Discovery and Biosynthesis of Bolagladins: Unusual Lipodepsipeptides from Burkholderia gladioli Clinical Isolates**

Article abstract of DOI:10.1002/anie.202009110

Two Burkholderia gladioli strains isolated from the lungs of cystic fibrosis patients were found to produce unusual lipodepsipeptides containing a unique citrate-derived fatty acid and a rare dehydro-β-alanine residue. The gene cluster responsible for the

Full text of DOI:10.1002/anie.202009110

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
International Edition Chemie
www.angewandte.org
Accepted Article
Title: Discovery and biosynthesis of bolagladins: unusual
lipodepsipeptides from Burkholderia gladioli clinical isolates
Authors: Yousef Dashti, Ioanna Nakou, Alex Mullins, Gordon Webster,
Xinyun Jian, Eshwar Mahenthiralingam, and Gregory Challis
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202009110
Link to VoR: https://doi.org/10.1002/anie.202009110

Angewandte Chemie International Edition
10.1002/anie.202009110
RESEARCH ARTICLE
Discovery and biosynthesis of bolagladins: unusual
lipodepsipeptides from Burkholderia gladioli clinical isolates
Yousef Dashti,[a]† Ioanna T. Nakou, Alex J. Mullins, Gordon Webster, Xinyun Jian,
[a]
[b]
[b]
[a, c]
Eshwar
Mahenthiralingam, and Gregory L. Challis[a, c, d]*
[
b]
[
a]
Dr. Y. Dashti, I. T. Nakou, Dr. X. Jian, Prof. G. L. Challis
Department of Chemistry
University of Warwick
Coventry CV4 7AL, United Kingdom
E-mail: g.l.challis@warwick.ac.uk
[b]
[c]
[d]
Dr. A. J. Mullins, Dr. G. Webster, Prof. E. Mahenthiralingam
Microbiomes, Microbes and Informatics Group, Organisms and Environment Division, School of Biosciences
Cardiff University
Cardiff, CF103 AT, United Kingdom
Dr. X. Jian, Prof. G. L. Challis
Warwick Integrative Synthetic Biology Centre
University of Warwick
Coventry CV4 7AL, United Kingdom
Prof. G. L. Challis
Department of Biochemistry and Molecular Biology
Monash University
Clayton, VIC 3800, Australia
†
Current address: The Centre for Bacterial Cell Biology, Biosciences Institute, Medical School
Newcastle University
Newcastle upon Tyne, NE2 4AX, United Kingdom
Supporting information for this article is given via a link at the end of the document.
Abstract: Two Burkholderia gladioli strains isolated from the lungs of
and lactoferrin also contributes to low iron availability in
mammals.[ As a consequence, microorganisms have evolved
several strategies for obtaining iron from the environment and
their hosts. One strategy involves the production of siderophores,
specialised metabolites with a high affinity for ferric iron.[4] In
Gram-negative bacteria, ferric-siderophore complexes are
typically transported into the periplasm by TonB-dependent outer
membrane receptors. Periplasmic binding proteins then shuttle
the ferric-siderophore complexes to the inner membrane, where
ATP-binding cassette (ABC) transporters transfer them into the
cytoplasm.[ Once in the cytoplasm, iron is released from the
ferric-siderophore complex by reduction to the ferrous form and/or
hydrolytic cleavage of the ligand.[5a]
Several investigations of pathogenic microorganisms have
identified a strong link between siderophore production and
virulence.[6] In the early stages of infection, siderophores play a
key role in sequestering iron from host tissues. The identification
of siderophores produced by pathogenic microorganisms is
therefore an important field of study because interference with
3]
cystic fibrosis patients were found to produce unusual
lipodepsipeptides containing a unique citrate-derived fatty acid and a
rare dehydro-β-alanine residue. The gene cluster responsible for their
biosynthesis was identified by bioinformatics and insertional
mutagenesis. In-frame deletions and enzyme activity assays were
used to investigate the functions of several proteins encoded by the
biosynthetic gene cluster, which was found in the genomes of about
50% of B. gladioli isolates, suggesting that its metabolic products play
an important role in the growth and/or survival of the species. The
Chrome Azurol S (CAS) assay indicated that these metabolites bind
ferric iron, which suppresses their production when added to the
growth medium. Moreover, a gene encoding a TonB-dependent ferric-
siderophore receptor is adjacent to the biosynthetic genes, suggesting
that these metabolites may function as siderophores in B. gladioli.
5]
Introduction
siderophore-mediated iron uptake is an attractive strategy for
attenuating virulence.[7] The discovery of novel siderophores is
also relevant to other therapeutic applications. For example,
Iron is an essential element for most organisms. In biological
systems, iron is mainly present in two oxidation states: ferrous
and ferric.[ The interconversion of the ferrous and ferric
oxidation states is essential for a variety of redox reactions and
electron transfer processes in cells.[2] However, despite the
relatively high abundance of iron in the Earth’s crust, it is not
readily bioavailable. This is because it is predominantly in the
ferric form, which has low aqueous solubility.[3] Moreover,
sequestration by proteins such as hemoglobin, ferritin, transferrin,
1]
desferrioxamine
B (marketed as Desferal), a siderophore
[8]
produced by numerous Streptomyces species, is used to treat
iron overload resulting from multiple blood transfusions and
aluminium toxicity due to dialysis. However, because Desferal is
administered by injection and has multiple side effects, orally-
available alternatives with fewer side effects are actively being
1
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202009110
RESEARCH ARTICLE
sought.[9] In addition to clinical uses, siderophores show promise
for environmental and biotechnological applications.[10]
of comparative bioinformatics analyses, targeted gene deletions,
and enzyme activity assays elucidated several key steps in the
biosynthesis of these unusual natural products, which we propose
based on several lines of evidence may function as siderophores.
Burkholderia is a genus of highly diverse Gram-negative bacteria.
Species belonging to this genus have been isolated from soil,
water, plants, animals and humans.[11] Several investigations
have shown that they are prolific producers of specialised
metabolites,[ and genome sequence analyses have highlighted
their underexplored biosynthetic potential.[13] To date, pathogenic
Burkholderia species have been reported to produce
siderophores belonging to four distinct structural classes
12]
Results and Discussion
Isolation and structure elucidation of the bolagladins
B. gladioli BCC0238 produces gladiolin and icosalide A1 when
(
ornibactins/malleobactins,
pyochelin,
cepabactin,
and
cepaciachelin; Figure 1).[ Members of the cepacia complex and
pseudomallei group produce one or more of these siderophores.
In contrast, Burkholderia gladioli does not produce any of these
metabolites and was long believed to employ siderophore-
independent mechanisms for iron acquisition.[15] However,
Hertweck and co-workers recently reported that three B. gladioli
environmental isolates produce the diazeniumdiolate siderophore
gladiobactin.[
14]
grown on a solid minimal medium containing glycerol as the sole
[12i, 17]
carbon source.
Two novel metabolites were identified by
UHPLC-ESI-Q-TOF-MS analysis of extracts from cultures of a
gladiolin non-producing mutant of B. gladioli BCC0238 (with an
in-frame deletion in the region of gbnD1 encoding the ER domain)
grown on BSM medium containing ribose as an additional carbon
source alongside glycerol. The metabolites were isolated as
amorphous solids from ethyl acetate extracts of 3-day cultures (1L
total volume) using semi-preparative HPLC, and their planar
structures were elucidated using a combination of HR-ESI-MS
and 1D/2D NMR experiments (Figure 2; Tables S2 and S3,
Figures S3-S14).
16]
We have recently discovered that B. gladioli BCC0238, isolated
from the sputum of a child with cystic fibrosis (CF), produces
several specialised metabolites. These include gladiolin, a novel
macrolide with potent activity against drug resistant
Mycobacterium tuberculosis clinical isolates,[12i] and icosalide
A1,[17] an asymmetric lipopeptodiolide antibiotic originally isolated
from an Aureobasidium species fungus,[18] but subsequently
shown to be produced by various strains of B. gladioli including
one associated with the fungus.[17, 19] Analysis of the B. gladioli
BCC0238 complete genome sequence identified several gene
clusters encoding cryptic nonribosomal peptide synthetase
The molecular formulae of the two metabolites, which we named
bolagladins A 1 and B 2, respectively, were established as
14+: 824.4288, found:
C
39
H
61
N
5
O
14 (calculated for C39
H
62
N
5
O
14+:
824.4284) and
8
C
40
H
63
N
5
O
14 (calculated for
40 64 5
C H N O
38.4444, found: 838.4447) using positive ion ESI-Q-TOF-MS
(
Figure S17). Four resonances with chemical shifts characteristic
of protons attached to C-2 in -amino acids ( 4.17, 4.79, 5.36,
and 5.52) correlated with signals attributable to exchangeable N-
H
(
NRPS), polyketide synthase (PKS) and hybrid NRPS-PKS
assembly lines.[
12g]
Here we report the discovery of bolagladins A
H protons (
H
8.17, 8.70, 9.91, and 9.99, respectively) in the
and B, novel lipodepsipeptides containing a unique citrate-
derived fatty acid and a rare dehydro--alanine residue, as the
metabolic products of one of these gene clusters. A combination
1
COSY spectrum of bolagladin A 1. Further analysis of H NMR
and COSY spectra identified four -amino acid spin systems,
which in conjunction with HSQC and HMBC data, were assigned
to a valine residue, a homoserine (Hse) residue and two serine
residues in the sequence Ser-Hse-Val-Ser (Figure 2). A 3
J
CH
correlation from the β-protons of the N-terminal Ser (N-Ser)
Figure 2. Structures of bolagladin A (1) and B (2). COSY and key HMBC
correlations are shown for bolagladin A.
Figure 1. Structure of ornibactins, malleobactin E, pyochelin, cepaciachelin and
cepabactin identified from pathogenic strains of Burkholderia.
2
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202009110
RESEARCH ARTICLE
residue (
Ser (C-Ser) residue (
H
4.73 and 4.90) to the carbonyl carbon of the C-terminal
170.0) established the presence of a
residue in the tetradepsipeptide (
of the Dba residue (
( 11.56) and the carbonyl carbon of a fatty acid residue (
165.1). This indicates that the Dba residue links the fatty acid
residue to the tetradepsipeptide. The fatty acid residue is very
unusual, with Z-configured double bonds spanning C2-C3 and
C11-C12 (based on CH=CH coupling constants of 11.5 and 13.5
Hz, respectively), and two carboxyl acid groups and a methoxy
group appended to its tail (Figure 2).
H
9.99) and the carbonyl carbon
C
168.3), and the N-H proton of Dba residue
C
tetradepsipeptide (Figure 2). Analysis of the COSY and HMBC
data also revealed a dehydro-β-alanine (Dba) residue, with a
coupling constant of 14.0 Hz between the  and  protons
indicating that the double bond is E-configured. The only other
natural products reported to contain a Dba residue are the
enamidonins isolated from Streptomyces species.[20] 2JCH
correlations were observed between the N-H proton of the N-Ser
H
C
Figure 3. Organization of the bolagladin biosynthetic gene cluster (bolA-T) and proposed pathway for bolagladin biosynthesis. The precise timing of O-methylation
of the citrate-derived starter unit for fatty acid biosynthesis remains to be established, but is postulated to be an early step. Abbreviations are as follows: A,
adenylation domain; TE, thioesterase domain; C
aminoacyl thioesters; C , bifunctional epimerization-condensation domain; FAS, fatty acid synthase. Acyl carrier proteins (ACPs) and Peptidyl carrier protein (PCP)
domains are shown as black circles.
, chain initiating condensation domain; L
C , chain elongating condensation domain utilizing two L-configured
I L
E
3
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202009110
RESEARCH ARTICLE
The absolute configurations of the -amino acid residues in
bolagladin A 1 were determined as L-Ser-D-Val-L-Hse-L-Ser
using Marfey’s method[21] (Figure S15). Comparison of the NMR
spectroscopic data for bolagladins A 1 and B 2 showed that the
Val residue in the former is substituted by an Ile residue in the
gbnD1_ER via insertional mutagenesis. LC-MS analysis
confirmed that the production of bolagladins A 1 and B 2 was
abolished in the mutant (Figure 4).
Putative functions were assigned to the proteins encoded by the
bolA-bolT genes flanking bolH on the basis of sequence analyses
(Table S4). This enabled us to propose a plausible pathway for
bolagladin biosynthesis (Figure 3). To validate this pathway
experimentally, we envisioned examining the effect of in-frame
deletions in key biosynthetic genes on bolagladin production.
However, this proved to be challenging and time consuming in B.
gladioli BCC0238 (see supporting information), so we screened
other genome-sequenced B. gladioli isolates containing the bol
locus (see below) to establish whether they (i) produce the
bolagladins and (ii) are amenable to construction of in-frame
deletions. B. gladioli BCC1622 (also isolated from a CF lung
infection) was found to meet both criteria and an analogous
gladiolin non-producing mutant of this strain (B. gladioli BCC1622
∆gbnD1_ER) was created to enable in-depth functional studies of
selected bolagladin biosynthetic genes. BolR shows sequence
similarity to citrate synthase, a key enzyme in the Krebs cycle,
which catalyzes the condensation of acetyl-CoA with
oxaloactetate and hydrolysis of the resulting thioester to form
citrate (Figure 5a).[ This suggests that citryl-CoA may serve as
the starter unit for assembly of the unusual fatty acid residue
incorporated into the bolagladins (Figure 3). To verify the function
of BolR, we overproduced it in E. coli as an N-terminal His6-fusion
and purified it to homogeneity (see Supporting Information).
Incubation of the purified protein with oxaloacetate (4) and acetyl-
CoA (5) for two hours at room temperature resulted
2
latter, consistent with the difference of CH in the molecular
formulae. The absolute configurations of the -amino acids in
bolagladin B (2) were similarly shown to be L-Ser-D-allo-Ile-L-
Hse-L-Ser by Marfey’s method (Figure S15), using a C3 column
instead of a C18 column to separate the Marfey’s derivatives of
D-Ile and D-allo-Ile (Fig. S15).[22]
Overall, the structures of bolagladins A 1 and B 2 are highly
unusual, containing a unique fatty acid residue with several polar
functional groups appended to its tail, linked via a rare Dba
residue to a depsitetrapeptide. We thus sought to develop an
understanding of bolagladin biosynthesis, focusing on the origin
of the starter unit for the fatty acid assembly and the mechanism
of Dba incorporation.
Bolagladin biosynthesis
Bioinformatics analysis of the B. gladioli BCC0238 genome
sequence identified the putative bolagladin (bol) biosynthetic
gene cluster (Figure 3 and Table S4), which contains a gene
25]
(
bolH) encoding a tetramodular NRPS. Sequence analysis of the
adenylation (A) domains in this NRPS indicated that modules 1-4
activate L-Ser, L-Val/L-Ile, L-Hse and L-Ser, respectively (Table
S5),[23] suggesting the NRPS assembles the depsipeptide portion
of the bolagladins. Similarly, phylogenetic analysis of the BolH
condensation (C) domains indicated that they belong to the
[
24]
I
following groups. Module 1: chain initiating (C – links external
acyl donor and L-configured acyl acceptor); modules 2 and 4 –
L
chain elongating ( C
L
– links L-configured acyl donor and
bifunctional condensation
– epimerizes acyl donor to D-configuration and
acceptor); and module
epimerization (C
3
–
/
E
links with L-configured acyl acceptor) (Figure 3). This is consistent
with the structure elucidation data, which indicate that the
bolagladins contain L-Ser, L-Hse and D-Val/D-allo-Ile.
To verify the involvement of this gene cluster in bolagladin
biosynthesis, we inactivated bolH in B. gladioli BCC0238
6
x10
5
x10
3
2
1
0
5
4
3
2
1
4
x10
8
6
4
2
0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Time (min)
16.50 16.75 17.00 17.25 17.50 17.75 18.00 18.25 18.50 18.75
Time (min)
Figure 5. (a) Reaction catalyzed by BolR. (b) Extracted ion chromatograms for
-
m/z = 130.9986, 808.1185, 191.0197, and 766.1079 corresponding to [M-H] for
Figure 4. Comparison of the extracted ion chromatograms for m/z = 824.4288
1) and 838.4444 (2) from UHPLC-ESI-Q-TOF MS analyses of culture extracts
from B. gladioli BCC0238 gbnD1_ER (black) and B. gladioli BCC0238
gbnD1_ER-bolH (red). Insertional mutagenesis in bolH confirmed the
involvement of the bol cluster in bolagladin production.
4, 5, 6 and 7 from negative ion mode UHPLC-ESI-Q-TOF-MS analyses of the
(
BolR-catalyzed condensation of oxaloacetate with acetyl-CoA (top panel) and a
negative control reaction containing BolR inactivated by boiling for 15 minutes
prior to addition (bottom panel). The measured m/z values for citrate and
coenzyme A were 191.0195 and 766.1076, respectively.

4
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202009110
RESEARCH ARTICLE
in complete conversion to products with molecular formulae
corresponding to citric acid (6) and coenzyme A 7 (Figure 5b).
To probe the role played by citrate in bolagladin biosynthesis, we
constructed an in-frame deletion in bolR. The resulting mutant
was unable to assemble bolagladins A and B, but produced a new
We postulated that BolS, which shows sequence similarity to S-
adenosyl-L-methione (SAM) dependent methyltransferases,
catalyzes methylation of the hydroxyl group in the citrate-derived
starter unit at some point during bolagladin biosynthesis. To test
this hypothesis, we constructed an in-frame deletion in bolS. The
production of bolagladins A 1 and B 2 was abrogated in the
resulting mutant and two new metabolites with the molecular
metabolite with the molecular formula C37
H
59
N
5
O
12 (calculated for
+
C
37
H
60
N
5
O12 : 766.4233, found: 766.4238; Figures 6 and S16).
59
N
5
O
14 (calculated for C38
60 5
H N O
14+: 810.4131,
The production level of this metabolite was insufficient for NMR
spectroscopic analysis. We therefore conducted LC-MS/MS
analyses, which indicated the metabolite has the same
formulae C38
found: 810.4124) and C39
H
H
61
5
N O14 (calculated for C39H N O
62 5
14+:
824.4288, found: 824.4296) were produced (Figures 7 and S16).
LC-MS/MS analyses indicated that these compounds are
depsipeptide core as bolagladin
B 2 (molecular formula
C
40
H
63
N
5
O
14
;
Figures and S16) and that the structural
6
2
congeners of 1 and 2, lacking a CH group from the fatty acid or
differences must lie in the fatty acid and/or Dba residues.
However, because the new metabolite contains three fewer
carbon atoms, four fewer hydrogen atoms and two fewer oxygen
atoms than bolagladin B, but the same number of nitrogen atoms,
these differences cannot be due to modification or loss of the Dba
residue. The most plausible structure for this metabolite is 3, in
which the fatty acid residue is derived from an oxaloacetyl-CoA
starter unit that is subsequently reduced (Figure 6). This suggests
that BolR may catalyze the condensation of acetyl-CoA with the
keto group of oxaloacetyl-CoA in bolagadin biosynthesis (Figure
S17), but further experiments will be required to confirm this.
Dba residues (Figure S17). Although production levels of these
metabolites were low, we were able to purify a sufficient quantity
of the bolagladin B congener for NMR spectroscopic analysis
(Table S6, Figures S18-S23). Differences were observed in the
1
3C chemical shifts of the resonances due to C-16, C-17, C-18, C-
19 and C-20 in the fatty acid residue and the signals attributed to
1
the O-methyl group in bolagladin B were absent from the H and
1
3C spectra. We therefore conclude that the two metabolites
accumulated in the bolS mutant are desmethyl-bolagladins A 8
and B 9. The low levels of 8 and 9 produced by the mutant suggest
O-methylation of the citrate-derived starter unit is an early step in
bolagladin biosynthesis, rather than a late-stage modification.
However, the precise timing of this reaction remains to be
determined.
Citrate (or its O-methylated derivative) is proposed to be
converted to the corresponding coenzyme A thioester by BolB,
which shows similarity to acyl-CoA synthetases (Figure 3 and
Table S4). Either BolM or BolP, both of which show similarity to

-ketoacyl-ACP synthase III (KAS III) enzymes that typically
initiate fatty acid biosynthesis in bacteria (Table S4),[ could then
26]
6
x10
4
3
2
1
6
x10
3
2
1
6
x10
1.5
1
.0
.5
0
1
4
15
16
17
18
19
20
Time (min)
14
15
16
17
18
19
20
Time (min)
Figure 7. (a) Structures of desmethyl-bolagladins A 8 and B 9 accumulated in
the bolS mutant of B. gladioli BCC1622 ∆gbnD1_ER. (b) Top panel: extracted
Figure 6. (a) Comparison of fragment ions observed for metabolite 3 and
bolagladin B (2) in MS/MS analyses (b) Extracted ion chromatograms (EICs)
from UHPLC-ESI-Q-TOF-MS analyses of extracts from B. gladioli BCC1622
+
ion chromatogram at m/z = 824.4288 (corresponding to [M+H] for 1) and
8
38.4444 (corresponding to [M+H]+ for 2) from UHPLC-ESI-Q-TOF MS
∆
gbnD1_ER and a bolR deletion mutant. From top to bottom, these are: EIC at
analyses of culture extracts from B. gladioli BCC1622 ∆gbnD1_ER. Bottom
+
m/z 824.4288 (corresponding to [M+H] for 1) and 838.4444 (corresponding to
+
panel extracted ion chromatogram at m/z = 810.4131 (corresponding to [M+H]
+
[
M+H] for 2) for the wild type strain; EIC at m/z 824.4288 and 838.4444 for the
for 8) and 824.4288 (corresponding to [M+H]+ for 9) from UHPLC-ESI-Q-TOF
MS analyses of culture extracts from bolS mutant of B. gladioli BCC1622
+
mutant; EIC at m/z 766.4233 (corresponding to [M+H] for 3) for the wild type
strain; and EIC at m/z 766.4233 for the mutant.
∆gbnD1_ER.
5
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202009110
RESEARCH ARTICLE
catalyse the elongation of citryl-CoA (or its O-methylated
derivative) with a malonyl group attached to the primary metabolic
fatty acid synthase (FAS) ACP (Figure 3). Interestingly, the
conserved active site Cys residue is mutated to Ser in BolM and
Thr in BolP. The functional significance of this is unclear, but an
analogous Cys to Ser mutation is observed in DpsC, a KAS III
homologue that has been reported to initiate assembly of the
daunorubicin polyketide chain using a propionyl-CoA starter
unit.[27] Further processing of the -ketothioester resulting from
elongation of (O-methyl)-citryl-CoA with malonyl-ACP by the
primary metabolic FAS would afford the saturated O-methyl-
citrate-primed fatty acyl-ACP thioester 10 (Figure 3). The 11, 12
and 2, 3 double bonds are likely introduced into this thioester by
BolF and BolL, which are similar to membrane-associated fatty
acid desaturases and acyl-ACP desaturases, respectively (Figure
analogous role in bolagladin biosynthesis (Figure 3). In-frame
deletion of bolN in B. gladioli BCC1622 ∆gbnD1 abolished
bolagladin production and it could not be restored by feeding β-
alanine to the mutant. This is consistent with the proposed roles
of BolO, BolC and BolN and rules out -alanine as an intermediate
in the biosynthetic pathway. The amino group of the -alanyl-BolC
thioester is proposed to be condensed with the citrate-primed fatty
acyl thioester by one of the KAS III homologues BolM or BolP
(Figure 3).
The sequence similarity of BolQ to acyl-CoA dehydrogenases
suggested it might be responsible for formation of the Dba residue
via desaturation of the N-acyl--alanyl-BolC thioester. To test this
hypothesis, we created an in-frame deletion in bolQ, which
abolished the production of bolagladins A and B and led to the
accumulation of two new metabolites with the molecular formulae
3
and Table S4), completing the assembly of the unusual fatty
39 63 5 65 5
C H N O14 and C40H N O14 (Figure 8). Purification and NMR
acid residue incorporated into the bolagladins.
spectroscopic analysis showed these metabolites are dihydro-
bolagladins A 11 and B 12, in which the Dba residue has been
replaced by -alanine (Tables S7 and S8, and Figures S25-S35).
This is consistent with the proposed function of BolQ as a N-acyl-
-alanyl thioester desaturase, although on the basis of these data
the possibility of BolQ converting the -alanine residue to Dba at
a later stage in bolagladin biosynthesis cannot be ruled out.
The Dba residue of the bolagladins is postulated to derive from L-
aspartate, which we propose undergoes adenylation of its -
carboxyl group catalyzed by BolO, followed by transfer onto BolC,
a putative freestanding acyl carrier protein (Figure 3). BolO shows
similarity to VinN,[28] which catalyzes adenylation of the -carboxyl
group of -methyl-aspartate and subsequent transfer to a
freestanding acyl carrier protein VinL in the biosynthesis of
vicenistatin. Moreover, the Asp230 and Ser299/Arg331 residues
in VinN, which are proposed to play a key role in recognition of
the -amino and -carboxyl groups of -methylaspartate,
respectively, are conserved in BolO (Fig. S24). In vicenistatin
biosynthesis, the pyridoxal phosphate (PLP)-dependent enzyme
VinO catalyses decarboxylation of the -methyl-iso-aspartyl-VinL
thioester to form the corresponding -methyl--alanyl thioester.[24]
We propose that BolN, which shows sequence similarity to BtrK,
a PLP-dependent enzyme that catalyzes decarboxylation of an
iso-glutamyl-ACP thioester in butirosin biosynthesis, plays an
The bol locus is widely conserved in B. gladioli
A local nucleotide BLAST search of 1318 genomes representing
Burkholderia, Paraburkholderia and Caballeronia species showed
B. gladioli is the only species containing the bolagladin
biosynthetic gene cluster (as indicated by the presence of
[29]
bolH). Read mapping of paired-end Illumina reads from 234 B.
gladioli strains against the bol gene cluster revealed that it is
present in 105 strains. However, there is evidence of gene
substitutions and deletions in the regions flanking the cluster
(
Figure 9). The bolU gene, encoding a putative TonB-dependent
ferric-siderophore outer membrane receptor, is absent from five
of the 105 B. gladioli genomes (BCC1837, BCC1843, BCC1861,
BCC1870 and BCC1871) containing the bolagladin biosynthetic
gene cluster and is present in 52% (67 of 129) of genomes lacking
it.
Biological function of the bolagladins
6
x10
Bolagladins A (1) and B (2) showed no activity at a concentration
of 64 g/mL in disc diffusion assays against any of the ESKAPE
panel of bacterial pathogens,[ Mycobacterium bovis BCG, or
Candida albicans. The 3-methoxy-1, 4-dicarboxylate moiety of the
bolagladins is reminiscent of ferric iron-chelating citrate residues
in several siderophores, such as achromobactin, vibrioferrin, and
staphyloferrin B.[31] Moreover, bolU flanking the left end of the
bolagladin biosynthetic gene cluster encodes a putative TonB-
dependent ferric-siderophore outer membrane receptor. We thus
hypothesised that the bolagladins could function as siderophores.
Although ferric complexes of the bolagladins were too labile to
observe in ESI-Q-TOF MS analyses, the chrome azurol S (CAS)
assay indicated they are able to sequester ferric iron, albeit at
higher concentrations than typical tris-hydroxamate siderophores
(Figure S36).[4]
1
1
0
0
0
.25
.00
.75
.50
.25
30]
6
x10
2
.0
.5
.0
.5
1
1
0
1
6.0
16.5
17.0
17.5
Time (min)
18.0
18.5
Figure 8. (a) Structures of dihydro-bolagladins A (11) and B (12) produced by
the bolQ mutant of B. gladioli BCC1622 ∆gbnD1_ER. (b) Extracted ion
chromatograms at m/z = 824.4288 and 838.4444 (in blue; corresponding to
+
[
8
(
M+H] for bolagladins A (1) and B (2), respectively), and 826.4444 and
40.4601 (in red, corresponding to [M+H]+ for dihydro-bolagladins A (11) and B
12), respectively) from UHPLC-ESI-Q-TOF MS analyses of culture extracts
from B. gladioli BCC1622 ∆gbnD1_ER and the bolQ mutant.
Siderophore production in microbes is usually regulated by iron
availability. In iron-deficient media, production is upregulated,
whereas in iron replete media it is suppressed. Accordingly, the
6
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202009110
RESEARCH ARTICLE
Figure 9. Conservation of the bolagladin biosynthetic gene cluster in B. gladioli. The gene cluster was extracted from six B. gladioli genome assemblies (BCC1622,
BCC0238, BCC1665, BCC1675, BCC1781, and BCC1837, all of which are CF lung infection isolates) that highlight the diversity of gene insertions, deletions, and
substitutions flanking the cluster. B. gladioli BCC1835 and BCC1838 were included as examples of strains lacking the bolagladin biosynthetic gene cluster. No
variation in the bolA-bolT genes was observed for strains containing the bolagladin biosynthetic gene cluster.
level of bolagladin production by B. gladioli BCC1622
gbnD1_ER decreased as increasing concentrations of ferric iron
Additional work is required to establish the genetic basis for this
high degree of metal ion tolerance.

were added to the medium (Figure S36). Combined with the
results of the CAS assay, these data indicate that the bolagladins
may function as siderophores in B. gladioli.
Conclusion
Siderophores often contribute to virulence in pathogenic bacteria.
Thus, we used a Galleria mellonella wax moth infection model to
investigate whether the bolagladins are a virulence factor in B.
gladioli. Ornibactin was shown to contribute to virulence in
Burkholderia cenocepacia using this and other models.[15, 32]
However, no attenuation of virulence was towards the wax moth
larvae was observed in the bolN mutant of B. gladioli BCC1622
Bacteria belonging to the Burkholderia genus are increasingly
being recognised as an underexplored source of novel natural
products.[34] Using a combination of carbon source modification
and inactivation of the biosynthetic pathway for gladiolin, which is
produced at high titre and interferes with the detection of lower
abundance metabolites, we have identified bolagladins A (1) and
∆gbnD1_ER, relative to the parental strain (Figure S37).
(
2) as the metabolic product of a cryptic nonribosomal peptide
A similar observation was reported for pyochelin deficient mutants
of B. cenocepacia H111,[32] leading to the hypothesis that some
Burkholderia siderophores may play a role in metal homeostasis
rather than virulence.[33] To investigate this hypothesis, B.
cenocepacia H111 pyochelin and ornibactin non-producing
mutants were grown alongside the wild type strain in media
containing various metal salts (10-50 μM). The metal salts did not
affect the wild type strain, but were toxic to the siderophore non-
producing mutants, indicating that pyochelin and ornibactin
protect B. cenocepacia against metal ion toxicity.[33] This finding
was further supported by supplementing the medium with
pyochelin and ornibactin, which reversed toxicity in the
siderophore non-producing mutants.[33]
biosynthetic gene cluster in B. gladioli. Similar approaches may
prove useful for identifying the products of other cryptic
Burkholderia biosynthetic gene clusters.
The bolagladins have very unusual structures, consisting of a
unique fatty acid residue with several polar functional groups
appended to its tail, linked via a very rare Dba residue to a
depsitetrapeptide. Identification of the bolagladin biosynthetic
gene cluster in B. gladioli BCC0238 and BCC1622 enabled us to
probe the origin of these unusual structural features using a
combination of detailed bioinformatics analysis, gene deletions
and enzyme activity assays. Based on these studies, we propose
that the fatty acid residue originates from a citryl-CoA starter unit.
To our knowledge, there is no precedent for the utilisation of citryl-
CoA as a starter unit in fatty acid or polyketide biosynthesis. We
also hypothesise that the Dba residue derives from loading of
aspartate onto a freestanding ACP. The resulting iso-aspartyl
thioester is proposed to undergo decarboxylation and N-acylation,
prior to being desaturated by a flavin-dependent dehydrogenase,
resulting in an N-acyl-Dba thioester starter unit for the NRPS that
assembles the depsitetrapeptide. While loading of aspartate / -
We thus compared the tolerance of the B. gladioli BCC1622
∆gbnD1_ER and ∆gbnD1_ER-bolN strains to salts of aluminium,
zinc, cobalt, copper, cadmium, nickel and lead at various
concentrations up to 100 μM. No decrease in metal ion tolerance
was observed for the bolagladin non-producing mutant relative to
parent strain and the results of these experiments indicated that
B. gladioli is much less susceptible to metal ion toxicity than B.
cenocepacia. Further experiments demonstrated that B. gladioli
methyl-aspartate onto
decarboxylation and N-acylation, is
a
standalone ACP, followed by
well-established
could readily grow in media containing 1 mM Ni , Al and Zn2+.
2
+
2+
a
7
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202009110
RESEARCH ARTICLE
[
[
7]
8]
J. A. Ferreras, J. S. Ryu, F. Di Lello, D. S. Tan, L. E. Quadri,
Nat. Chem. Biol. 2005, 1, 29-32.
A. L. Crumbliss, Handbook of Microbial Iron Chelates, CRC
Press, Boca Raton, FL, 1991.
R. J. Bergeron, G. M. Brittenham, The Development of Iron
Chelators for Clinical Use, CRC Press Boca Raton, FL,
mechanism for provision of -aminoacyl starter units to type I
modular polyketide synthases,[35] to our knowledge there is no
precedent for this mechanism being employed to provide such
starter units to NRPSs. Thus, our findings expand the scope of -
aminoacyl starter unit biosynthetic machinery, which may prove
useful in bioengineering approaches to natural product
diversification.
[9]
1994.
[
10]
a) J. B. Neilands, J. Biol. Chem. 1995, 270, 26723-26726;
b) E. Ahmed, S. J. M. Holmström, Microb. Biotechnol. 2014,
The bolagladin biosynthetic gene cluster is present in
approximately 45% of B. gladioli genomes, but was not found in
other Burkholderia species. This suggests the bolagladins play an
important role in the adaption of B. gladioli to its environmental
niches. Several lines of evidence indicate that the bolagladins
may function as siderophores, further challenging the assumption
7
, 196-208.
[11]
E. Mahenthiralingam, A. Baldwin, C. G. Dowson, J. Appl.
Microbiol. 2008, 104, 1539-1551.
a) L. P. Partida-Martinez, I. Groth, I. Schmitt, W. Richter, M.
Roth, C. Hertweck, Int. J. Syst. Evol. Microbiol. 2007, 57,
[
12]
2583-2590; b) N. Moebius, C. Ross, K. Scherlach, B. Rohm,
M. Roth, C. Hertweck, Chem. Biol. 2012, 19, 1164-1174; c)
X. Liu, S. Biswas, M. G. Berg, C. M. Antapli, F. Xie, Q. Wang,
M.-C. Tang, G.-L. Tang, L. Zhang, G. Dreyfuss, Y.-Q.
Cheng, J. Nat. Prod. 2013, 76, 685-693; d) J. B. Biggins, C.
D. Gleber, S. F. Brady, Org. Lett. 2011, 13, 1536-1539; e)
A. S. Eustáquio, J. E. Janso, A. S. Ratnayake, C. J.
O’Donnell, F. E. Koehn, Proc. Natl. Acad. Sci. U. S. A. 2014,
that B. gladioli employs siderophore-independent mechanisms for
iron acquisition.[
15-16]
While the mode of ferric iron binding to the
bolagladins remains to be established, it seems likely that the two
citrate-derived carboxy and methoxy groups in the unusual fatty
acid residue, and one or both of the side hydroxyl groups in the
depsipetide are involved. Siderophores are known to play roles in
virulence and metal ion tolerance in other Burkholderia species,
but a Galleria mellonella infection model did not provide evidence
that the bolagladins contribute to virulence in B. gladioli and
bolagladin-deficient mutants did not show reduced tolerance
towards metal ions. Further studies are therefore required to
develop a better understanding of the adaptive benefit the
bolagladins confer on B. gladioli.
111, E3376-E3385; f) K. Ishida, T. Lincke, S. Behnken, C.
Hertweck, J. Am. Chem. Soc. 2010, 132, 13966-13968; g)
M. R. Seyedsayamdost, J. R. Chandler, J. A. Blodgett, P.
S. Lima, B. A. Duerkop, K. Oinuma, E. P. Greenberg, J.
Clardy, Org. Lett. 2010, 12, 716-719; h) E.
Mahenthiralingam, L. Song, A. Sass, J. White, C. Wilmot, A.
Marchbank, O. Boaisha, J. Paine, D. Knight, Gregory L.
Challis, Chem. Biol. 2011, 18, 665-677; i) L. Song, M.
Jenner, J. Masschelein, C. Jones, M. J. Bull, S. R. Harris,
R. C. Hartkoorn, A. Vocat, I. Romero-Canelon, P. Coupland,
G. Webster, M. Dunn, R. Weiser, C. Paisey, S. T. Cole, J.
Parkhill, E. Mahenthiralingam, G. L. Challis, J. Am. Chem.
Soc. 2017, 139, 7974-7981; j) A. J. Mullins, J. A. H. Murray,
M. J. Bull, M. Jenner, C. Jones, G. Webster, A. E. Green,
D. R. Neill, T. R. Connor, J. Parkhill, G. L. Challis, E.
Mahenthiralingam, Nat. Microbiol. 2019, 4, 996-1005.
Q. Esmaeel, M. Pupin, N. P. Kieu, G. Chataigné, M. Béchet,
J. Deravel, F. Krier, M. Höfte, P. Jacques, V. Leclère,
Microbiologyopen 2016, 5, 512-526.
Acknowledgements
This research was supported by grants from the BBSRC
[
13]
(
BB/L021692/1 and BB/L023342/1) to E.M and G.L.C; A.J.M.,
G.W., E.M. and G.L.C. also acknowledge current funding from
BBSRC (BB/S007652/1 and BB/S008020/1). Y.D. was supported
by a grant from the MRC (MR/N501839/1 to G.L.C). I.T.N. was
supported by the BBSRC through the Midlands Integrative
Bioscience Doctoral Training Partnership (BB/M01116X/1). E.M.
and G.W. acknowledge funding from the Life Science Bridging
Fund (LSBF R2-004) for the HPLC analysis and instrumentation.
The Bruker MaXis Impact UHPLC-ESI-Q-TOF-MS instrument
used in this research was funded by the BBSRC (B/K002341/1 to
G.L.C.). G.L.C. was the recipient of a Wolfson Research Merit
Award from the Royal Society (WM130033).
[14]
a) J. M. Meyer, D. Hohnadel, F. Halle, J. Gen. Microbiol.
1989, 135, 1479-1487; b) J. M. Meyer, V. T. Van, A. Stintzi,
O. Berge, G. Winkelmann, Biometals 1995, 8, 309-317; c)
H. Stephan, S. Freund, W. Beck, G. Jung, J. M. Meyer, G.
Winkelmann, Biometals 1993, 6, 93-100; dI. Barelmann, J.
M. Meyer, K. Taraz, H. Budzikiewicz, in Z. Naturforsch. C,
1996, 51, 627-630; e) P. Darling, M. Chan, A. D. Cox, P. A.
Sokol, Infect. Immun. 1998, 66, 874-877; f) J. Franke, K.
Ishida, M. Ishida-Ito, C. Hertweck, Angew. Chem. Int. Ed.
2013, 52, 8271-8275; g) J. Franke, K. Ishida, C. Hertweck,
Chem. Eur. J. 2015, 21, 8010-8014.
A. T. Butt, M. S. Thomas, Front. Cell. Infect. Microbiol. 2017,
[
[
15]
16]
7, 460-460.
R. Hermenau, J. L. Mehl, K. Ishida, B. Dose, S. J. Pidot, T.
Keywords: natural products • genome mining • nonribosomal
peptide synthetase • fatty acid • biosynthesis
P. Stinear, C. Hertweck, Angew. Chem. Int. Ed. Engl. 2019,
58, 13024-13029.
[
17]
M. Jenner, X. Jian, Y. Dashti, J. Masschelein, C. Hobson,
D. M. Roberts, C. Jones, S. Harris, J. Parkhill, H. A. Raja,
N. H. Oberlies, C. J. Pearce, E. Mahenthiralingam, G. L.
Challis, Chem. Sci. 2019, 10, 5489-5494.
C. Boros, C. J. Smith, Y. Vasina, Y. Che, A. B. Dix, B.
Darveaux, C. Pearce, J. Antibiot. 2006, 59, 486-494.
B. Dose, S. P. Niehs, K. Scherlach, L. V. Florez, M.
Kaltenpoth, C. Hertweck, ACS Chem. Biol. 2018, 13, 2414-
[
[
1]
2]
M. Sánchez, L. Sabio, N. Gálvez, M. Capdevila, J. M.
Dominguez-Vera, IUBMB Life 2017, 69, 382-388.
S. C. Andrews, A. K. Robinson, F. Rodríguez-Quiñones,
FEMS Microbiol. Rev. 2003, 27, 215-237.
[
[
18]
19]
[
[
3]
4]
E. P. Skaar, PLoS Pathog. 2010, 6, e1000949.
B. Schwyn, J. B. Neilands, Anal. Biochem. 1987, 160, 47-
56.
2420.
[
5]
a) R. C. Hider, X. Kong, Nat. Prod. Rep. 2010, 27, 637-657;
b) J. P. Bellenger, T. Wichard, A. B. Kustka, A. M. L.
Kraepiel, Nat. Geosci. 2008, 1, 243-246.
[
20]
a) S. Koshino, H. Koshino, N. Matsuura, K. Kobinata, R.
Onose, K. Isono, H. Osada, J. Antibiot. 1995, 48, 185-187;
b) S. Son, S. K. Ko, S. M. Kim, E. Kim, G. S. Kim, B. Lee, I.
J. Ryoo, W. G. Kim, J. S. Lee, Y. S. Hong, J. H. Jang, J. S.
Ahn, J. Nat. Prod. 2018, 81, 2462-2469.
R. Bhushan, H. Bruckner, Amino Acids 2004, 27, 231-247.
S. Vijayasarathy, P. Prasad, L. J. Fremlin, R. Ratnayake, A.
A. Salim, Z. Khalil, R. J. Capon, J. Nat. Prod. 2016, 79, 421-
[
6]
a) J. Heesemann, K. Hantke, T. Vocke, E. Saken, A. Rakin,
I. Stojiljkovic, R. Berner, Mol. Microbiol. 1993, 8, 397-408;
b) J. J. Bullen, E. Griffiths, Iron and Infection: Molecular,
Physiological and Clinical Aspects, 2nd ed., John Wiley &
Sons, New York, 1999.
[
[
21]
22]
427.
8
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202009110
RESEARCH ARTICLE
[
23]
a) T. Stachelhaus, H. D. Mootz, M. A. Marahiel, Chem. Biol.
999, 6, 493-505; b) G. L. Challis, J. Ravel, C. A. Townsend,
1
Chem. Biol. 2000, 7, 211-224.
[
[
24]
25]
C. Rausch, I. Hoof, T. Weber, W. Wohlleben, D. H. Huson,
BMC Evol. Biol. 2007, 7, 78.
J. Kay, P. D. Weitzman, Krebs' citric acid cycle: half a
century and still turning, Biochemical Society, London,
1987.
[
[
26]
27]
R. Nofiani, B. Philmus, Y. Nindita, T. Mahmud,
MedChemComm 2019, 10, 1517-1530.
D. R. Jackson, G. Shakya, A. B. Patel, L. Y. Mohammed, K.
Vasilakis, P. Wattana-Amorn, T. R. Valentic, J. C. Milligan,
M. P. Crump, J. Crosby, S.-C. Tsai, ACS Chem. Biol. 2018,
13, 141-151.
[
[
28]
29]
A. Miyanaga, J. Cieslak, Y. Shinohara, F. Kudo, T. Eguchi,
J. Biol. Chem. 2014, 289, 31448-31457.
For the independent discovery of bolagladins in
Burkholderia gladioli pv. cocovenenans see: B. Dose, C.
Ross, S. P. Niehs, K. Scherlach, J. P. Bauer, C. Hertweck,
Angew. Chem. Int. Ed. 2020.
[
[
30]
31]
L. B. Rice, J. Infect. Dis. 2008, 197, 1079-1081.
J. Cheung, Michael E. P. Murphy, David E. Heinrichs,
Chem. Biol. 2012, 19, 1568-1578.
[
[
[
[
32]
33]
34]
35]
A. Mathew, L. Eberl, A. L. Carlier, Mol. Microbiol. 2014, 91,
805-820.
A. Mathew, C. Jenul, A. L. Carlier, L. Eberl, Environ.
Microbiol. Rep. 2016, 8, 103-109.
S. Kunakom, A. S. Eustaquio, J. Nat. Prod. 2019, 82, 2018-
2
037.
A. Miyanaga, F. Kudo, T. Eguchi, Curr. Opin. Chem. Biol.
016, 35, 58-64.
2
9
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202009110
RESEARCH ARTICLE
Entry for the Table of Contents
Bolagladins A and B were discovered as products of a cryptic nonribosomal peptide synthetase in two Burkholderia gladioli strains
isolated from the lungs of cystic fibrosis patients. These metabolites contain a unique citrate-primed fatty acid and a rare dehydro--
alanine residue. A combination of bioinformatics analyses, and genetic and biochemical experiments illuminated the mechanisms for
installation of these unusual structural features.
Institute and/or researcher Twitter usernames: @Challis_Group, @warwickchem
1
0
This article is protected by copyright. All rights reserved.