Polyketide synthase chemistry does not
direct biosynthetic divergence between
9- and 10-membered enediynes
Geoff P. Horsmana,1, Yihua Chena,1, Jon S. Thorsona,b, and Ben Shena,b,c,2
a
Division of Pharmaceutical Sciences, School of Pharmacy, bNational Cooperative Drug Discovery Group, and cDepartment of Chemistry, University of
Wisconsin, Madison, WI 53705
Enediynes are potent antitumor antibiotics that are classified as
9- or 10-membered according to the size of the enediyne core
structure. However, almost nothing is known about enediyne core
biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work
identified enediyne-specific polyketide synthases (PKSEs) that can
be phylogenetically distinguished as being involved in 9- versus
10-membered enediyne biosynthesis, suggesting that biosynthetic
divergence might originate from differing PKSE chemistries. Recent
in vitro studies have identified several compounds produced by the
PKSE and associated thioesterase (TE), but condition-dependent
product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered
enediyne core biosynthetic divergence as revealed by comparing
the products from three 9-membered and two 10-membered
PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic
function for 9- and 10-membered PKSEs by the production of a
heptaene metabolite from: (i) all five cognate PKSE-TE pairs in
Escherichia coli; (ii) the C-1027 and calicheamicin cognate PKSETEs in Streptomyces lividans K4-114; and (iii) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs
and TEs from different 9- and 10-membered enediyne biosynthetic
machineries are freely interchangeable, revealing that 9- versus
10-membered enediyne core biosynthetic divergence occurs
beyond the PKSE-TE level. These findings establish a starting point
for determining the origins of this biosynthetic divergence.
biosynthesis ∣ C-1027 ∣ thioesterase
E
nediyne natural products are some of the most potent antitumor antibiotics discovered (1), and neocarzinostatin (NCS)
and calicheamicin (CAL) enjoy clinical success in Japan (2)
and the United States (3), respectively. While their unique
mechanism of action (4, 5) and extraordinary biological activity
generate interest among clinicians, the unusual molecular architecture of the enediynes motivates elucidation of the biosynthetic
logic directing their assembly. Most structurally remarkable is
the enediyne core, which is composed of two acetylenic groups
conjugated by a double bond within either a 9- or 10-membered
ring (Fig. 1A). Enediyne biosynthesis has been studied most
extensively for the 9-membered enediyne C-1027, revealing a
convergent process in which several peripheral moieties are
appended to the enediyne core (6). Indeed, recent elucidation
of peripheral pathway biochemistry has enabled rational design
of novel enediyne analogs possessing altered biological activities
(7, 8). However, despite rapid progress toward understanding
peripheral moiety construction, the biosynthesis of the enediyne
core structure remains a mystery.
The enediynes are classified according to the enediyne core
ring size as either 9- or 10-membered, and how these two classes
biosynthetically diverge is an intriguing question. Several biosynthetic gene clusters for both 9- and 10-membered enediynes have
www.pnas.org/cgi/doi/10.1073/pnas.1003442107
been sequenced, as exemplified by C-1027 (9), NCS (10), and
maduropeptin (MDP) (11) for the 9-membered enediynes and
CAL (12) and dynemicin (DYN) (13) for the 10-membered
enediynes, respectively (Fig. 1A). Common to all clusters is a five
gene cassette (Fig. 1B, black) that encodes a unique enediynespecific iterative type I polyketide synthase (PKSE), a thioesterase (TE), and three additional genes of unknown function. The
PKSE generates an enediyne core precursor from iterative
condensation of malonyl-CoA (9); the high sequence identities
(41–68%, Table S1) and identical domain organization among
PKSEs (Fig. S1) suggest a common catalytic function and product. These data have inspired speculation that PKSEs from both
classes generate an early common intermediate, which is directed
to divergent fates by the action of 9- or 10-membered-specific
accessory enzymes (Fig. 2A) (14). This hypothesis is consistent
with the identification of 6–7 additional genes adjacent to the
core cassette that are common only among the 9-membered gene
clusters (Fig. 1B, gray). However, phylogenetic analysis of PKSEs
reveals that they group into two clades according to enediyne core
ring size. This ability to distinguish 9- versus 10-membered PKSEs
led to a predictive familial classification model for new enediyneencoding genes (14) and suggests that a possible catalytic difference between the two PKSE families may be responsible for
generating the respective core structures.
Indeed, recent evidence for a functional distinction between
9- and 10-membered PKSEs is consistent with PKSE-directed
biosynthetic divergence of the two enediyne families (Fig. 2B).
For instance, we previously reported that NcsE and SgcE—the
PKSEs involved in the biosynthesis of the 9-membered enediynes
NCS and C-1027, respectively—produced heptaene (1) when coexpressed with their associated TEs, NcsE10 and SgcE10 (15, 16).
In contrast, Liang and coworkers observed methyl hexaenone (2)
as the major product of the in vitro combination of CalE8 and
CalE7, the PKSE-TE pair from the 10-membered enediyne
CAL (17). This apparently inefficient keto-reduction, or “ketoreductase (KR) skipping,” by CalE8 in the final round of iterative
chain extension was therefore proposed as the first point of
biosynthetic divergence between 9- and 10-membered enediyne
cores (Fig. 2B).
However, subsequent reports using in vitro methods to decipher functional differences between 9- and 10-membered PKSEs
reveal more complex behavior. For example, Townsend and coworkers detected 1, 2, and several truncated polyketides from in
Author contributions: G.P.H., Y.C., and B.S. designed research; G.P.H. and Y.C. performed
research; G.P.H., Y.C., and B.S. analyzed data; J.T. contributed new reagents/analytic tools;
and G.P.H., Y.C., and B.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
G.P.H. and Y.C. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail: bshen@pharmacy.wisc.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1003442107/-/DCSupplemental.
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Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved May 17, 2010 (received for review March 16, 2010)
Fig. 1. Selected 9- and 10-membered enediynes whose biosynthetic gene clusters have been sequenced. (A) Chemical structures of the 9- (C-1027, NCS, and
MDP) and 10-membered (CAL, DYN, and ESP) enediynes with the enediyne core structures shown in bold. (B) Regions of enediyne gene clusters encoding
enediyne core-biosynthesis with genes common among both 9- and 10-membered enediynes shown in black and genes common only among the 9-membered
enediynes shown in gray. The pksE genes for 9- and 10-membered systems are named E and E8, respectively, while TE genes are named E10 and E7, respectively.
vitro analysis of CalE8 and CalE7, indicating partial KR function
in the final round of chain extension by CalE8 (dashed line in
Fig. 2B). The variety of products was interpreted to be a consequence of PKSE catalysis occurring in the absence of required
accessory proteins and suggested that the apparent catalytic difference between 9- and 10-membered PKSEs may be an artifact
of assay conditions (18). Indeed, by optimizing in vitro reaction
conditions, Liang and coworkers variably observed 1, 2, and an
unknown compound as major products from 10-membered
(CAL and DYN) and 9-membered (C-1027) PKSE-TE systems
(19). Although the assay could be modified to yield 1 as the major
product from each system, the condition-dependent product profiles of in vitro PKSE-TE systems caution against interpreting a
common catalytic function for all PKSEs and motivate further
study using more reproducible methods.
The difficulties with in vitro experimental systems for PKSE
studies contrast sharply with our previously reported in vivo system, in which expression of pksE-TE from either the C-1027 or
NCS biosynthetic machineries in either Escherichia coli or Streptomyces consistently provided 1 as the only major product (15).
The apparent advantage of in vivo methods may reflect the difficulty of reconstituting the large multifunctional PKSE megaenzyme in vitro in the absence of the mild and regenerative
conditions of the cell in vivo. We therefore elected to extend
the use of in vivo systems to compare 9- and 10-membered PKSEs
with the intent of more faithfully reproducing the conditions presented to the biosynthetic machinery in the native producers.
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We herein report that PKSE chemistry does not direct 9- versus
10-membered enediyne core biosynthetic divergence (Fig. 2A)
by comparing the products from three 9- (C-1027, NCS, MDP)
and two 10-membered (CAL, DYN) PKSE-TE systems under
identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9and 10-membered PKSEs by the production of 1 from: (i) all five
cognate PKSE-TE pairs in E. coli; (ii) the SgcE-SgcE10 and
CalE8-CalE7 cognate pairs in Streptomyces lividans K4-114;
and (iii) selected native producers of both 9- and 10-membered
enediynes. Finally, as a first step toward investigating the possible
role of protein–protein interactions in directing biosynthetic divergence between 9- and 10-membered enediynes, we demonstrated no pathway-specific fidelity of the PKSE-TE interaction.
These findings set the stage to further search for the origins of
biosynthetic divergence between 9- and 10-membered enediynes.
Results and Discussion
The first step toward comparing 9- and 10-membered PKSEs was
to prepare compatible E. coli constructs for coexpressing pksE and
TE from each of the five sequenced enediyne gene clusters: C1027 (sgcE-sgcE10), NCS (ncsE-ncsE10), MDP (mdpE-mdpE10),
CAL (calE8-calE7), and DYN (dynE8-dynE7) (Table S2). Coexpression of each cognate pair in a 50 mL E. coli culture and HPLC
analysis of the concentrated acetone extract revealed that 1 was
the only major product from each of the five PKSE-TE systems
(Fig. 3A, I–V). This compound was readily identified to be 1
Horsman et al.
by its distinctive absorption spectrum (Fig. S2) and expected mass
of 198.1 and confirmed to be identical to the previously characterized authentic standard (15). Productions of 1 by either the
9- or 10-membered PKSE-TE systems under the conditions
examined are very similar as exemplified by the estimated yields
of 1 at 1.3 0.5 mg∕L for CalE8-CalE7 and 2.8 1.0 mg∕L for
MdpE-MdpE10, respectively. Production was abolished when:
(i) the TE was excluded (Fig. 3A, VI) or (ii) a ketosynthase-inactivated SgcE-C211A enzyme (15) was used (SgcE KS°, Fig. 3A,
VII), showing that production is both TE- and PKSE-dependent.
Taken together, the in vivo data demonstrates a common catalytic
function for all PKSEs, implying that 9- versus 10-membered
enediyne pathway divergence is dictated by accessory enzymes,
as previously proposed (14, 18).
Having successfully established a common chemistry for 9- and
10-membered PKSE-TE systems in E. coli, we then showed that 1
is also produced by PKSE-TE expression in Streptomyces lividans
K4-114 (20), a host more closely related to the enediyne native
producers. Due to the condition-dependent product profile
observed using in vitro systems (17–19, 21), we reasoned that
the in vivo PKSE-TE chemistry may also be host-dependent,
and we therefore sought to examine production in a host other
than E. coli. Cognate PKSE-TE constructs for C-1027 (sgcEsgcE10) and CAL (calE8-calE7) were prepared (Table S2), and
expression of each construct in S. lividans K4-114 yielded 1
(Fig. 3B, I and II). This result further demonstrates a common
chemistry catalyzed by both 9- and 10-membered PKSE-TE pairs
in a host possessing cellular conditions that are presumably more
representative of the native producer strains.
Finally, a common PKSE-TE chemistry was ultimately verified
by the production of 1 in selected native producers of both 9- and
Horsman et al.
10-membered enediynes under enediyne-producing conditions.
Although 1 is produced from pksE-TE expression in both E. coli
and S. lividans, we could not definitively exclude the possibility
that production of 1 was an artifact of our simple two-enzyme
system in a heterologous host. We therefore carefully examined
the fermentation products of the native C-1027 producer S. globisporus C-1027. Strikingly, 1 is produced in S. globisporus at a
high level (∼14 mg∕L) (Fig. 3C, I). Production of 1 was shown
to be sgcE-dependent—it was abolished in a ΔsgcE strain of
S. globisporus C-1027, SB1005 (9) (Fig. 3C, II), and restored
by complementation of SB1005 with a functional copy of the sgcE
gene (Fig. 3C, III). Moreover, 1 was also produced independently
of C-1027, as demonstrated by its production in S. globisporus
SB1010, a strain unable to produce C-1027 due to an inactivated
epoxide hydrolase gene sgcF (Fig. 3C, IV and Fig. S3) (22).
Inspired by these findings, we examined another 9-membered enediyne producer Streptomyces carzinostaticus ATCC 15944 (NCS
producer) and two 10-membered enediyne native producers
Micromonospora echinospora ssp. calichensis (CAL producer)
and Actinomadura verrucosospora ATCC 39334 (esperamicin
(ESP) producer); all strains tested produced 1 (Fig. 3C, V–VII)
under enediyne-producing conditions (Fig. S3). This fascinating
observation further demonstrates that PKSEs from 9- and
10-membered systems utilize the same carbon chain extension
chemistry en route to different enediyne core structures.
Having established a common catalytic function for 9- and 10membered PKSE classes, we reasoned that pathway divergence
might involve protein–protein interactions between the PKSE
and pathway-specific accessory enzymes. As a first step in this
direction, we sought to probe the pathway-specific fidelity of
the PKSE-TE interaction, motivated in part by the observation
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Fig. 2. Two proposed origins of biosynthetic divergence between 9- and 10-membered enediynes. (A) Common PKSE chemistry based on current
in vivo studies: All PKSEs generate a common intermediate, which is processed by respective pathway-specific enzymes to furnish different core structures.
(B) Divergent PKSE chemistry based on previous in vitro studies: The phylogenetically related class of 9-membered PKSEs generate 1 via a β-hydroxy thioester,
while the 10-membered PKSEs may stall at the final round of iterative keto-reduction to yield 2 as a major product.
Fig. 3. HPLC chromatograms with UV detection at 370 nm showing production of 1 from selected: (A) recombinant E. coli strains coexpressing cognate pksE-TE
pairs; (B) recombinant S. lividans K4-114 strains coexpressing cognate and mismatched pksE-TE pairs; (C) native enediyne producers (I) S. globisporus C-1027,
(II) ΔsgcE mutant SB1005, (III) SB1011 (SB1005 complemented by expressing sgcE in trans), (IV) ΔsgcF mutant SB1010, (V) S. neocarzinostaticus ATCC 15944,
(VI) M. echinospora ssp. calichensis, and (VII) A. verrucosospora ATCC 39334; and (D) recombinant E. coli strains coexpressing mismatched pksE-TE pairs.
that both PKSEs (14) and TEs appear to phylogenetically cluster
according to enediyne structural class (Fig. S4). Although Liang
and coworkers showed that SgcE10 could substitute for CalE7 to
similarly liberate 2 from CalE8 (21), the combinatorial flexibility
afforded by our five PKSE and five TE constructs enables a more
thorough examination of pathway-specific effects by the convenient coexpression of any PKSE-TE combination. Significantly, all
five TEs were freely interchangeable with all five PKSEs to yield 1
from all 20 possible mismatched PKSE-TE combinations in
E. coli (Fig. S2), as exemplified by representative chromatograms
in Fig. 3D. We then verified this result in a different host,
S. lividans K4-114 (20), to ensure that any pathway-specific effects
were not host-dependent. Specifically, we prepared constructs of
sgcE-calE7 and calE8-sgcE10 (Table S2) for expression in S. lividans K4-114 and observed the production of 1 in both cases
(Fig. 3B, III and IV). The interchangeability of TEs between 9and 10-membered PKSE-TE systems clearly rules out the possibility that pathway-specific divergence originates from the PKSE-TE
interaction and further supports a unified view of PKSE catalysis.
In summary, we have presented definitive evidence that PKSE
chemistry does not direct 9- versus 10-membered enediyne core
divergence. We instead envision a universal PKSE-bound intermediate that is modified by pathway-specific accessory enzymes
en route to formation of the 9- or 10-membered enediyne core
structures (Fig. 2A). Thus, the phylogenetic divergence between
9- and 10-membered PKSEs may reflect enediyne core-specific
protein-protein interactions between the PKSE and corresponding accessory enzymes. This hypothesis is consistent with our ear11334 ∣
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lier observation that only the 9-membered pksE genes, but not
calE8, could restore C-1027 or NCS production in pksE null
mutants (15). As an important first step toward elucidating pathway-directing interactions between PKSEs and accessory enzymes,
we demonstrated the production of 1 by all possible PKSE-TE
combinations. This PKSE-TE promiscuity establishes that biosynthetic divergence between 9- and 10-membered enediyne cores
requires more than just these two enzymes. Our findings now provide a starting point from which to search for the origins of this
divergence.
The present study raises additional questions and possibilities
with respect to the reproducible production of 1 from a wide variety of host systems. The high yields from the native producers
raises interesting questions regarding the biological relevance
of this compound. For example, S. globisporus C-1027 produces
∼14 mg∕L of 1 and ∼1 mg∕L of C-1027 chromophore (9), which
corresponds to successful C-1027 production in only about 1 of
every 70 enzyme turnovers. Finally, the apparent ubiquity of 1 in
all enediyne producers provides a convenient phenotypic screen
to identify new enediyne-producing organisms, complementing
the genotypic screenings we have reported previously (14, 23).
Methods
General. Oligonucleotide synthesis and DNA sequencing was performed by
the University of Wisconsin Biotechnology Center. PCR was performed using
Pfx polymerase (Invitrogen, Carlsbad, CA), and 3′ A-overhangs were added
with Taq polymerase from Invitrogen for 10 min at 72 °C prior to subcloning
into the pGEM-T Easy vector from Promega (Madison, WI). Mass spectrometry
Horsman et al.
Coexpression of pksE-TE Constructs in E. coli. Various combinations of pksEand TE-containing plasmids (Table S2) were cotransformed into E. coli
BL21(DE3) and plated on LB agar plates containing the appropriate combination of two antibiotics for selection. Single colonies were picked and
grown overnight at 37 °C in LB and then transferred to 50 mL LB cultures
supplemented with the appropriate antibiotics. The cultures were grown
at 37 °C until the optical density at 600 nm (OD600 ) reached ∼0.2, at which
point the cultures were transferred to 18 °C incubators until they reached
OD600 ∼ 0.4 and then supplemented with 0.1 mM IPTG to induce gene expression. The cultures were then incubated for an additional 2 d prior to harvest.
Expression of pksE-TE Constructs in S. lividans K4-114. Various combinations of
pksE- and TE-containing plasmids (Table S2) were introduced into S. lividans
K4-114 by protoplast transformation according to standard protocols (24).
Single colonies of the resultant recombinant strains were used to inoculate
5 mL cultures of MYME media supplemented with 10 μg∕mL thiostrepton
and incubated at 30 °C for ∼5 days, and 1 mL of each starter culture was then
used to inoculate a 50 mL culture, which was in turn incubated at 30 °C
for ∼5–10 days.
CAL (25), and ESP (26) production, respectively. After 3 to 5 d fermentation,
the culture was centrifuged to collect the cell pellet. The supernatant was
adjusted to pH 4.0 using 1 M HCl and centrifuged again. The resulting precipitate was combined with the cell pellet and extracted with acetone. The
acetone extracts were subjected to HPLC as described below. Bioassays
against Micrococcus luteus ATCC 9431 to detect enediyne production were
performed as described previously (Fig. S3) (27).
Extraction and HPLC Analysis of Heptaene (1) from Recombinant Strains Coexpressing pksE-TE. Each 50 mL culture of the E. coli or S. lividans recombinant
strains coexpressing pksE-TE was acidified to pH ∼ 3 and centrifuged to pellet
the cells. The cell pellet was then resuspended in 20 mL acetone by vortexing.
The acetone extract was centrifuged to pellet the cell debris, and the supernatant was rotary evaporated to a volume of 1 mL, of which 100 μL was
subjected to HPLC analysis. HPLC was carried out on a Varian HPLC system
equipped with Prostar 210 pumps, a photodiode array detector, and an Alltech Alltima C18 column (5 μm, 4.6 × 250 mm, Grace Davison Discovery
Sciences, Deerfield, IL) using mobile phases A (water, 0.1% trifluoroacetic
acid) and B (methanol, 0.1% trifluoroacetic acid). A gradient elution program
was employed as follows: 60–100% B (0–15 min), 100% B (15–35 min),
100–60% B (35–35.5 min), 60% B (35.5–40 min). The identity of 1 produced
from the recombinant strains or the native enediyne producers was established by comparing its UV-Vis spectrum, mass spectrum, and HPLC retention
time with a previously characterized authentic standard of 1 (15).
Production, Extraction, and HPLC Analysis of Heptaene (1) from Native Enediyne
Producers. S. globisporus strains were cultured in A9 medium using a
two-step fermentation for C-1027 production as described previously (22).
S. carzinostaticus ATCC 15944, M. echinospora ssp. calichensis, and A. verrucosospora ATCC 39334 were cultured as previously described for NCS (10),
ACKNOWLEDGMENTS. We thank Dr. Y. Li, Institute of Medicinal Biotechnology,
Chinese Academy of Medical Sciences (Beijing), for the wild-type S. globisporus, and the Department of Chemistry, University of Wisconsin, Madison,
for support in obtaining the MS data. This work was supported by the
National Institutes of Health Grants CA78747 and CA113297 (to B.S.) and
CA84374 (to J.S.T.). G.P.H. is a Natural Sciences and Engineering Research
Council of Canada postdoctoral fellow.
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was performed using electron impact ionization on a Waters (Micromass)
AutoSpec mass spectrometer (Milford, MA). Strains, plasmids, primers,
detailed procedures for making the pksE and TE expression constructs, sequence comparison and phylogenetic analysis of PKSEs and TEs, and HPLC
analyses of 1 and bioassays for enediyne production are summarized in
Tables S1, S2, and S3 and Figs. S1, S2, S3, and S4 and provided in the online
supporting information.