Genome Mining of Oxidation Modules in trans-Acyltransferase Polyketide Synthases Reveals a Culturable Source for Lobatamides

Article abstract of DOI:10.1002/anie.201916005

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are multimodular megaenzymes that biosynthesize many bioactive natural products. They contain a remarkable range of domains and module types that introduce different substituents into growing polyketide chains. As one such modification, we recently reported Baeyer–Villiger-type oxygen insertion into nascent polyketide backbones, thereby generating malonyl thioester intermediates. In this work, genome mining focusing on architecturally diverse oxidation modules in trans-AT PKSs led us to the culturable plant symbiont Gynuella sunshinyii, which harbors two distinct modules in one orphan PKS. The PKS product was revealed to be lobatamide A, a potent cytotoxin previously only known from a marine tunicate. Biochemical studies show that one module generates glycolyl thioester intermediates, while the other is proposed to be involved in oxime formation. The data suggest varied roles of oxygenation modules in the biosynthesis of polyketide scaffolds and support the importance of trans-AT PKSs in the specialized metabolism of symbiotic bacteria.

Full text of DOI:10.1002/anie.201916005

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Accepted Article
Title: Genome mining of oxidation modules in trans-acyltransferase
polyketide synthases reveals a culturable source for lobatamides
Authors: Reiko Ueoka, Roy Meoded, Alejandro Gran-Scheuch, Agneya
Bhushan, Marco Fraaije, and Jörn Piel
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201916005
Angew. Chem. 10.1002/ange.201916005
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COMMUNICATION
Genome mining of oxidation modules in trans-acyltransferase
polyketide synthases reveals a culturable source for lobatamides
Reiko Ueoka+[a], Roy A. Meoded+[a], Alejandro Gran-Scheuch[b,c], Agneya Bhushan[a],
[
b]
[a]
Marco W. Fraaije , Jörn Piel*
[
a]
Dr. R. Ueoka[+], Dr. R. A. Meoded[+], Dr. A. Bhushan, Prof. Dr. J. Piel
Institute of Microbiology
ETH Zurich
Vladimir-Prelog-Weg 4, 8093 Zurich (Switzerland)
E-mail: jpiel@ethz.ch
[
b]
c]
A. Gran-Scheuch, Prof. Dr. M. W. Fraaije
Molecular Enzymology Group
University of Groningen
Nijenborgh 4, 9747AG, Groningen (The Netherlands)
A. Gran-Scheuch
[
Department of Chemical and Bioprocesses Engineering
Pontificia Universidad Catꢀlica de Chile
Avenida Vicuꢁa Mackenna 4860, 7820436, Santiago (Chile)
These authors contributed equally to this work.
[+
]
Abstract: Bacterial trans-acyltransferase polyketide synthases
trans-AT PKSs) are multimodular megaenzymes that biosynthesize
a)
Ox
CR
DH
KR
KS
(
KS
KS
many bioactive natural products. They contain a remarkable range of
domains and module types that introduce different substituents into
growing polyketide chains. As one such modification, we recently
reported Baeyer-Villiger-type oxygen insertion into nascent
polyketide backbones, generating malonyl thioester intermediates. In
this work, genome mining focusing on architecturally diverse oxidation
modules in trans-AT PKSs led us to the culturable plant symbiont
Gynuella sunshinyii that harbors two distinct modules in one orphan
PKS. The PKS product was revealed to be lobatamide A, a potent
cytotoxin previously only known from a marine tunicate. Biochemical
studies show that one module generates glycolyl thioester
intermediates, while the other is proposed to be involved in oxime
formation. The data suggest varied roles of oxygenation modules in
the biosynthesis of polyketide scaffolds and support the importance of
trans-AT PKSs in the specialized metabolism of symbiotic bacteria.
oocydin B 1
b)
oocydin A 2
pederin 3
toblerol A 4
c)
Bacterial complex polyketides belong to the most important
natural product classes of therapeutic value.[1] Two distinct
lobatamide A 5
salarin A 6
pateamine A 7
families of multimodular polyketide synthases (PKSs), termed cis-
and trans-acyltransferase (trans-AT) PKSs, generate most of
these compounds. The textbook biosynthetic model is
represented by the cis-AT PKS family, such as the erythromycin
PKS.[ These enzymes are usually composed of a limited set of
functionally different biosynthetic multi-domain modules that
elongate and modify intermediates, and give rise to contiguous
carbon chains carrying keto, hydroxyl, double-bond, and carbon
branch modifications. In contrast, the highly complex trans-AT
PKSs can accommodate a remarkable diversity of modules (>150
Figure 1. Oxygen incorporation into polyketide backbones. Moieties
derived from oxygen insertion are highlighted in orange. Oxygen atoms of
unknown biosynthetic origin are highlighted in blue. a) Mechanism for oxygen
insertion into growing polyketide backbones exemplified by OocK, a trans-acting
BV monooxygenase from the oocydin (oocydin B, 1) pathway. The enzyme
converts a β-ketothioester substrate to a malonyl derivative. b) Oocydin A (2),
pederin (3), and toblerol A (4) with carboxylate- and alcohol-type termini arising
from oxygen insertion and ester cleavage. c) Biosynthetically unassigned
polyketides harboring mid-chain oxygens: lobatamide A (5) from a tunicate,
salarin A (6) and pateamine A (7) from marine sponges.
1-2]
[
3]
module architectures are currently known ) and employ
integrated domains as well as free-standing, trans-acting
[
4]
enzymes with often poorly understood functions. The large
functional range of trans-AT PKSs suggests high biosynthetic
diversity outside the scope of canonical polyketides. Another
phenomenon of trans-AT PKSs is their dominant role in the
specialized metabolism of non-actinomycete symbiotic bacteria,
In previous work, we identified the insertion of oxygen into
growing polyketide chains as a non-canonical reaction by which
trans-AT PKSs diversify product skeletons.[ As shown for the
trans-acting flavin adenine dinucleotide (FAD)-dependent
oxygenase OocK from the oocydin pathway, the enzyme
performs a Baeyer-Villiger (BV) oxidation on a β-ketothioester
intermediate to generate a malonyl derivative (Figure 1A) that is
then further elongated. Preservation or hydrolysis of the new ester
moiety gives rise to oxygens within polyketide backbones
(oocydin B and haterumalides), carboxylate pseudostarters
(oocydin A) or terminal alcohols (introduced by the OocK
9]
with examples reported from fungi,[
5]
insects,
[6]
marine
[
7]
[8]
invertebrates, and plants. To fully access this biosynthetic
potential, insights into the function of trans-AT PKS assembly
lines and their components are needed to improve our ability to
identify alternative producers to
invertebrate sources, and provide novel tools for biosynthetic
engineering.
[
1]
predict their products,
[9]
homologs in the pederin[ and toblerol pathway, Figure 1B).
Mid-chain oxygens also occur in other polyketides (Figure 1C).[
Although these moieties could principally result from various
6]
[8]
10]
1
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COMMUNICATION
processes,[11] the presence of OocK homologs in diverse orphan
PKS biosynthetic gene clusters (BGCs) (Figures S1-S4) suggests
that nature employs oxygen insertion more widely to construct
unusual polyketide scaffolds. In this work, we applied a genome
mining strategy to investigate the wider scope of flavoprotein
monooxygenases acting during polyketide elongation. The data
revealed an architecturally distinct oxygen insertion module that
generates a glycolyl intermediate, as well as evidence for a third
module type involved in oxime formation. Both new modules are
used in the biosynthesis of lobatamides, potent cytotoxins
previously only known from marine tunicates, but identified here
from a culturable plant symbiont.
a)
4
kb
lbm F G
H
I
J K LMNO
A
B
0
0
C-A-PCP-KS -Ox-ACP-KS -
MT-ACP-KS-KR-ACP-KS
DH-ACP-C-A-PCP-
KS-DH-KR-ACP-KS
0
C
D
E
P Q R
KR-ACP-KS-Ox-MT-ACP-KS-DH-
KR-ACP-KS-DH-KR-MT
ACP-KS-DH-KR-ACP- KS-DH-
KS-ACP-KS-KR-ACP ACP-TE
b)
c)
8
9
We initiated our work by identifying OocK homologs and
other PKS-associated flavoprotein monooxygenases encoded in
orphan BGCs using GenBank, genome neighborhood,[12]
phylogenetic, and manual analyses (status: November 2019)
(
Figures S1-S4). These revealed 69 candidate trans-AT PKS
, molecular formula:C26H42N2O7
systems harboring such enzymes (Figures S1-S4). The
phylogram contained a large clade containing the functionally
related OocK, PedG, and TobD as well as homologs from
uncharacterized PKSs. Another functionally assigned clade bears
module-integrated oxygenase (Ox) domains for which the variant
from Burkholderia sp. FERM BP-3421 was shown to introduce an
epoxide unit in spliceostatin (Figure S1).[8] In addition, other non-
OocK clades mostly belong to Ox domains integrated in various
module types, suggesting further biosynthetic diversity. Focusing
on uncharacterized oxygenases (Figure S1), an orphan trans-AT
system (termed lbm PKS, Figure 2A) in the Gram-negative
bacterium Gynuella sunshinyii (NZ_CP007142) appeared an
intriguing candidate, since the PKS contains oxygenase modules
from two different unassigned clades (Figure S1, Table S1). G.
sunshinyii is an unusual halophilic root-associated plant
symbiont[ that was recently recognized as a talented producer
Figure 2. The lbm BGC and predicted polyketide structures. a) BGC
architecture with core PKS and accessory genes marked in dark- and light grey,
respectively. The domain architecture of the core PKSs is shown using the
following abbreviations: A, adenylation domain; C, condensation domain; DH,
0
dehydratase; ER, enoylreductase; KR, ketoreductase; KS, ketosynthase; KS ,
non-elongating KS; MT, methyltransferase; Ox, oxygenase; TE, thioesterase.
Numbers above KS domains correspond to consecutive KS and module
numbers. Ox domains are shown in grey. b) TransATor-predicted structure 8 of
the lbm product. High- and low-confidence regions are shown in black and grey,
respectively. c) Manually refined structure 9 used to find the natural product.
To search for the predicted compound, G. sunshinyii was
cultivated, and the extract was analyzed by ultra-high-
performance liquid chromatography/high-resolution spectrometry
(UHPLC-HRMS). Manual inspection of metabolite-related MS
features and their molecular formulae suggested from the high-
resolution masses showed a candidate ion peak at m/z 513.2230
13]
[14]
of diverse natural products. Its genome contains six trans-AT
PKS BGCs, the highest known number for any organism. The two
integrated Ox domains are located in the PKS proteins LmbA and
LmbC, and will be referred to as LmbA-Ox and LmbC-Ox,
+
[M+H] (Figure S5). This corresponded to a molecular formula of
respectively. Additional features in these modules are
a
C H N O (calc. 513.2231). This formula was closest to that of
2
7
32
2
8
methyltransferase (MT) in the LmbC-Ox module, suggesting α-C-
26 42 2 7
the predicted structure (C H N O ) and appeared a good
0
methylation, and a predicted non-elongating KS (KS ) in the
candidate for the lbm product. No other orphan PKSs in the
genome were predicted to account for a similar chemical formula
that contained two nitrogen atoms (i.e., PKSs that contained two
NRPS modules).[ To isolate the compound, bacterial pellets
from a 2 L culture of G. sunshinyii culture were extracted with
acetone. MS-guided fractionations using reversed phase-high
performance liquid chromatography (RP-HPLC) provided pure
LmbA-Ox module, suggesting a moiety introduced by the
upstream module is further modified. This upstream module is
located at the N-terminus of LbmA and resembles an NRPS
loading module with predicted[15] glycine specificity. A C-terminal
TE domain is encoded on the PKS gene lmbE at the downstream
end, overall suggesting collinearity between the PKS gene order
and biosynthetic events.
14]
1
compound 5. The H NMR and HSQC data showed the presence
of a doublet aliphatic methyl, one methyl connected to an sp2
carbon, one methoxy group, three oxymethines, and 11 methines
As guidance for the targeted isolation of the lbm polyketide,
we analyzed the PKS using the recently developed automated
prediction tool TransATor.[3] This web application suggests
chemical structures for trans-AT PKSs from phylogenetically
inferred[ KS substrates. In agreement with the unusual PKS
architecture, the TransATor output 8 (Figure 2B) contained
extended regions of low confidence. After removal of a methyl
group from the five-bonded C18, the structure was refined by
closer inspection of the modular architecture. Since TransAtor
2
connected to sp carbons (Figure S6). Analysis of 2D NMR data
including COSY, HMBC, and HSQC determined the structure as
that of lobatamide A (Figure 3A, Figures S7-S9). Lobatamides are
+
potent vacuolar (H )-ATPase inhibitors interfering with tumor
15]
[17]
metastasis. As they were previously known only from a marine
[18]
invertebrate, the tunicate Aplidium lobatum , G. sunshinyii
represents the first culturable source for these compounds.
+
Lobatamides belong to a diverse group of benzolactone (H )-
ATPase inhibitors (Figure 3A) with as-yet unknown PKSs and
isolated from a remarkable range of organisms, including a
can predict intermediates only for modules that have
a
downstream KS, no elongation was predicted for the terminal
module. A terminal C2 unit was therefore added based on cis-AT
PKS rules. To account for the N-terminal NRPS module of LbmA,
we suspected glycine for biosynthetic initiation. This replacement
also removed an exomethylene group in the TransATor structure
that was unlikely because polyketide β-branching components[16]
were not found in the BGC. These modifications resulted in the
hypothetical structure 9 (Figure 2C) as a basis for the analytical
work.
[19]
[20]
sponge (salicylihalamides),
a
and
fungus (CJ-12,950),
Pseudomonas strain
[21]
myxobacteria (apicularens),
a
[22]
(
oximidines). Their related structures and the identification of
bacterial sources suggest that all compounds are of prokaryotic
origin. For an expansion of this family, see the study on
[23]
necroximes conducted concurrently with the work here.
Structure 5 features an internal ester that is reversed in
comparison to oocydin, pederin, and toblerol biosynthesis. To
2
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COMMUNICATION
investigate the role of the LmbC-Ox module in its formation, we
cloned the region encoding the Ox domain into a pET28a variant
a)
(
6
see SI) for expression in E. coli as an N-terminally His -tagged
*
protein (Figure S10). Purified LbmC-Ox was obtained as a soluble
yellow protein indicating bound FAD. As the LmbC-Ox module
contained an additional MT domain, we suspected the Ox domain
to accept either a β-ketothioester or an α-methyl-β-ketothioester,
depending on whether LmbC-Ox acts before or after C-
methylation. We initially assayed the enzyme with the
unmethylated thioester 13 as a simplified surrogate of an acyl
lobatamideA 5
apicularen A10
oximidine I 11
salicylihalamideA 12
b)
1
3
14
15
1
.2×109
×108
9
8
6×10
3×10
carrier
protein
(ACP)-bound
4’-phosphopantetheinyl
8
intermediate. Assay mixtures with test substrate, LbmC-Ox, and
NADPH were incubated for 90 min at room temperature and then
extracted with ethyl acetate. UHPLC-HRMS analysis suggested
two new products in the assay mixture, both with a mass
difference of +16 Da relative to the substrate (Figure 3B). Isolation
and NMR-based structure elucidation of the products (Figures
S12-17, Table S4) revealed that the two isomers carry the
inserted oxygen either at the γ (Figure 3B, product 14) or the β
position (product 15). The less abundant thioester 15 is
noteworthy because it exhibits a reversed ester moiety as
compared to the OocK/PedG/TobD products and corresponds to
the topology present in lobatamides. Suspecting that the major
isomer 14 was an aberrant byproduct due to the choice of the
surrogate substrate, we also synthesized thioester 16 harboring
the predicted methyl group at the α position (Figure 3C, Figure
S18). Repetition of the enzyme assay with this new test substrate
produced one product peak (Figure 3C). Isolation and structure
elucidation identified it as thioester 17 (Figure 3C, Figure S19-24,
Table S5). The result from the biochemical study matches the
internal ester topology at C-10 of lobatamides, i.e., facing the
same direction as the C-24 ester created by macrolactonization.
In lieu of knock-out studies that were so far unsuccessful or of
heterologous whole-cluster expression, the unusual function of
LmbC-Ox and its location within the PKS reasonably link the lbm
BGC to lobatamide.
0
1
0
15
20
25
30
t / min
c)
16
17
4
3
2
1
×109
×109
×109
×109
0
1
0
15
20
25
30
t / min
Figure 3. Selected benzolactone enamide polyketides and enzymatic
assays with LbmC-Ox. a) Benzolactone enamides with the moiety derived from
oxygen insertion highlighted with an asterisk. b-c) UHPLC-HRMS data showing
the extracted ion chromatograms (EIC) of assay mixtures, including the boiled-
enzyme negative control (upper/grey) and test reaction using all components
lower/black). b) EIC for 13 and 13+[ O] (calc. for [M+H]+ as 246.1158, and
16
(
2
62.1108, respectively). For the mass spectrum of the new product 15 see
Figure S11. c) EIC for 16 and 16+[ O] (calc. for [M+H]+ as 260.1315 and
16
2
76.1264, respectively). Mass spectrum of product 17 is shown in Figure S19.
Comparing the PKS-associated enzymes OocK and LbmC-
Ox to a wider range of previously characterized class B
flavoprotein monooxygenases (Figure S25), BV oxidation activity
appears more prevalent in the LmbC-Ox than the OocK clade.[
To further explore their biocatalytic potential, we tested 37
substrates not resembling polyketide intermediates and harboring
diverse functional groups (Figures S26-S27, Table S6). When
comparing the free-standing OocK with the excised LbmC-Ox, the
latter exhibited greater thermostability and a broad substrate
scope, accepting 12 of the 37 test compounds (18-26, Figure 5).
Both enzymes accept NADPH as hydride donor, while OocK also
displays activity with NADH (Table S7). These results suggest
that the two enzymes can be used as biocatalysts for various
oxidations, in addition to showing potential as PKS engineering
tools to introduce non-standard moieties into polyketides.
25]
The results suggest an overall biosynthetic model for
lobatamides as shown in Figure 4. A glycine starter would be the
source of the oxime moiety, and oxygen insertion and
macrolactonization generate the ester groups at C-10 and C-24,
respectively. The salicylate group generated by the terminal four
modules is also
a feature of all other members of the
benzolactone family, which currently lack known BGCs. However,
similar tetramodular series that generate benzene moieties are
also known from the otherwise unrelated psymberin and legioliulin
PKSs.[
24]
3
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Angewandte Chemie International Edition
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COMMUNICATION
1
KS0
2
3
4
5
6
7
8
9
10
11 12
13
DH
Ox
DH
DH KR
cMT
DH
A
Ox
0
MT
KR
0
DH
A
KR
KR
cMT
KR
KR
KR
DH
TE
C
KS
KS
KS
C
KS
KS
KS
KS
KS
KS
KS KS
KS
?
A:
C:
DH:
KS:
KS :
KR:
adenylation
condensation
dehydratase
ketosynthase
non-elongating KS
ketoreductase
0
cMT: C-methyltransferase
TE:
Ox:
thioesterase
FAD dependent oxygenase
acylcarrier protein
-
:
lobatamideA 5
Figure 4. Biosynthetic model for lobatamide A. Ox domains and the corresponding polyketide moieties (proposed for the oxime) are highlighted in color. Consecutive
KS numbers are shown above the domains.
modify the carbon backbone itself. Our results also exemplify how
Accepted by
OocK and LbmC-Ox
Only accepted by LbmC-Ox
genome mining can pave the way to finding microbial sources for
bioactive compounds that had previously been described only
from marine invertebrates. The production of the tunicate
metabolite lobatamide A by G. sunshinyii further validates this
2
2
27
28
plant symbiont as
metabolites.
a rich source of bioactive specialized
1
1
8
9
2
2
3
4
Experimental Section
See supporting information.
2
2
0
1
25
26
2
9
Acknowledgements
We thank Young Ryun Chung and Dmitri Mavrodi for discussions
on the biology of G. sunshinyii. This project was funded by the
SNSF (grant IDs 205321_165659 and 205320_185077), and the
European Research Council (ERC) under the European Union’s
Horizon 2020 research and innovation programme (grant
agreement No 742739).
Figure 5. Structures of compounds deviating from polyketides intermediates
that were accepted by LbmC-Ox and OocK or by LbmC-Ox alone. For products,
see Figure S26.
In conclusion, our data support the existence of at least two
further module types in trans-AT PKSs that permit oxidative
modifications of polyketide core structures. In addition to the
topologically related oxygen insertions catalyzed by modules
utilizing trans-acting OocK/PedG/TobD-type enzymes (clade I
modules, Figure S1), we identified here a distinct, module-
integrated Ox domain that installs esters of the opposite
orientation (clade II modules, Figure S1). It is unknown whether
all members of one oxygenase clade generate the same ester
topology. However, in line with a potentially predictive uniform
Keywords: bacterial natural products • marine natural products •
biosynthesis • polyketides
[
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Angewandte Chemie International Edition
10.1002/anie.201916005
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O atom where art thou. The classic model of modular polyketide synthases (PKSs) involves a small set of reactions generating
combinatorially substituted carbon chains. We provide evidence for architecturally diverse PKS modules that diversify polyketides by
oxygenation. Studying an orphan PKS with two oxygenase modules from a plant symbiont reveals lobatamides, in-chain oxygenated
and oxime-bearing cytotoxins previously only known from a tunicate.
6
This article is protected by copyright. All rights reserved.