Classification, prediction, and verification of the regioselectivity of fungal polyketide synthase product template domains

Article abstract of DOI:10.1074/jbc.M110.128504

The fungal iterative nonreducing polyketide synthases (NRPKSs) synthesize aromatic polyketides, many of which have important biological activities. The product template domains (PT) embedded in the multidomain NRPKSs mediate the regio-selective cyclization of the highly reactive polyketide backbones and dictate the final structures of the products. Understanding the sequence-activity relationships of different PT domains is therefore an important step toward the prediction of polyketide structures from NRPKS sequences and can enable the genome mining of hundreds of cryptic NRPKSs uncovered via genome sequencing. In this work, we first performed phylogenetic analysis of PT domains from NRPKSs of known functions and showed that the PT domains can be classified into five groups, with each group corresponding to a unique product size or cyclization regioselectivity. Group V contains the formerly unverified PT domains that were identified as C6-C11 aldol cyclases. The regioselectivity of PTs from this group were verified by product-based assays using the PT domain excised from the asperthecin AptA NRPKS.Whencombined with dissociated PKS4 minimal PKS, or replaced the endogenous PKS4 C2-C7 PT domain in a hybrid NRPKS, AptA-PT directed the C6-C11 cyclization of the nonaketide backbone to yield a tetracyclic pyranoanthraquinone 4. Extensive NMR analysis verified that the backbone of 4 was indeed cyclized with the expected regioselectivity. The PT phylogenetic analysis was then expanded to include ～100 PT sequences from unverified NRPKSs. Using the assays developed for AptA-PT, the regioselectivities of additional PT domains were investigated and matched to those predicted by the phylogenetic classifications.

Full text of DOI:10.1074/jbc.M110.128504

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 30, pp. 22764–22773, July 23, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Classification, Prediction, and Verification of the
Regioselectivity of Fungal Polyketide Synthase Product
□
S
Template Domains*
Received for publication, March 29, 2010, and in revised form, May 14, 2010 Published, JBC Papers in Press, May 17, 2010, DOI 10.1074/jbc.M110.128504
‡§1
Yanran Li (
ᴢႷ✊
)‡, Wei Xu (
)‡, and Yi Tang (
)
૤༩
ᕤӳ
From the Departments of ‡Chemical and Biomolecular Engineering and §Chemistry and Biochemistry, University of California,
Los Angeles, California 90095
polyketide synthases (PKSs)2 and nonribosomal peptide syn-
thetases (NRPSs) are particularly abundant. Many of the
encoded pathways are silent under laboratory culturing condi-
tions, and as a result, the metabolites associated with these
pathways have not been observed or isolated (7). Mining these
uncharacterized pathways is therefore an important objective
toward discovery of new bioactive molecules and novel enzy-
matic machineries (8).
The fungal iterative nonreducing polyketide synthases
(NRPKSs) synthesize aromatic polyketides, many of which have
important biological activities. The product template domains
(PT) embedded in the multidomain NRPKSs mediate the regio-
selective cyclization of the highly reactive polyketide back-
bones and dictate the final structures of the products. Under-
standing the sequence-activity relationships of different PT
domains is therefore an important step toward the prediction of
polyketide structures from NRPKS sequences and can enable
the genome mining of hundreds of cryptic NRPKSs uncovered
via genome sequencing. In this work, we first performed phylo-
genetic analysis of PT domains from NRPKSs of known func-
tions and showed that the PT domains can be classified into five
groups, with each group corresponding to a unique product size
or cyclization regioselectivity. Group V contains the formerly
unverified PT domains that were identified as C6-C11 aldol
cyclases. The regioselectivity of PTs from this group were veri-
fied by product-based assays using the PT domain excised from
the asperthecin AptA NRPKS. When combined with dissociated
PKS4 minimal PKS, or replaced the endogenous PKS4 C2-C7
PT domain in a hybrid NRPKS, AptA-PT directed the C6-C11
cyclization of the nonaketide backbone to yield a tetracyclic pyr-
anoanthraquinone 4. Extensive NMR analysis verified that the
backbone of 4 was indeed cyclized with the expected regioselec-
tivity. The PT phylogenetic analysis was then expanded to
include ϳ100 PT sequences from unverified NRPKSs. Using the
assays developed for AptA-PT, the regioselectivities of addi-
tional PT domains were investigated and matched to those pre-
dicted by the phylogenetic classifications.
One method of predicting the structures of polyketides en-
coded in cryptic pathways is by analyzing the sequence of the
PKSs. This method has been successfully used in the prediction
of bacterial polyketides by examining the linear arrangement of
catalytic sites along the modular type I PKSs (9, 10). However,
this method is not readily applicable to fungal polyketides
because of the differences in enzyme architectures and more
importantly, the lack of the colinearity rule. Fungal PKSs are
large megasynthases that assemble single copies of catalytic
domains in one polypeptide. The domains function in an itera-
tive fashion and use built-in programming rules to dictate chain
length, cyclization, reductive tailoring, and other structural fea-
tures (11). Therefore, understanding the relationships between
protein sequence and catalytic activity, as well as substrate
specificity, of each domain is essential to unlocking the biosyn-
thetic potential of fungal PKSs.
Among the different families of fungal iterative PKSs,
the nonreducing PKSs (NRPKSs) are responsible for the syn-
thesis of a number of important metabolites, including the
well-known mycotoxin aflatoxin (12). Other representa-
tive products of NRPKSs include pigment precursors
(1,3,6,8-tetrahydroxylnaphthalene, THN), compounds with
antibiotic (viridicatumtoxin) and anticancer (bikaverin) prop-
erties. Polyketides produced from NRPKSs are aromatic com-
pounds with sizes ranging from monocyclic aromatics to those
containing multiple fused rings. Starting from the N terminus
of the megasynthase, the following domains are typically found
in an NRPKS: a starter unit:ACP transacylase (SAT) that selects
the starter unit (13); a ketosynthase (KS) that catalyzes repeated
decarboxylative condensation to elongate the polyketide back-
bone; a malonyl-CoA:ACP transacylase (MAT) that selects and
transfers the extender unit malonyl-CoA; a product template
Natural products such as polyketides and nonribosomal
peptides isolated from filamentous fungi have played indis-
pensable roles in human health care (1). During the past
decade, advances in rapid DNA sequencing techniques have
enabled the complete genome sequencing of many fungi spe-
cies (2–6). One important outcome of the sequencing efforts is
the revelation of large numbers of natural product biosynthetic
pathways encoded in these organisms. Among them, putative
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant 1R01GM085128 and a David and Lucile Packard Fellowship (to Y. T.). 2 The abbreviations used are: PKS, polyketide synthase; DTT, dithiothreitol;
S
□
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S7 and Tables S1–S3.
NRPKS, nonreducing polyketide synthase; PT, product template; NRPS,
nonribosomal peptide synthetases; THN, 1,3,6,8-tetrahydroxylnaphtha-
lene; HMBC, heteronuclear multiple bond correlation; HMQC, hetero-
nuclear multiple quantum coherence.
1 To whom correspondence should be addressed: University of California, Los
Angeles, CA 90095. E-mail: yitang@ucla.edu.
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Fungal Polyketide Cyclization
FIGURE1. A, biosynthesisoffungalaromaticpolyketides. C2-C7, C4-C9/C2-C11, andC6-C11/C4-C13arecommonlyobservedF-modefoldingpatternscatalyzed
by fungal NRPKSs. Product template domains that mediate C2-C7 and C4-C9/C2-C11 folding modes have been characterized in the study of PKS4 and PksA,
respectively. TE/CLCdomainscatalyzethecyclizationofthelastringandoff-loading. IntheabsenceofPT-mediatedcyclization, thePKS4minimalPKSproduces
spontaneously cyclized products 2 and 3. B, C6-C11/C4-C13 cyclization regioselectivity has been observed in various fungal aromatic polyketide such as
asperthecin, emodin, chrysophanol, and hypomycetin.
(PT) domain that controls the immediate cyclization regios- units. As shown in Fig. 1, the three most commonly observed
electivity of the reactive polyketide backbone (14); an acyl-car- F-mode folding patterns are C2-C7, C4-C9, and C6-C11.
rier protein (ACP) that serves as the tether of the growing and Recently, the PT domain has been shown to be responsible
completed polyketide via its phosphopantetheinyl arm (15); for this important first step in tailoring the completed
and a thioesterase/Claisen-like cyclase (TE/CLC) domain that polyketide backbone. Using dissected domains, Townsend
cyclizes and releases the product (16). Other domains such as and co-workers demonstrated that the PT from PksA (from
methyltransferase (MT) and reductase (R) can be found in sub- Aspergillus parasiticus) can regioselectively cyclize a hex-
clades of the NRPKSs (6). Establishment of domain boundaries anoyl-primed octaketide via C4-C9 and C2-C11 aldol con-
using computation tools such as the Udwary-Merski algorithm densations en route to norsolorinic acid, the key intermedi-
(UMA) (17), and dissection of domains (12, 18–20), have been ate of aflatoxin biosynthesis (12) (Fig. 1A). The structural
used to verify the function of individual domains. Expression of basis of PksA-PT has also been elucidated to confirm its
intact megasynthases in heterologous hosts such as Aspergillus catalytic role as an aldol cyclase (14). Ma et al. (23) have
oryzae (21, 22) and Escherichia coli (23) has been used to recon- reconstituted the intact bikaverin synthase PKS4 (from Gib-
stitute the activities of the entire megasynthase through struc- berella fujikuroi) in E. coli to synthesize the C2-C7 cyclized
tural determination of the polyketide products.
nonaketide SMA76a. Systematic removal of individual
Because the poly-␤-ketone backbone of a newly synthe- domains from the intact PKS4 was then conducted to verify
sized polyketide is extremely reactive, the NRPKSs must the functional roles of the TE and PT domains (18, 19).
suppress spontaneous cyclization and control the regioselec- Removal of the TE/CLC domain (named PKS4–99) produced
tivity of the first cyclization step to afford the F-mode folding the C2-C7 cyclized SMA93 1 and confirmed the role of the
of the fungal aromatic polyketides (24). In this mode, the first TE/CLC in catalyzing the C1-C10 Claisen reaction (18) (Fig.
aromatic ring contains two intact acetate units, whereas the 1A). Further excision of the PT domain from PKS4–99 abol-
two bridging carbons are derived from two different acetate ished the production of 1 and led to the biosynthesis of aber-
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Fungal Polyketide Cyclization
rantly cyclized products 2 and 3, confirming the role of the PT son correction method (34) and are in the units of the number
domain in catalyzing the C2-C7 aldol condensation (19).
The commonly observed C6-C11 F-mode cyclization is
exemplified by the model product emodin and related com-
pounds (25–28) (Fig. 1B). NRPKSs that synthesize C6-C11
cyclized aromatic polyketides include AptA and AN0150
involved in the synthesis of asperthecin and emodin in Asper-
gillus nidulans, respectively (25, 26), as well as ACAS that syn-
thesizes atrochrysone in Aspergillus terreus (Fig. 1A) (29). A
common feature observed among these PKSs is the lack of a
fused TE/CLC domain. For ACAS, a discrete ␤-lactamase-type
TE (ACTE) is employed to release the product from ACAS (29).
Unlike the PT domains from PksA and PKS4, the activities of
the PT domains from C6-C11 regiospecific NRPKSs have not
been verified.
of amino acid substitutions per site.
Cloning of the Stand-alone PT Domains—Primers used to
amplify AptA-PT, which contains one intron, are listed in
supplemental Table S1. The first exon was amplified by PCR
from A. nidulans genomic DNA with the forward primer
AptA-PT-f and reverse primer AptA-PT-i-r; and the second
exon was amplified using the primer AptA-PT-i-f and the
reverse primer AptA-PT-r. Splice by overlapping extension
PCR (SOE-PCR) was used to fuse the two pieces to synthesize a
DNA fragment encoding AptA-PT flanked with NdeI and NotI.
The DNA fragment of AptA-PT was inserted into pET24 vector
digested with NdeI and NotI to yield pYR94. The PT domains of
AN0150 and VrtA were cloned using similar methods, and the
primers are listed in supplemental Table S1. Following ampli-
fication, the gene encoding AN0150-PT was inserted into
pET24 vector digested with NdeI and NotI to yield pYR160, and
the gene encoding VrtA-PT was inserted into pET28 vector
digested with NdeI and EcoRI to yield pYR149.
Because of the important roles played by PT domains in con-
trolling the polyketide structures, being able to precisely pre-
dict the cyclization regioselectivity of PT domains is an impor-
tant step toward suggesting the potential products of NRPKS.
Toward this end, we sought to 1) establish a sequence-activity
relationship through bioinformatics analysis; 2) establish a sim-
ple experimental platform to assay the activities of PT domains;
and 3) use the experimental platform to confirm the predicted
regioselectivity of PT domains from cryptic NRPKSs.
Construction of PKSH1—Primers used to amplify AptA-PT
and PKS4-ACP are listed in supplemental Table S1. AptA-PT
and PKS4-ACP were amplified and fused together by SOE-
PCR to synthesize a DNA fragment encoding AptA-PT-PKS4-
ACP flanked with NotI. pWJ279 is derived from pET28 and
encodes the N-terminal hexahistidine tagged PKS4-SAT-KS-
MAT between NdeI and NotI. The DNA fragment of AptA-PT-
PKS4-ACP flanked by NotI was inserted into the NotI-digested
pWJ279 to yield pYR123.
EXPERIMENTAL PROCEDURES
Materials—[1,2,3-13C]Malonate was purchased from Cam-
bridge Isotope Laboratories. CoA was purchased from Sigma
Aldrich. A. nidulans FGSC A4 and A. niger ATCC 1015 were
obtained from NRRL. The E. coli strain XL-1 Blue utilized for
DNA manipulation and BL21(DE3) used for protein expression
were purchased from Stratagene. The protein expression strain
BAP1 described by Pfeifer et al. (30) was from Prof. Chaitan
Khosla.
Expression and Purification of Proteins—Expression of stand-
alone PT domains and full megasynthases follow identical pro-
cedures. The example of AptA-PT is detailed here. pYR94 was
used to transform the BL21(DE3) for protein expression. The
cells were cultured at 37 °C and 250 rpm in 500 ml LB medium
supplemented with 35 ␮g/ml kanamycin to a final OD600
between 0.4 and 0.6. The culture was then incubated on ice for
10 min before addition of 0.1 M isopropylthio-␤-D-galactoside
(IPTG) for protein expression. The cells were further cultured
at 18 °C for 12–16 h. The cells were then harvested by centrifu-
gation (3,500 rpm, 15 min, 4 °C), resuspended in ϳ25 ml lysis
buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 10 mM imidazole)
and lysed by sonication on ice. Cellular debris was removed by
centrifugation (15,000 rpm, 30 min, 4 °C), and Ni-NTA agarose
resin was then added to the supernatant (1–2 ml/liter of cul-
ture). The suspension was swirled at 4 °C for 2 h and loaded into
a gravity column. The protein-bound resin was washed with
one column volume of 10 mM imidazole in buffer A (50 mM
Tris-HCl, pH 7.9, 2 mM EDTA, 2 mM DTT), followed by 0.5
column volume of 20 mM imidazole in buffer A. The protein
was then eluted with 250 mM imidazole in buffer A. Purified
AptA-PT was concentrated and exchanged into buffer Aϩ10%
glycerol with the centriprep filter devices (Amicon), aliquoted
and flash frozen. Protein concentration was determined with
the Bradford assay using bovine serum albumin as a standard.
In Vitro Reconstitution of PKSH1—The reactions were per-
formed at 100 ␮l scale in 100 mM phosphate buffer (pH 7.4) in
the presence of 100 mM sodium malonate, 2 mM DTT, 7 mM
General Technique for DNA Manipulation—The A. nidulans
and A. niger genomic DNA were prepared using the ZYMO
(Orange, CA) ZR fungal/bacterial DNA kit according to sup-
plied protocols. PCR reactions were performed with the Phu-
sion high-fidelity DNA polymerase (NEB) and Platinum Pfx
DNA polymerase (Invitrogen). The PCR products are cloned
into the pCR-blunt vector obtained from Invitrogen. Restric-
tion enzymes (NEB) and T4 ligase (Invitrogen) were used to
digest and ligate the DNA fragments. The primers used to
amplify the genes were synthesized by IDT and Operon.
Phylogenetic Analysis of PT Domains—The 128 PT se-
quences were obtained from National Center for Biotechnology
Information (NCBI) through blasting the PT domains and KS
domains of PKS4, ACAS and PksA. The sequence for VrtA
from Penicillium aethiopicum was provided by Y. H. Chooi. All
of these 129 NRPKSs belong to subclades I and II as defined in
Ref. (6). PT domains of each NRPKS were carefully extracted
according to the boundary of PksA-PT (12). The PT sequences
were aligned with CLUSTALX 2.0 (31), and phylogenetic anal-
yses were conducted using MEGA version 4 (32) by means of
the boot strap minimum evolution method. The evolutionary
history was inferred using the Minimum Evolution method MgCl2, 20 mM ATP, 5 mM coenzyme A, 20 ␮M MatB, and 10 ␮M
(33). The evolutionary distances were computed using the Pois- PKSH1. The reactions were terminated with 1 ml of 99% ethyl
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Fungal Polyketide Cyclization
acetate (EA)/1% acetic acid (AcOH). The organic phase was clades I and II, subclade III NRPKSs were not included in this
separated, evaporated to dryness, redissolved in 20 ␮l of DMSO study. The subclades I and II PT sequences were aligned and
and analyzed with a Shimadzu 2010 EV Liquid Chromatogra- phylogenetically analyzed by means of the boot strap minimum
phy Mass Spectrometer by using both positive and negative evolution method (32). The resulting phylogenetic tree shown
electrospray ionization and a phenomenex Luna 5 ␮m, 2.0 ϫ in Fig. 2 clearly divides these NRPKSs into five major groups.
100 mm C18 reverse-phase column. Compounds were sepa-
Group I includes the NRPKSs that synthesize compounds
rated on a linear gradient of 5% acetonitrile (CH3CN, v/v) in containing a single aromatic ring, exemplified by the recently
water (0.1% formic acid) to 95% CH3CN in water (0.1% formic discovered orsellinic acid synthase AN7909 from A. nidulans
acid) over 30 min with a flow rate of 0.1 ml/min.
(26, 35). Group I also consists of the NRPKSs that are involved
In Vitro Characterization of the Stand-alone PT Domains— in the synthesis of the aromatic portions of the resorcylic
The reactions were performed at 100 ␮l scale in 100 mM phos- acid lactones, including the zearalenone PKS13 (36, 37), the
phate buffer (pH 7.4) in the presence of 100 mM sodium malo- hypothemycin Hpm3 (38, 39), and the radicicol RDC1/RADS2
nate, 2 mM DTT, 7 mM MgCl2, 20 mM ATP, 5 mM coenzyme A, (38, 40). Primed by different starter units, Group I NRPKSs
20 ␮M MatB, 10 ␮M PKS4 KS-MAT didomain, 50 ␮M PKS4- synthesize a tetraketide backbone and the PT domains perform
ACP, and 50 ␮M PT domain. The reactions were terminated, the regioselective C2-C7 cyclization to yield the aromatic ring.
extracted, and analyzed with the same procedure as in the in Group II contains most of the known THN synthases (THNSs).
vitro reconstitution of PKSH1.
The PT domain of this group catalyzes the cyclization of pen-
Large Scale Synthesis of 4 and NMR Characterization— taketide backbones via C2-C7 aldol condensation, followed by
To obtain a sufficient quantity of 4, the 100 ␮l in vitro assay TE/CLC catalyzed cyclization of the second ring and product
was scaled up to 100 ml. The reaction mixture was shaken release (41).
gently at room temperature and the reaction progress was
The remaining three groups contain NRPKSs that synthe-
monitored by HPLC. After ϳ48 h when the product level had size longer polyketide chains and multiply fused-ring struc-
reached a plateau, the reaction mixture was extracted three tures. Group III contains NRPKSs that cyclize first ring via
times with equal volume of EA (1% AcOH). The resultant C2-C7 regioselectivity, such as the nonaketide synthase PKS4
organic extracts were combined and evaporated to dryness, (23, 42) and the aurofusarin heptaketide synthase PKS12 from
washed with methanol, redissolved in DMSO, and purified G. zeae (43). An interesting member of the phylogenetic tree is
by reverse-phase HPLC (Alltech Apollo 5 ␮m, 250 mm ϫ 4.6 the WA synthase that synthesizes THN from A. nidulans. This
mm) on a linear gradient of 45 to 95% CH3CN (v/v) over 15 NRPKS is a member of Group III instead of Group II. This
min and 95% CH3CN (v/v) for 15 min in water (0.1% triflu- classification is consistent with previous biochemical analy-
oroacetic acid) at a flow rate of 1 ml/min. The heteronuclear sis, which showed that synthesis of THN by WA is a result of
multiple quantum coherence (HMQC) and heteronuclear chain shortening of the heptaketide product YWA1 (44).
multiple bond correlation (HMBC) spectra of 4 were per- The well-studied PksA and other NRPKSs that are involved
formed on the Bruker DRX-600 spectrometer using DMSO-d6 in the synthesis of C4-C9/C2-C11 cyclized aromatic
1
as the solvent, the H spectrum was performed on a Bruker polyketides are clearly separated into Group IV. This group
DRX-500 spectrometer, and 13C spectrum was performed on a contains other known aflatoxin synthases that are analogues of
Bruker ARX-500 spectrometer.
PksA (45, 46), as well as the heptaketide synthase cercosporin
Construction, Purification, and Reconstitution of the PKSH2— synthase CTB1 (47). Within this group, NRPKSs primed with
Primers used to amplify An03g05440-PT and PKS4-ACP are different starter units are clearly divergent in sequence, as illus-
listed in supplemental Table S1. An03g05440-PT was first fused trated by the phylogenetic distance between the acetate-primed
to PKS4-ACP through SOE-PCR to yield the DNA fragments CTB1 and the hexanoate-primed aflatoxin synthases. Lastly,
encoding PT-ACP flanked with NotI. With the same procedure Group V consists of AptA, AN0150, and ACAS, which puta-
as in constructing PKSH1, the DNA fragment of An03g05440- tively cyclize the nascent polyketide via C6-C11/C4-C13 regio-
PT-PKS4-ACP flanked by NotI was inserted into the NotI-di- selectivity. Because all members of this group lack the C ter-
gested pWJ279 to yield pYR243. pYR243 was expressed and minus TE/CLC domain, the Group V PT domains appear to be
purified with the same procedure as PKSH1. Small scale in vitro responsible for all the enzyme-catalyzed polyketide cyclization
assays of 100 ␮l were performed at room temperature and ana- reactions.
lyzed by LC-MS.
Validating the Group V PT Domain Activities—Our anal-
ysis with a limited number of PT domains indicated that
cyclization specificities can be readily correlated and classi-
RESULTS
Phylogenetic Analysis of PTs—To establish a sequence-activ- fied based on protein sequence. To validate this phylogenetic
ity relationship, we performed a phylogenetic analysis of PT classification of PT functions, we attempted to confirm the pre-
domains from NRPKSs that have been associated with known dicted but unverified C6-C11/C4-C13 cyclization regioselec-
fungal aromatic polyketides (supplemental Table S2). A previ- tivity of Group V PT domains. To do so, we hypothesized that
ous phylogenetic analysis of the KS sequences classified the PT domains can be extracted from the native megasynthase and
NRPKSs into subclades I and II that consist of PKS4 and PksA; functionally combined with a well-characterized heterologous
and a subclade III of which the PKSs all contain a MT domain minimal PKS. By analyzing the structure of the product synthe-
embedded between ACP and TE/CLC domains (6). Because of sized by the hybrid components, the regiospecificity of the
this architectural difference and phylogenetic distances to sub- unknown PT domain can be deciphered. This approach may be
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Fungal Polyketide Cyclization
FIGURE2. PhylogeneticanalysisofPTdomainsfrom30NRPKSsthathavebeenrelatedtoknownaromaticpolyketides. TheNRPKSshavebeenclassified
intofivemajorgroups:groupI, C2-C7monocyclicPKSs;groupII, THNsynthases(C2-C7bicyclicPKSs);groupIII, C2-C7multicyclicPKSs;groupIV, C4-C9PKSs;and
group V, C6-C11 PKSs. The evolutionary distances are in the units of the number of amino acid substitutions per site. The sum of branch length is 10.38. The tree
is drawn to scale (see scale bar), with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.
especially useful for NRPKSs of which the intact megasynthase synthase MatB (48) from Rhizobium trifolii to generate the
or the endogenous minimal PKS domains cannot be solubly malonyl-CoA extender units from malonate, ATP and CoA.
expressed or functionally reconstituted.
The reaction was allowed to proceed for 12 h, after which the
The G. fujikuroi PKS4 is well-suited to serve as the template organic products were extracted, dried, and analyzed by LC-MS
for PT domain swapping because (i) the megasynthases can be (Fig. 3B). As expected, the minimal PKS alone produced
expressed at high levels and the minimal PKS (KS-MAT, ACP) polyketides that are not cyclized in accordance with the
can produce ample amounts of the nonaketide products in F-modes, including 2 and 3 (supplemental Fig. S2). In contrast,
vitro, which facilitates product structure elucidation; (ii) the in the presence of AptA-PT, the spontaneous cyclization modes
minimal PKS domain does not significantly influence the cycli- were suppressed and a predominant product that corresponds
zation regioselectivity of the nascent polyketide (19). We there- to a new compound 4 (retention time, RT ϭ 25.5 min) was
Ϫ
fore anticipated that a heterologous PT could dictate the cycli- produced (Fig. 3B). Compound 4 has a parent ion peak [M-H]
zation outcome; and (iii) other built-in cyclization domain, at m/z 337, which is consistent with a nonaketide backbone that
such as the TE/CLC domain can be removed from the assembly has undergone three dehydrations and one oxidation. The
line without affecting the product elongation and turnover. nonaketide backbone of 4 was further confirmed when an
Hence, we can attribute all cyclization steps to that of the trans- increase of 9 mu was observed for 4 upon using [2-13C]ma-
planted PT domain.
lonate as the substrate of MatB (supplemental Fig. S3). The UV
We chose to study the PT domain from the asperthecin absorbance of 4 displays ␭max at 247, 315, 474 nm, which sug-
synthase AptA (AptA-PT) (25) of Group V. The gene encod- gests that this compound contains a highly conjugated chro-
ing AptA-PT was amplified from aptA in accordance with mophore (supplemental Fig. S3).
the boundaries of the crystallized PksA PT domain (12)
We next tested whether the AptA-PT domain can function-
(supplemental Fig. S1). AptA-PT was then expressed from ally replace the PKS4 PT domain in PKS4 to create a hybrid
E. coli strain BL21(DE3) and purified to a final yield of 13 megasynthase. Using the TE-less PKS4 (PKS4–99) as the tem-
mg/liter. To assay the regioselectivity, AptA-PT was com- plate and SOE PCR, we constructed PKSH1 in which the orig-
bined with the PKS4 minimal PKS. We used the malonyl-CoA inal PT domain was replaced with the AptA-PT (Fig. 3A).
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Fungal Polyketide Cyclization
FIGURE 3. A, construction and SDS-PAGE analysis of PKSH1. PKSH1 (196 kDa) is constructed through domain shuffling of PT domain between PKS4 and AptA
using PKS4–99 as a template. For SDS-PAGE: lane M, bench mark ladder; lane E, Ni-NTA-purified PKSH1. B, LC-MS (325 nm) analysis of the polyketides
synthesized by PKSH1 in vitro. Trace i, PKS4 minimal PKS alone synthesizes nonaketides 2 and 3, which are derived from spontaneous cyclization of the
backbone; trace ii, PKS4 minimal PKS domains and AptA-PT interact efficiently to afford a new nonaketide 4 as the predominant product; trace iii, parent
megasynthase PKS4–99 synthesizes 1 as a major product; and trace iv, PKSH1 synthesizes 4 as the predominant product.
PKSH1 was expressed in E. coli strain BAP1, which contains a
Elucidation of the Structure of 4—To determine the structure
chromosomally encoded copy of the broadly specific phospho- and cyclization pattern of 4, we scaled up the in vitro assay with
pantetheinyl transferase, Sfp (30). This hybrid megasynthase PKSH1 with [12C]malonate that is enriched with 10% of 1,2,3-
was soluble-expressed in BAP1, and was purified with nickel 13C-malonate. The 13C-enriched 4 was purified from the crude
affinity chromatography to near homogeneity (ϳ2 mg/liter) extract through reverse-phase HPLC and thoroughly charac-
(Fig. 3A). Interestingly, when PKSH1 was assayed for product terized by NMR spectroscopy. Seven signals were observed in
formation in vitro, a similar product profile was observed com- the 1H NMR spectrum including one methyl (␦H ϭ 2.29 ppm),
pared with the reaction mixture containing the dissociated four aromatic (␦H ϭ 6.65, 6.81, 7.16, 7.69 ppm), and two phe-
enzymatic components. Whereas PKS4–99 produced the nolic protons (␦H ϭ 12.11, 13.74 ppm). A spin system of CH
expected C2-C7 cyclized 1 as the main product, PKSH1 synthe- (␦H ϭ 6.65 ppm) -CH (␦H ϭ 7.16 ppm) was observed with a
sized 4 as the predominant product (Fig. 3B). This result indi- coupling constant of JHH ϭ 2.4 Hz. The 13C NMR spectrum
cates that while the hybrid megasynthases remain functional to (Table 1, supplemental Fig. S4) showed 18 signals, each appear-
produce polyketides, the regioselectivity of the cyclization steps ing as doublets due to isotopic enrichment. The signals include
is completely altered upon replacement of the PT domain. The those that are associated with a quinone (␦C ϭ 181.4 and ␦C ϭ
robust turnover of products by PKSH1 further confirms the 188.4 ppm). An additional carbonyl peak (␦C ϭ 158.1 ppm) was
dexterity of PKS4 toward domain engineering experiments.
observed and is indicative of a pyrone moiety. 1H-13C HMQC
JULY 23, 2010•VOLUME 285•NUMBER 30
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Fungal Polyketide Cyclization
(supplemental Fig. S4) correlations enabled the assignment of shown in Fig. 4. Importantly, this verified that AptA-PT indeed
the four hydrogen atoms to the directly bonded carbon atoms catalyzes the cyclization of polyketide backbone via C6-C11
(8-C/H, 10-C/H, 14-C/H, and 16-C/H). The coupling constants aldol condensation. Cyclization of the second ring via C4-C13 is
calculated from the 13C spectrum allowed establishment of most likely to be also facilitated by this PT domain. Closure
nine sets of neighboring carbons. Together with the remaining of the third ring via C2-C15 is likely spontaneous, which was
one-dimensional and two-dimensional NMR data, including proposed for the ACAS (29) and is commonly observed for
1H-13C HMBC (supplemental Fig. S4) correlations, the struc- most bacterial anthraquinone polyketides (49). Finally, a spon-
ture of 4 was elucidated as an ␣-pyranoanthraquinone as shown taneous C1-O17 esterification reaction takes place to form the
in Fig. 4.
␣-pyrone fourth ring and concomitant product release, fol-
Based on the structure and the [1,2-13C]acetate incorpora- lowed by second ring oxidation to yield 4 (Fig. 4). To generalize
tion pattern of 4, the cyclizations steps catalyzed by PKSH1 are this finding, we further dissected the PT domain of A. nidulans
AN0150 (26) as a second example of Group V PTs (sup-
plemental Fig. S1). When combined, PKS4 minimal PKS and
TABLE 1
AN0150-PT also synthesized 4 as the predominant product
NMR spectral data for 4 in DMSO-d6
The 1H and 13C NMR spectrum were recorded at 500 MHz, the 1H-13C HMBC and
(supplemental Fig. S5).
HMQC spectra were collected at 600 MHz.
Therefore, we have functionally verified the regioselectivity
No.
13C ␦
JCC
1H ␦
1H-13C HMBC
of Group V PT domains through heterologous recombination
with PKS4 minimal PKS, both as dissociated enzymes and as a
hybrid megasynthase. Together, the C2-C7, C4-C19, and the
C6-C11 PT domains represent the three known fungal “cycla-
ses” reconstituted so far. It is interesting to note that the AptA
PT domain functions efficiently on a nonaketide backbone,
while asperthecin was predicted to derive from an octaketide
backbone (25). Thus the PT domains may also be functional
when paired with minimal PKSs that are of different chain
length specificities.
ppm
Hz
ppm (m, area, JHH (Hz))
1
158.1
110.3a
164.8
113.7
188.4
109.3
164.5
108.5
165.5
108.8
134.9
181.1a
137.1
115.0
145.7
104.2
160.0
19.4
75.2
75.7
64.8
63.9
59.6
59.6
69.8
69.3
61.7
62.0
54.4
54.6
62.6
63.4
53.4
53.3
51.8
51.1
2
3
13.74 (s, 1H)
2, 4
4
5
6
7
12.11 (s, 1H)
6, 7, 8
6, 10
8
6.65 (d, 1H, 2.4)
9
10
11
12
13
14
15
16
17
18
7.16 (d, 1H, 2.4)
6, 8, 12
Prediction of PT Regioselectivity from NRPKS of Unknown
Function—Given that all five groups of PT functions have
been functionally verified, the phylogenetic tree may there-
fore serve as a predictive tool of product structures synthe-
sized from NRPKSs. To do so, we included 99 additional PT se-
quences extracted from NRPKSs of
7.69 (s, 1H)
6.81 (s, 1H)
2.29 (s, 3H)
2, 4, 12, 16
2, 14, 17
16, 17
a Weak signals and were observable in the 1H-13C HMBC.
unknown functions (supplemen-
tal Table S3) into the alignment.
The inclusion of these sequences
did not significantly influence the
phylogenetic classification and
most of the sequences fall into
these five groups discussed above
(supplemental Fig. S6).
To test whether the PT regio-
selectivity agrees with the phylo-
genetic classification, we chose to
functionally test a PT domain ex-
tracted from an unknown NRPKSs.
The PT domain chosen was that
of An03g05440 from A. niger (as-
signed as e_gw1_14.257 in A. niger
ATCC 1015). The An03g05440-PT
is located within group III, yet
belongs to a branch that does not
contain any functionally verified
NRPKS. The gene fragment encod-
ing the An03g05440-PT was ampli-
FIGURE 4. The biosynthesis of 4 and the 1H-13C HMBC correlations of 4. 4 is synthesized from a nonaketide
backbone synthesized by the PKS4 minimal PKS domains, followed by the AptA-PT-mediated C6-C11 and
C4-C13 cyclizations as shown in this study. Cyclization of the third ring via C2-C15 cyclization is proposed to
occur spontaneously. The proposed, spontaneous C1-O17 esterification forms the forth ring and releases the
product, followed by second ring oxidation to afford 4.
fied from the A. niger genomic DNA
and replaced pks4 PT in the
PKS4–99 template to afford the
hybrid megasynthase PKSH2. PKSH2
22770 JOURNAL OF BIOLOGICAL CHEMISTRY
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Fungal Polyketide Cyclization
between protein sequence and
cyclization regioselectivity. The five
groups cover the known cycliza-
tions modes of C2-C7, C4-C9, and
C6-C11. The PT domains that
control the C2-C7 aldol cycliza-
tions were further grouped sepa-
rately based on the ring sizes of the
final products. Through verification
of the regioselectivities of the Group
V PT domains, we established a
straightforward experimental ap-
proach to assay the function of PT
domains. The assay is based on het-
FIGURE 5. A, construction and SDS-PAGE analysis of PKSH2. PKSH2 (193 kDa) is constructed through domain
shuffling of PT domain between PKS4 and An03g05440 using PKS4–99 as a template. For SDS-PAGE: lane M,
bench mark ladder; lane E,, Ni-NTA-purified PKSH2. B, LC-MS (325 nm) analysis of the polyketides synthesized
by PKSH2 in vitro. Trace i, the parent megasynthase PKS4–99 synthesizes 1 as a major product; and trace ii,
PKSH2 also synthesizes 1 as the major product, confirming the phylogenetic prediction of An03g05440-PT as erologous combinations of target
a C2-C7 aldol cyclase.
PT domains with an NRPKS mini-
mal PKS. Combining the bioinfor-
matic and the simple biochemical approaches, we were able
to predict and confirm the activities of two NRPKS PT
domains uncovered from genome sequencing. Similarly,
fundamental understanding of sequence-activity relation-
ships of domains that control chain length, such as the KS,
can provide additional insights into the structures of
polyketides produced by cryptic NRPKSs.
was similarly expressed in BAP1, and purified to a final yield of
ϳ1.5 mg/liter (Fig. 5A). PKSH2 was assayed in vitro and the
products were analyzed with LC-MS (Fig. 5B). Similar to
PKS4–99, PKSH2 synthesized 1 as the major product with sim-
ilar yields. This confirms An03g05440-PT indeed directs the
cyclization of the nonaketide backbone via C2-C7 regioselec-
tivity and suggests that the parent NRPKS is responsible for
the synthesis of a product that contains this structural fea-
ture. In accordance with the recent review on the secondary
metabolites synthesized by A. niger (50), the C2-C7
An03g05440 may be involved in the synthesis of the YWA1
analogues such as fonsecin, rubrofusarin, or the dimeric auras-
perone B.
The assay developed here is highly robust and the dominant
polyketide produced reflects the regioselectivity of the target
PT domains. The heterologous PT domains were able to inter-
act compatibly with the PKS4 ACP domain, illustrating this key
protein-protein interaction is not specific between endogenous
partners. The mix and match of minimal PKS components with
different PT domains are reminiscent of the combinatorial bio-
synthesis experiments performed with the bacterial type II
PKSs and cyclases (49, 54). In these studies, the cyclases were
found to be broadly specific and can be combined with different
type II minimal PKSs to generate a number of regioselectively
controlled unnatural aromatic polyketides. Our work here indi-
cates that this approach can also be extended to fungal iterative
type I PKSs to synthesize different compounds cyclized via the
F-modes.
Viridicatumtoxin is a tetracycline-like fungal aromatic
polyketide (supplemental Fig. S7). Recently, the gene cluster has
been located in P. aethiopicum and the NRPKS involved was
shown to be vrtA (51). Previous 13C enrichment study of viridi-
catumotoxin led to two possible cyclization pathways (52),
which can originate from either C6-C11 or C8-C13 first ring
cyclization (supplemental Fig. S7). Our phylogenetic analysis
finds VrtA PT domain is located in Group V (supplemen-
tal Fig. S6). To clarify the regioselectivity of VrtA, we dissected
VrtA-PT (supplemental Fig. S1) and assayed it with PKS4 min-
imal PKS domains in trans. As expected from phylogenetic pre-
diction, 4 was synthesized as a major product (supplemen-
tal Fig. S7). This result indicates that the formation of the
viridicatumotoxin aglycon initiates with the C6-C11 regio-
selective cyclization.
The construction of functional, hybrid NRPKSs demon-
strates modularity of the domains in fungal PKSs, in that key
domains can be readily swapped without major compromises
to megasynthase structure, activity, and processivity. Such
modularity provides further evidence that NRPKS domains
may be evolutionarily derived from the juxtaposition of disso-
ciated catalytic domains. The dimeric PT domains likely play a
key role in maintaining the overall structural integrity of the
DISCUSSION
With an increasing number of sequenced genomes from fil- megasynthases. Our results therefore suggest the overall folds
amentous fungi, it has become apparent that these organisms of the different PT domains from different groups may be sim-
are “underachievers” as natural product producers. A majority ilar, thereby maintaining the endogenous communication and
of sequenced biosynthetic gene clusters are silent under labo- protein-protein interactions between the upstream KS-AT
ratory conditions and as a result, the metabolites associated didomain and the downstream ACP domain. We compared
with these pathways have remained cryptic. Therefore, being the sequences of Group V PT domains to those from the other
able to predict structures or partial structures of compounds groups and found the sequential similarities between different
based on sequence analysis of pathways is an important goal groups are moderate. Group V PT domains show no more than
toward mining the fungal biosynthetic potential (53). In this 30% identities and 40% similarities to members of the other
work, we used phylogenic analysis of the PT domains to classify groups. For example, AptA-PT and PksA-PT share only 19%
fungal NRPKSs into five groups and showed a correlation identity and 33% similarity, even though the final products are
JULY 23, 2010•VOLUME 285•NUMBER 30
JOURNAL OF BIOLOGICAL CHEMISTRY 22771

Fungal Polyketide Cyclization
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Structural analysis of C2-C7 and C6-C11 PT domains, and
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Whereas using dissociated catalytic domains can provide a
rapid method of assaying PT activities, using hybrid megasyn-
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combinatorial biosynthesis applications. First, the simplicity of
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JULY 23, 2010•VOLUME 285•NUMBER 30
JOURNAL OF BIOLOGICAL CHEMISTRY 22773

Enzymology:
Classification, Prediction, and Verification
of the Regioselectivity of Fungal Polyketide
Synthase Product Template Domains
Yanran Li, Wei Xu and Yi Tang
J. Biol. Chem. 2010, 285:22764-22773.
doi: 10.1074/jbc.M110.128504 originally published online May 17, 2010
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