An artificial pathway to 3,4-dihydroxybenzoic acid allows generation of new aminocoumarin antibiotic recognized by catechol transporters of E. coli

Article abstract of DOI:10.1016/j.chembiol.2010.12.016

An artificial operon was synthesized, consisting of the genes for chorismate pyruvate-lyase of E. coli and for 4-hydroxybenzoate 3-hydroxylase of Corynebacterium cyclohexanicum. This operon, directing the biosynthesis of 3,4-dihdroxybenzoate, was expressed in the heterologous expression host Streptomyces coelicolor M512, together with a modified biosynthetic gene cluster for the aminocoumarin antibiotic clorobiocin. The resulting strain produced a clorobiocin derivative containing a 3,4-dihdroxybenzoyl moiety. Its structure was confirmed by MS and NMR analysis, and it was found to be a potent inhibitor of the gyrases from Escherichia coli and Staphylococcus aureus. Bioassays against different E. coli mutants suggested that this compound is actively imported by catechol siderophore transporters in the cell envelope. This study provides an example of the structure of a natural product that can be rationally modified by synthetic biology.

Full text of DOI:10.1016/j.chembiol.2010.12.016

Chemistry & Biology
Article
An Artificial Pathway to 3,4-Dihydroxybenzoic Acid
Allows Generation of New Aminocoumarin Antibiotic
Recognized by Catechol Transporters of E. coli
Silke Alt,1 Nadja Burkard,2 Andreas Kulik,3 Stephanie Grond,2 and Lutz Heide1,
*
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universitat Tubingen, Auf der Morgenstelle 8,
1
¨
¨
72076 Tu¨ bingen, Germany
2
¨
¨
¨
Institut fu¨ r Organische Chemie, Eberhard-Karls-Universitat Tubingen, Auf der Morgenstelle 18, 72076 Tubingen, Germany
3Mikrobiologisches Institut, Eberhard-Karls-Universita¨ t Tu¨ bingen, Auf der Morgenstelle 28, 72076 Tu¨ bingen, Germany
*Correspondence: heide@uni-tuebingen.de
DOI 10.1016/j.chembiol.2010.12.016
SUMMARY
The aminocoumarin antibiotics are characterized by their
3-amino-4,7-dihydroxycoumarin moiety (Heide, 2009a). This
An artificial operon was synthesized, consisting of family of antibiotics comprises potent DNA gyrase inhibitors,
including clorobiocin and the structurally related novobiocin (Fig-
ure 1). These compounds interact with the subunit B of bacterial
the genes for chorismate pyruvate-lyase of E. coli
and for 4-hydroxybenzoate 3-hydroxylase of Coryne-
bacterium cyclohexanicum. This operon, directing
the biosynthesis of 3,4-dihdroxybenzoate, was ex-
pressed in the heterologous expression host Strepto-
myces coelicolor M512, together with a modified
biosynthetic gene cluster for the aminocoumarin anti-
DNA gyrase and inhibit ATP-dependent supercoiling of DNA,
which is essential for transcription and DNA replication in
bacteria. The therapeutic potential of the aminocoumarins lies
especially in their very high affinity to gyrase. Their equilibrium
dissociation constants (KD) for gyrase is in the 10 nM range,
i.e., much lower than that of modern fluoroquinolones (Maxwell
biotic clorobiocin. The resulting strain produced
a clorobiocin derivative containing a 3,4-dihdroxy-
benzoyl moiety. Its structure was confirmed by MS
and Lawson, 2003). Even though clorobiocin is a more potent
inhibitor of DNA gyrase, only novobiocin (Albamycin) has been
licensed for clinical use in human infections with Gram-positive
and NMR analysis, and it was found to be a potent bacteria such as methicillin-resistant Staphylococcus aureus
strains (Maxwell and Lawson, 2003). However, due to its poor
solubility in water and its low activity against Gram-negative
inhibitor of the gyrases from Escherichia coli and
Staphylococcus aureus. Bioassays against different
E. coli mutants suggested that this compound is
actively imported by catechol siderophore trans-
porters in the cell envelope. This study provides an
example of the structure of a natural product that
can be rationally modified by synthetic biology.
pathogens, clinical application of novobiocin remains restricted.
Over the last years, the biosynthetic gene clusters of five
different aminocoumarin antibiotics have been cloned and
sequenced (Heide, 2009a), and this group of antibiotics has
become a successful example for the generation of new anti-
biotics by genetic techniques, e.g., metabolic engineering,
mutasynthesis, and chemoenzymatic synthesis (Heide, 2009b).
Structurally, clorobiocin is composed of three moieties: the
INTRODUCTION
acylated noviose deoxysugar (Ring C), the 3-amino-4,7-dihy-
droxycoumarin which is chlorinated at 80-position (Ring B), and
The first experiment showing that a new antibiotic can be the 3-dimethylallyl-4-hydroxybenzoyl moiety (Ring A). These
produced by genetic engineering, i.e., by the combination of three moieties are linked by a glycosidic and an amide bond (Fig-
biosynthetic genes from different organisms, was published in ure 1). X-ray crystallographic studies have shown that Ring B and
1985 (Hopwood et al., 1985). Since then, the number of biosyn- Ring C are essential for the interactions of clorobiocin (as well as
thetic genes and gene clusters available for such experiments, novobiocin) with gyrase, while Ring A is much less involved in the
and the genetic techniques available for recombination and binding of the antibiotic to the target (Maxwell and Lawson, 2003).
expression have expanded greatly, and many new bioactive Therefore, it appears possible to vary the structure of Ring A
compoundshavebeengeneratedbygeneticengineeringofmicro- without severely affecting the DNA gyrase inhibitory activity.
organisms (Hopwood, 2009a, 2009b). Advances in the methods
A principal shortcoming of the aminocoumarin antibiotics is
of chemical DNA synthesis now allow to readily adapt the codon their poor activity against Gram-negative organisms which is
usage in a given gene to the codon usage of different expression partly due to their poor permeation across the outer membrane
hosts. This greatly expands the possibilities for the generation of these organisms (Tropp et al., 1995). Gram-negative bacteria
of new bioactive compounds by the combination of genes from have efficient iron acquisition systems which consist of outer
very different organisms in a suitable host strain using a synthetic membrane proteins that bind iron complexes of specific sidero-
biology approach. The present paper reports the rational design phores and facilitate their active transport into the cell (Neilands,
of a structurally modified antibiotic by such a strategy.
1995). Many siderophores, like enterobactin of Escherichia coli,
304 Chemistry & Biology 18, 304–313, March 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Artificial Pathway to a New Coumarin Antibiotic
Ring C
Ring B
Ring A
product formation
[nmol s-1 (mg protein)-1]
NovL
CloL
CouL
SimL
OH
H
N
O
C
Ring A
CH3
HOOC
5.1
3.6
0.70
0.75
O
O
O
OH
O
H3CO
OH
CH3
CH3
novobiocin
3,4-DHBA
OH
O
H2N
HOOC
OH
OH
1.3
1.5
0.65
0.66
OH
H
N
O
C
O
CH3
CH3
O
O
O
O
O
OH
H3CO
caffeic acid
Cl
clorobiocin
0.13
< 0.01 < 0.01
0.19
HOOC
OH
OH
OH
H3C
N
H
O
OH
OH
OH
OH
HOOC
HOOC
OH
HOOC
OH
OH
HOOC
Figure 1. The Aminocoumarin Antibiotics Novobiocin and Clorobio-
cin
OH
OH
2,3-DHBA
3,4-DH-phenyl-
acetic acid
3,4-DH-phenyl-
propionic acid
3,4-DH-
mandelic acid
contain catechol (= o-diphenol) motifs. Their hydroxyl groups are
responsible for chelating the Fe3+ ion (Neilands, 1995). The bacte-
rial iron uptake mechanisms offers a possibility to overcome
product formation with all four enzymes
< 0.01 nmol s-1 mg-1
membrane-associated drug resistance by
a Trojan horse
¨
approach (Miethke and Marahiel, 2007; Mollmann et al., 2009).
Ithasbeenshownthatbeta-lactamantibioticstowhichacatechol
moiety had been chemically attached were transported by side-
rophore transporters into the Gram-negative cell, resulting in an
Figure 2. Substrate Tolerance of Different Aminocoumarin Acyl
Ligases
Activity of different aminocoumarin acyl ligases with acyl substrates containing
catechol motifs. Amide bond formation was determined with 3-amino-4,7-
dihydroxy-8-methyl-coumarin as amino substrate in the presence of ATP
and Mg2+, as described in the Experimental Procedures.
¨
enhanced antibacterial activity (Mollmann et al., 2009). In the
present study, we attempted to develop a clorobiocin derivative
with a catechol moiety instead of the genuine Ring A, using
a synthetic biology approach, and to test whether it would be
transported into the cell under involvement of catechol sidero-
phore transporters. We (1) isolated the gene cluster for clorobio-
cin from a Streptomyces strain; (2) deleted the pathway to the
genuine Ring A moiety of this antibiotic from the cluster by gene
inactivation; (3) constructed a new, artificial biosynthetic pathway
to a catechol moiety by combining one gene from the Gram-
negative Escherichia coli and one gene from the Gram-positive
Corynebacterium cyclohexanicum into a synthetic operon; (4) ex-
pressed these genes from three different organisms in a fourth
organism, i.e., Streptomyces coelicolor M512, as heterologous
host. This genetic strategy resulted in the production of the
desired compound, i.e., a clorobiocin derivative with a catechol
(= o-diphenol) moiety. We could provide evidence that this
compound was transported across the cell envelope of E. coli
under involvement of TonB-dependent transporters, i.e., bacte-
rial siderophore transporters which recognize catechol moieties.
(Figure 1) with an acyl moiety containing a catechol structure.
We considered six different acids which contain catechol motifs
(Figure 2). Previous mutasynthetic experiments, aimed at the
replacement of Ring A by other acyl groups, had shown that
the success of those experiments is dependent primarily on
the acceptance of the acyl substrate by the aminocoumarin
acyl ligase (= amide synthetase) which attaches the acyl moiety
to the 3-amino group of the aminocoumarin moiety (Anderle
et al., 2007; Galm et al., 2004a). The later biosynthetic steps,
i.e., glycosylation and final tailoring steps, appeared not to be
affected by modifications of the structure of the acyl moiety.
We therefore tested the acceptance of the six acyl substrates
with catechol motifs (Figure 2) by four different aminocoumarin
acyl ligases, i.e., NovL, CloL, CouL, and SimL, obtained from
the biosynthetic gene cluster of novobiocin, clorobiocin, cou-
mermycin A1, and simocyclinone D8, respectively. These four
enzymes were expressed and purified as described previously
(Galm et al., 2004a; Luft et al., 2005; Schmutz et al., 2003;
Steffensky et al., 2000a). Aminocoumarin acyl ligase activity
was tested using 3-amino-4,7-dihydroxy-8-methyl-coumarin
as amino substrate and the six catechols as acyl substrates in
an in vitro assay for amide formation (see Experimental Proce-
dures). The identity of the resulting compounds was confirmed
RESULTS
Investigation of the Substrate Tolerance of Different
Aminocoumarin Acyl Ligases for Acyl Substrates
with Catechol Moieties
The aim of our study was the replacement of the genuine 3-di- by LC-MS. As shown in Figure 2, the best accepted catechol
methylallyl-4-hydroxybenzoyl moiety (= Ring A) in clorobiocin substrate was 3,4-dihydroxybenzoic acid (3,4-DHBA). In case
Chemistry & Biology 18, 304–313, March 25, 2011 ª2011 Elsevier Ltd All rights reserved 305

Chemistry & Biology
Artificial Pathway to a New Coumarin Antibiotic
of CloL, amide bond formation with this substrate reached 42% feeding of this compound in order to supply 3,4-DHBA for ami-
of the reaction velocity observed with the genuine substrate Ring nocoumarin antibiotic formation.
A. Caffeic acid was not accepted by CloL but by SimL, albeit only
to a low extent. The other four catechols were not accepted by Creating an Artificial Pathway to 3,4-Dihydroxybenzoic
any of the investigated enzymes. We therefore concentrated Acid
further experiments on 3,4-DHBA and the aminocoumarin acyl 3,4-DHBA can be formed by the well-characterized 4-hydroxy-
ligase of clorobiocin biosynthesis, i.e., CloL.
benzoate-3-hydroxylase PobA of Corynebacterium cyclohexani-
cum (Figure 3) (Fujii and Kaneda, 1985; Huang et al., 2008). This
44 kDa flavoprotein monooxygenase is involved in catabolic
processes, e.g., lignin degradation. In all Gram-positive bacteria
it uses NADH in order to provide the required reduction equiva-
Inactivation of cloQ in the Biosynthetic Gene Cluster
of Clorobiocin, and Heterologous Expression
of the Modified Cluster
In order to replace Ring A in clorobiocin with 3,4-DHBA, the lents. However, the substrate of PobA, 4-hydroxybenzoic acid
biosynthesis of Ring A had to be abolished to avoid competition (4HBA), is not expected to be present in Streptomyces in high
between the genuine and the artificial acyl moiety as precursor in concentrations. 4HBA is an intermediate of ubiquinone biosyn-
the antibiotic biosynthesis. The first committed step in Ring A thesis. In most organisms, it is formed by degradation of tyrosine
biosynthesis is the prenylation of 4-hydroxyphenylpyruvate (Meganathan, 2001). However, Gram-negative bacteria such as
under catalysis of CloQ (Pojer et al., 2003). Therefore, we carried E. coli derive 4HBA directly from chorismate by elimination of the
out an inactivation of the gene cloQ within the biosynthetic gene enol-pyruvyl side chain under catalysis of chorismate pyruvate
cluster of clorobiocin. lyase, a 19 kDa protein encoded by the gene ubiC (Figure 3)
The heterologous expression cosmid clo-BG1, containing the (Siebert et al., 1992). The reaction does not require cofactors.
clorobiocin biosynthetic gene cluster, had been prepared previ- By heterologous expression of ubiC in the chloroplasts of plants,
´
ously (Eustaquio et al., 2005). It contains the FC31 attachment which do not contain an ortholog of this gene, very large amounts
site for stable integration into the genome of different Strepto- of 4HBA could be generated without any detrimental effect on
myces host strains. Using Red/ET-mediated recombination, growth (Viitanen et al., 2004). Also Streptomyces genomes do
we replaced the coding sequence of cloQ with an apramycin not contain an ortholog of ubiC. An expression of both ubiC
resistance gene (see Experimental Procedures). The resistance and pobA therefore presented an attractive possibility to
cassette was subsequently removed by restriction digestion generate 3,4-DHBA in our Streptomyces strains in vivo.
and religation, using XbaI and SpeI sites introduced into the
The GC content of ubiC of E. coli is 53%, much lower than the
PCR primer sequence as described previously (Gust, 2009). average content in genes of the GC-rich Streptomyces. There-
This cloQ-deficient gene cluster was integrated into the genome fore, the sequence of ubiC was modified in order to adapt it to
the codon preference of Streptomyces. The Gene Designer
of S. coelicolor M512, resulting in S. coelicolor M512(clo-SA2).
Cultivation of this strain in clorobiocin production medium did program (DNA 2.0, Menlo Park, CA) was employed for this
not result in production of clorobiocin, while corresponding purpose, using the codon usage table of Streptomyces coeli-
heterologous expression strains containing the intact cluster color. The same was done for the gene pobA of Corynebacte-
´
produce this antibiotic (Eustaquio et al., 2005; Flinspach et al., rium cyclohexanicum, although fewer modifications were
2010). However, feeding of Ring A (1 mg per 80 ml culture required for this gene from a GC-rich species. The two genes
medium) restored production of clorobiocin, as confirmed by were translationally coupled in order to increase translation effi-
HPLC and HPLC-MS analysis in comparison to an authentic ciency and to facilitate co-regulation. The nucleotide sequence
reference compound. Therefore, the inactivation of cloQ had of the synthetic ubiC/pobA construct is given in Figure S1 (avail-
led to the abolishment of the production of Ring A, but had not able online). After commercial synthesis, this DNA fragment
affected the subsequent steps of clorobiocin biosynthesis.
was cloned into the Streptomyces expression vector pUWL201
(Doumith et al., 2000), placing it under control of the strong
constitutive ermE* promoter. The resulting plasmid, pSA11,
was introduced into S. coelicolor(clo-SA2) by protoplast trans-
Mutasynthetic Experiments with 3,4-DHBA
and Caffeic Acid
Feeding of different amounts of 3,4-DHBA to cultures of S. coeli- formation. Thereby, all genes required for the biosynthesis of
color M512(clo-SA2) did not lead to the formation of a clorobiocin the desired compound were assembled in the heterologous
derivative, as shown in HPLC and LC-MS analysis. In a parallel expression strain (Figure 3).
experiment, we also fed caffeic acid. In these experiments,
we additionally introduced a SimL expression plasmid, pSH2 Production of Novclobiocin 401 by Streptomyces
(Anderle et al., 2007), since SimL but not CloL was able to accept coelicolor(clo-SA2) Harboring Plasmid pSA11
caffeic acid (Figure 2). Also these experiments remained unsuc- Strain S. coelicolor(clo-SA2) harbouring pSA11 was cultivated in
cessful. However, we noticed that caffeic acid was clearly de- three different media: (1) the complex clorobiocin production
tected in the cultures after feeding, while no detectable amounts medium described for the wild-type producer strain Strepto-
of 3,4-DHBA were present in the cultures already one day after myces roseochromogenes var. oscitans DS 12.976 (Mancy
feeding. We speculated that 3,4-DHBA may rapidly be oxidized et al., 1974); (2) the complex GYM medium described for S. coeli-
in the medium, or may quickly be metabolized by catabolic path- color A3(2) fermentation (Shima et al., 1996); (3) the chemically
ways similar to those described in Corynebacterium (Merkens defined medium (CDM) developed for novobiocin production in
et al., 2005). We therefore considered whether a continuous the wild-type producer strain Streptomyces niveus (Kominek,
in vivo biosynthesis of 3,4-DHBA might be superior to an external 1972). Ethyl acetate extracts from cultures in the three media
306 Chemistry & Biology 18, 304–313, March 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Artificial Pathway to a New Coumarin Antibiotic
OH
H
N
O
C
S. roseochromogenes
CH3
O
H3C
O
O
O
OH
O
H
host strain S. coelicolor M512
H3C
Cl
O
OH
OH
clorobiocin
HOOC
OH
OH
+
NH2
C
O
H3C
ATP
N
H
ΔcloQ
HO
O
O
CloL
OH
H
N
O
C
OH
OH
clorobiocin gene
cluster
HO
O
O
OH
H
O
C
E. coli
N
OH
OH
C. cyclohexanicum
CH3
O
H3C
O
O
O
O
H
COOH
COOH
COOH
H3C
Cl
O
PobA
UbiC
CH2
OH
C
O
novclobiocin 401
OH
O
COOH
H3C
OH
3,4-DHBA
OH
4-hydroxybenzoic
acid
OH
N
H
chorismic
acid
Figure 3. Strategy for the Generation of a Clorobiocin Derivative Containing a 3,4-Dihydroxybenzoyl Moiety
The sequence of the synthetic ubiC/pobA operon is shown in Figure S1.
were analyzed by HPLC and LC-MS. The formation of a new medium (64 mg/ml). In this case, the new metabolite clearly rep-
compound with the expected molecular ion of m/z 643 [M-H]- resented a major compound in the extract (Figure 4A). Prepara-
was detected in all media. The amount of this compound was tive isolation of this metabolite from 2 l of culture resulted in
moderate using the two complex media (4 and 7 mg/ml, respec- 18 mg of pure compound (Figure 4B). This new clorobiocin deriv-
tively), but was much higher using the chemically defined ative was termed novclobiocin 401.
A
mAU
C
culture extract
mass spectrometric fragmentation of
novclobiocin 401
800
600
400
200
0
novclobiocin 401
- pyrrol - noviose
226
507
- CO2
362
318
OH
H
N
O
C
OH
OH
CH3
O
O
O
O
H
3CO
0
5
10
15
20 min
Cl
CH3
Intens.
[M-H]- 643
5
OH
O
x10
H3
C
N
H
507.1
1.2
1.0
0.8
0.6
0.4
0.2
0.0
B
mAU
purified compound
O
3000
novclobiocin 401
225.8
2000
1000
0
361.9
317.9
-MS2(643.0)
0
5
10
15
20 min
100
200
300
400
500
600
m/z
Figure 4. HPLC and LC-MS Analysis
(A) HPLC chromatogram of a culture extract from the heterologous producer strain Streptomyces coelicolor M512(clo-SA2)/pSA11 cultivated in CDM medium.
(B) Purified compound isolated from cultures in CDM medium.
(C) Mass spectrometric fragmentation of novclobiocin 401 obtained by selected ion monitoring chromatograms. LC-ESI-MS mass scans were performed in
negative mode for m/z 643. The suggested fragmentation scheme for the compound is shown.
Chemistry & Biology 18, 304–313, March 25, 2011 ª2011 Elsevier Ltd All rights reserved 307

Chemistry & Biology
Artificial Pathway to a New Coumarin Antibiotic
Structure Elucidation of Novclobiocin 401
Gyrases (supercoiling assays)
A
LC-MS analysis in negative mode showed the presence of the
molecular ion of m/z 643 [M-H]-, corresponding to the expected
molecular mass of 644 for novclobiocin 401. MS/MS analysis
(Figure 4C) showed a fragmentation pattern corresponding to
that identified in previous mass spectrometric studies of amino-
coumarin antibiotics (Kammerer et al., 2004). High resolution
ESI-MS in positive mode showed a molecular ion of m/z =
645.14858, which is in agreement with the calculated value of
m/z = 645.14818 (C30H30N2O12Cl [M+H]+; D 0.62 ppm).
Novclobiocin 401 concentrations [μM]
C
0
0.13 0.06 0.03 0.02 0.008 0.004 0.002 0.001
Unidimensional (1H NMR, 13C NMR) and multidimensional
(1H-1H COSY, 1H HSQC, and 1H HMBC) NMR spectroscopy
confirmed the expected structure. Chemical shifts for the
substituted pyrrole, the deoxysugar and the aminocoumarin
Topoisomerases IV (decatenation assays)
Novclobiocin 401 concentrations [μM]
B
´
moiety were in accordance to those of clorobiocin (Eustaquio
et al., 2003). The chemical shifts and coupling patterns of the
protons in 1H NMR spectra of novclobiocin 401 showed the
catechol structure of the new acyl moiety. A strong high-field
C
0
50
40
30
20
10
5
2.5
1.25
13C NMR shift for carbon C-2 (dC = 116.1) and C-4 (dC
=
=
150.8) in novclobiocin 401 compared to clorobiocin (dC-2
130.9, dC-4 = 161.0) indicated a new hydroxyl substitution in
ortho-position (C-3). Additionally, the NMR spectra of novclo-
biocin 401 lack all proton and carbon NMR signals of the dime-
thylallyl group of the native clorobiocin. Full comparative 1H
and 13C NMR spectroscopic data of novclobiocin 401 and clor-
obiocin as well as 1H-1H COSY, 1H HSQC and 1H HMBC corre-
lations from 2D NMR experiments for novclobiocin 401 are
given in Table S1, Figures S2 and S3, and Supplemental Exper-
imental Procedures.
Figure 5. Determination of the Inhibitory Activity by Supercoiling
and Decatenation Assays
Supercoiling (A) and decatenation (B) assays with E. coli and S. aureus DNA
gyrase and topoisomerase IV. The first lane labeled with C contains control
assays without enzyme. The lane labeled 0 contains assays with addition of
3 ml solvent (5% aqueous DMSO). The following lanes contain assays to which
the indicated amount of antibiotic, dissolved in 5% DMSO, has been added. In
the supercoiling assays, the lower band shows supercoiled pBR322 DNA,
formed under catalysis of DNA gyrase. In the decatenation assays, the lower
band shows decatenated kinetoplast DNA, formed under catalysis of topoiso-
merase IV.
Inhibitory activities against E. coli and S. aureus
DNA Gyrase and Topoisomerase IV
The new compound was investigated in vitro for its inhibitory
effects on E. coli and S. aureus DNA gyrase and topoisomerase
IV in comparison with the natural antibiotics clorobiocin and
novobiocin. Two different assays were used: a DNA gyrase
supercoiling assay and a topoisomerase IV decatenation assay.
The 50% inhibitory concentrations (IC50s) of novclobiocin 401 Construction of E. coli Mutants for Investigation of
were determined as 0.006 mM for S. aureus DNA gyrase and Antibiotic Import by Catechol Siderophore Transporters
0.03 mM for E. coli DNA gyrase (Figure 5). These inhibitory As reported above, E. coli gyrase is equally sensitive to clorobio-
concentrations are identical to those observed with clorobiocin cin and novclobiocin 401. A difference in the antibacterial activity
and lower than those observed with the clinically used novobi- of these two compounds may therefore be primarily caused by
ocin (inhibitory concentration: 0.01 mM and 0.08 against the either increased influx or decreased efflux. In order to observe
two gyrases, respectively). This clearly proves that the replace- differences in influx without interferences by differences in efflux,
ment of the genuine Ring A moiety with a 3,4-DHBA moiety we inactivated the gene tolC which codes for an essential part of
had not affected the potency of the compound as gyrase the AcrAB/TolC and AcrAD/TolC drug efflux pumps of E. coli
inhibitor.
DNA gyrase inhibitors of the fluoroquinolone class also inhibit
(Blair and Piddock, 2009).
E. coli possesses three outer membrane transporters for active
topoisomerase IV which is very similar to gyrase (Maxwell and import of catechol siderophores, i.e., Fiu, Cir, and FepA (Braun
Lawson, 2003). Thus, we examined whether novclobiocin 401 and Hantke, 2001). All three transporters receive the energy for
also inhibits the decatenation activity of E. coli and S. aureus active transport from the periplasmic binding protein TonB.
topoisomerase IV. However, just as novobiocin and clorobiocin Expression of these proteins is upregulated under conditions of
the new compound inhibited topoisomerase IV from S. aureus iron starvation. Bacterial growth during an infection in the human
and E. coli only at much higher concentrations than required body represents a condition of extreme iron starvation (Chu et al.,
for gyrase inhibition (IC50 values: 35 mM for S. aureus topoiso- 2010). For functional studies of catechol siderophore transport in
merase IV and >50 mM for E. coli topoisomerase IV) (Figure 5). E. coli, conditions of iron starvation are usually created by (1)
Therefore, novclobiocin 401 is expected to act primarily as mutation of entC which abolishes the biosynthesis of the impor-
gyrase inhibitor in both S. aureus and E. coli.
tant siderophore enterobactin (Crosa and Walsh, 2002); (2)
308 Chemistry & Biology 18, 304–313, March 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Artificial Pathway to a New Coumarin Antibiotic
Figure 6. Determination of the Antibacterial
Activity by Disk Diffusion Assays
E. coli (ΔtonB/ΔtolC/ΔentC)
E. coli (ΔtolC/ΔentC)
The antibacterial activity of clorobiocin, novo-
biocin, and novclobiocin 401 was determined
against E. coli mutants with and without active
TonB. To improve visualization of the inhibition
zones, living cells were stained with 2,3,5-tri-
phenyltetrazolium chloride.
clorobiocin
novobiocin
DtonB/DtolC/DentC triple mutant, novo-
biocin and clorobiocin gave the same
MIC values as observed against the
DtolC/DentC double mutant (23 and
12 mg/ml, respectively). However, novclo-
novclobiocin
401
addition of the iron chelator 2,20-bipyridyl to the culture medium biocin 401 showed much less activity against the tonB-deficient
¨
(Alves et al., 2010; Mollmann et al., 2009). We therefore decided triple mutant (MIC: 47 mg/ml) than against the double mutant with
active tonB (MIC: 6 mg/ml). This shows again that TonB-depen-
to use DentC mutants and 2,20-bipyridyl containing media.
The involvement of the catechol siderophore transporters Fiu, dent transport is important for the antibacterial activity of novclo-
Cir, and FepA in the uptake of novclobiocin 401 can be investi- biocin 401, but not of novclobiocin and clorobiocin.
gated by comparing mutants with and without active TonB for
Against the wild-type strain E. coli K-12, the MICs of novobi-
their sensitivity to clorobiocin and novclobiocin 401. As ocin, clorobiocin and novclobiocin 401 resulted as 375, 47,
described above, TonB energizes all three catechol transporters. and 95 mg/ml. Therefore, against the wild-type novclobiocin
Due to these considerations, we decided to generate an E. coli 401 was less active than its parent compound clorobiocin (see
DtolC/DentC mutant as well as an E. coli DtonB/DtolC/DentC Discussion). The poor activity of aminocoumarins against wild-
mutant and to compare their sensitivity to clorobiocin and nov- type strains of E. coli is in accordance with previous observations
clobiocin 401. Single gene in-frame deletion mutants of entC, (Anderle et al., 2008; Galm et al., 2004b).
tolC, and tonB in E. coli K-12 were available from the Keio collec-
tion (Baba et al., 2006). Using these strains, we generated E. coli DISCUSSION
DtolC/DentC and E. coli DtonB/DentC double mutants as well as
an E. coli DtonB/DtolC/DentC triple mutant by Red/ET-mediated The present study provides an example for the rational design of
recombination (see Experimental Procedures). The correct a structurallymodifiedantibioticby utilizing synthetic biology prin-
genotype of the mutants was verified by PCR analysis of the ciples. Ourengineeringstrategy involved genes fromfour different
genomic DNA of the mutant.
organisms: (1) the Gram-negative organism E. coli as the source
of ubiC, which codes for an enzyme of an anabolic pathway; (2)
the Gram-positive organism Corynebactericum cyclohexanicum
Antibacterial Activity of Novclobiocin 401
The antibacterial activity of novclobiocin 401, in comparison to as the source of pobA, coding for an enzyme of a catabolic
clorobiocin and novobiocin, was first tested in disk diffusion pathway; (3) Streptomyces roseochromogenes as the source of
assays with the E. coli DtolC/DentC and the E. coli DtonB/ the biosynthetic gene cluster of clorobiocin, which was modified
DtolC/DentC mutants. To improve visualization of the inhibition by deletion of cloQ; (4) Streptomyces coelicolor M512 as heterol-
zones, living cells on the agar plates were stained with 2,3,5-tri- ogous expression host. S. coelicolor M512 (Floriano and Bibb,
phenyltetrazolium chloride. The results are shown in Figure 6. 1996) is a derivative of the strain S. coelicolor A3(2). Strain M512
Against the mutant with intact TonB, novclobiocin 401 shows does not produce three of the genuine antibiotics of strain A3(2),
approximately 2-fold higher activity than clorobiocin, and i.e., methylenomycin, actinorhodin, and undecylprodigiosine,
4-fold higher activity than novobiocin. In contrast, against the which facilitates the detection and isolation of heterologously
E. coli mutant with deleted tonB novclobiocin 401 is approxi- produced compounds. The modified clorobiocin gene cluster
mately 10-fold less active than clorobiocin. This shows that was stably integrated into the chromosome of this organism,
TonB-dependent transport plays an important role in the anti- and the synthetic operon containing ubiC and pobA under control
bacterial activity of novclobiocin 401 but not in the activity of of the strong constitutive ermE* promoter was expressed from
clorobiocin and novobiocin.
a replicative plasmid. This readily resulted in the production of
We also determined the minimal inhibitory concentrations in the desired compound novclobiocin 401. The yield of novclobio-
a liquid culture, following the procedure described by Wiegand cin 401 exceeded that of clorobiocin reported for both the wild-
et al. (2008), but including 50 mM 2,20-bipyridyl into the culture type and for a heterologous producer strain (Eustaquio et al.,
´
media. Under these conditions, the minimal inhibitory concentra- 2005). Even without extensive optimization experiments, novclo-
tions of novobiocin and clorobiocin against E. coli DtolC/DentC biocin 401 represented a major compound in the culture extract
double mutants were 23 and 12 mg/ml, respectively. The (Figure 4A) which facilitated the preparative isolation of the
MIC of novclobiocin 401 was 6 mg/ml, showing again that novclo- compound. Recently, improved heterologous expression strains
biocin 401 had a higher antibacterial activity than its parent have been derived from S. coelicolor and may offer a benefit in
compound clorobiocin against this mutant. Against the future experiments (Gomez-Escribano and Bibb, 2010).
Chemistry & Biology 18, 304–313, March 25, 2011 ª2011 Elsevier Ltd All rights reserved 309

Chemistry & Biology
Artificial Pathway to a New Coumarin Antibiotic
The production of novclobiocin 401 did not result in any may therefore aim at a reduced efflux, either by modification of
impairment of growth of the heterologous producer strain. the drug or by combination with an efflux pump inhibitor, and
Apparently, both the diversion of chorismate for the production at a further improvement of active import, e.g., by inclusion of
of 3,4-DHBA and the accumulation of the potent gyrase inhibitor a 2,3-DHBA moiety which is the catechol moiety present in en-
novclobiocin 401 were tolerated well. Self-resistance of the terobactin. The chemical synthesis of a clorobiocin derivative
heterologous producer strain was expected, as the clorobiocin with a 2,3-DHBA moiety is not straightforward, since a clorobio-
gene cluster, expressed in S. coelicolor M512, contains an cin derivative lacking Ring A (e.g., containing only Rings B and C,
´
aminocoumarin resistance gene (Eustaquio et al., 2005).
Figure 1) can not be obtained from any producer strain, due to
The genetic engineering experiment resulted in a clorobiocin the fact that Ring A is the starter moiety for clorobiocin biosyn-
derivative in which the genuine Ring A (Figure 1) is replaced thesis. The de novo chemical synthesis of an entire aminocou-
with a 3,4-DHBA moiety. In vitro investigation of the inhibitory marin antibiotic is a complicated multistep procedure (Laurin
effect of this compound on the gyrases of E. coli and S. aureus et al., 1999). Generation of a clorobiocin derivative with a 2,3-
showed no change of activity in comparison to the parent DHBA moiety by genetic engineering depends on the identifica-
compound clorobiocin. This confirms that the structure of tion of an aminocoumarin acyl ligase which, in contrast to the
Ring A is of little importance for the interaction of aminocoumarin presently known enzymes, can accept 2,3-DHBA as substrate
antibiotics with their principal target, and can be modified to (Figure 2). A search for such an aminocoumarin acyl ligase is
introduce desirable motifs into the molecule. It should be noted currently under way in our laboratory.
that the IC50 of novclobiocin 401 against the gyrases of E. coli
and Staphylococcus aureus is two to three orders of magnitude
lower than that of modern fluoroquinolones (Takei et al., 2001),
confirming the strong potency of aminocoumarins.
SIGNIFICANCE
The present study provides an example that the rational
modification of the structure of an antibiotic can be achieved
by a synthetic biology approach. Specifically, we planned to
replace a structural moiety which was not required for anti-
biotic activity by a different moiety which would serve as an
‘‘import handle,’’ facilitating the import of the antibiotic into
the bacterial cell by catechol siderophore transporters. Our
study provides a proof-of-principle for this strategy. It
confirms earlier results that catechol siderophore trans-
porters can be used in a ‘‘Trojan horse’’ approach for the
active import of an antibiotic. We achieved the incorporation
of a catechol motif into the structure of the antibiotic utilizing
a synthetic biology strategy. Such strategies are promising
for the generation of new bioactive molecules in future.
The growing number of sequenced biosynthetic gene clus-
ters and microbial genomes provides a rich pool for enzymes
which can be reassembled to generate artificial biosynthetic
pathways. In the present study, genes from three different
organisms were combined in a fourth organism, resulting
in the biosynthesis of the desired antibiotic. DNA synthesis
was used to adapt the codon usage of two of the introduced
genes to the requirement of the expression host. We used
a genetically engineered and fully sequenced expression
host which is amenable to rapid genetic manipulation
and devoid of several interfering biosynthetic pathways.
Considering the current advances in the technologies for
DNA sequencing, DNA synthesis, DNA manipulation, and
engineering of expression hosts, many new bioactive
compounds may be accessible in future by the generation
of artificial biosynthetic pathways. The present study
provides an example for such an approach.
Novclobiocin 401, containing a catechol motif, showed higher
antibacterial activity than its parent compound clorobiocin
against E. coli mutants which were defective in their TolC-depen-
dent efflux pumps. When additionally tonB was deleted, the
activity of novclobiocin 401 was reduced eightfold (as calculated
from the MIC values), while the activity of clorobiocin remained
unchanged. TonB supplies the energy for the catechol sidero-
phore transporters Cir, Fiu, and FepA, and therefore this result
strongly suggests that these transporters are involved in the
active import of novclobiocin 401. Previous experiments showed
that especially Cir and Fiu have a broad substrate specificity and
can import catechol compounds with quite different structures
(Heinisch et al., 2003).
Uptake of drug-siderophore conjugates by Cir and Fiu has
been demonstrated previously, especially for cephalosporin-
¨
catechol conjugates (Heinisch et al., 2003; Mollmann et al.,
2009). In these experiments, the catechol moiety conjugated to
the antibiotic added considerably to the molecular weight of
the drug; often the size of the catechol moiety exceeded the
size of the antibiotic moiety. In the present study, a preexisting
moiety (i.e., Ring A) of the antibiotic was replaced by a catechol
moiety, and the molecular weight of the new catechol compound
(645 Da) was lower than that of the parent compound clorobiocin
(697 Da).
Though novclobiocin 401 was transported into E. coli cells
under involvement of TonB-dependent transporters, it did not
show higher activity than clorobiocin against wild-type and
DentC mutants of E. coli. The MIC values of clorobiocin and nov-
clobiocin 401 against DentC mutants were 23 and 95 mg/ml,
respectively. Under identical conditions, the MICs against
DentC/DtolC double mutants were 12 and 6 mg/ml, respectively.
These results suggest that novclobiocin 401 is more rapidly ex-
ported than clorobiocin by TolC-dependent drug efflux pumps.
The introduction of a catechol moiety into the clorobiocin mole-
cule therefore had the desired effect to facilitate active import by
catechol siderophore transporters, but also the undesired effect
to accelerate TolC-dependent efflux. Future attempts to improve
the activity of aminocoumarins against Gram-negative organism
EXPERIMENTAL PROCEDURES
Chemicals
Novobiocic acid was isolated from Streptomyces spheroides AM1T2 (Steffen-
sky et al., 2000b); 3-amino-4,7-dihydroxy-8-methyl-coumarin (Ring B) was
kindly provided by Pharmacia & Upjohn, Inc. (Kalamazoo, MI). 3-Dimethyl-
allyl-4-hydroxybenzoic acid (Ring A) was obtained by hydrolysis of novobiocin
(Kominek and Meyer, 1975). Novobiocin and catechol compounds were
310 Chemistry & Biology 18, 304–313, March 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Artificial Pathway to a New Coumarin Antibiotic
purchased from Sigma-Aldrich. Clorobiocin was kindly provided by A. Maxwell
(John Innes Centre, Norwich, UK).
Streptomyces coelicolor (DNA2.0 Gene Designer Software). The two genes
were linked by translational coupling and flanked by SpeI and HindIII restriction
sites. The DNA fragment was commercially synthesized by DNA2.0 (Menlo
Park, CA) and provided in the vector pJ201. The nucleotide sequence of the
synthetic ubiC/pobA DNA fragment is shown in Figure S1.
Bacterial Strains, Plasmids, Cosmids, and Culture Conditions
The E. coli and Streptomyces strains, plasmids, and cosmids used in this study
are listed in Table S2. E. coli strains were cultivated in liquid or on solid LB
medium at 37ꢀC. Streptomyces strains were routinely precultivated in liquid
TSB medium (BD Bioscience) at 30ꢀC for 2 days. The cultivation was continued
in distillers’ solubles medium (Mancy et al., 1974), GYM medium (Shima et al.,
1996), and CDM medium (Kominek, 1972) for antibiotic production at 30ꢀC for
5–8 days. Standard methods for cultivation, DNA isolation and manipulation
were performed as described by Sambrook and Russell, (2001) and Kieser
et al. (2000).
Construction of the Plasmid pSA11
The synthetic ubiC/pobA fragment was isolated from the pJ201 vector
(DNA2.0) by restriction digest with SpeI and HindIII and cloned into
pUWL201 (Doumith et al., 2000) which contains the constitutive ermE*
promoter for foreign gene expression. Transformation of pSA11 into the inte-
gration mutant S. coelicolor M512(clo-SA2) was carried out by PEG-mediated
protoplast transformation (Kieser et al., 2000). Transformed colonies appeared
after 5 days at 30ꢀC.
Assay for Aminocoumarin Acyl Ligase
Production and Purification of Novclobiocin 401
The assay contained 2 mM of the respective acyl substrate (3,4-dihydroxyben-
zoic acid, 2,3-dihydroxybenzoic acid, caffeic acid, 3,4-dihydroxyphenylacetic
acid, 3,4-dihydroxypropionic acid or 3,4-dihydroxymandelic acid), 2 mM 3-
amino-4,7-dihydroxy-8-methyl-coumarin (Ring B), 5 mM ATP, 5 mM MgCl2,
10 mM ascorbic acid, 100 mm Tris-HCl (pH 8.0) and 10 mg of the respective
enzyme in a final volume of 100 ml. The reaction was carried out for 45 min
at 30ꢀC and stopped by addition of 5 ml 1.5 M trichloroacetic acid. The reaction
mixture was extracted with 100 ml ethyl acetate. The organic layer was used for
analysis. After evaporation of the solvent and dissolution in methanol, the
sample was analyzed by HPLC with a Multosphere RP18-5 column (250 3
4 mm, 5 mm; Agilent) at a flow rate of 1 ml$min-1. A linear gradient from 60%
to 100% solvent B (solvent A H2O/HCOOH 99:1; solvent B MeOH/HCOOH
99:1) over 30 min was used. UV detection was carried out at 330 nm. Novobio-
cic acid was used as standard.
S. coelicolor M512(clo-SA2)/pSA11 was precultivated in TSB medium (BD
Bioscience) with 50 mg/ml thiostrepton for 3 days and then used to inoculate
40 flasks, each containing 50 ml CDM production medium (Kominek, 1972)
with 50 mg/ml thiostrepton. The cultivation was carried out for 5 days at
30ꢀC and 210 rpm. The culture was adjusted to pH 4 with hydrochloric acid
and extracted twice with an equal volume of ethyl acetate. The organic layer
was evaporated to dryness. The residue was dissolved in methanol and puri-
fied by HPLC with a Multosphere column 120 RP18-5 (250 3 20 mm, 5 mm;
C&S Chromatographie Service Du¨ ren, Germany) at a flow rate of 2 ml/min. A
linear gradient from 70% to 100% solvent B in solvent A over 36 min was
used (solvent A H2O/HCOOH 99:1; solvent B MeOH: HCOOH 99:1).
For analytical purposes, 1 ml bacterial culture was acidified with HCl to pH 4
and extracted with equal volume of ethyl acetate. After evaporation of the
solvent, the residue was redissolved in 100 ml methanol. Eighty microliters
was analyzed by HPLC with a Multosphere RP18-5 column (250 3 4 mm,
5 mm; Agilent) at a flow rate of 1 ml/min. A linear gradient from 60% to 100%
solvent B over 23 min was used. UV detection was carried out at 280 nm.
Inactivation of cloQ in Cosmid clo-BG1 and Heterologous Expression
In cosmid clo-BG1 (Eusta´ quio et al., 2005) cloQ was replaced by Red/ET-
mediated recombination with an apramycin-resistance (aac(3)IV) cassette
that was flanked by XbaI and SpeI recognition sites. For replacement of
cloQ, the cassette was generated by PCR using pUG019 (Eusta´ quio et al.,
2005) as template and the primers cloQ_f (50-GGC GCG CCC ATT GCT CAC
CGT CTT ACC GAC ACC GTC CTT ATT CCG GGG ATC TCT AGA TC-30)
and cloQ_r (50-TCC CAT GGT CGA TTC CGT GTG TTG GTG AAG TGC GCG
CAG ACT AGT CTG GAG CTG CTT C-30). Underlined letters indicate the
XbaI and SpeI restriction sites. PCR amplification was performed in 50 ml
volume with 100 ng template, 0.25 mM dNTPs, 50 pmol of each primer, and
5% (v/v) DMSO with the Expand High Fidelity PCR system (Roche Molecular
Biochemicals): denaturation at 94ꢀC for 2 min, then 10 cycles with denatur-
ation at 94ꢀC for 45 s, annealing at 50ꢀC for 45 s, and elongation at 72ꢀC for
90 s, followed by 15 cycles with annealing at 55ꢀC for 45 s, and the last elon-
gation step at 72ꢀC for 5 min. The PCR product was introduced by electropo-
ration into E. coli BW25113/pIJ790 harboring cosmid clo-BG1 (Eusta´ quio
et al., 2005). The resulting modified cosmid was isolated, transformed into
the nonmethylating strain E. coli ET12567, reisolated, and digested with
XbaI and SpeI to remove the apramycin-resistance cassette. Religation over-
night at 4ꢀC gave the cosmid clo-SA2.
DNA Gyrase and Topoisomerase IV Activity Assays
Relaxed pBR322 DNA and kDNA (from Crithidia fasciculata), DNA gyrase and
topoisomerase IV enzymes from E. coli and S. aureus were obtained from In-
spiralis (Norwich, UK). DNA gyrase and topoisomerase IV holoenzymes were
reconstituted by mixing approximately equimolar amounts of recombinant
E. coli and S. aureus GyrA and GyrB and ParC and ParE subunits, respectively.
DNA gyrase activity was measured by a supercoiling assay that monitored the
ATP-dependent conversion of relaxed pBR322 DNA to the supercoiled form.
Topoisomerase IV activity was measured by a decatenation assay that moni-
tored the ATP-dependent unlinking of DNA minicircles from kDNA. Details of
the assay procedures are described in the Supplemental Experimental
Procedures.
Agar Diffusion Tests
Agar plates (40 ml medium) were prepared by suspending 1 ml of a culture of
the respective E. coli mutant (overnight culture in LB medium, OD600 1.2) and
50 mM 2,20-bipyridyl in melted Mueller-Hinton agar (Roth). To paper disks of
7 mm diameter, different amounts of the respective antibiotic, dissolved in
15 ml methanol, were applied. These disks were placed on the agar plates
which were incubated at 37ꢀC for 16 hr. For visualization of living cells, 5 ml
of 0.5% aqueous 2,3,5-triphenyltetrazolium chloride (Roth) were added to
the plates, and after further incubation for 10 min, the inhibition zones were
determined.
The cosmid clo-SA2 isolated from E. coli ET12567 was introduced into
S. coelicolor M512 by PEG-mediated protoplast transformation (Kieser
et al., 2000). Clones resistant to kanamycin were selected. Feeding experi-
ments were carried out by addition of 1 mg Ring A (3-dimethylallyl-4-hydrox-
ybenzoic acid) dissolved in 100 ml ethanol to 80 ml S. coelicolor M512(clo-SA2)
´
culture in distillers’ solubles medium (Eustaquio et al., 2003).
For mutasynthesis experiments, mg of the respective catechol
3
compounds were dissolved in 100 ml ethanol and added to 80 ml of the culture
of the cloQ-defective strain S. coelicolor M512(clo-SA2) in distillers’ solubles
medium one day after inoculation. After 5–8 days cultivation at 30ꢀC and
210 rpm, the cultures were extracted and analyzed by HPLC described below.
Generation of E. coli Mutants
E. coli double and triple mutants were generated from E. coli K-12 MG1655
mutants obtained from the Keio collection (Baba et al., 2006). The gene entC
was replaced in E. coli JW5195-1/pIJ790 and JW5503-1/pIJ790 using Red/
ET-mediated recombination (Gust et al., 2003). An apramycin resistance
cassette [acc(3)IV] was amplified from plasmid pIJ773 (Gust et al., 2003).
The primers used for PCR were as follows: entC_f: (50-TCA TTA TTA AAG
CCT TTA TCA TTT TGT GGA GGA TGA TAT GAT TCC GGG GAT CCG TCG
ACC-30) and entC_r (50-CCG GCC AGC GGG TGA ATG GAA TGC TCA TCC
TCG CTC CTT ATG TAG GCT GGA GCT GCT TC-30). Italic letters represent
Design of the Synthetic Gene Operon
The nucleotide sequences of the genes pobA coding for a 4-hydroxybenzoate
hydroxylase from Corynebacterium cyclohexanicum (GenBank AB210281)
and ubiC coding for a chorismate pyruvate-lyase from Escherichia coli
536 (GenBank NC_008253) were redesigned using the codon preference of
Chemistry & Biology 18, 304–313, March 25, 2011 ª2011 Elsevier Ltd All rights reserved 311

Chemistry & Biology
Artificial Pathway to a New Coumarin Antibiotic
39 nucleotide homologous extensions for Red/ET-mediated recombination.
The gene tolC was replaced in E. coli JW5195-1/pIJ790 using Red/ET-medi-
ated recombination (Gust et al., 2003). For this purpose, a streptomycin resis-
tance cassette [aadA] was amplified from plasmid pIJ778 (Gust et al., 2003)
using the primer pair tolC_f (50-GAT CGC GCT AAA TAC TGC TTC ACC
ACA AGG AAT GCA AAT GAT TCC GGG GAT CCG TCG ACC-30) and tolC_r
(50-ACG TTC AGA CGG GGC CGA AGC CCC GTC GTC GTC ATC ATG TAG
GCT GGA GCT GCT TC-30). The genotype of the resulting mutants was
confirmed by PCR with chromosomal DNA.
Flinspach, K., Westrich, L., Kaysser, L., Siebenberg, S., Gomez-Escribano,
J.P., Bibb, M., Gust, B., and Heide, L. (2010). Heterologous expression of
the biosynthetic gene clusters of coumermycin A1, clorobiocin and capraza-
mycins in genetically modified Streptomyces coelicolor strains. Biopolymers
93, 823–832.
Floriano, B., and Bibb, M. (1996). AfsR is a pleiotropic but conditionally
required regulatory gene for antibiotic production in Streptomyces coelicolor
A3(2). Mol. Microbiol. 21, 385–396.
Fujii, T., and Kaneda, T. (1985). Purification and properties of NADH/NADPH-
dependent p-hydroxybenzoate hydroxylase from Corynebacterium cyclohex-
anicum. Eur. J. Biochem. 147, 97–104.
SUPPLEMENTAL INFORMATION
Galm, U., Dessoy, M.A., Schmidt, J., Wessjohann, L.A., and Heide, L. (2004a).
In vitro and in vivo production of new aminocoumarins by a combined
biochemical, genetic, and synthetic approach. Chem. Biol. 11, 173–183.
Supplemental Information includes Supplemental Experimental Procedures,
three figures, and two tables and can be found with this article online at
doi:10.1016/j.chembiol.2010.12.016.
Galm, U., Heller, S., Shapiro, S., Page, M., Li, S.M., and Heide, L. (2004b).
Antimicrobial and DNA gyrase-inhibitory activities of novel clorobiocin deriva-
tives produced by mutasynthesis. Antimicrob. Agents Chemother. 48, 1307–
1312.
ACKNOWLEDGMENTS
We are grateful to K. Hantke (Tu¨ bingen University) and U. Mo¨ llmann (Hans
Kno¨ ll Institut, Jena) for their valuable advice and support. This work was sup-
ported by a grant from the Deutsche Forschungsgemeinschaft (SFB 766).
Gomez-Escribano, J.P., and Bibb, M. (2010). Engineering Streptomyces
coelicolor for heterologous expression of secondary metabolite gene clusters.
Microb. Biotechnol., in press. Published online October 26, 2010. doi:10.1111/
j.1751–7915.2010.00219.x.
Received: November 2, 2010
Revised: December 1, 2010
Accepted: December 2, 2010
Published: March 24, 2011
Gust, B. (2009). Cloning and analysis of natural product pathways. Methods
Enzymol. 458, 159–180.
Gust, B., Challis, G.L., Fowler, K., Kieser, T., and Chater, K.F. (2003). PCR-tar-
geted Streptomyces gene replacement identifies a protein domain needed for
biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci.
USA 100, 1541–1546.
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