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Discovery of a new peptide natural product by
Streptomyces coelicolor genome mining
Sylvie Lautru1, Robert J Deeth1, Lianne M Bailey1,2 & Gregory L Challis1
Analyses of microbial genome sequences reveal numerous
examples of gene clusters encoding proteins typically involved
in complex natural product biosynthesis but not associated
with the production of known natural products1–3. In
Streptomyces coelicolor M145 there are several gene clusters
encoding new nonribosomal peptide synthetase (NRPS)
systems not associated with known metabolites. Application
of structure-based models for substrate recognition by NRPS
adenylation domains4–6 predicts the amino acids incorporated
into the putative peptide products of these systems7,3, but the
accuracy of these predictions is untested. Here we report
the isolation and structure determination of the new trishydroxamate tetrapeptide iron chelator coelichelin from
S. coelicolor using a genome mining approach guided by
substrate predictions for the trimodular NRPS CchH, and we
show that this enzyme, which lacks a C-terminal thioesterase
domain, together with a homolog of enterobactin esterase
(CchJ), are required for coelichelin biosynthesis. These results
demonstrate that accurate prediction of adenylation domain
substrate selectivity is possible and raise intriguing mechanistic
questions regarding the assembly of a tetrapeptide by a
trimodular NRPS.
Among the putative secondary metabolic gene clusters identified in
the S. coelicolor genome sequence that do not direct production of a
known metabolite3, the cch cluster (Fig. 1a) is proposed to encode an
NRPS system that catalyzes assembly of a new ferric-iron chelating
peptide coelichelin7. Sequence analysis of the NRPS CchH encoded
within this cluster shows that it contains three modules, which,
according to the ‘colinearity rule’ for NRPS8, implies that CchH
assembles a tripeptide product (Fig. 1b)7. CchH is unusual because
it does not contain a thioesterase or reductase domain, present at the
C terminus of the last module in most other NRPS9, to catalyze
cleavage of the thioester bond between the fully assembled peptide and
the last peptidyl carrier protein (PCP) domain7. Indeed, genes
encoding homologs of such thioesterases (TEs) or NADH-dependent
reductases are not found anywhere in or adjacent to the cch cluster7.
Our predictive model for amino acid recognition by A domains
hypothesizes that modules 1, 2 and 3 of CchH recognize and
selectively activate L-d-N-formyl-d-N-hydroxyornithine (L-hfOrn),
L-threonine (L-Thr) and L-d-N-hydroxyornithine (L-hOrn), respectively5,7, and suggests two alternative structures (1 and 2) for the
putative tripeptide product of this NRPS (Fig. 1c)7.
These structural predictions proved critical for identifying culture
conditions in which the cch cluster is expressed and for designing a
strategy to identify the product of CchH. The predicted hydroxamic
acid functional groups in coelichelin suggest that it functions as a
siderophore in S. coelicolor, implying that an iron-deficient medium is
required for expression of the cluster and that coelichelin could be
identified by converting it to a ferric-hydroxamate complex, which
should be easily detected by UV-vis spectroscopy. These insights
successfully guided the identification of coelichelin using a strategy
combining gene knockouts with comparative metabolic profiling.
Thus, we inactivated cchH in S. coelicolor M145 to create S. coelicolor
W5. We identified a compound, which forms a complex with ferric
iron, lacking in the W5 strain but present in the M145 strain by
comparative HPLC analysis of culture supernatants from the two
strains grown under iron-deficient conditions (Supplementary Methods online). Addition of ferric iron to the culture medium suppressed
production of this compound confirming that it is only produced
under iron-deficient culture conditions. The lmax of 435 nm for ferricoelichelin suggested that it was a tris-hydroxamate complex. We
partially purified ferri-coelichelin from the culture supernatant of
S. coelicolor M145 by semipreparative HPLC and, after removal of the
ferric iron by treatment with 8-hydroxyquinoline, we purified desferricoelichelin to homogeneity by a further semi-preparative HPLC step.
We determined the structure of coelichelin (3, Fig. 1d) using similar
methods to those reported for exochelin MS10, exochelin MN11 and
ornibactin12. A combination of high resolution and tandem mass
spectrometric analysis of desferri-coelichelin and one- and twodimensional high-field NMR analysis (1H, DQF-COSY, TOCSY,
HMBC and ROESY) of gallium-coelichelin established the identity
of the amino acid residues and their sequence (Fig. 2 and Supplementary Methods online). We determined the relative stereochemistry of the four a-carbons in coelichelin using molecular modelling in
combination with inter-residue distances and dihedral angles calculated for Ga-coelichelin from the ROESY and 1H NMR data, respectively. The relative configuration of the threonine a- and b-carbons
was determined by acid-promoted hydrolysis of Ga-coelichelin, followed by conversion of the liberated threonine to its N-trifluoroacetyl
1Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK. 2Present address: Department of Biological Sciences, University of Warwick, Coventry CV4
7AL, UK. Correspondence should be addressed to G.L.C. (G.L.Challis@warwick.ac.uk).
Received 13 June; accepted 18 August; published online 11 September 2005; doi:10.1038/nchembio731
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Figure 1 Organization of coelichelin NRPS and biosynthetic gene cluster. (a) The organization of the cch gene cluster highlighting in gray the genes
inactivated in this work. (b) The module and domain organization of the NRPS encoded by cchH. (c) The structures 1 and 2 previously suggested for two
hypothetical products of CchH. (d) The experimentally determined structure of coelichelin 3. The domains of the NRPS in b are labeled as follows: A,
adenylation; E, epimerization; C, condensation. The structures of the amino acids predicted to be incorporated into coelichelin by each A domain (shaded
in gray) are illustrated attached as thioesters to the adjacent PCP domains (represented as black spheres) in b. The reactions catalyzed by each domain
are illustrated in Figure 3 and discussed in the text.
expressed the cch cluster in the heterologous host Streptomyces
fungicidicus B-547713, which we found is easily transformed by
intergenic conjugation14. Comparative HPLC analysis of cultures
grown in iron-deficient medium showed that wild-type S. fungicidicus
does not produce 3, whereas three independent transformants containing the cch cluster integrated into the S. fungicudicus chromosome
all produce significant quantities of 3 (Supplementary Methods
online), providing strong evidence that CchH is the only NRPS
required for the biosynthesis of 3 in S. coelicolor. We have also
shown that a virtually identical cluster of genes resides, within a
different genomic context, in the chromosome of Streptomyces ambofaciens ATCC23877 and that this cluster directs coelichelin production
(S.L., F. Barona-Gómez, F.-X. Francou, J.-L. Pernodet and G.L.C,
unpublished data), providing further evidence that the cch cluster is
sufficient for coelichelin biosynthesis.
The carboxyl group in the hOrn residue of 3 led us to hypothesize
that a specific hydrolase must be required to release coelichelin from
CchH. Because no C-terminal TE domain, normally utilized by NRPS
for hydrolytic release of their peptide products9, is present in CchH,
we examined the requirement of cchJ for coelichelin biosynthesis. CchJ
is similar to enterobactin esterase, which is not required for
isopropyl ester derivative and comparison by chiral gas chromatography with authentic standards (Supplementary Methods online). The
well-known usual preference of NRPS adenylation domains for
L-amino acid substrates9, and the presence of epimerization (E)
domains, which catalyze inversion of stereochemistry at the a-carbons
in L-amino acids substrates of NRPS9, in modules 1 and 2, but
not module 3 of CchH, led us to conclude that the absolute stereochemistry of coelichelin is D-hfOrn-D-allo-Thr-L-hOrn-D-hfOrn.
Our results are consistent with the requirement of CchH for
assembly of 3 from the amino acids L-hfOrn, L-Thr and L-hOrn—as
our model for amino acid recognition by NRPS A domains predicts5,7.
It is important to note that such an accurate prediction could not have
been made using another model because it does not discriminate
between A domains activating Orn, hOrn and hfOrn4. The experimentally determined structure 3 is very similar to the predicted
structure 27, differing only in the addition of a second D-hfOrn
residue to the a-amino group of the L-hOrn residue (Fig. 1c,d).
This could not have been predicted from sequence analysis of CchH
because the assembly of a tetrapeptide by a trimodular NRPS
was without literature precedent. To rule out the involvement of a
second NRPS (in addition to CchH) in the biosynthesis of 3, we
a
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© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
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Figure 2 Structure elucidation of coelichelin. (a) Assignment of the y ions (where the charge is retained in the COOH-terminal fragment of the peptide)
observed in the product ion spectrum resulting from tandem mass spectrometry of (M + H)+ at m/z ¼ 566 (a product ion with m/z ¼ 250 is also observed
corresponding to loss of both hfOrn residues). (b) Spin systems identified in Ga-coelichelin by TOCSY and COSY. (c) Correlations observed in the HMBC
(solid lines) and ROESY (dashed lines) spectra of Ga-coelichelin that establish the sequence. Inter-residue aCH-NH distance constraints calculated from
the ROESY data are given in Å. The relative stereochemistry determined by modeling the Ga-coelichelin complex is also illustrated. (d) Calculated structure
of the only diastereomer of Ga-coelichelin consistent with the inter-residue distances derived from the ROESY data and dihedral angles derived from the
1H NMR spectrum. The inter-residue distances in the model corresponding to the experimental distances are illustrated in the same colors in c and d.
2
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Figure 3 Model for coelichelin biosynthesis. Each of the transformations in stages 1–5 is discussed in the text. The domains that participate in each stage
are highlighted in gray. PCP1, PCP2 and PCP3 are the PCP domains in modules 1, 2 and 3 of CchH, respectively.
enterobactin biosynthesis15. Instead, enterobactin esterase hydrolytically cleaves enterobactin into three 2,3-dihydroxybenzoyl-L-serine
subunits and is proposed to play a role in recovery of iron from the
ferric-enterobactin complex16. We replaced cchJ on the chromosome
of S. coelicolor M145 to generate S. coelicolor W6. No coelichelin was
detected in culture supernatants or lysed cells of this mutant grown
under iron-deficient conditions (Supplementary Methods online),
indicating that CchJ is required for coelichelin biosynthesis, consistent
with it acting as the thioesterase that hydrolytically releases the fully
assembled tetrapeptidyl thioester from the last PCP domain of CchH.
Taken together our results suggest a model for the assembly of the
tetrapeptide 3 by the trimodular NRPS CchH and the esterase CchJ
(Fig. 3). In the first step, L-hfOrn, L-Thr and L-hOrn are bound and
converted to aminoacyl adenylates by the adenylation domains of
modules 1, 2 and 3, respectively. Each aminoacyl adenylate is transferred onto the adjacent PCP domain to form the corresponding
aminoacyl thioesters. The E domains within modules 1 and 2 catalyze
epimerization of the a-stereocenters in the corresponding aminoacyl
thioesters (alternatively, this may occur after each condensation
reaction). In the second stage, the condensation (C) domain of
module 2 catalyzes nucleophilic attack of the hfOrn carbonyl carbon
with the threonine amino group to form hfOrn-Thr attached to the
PCP2 domain. A second condensation between the d-amino group of
hOrn and hfOrn-Thr, catalyzed by the C domain of module 3, yields
the tripeptide D-hfOrn-D-allo-Thr-L-hOrn attached to the PCP3
domain. In the third stage, a second molecule of hfOrn is attached
to the PCP1 domain and the E domain catalyzes epimerization of the
a-stereocenter in the resulting aminoacyl thioester. Nucleophilic attack
of the hOrn a-amino group in D-hfOrn-D-allo-Thr-L-hOrn-PCP3 on
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D-hfOrn-PCP1
is catalyzed by the C domain of either module 2 or
module 3 in the fourth stage, to give the fully assembled tetrapeptidyl
thioester attached to the PCP3 domain. Finally, 3 is released from
the NRPS by CchJ-catalyzed hydrolysis. Premature release of the
tripeptide D-hfOrn-D-allo-Thr-L-hOrn is probably prevented by high
selectivity of CchJ for the tetrapeptidyl thioester.
In our model, the A, PCP and E domains of module 1 and the
C domain of either module 2 or module 3 of CchH are used
iteratively, and several other domains are skipped in the second
iteration of the assembly process. Although iterative domain or
module use17–21, or module skipping22, is proposed for other NRPS
systems, the trimodular NRPS CchH uniquely employs both processes
in the assembly of its tetrapeptide product. Utilization of a separately
encoded esterase, which is similar to enterobactin esterase rather than
to thioesterases (both members of the a, b-hydrolase superfamily23,24),
for product release is another unique feature of the coelichelin
biosynthetic assembly line.
In conclusion, we have demonstrated that accurate predictions of
amino acids incorporated into new nonribosomal peptides can be
made from the sequences of NRPS catalyzing their assembly and
that such predictions usefully guide the discovery of new nonribosomal peptides by genome mining. Our results also reveal new
mechanistic possibilities for NRPS, suggesting that there may be
even greater catalytic versatility in their assembly-line enzymology
than previously thought.
METHODS
Inactivation of cchH. A 5.7-kb KpnI fragment of cosmid SCF34 from the
S. coelicolor M145 ordered cosmid library25 was subcloned into KpnI-digested
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pIJ2925. A 2.5-kb KpnI-EcoRI fragment from this construct was subcloned into
KpnI and EcoRI-digested pKC113226. Escherichia coli ET12567 containing
pUZ8002 was transformed with the resulting plasmid, which was subsequently
transferred by conjugation into S. coelicolor M145, selecting for apramycin
resistance, using a standard procedure14. Four apramycin-resistant transconjugants were analyzed by southern hybridization for correct integration of the
disruption plasmid into the cchH gene. One of these mutants was selected for
further analysis and named S. coelicolor W5.
Inactivation of cchJ. The recently developed REDIRECT technology was used
to replace cchJ with the aac(3)IV gene in S. coelicolor M14527,28. Cosmids and
strains were provided by PBL Biomedical Laboratories. In the SCF34 cosmid,
cchJ was replaced by the apramycin resistance cassette from pIJ773. The
‘disruption’ oligonucleotides used for the amplification of the apramycinresistance cassette with the Expand high-fidelity DNA polymerase (Roche) were
5¢TAGTCTTCCGGATTTCCGCTCCACAAGGGGACGTTCATGATTCCGGGG
ATCCGTCG ACC3¢ (forward primer), and 5¢GCGGACCGGGGCCCGCCGGG
CCGGTCGCGGCCGCCCCTATGTAGGCTGGAGCTGCTTC3¢ (reverse primer;
bases identical to flanking regions of cchJ are underlined). The mutagenized
cosmid was introduced into S. coelicolor M145 by RP4-based conjugation from
E. coli ET12567 containing pUZ8002 and selected for using apramycin14.
Double crossover exconjugants were identified by their sensitivity to kanamycin,
and inactivation of cchJ in S. coelicolor W6 was verified by PCR using the
primers 5¢TCAGGCGTCCGCGGTCCG GG3¢ and 5¢CGCGCGGGGCGTCGCC
AAGC3¢.
Heterologous expression of the cch cluster in S. fungicidicus B-547713. No
orthologs of genes in the S. coelicolor cch cluster could be detected in
S. fungicidicus by PCR. Coelichelin could not be detected in the culture supernatant of S. fungicidicus grown in iron-deficient medium (see further on and
Supplementary Methods online). The bla gene of SCF34 was replaced by the
4.9-kb DraI-BsaI fragment of pIJ787 using PCR targeting to create SCF3478728. The aac(3)IV gene was amplified using Expand High Fidelity Polymerase
(Roche) by PCR using the primers 5¢CACGGTCGGGCCCTGCCGGG
TTGCGGGTCCTGCGCGGCCAAGCTTGTACGGCCCACAGA-3¢ and 5¢CTG
CAGCTCGTAGCGGTCCTGGAGGTAGATGCCGCTGTTAAGCTTCGACTAC
CTTGGT-3¢ from the gel-purified 1-kb EcoRI fragment of pSPM83 (provided
by J.-L. Pernodet, University of Paris-Sud 11). This PCR product was used to
replace Sco0500 to Sco0505 in SCF34-787 by PCR targeting27,28. The resulting
cosmid was introduced into E. coli ET12567/pUZ8002 by electroporation and
conjugal transfer of the cosmid to S. fungicidicus B-5477 was carried out using a
standard procedure14. Three apramycin resistant transconjugants were isolated
and the presence of the cluster in the chromosome was confirmed by PCR.
HPLC analysis. A previously described iron-deficient medium29 was used to
grow S. coelicolor M145, W5, W6, wild type S. fungicidicus B-5477 and
S. fungicidicus B-5477 containing the cch cluster at 30 1C in the dark for
4 days. Cultures were centrifuged and supernatants were concentrated using a
rotary evaporator. Dry extracts were redissolved in the minimum amount of
water required and ferri-coelichelin was formed by addition of FeCl3. Before
HPLC injection, the concentrated supernatants were filtered using a Vivaspin
0.5 ml concentrator (10,000 MWCO). Samples were analyzed on a Supelco
Discovery HSF5 column (150 4.6 mm, 5 mm) eluting with isocratic 10 mM
ammonium carbonate (pH 7.0)/MeOH 9:1 at 1 ml min1. Ferric-tris-hydroxamate siderophores were detected by monitoring absorbance at 435 nm.
Under these conditions, ferricoelichelin elutes with a retention time of approximately 2.8 min. The identity of ferricoelichelin was confirmed by ESI-MS.
Purification of coelichelin. Large-scale cultures of S. coelicolor M145 were
grown, and concentrated crude culture supernatants containing ferricoelichelin were prepared as described in the previous sections. Ferri-coelichelin
was purified from the crude culture supernatant on a semi-preparative Supelco
Discovery HSF5 column (150 10 mm, 5 mm) using isocratic elution with
10 mM ammonium carbonate (pH 7.0)/MeOH 9:1 at 5 ml min1. Absorbance
at 435 nm was used to monitor elution of ferri-coelichelin. The ferric iron was
removed from ferri-coelichelin using an excess of 8-hydroxyquinoline. The
ferric-8-hydroxyquinoline complex was removed by extraction into dichloromethane12. Because of the instability of desferri-coelichelin, the next
4
purification step was carried out without delay. Desferricoelichelin was purified
on a Phenomenex Synergy Fusion-RP column (250 10 mm, 4 mm) eluting
with isocratic 10 mM ammonium carbonate (pH 7.0) at 5 ml min–1.
Absorbance was monitored at 200 nm. Desferricoelichelin was collected and
immediately freeze-dried. Ga-coelichelin was prepared following an established
protocol12 and purified to homogeneity using the semipreparative Synergy
Fusion-RP column and the same conditions described above for ferricoelichelin. The chromatogram for the purification of Ga-coelichelin is shown
in the Supplementary Methods online.
Structure elucidation of coelichelin. Spectroscopic, computational and analytical methods used in the structure determination of coelichelin are described
in the Supplementary Methods online.
Note: Supplementary information is available on the Nature Chemical Biology website.
ACKNOWLEDGMENTS
P. Grice (Department of Chemistry, University of Cambridge, Cambridge, UK)
and A. Clarke (Department of Chemistry, University of Warwick, Coventry, UK)
are very gratefully acknowledged for assistance with recording and processing
of NMR data. We thank A. Giannakopulos and P.J. Derrick (Department of
Chemistry, University of Warwick, Coventry, UK) for assistance with ESI-FT-ICRMS and A. Lubben (Bruker Daltonics, Coventry, UK) for assistance with ESI-MSMS and ES-TOF-MS. J.L. Pernodet (University of Paris-Sud 11) is gratefully
acknowledged for the gift of the plasmid pSPM83. J. Irwin is gratefully
acknowledged for assistance with the GC analysis. F. Barona-Gómez, C. Corre,
C. Blindauer and J.P. Rourke (Department of Chemistry, University of Warwick,
Coventry, UK) are acknowledged for helpful discussions. This work was
supported by grants from the UK Biotechnology and Biological Sciences Research
Council and Wellcome Trust. S.L. thanks the EU for a Marie Curie fellowship.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
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