Genome-Based Identification of a Plant-Associated Marine Bacterium as a Rich Natural Product Source

Article abstract of DOI:10.1002/anie.201805673

The large number of sequenced bacterial genomes provides the opportunity to bioinformatically identify rich natural product sources among previously neglected microbial groups. Testing this discovery strategy, unusually high biosynthetic potential was suggested for the Oceanospirillales member Gynuella sunshinyii, a Gram-negative marine bacterium from the rhizosphere of the halophilic plant Carex scabrifolia. Its genome contains numerous unusual biosynthetic gene clusters for diverse types of metabolites. Genome-guided isolation yielded representatives of four different natural product classes, of which only alteramide A was known. Cytotoxic lacunalides were identified as products of a giant trans-acyltransferase polyketide synthase gene cluster, one of six present in this strain. Cytological profiling against HeLa cells suggested that lacunalide A disrupts CDK signaling in the cell cycle. In addition, chemical studies on model compounds were conducted, suggesting the structurally unusual ergoynes as products of a conjugated diyne–thiourea cyclization reaction.

Full text of DOI:10.1002/anie.201805673

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Accepted Article
Title: Genome-based identification of a plant-associated marine
bacterium as a rich natural product source
Authors: Reiko Ueoka, Agneya Bhushan, Silke I. Probst, Walter M.
Bray, R. Scott Lokey, Roger G. Linington, and Jörn Piel
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201805673
Angew. Chem. 10.1002/ange.201805673
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COMMUNICATION
Genome-based identification of a plant-associated marine
bacterium as a rich natural product source
[
a]
[a]
[a]
[b]
[b]
Reiko Ueoka , Agneya Bhushan , Silke I. Probst , Walter M. Bray , R. Scott Lokey , Roger G.
[c]
[a]
Linington , and Jörn Piel*
Abstract: The large number of sequenced bacterial genomes
provides the opportunity to bioinformatically identify rich natural
product sources among previously neglected microbial groups.
Testing this discovery strategy, unusually high biosynthetic potential
was suggested for the Oceanospirillales member Gynuella sunshinyii,
a Gram-negative marine bacterium from the rhizosphere of the
halophilic plant Carex scabrifolia. Its genome contains numerous
unusual biosynthetic gene clusters for diverse types of metabolites.
Genome-guided isolation yielded representatives of four different
natural product classes, of which only alteramide A was known.
Cytotoxic lacunalides were identified as products of a giant trans-
acyltransferase polyketide synthase gene cluster, one of six present
in this strain. Cytological profiling against HeLa cells suggests that
lacunalide A disrupts CDK signaling in the cell cycle. In addition,
chemical studies on model compounds were conducted, suggesting
the structurally unusual ergoynes as products of a conjugated diyne-
thiourea cyclization reaction.
Here we investigated whether combining BGC-based
dereplication with an analysis of BGC frequencies in poorly
explored taxonomic and ecological groups would permit the
discovery of talented producers that generate chemical novelty. A
global analysis of BGC frequencies per organism suggested as
top candidate the plant-associated marine bacterium Gynuella
sunshinyii YC6258.5 Its chemical richness was confirmed by
characterizing four different types of natural products, of which
6
only one was previously described. Among the natural products
with a new chemical scaffold are the cytotoxic lacunalides, which
are among the largest known bacterial polyketides generated by
a trans-acyltransferase polyketide synthase (trans-AT PKS)7.
We initiated our study by analyzing BGC frequencies in all
finished bacterial genomes present in the AntiSMASH database.8
Actinomycetes, the best-studied bacterial natural product
resource, were excluded to result in 2420 genomes as input for
the analysis. Using a Python script, BGC numbers were assessed
per BGC type, including nonribosomal peptide synthetases
(NRPSs), type I PKSs belonging to the cis-AT and trans-AT family,
type II PKSs, type III PKSs, and PKS or NRPS hybrids (Fig. 1,
Table S1).
The biosynthetic richness of bacteria provides an important
basis of pharmaceutical discovery and has resulted in numerous
drugs.1 While earlier screening efforts mostly focused on few
bacterial groups known as "talented producers", genomic data
suggest capacity for natural product biosynthesis in many
chemically unexplored taxa.2 Generally, natural product
structures correlate well with the genes present in biosynthetic
gene clusters (BGCs). In-silico prediction and dereplication of
biosynthetic products is therefore a powerful strategy to detect
metabolic novelty and to guide natural product isolation.3 This
approach is validated by successful BGC-based discoveries of
compounds from virtually every major natural product class.4
[
a]
Dr. Reiko Ueoka, Agneya Bhushan, Silke I. Probst, Prof. Dr. Jörn
Piel.
Institute of Microbiology
Eigenössische Technische Hochschule (ETH) Zurich
Vladimir-Prelog-Weg 1-5/10
Figure 1. Analysis of BGC frequencies in complete bacterial genomes present
in the AntiSMASH database. Actinomycetes were excluded from this analysis.
8
093 Zurich
Switzerland
E-mail: jpiel@ethz.ch
Dr. Walter M. Bray, Prof. Dr. R. Scott Lokey
Chemistry & Biochemistry Department
University of California Santa Cruz
"Type I PKS" includes mono- and multimodular cis-AT PKSs, "PKS or NRPS
hybrids" refers to hybrids of PKSs and/or NRPS with other natural product
enzyme families. The top 12 entries are shown. For a list of the top 100 strains,
see Table S1.
[
[
b]
c]
1
156 High Street
95064 Santa Cruz, California
United States
Among the 100 top hits with the highest total BGC numbers
were strains belonging to several recognized talented producer
taxa: myxobacteria, cyanobacteria, Photorhabdus and
Xenorhabdus, Burkholderia and relatives, bacilli, and clostridia.
However, at position 9 in the list and being the single exception
among the top 18 organisms was Gynuella sunshinyii, a halophilic
organism of the chemically poorly studied order Oceanospirillales.
This bacterium was isolated from the rhizosphere of the halophyte
Prof. Dr. Roger Linington
Department of Chemistry
Simon Fraser University
8
888 University Drive
Bumaby, BC V5A 1S6
Canada
Supporting information for this article is given via a link at the end of
the document.
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5
2
Carex scabrifolia growing in a marine tidal flat. Among a total of
6 PKS and NRPS BGCs present in its genome, 13 have < 50%
absence of one methyl group attached to an sp carbon and the
2
1
presence of one additional methine connected to an sp carbon,
similarities to functionally assigned counterparts based on a
search using the MIBiG database of characterized BGCs (Table
S2).9 Particularly noteworthy is the number of trans-AT PKS
clusters (Fig. 1, Table S1-2), which includes two PKSs and three
hybrid NRPS-PKS systems. In addition, by manual analysis we
identified a further trans-AT PKS-NRPS hybrid system (pks5),
overlooked by AntiSMASH, making the six BGCs in G. sunshinyii
to our knowledge the highest number of trans-AT PKS loci in a
single genome reported to date. Trans-AT PKSs are a widespread
but poorly explored family of natural product enzymes with high
potential for novelty.7 We also conducted manual homology
searches using a collection of non-standard natural product
enzymes. This search revealed for JamB, a cyanobacterial
as compared to 1 (Fig. S15-16). 2D NMR analysis revealed that
C-20 carries a hydrogen instead of the C-53 methyl group in 1
(Figure S17-19). The geometry of the double bounds was
determined from coupling constants and NOESY/ROESY data
(Table S4-5, Fig. S20-21). The large coupling constants of H-2/H-
3
3
3 ( JH2,H3 = 15.7 Hz in 1 and 2), H-20/H-21 ( JH20,H21 = 15.6 Hz in
2), and H-38/H-39 ( JH38,H39 = 15.2 Hz in 1 and 3JH38,H39 = 15.3 Hz
in 2) were consistent with trans double bonds. NOESY/ROESY
correlations between H-3 and H-5 and between H-6 and H-51 in
1 and 2 showed 4E stereochemistry, and NOESY correlations
between H-19 and H-21 and between H-22 and H-53 in 1
revealed 20E stereochemistry. The relative stereochemistry of the
hydroxyl and methyl groups in 1 was determined by application of
3
1
0
13
14
alkyne-forming desaturase from the jamaicamide pathway,
a
Kishi's universal database method and J-based analysis.
small BGC containing two homologs (Table S2). Based on its
large number of unassigned BGCs, unusual habitat, and
taxonomic distance to known talented producer groups, we
selected G. sunshinyii for further chemical analysis focusing first
on the trans-AT PKS clusters (Table S2). An initial MIBiG analysis
of the clusters suggested for pks1 and pks4 congeners of
cytotoxic phormidolides11 and tartrolon-type antibiotics12,
respectively, while those of pks2, pks3, pks5, and pks6 lacked
architecturally similar clusters in any other sequenced bacterium
in the GenBank database (Table S2). Intriguingly, with a size of
ca. 129 kb and 25 encoded PKS modules, pks3 was one of the
largest known trans-AT PKS gene cluster at the time of writing,
suggesting a structurally distinct polyketide (Fig. 2 Table S3).
Small  values (66.5 ppm in CD OD) suggested anti/anti-
C
3
relationship in C7-C17. The relative configuration of C-17-19 was
suggested to be syn/anti or anti/syn by comparison of 13C
chemical shifts of C-52 in 1 to that of Kishi´s database (10.6 ppm
in CD
relationship in C-17-19 (Fig. S20, S22-23). Finally, the small 13
chemical shift of C-54 in 7 suggested syn/syn relationship of C-
41-43 (6.8 ppm in CD OD). The absolute stereochemistry of all
13 hydroxyl groups
7S,9R,11S,13R,15S,17R,19S,23R,27R,31R,41S,43R,49R
3
OD). NOESY data and J-based analysis showed anti/syn
C
3
was
suggested
to
be
on
the basis of both the predicted KS substrate specificity and
15
ketoreductase (KR) domain analysis (Tables S4 and S7). While
the KS- and KR-based predictions were generally in agreement,
for the fifth KS in the lacunalide PKS assembly line, designated
as LcnKS18 (17R, Fig. 2) and its preceding KR (LcnC_KR4, 17S),
contradicting configurations were predicted; however, the above-
mentioned NMR analysis suggested 17R stereochemistry.
Therefore, the absolute configuration of the two methyl groups
was suggested to be 18R,42S. Lacunalides are novel 42-
membered macrolactones with a poly-1,3-hydroxyl moiety. Their
structures match well the KS-based prediction (Fig. 2, Fig. S6).
The antibiotic desertomycins/oasomycins, isolated from
actinomycetes, also contain 42- or 44-membered macrolactone
Trans-AT PKSs are extraordinarily diverse multimodular
enzymes, in which each module carries a ketosynthase (KS)
domain that elongates a polyketide intermediate. We have
previously developed a method that predicts polyketide moieties
from trans-AT KS sequences and thus provides insights into the
4c
collective polyketide core structure . Based on such analysis, we
were able to predict a structure for pks3 (Table S4). To search for
compounds with a matching predicted formula, we cultivated G.
sunshinyii and analyzed culture extracts by liquid chromatography
mass spectrometry (LCMS). High resolution-electrospray
ionization mass spectrometry (HR-ESIMS) showed the presence
16
with a poly-1,3-hydroxyl moiety, but are otherwise distinct and
biosynthesized by cis-AT PKSs.17
+
of a candidate ion peak at m/z 971.7002 [M+H] with a suggested
chemical formula to be C54
H
99
O
14, close to that of the predicted
18) (Fig. S1). Targeted MS-guided isolation by
reversed phase-high performance liquid
To test for activity, we subjected 1 to cytotoxicity and anti-
bacterial assays. Lacunalide was found to be cytotoxic towards
HeLa cells (IC50 = 1 µM) and the Gram-positive bacteria
Arthrobacter crystallopoietes and Bacillus subtilis with minimal
inhibitory concentration values of 0.32 µM and 8 µM respectively.
106
structure (C54H O
repetitive
chromatography (RP-HPLC) yielded the pure compound, named
lacunalide A (1) (Fig. 3). Further analysis of the bacterial extracts
provided another congener, lacunalide B (2) (Fig. 2).
1
The H NMR and HSQC data of 1 suggested the presence of
2
three doublet methyls, two methyls connected to sp carbons, six
protons connected to sp2 carbons and many hydroxymethines
(
(
Fig. S2-3). Detailed 2D NMR analysis revealed five substructures
Fig. S4-6). Connection of these substructures was determined by
methanolysis of
desorption/ionization-(MALDI-)MS analysis of the resulting
1
and subsequent matrix-assisted laser
2
product (7, Fig. S7-13). Lacunalide B (2) had a predicted
molecular formula of C53
for 1 (Fig. S14). The H NMR and HSQC spectra suggested the
H
96
O
14, which was one CH
2
unit less than
1
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Figure 2. a) The lcn BGC. Black: core PKS genes; dark grey, accessory genes; light grey, resolvase gene. b) Biosynthetic model of lacunalide A. Each circle
corresponds to a functional domain. Small black circles are acyl carrier protein (ACP) domains. AH, acyl hydrolase; AT, acyltransferase; DH, dehydratase; DH*,
0
predicted non-functional DH; ER, enoyl reductase; KR?, proposed ketoreductase; KS , non-elongating KS; MT, methyltransferase; TE, thioesterase. Numbers
above KS domains correspond to consecutive KS and module numbers. In the chemical structure, wedged bonds specify the stereochemistry based on KS, KR,
and NMR analysis, non-wedged stereo bonds refer to the stereochemistry based on KS and NMR analysis (for C-17), on KS analysis (for C-27 and C-31), and
on NMR analysis (for methyl groups).
No activity towards Gram-negative bacteria was observed up to
concentrations of 40 µM. For further insights, we subjected 1 to
cytological profiling against HeLa cells.18 This assay predicts
compound modes of action (MOA) by using high-content imaging
to measure hundreds of size and shape features on a per-cell
basis. Cytological profiles are determined for each test compound
by comparing variations in feature values between drug-treated
and control cell populations; these profiles are compared to those
from annotated control compounds to afford predictions about
mechanisms of action. 1 gave a strong cytological profile at
concentrations of 0.195 M and above in a region of the cluster
diagram associated with G1/S stall (Fig. S24-25). This profile was
located in
compounds associated with inhibition of CDK (roscovitine, H9,
cerulenin, and the olomoucine derivative N9-
isopropylolomoucine; Fig. S24-25), suggesting that lacunalide A
may disrupt CDK signaling.
a separate sub-cluster including four control
Figure 3. Structures of natural products produced by G. sunshinyii. Lacunalides
A (1) and B (2), sunshinamide (3), ergoynes A (4) and B (5), and alteramide A
(6).
We next wished to cover a wider variety of natural product
classes by focusing on the unassigned nonribosomal peptide
synthase (NRPS) gene cluster nrps6 (Table S2, S8). LC-HRMS
analysis of culture extracts suggested a candidate ion peak for a
compound with two sulfur atoms, which agreed with the presence
of two cysteines in the predicted structure based on an analysis
of the NRPS adenylation (A) domains (Fig. 4 and S26). The
To determine the actual structure, 3 was isolated by repetitive
MS-guided RP-HPLC. H NMR and 2D NMR data showed the
presence of a phenylalanine, two cysteines connected via a
disulfide bond, and a fatty acid carrying oxygen at the γ-carbon
1
(
Fig. S27-31B, Table S9). The length of the fatty acid chain was
2
determined by ESI-MS analysis (Fig. S31B-32), and the
connection of each component was deduced from ROESY data
compound (3) had a predicted molecular formula of C25
H
35 5
O N
3
S
2
(
Fig. S31C, 33). Finally, the ester bond between C-3 and C-1'''
was suggested by its molecular formula. The absolute
configuaration of amino acids was determined by Marfey's
+
(m/z 522.2083 [M+H] ), as determined by HR-ESIMS (Fig. S26).
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analysis,19 using the acid hydrolysate of 3 and of its desthio
and H-5' revealed 10E stereochemistry in 4, and a NOESY cross
peak between H-10 and H-12 showed 10Z stereochemistry in 5
(Fig. S45).
20
derivative generated by reaction with Raney-Nickel (Fig. S34).
The amino acids in 3 were assigned to be -Phe and two -Cys,
D
L
which were in good agreement with the predicted stereochemistry
based on the presence of an epimerase domain in the second
NRPS module (Fig. 4). Compound 3, named sunshinamide, is a
new cyclodepsitripeptide, for which the closest structural hits in
Scifinder are the fungus-derived beauveriolides.21 Although
numerous beauveriolide derivatives were synthesized,
sunshinamide was novel for its two serial cysteines connected via
a disulfide bond. In our assays, 3 showed cytotoxic activity against
HeLa cells with an IC50 value of 0.59 M.
Since AntiSMASH had not detected a candidate for the
ergoyne BGC, we manually searched for matching genes.
Interestingly, we only found genes encoding homologs of EgtB
and EgtD, two of the five enzymes involved in ergothioneine
22
biosynthesis (Table S11), but further natural product genes
were missing. However, the genome contains an additional small
cluster candidate located 3.5 Mbp away from the ergothioneine
locus harboring the JamB-type10 acetylenase genes, as well as
23
genes for a fatty acyl ligase, and an ACP (Table S12). Since the
putative BGCs for the two structural ergoyne moieties are
disconnected, we speculated whether 4 and 5 might be products
of
a spontaneous reaction of a tetrayne precursor and
ergothioneine (Fig. 5a). To investigate this hypothesis, chemical
transformation of model compounds was performed (Fig. 5).
Synthesized product 9 (Fig. S46-47) was treated with 2-
mercaptoimidazole in the presence of K CO at room temperature.
2 3
Analysis of LCMS data showed the production of a major
component with a structure 10 determined by 2D NMR (Fig. S48-
52, Table S13). Detailed analysis of LCMS data showed trace
amount of other products with the same mass as for 10 (Fig. S53).
Therefore, the reaction mixture was heated to overcome potential
steric and electronic effects of the phenyl group.
Figure 4. a) The ssa BGC. Black: core NRPS gene, dark grey: accessory genes,
light grey: genes putatively not involved in sunshinamide biosynthesis. b)
Biosynthetic model of sunshinamide (3). A, adenylation domain; C,
condensation domain; E, epimerization domain; Small black circles correspond
to peptide carrier proteins.
The discovery of new bioactive natural products from two
different families encouraged us to search for additional
compounds in G. sunshinyii extracts. Detailed analysis of the LC-
HRMS data suggested another sulfur-containing metabolite, but
2
the MS data did not match to sunshinamide or to any other typical
peptide fragmentation pattern (Fig. S36-37). This compound was
highly labile during MS-guided isolation, but careful choice of the
solvents during the extraction and rapid separation in the dark
yielded a fraction enriched with two congeners, named ergoyne A
Figure 5. a) Proposed biosynthetic pathway of ergoynes A (5, 10E) and B (6,
10Z). b) Chemical investigation of ergoyne biosynthesis with model compounds.
(
4) and B (5). Both compounds had a predicted molecular formula
+
of C27H
33 4
O N
3
S (m/z 496.2251 [M+H] ), as determined by HR-
ESIMS (Fig. S36). 2D NMR analysis revealed
resembling ergothioneine, an unusual antioxidant thiourea-
containing amino acid reported from several microorganisms that
has been proposed as a vitamin (Fig. S38-43, Table S10). In
addition to the ergothioneine portion, the data supported a highly
conjugated, lipid-like moiety containing an isopentyl branch (Fig.
S38-43). A diyne moiety in the chain was determined by HMBC
a moiety
Accumulated products were isolated by RP-HPLC, and the
structure was determined by 2D NMR as 11 and its geometrical
isomer 12 (Fig. S54-59, Table S14). This result supports the
biosynthetic hypothesis in Fig. 5a. The mixture of 11 and 12
showed cytotoxicity against HeLa cell line with an IC50 value of
22
5.95 M.
C
correlations from H-4, H-5, and H-10 to four carbons ( 72-83
In conclusion, a genome mining strategy targeting BGC
ppm, Fig. S41-43). The ergothioneine and fatty acid-like unit were
connected by HMBC correlations from H-13 and H-5' to C-6', and
by 15N-HMBC correlations from H-10, H-12, and H-5' to a nitrogen
frequencies and uniqueness in phylogenetically and ecologically
unusual producers revealed G. sunshinyii as rich source of
bioactive metabolites (Fig. 3). Our analysis showed that the BGC
number is even higher than initially suggested by automated
prediction. Chemical studies on some of the metabolites identified
N
( 177.2 ppm, Fig. S41-44). A NOESY cross peak between H-10
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Angewandte Chemie International Edition
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COMMUNICATION
natural products with three distinct, novel scaffolds in addition to
a known compound, alteramide A (detailed in Fig. S60-64). This
includes the structurally and chemically intriguing ergoynes and
the complex lacunalides with cytotoxic and antibacterial activity
as products of a giant trans-AT PKS system. Further investigation
on other BGCs in G. sunshinyii is ongoing. Since the producer
studied here is the only known strain in the genus Gynuella,
targeted isolation of additional members from similar habitats
could be an attractive strategy for drug discovery based on new
producer taxa.
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Acknowledgements
We thank R. A. Meoded and Y. R. Chung for insightful discussion.
J.P. acknowledges funding from the ERC (Advanced Grant
SynPlex) and the SNF (NRP72 "Antimicrobial Resistance"). R.U.
was a fellowship recipient of the Alexander von Humboldt
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Angewandte Chemie International Edition
10.1002/anie.201805673
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Let the sunshine in: A comparison of
all published bacterial genomes was
conducted to identify novel prolific
natural product sources among
ecologically and phylogenetically
unusual bacteria. This suggested the
plant root-associated halophilic
bacterium Gynuella sunshinyii as a
top candidate. Its richness was
confirmed in an extensive study of its
chemical repertoire, revealing diverse
metabolites with new scaffolds and
promising activities.
Reiko Ueoka, Agneya Bhushan, Silke I.
Probst, Walter M. Bray, R. Scott Lokey,
Roger G. Linington, and Jörn Piel*
Page No. – Page No.
Genome-based identification of a
plant-associated marine bacterium as
a rich natural product source
This article is protected by copyright. All rights reserved.