Krisynomycins, Imipenem Potentiators against Methicillin-Resistant Staphylococcus aureus, Produced by Streptomyces canus

Article abstract of DOI:10.1021/acs.jnatprod.0c00294

A reinvestigation of the acetone extract of the strain CA-091830 of Streptomyces canus, producer of the imipenem potentiator krisynomycin, resulted in the isolation of two additional analogues, krisynomycins B (1) and C (2), with different chlorination patterns. Genome sequencing of the strain followed by detailed bioinformatics analysis led to the identification of the corresponding biosynthetic gene cluster (BGC) of this cyclic nonribosomal peptide family. The planar structure of the new molecules was determined using HRMS, ESI-qTOF-MS/MS, and 1D and 2D NMR data. Their absolute configuration was proposed using a combination of Marfey's and bioinformatic BGC analyses. The krisynomycins displayed weak to negligible antibiotic activity against methicillin-resistant Staphylococcus aureus (MRSA), which was significantly enhanced when tested in combination with sublethal concentrations of imipenem. The halogenation pattern plays a key role in the antimicrobial activity and imipenem-potentiating effects of the compounds, with molecules having a higher number of chlorine atoms potentiating the effect of imipenem at lower doses.

Full text of DOI:10.1021/acs.jnatprod.0c00294

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Article
Krisynomycins, Imipenem Potentiators against Methicillin-Resistant
Staphylococcus aureus, Produced by Streptomyces canus
́
́
́
Mercedes Perez-Bonilla,* Daniel Oves-Costales, Ignacio Gonzalez, Mercedes de la Cruz, Jesus Martín,
Francisca Vicente, Olga Genilloud, and Fernando Reyes*
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ABSTRACT: A reinvestigation of the acetone extract of the strain
CA-091830 of Streptomyces canus, producer of the imipenem
potentiator krisynomycin, resulted in the isolation of two
additional analogues, krisynomycins B (1) and C (2), with
different chlorination patterns. Genome sequencing of the strain
followed by detailed bioinformatics analysis led to the
identification of the corresponding biosynthetic gene cluster
(BGC) of this cyclic nonribosomal peptide family. The planar
structure of the new molecules was determined using HRMS, ESI-
qTOF-MS/MS, and 1D and 2D NMR data. Their absolute
configuration was proposed using a combination of Marfey’s and
bioinformatic BGC analyses. The krisynomycins displayed weak to
negligible antibiotic activity against methicillin-resistant Staph-
ylococcus aureus (MRSA), which was significantly enhanced when tested in combination with sublethal concentrations of imipenem.
The halogenation pattern plays a key role in the antimicrobial activity and imipenem-potentiating effects of the compounds, with
molecules having a higher number of chlorine atoms potentiating the effect of imipenem at lower doses.
ntimicrobial resistance (AMR) threatens the effective
insensitive penicillin-binding protein (PBP2a) allows MRSA
to proliferate in the presence of typically inhibitory
concentrations of β-lactam antibiotics.10 Nonetheless, those
compounds that interfere with the stability of PBP2a render
MRSA susceptible to β-lactam antibiotics. Consequently,
another strategy to address emerging resistance is the
identification of molecules that could potentiate or enhance
the activity of existing antibacterial agents. Probably, the best
known combinations with β-lactam antibiotics are the drugs
amoxicillin-clavulanic acid, ampicillin-sulbactam, and piper-
acillin-tazobactam, but other molecules that potentiate the β-
lactam antibiotic activity have also been reported.11 The cyclic
depsipeptide krisynomycin and the lipoglycopeptide actino-
carbasin are also among the compounds that potentiate the
activity of imipenem against the MRSA strain COL.12 Other
imipenem potentiators against MRSA isolated from micro-
organisms are cyslabdan, cyslabdans B and C,13,14 and the
xanthoradones A, B, and C.15
A
prevention and treatment of an ever-increasing range of
infections caused by bacteria, parasites, viruses, and fungi.1
Although AMR occurs naturally over time, this process is being
accelerated by the misuse and overuse of antibiotics, as well as
poor infection prevention and control.1,2 It has been estimated
that AMR will cause around 10 million deaths a year by 2050,
being the major cause of death in comparison with other major
health problems, such as cancer.3
Nature has been historically the source of a considerable
number of drugs developed from microorganisms of varied
environments.4,5 In fact, actinomycetes, and Streptomyces in
particular, have been for decades one of the most significant
sources for the discovery of new antibiotics, with a number of
drugs successfully introduced in the market and still used today
in clinical practice.6,7
Peptides, and notably natural cyclic peptides such as
vancomycin, daptomycin, and polymyxin B, have gained
special attention due to their remarkable variety of structures
and valuable functions. To date, more than 40 cyclic peptide
drugs are currently on the market, and approximately one new
cyclopeptide drug enters the market annually on average.8
Methicillin-resistant Staphylococcus aureus (MRSA) is a
common cause of infection in hospitals and the community.
β-Lactam antibiotics are the most successful chemical class of
antibiotics used to treat infections in humans.9 However, the
acquisition of a non-native gene encoding a β-lactam-
Received: March 17, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
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1
Table 1. H (500 MHz) and 13C (125 MHz) NMR Data of Compounds 1−3 in DMSO-d6
1
2
3
ab
,
ab
,
residue
position
δC, mult.
δH, mult. (J in Hz)
δC, mult.
δH, mult. (J in Hz)
δC, mult.
δH, mult. (J in Hz)
malonic acid
1
2
3
4
5
169.3, C
42.1, CH2
165.2, C
171.9, C
48.0, CH
170.3, C
43.1, CH2
166.5, C
172.3, C
48.3, CH
3.18, d (5.5)
43.4, CH2
3.18, br s
3.09, m
Ala
171.9, C
49.3, CH
4.39, m
4.38, m
4.33, m
NH
6
7
8
NH
9
10
11
12
13
NH
14
8.26 d (7.4)
1.20, d (7.1)
8.27, m
1.20, d (7.0)
8.44, m
1.21, d (7.0)
18.0, CH3
170.5, C
57.3, CH
19.4, CH3
58.6, CH
18.1, CH3
171.0, C
57.6, CH
Val 1
4.20, m
7.88, d (8.6)
2.00, m
0.84, m
0.83, m
4.21, m
7.89, m
2.00, m
0.82, m
0.83, m
4.19, m
8.12, m
2.01, m
0.81, m
0.82, m
30.4, CH
18.8, CH3
17.7, CH3
167.7, C
31.7, CH
20.2, CH3
19.1, CH3
30.4, CH
19.2, CH3
18.0, CH3
168.0, C
Ser 1
Cya
Pro
51.4, CH
4.48, dd (11.9, 7.1)
8.04, d (7.1)
4.16, m
52.7, CH
64.8, CH2
4.49, m
8.05, d (7.0)
4.17, m
51.7, CH
4.50, m
8.05, d (6.8)
4.18, m
63.5, CH2
63.7, CH2
4.10, m
4.10, m
4.08, m
15
16
NH
17
171.2, C
47.2, CH
171.3, C
47.5, CH
5.17, ddd (12.7, 7.5, 5.3)
8.42, d (7.5)
3.42, m
48.6, CH
54.0, CH2
5.16, m
8.41, d (7.6)
3.42, m
5.13, m
8.37, d (7.3)
d
52.6, CH2
52.8, CH2
2.90, br d (12.7)
2.90, m
2.95, m
18
19
20
173.7, C
61.0, CH
29.2, CH2
173.7, C
61.2, CH
29.4, CH2
4.42, m
1.95, m
1.51, m
1.81, m
1.48, m
4.26, m
3.92, m
62.3, CH
30.6, CH2
4.42, m
1.96, m
1.52, m
1.82, m
1.50, m
4.23, m
3.91, m
4.39, m
1.95, m
1.48, m
1.80, m
1.45, m
4.20, m
3.91, m
21
22
23.7, CH2
47.3, CH2
25.1, CH2
48.7, CH2
23.9, CH2
47.5, CH2
Trp
23
24
NH
25
171.7, C
56.7, CH
171.7, C
56.8, CH
4.01, m
8.51, d (6.6)
3.20, m
58.0, CH
26.2, CH2
4.01, m
8.57, d (6.8)
3.21, m
4.02, m
8.50, d (6.8)
3.18, m
24.9, CH2
25.2, CH2
2.92, br d (13.2)
2.91, m
2.91, m
26
110.4, C
112.1, C
112.2, C
27
124.0, CH
7.34, d (2.2)
10.83, s
126.6, CH
7.43, br s
11.22, s
125.5, CH
7.41, br s
11.23, br s
NH (indole)
28
29
30
31
32
33
34
35
NH
36
135.9, C
132.7, C
115.6, C
121.5, CH
120.6, CH
118.4, CH
128.8, C
132.8, C
115.8, C
120.4, CH
119.4, CH
117.4, CH
129.0, C
170.9, C
55.3, CH
111.2, CH
120.7, CH
118.2, CH
117.9, CH
126.6, C
7.30, d (7.9)
7.02, t (7.9)
6.91, t (7.9)
7.51, d (7.9)
7.11, d (7.7)
6.91, t (7.7)
7.49, d (7.7)
7.11, d (7.7)
6.91, t (7.7)
7.50, d (7.7)
prenyl Trp
171.2, C
55.3, CH
4.38, m
7.66, d (8.6)
3.68, m
56.7, CH
29.3, CH2
4.37, m
7.70, m
3.69, m
3.02, m
4.38, m
7.69, m
3.63, br d (12.9)
3.01, m
27.9, CH2
28.2, CH2
3.01, dd (14.6, 11.1)
37
110.2, C
112.1, C
38
125.7, CH
7.61, br s
10.71, s
127.0, CH
7.61, br s
10.72, s
127.3, CH
7.72, br s
10.99, br s
NH (indole)
39
40
41
42
43
44
136.8, C
133.2, C
133.4, C
113.8, C
119.8, CH
119.5, CH
132.4, C
126.3, C
109.4, CH
120.4, CH
118.4, CH
133.1, C
7.16, d (7.5)
6.92, t (7.5)
6.72, d (7.5)
110.7, CH
121.7, CH
119.7, CH
132.7, C
7.15d (7.2)
6.92, t (7.2)
6.72, d (7.2)
7.03, d (7.8)
6.74, d (7.8)
124.1, C
124.2, C
B
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Table 1. continued
1
2
3
ab
,
ab
,
residue
position
δC, mult.
δH, mult. (J in Hz)
δC, mult.
δH, mult. (J in Hz)
δC, mult.
δH, mult. (J in Hz)
45
31.4, CH2
3.83, dd (15.1, 6.2)
3.72, m
5.33, m
32.8, CH2
3.83, dd (16.6, 6.2)
3.72, m
5.33, m
31.0, CH2
3.82, m
3.72, m
5.30, m
46
47
48
49
50
51
NH
52
53
123.9, CH
130.9, C
17.6, CH3
25.3, CH3
170.7, C
125.2, CH
131.0, C
19.0, CH3
26.7, CH3
170.9, C
123.5, CH
131.8, C
17.9, CH3
25.5, CH3
171.3, C
1.70, s
1.70, s
1.70, s
1.70, s
1.71, s
1.71, s
Val 2
59.5, CH
4.08, m
7.42, d (8.6)
1.98, m
0.84, m
0.81, m
60.8, CH
4.09, m
7.41, m
1.98, m
0.87, m
0.81, m
59.6, CH
4.09, m
7.48, m
2.00, m
0.84, m
0.78, m
30.4, CH
18.8, CH3
17.9, CH3
171.8, C
31.7, CH
20.4, CH3
19.5, CH3
30.6, CH
19.1, CH3
18.3, CH3
172.1, C
54
55
Leu
56
NH
57
58
59
50.8, CH
4.38, m
7.51, m
1.50, m
1.58, m
0.81, m
0.84, m
52.2, CH
4.38, m
7.51, m
1.50, m
1.59, m
0.82, m
0.85, m
51.1, CH
4.37, m
7.55, d (7.2)
1.49, m
1.57, m
0.81, m
c
39.8, CH2
23.9, CH
21.1, CH3
22.7, CH3
169.3, C
41.2, CH2
25.3, CH
22.5, CH3
24.1, CH3
169.2, C
, CH2
24.1, CH
21.4, CH3
22.9, CH3
169.5, C
60
61
0.84, m
Ser 2
62
NH
63
55.1, CH
4.15, m
7.55, m
3.78, m
3.72, m
56.4, CH
4.16, m
7.58, m
3.78, m
3.73, m
4.95, br s
55.2, CH
4.17, m
7.67, m
3.77, m
3.72, m
4.97, m
60.2, CH2
61.6, CH2
60.4, CH2
OH
4.95, br s
a
b
c
CH and CH2 were assigned based on HSQC correlations. Quaternary carbons were assigned based on HMBC correlations. Obscured by solvent
d
peak. Obscured by water peak.
The planar structure of krisynomycin has been previously
reported,12 but no isolation procedures or spectroscopic data
have been so far disclosed for this molecule. The krisynomycin
producer strain CA-091830, available from MEDINA’s
collection, was used for cultivation and isolation of this
compound and other congeners. LC-HRMS analysis of the
extract revealed the presence of krisynomycin and two new
structurally related components in the chromatographic profile,
which prompted a more detailed investigation of its
composition. Extraction of the culture broth followed by a
chromatographic fractionation of the extract led to the
isolation of the new krisynomycins B and C (1 and 2) and
krisynomycin (3). Recent advances in biosynthetic gene cluster
(BGC) analysis are providing very valuable information from
the assembly line enzymology of natural products, such as
polyketides and nonribosomal peptides, which is effectively
used to support the determination of the stereochemistry of
these compounds.16,17 As an example, the stereochemistry of
the lipodepsipeptide BII-Rafflesfungin was determined using
the combined information obtained from Marfey’s analysis and
the BGC.17 Hence, a bioinformatic analysis of the
krisynomycin BGC was employed to support the proposed
absolute configuration of the krisynomycin family. Structural
elucidation, including the absolute configuration of the three
compounds using a combination of NMR spectroscopy and
Marfey’s and BGC analyses together with their activity as
imipenem potentiators, is reported.
amorphous solid. A molecular formula of C63H86N12O18S was
deduced from the (+)-ESI-TOF analysis ([M + H]+
1331.5981, Δ +0.0004). The H NMR spectrum of 1 in
1
DMSO-d6 displayed signals that were attributable to a peptide,
including amide hydrogens (N−H) around 8 ppm and signals
of α-amino acid hydrogens between 4 and 5 ppm (Table 1).
Interpretation of the 2D NMR data (COSY, TOCSY, HSQC,
and HMBC) allowed us to identify the presence of Ala, 2 ×
Val, 2 × Ser, Cya, Pro, Trp, Prenyl-Trp, and Leu residues as
well as the cyclic nature of the depsipeptide, which was
confirmed later by HRMS fragmentation. In addition to these
common amino acids, a signal at δH 3.18 (H2-2) assigned to a
methylene group coupled to two carbonyls at δC 169.3 (C-1)
and 165.2 (C-3) in the HMBC spectrum accounted for the
presence of a malonic acid residue. A characteristic feature of
the compound was an olefinic hydrogen at δH 5.33 (m, H-46)
1
in the H NMR spectrum, attributed to the presence of a
trisubstituted double bond, which was also evidenced by the
sp2 signals at δC 123.9 (C-46) and 130.9 (C-47) in the 13C
NMR spectrum. Signals of two singlet methyl groups at δH
1.70 (6H, s, H3-48 and H3-49) suggested that the double bond
was substituted by two methyl groups. Finally, the methylene
hydrogens at δH 3.72 and 3.83 (H2-45), coupled in the COSY
spectrum to H-46, corroborated the presence of a prenyl unit.
Correlations in the HMBC spectrum between H2-45 and the
carbons C-42, C-43, C-46, and C-47 indicated that the prenyl
unit was linked to C-43 of one of the tryptophan residues.
The connections between the different amino acids were
first inferred from HMBC correlations involving the NH
hydrogens of the peptidic bonds (Figure 1). HRMS
fragmentation data additionally supported the amino acid
RESULTS AND DISCUSSION
■
Structural Elucidation of the Planar Structure of
Krisynomycins. Compound 1 was obtained as a white
C
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based on its (+)-ESI-TOF analysis ([M + H]+ 1399.5208, Δ
−0.0011). Its NMR data were similar to those of compound 2,
with the major difference being the disappearance of the signal
of the aromatic hydrogen H-40 in the Prenyl-Trp residue and a
change in the multiplicity of H-41 from triplet to doublet with
respect to 1 and 2. These changes were in agreement with the
location of the additional chlorine atom in krisynomycin at C-
40. Consequently, this carbon underwent a small downfield
shift to 113.8 ppm (Table 1). COSY and HMBC correlations
corroborated the position of this second chlorine atom at C-40
of the Prenyl-Trp residue (Figure S2). NMR data confirmed
the planar structure previously described for krisynomycin.12
Marfey’s Analysis. Once the planar structures of the three
compounds were established, Marfey’s analysis was used to
determine the absolute configuration of each amino acid
residue.18 Acid hydrolysis of the cyclic depsipeptides followed
by LC-MS analysis of the hydrolysates after derivatization with
N-(2,4-dinitro-5-fluorophenyl)-D/L-valinamide (D/L-FDVA,
Marfey’s reagent) and comparison with the retention times
and mass spectra obtained for standards revealed the presence
of L-Ala, D-Val, L-Val, L-Cya, L-Pro, two units of L-Ser, and D-
Leu (Figures S4−S7). The determination of the absolute
configuration of the Trp-containing units was unsuccessful,
even if phenol19 or thioglycolic acid20,21 was added as
protective agent in the acid hydrolysis to prevent the
degradation of tryptophan. Moreover, the presence of both
D- and L-Val was detected, preventing the determination of the
absolute configuration of the krisynomycins due to the location
of the two residues of this amino acid in the three molecules.
In Silico Analysis of the Krisynomycins’ Biosynthetic
Gene Cluster. The genome of Streptomyces canus CA-091830
was sequenced and assembled employing a combination of
PacBio and Illumina NovaSeq 6000 (Macrogen, Seoul, South
Korea). All contigs were analyzed using antiSMASH,22
revealing 26 regions potentially encoding secondary metabolite
gene clusters. The krisynomycins are cyclic nonribosomal
depsipeptides displaying three specific features, which we
envisioned could guide us in the identification of their BGC:
prenylation of one Trp residue at position C-43, chlorination
of one or two Trp residues at positions C-29/C-40, and the
presence of Cya as one of the units in the NRPS-synthesized
backbone. Additionally, analysis of the peptide backbone
indicated that the NRPS system should comprise 10 modules,
assuming a canonical sequential assembly of the amino acid
units.23 Careful examination of the NRPS-containing regions
identified by antiSMASH allowed us to identify a single BGC
spanning 50 kb, which fulfilled the above requirements (Figure
3 and Table S7). Thus, kriC encodes a pyridoxal-phosphate-
dependent enzyme (pfam00291), which shows homology to
ANS62969.1 (47% identity, 57% similarity) and AAM06667.1
(37% identity, 51% similarity), two biochemically character-
ized cysteate synthases from Streptomyces lincolnensis and
Methanosarcina acetivorans, respectively,24,25 and therefore is
proposed to catalyze the formation of cysteate from O-
phospho-L-serine and sulfite. kriE encodes a FADH2-depend-
ent (pfam04820) tryptophan halogenase, which shows high
homology (64% identity, 77% similarity) to RebH, the
biochemically characterized tryptophan 7-halogenase from
rebeccamycin biosynthesis.26 Unlike as is seen for the
rebeccamycin cluster, a flavin reductase to generate FADH2
cannot be found within the krisynomycin BGC putative
boundaries. Presumably one of the five flavin reductases that
we have identified, encoded elsewhere in the genome of
1
Figure 1. H−1H COSY and key HMBC correlations for 1.
sequence. Hydrolysis of 1 under basic conditions yielded the
linear peptide (Figure 2). Acquisition of the MS2 spectrum of
the protonated linear peptide ([M + H]+ 1349.6058) led to the
identification of several key fragments that confirmed the
structure proposed by NMR analysis. The first fragmentation
originated the ion at m/z 1244.5665 ([M + H]+) accounting
for the residue Malonate-Ala-Val-Ser-Cya-Pro-Trp-Pre-Trp-
Val-Leu by loss of the terminal Ser amino acid. Subsequent
losses of Leu and Val originated the fragments at m/z
1131.4823 and 1032.4096. Cleavage of the amide bond
between both Trp units and loss of the Pre-Trp residue
originated the ion at m/z 778.2696 ([M + H]+). The rest of
the peptide could be sequenced by successive losses of the
amino acids Trp, Pro, and Cya, originating the ions at m/z
592.1910, 495.1361, and 344.1475, respectively. The amino
acid sequence was also confirmed by the ions at m/z 855.4751,
758.4234, 572.3419, 318.2045, and 219.1348 by successive
cleavage of the amide bonds between the amino acids Pro-Trp-
Pre-Trp-Val-Leu-Ser as indicated in Figure 2. Compound 1
was named krisynomycin B.
Compound 2 was obtained as a white amorphous solid. A
molecular formula of C63H85ClN12O18S was deduced from the
(+)-ESI-TOF analysis ([M + H]+ 1365.5609, Δ −0.0022),
suggesting that 2 was a chlorinated version of 1. The major
difference between the 1H NMR data of both compounds was
the absence of an aromatic hydrogen in one of the Trp
residues. The absence of the signal corresponding to H-29, the
multiplicity observed for H-30 to H-32 in 2, and correlations
observed in the COSY spectrum between H-30/H-31 and H-
31/H-32 located the chlorine atom at C-29 of the Trp residue
(Figure S1), whose 13C signal did not display correlations in
the HSQC spectrum. Consequently, C-29 experienced a small
downfield shift to δC 115.6 (Table 1). Compound 2 was
named krisynomycin C.
Krisynomycin (3) was also obtained as a white amorphous
solid. A molecular formula of C63H84Cl2N12O18S was assigned
D
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Figure 2. Fragments observed for 1 by MSn.
Figure 3. Putative krisynomycins BGC and domain organization of the NRPS proteins. The adenylation domains are labeled with the antiSMASH
and PRISM amino acid specificity prediction. Asterisks indicate a mismatch between the prediction and the amino acids incorporated into the
compounds.
Streptomyces canus CA-091830, is responsible for the NADH-
dependent reduction of FAD to provide the FADH2 for the
halogenation at C-7 of L-tryptophan. kriH encodes a 370 aa
protein that belongs to the ABBA-type family of aromatic
prenyltransferases. Actinobacterial prenyltransferases catalyzing
the prenylation of L-tryptophan at different positions have been
described recently, including IsaA from Streptomyces sp.
MBT28, which catalyzes the addition of a prenyl unit to
position C-7 of L-Trp,27 IptA from Streptomyces sp. SN-593,
which catalyzes prenylation of L-Trp at C-6,28 SCO7467 from
E
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Figure 4. Proposed biosynthesis of the krisynomycins.
Streptomyces coelicolor A3(2), which catalyzes prenylation of L-
Trp at C-5,29 and CymD from Salinispora arenicola, which
promotes the addition of a prenyl unit to position N-1 of L-
Trp.30 Fungal prenyltransferases catalyzing prenylation at C-4
of L-Trp and indole derivatives have also been character-
ized.31,32 However, to the best of our knowledge, no bacterial
prenyltransferase catalyzing the transfer of a prenyl unit to
position C-4 of L-Trp has been described. KriH shows
relatively low levels of homology to IsaA (23% identity, 35%
similarity), IptA (21% identity, 37% similarity), SCO7467
(24% identity, 38% similarity), or FgaPT2 (11% identity, 24%
similarity). It could be hypothesized that KriH catalyzes
prenylation of L-tryptophan at C-4 to afford 4-prenyl-L-Trp,
which could then be employed by the NRPS to generate the
krisynomycins. 4-Prenyl-L-Trp might also be the substrate for
KriE, thus generating 7-chloro-4-prenyl-L-Trp, which in turn
could be used by the NRPS to afford krisynomycin.
Additionally, KriE is likely to be able to employ L-Trp as
substrate to afford 7-chloro-L-Trp, which would be required in
the biosynthesis of krisynomycin and krisynomycin C.
Alternatively, chlorination of L-Trp by KriE at C-7 might
take place prior to the prenylation at C-4 catalyzed by KriH
(Figure S8).
On the other hand, kriF and kriG encode two multimodular
nonribosomal peptide synthetases encompassing 10 modules.
Adenylation domain specificity was analyzed using both
antiSMASH22 and PRISM,33 which predicted a peptide
backbone comprising Ser, Val, Ser, Asp/Asn, Pro, unknown/
Phe, unknown/Trp, Val, unknown/Phe, and Ser (Figure 3).
Several discrepancies are found between the bioinformatic
prediction and the structures of krisynomycins. The
adenylation domain of module 1 is predicted to incorporate
Ser, but instead the Ala residue was found at the N-terminus in
all three krisynomycins. The prediction for the adenylation
domain of module 4 is either Asp (antiSMASH) or Asn
(PRISM), yet Cya is selected in all cases. No prediction is
obtained for the adenylation domains of modules 6 and 7 by
antiSMASH, and Phe and Trp are suggested by PRISM. It is
likely that these two adenylation domains have a relaxed
substrate specificity, and so module 6 might be able to load L-
tryptophan (to afford krisynomycin B) or 7-chloro-L-
tryptophan (to generate krisynomycin and krisynomycin C),
whereas module 7 might be able to load 4-prenyl-L-tryptophan
(to afford krisynomycins B and C) or 7-chloro-4-prenyl-L-
tryptophan (to generate krisynomycin). Finally, the adenyla-
tion domain of module 9 is predicted to incorporate Phe
(PRISM), but instead the Leu residue was found in all
krisynomycins. These mismatches between the bioinformatic
predictions and the substrates actually selected by the
adenylation domains stress the importance of further refine-
ment of the available domain-analysis tools.
Importantly, two epimerization domains are found in
modules 8 and 9, which is in agreement with both the
antiSMASH analysis and a phylogenetic study of the amino
acid sequences from the condensation domains, which
indicates that the C-domains from modules 9 and 10 belong
34
D
to the CL subtype and therefore catalyze the condensation
of an upstream T-loaded D-amino acid with a downstream T-
loaded L-amino acid. All remaining C-domains belong to the
LCL subtype. Taking these observations together and the MS/
MS, NMR, and Marfey’s analysis data, revealing the presence
of both D- and L-Val, we propose the amino acid sequence L-
Ala, L-Val, L-Ser, L-Cya, L-Pro, L-Trp/7-Cl-L-Trp, 4-prenyl-L-
Trp/7-Cl-4-prenyl-L-Trp, D-Val, D-Leu, L-Ser as the backbone
synthesized by the NRPS and found in the structures of the
three krisynomycins (Figure 4). It is worth noting that the
hypothetical krisynomycin derivative that would be obtained
with the selection and loading of L-Trp and 7-Cl-4-prenyl-L-
Trp by modules 6 and 7, respectively, has not been isolated nor
even detected by LC-MS in the culture extracts.
Finally, the T-LCL didomain from module 1 is likely to be
involved in the condensation of a malonic acid unit into the N-
terminus alanine residue. It could be speculated that the
malonate could be ACP-, CoA-, or AMP-mediated transferred
into the first T domain from module 1 and then condensed
L
with the free NH2 group of the alanine residue by the CL
domain from the same module.
Antibacterial Activity. As krisynomycin has been
previously reported as an imipenem synergistic compound,12
the efficiency of the combined treatment of krisynomycins B
and C together with imipenem against MRSA was also
investigated using the liquid microdilution method. Neither of
the krisynomycins B and C showed antibacterial activity when
tested alone (MICs above 128 μg/mL, Table 2) compared to
F
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number of halogenated metabolites have been isolated from
living organisms, such as the antibiotics chloramphenicol and
vancomycin, the antitumor agent rebeccamycin, whose biosyn-
thesis also involves a regioselective enzymatic halogenation of
tryptophan,39,40 and the potential anticancer agent salinispor-
amide A.41
In conclusion, krisynomycins were shown to be novel
natural products that act as synergistic agents of imipenem
against MRSA. In addition to potentiating imipenem activity
against MRSA, krisynomycins might also potentiate the
antibacterial activity of other carbapenems and be effective
against MRSA or other pathogenic clinical isolates. Con-
sequently, they could constitute the starting point for the
development of improved agents for combination therapies
and expand the already broad active spectrum of imipenem.
Table 2. Potentiation of Antimicrobial Activity against
MRSA MB5393 by Krisynomycins 1−3
a
MIC (μg/mL) against MRSA MB5393
no
imipenem
4 μg/mL
imipenem
2 μg/mL
imipenem
1 μg/mL
imipenem
compound
krisynomycin B
(1)
>128
8−16
16−32
16−32
krisynomycin C
(2)
>128
2−4
4−8
4−8
krisynomycin
(3)
16−32
0.25−0.5
0.5−1
0.5−1
a
MIC imipenem = 16−32 μg/mL.
the moderate activity shown by krisynomycin (MIC 16−32
μg/mL, Table 2). However, all three compounds potentiated
to a certain extent imipenem activity when methicillin-resistant
S. aureus was exposed to different subinhibitory concentrations
of the drug (4, 2, and 1 μg/mL) in the presence of different
concentrations of krisynomycins. The combination of
krisynomycin C and krisynomycin B with imipenem at 4 μg/
mL displayed an activity 8 to 32 times lower (MICs of 2−4
and 8−16 μg/mL, respectively) than that of the mixture of
krisynomycin with imipenem at 4 μg/mL (MIC 0.25−0.5 μg/
mL) (Figure S29). A similar behavior was observed when the
compounds were combined with imipenem concentrations of
1 and 2 μg/mL (Table 2). Our results therefore confirm that
an increased number of chlorine atoms in the krisynomycin
skeleton plays an important role on the potentiating effect of
imipenem activity by the krisynomycin family. These findings
are in line with the beneficial effects that the introduction of
halogen atoms seem to have in the development of new
drugs.35
In summary, krisynomycins are nonribosomal cyclic
depsipeptides showing two specific structural features:
prenylation and chlorination in the tryptophan amino acid
units. Prenylation is a common modification of natural
products from microbial and plant origin that contributes to
their structural diversity. This functional group appears in
many cases to provide a higher level of bioactivity in
comparison with the nonprenylated precursors, often by
increasing affinity for biological membranes and interactions
with cellular targets.36 As mentioned above, an explosion in the
number of identified indole prenyltransferases has occurred in
recent times,36,37 although to the best of our knowledge this is
the first time that a bacterial prenyltransferase catalyzing the
addition of a prenyl unit to position C-4 of L-tryptophan is
described.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using a Jasco P-2000 polarimeter. UV spectra were
obtained with an Agilent 1100 DAD. IR spectra were recorded on a
JASCO FT/IR-4100 spectrometer equipped with a PIKE MIRacle
single reflection ATR accessory. NMR spectra were recorded in
DMSO-d6 on a Bruker Avance III spectrometer (500 and 125 MHz
for H and 13C NMR, respectively) equipped with a 1.7 mm TCI
1
MicroCryoProbe (Bruker Biospin). Chemical shifts were reported in
ppm using the signals of the residual solvent as internal reference (δH
2.50 and δC 39.51). LC-MS and LC-HRMS analyses were performed
as previously described using an ionization voltage of 75 eV.42
Microbial Isolation. Streptomyces canus CA-091830 was isolated
from a sand sample collected in 1997 in the Kalahari Desert in South
Africa. The sand sample was subjected to a dry heat pretreatment at
100 °C for 1 h and serially diluted and plated on soil extract
medium.43
16S rRNA Sequencing and Phylogenetic Analysis. The
almost-complete 16S rRNA gene sequence (1514 bp) of strain CA-
091830 was compared with those deposited in public databases and
the EzBiocloud server (https://www.ezbiocloud.net/44). The strain
exhibited the highest identity (99.31%.) with Streptomyces canus DSM
40017T (KQ948708), using EzBiocloud and GenBank sequence
similarity searches and homology analysis.
Phylogenetic and molecular evolutionary analyses were conducted
using MEGA version X.45 Multiple alignment was carried out using
CLUSTALX,46 integrated in the software. The phylogenetic analysis,
based on the neighbor-joining method47 using matrix pairwise
comparisons of sequences corrected with the Jukes and Cantor
algorithm,48 shows that the strain is closely related to the type strain S.
canus DSM 40017T and can be proposed as a new strain of this
species (Figure S9). The morphological and 16S rRNA gene sequence
data were indicative that strain CA-091830 was a member of the
genus Streptomyces, and the strain was referred to as Streptomyces
canus CA-091830.
An additional feature in the krisynomycins is the
chlorination at position C-7 of L-tryptophan to produce 7-Cl-
L-Trp and 7-Cl-4-prenyl-L-Trp in krisynomycin and krisyno-
mycin C. Significantly, a large number of drugs and drug
candidates in clinical development are halogenated structures.
In fact, insertion of halogen atoms has been used in hit-to-lead
or lead-to-drug optimization processes to increase membrane
permeability and, therefore, improve the oral absorption of
drug candidates. On one hand, previous studies have shown
that halogen substituents influence the biomolecular inter-
actions not only because of steric aspects but also as a result of
the modification of thermodynamic parameters, with ΔG being
the parameter that best correlates with protein affinity (Kd).
Additionally, although sensible for the position of the halogen,
dihalogenated peptides led to more stable complexes (lowest
Fermentation of the Producing Microorganism. The first
seed culture of the strain CA-091830 was prepared by inoculating 10
mL of seed medium, which consists of soluble starch (20 g/L),
dextrose (10 g/L), NZ amine EKC (Sigma) (5 g/L), Difco beef
extract (3 g/L), Bacto peptone (5 g/L), yeast extract (5 g/L), and
CaCO3 (1 g/L), adjusted to pH 7.0 with NaOH before addition of
CaCO3, in a 40 mL tube with 0.5 mL of a frozen inoculum stock of
the producing strain and incubating the tube at 28 °C with shaking at
220 rpm for 48 h. A second seed culture was prepared by inoculating
50 mL of seed medium in two 250 mL flasks with 2.5 mL of the first
seed culture. A 5% aliquot of the second seed culture was transferred
to each of the eight flasks (500 mL) containing 150 mL of the
production CLA medium consisting of lactose monohydrate (Panreac
141375) (40 g/L), corn meal (Sigma-C6304) (40 g/L), and yeast
autolysate (Sigma 43750) (5 g/L), adjusted to pH 7.0. The flasks
were incubated at 28 °C for 7 days in a rotary shaker at 220 rpm and
70% humidity before harvesting.
ΔG) with subsequent improvement in protein affinity.35,38
A
G
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Isolation and Identification of Metabolites. A culture (16 ×
125 mL, 2 L) of the strain was obtained in CLA medium. Acetone was
added to the fermentation flasks (1:1, 125 mL), and they were shaken
hydrolysis product of compound 1 were as follows: L-Cya: 2.99, L-Ser:
5.28, L-Pro: 11.21, L-Ala: 11.83, D-Val: 11.83, D-Leu: 16.33, and L-Val:
21.83.
Genomic DNA Isolation, Sequencing, and Bioinformatic
Analysis. Genomic DNA of strain CA-091830 was extracted and
purified as previously described from cultures grown in the same seed
medium employed for the production of krisynomycins.49 The
genome of strain CA-091830 was sequenced and assembled de novo
using a combined strategy of Illumina NovaSeq6000 S4 and PacBio
sequencing (Macrogen, Seoul, Korea). Eleven contigs with a total
length of 9 895 814 bp were obtained. Identification of potential
BGCs was carried out with antiSMASH, and annotation of the ORFs
was performed employing the translated sequences and the BLASTP
algorithm at the NCBI Web site. The krisynomycins’ BGC was
analyzed using both antiSMASH22 and PRISM.33 Bioinformatic
analysis of the condensation domains of the NRPS proteins KriF and
KriG was carried out using a phylogenetic analysis employing their
amino acid sequences together with the amino acid sequences of 525
known condensation domains.34
Antibacterial Activity Bioassay. MRSA COL MB5393 is a
hospital-acquired penicillinase-negative strain widely used in S. aureus
methicillin resistance and virulence studies. The broth dilution
method was used to determine MIC values, and they were defined as
the lowest concentration of compound that either singly or in
combination with imipenem inhibited ≥90% of the growth of MRSA
strain after overnight incubation. Bioassay was performed in triplicate
on three different days. The antibacterial activities of the compounds
were evaluated using sequential 2-fold serial dilutions in DMSO to
provide 20 concentrations starting at 128 μg/mL for all the assays.
The Genedata Screener software (Genedata, Inc., Basel, Switzerland)
was used to process and analyze the data as well as to calculate the RZ
factor, which predicts the robustness of the assay.50,51 The RZ factor
obtained from all experiments ranged between 0.90 and 0.97.
The MRSA COL MB 5393 strain was grown in a shaken culture
(220 rpm) of brain heart infusion broth (BHI, 37 g/L) incubated
overnight at 37 °C. The bacterial inoculum was then diluted to obtain
the corresponding equivalent for the assay of approximately 1.1 × 106
CFU/mL. For the assay, 90 μL/well of diluted inoculum was mixed
with 1.6 μL/well of each compound dissolved in DMSO and 8.4 μL/
well of four different concentrations of imipenem starting at 4 μg/mL
(subminimal inhibitory concentration). Vancomycin was included as
positive control on each plate. The absorbance at T0 (zero time) was
measured at 612 nm with an EnVision multimode plate spectropho-
tometer, and immediately thereafter, the plates were incubated at 37
°C for 18 h. After this period, the assay plates were shaken using a
Micromix-5 agitator, and once again the absorbance at Tf (final time)
was measured at 612 nm. The percentage of growth inhibition was
calculated according to previously described methodology.52
in a Kuhner at 220 rpm for 1 h. After that, the mixture was centrifuged
̈
(5 min, 8500 rpm) and filtered under vacuum, and the pellet was
discarded. Acetone was evaporated under a nitrogen stream until the
original volume of fermentation (2 L) to get the crude extract. This
aqueous residue was loaded onto an SP207ss resin column (65 g, 32
× 100 mm) and eluted with an acetone−H2O stepped gradient (10/
90 for 6 min, 20/80 for 6 min, 40/60 for 6 min, 60/40 for 6 min, 80/
20 for 6 min, and 100/0 for 12 min, 10 mL/min, 20 mL/fraction) in a
Teledyne CombiFlash RF instrument to give 21 fractions. Fraction 16
(obtained with 80% acetone) was subjected to preparative reversed-
phase HPLC (Kinetex PFP Phenomenex, 21.2 × 250 mm, 5 μm, 14
mL/min, UV detection at 210 and 280 nm, 8.75 mL/fraction) using
H2O (solvent A) and CH3CN (solvent B). Elution was carried out
using isocratic conditions of 5% B for 5 min and then a linear gradient
from 5% to 70% B in 52 min, yielding 73 fractions. Subfraction 38 was
further purified by semipreparative reversed-phase HPLC (Zorbax
RX-C8, 9.4 × 250 mm, 5 μm, 3.6 mL/min, UV detection at 210 and
280 nm, 1.8 mL/fraction) using H2O + 0.1% TFA (solvent A) and
CH3CN + 0.1% TFA (solvent B). Elution was carried out using
isocratic conditions of 25% B for 5 min followed by a linear gradient
from 25% B to 75% B in 35 min to yield 1 (tR 24.5 min, 0.3 mg).
Fraction 17, also obtained with 80% acetone, was subjected to
preparative reversed-phase HPLC under the same conditions used for
fraction 16. Subfraction 41 was further purified using the conditions
described above for subfraction 38 to yield 2 (tR 26 min, 0.5 mg) and
3 (tR 28 min, 0.5 mg).
Krisynomycin B (1): white amorphous solid; [α]2D5 −15.8 (c 0.07,
MeOH); UV (DAD) 210, 290 nm; IR (ATR) νmax 3074, 2966, 2927,
2870, 1623, 1593, 1532, 1510, 1434, 1240, 1203, 1182, 1134, 829
cm−1; 1H and 13C NMR data see Table 1; (+)-HRESIMS m/z
1331.5981 [M + H]+ (calcd for C63H87N12O18S, 1331.5977).
Krisynomycin C (2): white amorphous solid; [α]2D5 −2.1 (c 0.07,
MeOH); UV (DAD) 210, 290 nm; IR (ATR) νmax 3076, 2968, 2924,
2867, 1669, 1622, 1593, 1532, 1510, 1434, 1239, 1203, 1181, 1134,
1
831 cm−1; H and 13C NMR data see Table 1; (+)-HRESIMS m/z
1365.5607 [M + H]+ (calcd for C63H85ClN12O18S, 1365.5587).
Krisynomycin (3): white amorphous solid; [α]2D5 −12.4 (c 0.13,
MeOH); UV (DAD) 210, 290 nm; IR (ATR) νmax 3067, 2969, 2922,
1
2873, 1675, 1531, 1437, 1200, 1178, 1134, 837 cm−1; H and 13C
NMR data see Table 1; (+)-HRESIMS m/z 1399.5206 [M + H]+
(calcd for C63H84Cl2N12O18S, 1399.5197).
Marfey’s Analysis of Compound 1. A sample (100 μg) of
compound 1 was dissolved in 0.2 mL of 6 N HCl and heated at 110
°C for 16 h. The crude hydrolysate was evaporated to dryness under a
N2 stream, and the residue was dissolved in 100 μL of water. This
solution was divided into two 50 μL aliquots. To each aliquot of the
hydrolysate and to an aliquot (50 μL) of a 50 mM solution of each
amino acid (D, L, or a DL mixture) were added 20 μL of a 1 M
NaHCO3 solution and a 1% (w/v) solution (100 μL) of D- or L-
FDVA (Marfey’s reagent, N-(2,4-dinitro-5-fluorophenyl)-L-valina-
mide). The reaction mixtures were incubated at 40 °C for 60 min.
The reactions were quenched by addition of 20 μL of 1 N HCl, and
the crude mixtures were diluted with 200 μL of acetonitrile and
analyzed by LC/MS on an Agilent 1260 Infinity II single quadrupole
LC/MS instrument. Separations were carried out on an Atlantis T3
column (4.6 × 100 mm, 5 μm) maintained at 40 °C. A mixture of two
solvents, A (10% CH3CN, 90% H2O) and B (90% CH3CN, 10%
H2O), both containing 1.3 mM trifluoroacetic acid and 1.3 mM
ammonium formate, was used as the mobile phase under a linear
gradient elution mode (isocratic 25% B for 2 min, 25−45% B in 27
min, isocratic 45% B for 5 min) at a flow rate of 1 mL/min.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00294.
■
sı
1D and 2D NMR spectra and MS/MS fragmentation
data of krisynomycins, together with HPLC chromato-
grams of Marfey’s analysis of compound 1 and
additional information on the biosynthetic pathway
(PDF)
AUTHOR INFORMATION
Corresponding Authors
■
́
́
Mercedes Perez-Bonilla − Fundacion MEDINA, Centro de
Retention times (min) for the derivatized (D-FDVA) amino acid
standards under the reported conditions were as follows: D-Cya: 2.74;
L-Cya: 2.88, D-Ser: 4.32, L-Ser: 5.21, D-Ala: 7.16, L-Ala: 11.81, D-Pro:
7.75, L-Pro: 11.18, D-Val: 11.81, L-Val: 21.79, D-Leu: 16.20, L-Leu:
27.90, D-Trp: 16.20, L-Trp: 22.40. Retention times (min) for the
observed peaks in the HPLC trace of the D-FDVA-derivatized
́
Excelencia en Investigacion de Medicamentos Innovadores en
́
Andalucıa, 18016 Armilla, Granada, Spain; Phone: +34
958993965; Email: mercedes.perez@medinaandalucia.es
́
Fernando Reyes − Fundacion MEDINA, Centro de Excelencia
́
́
en Investigacion de Medicamentos Innovadores en Andalucıa,
H
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Jacob, C.; Lee, S. S.; Lin, M. M.; Michels, A. A.; Ogawa, A. M.;
Painter, R. E.; Parish, C. A.; Park, Y.-W.; Benton-Perdomo, L.; Petcu,
M.; Phillips, J. W.; Powles, M. A.; Skorey, K. I.; Tam, J.; Tan, C. M.;
Young, K.; Wong, S.; Waddell, S. T.; Miesel, L. Antimicrob. Agents
Chemother. 2012, 56, 4662−4670.
18016 Armilla, Granada, Spain; orcid.org/0000-0003-
1607-5106; Phone: +34 958993965, ext. 7006;
Email: fernando.reyes@medinaandalucia.es
Authors
(13) Fukumoto, A.; Kim, Y.-P.; Hanaki, H.; Shiomi, K.; Tomoda, H.;
Omura, S. J. Antibiot. 2008, 61, 7−10.
́
Daniel Oves-Costales − Fundacion MEDINA, Centro de
́
Excelencia en Investigacion de Medicamentos Innovadores en
(14) Koyama, N.; Tokura, Y.; Takahashi, Y.; Tomoda, H. Acta
Pharm. Sin. B 2011, 1, 236−239.
́
Andalucıa, 18016 Armilla, Granada, Spain
́
́
Ignacio Gonzalez − Fundacion MEDINA, Centro de Excelencia
(15) Yamazaki, H.; Omura, S.; Tomoda, H. J. Antibiot. 2010, 63,
329−330.
́
́
en Investigacion de Medicamentos Innovadores en Andalucıa,
18016 Armilla, Granada, Spain
(16) Fischbach, M. A.; Walsh, C. T. Chem. Rev. 2006, 106, 3468−
3496.
́
Mercedes de la Cruz − Fundacion MEDINA, Centro de
́
Excelencia en Investigacion de Medicamentos Innovadores en
(17) Sinha, S.; Nge, C.-E.; Leong, C. Y.; Ng, V.; Crasta, S.; Alfatah,
M.; Goh, F.; Low, K.-N.; Zhang, H.; Arumugam, P.; Lezhava, A.;
Chen, S. L.; Kanagasundaram, Y.; Ng, S. B.; Eisenhaber, F.;
Eisenhaber, B. BMC Genomics 2019, 20, 374.
(18) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596.
(19) Muramoto, K.; Kamiya, H. Anal. Biochem. 1990, 189, 223−230.
(20) Matsubara, H.; Sasaki, R. M. Biochem. Biophys. Res. Commun.
1969, 35, 175−181.
(21) Yokote, Y.; Murayama Arai, K.; Akahane, K. Anal. Biochem.
1986, 152, 245−249.
(22) Blin, K.; Shaw, S.; Steinke, K.; Villebro, R.; Ziemert, N.; Lee, S.
Y.; Medema, M. H.; Weber, T. Nucleic Acids Res. 2019, 47, W81−
W87.
́
Andalucıa, 18016 Armilla, Granada, Spain
́
́
Jesus Martín − Fundacion MEDINA, Centro de Excelencia en
́
́
Investigacion de Medicamentos Innovadores en Andalucıa,
18016 Armilla, Granada, Spain
́
Francisca Vicente − Fundacion MEDINA, Centro de Excelencia
́
́
en Investigacion de Medicamentos Innovadores en Andalucıa,
18016 Armilla, Granada, Spain
́
Olga Genilloud − Fundacion MEDINA, Centro de Excelencia
́
́
en Investigacion de Medicamentos Innovadores en Andalucıa,
18016 Armilla, Granada, Spain
Complete contact information is available at:
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(23) Amoutzias, G. D.; Van de Peer, Y.; Mossialos, D. Future
Microbiol. 2008, 3, 361−370.
(24) Graham, D. E.; Taylor, S. M.; Wolf, R. Z.; Namboori, S. C.
Biochem. J. 2009, 424, 467−478.
Notes
The authors declare no competing financial interest.
(25) Wang, M.; Chen, D.; Zhao, Q.; Liu, W. J. Org. Chem. 2018, 83,
7102−7108.
ACKNOWLEDGMENTS
■
(26) Yeh, E.; Garneau, S.; Walsh, C. T. Proc. Natl. Acad. Sci. U. S. A.
2005, 102, 3960−3965.
The polarimeter, HPLC, IR, and NMR equipment, and plate
reader used in this work were purchased via grants for scientific
and technological infrastructures from the Ministerio de
(27) Wu, C.; Du, C.; Gubbens, J.; Choi, Y. H.; van Wezel, G. P. J.
Nat. Prod. 2015, 78, 2355−2363.
́
Ciencia e Innovacion [Grant Nos. PCT-010000-2010-4
(28) Takahashi, S.; Takagi, H.; Toyoda, A.; Uramoto, M.; Nogawa,
T.; Ueki, M.; Sakaki, Y.; Osada, H. J. Bacteriol. 2010, 192, 2839−2851.
(29) Ozaki, T.; Nishiyama, M.; Kuzuyama, T. J. Biol. Chem. 2013,
288, 9946−9956.
(NMR), INP-2011-0016-PCT-010000 ACT6 (polarimeter,
HPLC, and IR), and PCT-01000-ACT7, 2011-13 (plate
reader)].
(30) Roose, B. W.; Christianson, D. W. Biochemistry 2019, 58,
3232−3242.
REFERENCES
■
̈
(31) Unsold, I. A.; Li, S.-M. Microbiology 2005, 151, 1499−1505.
(1) World Health Organization. Key Facts, 2018 (accessed March
17, 2020) http://www.who.int/news-room/fact-sheets/detail/
antimicrobial-resistance.
(32) Ding, Y.; Williams, R. M.; Sherman, D. H. J. Biol. Chem. 2008,
283, 16068−16076.
(33) Skinnider, M. A.; Merwin, N. J.; Johnston, C. W.; Magarvey, N.
A. Nucleic Acids Res. 2017, 45, W49−W54.
(2) Harbarth, S.; Balkhy, H. H.; Goossens, H.; Jarlier, V.; Kluytmans,
J.; Laxminarayan, R.; Saam, M.; Van Belkum, A.; Pittet, D. Antimicrob.
Resist. Infect. Control 2015, 4, 49.
(3) O’Neill, J. Antimicrobial Resistance: Tackling a crisis for the
health and wealth of nations. The Review on Antimicrobial Resistance
chaired by Jim O’Neill. December 2014 (accessed March 17, 2020);
h t t p s : / / a m r - r e v i e w . o r g / s i t e s / d e f a u l t / fi l e s /
A M R % 2 0 R e v i e w % 2 0 P a p e r % 2 0 -
%20Tackling%20a%20crisis%20for%20the%20health%20and%20
wealth%20of%20nations_1.pdf.
(4) Berdy, J. J. Antibiot. 2005, 58, 1−26.
(5) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629−661.
(6) Baltz, R. H. Curr. Opin. Pharmacol. 2008, 8, 557−563.
(7) Genilloud, O. Nat. Prod. Rep. 2017, 34, 1203−1232.
(8) Abdalla, M. A.; McGaw, L. J. Molecules 2018, 23, 2080.
(9) Larrull, L. I.; Testero, S. A.; Fisher, J. F.; Mobashery, S. Curr.
Opin. Microbiol. 2010, 13, 551−557.
(34) Rausch, C.; Hoof, I.; Weber, T.; Wohlleben, W.; Huson, D. H.
BMC Evol. Biol. 2007, 7, 78.
(35) Hernandez, M. Z.; Cavalcanti, S. M. T.; Moreira, D. R. M.; de
Azevedo Junior, W. F.; Leite, A. C. L. Curr. Drug Targets 2010, 11,
303−314.
(36) Tello, M.; Kuzuyama, T.; Heide, L.; Noel, J. P.; Richard, S. B.
Cell. Mol. Life Sci. 2008, 65, 1459−1463.
(37) Tanner, M. E. Nat. Prod. Rep. 2015, 32, 88−101.
(38) Memic, A.; Spaller, M. R. ChemBioChem 2008, 9, 2793−2795.
́
́
(39) van Pee, K.-H.; Patallo, E. P. Appl. Microbiol. Biotechnol. 2006,
70, 631−641.
(40) Zhu, X.; De Laurentis, W.; Leang, K.; Herrmann, J.; Ihlefeld,
́
K.; van Pee, K.-H.; Naismith, J. H. J. Mol. Biol. 2009, 391, 74−85.
(41) Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.;
Jensen, P. R.; Fenical, W. Angew. Chem., Int. Ed. 2003, 42, 355−357.
(42) Martín, J.; Crespo, G.; Gonzalez-Menendez, V.; Perez-Moreno,
́
́
́
(10) Peacock, S. J.; Paterson, G. K. Annu. Rev. Biochem. 2015, 84,
577−601.
(11) Foster, T. J. Trends Microbiol. 2019, 27, 26−38.
(12) Therien, A. G.; Huber, J. L.; Wilson, K. E.; Beaulieu, P.; Caron,
A.; Claveau, D.; Deschamps, K.; Donald, R. G.; Galgoci, A. M.;
Gallant, M.; Gu, X.; Kevin, N. J.; Lafleur, J.; Leavitt, P. S.; Lebeau-
́
́
́
G.; Sanchez-Carrasco, P.; Perez-Victoria, I.; Ruiz-Perez, L. M.;
́
Gonzalez-Pacanowska, D.; Vicente, F.; Genilloud, O.; Bills, G. F.;
Reyes, F. J. Nat. Prod. 2014, 77, 2118−2123.
(43) Hayakawa, M.; Otoguro, M.; Takeuchi, T.; Yamazaki, T.;
Iimura, Y. Antonie van Leeuwenhoek 2000, 78, 171−185.
I
https://dx.doi.org/10.1021/acs.jnatprod.0c00294
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
(44) Yoon, S. H.; Ha, S. M.; Kwon, S.; Lim, J.; Kim, Y.; Seo, H.;
Chun, J. Int. J. Syst. Evol. Microbiol. 2017, 67, 1613−1617.
(45) Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S.
Mol. Biol. Evol. 2013, 30, 2725−2729.
(46) Thompson, J. D.; Gibson, T. J.; Plewniak, F.; Jeanmougin, F.;
Higgins, D. G. Nucleic Acids Res. 1997, 25, 4876−4882.
(47) Saitou, N.; Nei, M. Mol. Biol. Evol. 1987, 4, 406−425.
(48) Jukes, T. H.; Cantor, C. Evolution of Protein Molecules, in
Mammalian Protein Metabolism; Academic Press, 1969.
(49) Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hopwood,
D. A. Preparation and Analysis of Genomic and Plasmid DNA in
Practical Streptomyces Genetics; John Innes Foundation, Norwich,
2000.
(50) Zhang, J. H.; Chung, T. D.; Oldenburg, K. R. J. Biomol.
Screening 1999, 4, 67−73.
̈
(51) Kummel, A.; Gubler, H.; Gehin, P.; Beibel, M.; Gabriel, D.;
Parker, C. N. J. Biomol. Screening 2010, 15, 95−101.
́
(52) Martín, J.; da S. Sousa, T.; Crespo, G.; Palomo, S.; Gonzalez, I.;
Tormo, J. R.; de la Cruz, M.; Anderson, M.; Hill, R. T.; Vicente, F.;
Genilloud, G.; Reyes, F. Mar. Drugs 2013, 11, 387−398.
J
https://dx.doi.org/10.1021/acs.jnatprod.0c00294
J. Nat. Prod. XXXX, XXX, XXX−XXX