Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic enzyme into echinomycin biosynthetic pathway for rational design and synthesis of potent antibiotic unnatural natural product

Article abstract of DOI:10.1021/ja902261a

Natural products display impressive activities against a wide range of targets, including viruses, microbes, and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable toxicity. Not only can engineering Escherichia co

Full text of DOI:10.1021/ja902261a

Published on Web 06/10/2009
Escherichia coli Allows Efficient Modular Incorporation of
Newly Isolated Quinomycin Biosynthetic Enzyme into
Echinomycin Biosynthetic Pathway for Rational Design and
Synthesis of Potent Antibiotic Unnatural Natural Product
Kenji Watanabe,*,† Kinya Hotta,‡ Mino Nakaya,§ Alex P. Praseuth,|
Clay C. C. Wang,⊥ Daiki Inada,§ Kosaku Takahashi,# Eri Fukushi,∇ Hiroki Oguri,§,O
and Hideaki Oikawa*,§
Research Core for Interdisciplinary Sciences, Okayama UniVersity, Okayama 700-8530, Japan,
Department of Biological Sciences, National UniVersity of Singapore, Singapore 117543, Singapore,
DiVision of Chemistry, Graduate School of Science, Hokkaido UniVersity, Sapporo 060-0810, Japan,
BioPharmaceuticals Formulation DeVelopment, Allergan, Inc., 2525 Dupont DriVe, IrVine,
California 92612, Department of Pharmacology and Pharmaceutical Sciences, UniVersity of Southern
California, Los Angeles, California 90033, DiVision of Applied Bioscience, Graduate School of
Agriculture, Hokkaido UniVersity, Sapporo 060-8589, Japan, The Analytical Core facility of NMR
and Mass spectrometry, School of Agriculture, Hokkaido UniVersity, Sapporo 060-8589, Japan, and
CreatiVe Research InitiatiVe ‘Sousei’ (CRIS), Hokkaido UniVersity, Sapporo 001-0021, Japan
Received March 23, 2009; E-mail: kenji55@cc.okayama-u.ac.jp; hoik@sci.hokudai.ac.jp
Abstract: Natural products display impressive activities against a wide range of targets, including viruses,
microbes, and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable
toxicity. Not only can engineering Escherichia coli for plasmid-based pharmacophore biosynthesis offer
alternative means of simple and easily scalable production of valuable yet hard-to-obtain compounds, but
also carries a potential for providing a straightforward and efficient means of preparing natural product
analogs. The quinomycin family of nonribosomal peptides, including echinomycin, triostin A, and SW-
163s, are important secondary metabolites imparting antibiotic antitumor activity via DNA bisintercalation.
Previously we have shown the production of echinomycin and triostin A in E. coli using our convenient and
modular plasmid system to introduce these heterologous biosynthetic pathways into E. coli. However, we
have yet to develop a novel biosynthetic pathway capable of producing bioactive unnatural natural products
in E. coli. Here we report an identification of a new gene cluster responsible for the biosynthesis of SW-
163s that involves previously unknown biosynthesis of (+)-(1S, 2S)-norcoronamic acid and generation of
aliphatic side chains of various sizes via iterative methylation of an unactivated carbon center. Substituting
an echinomycin biosynthetic gene with a gene from the newly identified SW-163 biosynthetic gene cluster,
we were able to rationally re-engineer the plasmid-based echinomycin biosynthetic pathway for the
production of a novel bioactive compound in E. coli.
Introduction
different organisms through recent genome and metagenome
sequencing efforts.1 However, many organisms are difficult to
With the growing global threat of multidrug-resistant mi-
crobes and increasing economic and environmental concerns
on synthetic production of natural products and their analogs,
there is an increasing interest in developing alternate platforms
for the discovery, production and development of pharmaceuti-
cally valuable compounds. To date, many biosynthetic gene
clusters encoding polyketide synthases (PKSs), nonribosomal
peptide (NRP) synthetases (NRPSs), mixed PKS-NRPSs and
associated auxiliary enzymes have been found in various
culture in large scale,2 and some are impractical to handle due
to pathogenicity. Others produce natural products at very low
level or maintain their gene clusters silent under conventional
culture conditions.3 One way to exploit the potential of these
organisms and the genomic information for drug discovery and
development is to establish a heterologous production system
using a convenient host organism capable of expressing the
exogenous genes and biosynthesizing the desired compounds.
Several organisms, including certain Streptomyces strains and
yeast, have been used as such hosts,4 but Escherichia coli carries
† Okayama University.
‡ National University of Singapore.
§ Graduate School of Science, Hokkaido University.
| Allergan, Inc.
(1) Bode, H. B.; Muller, R. Angew. Chem., Int. Ed. 2005, 44, 6828–46.
(2) Riesenfeld, C. S.; Schloss, P. D.; Handelsman, J. Annu. ReV. Genet.
2004, 38, 525–52.
⊥ University of Southern California.
# Graduate School of Agriculture, Hokkaido University.
∇ School of Agriculture, Hokkaido University.
O Creative Research Initiative ‘Sousei’ (CRIS), Hokkaido University.
(3) Wenzel, S. C.; Mu¨ller, R. Curr. Opin. Biotechnol. 2005, 16, 594–
606.
(4) Zhang, H.; Wang, Y.; Pfeifer, B. A. Mol. Pharm. 2008, 5, 212–25.
9
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A R T I C L E S
Watanabe et al.
certain advantages over others. First, a wealth of tools are
available for incorporation, controlling expression, and modi-
fication of foreign genes in E. coli, such as plasmids with various
useful properties, including plasmid compatibility and select-
ability. Additionally, methods of manipulating E. coli and its
vectors are well-established and straightforward to perform.
Furthermore, E. coli is forgiving in expressing exogenous genes
and readily grows with a very short doubling time in a simple,
inexpensive growth medium without requiring particularly
difficult-to-obtain culture conditions. Although E. coli is not
known for its ability to produce diverse secondary metabolites,
the fact that E. coli is naturally capable of producing siderophore
NRPs5 and a PK-NRP hybrid molecule colibactin6 suggests that
its metabolic system is not entirely foreign to natural product
biosynthesis. To introduce a heterologous biosynthetic pathway
into E. coli, we chose a plasmid-based system. In this approach,
plasmids are used to assemble and deliver genes necessary to
reconstitute the target biosynthetic pathway into the host.
Although this approach demands initial efforts of constructing
plasmids bearing multiple genes of the target biosynthetic
pathway, we have lowered the hurdle significantly by developing
a cloning method that exploits iterative ligation of DNA
fragments bearing compatible cohesive-ends. This led to the
first successful production of a bioactive NRP natural product
echinomycin 1 and triostin A 2 in E. coli (Figure 1).7
In parallel with the development of the E. coli-based system
for heterologous production of secondary metabolites, we are
also trying to identify new biosynthetic pathways that give rise
to unique chemical structures found among natural products.
Such knowledge is crucial in broadening the scope of combi-
natorial biosynthesis of various natural products and their
analogs. Discovery of new enzymes that are capable of
performing different transformations permits expansion of the
toolbox with which we can devise novel biosynthetic pathways
for production of interesting new compounds. Combined with
the advantages of using E. coli as a heterologous production
host described earlier, E. coli-based plasmid-borne heterologous
secondary metabolite biosynthetic system can not only facilitate
the reconstitution of various biosynthetic gene clusters but also
provide a flexible platform suitable for fast and simple rational
redesigning of biosynthetic pathways through modular reas-
sembling of the biosynthetic genes from various biosynthetic
systems. Here, to put this concept to work, we have identified
a new biosynthetic gene cluster responsible for the biosynthesis
of the SW-163 series of NRPs involving several previously
uncharacterized enzymatic transformations. Then we took this
new knowledge and applied it to our previously established E.
coli-based plasmid-borne NRP biosynthetic system to achieve
a rationally engineered biosynthesis of unnatural bioactive NRP
we named ecolimycin C, a hybrid compound of 2 and SW-163
C 3.
Figure 1. Chemical structures of quinomycin antibiotics.
and its intermediate 2 in E. coli.7 Originally identified and
isolated from Streptomyces strains, both compounds were
categorized as quinomycin antibiotics because of their quinoxa-
line or quinoline chromophores attached to the C2-symmetric
bicyclic depsipeptide core.8 This family of antibiotics is well-
characterized, and exhibits potent antibacterial, anticancer and
antiviral activities.9 While both compounds bis-intercalate into
GC-rich DNA sequences, 1 and 2 exhibit distinct sequence
preference. Now we have isolated an additional family of
quinomycin antibiotics, SW-163C 3, D 4, E 5, F 6 and G 710,11
from Streptomyces sp. SNA15896 (Figure 1). All of them carry
a pair of 3-hydroxyquinaldic acid (HQA, 8) chromophores along
with a nonproteinogenic amino acid, norcoronamic acid (NCA),
whose biosynthetic mechanism has not been described to date.
The absolute configuration of 4, and hence the SW-163s, was
determined by establishing the absolute configuration of the
NCA residue to be (+)-(1S, 2S)-norcoronamic acid [(+)-NCA,
9].11 Also, like in 1, compounds 4, 5, 6 and 7 all possess a
Results
Isolation of SW-163 Biosynthetic Gene Cluster. Previously,
we reported the total biosynthesis of two antitumor NRPs, 1
(5) Valdebenito, M.; Crumbliss, A. L.; Winkelmann, G.; Hantke, K. Int.
J. Med. Microbiol 2006, 296, 513–20.
(8) Martin, D. G.; Mizsak, S. A.; Biles, C.; Stewart, J. C.; Bacynsky, L.;
Meulman, P. A. J. Antibiot. (Tokyo) 1975, 28, 332–6.
(9) Sieber, S. A.; Marahiel, M. A. Chem. ReV. 2005, 105, 715–38.
(10) Kurosawa, K.; Takahashi, K.; Tsuda, E. J. Antibiot. (Tokyo) 2001,
54, 615–21.
(6) Nougayre`de, J. P.; Homburg, S.; Taieb, F.; Boury, M.; Brzuszkiewicz,
E.; Gottschalk, G.; Buchrieser, C.; Hacker, J.; Dobrindt, U.; Oswald,
E. Science 2006, 313, 848–51.
(7) Watanabe, K.; Hotta, K.; Praseuth, A. P.; Koketsu, K.; Migita, A.;
Boddy, C. N.; Wang, C. C.; Oguri, H.; Oikawa, H. Nat. Chem. Biol.
2006, 2, 423–8.
(11) Nakaya, M.; Oguri, H.; Takahashi, K.; Fukushi, E.; Watanabe, K.;
Oikawa, H.; Koketsu, K. Biosci. Biotechnol. Biochem. 2007, 71, 2969–
76.
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Figure 2. Biosynthetic cluster of SW-163 from Streptomyces sp. SNA15896. (a) Organization of the SW-163 biosynthetic gene cluster. (b) Deduced functions
of the ORFs identified within the SW-163 biosynthetic gene cluster based on the percentage sequence similarity/identity to known proteins determined using
BLAST.
central thioacetal cross-bridge formed by the chemical rear-
rangement of a disulfide bridge catalyzed by a post-NRPS
modification enzyme, methyltransferase.7 Most interestingly,
however, the thioacetal bond is appended by a distinct alkyl
side chain in these compounds, each varying by a single carbon
unit.
On the basis of the results from our feeding experiments using
isotope-labeled substrates14 in conjunction with the previous
BLAST12 analyses of the genes from the SW-163, thiocoraline13
and echinomycin7 biosynthetic clusters, biosynthesis of 8 is
thought to proceed similarly as previously proposed for the
biosynthesis of quinoxaline-2-carboxylic acid (QXC 10, see
Figure 4b). That is, the biosynthesis proceeds via the initial
ꢀ-hydroxylation of L-tryptophan to form (2S, 3S)-ꢀ-hydrox-
ytryptophan 11, followed by the formation of ꢀ-hydroxykynure-
nine 12. Transamination of 12 by Swb1 can generate 13, and
oxidation and dehydration of 13 by Swb2 can afford 8 (Figure
3a).
Closely resembling the echinomycin biosynthetic gene cluster,
the SW-163 biosynthetic gene cluster (Figure 2) was identified
and isolated from the total DNA of Streptomyces sp. SNA15896
(Supporting Information Experimental Procedures). DNA se-
quence analysis of this 38 kilobase-long cluster revealed the
presence of fifteen genes considered to be involved directly in
the biosynthesis of SW-163s: seven genes (swb1, swb2, swb10,
swb11, swb13, swb14 and swb18) likely forming the pro-
posed SW-163 primer unit 8 (Figure 3a), three genes (swb6,
swb7, swb9) assembling 9 (Figure 3b), and five genes (swb8,
swb12, swb16, swb17, swb20) constructing and modifying the
peptide backbone (Figure 3c). The cluster also contains a
presumed resistance gene (swb15) and other genes, including
swb3, swb4, swb5 and swb19, presumably involved in the
regulation of biosynthesis or export of the products.
Despite the structural similarity of 9 to coronamic acid, whose
biosynthesis involves a cryptic chlorination step,15 no homo-
logues of the coronamic acid biosynthetic genes were identified
(12) McGinnis, S.; Madden, T. L. Nucleic Acids Res. 2004, 32, W20–5.
(13) Lombo, F.; Velasco, A.; Castro, A.; de la Calle, F.; Brana, A. F.;
Sanchez-Puelles, J. M.; Mendez, C.; Salas, J. A. ChemBioChem 2006,
7, 366–76.
(14) Koketsu, K.; Oguri, H.; Watanabe, K.; Oikawa, H. Org. Lett. 2006,
8, 4719–22.
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Watanabe et al.
Figure 3. Proposed mechanism for the SW-163 biosynthesis. (a) Proposed pathway for the biosynthesis of HQA 8. (b) Proposed mechanism for the formation
of (+)-(1S, 2S)-norcoronamic acid [9 (+)-NCA]. (c) Proposed pathway for the biosynthesis of the octadepsipeptide core. (d) Proposed mechanism for the
formation of a thioacetal bridge. (e) Proposed mechanism for the formation of a series of alkyl substituents differing by one carbon unit. NRPS domain name
abbreviations are: A, adenylation; C, condensation; E, epimerization; M, N-methyltransferase; M*, inactive methylation; T, thiolation; and TE, thioesterase.
Other abbreviations are: Ado-CH3, 5′-deoxyadenosine; AMP adenosine 5′-monophosphate; HQA, 3-hydroxyquinaldic acid; MeCbl, methylcobalamine; (+)-
NCA, (+)-(1S, 2S)-norcoronamic acid; SAM, S-adenosyl-L-methionine; and SAH, S-adenosyl-L-homocysteine.
in the SW-163 biosynthetic gene cluster. Although the mech-
anism of biosynthesis of 9 remains unclear, based on previous
reports of a radical SAM dehydrogenase and lysine 2,
3-aminomutase,16,17 we hypothesize that predicted radical SAM
protein Swb7 and pyridoxal-5′-phosphate (PLP)-dependent
aminotransferase Swb6 can work together closely to form 9 from
L-Val via radical cyclopropanation (Figure 3b).
bimodular NRPSs, Swb16 and Swb17. The terminal TE domain
in Swb17 likely homodimerizes and cycloreleases the peptide
chain (Figure 3c).18 Following cyclization of the linear peptide,
an oxidoreductase Swb20 presumably catalyzes an oxidation
reaction to generate the disulfide bond in 3. Subsequently, the
disulfide bridge in 3 can be converted to a thioacetal bridge to
form 4 (Figure 3d) by a predicted methyltransferase Swb8,
homologue of the SAM-dependent methyltransferase Ecm18 that
catalyzes the equivalent step in the echinomycin biosynthetic
pathway.7
Assembling of the SW-163 peptide scaffold from L-Ser,
L-Ala, L-Cys and 9 appears to be accomplished by two
(15) Vaillancourt, F. H.; Yeh, E.; Vosburg, D. A.; O’Connor, S. E.; Walsh,
C. T. Nature 2005, 436, 1191–4.
(16) Frey, P. A.; Magnusson, O. T. Chem. ReV. 2003, 103, 2129–48.
(17) Yokoyama, K.; Numakura, M.; Kudo, F.; Ohmori, D.; Eguchi, T.
J. Am. Chem. Soc. 2007, 129, 15147–55.
(18) Trauger, J. W.; Kohli, R. M.; Mootz, H. D.; Marahiel, M. A.; Walsh,
C. T. Nature 2000, 407, 215–8.
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Figure 4. Biosynthesis of a novel antibiotic ecolimycin C 14 in E. coli. (a) Chemical synthesis of the extender unit 9 fed to E. coli for the biosynthesis of
14. a, (i) SOCl2, ClCH2CH2Cl, (ii) cat. RuCl3, NaIO4, CH3CN-H2O; b, dibenzyl malonate, NaH, 1,2-dimethoxyethane; c, 1 M NaOH, MeOH; d,
diphenylphosphoryl azide, Et3N, t-BuOH; e, (i) H2, 10% Pd/C, EtOAc, (ii) 6 M HCl, MeOH. Product yield of each chemical reaction step is reported in
1
percentages. (b) Schematics of the pathway for the E. coli biosynthesis of 14. Abbreviation: QXC, quinoxaline-2-carboxylic acid. (c) LC-MS data, (d) H
NMR spectrum and (e) HMBC spectrum of 14. Important correlations (in red) are 1: δ 1.05 (NCA-CH3) and 46.4 (NCA-CR); 2: δ 1.18 (NCA-CH2) and 46.4
(NCA-CR); 3: δ 1.51 (NCA-CH2) and 46.4 (NCA-CR); 4: δ 1.51 (NCA-CH2) and 170.1 (NCA-CO); 5: δ 2.01 (NCA-CH) and 46.4 (NCA-CR).
Lastly but most notably, we propose that Swb9, a predicted
radical SAM protein, performs the further iterative methylation
of the thioacetal bridge. With the finding of Swb9, we are able
to come up with a more plausible mechanism for the iterative
methylation of the thioacetal bridge (Figure 3e) than our
previously proposed mechanism.7 Using the well-established
method19 for isotopically enriching the SAM-derived methyl
groups with a labeled L-methionine, we were able to observe
that, in addition to the four N-methyl groups of the cyclic
depsipeptide core, every proton in the S-alkyl chains of 4, 5, 6
and 7 became deuterated upon feeding of [methyl-D3]-L-
methionine to the SW-163-producing host (Supporting Informa-
tion Figure 1). These results were consistent with the S-alkyl
side chains formed by stepwise transfer of a methyl group
derived from SAM by a methyltransferase. As proposed for the
methylation during the fosfomycin biosynthesis,20 a radical SAM
protein can perform a methyl transfer reaction on a nonactivated
(20) Woodyer, R. D.; Li, G.; Zhao, H.; van der Donk, W. A. Chem.
Commun. 2007, 359–61.
(19) Rawlings, B. J. Nat. Prod. Rep. 1997, 14, 523–56.
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carbon at the cost of one molecule of SAM and methylcobal-
amine (MeCbl). We hypothesize that this unique radical SAM
methyltransferase can iteratively methylate 4 to generate ethyl-
5, i-propyl- 6, and sec-butyl 7 derivatives (Figure 3e). Further
studies to support this idea are currently underway.
E. coli Biosynthesis of Novel NRP with Antibiotic Activity.
To further challenge the versatility of our system, we aimed to
biosynthesize in ViVo in E. coli a new, bioactive compound.
For this purpose, as an initial attempt we chose to replace L-Val
in the depsipeptide scaffold of 2 with 9 to yield a hybrid
molecule 14 we termed ecolimycin C. The crystal structure of
the complex between 2 and its DNA substrate showed L-Val
minimally participating in the DNA recognition.21 Also, the fact
that the SW-163 antibiotics carry this moiety indicated that the
new compound would most likely retain antibiotic activity.
To convert the triostin A biosynthetic pathway into a
biosynthetic pathway for 14, Ecm7 was replaced with Swb17,
a bimodular NRPS showing high production level in E. coli
(Supporting Information Figure 2a) that contains the adenylation
domain capable of accepting 9 as an extender unit and the
thioesterase domain capable of homodimerizing and cyclore-
leasing the peptide chain containing 9. To minimize plasmid
instability, gene assemblies were kept to a minimal length while
maintaining as many genes within a contiguous metabolic
pathway on a single plasmid as possible; the genes for the
biosynthesis of 10 were put together on one plasmid (pKW532),
two megasynthetases Ecm6 and Swb17 on another (pKW755,
Supporting Information Figure 3a), and the rest of the genes
on the third plasmid (pKW539, Supporting Information
Figure 3b). Also, to prevent potential premature termination and
mRNA degradation22 while transcribing an extremely long
polycistronic gene assembly, all genes were assembled in a
multimonocistronic fashion.
Because 14 may possess antibiotic activity, the growth of E.
coli can be hampered by its accumulation in the cytosol. Thus,
a self-resistance mechanism was included to circumvent this
potential problem. Previously, the echinomycin resistance-
conferring gene ecm16, a close homologue of the daunorubicin
resistance-conferring gene drrC that provides nondestructive
resistance against daunorubicin in S. liVidans and E. coli,23 was
used successfully for our E. coli in ViVo production of 1 and
2.7 Considering this previous report, along with the observation
that the expression level of swb15 in E. coli (Supporting
Information Figure 2b) was comparable to that of ecm16, we
reasoned that either Ecm16 or Swb15, UvrA-like protein with
high (90%) sequence homology to Ecm16 (Figure 2b), can
provide E. coli resistance against 14. To ascertain the effective-
ness of the two genes on the culture viability and titer of 14,
two separate constructs were prepared to include either ecm16
(pKW539) or swb15 (pKW756) in the expression system
(Supporting Information Figure 3b). However, since there was
almost no difference in the titer between the two systems, Ecm16
was used throughout the studies.
Figure 5. Halo-formation assay for assessing the antibiotic activity of 2,
3 and 14. A tryptic soy agar plate plated with B. subtilis was spotted with
(a) 2, (b) 3 and (c) 14. Each well contained the corresponding compound
at an incremental amount of 2: 1.0 ng; 3: 2.0 ng; 4: 4.0 ng; 5: 8.0 ng and
6: 16 ng. Well 1 is a negative control.
coli culture extract to a series of chromatographic steps to obtain
purity necessary for collecting well-resolved 2D NMR spectra,
the final yield of 14 was 0.6 mg per liter of culture. The expected
mass of [M + H]+ ion at m/z ) 1083.3849 was obtained on
the purified 14 by FAB-HR-MS (Figure 4c and Supporting
Information Table 3). The compound was characterized further
by 1H, 13C and 2D NMR (Figure 4d,e and Supporting Informa-
tion Table 3, Figures 4 and 5) to reveal a characteristic resonance
at δ 9.50 (s, 1 H, QXC H-3′). Ultimately, total correlated
spectroscopy (TOCSY) (Supporting Information Figure 5b)
confirmed unambiguously that our engineered bacterium was
capable of producing 14. Furthermore, the antibiotic activity
of this new hybrid NRP compound was established to be as
effective if not more potent than 3 as determined by halo
formation assay against Bacillus subtilis (Figure 5).
Discussion
The E. coli-based biosynthetic system clearly exhibits its ease
of manipulation when one attempts to introduce planned
modifications into the target biosynthetic pathway. Taking full
advantage of these features and challenging our plasmid-borne
NRP biosynthetic system further, we decided to combine two
NRP biosynthetic pathways from two different sources to
biosynthesize a novel hybrid NRP which we named ecolimycin
C 14. To prepare this hybrid molecule, we applied part of the
biosynthetic gene cluster for the quinomycin antibiotics
SW163C-G that we have newly discovered to our plasmid-borne
triostin A biosynthetic pathway. Apart from the very interesting
thioacetal bridge alkyl side chain formation that appears to be
accomplished by unprecedented iterative methylation of non-
activated carbon centers by a radical SAM methyltransferase
Swb9 (Figure 3e), the SW-163 biosynthetic pathway involves
the production of nonproteinogenic amino acid 9 (Figure 3b)
and its incorporation into the homodimeric cyclic depsipeptide
core by the NRPS Swb17 (Figure 3c). By replacing Ecm7 in
the triostin A biosynthetic pathway with Swb17, we envisioned
the production of 14, which carries the chromophores of 2 while
having a sterically more constrained, R-branched amino acid
residue of 3 in its backbone (Figure 3b). The HQA-to-QXC
and L-Val-to-(+)-NCA switch seemed modest enough to
preserve the antibiotic activity of the parent compounds 2 and
3, yet different enough to see possible alterations in the substrate
DNA affinity and/or sequence selectivity.24
BL21 (DE3) was transformed with three plasmids pKW532,
pKW755 and pKW539, and subjected to shake-flask cultivation
for seven days in M9 minimal medium supplemented with
chemically synthesized 9 (Figure 4a). After subjecting the E.
Although it was necessary to feed E. coli chemically
synthesized 9 to accomplish the biosynthesis of 14, the observed
titer of 14 was significantly higher than that of 1 or 2 without
requiring feeding of 10. This unexpected high yield of an
unnatural NRP could be due to higher expression level of swb17
(21) Wang, A. H.; Ughetto, G.; Quigley, G. J.; Hakoshima, T.; van der
Marel, G. A.; van Boom, J. H.; Rich, A. Science 1984, 225, 1115–
21.
(22) Sørensen, H. P.; Mortensen, K. K. J. Biotechnol. 2005, 115, 113–28.
(23) Lomovskaya, N.; Hong, S. K.; Kim, S. U.; Fonstein, L.; Furuya, K.;
Hutchinson, R. C. J. Bacteriol. 1996, 178, 3238–45.
(24) Fox, K. R.; Gauvreau, D.; Goodwin, D. C.; Waring, M. J. Biochem.
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(Supporting Information Figure 2a). Also, lower metabolic
turnover of the building block 9 may have helped boost the
yield further. Nevertheless, this result indicates that, in addition
to Ecm7, another NRPS Swb17 can also accept, elongate,
homodimerize and cyclorelease unnatural peptide chain. Fur-
thermore, Ecm17 exhibited relaxed substrate specificity by
accepting the unnatural intermediate to 14. Most importantly,
however, our result demonstrates that two NRPSs from differing
origin were able to establish productive in ViVo intermodular
interactions and produce 14 in E. coli. The fact that 14 was
obtained at a comparable or better yield than the parent
compounds in E. coli by a simple substitution of an enzyme in
the corresponding heterologous biosynthetic pathway without
any extensive modifications of the pathway nor its constituents
is especially encouraging for the future prospect of our system.
In addition, the efficiency and versatility of the E. coli platform
can be improved further by incorporating various technologies
currently being investigated, including the development of
efficient fed-batch fermentation protocols,25 use of quantitative
metabolic flux analyses for identifying the possible routes to
improving the system,26 application of directed evolution for
improving the protein-protein interactions among foreign
enzymes and their domains,27 and manipulation of the tran-
scriptional machineries for tailoring the global cellular phenotype
for improved heterologous secondary metabolite production.28
Taken together, we believe that our results have laid down the
foundation for the future development and refinement of E. coli
as a flexible, robust platform for fast and simple large-scale
production of pharmaceutically useful molecules and their
analogs in an environmentally friendly fashion from simple
carbon and nitrogen sources.
Acknowledgment. This paper is dedicated to the memory of
Professor Akitami Ichihara (1934-2009), for his pioneering studies
in bioorganic chemistry, including total synthesis of tautomycin.
We thank DAIICHI SANKYO Co., Ltd. for providing a sample of
Streptomyces sp. SNA15896. This study was supported by Special
Coordination Funds for Promoting Sciences and Technology of the
Ministry of Education, Sport, Culture, Science and Technology of
Japan (K.W.), by National Institute of General Medical Sciences
grant GM 075857-01 (C.C.C.W. and K.W.), by American Cancer
Society grant RSG-06-010-01-CDD (C.C.C.W. and K.W.), by
Novozymes Japan Research Foundation (K.W.) and by Northern
Advancement Center for Science and Technology Foundation
(K.W.).
Supporting Information Available: Experimental procedures,
two tables of DNA sequence information for the oligonucle-
otides, a figure for mass spectra of 3, 4, 5, 6, 7 isolated from
labeled methionine-feeding experiment, and a table and two
figures of spectrometric data on the E. coli-produced 14. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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