Two novel cyclic depsipeptides Xenematides F and G from the entomopathogenic bacterium Xenorhabdus budapestensis

Article abstract of DOI:10.1038/s41429-019-0203-y

Two novel depsipeptides xenematides F and G (1, 2), were isolated from entomopathogenic Xenorhabdus budapestensis SN84 along with a known compound xenematide B. The structures of the two new molecules were elucidated using NMR, MS and Marfey’s method. The xenematide G (2) contains α-aminoheptanoic acid, a non-protein amino acid that is rarely found in secondary metabolites from entomopathogenic bacteria. Xenematides F and G were tested for antibacterial activity. Xenematide G (2) exhibited moderate antibacterial activity.

Full text of DOI:10.1038/s41429-019-0203-y

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0203-y
ARTICLE
Two novel cyclic depsipeptides Xenematides F and G from the
entomopathogenic bacterium Xenorhabdus budapestensis
1
Xuedong Xi1 Xingzhong Lu1 Xiaodong Zhang1 Yuhui Bi1 Xiaochun Li1 Zhiguo Yu
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Received: 9 January 2019 / Revised: 29 May 2019 / Accepted: 5 June 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
Two novel depsipeptides xenematides F and G (1, 2), were isolated from entomopathogenic Xenorhabdus budapestensis
SN84 along with a known compound xenematide B. The structures of the two new molecules were elucidated using NMR,
MS and Marfey’s method. The xenematide G (2) contains α-aminoheptanoic acid, a non-protein amino acid that is rarely
found in secondary metabolites from entomopathogenic bacteria. Xenematides F and G were tested for antibacterial activity.
Xenematide G (2) exhibited moderate antibacterial activity.
Introduction
from Xenorhabdus budapestensis NMC-10 designated as
GP-19 and EP-20 was active against Verticillium dahlia and
Phytophthora capsici, respectively [11]. Rhabdopeptides/
xenortide-like peptides were recently identified and
show remarkable activity against trypanosoma and plas-
modium [12, 13]. A derivative of the cyclic depsipeptide,
xenematide A, exhibits antibacterial activity against both
Gram-negative and Gram-positive bacteria [9].
Natural product research has strongly benefited in recent
years from progress in sequencing technology and analy-
tical chemistry [14, 15]. Many gene clusters encoding
enzymes involved in natural product biosynthesis have been
identified. Because the isolation and identification of new
natural products using traditional methods demands a great
deal of time, effort, and resources, the use of PCR to target
the genomes of strains of interest has been increasingly used
as alternative approach to discover new natural products
[16–18]. In some studies, the biosynthesis genes have been
exploited as highly relevant indicators of natural product
production [19, 20]. Meanwhile, the use of real-time PCR
using primers with specific melting temperatures corre-
sponding to specific sequences had been shown to be a
specific and highly efficient method to screen strains with
identical natural product profiles [21].
Members of the genus Xenorhabdus are symbionts of
Steinernema nematodes and are important pathogens of
insects [1]. After nematodes invade the host insect, ento-
mopathogenic Photorhabdus spp. and Xenorhabdus spp.
are released into the haemolymph of the insect host. Next,
structurally diverse compounds produced by these bacteria
are released into the insect to interfere with its metabolic
processes [2]. Photorhabdus and Xenorhabdus play key
roles in regulating bacteria–insect–nematode interactions,
inducing the nematode infection process and protecting
dead insects from fungal invasion [3, 4]. Several peptides,
including the highly polar PAX (for peptide antimicrobial
from Xenorhabdus) peptides [5], GameXPeptides [6],
szentiamide [7], xentrivalpeptides [8] and xenematide
[9, 10] have been identified and isolated from Xenorhabdus
spp. In a previous study, five members of the PAX family
showed significant activity against plants and human fungal
pathogens, and moderate activity against several bacteria
and yeast [5]. Two novel antimicrobial peptides isolated
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0203-y) contains supplementary
material, which is available to authorized users.
The abundant production and significant activity of
peptides prompted us to identify and isolate new peptides
from Xenorhabdus. During our search for novel bioactive
natural products in entomopathogenic nematodes [22, 23],
a previously identified molecule, xenematide C, was
isolated from X. budapestensis SN19 together with new
xenematide E, and xenematide C exhibited moderate
* Zhiguo Yu
zyu@syau.edu.cn
1
Department of Plant Protection, Shenyang Agricultural University,
110866 Liaoning Province, China

X. Xi et al.
activity (EC50 = 22.71 μg ml−1) against Botrytis cinerea
[24]. Considering the great effort expended in discovering
the xenematide C-producing strain X. budapestensis SN19,
as well as the limited yield and derivation of xenematides
from strain SN19, in this study, we improved a method by
real-time PCR to screen isolates of Xenorhabdus spp. for
their ability to produce bioactive peptides. We demonstrated
the utility and effectiveness of our method for identifying
new xenematides from target producers. We have also
discovered new cyclic depsipeptides xenematides F and G,
the fermentation, isolation, structural elucidation and anti-
microbial activity are described.
The CH2Cl2 extract was subjected to silica gel chro-
matography that was eluted stepwise with CH2Cl2:MeOH
(100:0, 50:1, 20:1, 10:1, and 0:100, 3 l each) as the
mobile phase to produce five fractions, F1–F5. Fraction
F2 (1.3476 g) was subjected to gel chromatography on
Sephadex LH-20 and was eluted with MeOH to
yield xenematide C (426.5 mg) and a mixture of com-
pounds. The mixture was subsequently purified by
reverse-phase semi-preparative HPLC by applying a
MeOH–H2O gradient (with 0.1% HCOOH added to
both solvents) from 20% to 80% using a flow rate of
3.6 ml min−1 for 35 min. UV detection was performed
at 210 nm. Xenematide B (36 mg), xenematides F
(1, 11 mg), and G (2, 7.2 mg) eluted at 16.7, 20.5, and
27.9 min, respectively.
Materials and methods
General experimental procedures
NMR spectra, HPLC and LC–MS analysis
UV spectra were recorded on a Thermo ND-1000
spectrophotometer, and IR spectra on a Bruker IFS-55.
Preparation of a genomic DNA (gDNA) library, real-time
PCR, DNA sequencing, phylogenetic analysis, as well as
production of xenematide C is described in the Supple-
mentary Methods.
Optical rotations were measured with an ATAGO AP-300
polarimeter. NMR spectra were recorded on a 600 MHz
Bruker NMR spectrometer at room temperature. Carbon
signals and the residual proton signals of DMSO-d6
(δC 39.50 and δH 2.50, respectively) were used for cali-
bration. HPLC analysis was performed on an Agilent
1260 Infinity LC system coupled with a C18 column
(Agilent ZORBAX Eclipse XDB, 4.60 × 250 mm, 5 μm).
Fermentation and extraction
Marfey’s method was performed on
a Shimadzu
A two-staged fermentation was performed as previously
described [25]. For both stages, M medium (6.13 g glucose,
21.29 g peptone, 1.50 g MgSO4·7H2O, 2.46 g (NH4)2SO4,
0.86 g KH2PO4, 1.11 g K2HPO4, and 1.72 g NaSO4 in a
final volume of 1 l H2O, pH 7.0) was used. A 250 ml flask
containing 50 ml of M medium was inoculated with 100 μl
of a bacterial solution and incubated with shaking (200 rpm)
at 28 °C for 18 h to prepare the seed culture. Sixty 2-l flasks,
each containing 400 ml of M medium, were subsequently
inoculated with 20 ml of seed culture each and were allowed
to ferment for 120 h under identical conditions.
LC-2010A HT HPLC system coupled with a C18 column
(Agilent ZORBAX Eclipse XDB, 4.60 × 150 mm, 5 μm).
Semi-preparative HPLC was performed on an Agilent
1260 series system coupled with
a C18 column
(Agilent ZORBAX Eclipse XDB, 9.4 × 250 mm, 5 μm).
High-resolution mass spectra were acquired using an
Agilent 6500 series Q-TOF spectrometer. Column chro-
matography was performed using silica gel (100–200
mesh) and Sephadex LH-20 (GE Healthcare). The HPLC
analysis of crude extracts containing xenematide C was
performed using a 25 min solvent gradient (1 ml min−1
)
The production culture was centrifuged at 4723×g and
4 °C for 45 min to remove bacterial cells. The remaining
broth was extracted with 4% XAD-16 (Sigma) resin for 4 h
at room temperature with agitation. The resin was subse-
quently harvested by centrifugation and eluted four times
with MeOH. The combined eluates were then concentrated
under reduced pressure to obtain a dried extract.
from 10% to 75% CH3CN in H2O, and xenematide C
eluted at a retention time of 16.0 min. The peak areas at
210 and 280 nm were used to quantify xenematide C
production on the basis of calibration curves with
authentic xenematide C standards.
LC–MS was carried out on an Agilent 1260 Infinity LC
coupled to a 6230 TOF equipped with an Agilent Extend
C18 column (50 × 2.1 mm, 1.7 μm). Liquid chromatography
Isolation of metabolites
was performed using
a
17.0 min solvent gradient
(0.2 ml min−1) at 50% CH3CN in H2O containing 0.1%
formic acid. Xenematide C eluted at a retention time of
1.5 min. The extracted ion (m/z 624.2824) for the [xene-
matide C+H]+ ion verified xenematide C production.
Xenematide F (1): Colourless solid; UV (MeOH) λmax
The dried extract was re-dissolved in 50% MeOH (600 ml).
The solution was extracted four times with an equal volume
of CH2Cl2, after which the CH2Cl2 extract was collected
and concentrated on a rotary evaporator under vacuum at
35 °C to yield 16.827 g of solid brown residue.
(log ε) 226 (3.51), 283 (2.84) nm; IR νC=O 1735 cm−1
,

Two novel cyclic depsipeptides Xenematides F and G from the entomopathogenic bacterium Xenorhabdus. . .
νC–O–C 1168, 1096 cm−1, νC=O 1636(amide I) cm−1, δNH
HSQC, and HMBC spectra are available as Supporting
Information (Figs. S12–S16); HRESIMS m/z 590.2998
[M+H]+ (calculated for C32H40N5O6: 590.2978).
1538(amide II) cm−1; ½αŠ20 + 34.00 (c 1.00, MeOH); for 1H
D
and 13C NMR data (see Table 1); 1H and 13C NMR, COSY,
Table 1 NMR spectroscopic data (600 MHz (1H), 150 MHz (13C), d6-DMSO) for xenematides F and G (1, 2)a
Position
1
2
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
β-Ala
1
2
169.3, qC
34.0, CH2
169.3, qC
34.0, CH2
2.41, m
2.40, m
2.49, m
3.29m
2.49, m
3
34.5, CH2
3.31, m
34.6, CH2
3.41, m
3.41m
NH
7.38, t (6.0)
7.37, t (6.0)
OCH3
Trp
1
2
3
171.1, qC
54.5, CH
25.8, CH2
171.1, qC
54.4, CH
25.8, CH2
4.15, m
4.16, m
2.81, m
2.80, m
3.15, dd (15.1, 3.3)
3.15, dd (15.1, 3.3)
4
110.5, qC
127.0, qC
117.9, CH
118.2, CH
120.9, CH
111.3, CH
136.0, qC
110.5, qC
127.0, qC
117.9, CH
118.2, CH
120.9, CH
111.3, CH
136.0, qC
5
6
7.46, d (7.9)
6.97, t (7.9)
7.06, t (7.7)
7.33, d (8.1)
7.47, d (7.9)
6.96, t (7.8)
7.05, t (7.8)
7.33, d (8.1)
7
8
9
10
11/NH
12
10.66, brs
10.67, brs
123.0, CH
171.6, qC
6.81, d (1.6)
8.68, d (7.6)
123.0, CH
171.6, qC
6.83, d (2.4)
8.65, d (7.2)
NH
1
Leu (1) α-amino-heptanoic acid (2)
Phe (3)
2
3
54.8, CH
44.1, CH2
4.45, m
54.9, CH
34.8, CH2
4.45, m
2.20, m
2.27, m
1.48, m
1.48, m
1.24, m
1.24, m
1.24, m
1.24, m
0.85, t (7.2)
2.10, dd(13.6, 6.9)
2.17, dd(13.6, 7.5)
1.96, m
4
5
6
25.5, CH
22.3, CH3
22.3, CH3
25.0, CH2
30.9, CH2
21.8, CH2
13.9, CH3
0.88, d (6.7)
0.87, d (6.6)
7
8
9
NH
1
8.86, d (6.7)
8.85, d (6.6)
Thr
170.6, qC
53.9, CH
71.8, CH
16.3, CH3
170.6, qC
53.9, CH
71.9, CH
16.2, CH3
2
4.65, dd (9.3, 1.6)
5.10, m
4.65, dd (9.4, 1.9)
5.10, m
3
4
1.10, d (6.2)
7.96, d (9.4)
1.08, d (6.6)
7.95, d (9.6)
NH
OH
1
PAA
172.2, qC
35.6, CH2
172.9, qC
35.6, CH2
2
2.81, m
2.81, m
2.80, m
2.80, m
3
137.0, qC
128.2, CH
128.9, CH
126.5, CH
137.0, qC
128.2, CH
128.9, CH
126.5, CH
4/8
5/7
6
7.22, m
7.17, m
7.22, m
7.22, m
7.17, m
7.22 m
PAA 2-phenylacetic acid
1
aAssignments made on the basis of H–1H COSY, HSQC, and HMBC experiments

X. Xi et al.
Xenematide G (2): Colourless solid; UV (MeOH) λmax
(log ε) 229 (3.72), 283 (3.25) nm; IR νC=O 1736 cm−1, νC–O–C
1169, 1096 cm−1, νC=O 1637(amide I) cm−1, δNH 1537(amide
strains. The effect of different dilutions of the compounds
(up to 128 μg ml−1) on growth was assessed after 18 h incu-
bation at 37 °C using a Thermo Multiskan GO plate reader at
OD625. The MIC value was determined as the lowest con-
centration showing no growth compared to the control.
II) cm−1; ½αŠ20 + 35.00 (c 1.00, MeOH); for 1H and 13C NMR
D
1
data (see Table 1); H and 13C NMR, COSY, HSQC, and
HMBC spectra are available as Supporting Information
(Figs. S17–S21); HRESIMS m/z 604.3147 [M + H]+
(calculated for C33H42N5O6: 604.3135).
Results
Determination of the absolute amino acid
configurations
Development of a strain prioritisation method by
real-time PCR for cyclic depsipeptide discovery
Compounds 1 and 2 (1 mg each) were hydrolysed with 6 M
HCl (1 ml) at 120 °C for 3 h. The solutions were then eva-
porated to dryness, and the residues were re-dissolved in 1 ml
of water and dried on a rotary evaporator. Next, 100 μl of 1 M
sodium bicarbonate was added to each residue to form solu-
tions. Amino acid standards were also individually dissolved
in 100 μl of 1 M sodium bicarbonate at concentrations of
1 mg ml−1. The D, L-aminoheptanoic acid were purchased
from Aladdin Biochemical Technology and J&K Scientific.
Thirty-five microlitres of Nα-(2,4-dinitro-5-fluorophenyl)-
L-alaninamide (L-FDAA, Marfey’s Reagent, for threonine,
leucine and tryptophan) [26] and 60 μl Nα-(2,4-dinitro-5-
fluorophenyl)-D/L-leucinamide (D/L-FDLA, advanced Mar-
fey’s reagent, for α-aminoheptanoic acid) [27] in acetone
(10 mg ml−1) was added to each solution. The solutions were
incubated in a water bath at 55 °C for 1 h. After cooling to
room temperature, each solution was dissolved in MeCN for
HPLC analysis. At 26 °C, MeCN/H2O containing 0.1%
HCOOH was used as mobile phase using the following gra-
dient: 0–5 min, 20% MeCN; 5–35 min, 20–60% MeCN;
35–40 min, 60–100% MeCN and 40–50 min, 100–20%
MeCN. The flow rate was 1 ml min−1 with UV detection at
340 nm. The HPLC results for the amino acids were com-
pared to the results of the standard samples [26].
Real-time PCR was performed to detect the fluorescence
intensity of the target DNA. Melting curve analysis reveals
the melting temperature (Tm) corresponds to the length, base
composition, and nucleotide mismatch of PCR products and
can be used as specificity indicator [30]. Without the need
for gel electrophoresis, evaluating the Tm value of PCR
products become a key factor in accelerating the discovery
of natural products. We used SYBR Green I to perform real-
time PCR with the genomic DNA (gDNA) samples of a
Xenorhabdus genus strain collection for strain prioritisation,
as it enabled us to perform a melting curve analysis of
the PCR products. We isolated the xenematide producer
X. budapestensis SN19 in our preliminary work, which
produces a known xenematide (xenematide C). Because we
expended a great deal of effort in isolating the xenematide,
and the current repertoire of xenematide derivatives is
currently limited, we screened our Xenorhabdus sp. strain
collection at the Natural Product Laboratory of SYAU for
newly identified xenematide C producers.
The biosynthetic domain responsible for xenematide pro-
duction in X. nematophila ATCC19061 was previously
reported by Jason M. Crawford [10], and NRPS XNC1_2713
was indeed responsible for xenematide biosynthesis. The
condensation (C) domain is considered to be an active site
involved in xenematide biosynthesis. The gene sequences of
active sites C1 and C2 were chosen to design the primer
sets Xenf1/Xenr1, Xenf2/Xenr2 and Xenf6-JB/Xenr6-JB
(Figs. S1and S2). Three primer sets were predicted to produce
PCR amplicon sizes of 131, 90 and 135 bp. As the target
fragments were relatively short, the specificity cut off was set
at Tm 0.5 °C for each of the target amplicons. Four primers
would specifically narrow the scope of our search among the
correct hits and the relatively short target fragments could
effectively save PCR time.
Antifungal and antibacterial assays
Antifungal assays was performed with B. cinerea using
previously described methods [28]. Minimum inhibitory
concentrations (MICs) against bacteria were determined as
previously reported [29]. The antibacterial activity of the two
compounds was evaluated against Staphylococcus aureus
ATCC 25923, Bacillus subtilis NCTC 2116, Bacillus cereus
ATCC 10702, Pseudomonas aeruginosa A3, Shigella dys-
enteriae JS11910 and Salmonella enteritidis 1891. Strepto-
mycin and Gentamycin were used as positive controls. The
assays were performed in a 96-well microtiter plate format in
duplicate, with two independent cultures for each strain. The
two compounds were dissolved in DMSO (Sigma) and added
to the cultures in wells to give a final concentration of DMSO
of 2% that did not affect the growth of any of the tested
Strain prioritisation by real-time PCR for four
conserved nucleotide sequences, affording two
cyclic depsipeptide producers
The gDNA of the 47 strains was individually prepared and
used as template for PCR with each of four sets of primers.

Two novel cyclic depsipeptides Xenematides F and G from the entomopathogenic bacterium Xenorhabdus. . .
Each amplification assay included one blank reaction (i.e.,
no template DNA) as a negative control and a reaction with
strain SN19 gDNA as a positive control (Figure S6A–C).
As summarised in Figs. S6D, 2 of the 47 assayed strains
(as well as strain SN19) were identified as putative hits using
the primer sets Xenf1/Xenr1, Xenf2/Xenr2 and Xenf6-JB/
Xenr6-JB. The primer sets Xenf1/Xenr1 and Xenf2/Xenr2
were designed based on highly conservative sequences to
ensure high-precision matching with XNC1_2713-C2. The
Xenf6-JB/Xenr6-JB primers set was designed to match
XNC1_2713-C1. Sequencing of the resultant PCR amplicons
Fig. 1 Structures of compounds 1 and 2
revealed that strains SN18, SN19 and SN84 were likely
correct hits.
spectroscopic data (Table S3). Xenematide B has been pre-
Two identified positive strains produce xenematides
and harbour xenematide biosynthetic sequences
viously isolated from X. nematopila and was reported without
NMR spectroscopic data [10]. The 1H and 13C NMR, COSY,
HSQC, and HMBC spectra of xenematide B are available as
Supporting Information (Figs. S7–S11).
To validate the production of xenematide C by these strains,
all 10 strains which were preliminary or partly confirmed by
three pairs of primers were evaluated in fermentation assays
and assessed for xenematide C production. Two strains SN18
and SN84 were confirmed as xenematide producers (Fig. S3).
DNA sequencing of the resultant PCR amplicons confirmed
that each of the target genes was highly homologous (>85%)
to sequences from known xenematide producers (Fig. S4).
The hit strains were next subjected to polyphasic taxonomy
studies. A polyphasic taxonomy study was performed using
the 16S rRNA gene and ERIC-PCR on two hits. ERIC-PCR
is a DNA polymorphism assay based on amplification of
random DNA segments with single primers of arbitrary
nucleotide sequence. DNA polymorphism analysis with
ERIC-PCR can clearly distinguish microbial intraspecific
differences [31]. While SN18 and SN84 were assigned as X.
budapestensis by the phylogenetic tree based on the 16S
rRNA gene (Fig. S5A), the intraspecific identification via
ERIC-PCR revealed that SN18 was distinct from SN19 and
SN84 (Fig. S5B). As determined by ERIC-PCR, the SN84
was similar to SN19, despite being isolated from different soil
samples (Fig. S5B and Table S2).
The molecular formula of 1 was determined to be
1
C32H39N5O6 by HRESIMS analysis. The H and 13C NMR
spectra of 1 were similar to that of xenematide C, showing
identical signals for a β-alanine residue, a threonine residue, a
tryptophan residue, and a phenylacetyl (PAA) group [10]
1
(Table 1). However, further inspection of the H and 13C
NMR spectra of 1 revealed signals from an isopropyl unit
(δH 1.96, 0.88, and 0.87; δC 25.5, 22.3, and 22.3) which were
not present in xenematide C. The isopropyl unit gave a clue to
the presence of a leucine residue in 1. Furthermore, the
phenylalanine residue signals observed in spectra of xene-
matide C were not present in 1, suggesting that the pheny-
lalanine residue in xenematide C was substituted by a leucine
residue in 1. This was further supported by key correlations
observed in HMBC and COSY data (Fig. 2) and an accurate
mass of m/z 590.2998 measured for the [M + H]+ ion. Based
on these data, amino acid sequence of 1 was determined as
PAA-Thr-Leu-Trp-β-Ala. The absolute configurations of the
amino acids were determined using advanced Marfey’s
method as L-threonine, L-leucine, and D-tryptophan. Com-
pound 1 was given the name xenematide F.
HRESIMS analysis indicated that compound 2 possessed
1
Analysis and structural identification of three
the molecular formula C33H41N5O6. The H and 13C NMR
xenematides in strain SN84
spectra of 2 (Table 1) were similar to those of 1. Both 2 and 1
have a β-alanine residue, a threonine residue, a tryptophan
residue, and a PAA group as building blocks. The primary
difference between 1 and 2 is that the spectra of 2 showed
signals for an n-butyl group (δH 1.48, 1.24, 1.24 and 0.85; δC
30.9, 25.0, 21.8 and 13.9) instead of the isopropyl group
observed in spectra of 1. This suggested that the leucine
residue in 1 was replaced by α-aminoheptanoic acid in 2. This
was further supported by key correlations observed in HMBC
and COSY experiments (Fig. 2). Absolute configurations of
tryptophan and threonine were determined using Marfey’s
method as D-tryptophan and L-threonine. Since 2 and 1 are
Because of the richness of suspect xenematides we previously
observed in strain SN84 and because of the experience we
acquired in discovering xenematides in strain SN19, we chose
strain SN84 as the strain to ferment and characterise xene-
matides from. An XAD extract was obtained from a liquid
culture of strain SN84 (24 l). From the extract, two novel
xenematides, named F and G (Fig. 1), and a known xene-
matide (xenematide B; Fig. S7) were isolated and purified
using HPLC and gel chromatography. Xenematide B was
readily identified based on its molecular mass and NMR

X. Xi et al.
Fig. 2 Key COSY (bold) and
HMBC (arrows) correlations
supporting the structures of
xenematides F and G (1, 2)
Table 2 Antibacterial activity of
xenematide B, F and G
Compound
Average MIC (μg/ml)a
S. aureus
B. subtilis
B. cereus
P. aeruginosa
S. dysenteriae
S. enteritidis
Xenematide B
Xenematide F
Xenematide G
Streptomycin
Gentamycin
>128
>128
>128
4
64
>128
16
2
>128
>128
>128
>128
32
>128
32
16
4
>128
>128
>128
16
>128
64
>128
2
0.25
8
4
16
2
aAverage of two independent replicates
both dextrorotatory and are co-occurring metabolites, it was
assumed that they share the same biogenic origins. The ste-
reochemistry of α-aminoheptanoic acid is determined by
comparison with the control of D,L-aminoheptanoic acid using
the advanced Marfey’s method. The absolute configuration
of the α-aminoheptanoic acid in 2 was L-configuration
(Fig. S24). Compound 2 was given the name xenematide G.
the antibiotic daptomycin [32] or the immunosuppressant
cyclosporine A [33]. In this study, two xenematide producers
were identified, and two novel cyclic peptides were purified
and structurally characterised. As a key depsipeptide node, the
xenematide F (HRESIMS m/z 590.29) was predicted within
subnetwork [18]. We isolated the pure compound and pro-
vided the NMR data and formula of xenematide F for the first
time. As the first reported bioactivity xenematide, xenematide
A has an MIC value of 10 µg ml−1 against Bacillus subtilis,
Pseudomonas fluorescens, Pseudomonas syringae, Erwinia
amylovora, and Ralstonia solanacearum [17]. In our study,
the xenematide G has MIC value of 16 μg/ml against
B. subtilis which is similar to xenematide A. In the mean
time, the xenematide G contains α-aminoheptanoic acid, a
non-protein amino acid that is rarely found in secondary
metabolites from entomopathogenic bacteria. The special
amino acid may provide a structural foundation for the novel
bioactivity of xenematide G.
An improved real-time PCR method was used to prior-
itise the most promising strains from a microbial strain
collection for xenematide production. The high-throughput
and short extension time advantages of this method allowed
the time of discovery and cost associated with traditional
approaches to be decreased. Thus, the biosynthetic rela-
tionship of peptides and their producing NRPSs has become
a focus of research [34]. Based on the modification of
NRPSs, new NRPSs, peptides, peptide derivatives, func-
tionalizable peptides and peptide-based compounds could
Antimicrobial susceptibility testing
Xenematide B, F and G were tested for antifungal activity
against B. cinerea. The result show that three compounds
demonstrated no notable activity. Xenematide has been
reported to show antibacterial activities against a panel of
pathogenic bacteria [9]. Therefore, the antimicrobial activ-
ities of xenematide B, F and G were tested against six
pathogenic bacteria as shown in Table 2. The result suggest
that xenematide B show insignificant bioactivity. Xenema-
tide F show potent bioactivity against P. aeruginosa with
MIC value of 32 μg ml−1. Its congener xenematide G has
MIC value of 16 μg ml−1 against B. subtilis, which is similar
to xenematide A [9].
Discussion
Peptides derived from NRPSs represent an important class of
pharmaceutically relevant drugs. The prominent examples are

Two novel cyclic depsipeptides Xenematides F and G from the entomopathogenic bacterium Xenorhabdus. . .
potentially be created [35]. As strain banks expand with
microbial isolates, efficient molecular biology techniques,
such as real-time PCR should be introduced to accelerate
natural product discovery [36]. The primers were designed
to amplify relatively short target fragments (~100 bp) to
effectively shorten the required assay time without sacrifi-
cing accuracy. Based on the principle of real-time PCR,
relatively short target fragments can also avoid instances
where the Ct value appears too late [30].
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Acknowledgements This work are supported by Liaoning Province
Fund for Nature (No. 01032017001), Shenyang Agricultural Uni-
versity Postdoctoral Fund (No. 770215012) and Shenyang Agri-
cultural University Introducing Talent Fund (No. 880415016).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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