The fumagillin biosynthetic gene cluster in Aspergillus fumigatus encodes a cryptic terpene cyclase involved in the formation of β-trans-bergamotene

Article abstract of DOI:10.1021/ja312503y

Fumagillin 1 is a meroterpenoid from Aspergillus fumigatus that is known for its anti-angiogenic activity by binding to human methionine aminopeptidase 2. The genetic and molecular basis for biosynthesis of 1 had been an enigma despite the availability of the A. fumigatus genome sequence. Here, we report the identification and verification of the fma gene cluster, followed by characterization of the polyketide synthase and acyltransferase involved in biosynthesis of the dioic acid portion of 1. More significantly, we uncovered the elusive β-trans-bergamotene synthase in A. fumigatus as a membrane-bound terpene cyclase.

Full text of DOI:10.1021/ja312503y

Communication
pubs.acs.org/JACS
The Fumagillin Biosynthetic Gene Cluster in Aspergillus fumigatus
Encodes a Cryptic Terpene Cyclase Involved in the Formation of β-
trans-Bergamotene
†
†
‡
†
‡
,†,§
Hsiao-Ching Lin, Yit-Heng Chooi, Sourabh Dhingra, Wei Xu, Ana M. Calvo, and Yi Tang*
†
§
Department of Chemical and Biomolecular Engineering and Department of Chemistry and Biochemistry, University of California,
Los Angeles, California 90095, United States
‡
Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115, United States
S Supporting Information
*
ABSTRACT: Fumagillin 1 is a meroterpenoid from
Aspergillus fumigatus that is known for its anti-angiogenic
activity by binding to human methionine aminopeptidase
2. The genetic and molecular basis for biosynthesis of 1
had been an enigma despite the availability of the A.
fumigatus genome sequence. Here, we report the
identification and verification of the fma gene cluster,
followed by characterization of the polyketide synthase and
acyltransferase involved in biosynthesis of the dioic acid
portion of 1. More significantly, we uncovered the elusive
β-trans-bergamotene synthase in A. fumigatus as a
membrane-bound terpene cyclase.
Figure 1. Fumagillin 1 and related compounds.
free extracts from the ovalicin-producing fungus Pseudeurotium
1
2,13
ovalis,
while the rearrangement of 3 to ovalicin have been
probed by isotope incorporation studies in the context of ovalicin
14−17
biosynthesis.
Nevertheless, the genetic and molecular bases
odern genome sequencing technologies have significantly
for the biosynthesis of 3, 2 and ultimately 1 have remained
unknown. While genome sequencing of A. fumigatus Af293
provided initial hope that the gene cluster of 1 can be located, it
was soon realized that there is no terpene cyclase homologue
M
improved our abilities in identifying biosynthetic gene
clusters of natural products. However, gene cluster assignment
relying on homology search of enzymes catalyzing key
biosynthetic transformations can sometimes fail, largely due to
nature’s flexibility in using seemingly unrelated enzymes to
catalyze these reactions. In these cases, the link between the
natural product and the gene cluster cannot be easily established
despite a sequenced and annotated genome. One example is the
meroterpenoid fumagillin 1 produced by the pathogenic fungus
18
present in the entire genome. Because of this highly
unexpected finding, the gene cluster has not been identified 8
years after the sequencing report. More importantly, the lack of a
terpene cyclase candidate in A. fumigatus suggests that the
cyclization of FPP into 3 must be catalyzed by a noncanonical
terpene cyclase that is concealed from common homology search
strategies. Cryptic terpene cyclases in other fungal meroterpe-
1
Aspergillus fumigatus. Since its discovery in the 1950s, 1 and
19
related compounds have been intensely studied because of their
noid gene cluster have been noted. Here we describe the
verification of the gene cluster of 1 and identification of an
integral membrane protein as the terpene cyclase responsible for
the formation of 3 from FPP.
2
3
potential use in treating amebiasis and microsporidiosis, and
recently for their anti-angiogenic properties from the irreversible
inhibition of human type 2 methionine aminopeptidase
4
,5
(
MetAP2). Structurally, 1 consists of a highly oxygenated
Since the dioic acid moiety in 1 mostly likely originates from a
reduced polyketide pathway, we scanned all the highly reducing
PKS (HR-PKS) genes in the A. fumigatus genome and their
neighboring genes. Our attention was drawn to a cluster (hereby
named fma) containing HR-PKS gene AFUA_8G00370
(Af370) (Table S1). Most notably, the cluster encodes a
MetAP-2 (AFUA_8G00410 or Af410) and a type 1 MetAP.
cyclohexane-containing terpenoid (fumagillol, 2) that is
6
esterified with a decatetraenedioic (dioic) acid. The intriguing
structure of 1 has served as a total synthesis target for the past 40
7
years. 2 has also been remodeled to yield new structures via a
8
reaction discovery approach. Semisynthetic analogues of 1 with
modified ester group have undergone clinical trials for treating
4
9
Since 1 is known to inhibit eukaryotic MetAP-2, we postulated
different cancers. Hence, elucidating the biosynthesis of 1 can
that Af410 may encode a self-resistant enzyme for A. fumigatus
toward 1. Indeed, an additional housekeeping MetAP-2
have important biomedical ramifications.
Based on the feeding study of a structurally related compound
10
(
AFUA_2G01750) was found in A. fumigatus, which is well-
ovalicin (Figure 1), 2 is likely derived from the sesquiterpene β-
trans-bergamotene 3, which can also be isolated from A.
1
1
fumigatus. The enzymatic mechanism for the formation of 3
Received: December 21, 2012
Published: March 14, 2013
from farnesyl-PP (FPP) has been previously studied using cell-
©
2013 American Chemical Society
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Communication
Figure 3. In vitro verification of the functions of fma-PKS and fma-AT
in the biosynthesis of 4 and 5. (A) fma-PKS synthesized the C12
polyketide product that is hydrolyzed to yield 4 by fma-AT. (B)
Acylation of 2 (1 mM) yielded 5 by fma-PKS and -AT.
22
23
biosynthesis of lovastatin and chaetoviridin, respectively.
To examine the role of the enzyme encoded by Af380 (named
fma-AT), the 6xHis-tagged enzyme was solubly expressed from
BJ5464-NpgA and purified to homogeneity (Figure S2A).
Coincubation of fma-PKS and -AT with malonyl-CoA and
NADPH resulted in the formation of a single yellow compound 4
that can be extracted into the organic phase (Figure 3A). LC-MS
analysis of 4 yielded a mass of 190.1 (C H O ), consistent with
Figure 2. Genetic verification of fma-PKS in the biosynthesis of 1. (A)
The fma gene cluster. The numbers are part of the locus tag
AFUA_8G00###, e.g., 370 is AFUA_8G00370. Genes are color-
coded for polyketide biosynthesis (green), terpene cyclization (orange),
tailoring modification (blue), self-resistance gene (red), and unknown/
unassigned function (white) (see Table S1). (B) HPLC analysis of
metabolites extracted from wild type and ΔAf370 strain showing loss of
1
and restored production of 1 upon feeding of 5 to ΔAf370. (C) LC-
12
14
2
MS analysis of the ΔAf370 strain showing the accumulation of 2.
that of dodecapentaenoic acid (Figure 3A). Repeating the assay
13
with [2- C]-malonate and a MatB-dependent in situ malonyl-
CoA regeneration system resulted in an increase of the m/z of 4
by 6 mu, which is a further confirmation of 4 as a hexaketide.
Although 4 can be detected in the culture of BJ5464-NpgA co-
expressing fma-PKS and -AT (Figure S4), the compound readily
degraded and is unstable upon purification.
conserved among all sequenced aspergilli. A number of genes in
the vicinity of the Af370 and Af410 also encode enzymes that are
consistent with the structure of 1 and 2, including four oxidases
(
to initiate oxidative rearrangement of 3, to introduce the bis-
epoxide, to insert the C4,C5-syn-diol, and to oxidize the
polyketide methyl terminus); a methyltransferase that may O-
methylate C4-OH; and an α,β-hydrolase that may release or
transfer the polyketide from HR-PKS to 2 (Table S1). Thus,
despite the absence of a canonical terpene cyclase, we
hypothesized that the fma genes in Table S1 likely comprise
the target cluster.
To examine if fma-AT can transfer the polyketide product of
fma-PKS to 2, we performed the fma-PKS turnover assay in the
presence of fma-AT and 2. Interestingly, 2 can be converted to a
single new product 5 with the mass of 454.3 (m/z 477.1 [M
+
+Na] ), corresponding to that of 2 acylated with 4. To verify the
structure of 5, 2.5 mg of 2 was supplemented to a 50 mL yeast
culture co-expressing fma-PKS and -AT (Supporting Informa-
tion (SI)). Following 2 days of culturing, 1.1 mg of 5 can be
purified from the culture, which allowed complete NMR
characterization of 5 by 1D and 2D NMR, and confirmed the
structure to be that shown in Figure 3B. To verify 5 is a true on-
pathway intermediate in the biosynthesis of 1, ∼40 μg/mL of 5
was supplied to the culture of ΔAf370 strain. Following 2 days of
culturing, restoration of the biosynthesis of 1 was observed
(Figure 2B). Collectively, our genetic and biochemical studies on
fma-PKS and -AT confirm the involvement of these enzymes in
biosynthesis of 1, and provide the first evidence for this being the
authentic fma gene cluster.
We first deleted Af370 encoding the HR-PKS (named fma-
KU80
PKS) in A. fumigatus CEA17 akuB
strain (pyrG89,
KU80
ΔakuB
), which is deficient in non-homologous end
2
0
joining. In comparison to the isogenic control strain, the
metabolic profile of ΔAf370 analyzed by LC-MS showed the
complete abolishment of 1 (Figure 2B) and the accumulation of
2
(Figure 2C), which is barely detectable in the isogenic strain.
To further prove the role of fma-PKS, the entire 264 kDa enzyme
was expressed from Saccharomyces cerevisiae BJ5464-NpgA,
which has been used for soluble expression of large fungal
2
1
2+
PKSs. The 6xHis-tagged enzyme was purified by Ni -affinity
chromatography (Figure S2A). In vitro reaction with malonyl-
CoA and NADPH resulted in a yellow product that cannot be
extracted using ethyl acetate with 1% acetic acid (Figure S2B).
This result hinted that a conjugated polyunsaturated fatty acyl
product may be synthesized and remain attached to the fma-PKS
in the absence of a releasing enzyme. Since the product of fma-
PKS is to be transacylated to C5-hydroxyl of 2, we hypothesized
that a dedicated acyltransferase may be involved.
In light of the gene cluster identification and above
biochemical findings, a putative pathway for 1 is proposed as
shown in Figure 4. The terpenoid origin of the carbon backbone
6,10,11
of 1 via 3 has been previously established.
3 is cyclized from
FPP via nerolidyl diphosphate (NPP) followed by a bisabolyl
1
2,13
cation intermediate.
However, the previously proposed
6
2
intermediate 9 (Figure 4, route 1) was ruled out by a later H
In the fma gene cluster, a putative α/β hydrolase is encoded
immediately downstream of Af370 by AFUA_8G00380 (Af380),
which could be involved in PKS product release. Alternatively,
the polyketide chain may also be transferred directly to 2 by an
acyltransfease, such as LovD and CazE involved in the
NMR study via incorporation of deuterated mevalonates into
16
ovalicin. Furthermore, the proposed intermediate 10 has been
14
synthesized, but an attempt to incorporate the isotope-labeled
17
10 to ovalicin by feeding to P. ovalis was unsuccessful. Thus, the
biosynthesis of 3 may proceed via an alternative pathway, such as
4
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Journal of the American Chemical Society
Communication
Figure 4. Putative biosynthetic pathway of 1.
2
7
that in route 2, in which an iron-dependent oxygenase, such as
AFUA_8G00480, can hydroxylate 3 at C8, followed by
rearrangement of the oxo-Fe intermediate to yield the epoxide
tabacum epi-aristolochene synthases. Indeed, homology to the
α-helical structures of FPP synthase can also be detected for
Af520 (Figure S5). We therefore reasoned that Af520 may
encode a new type of terpene cyclases (named fma-TC) that may
be responsible for the cyclization of FPP to 3.
11 directly. A second epoxidation step, dihydroxylation, and O-
methylation afford 2. The conversion of 2 to exclusively 5 in the
presence of fma-PKS and -AT, instead of the shorter
decatetraenoate ester, is surprising. This suggests that formation
of the dioic acid moiety in 1 may proceed via an oxidative
cleavage of the terminal alkene in 5 to yield the dioic acid in 1
To investigate the involvement of fma-TC in the biosynthesis
of 1, we inactivated the gene through homologous recombina-
BKU80
tion in A. fumigatus aku
strain. Indeed, the resulting ΔAf520
mutant lost the abilities to produce 1 compared to the isogenic
control strain (Figure 5A). When 3 or 5 was supplied to the
ΔAf520 strain at 100 or 40 mg/L, respectively, production of 1
was restored (Figure 5A), thereby directly linking fma-TC to the
generation of 3 in A. fumigatus. Next, we cloned the cDNA of
fma-TC (for construction of the correct cDNA, see SI) into a
yeast 2μ expression vector and transformed it into BJ5464-NpgA.
We reasoned that the recombinant fma-TC may be able to utilize
the endogenous yeast FPP to produce 3. Gratifyingly, after 4 days
of culturing followed by extraction with hexane, we were able to
detect the formation of a highly hydrophobic compound that had
the same mass (204.2) as 3 using GC-MS (Figure 5B) from the
yeast strain. In contrast, a control strain that does not express
fma-TC did not produce any detectable amount of 3. Larger
scale culturing enabled us to isolate the compound at a purified
titer of 46 mg/L. GC-MS fragmentation (Figure 5E) and NMR
analyses (SI) confirmed that the yeast-derived compound is
(
(
Figure 4). Oxidative cleavage of alkene by a single oxygenase
either via a mono- or dioxygenase mechanism) is well-
documented, as exemplified by the carotenoid cleavage oxy-
2
4
genases. We propose the terminal alkene in 5 can undergo
oxidative C−C bond cleavage through epoxidation to yield 6,
then hydrolysis to afford a vicinal diol 7, which can undergo
cleavage to yield the aldehyde 8 and acetaldehyde. Final
oxidation of 8 then yields 1.
Identification of the fma gene cluster enabled us to take a
deeper look into the nearby genes for a possible terpene cyclase
that may synthesize 3. The locus AFUA_8G00520 (Af0520) was
originally annotated as encoding an integral membrane protein
with 173 amino acids. Further conserved domain analysis and re-
annotation revealed that the region encodes a 267-residue
protein that contains a PFAM01040 UbiA prenyltransferase (p-
hydroxybenzoic acid oligoprenyltransferase) domain (SI).
However, Af0520 exhibits only low homology (14−19% protein
identity/∼30% similarity) to the E. coli UbiA, S. cerevisiae COQ2
1
1,14
identical to 3.
Lastly, we prepared microsomal fractions from
the yeast strain that overexpressed fma-TC. When provided with
1 mM FPP and 5 mM MgCl , we were able to detect nearly sole
(
UbiA homologue), and other known fungal membrane-bound
2
25
prenyltransferases, and has almost no homology to the recently
discovered membrane-bound cyclase involved in biosynthesis of
pyripyropene A and 3,5-dimethylorsellinic acid (DMOA)-
formation of 3 in vitro. With ∼250 μg/mL microsomal protein,
the apparent rate of formation of 3 was estimated to be ∼4 μM/
1
9
27,28
min. Like UbiA,
the fma-TC-containing microsomes were
26
2+
derived meroterpenoids. Protein structure prediction indicates
that Af520 is comprised of six transmembrane helices (Figure
S5). Although they share only minimal homology, the E. coli
UbiA prenyltransferase has been shown to be structurally related
to Class I terpene synthases, both of which belong to all-α-helical
proteins, and a 3D model for UbiA has been built based on N.
unable to convert FPP into 3 in the absence of Mg . Collectively,
these results confirmed that the membrane-bound fma-TC is
indeed the terpene cyclase that produces 3 as an intermediate for
1 in A. fumigatus.
Based on the type of catalytic reaction and protein fold,
terpene cyclases can be generally categorized in the Class I
4
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ASSOCIATED CONTENT
Supporting Information
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*
S
AUTHOR INFORMATION
Corresponding Author
yitang@ucla.edu
Notes
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The authors declare no competing financial interest.
(
ACKNOWLEDGMENTS
H.-C.L. is supported by National Science Council of Taiwan
102-2917-I-564-008). This work was supported by NIH
R01GM085128 and 1DP1GM106413 to Y.T., and by Northern
Illinois University to A.M.C. NMR instrumentation was
supported by the NSF equipment grant CHE-1048804. We
deeply thank Prof. David Cane for enlightening discussions on
the biosynthetic route. We thank Prof. John Porco Jr. and Dr.
Alexander Grenning of CMLD-BU for the generous gifts of 1 and
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4
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