. S4†). Notably, each gene was flanked
by aprY, aprR, and aprT homologs, suggesting that they are
involved in the biosynthesis of 1 or its congeners. The number
of core peptide repeats of each AprA ortholog varies from one
to eight. The third and the sixth tyrosine residues were strictly
conserved among each repeat (Fig. S5†). The first phenyl-
alanine was also conserved, indicating the importance of these
three residues in the cyclization process. On the other hand, in
some cases, the second, the forth, and the fifth residues were
substituted with phenylalanine, histidine, and asparagine,
respectively (Fig. S5†). This observation indicated that these
positions did not affect cyclization and could be substituted
for structural diversification.
The highly-strained 14-membered paracyclophane ring of 1
has limited conformational flexibility and is rotationally
1
4
restricted, thus allowing us to elucidate its relative stereo-
chemistry by 1D- and 2D-NMR analyses. Both H1 and H2 were
Fig. 2 LC-MS profiles of the metabolites extracted from transformants.
Chromatograms were extracted at m/z 810.
40 | Org. Biomol. Chem., 2019, 17, 39–43
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Fig. 3 Stereochemical analysis of 1. (A) Newman projection of 3-phe-
nyllactic acid moiety of 1. (B) Key ROESY correlations.
3
observed as broad singlets, suggesting a small JHH value
between these protons (Fig. 3A). On the basis of this obser-
vation, the relative stereochemistry at C1 and C2 can be
deduced as (1R,2S) or (2R,1S). In addition, the NOEs between
H16 and 15-NH, H16 and H18, and H15 and H22 clearly indi-
cated that the relative configuration at C15 and C16 was either
all-S or all-R (Fig. 3B).
For determination of the absolute configuration, 1 was sub-
jected to a Pd-mediated hydrogenolysis, followed by hydrolysis
with 6 M HCl to give a mixture of 3-phenyllactic acid and
amino acids (Scheme 2). A mixture of amino acids were then
α
converted with N -(5-fluoro-2,4-dinitrophenyl)-L-leucinamide
1
5–17
(
L-FDLA) to yield L-FDLA derivatives.
LC-MS analysis
Fig. 4 LC-MS profiles of the constituents of 1. (A) L-FDLA derivatized
Tyr, (B) Thr, (C) Gly, and (D) chiral LC-MS analysis with authentic (R)- and
revealed that the retention times of the two constituent amino
acids Tyr and Thr from 1 were identical to those of the L-Tyr-
(
S)-phenyllactic acid. Chromatograms were extracted at m/z 476 for
and L-Thr-derivatives, respectively (Fig. 4A and B). L-FDLA-Gly FDLA-Tyr, m/z 414 for FDLA-Thr, m/z 370 for FDLA-Gly, and m/z 165
for phenyllactic acid.
was also observed (Fig. 4C). The molar ratio of L-Tyr and Gly
was roughly estimated to be 3 : 1 by comparing the peak areas
of FDLA derivatives (Fig. S6†). These results are consistent
with the ribosomal synthesis of the precursor peptide AprA.
The hydrolysate was also analyzed by chiral HPLC without deri-
vatization. The retention time of 3-phenyllactic acid from 1
was identical to that of commercial (R)-3-phenyllactic acid,
thus confirming the stereochemistry at C1 to be R (Fig. 4D).
Together with the relative configuration as described above,
The biosynthesis of 1 starts with transcription and trans-
lation of the structural gene aprA. The synthesized precursor
peptide AprA contains eight repeats of the core sequence
FYYTGY. Each repeat is flanked by KR, which is a target site of
Golgi protease, KexB, suggesting that AprA undergoes a similar
proteolytic process to that known in the biosynthesis of usti-
1
8
loxin B. AprA is also modified by AprY and AprR to furnish 1.
However, there is no information concerning the order of
these post-translational modifications. As discussed above, the
bicyclic structure of 1 is likely synthesized by the single
enzyme AprY. Considering the relatively small size of AprY and
that two C–O bonds were generated in the same stereochemi-
cal course, formation of these two bonds might be successively
catalyzed in the same active site of the enzyme. In ustiloxin
biosynthesis, initial hydroxylation and subsequent cyclization
the absolute structure of 1 was determined as shown in Fig. 1.
1
1
gave a macrocyclic system. This transformation accompanied
hydroxylation at the C position of L-Tyr. Similarly, macrocycli-
β
zation of 1 may accompany an α-hydroxylation–dehydration
sequence to give imine 2, which is readily hydrolyzed to yield
putative ketone intermediate 3. We tempted to explain that the
reductase AprR may be required for the final reduction to yield
1
(Scheme 1). As AprT shows homology to major facilitator
superfamily protein (MFS_1, Pfam ID: PF07690), it is likely
that this protein is not involved in biosynthesis but rather
exports the product.
4
Scheme 2 (a) HCOONH , 10% Pd/C, MeOH, 70 °C; (b) 6 M HCl, 110 °C.
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Besides RiPPs produced by Ascomycetes such as ustiloxin B
and phomopsin A, plants also produce cyclopeptides that are
J. P. Klinman, O. P. Kuipers, A. J. Link, W. Liu,
M. A. Marahiel, D. A. Mitchell, G. N. Moll, B. S. Moore,
R. Müller, S. K. Nair, I. F. Nes, G. E. Norris, B. M. Olivera,
H. Onaka, M. L. Patchett, J. Piel, M. J. Reaney, S. Rebuffat,
R. P. Ross, H. G. Sahl, E. W. Schmidt, M. E. Selsted,
K. Severinov, B. Shen, K. Sivonen, L. Smith, T. Stein,
R. D. Süssmuth, J. R. Tagg, G. L. Tang, A. W. Truman,
J. C. Vederas, C. T. Walsh, J. D. Walton, S. C. Wenzel,
J. M. Willey and W. A. van der Donk, Nat. Prod. Rep., 2013,
30, 108–160.
1
4
structurally related to 1. Cyclopeptides, such as mauritine A,
1
4,19,20
21
sanjoinin A,
and ophiorrhisine A, possess 14-mem-
bered paracyclophane rings similar to that of 1 (Fig. S7†).
Although DUF3328 proteins do not exist in plants, other oxi-
dases that operate with a similar mechanism might be
involved in the biosynthesis of these compounds. Future
studies focusing on the mechanism of DUF3328 proteins may
contribute to studies of similar cyclopeptides from other
genera.
2 H. E. Hallen, H. Luo, J. S. Scott-Craig and J. D. Walton,
Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 19097–
19101.
Conclusions
3 S. Ramm, B. Krawczyk, A. Mühlenweg, A. Poch, E. Mösker
and R. D. Süssmuth, Angew. Chem., Int. Ed., 2017, 56, 9994–
In this study, we achieved the heterologous production of 1
and determined its absolute structure, showing that DUF3328
protein AprY was involved in formation of the characteristic
bicyclic structure. Together with our previous results on usti-
loxin B, the present findings established the common strategy
for macrocyclization of RiPPs produced by filamentous fungi.
In both cases, oxidative cyclization catalyzed by UstY homologs
accompanies a C–O bond formation between the phenol and
9
997.
4
5
6
N. S. van der Velden, N. Kälin, M. J. Helf, J. Piel,
M. F. Freeman and M. Künzler, Nat. Chem. Biol., 2017, 13,
833–835.
M. Umemura, N. Nagano, H. Koike, J. Kawano, T. Ishii,
Y. Miyamura, M. Kikuchi, K. Tamano, J. Yu, K. Shin-ya and
M. Machida, Fungal Genet. Biol., 2014, 68, 23–30.
M. Umemura, H. Koike, N. Nagano, T. Ishii, J. Kawano,
N. Yamane, I. Kozone, K. Horimoto, K. Shin-ya, K. Asai,
J. Yu, J. W. Bennett and M. Machida, PLoS One, 2013, 8,
e84028.
β
C of an amino acid moiety. Compared with the macrocycliza-
tion of ustiloxins, which requires three enzymes (two
DUF3328s and one tyrosinase), macrocyclization of 1 would be
more feasible for biochemical characterization, as it requires
only one or two enzymes (AprY and/or AprR). However, pre-
liminary attempts to reconstitute this enigmatic reaction were
unsuccessful. Further studies are currently underway in our
group.
7
8
9
T. Tsukui, N. Nagano, M. Umemura, T. Kumagai, G. Terai,
M. Machida and K. Asai, Bioinformatics, 2015, 31, 981–
985.
W. Ding, W. Q. Liu, Y. Jia, Y. Li, W. A. van der Donk and
Q. Zhang, Proc. Natl. Acad. Sci. U. S. A., 2016, 113, 3521–
3
R. D. Johnson, G. A. Lane, A. Koulman, M. Cao, K. Fraser,
D. J. Fleetwood, C. R. Voisey, J. M. Dyer, J. Pratt,
M. Christensen, W. R. Simpson, G. T. Bryan and
L. J. Johnson, Fungal Genet. Biol., 2015, 85, 14–24.
526.
Conflicts of interest
There are no conflicts to declare.
1
1
0 N. Nagano, M. Umemura, M. Izumikawa, J. Kawano,
T. Ishii, M. Kikuchi, K. Tomii, T. Kumagai, A. Yoshimi,
M. Machida, K. Abe, K. Shin-Ya and K. Asai, Fungal Genet.
Biol., 2016, 86, 58–70.
1 Y. Ye, A. Minami, Y. Igarashi, M. Izumikawa, M. Umemura,
N. Nagano, M. Machida, T. Kawahara, K. Shin-Ya, K. Gomi
and H. Oikawa, Angew. Chem., Int. Ed., 2016, 55, 8072–
Acknowledgements
This work was financially supported by Grants-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan (JSPS KAKENHI Grants
(
(
15H01835 (H. O.), 16H06446 (A. M.), 17K15263 & 17H05425
T. O.), 17H05456 (M. U.)). We are grateful to DAICEL Corp. for
8075.
1
2 K. Tagami, A. Minami, R. Fujii, C. Liu, M. Tanaka,
K. Gomi, T. Dairi and H. Oikawa, ChemBioChem, 2014, 15,
the use of chiral column.
2076–2080.
1
3 T. Ugai, A. Minami, R. Fujii, M. Tanaka, H. Oguri, K. Gomi
and H. Oikawa, Chem. Commun., 2015, 51, 1878–1881.
References
1
P. G. Arnison, M. J. Bibb, G. Bierbaum, A. A. Bowers, 14 T. Gulder and P. S. Baran, Nat. Prod. Rep., 2012, 29, 899–
T. S. Bugni, G. Bulaj, J. A. Camarero, D. J. Campopiano, 934.
G. L. Challis, J. Clardy, P. D. Cotter, D. J. Craik, M. Dawson, 15 P. Marfey, Carlsberg Res. Commun., 1984, 49, 591–596.
E. Dittmann, S. Donadio, P. C. Dorrestein, K. D. Entian, 16 K. Fujii, Y. Ikai, T. Mayumi, H. Oka, M. Suzuki and
M. A. Fischbach, J. S. Garavelli, U. Göransson,
C. W. Gruber, D. H. Haft, T. K. Hemscheidt, C. Hertweck, 17 K. Fujii, Y. Ikai, H. Oka, M. Suzuki and K. Harada, Anal.
C. Hill, A. R. Horswill, M. Jaspars, W. L. Kelly, Chem., 1997, 69, 5146–5151.
K. Harada, Anal. Chem., 1997, 69, 3346–3352.
42 | Org. Biomol. Chem., 2019, 17, 39–43
This journal is © The Royal Society of Chemistry 2019

View Article Online
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1
1
8 A. Yoshimi, M. Umemura, N. Nagano, H. Koike, 20 R. Tschesche and H. Last, Tetrahedron Lett., 1968, 2993–2998.
M. Machida and K. Abe, AMB Express, 2016, 6, 9.
21 T. Onozawa, M. Kitajima, N. Kogure, N. Peerakam,
D. Santiarworn and H. Takayama, J. Nat. Prod., 2017, 80,
2156–2160.
9 R. Tschesche and I. Reutel, Tetrahedron Lett., 1968, 3817–
3
818.
This journal is © The Royal Society of Chemistry 2019
Org. Biomol. Chem., 2019, 17, 39–43 | 43