Dehydropropylpantothenamide isolated by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of animal cells

Article abstract of DOI:10.1007/s11418-017-1161-y

A new amide, named dehydropropylpantothenamide (1), was obtained by a co-culture of Nocardia tenerifensis IFM 10554^T in the presence of the mouse macrophage-like cell line J774.1 in modified Czapek-Dox (mCD) medium. Compound 1 was synthesized from d-pantothenic acid calcium salt in 6 steps. The absolute configuration of natural compound 1 was determined by comparisons of the optical rotation and CD spectra of synthetic 1. In the present study, a new method for producing secondary metabolites was demonstrated using a “co-culture” in which the genus Nocardia was cultured in the presence of an animal cell line.

Full text of DOI:10.1007/s11418-017-1161-y

Journal of Natural Medicines (2018) 72:280–289
https://doi.org/10.1007/s11418-017-1161-y
ORIGINAL PAPER
Dehydropropylpantothenamide isolated by a co‑culture of Nocardia
T
tenerifensis IFM 10554 in the presence of animal cells
1
1
2
1
2
2
Yasumasa Hara · Midori A. Arai · Kanae Sakai · Naoki Ishikawa · Tohru Gonoi · Takashi Yaguchi ·
1
Masami Ishibashi
Received: 24 November 2017 / Accepted: 30 November 2017 / Published online: 5 December 2017
©
The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature 2017
Abstract
T
A new amide, named dehydropropylpantothenamide (1), was obtained by a co-culture of Nocardia tenerifensis IFM 10554
in the presence of the mouse macrophage-like cell line J774.1 in modiꢀed Czapek-Dox (mCD) medium. Compound 1 was
synthesized from d-pantothenic acid calcium salt in 6 steps. The absolute conꢀguration of natural compound 1 was deter-
mined by comparisons of the optical rotation and CD spectra of synthetic 1. In the present study, a new method for producing
secondary metabolites was demonstrated using a “co-culture” in which the genus Nocardia was cultured in the presence of
an animal cell line.
Keywords Actinomycetes · Nocardia · Co-culture · Amide
Introduction
Actinomycetes of the genus Nocardia are Gram-positive
bacteria found in the lungs, skin, brain, and other organs
and cause infections in immunocompromised patients. When
bacteria of the genus Nocardia infect the human body, they
are attacked by immune cells such as macrophages. How-
ever, the activation of biosynthesis genes by mimicking this
phenomenon, namely, an infection state for the isolation of
new secondary metabolites, has not yet been investigated.
Therefore, we herein examined a new method for produc-
ing secondary metabolites using a “co-culture” in which the
genus Nocardia is cultured in the presence of an animal cell
line.
Actinomycetes produce many secondary metabolites with
diverse structures and activities [1], and many of these
metabolites produce antibiotics including streptomycin [2],
erythromycin [3], and kanamycin [4]. Many biosynthesis
genes on the genomes of actinomycetes are involved in the
production of secondary metabolites; however, a large pro-
portion is considered to be cryptic genes that are not acti-
vated to produce secondary metabolites [5, 6]. In order to
obtain new secondary metabolites, it is important to activate
these cryptic gene clusters. Recent studies have attempted to
produce secondary metabolites by stimulating gene expres-
sion using several methods such as culture conditions [7],
drug stimulation [8], the forced expression of genes [9], and
combined cultures [10–12].
We describe the isolation and structural elucidation of a
new compound (1) isolated in modiꢀed Czapek-Dox (mCD)
[13] medium by a co-culture of Nocardia tenerifensis IFM
T
10554 in the presence of J774.1. Five known steroids were
T
also isolated from N. tenerifensis IFM 10554 , and the
results obtained revealed that two out of the ꢀve compounds
were obtained only under co-culture conditions in DMEM
with 10% FBS medium.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11418-017-1161-y) contains
supplementary material, which is available to authorized users.
*
Masami Ishibashi
mish@chiba-u.jp
Results and discussion
1
2
Graduate School of Pharmaceutical Sciences, Chiba
Strains for the co-culture study were initially selected from
University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
7
6 strains belonging to the genus Nocardia obtained from
Medical Mycology Research Center, Chiba University, 1-8-1
Inohana, Chuo-ku, Chiba 260-8673, Japan
the Medical Mycology Research Center, Chiba University.
Vol:.(1234567890)
1
3

Journal of Natural Medicines (2018) 72:280–289
281
Biosynthetic gene clusters were examined in a gene
analysis of the genus Nocardia with antiSMASH [14]. A
phylogenetic tree was prepared with two analytical soft-
ware packages, Clustal X [15] and MEGA [16], using
the nocobactin-related biosynthetic gene [17] group, a
pathogenic factor, as an index. Strains were classiꢀed
into ꢀve clades. Based on these clades and the number
of biosynthetic gene clusters, six strains (N. altamirensis
T
T
Fig. 1 Structure of 1
IFM 10819 , N. mexicana IFM 10801 , N. tenerifensis
T
T
IFM 10554 , N. terpenica IFM 0706 , N. mexicana IFM
T
T
Table 1 1H-and 13C-NMR chemical shifts for dehydropropylpantoth-
enamide (1)
1
0801 , and N. vulneris NBRC 108936 ) were selected.
Bacteria of the genus Nocardia receive supplementation
with macrophages when they infect the human body [18].
Therefore, a mouse macrophage-like cell line (J774.1) was
selected as an animal cell line for co-culturing in order to
reconstruct the initial infection state.
Position
δH
δC
1
2
3
4
5
6
1
2
3
1
2
3
2
171.8
78.1
39.5
71.3
20.8
20.7
167.9
100.5
133.7
41.0
22.9
11.3
4.18 (d, 4.1)
Morphological changes in and the cell viability of
J774.1 were investigated using a co-culture with the six
selected Nocardia strains. All six strains caused morpho-
logical changes in J774.1 and ultimately led to cell death,
suggesting an interaction between the strain and cell line.
Culture conditions for the six selected strains were
examined in the presence or absence of J774.1 using a
combination of various media mCD, nutrient broth (NB)
3.53, 3.60 (d, 11.2)
0.99 (s, 3H)
1.06 (s, 3H)
’
’
’
”
”
”
5.01 (d, 9.0)
7.32 (dd, 9.0, 10.7)
3.27 (dt, 7.6, 6.2, 2H)
1.54 (m, 2H)
0.94 (d, 7.6, 3H)
3.61 (brs)
2.60 (brs)
11.9 (d, 10.7)
5.42 (brs)
[
19], Waksman [20], Yeast-Malt-Glucose (YMG) [21],
DMEM, and DMEM with 10% FBS media], temperatures
-OH
(
28 or 37 °C), air compositions (atmosphere or 5% CO ),
2
2
4-OH
containers (50-mL Erlenmeyer ꢁask or 75-cm cell culture
1
1
-NH
’-NH
ꢁ
ask), and shaking conditions (static or rotary shaking
culture).
1
13
LC–MS analyses of the extracts of culture broths obtained
Measured in CDCl3. 600 MHz ( H), 150 MHz ( C) δ in ppm. J in
Hz
under the various conditions described above revealed that
multiple peaks were selectively noted for the extract of N.
T
tenerifensis IFM 10554 cultured in the presence of J774.1.
Thus, this strain was selected for further study. The ratio of
Compound (1) was revealed to have the molecular for-
mula C H N O by high resolution ESIMS (obsd. m/z
T
the cell numbers of N. tenerifensis IFM 10554 and J774.1
1
2
22
2
4
+
was also examined under various conditions (1:1, 5:1, 10:1,
539.3080 [2 M + Na] , calcd for C H N O Na, 539. 3057).
24 44 4 8
1
5
0:1, and 100:1) in order to demonstrate that selective
The H-NMR spectrum of 1 measured in CDCl showed
3
LCMS peaks were present for the extracts obtained from
the culture at a ratio of 10:1 for mCD medium and 5:1 for
DMEM with 10% FBS medium.
one Z-oleꢀn [δ 7.32 (1H) and 5.01 (1H); J = 9.0 Hz], one
H
oxymethine [δ 4.18 (1H)], one oxymethylene [δ 3.53
H
H
(1H), 3.60 (1H)], two other methylene [δ 3.27 (2H) and
H
A large-scale co-culture (7.3 L) of N. tenerifensis IFM
1.54 (2H)], three methyl [δ 1.06 (3H), 0.99 (3H) and 0.94
H
T
1
0554 and J774.1 in mCD medium at a cell number ratio
(3H, J = 7.6 Hz)], two NH [δ 11.9 (1H) and 5.42 (1H)],
H
2
of 10:1 was performed at 28 °C in 175-cm cell culture
and two OH signals [δ 3.61 (1H) and 2.60 (1H)] (Table 1).
H
1
3
ꢁ
asks under static conditions for 2 weeks in atmospheric air.
The C-NMR spectrum of 1 measured in CDCl showed
3
After centrifugation of the culture broth, the supernatant and
methanol extract of mycelia were combined and subjected
to partition between ethyl acetate (EtOAc) and water. The
EtOAc fraction was subjected to fractionation with ODS
twelve peaks. The cross-peaks observed in the 2D TOCSY
data of 1 suggested the presence of two partial structures,
A and B (Fig. 2). The HMBC spectrum of 1 showed cor-
relations from the methylene protons (H -1″, δ 3.27) of
2
H
column chromatography (the MeOH–H O system), followed
partial structure B to the carbonyl carbon (C-1′, δ 167.9),
2
C
by reversed-phase HPLC separation (50% MeOH) to give a
which also correlated with the Z-oleꢀnic protons [H-2′ (δ
H
new compound (1), named dehydropropylpantothenamide
5.01) and H-3′ (δ 7.32)] of partial structure A, suggesting
H
(
Fig. 1).
that partial structure A was connected to partial structure
1
3

282
Journal of Natural Medicines (2018) 72:280–289
was further supported by comparisons of spectral data with
those of CJ15, 801 [22] having a related structure. In order
to elucidate the absolute stereochemistry of 1, its synthe-
sis was examined as described in Scheme 1. Two hydroxy
groups of D-pantothenic acid calcium salt (2) were protected
with p-anisaldehyde dimethyl acetal to give 3. After the con-
version of 3 to its methyl ester 4, the treatment of 4 with
Dess-Martin periodinane [23] aꢂorded Z-oleꢀn (5), and the
hydrolysis of 5 gave acid 6. Compound 6 was subjected to
amidation with propylamine to give 7, and deprotection of
the acetal group of 7 aꢂorded 1. The optical rotation and CD
spectrum of natural compound 1 corresponded well with
those of synthetic 1 (Fig. 4). Therefore, the conꢀguration at
position 2 was elucidated as the R conꢀguration. An HPLC
examination revealed that compound 1 was preferentially
produced under co-culture conditions in the presence of
J774.1 only, while it was not produced under single culture
conditions in the absence of J774.1 (Fig. 5).
A
B
Fig. 2 Partial structures, a and b, for 1
A large-scale co-culture (8.0 L) of N. tenerifensis IFM
T
1
0554 and J774.1 in DMEM with 10% FBS medium at
2
a cell number ratio of 5:1 was performed in 175-cm cell
culture ꢁasks under static conditions at 37 °C for 24 h under
C
D
5
% CO . After centrifugation of the culture broth, the super-
2
natant and methanol extract of mycelia were combined and
subjected to partition between EtOAc and water. The EtOAc
fraction was treated by reversed-phase HPLC separation
Fig. 3 Partial structures, c and d, and HMBC correlations for 1
(
10–100% MeOH gradient) to give ꢀve known compounds.
B through the carbonyl carbon C-1′ to give partial struc-
They were identiꢀed as 1, 4-androstadine-16α,17β-diol-
3-one (8) [24], 4-androstene-16α,17β-diol-3-one (9) [25],
1,4-androstadine-3,17-dione (10) [26], 4-androstene-
3,17-dione (11) [27], and cholesteryl oleate (12) [28], based
on analyses of NMR and ESIMS data. Comparisons of
HPLC data revealed that 8 and 9 were produced only under
co-culture conditions in the presence of J774.1 in DMEM
with 10% FBS medium (Fig. 6) and were not produced in
the absence of J774.1.
ture C (Fig. 3). The HMBC correlation observed from two
3
methyl protons [H -5 (δ 0.99) and H -6 (δ 1.06)] to sp
3
H
3
H
3
quaternary carbon [C-3 (δ 39.5)] and two sp oxygenated
C
carbons [C-2 (δ 78.1) and C-4 (δ 71.3)] suggested the
C
C
presence of partial structure D (Fig. 3). The remaining car-
bonyl group (δ 171.8) had to be located at the C-1 position,
C
connecting partial structures C and D, through the process
of elimination to construct the complete structure 1, which
Scheme 1 Synthesis of 1
1
3

Journal of Natural Medicines (2018) 72:280–289
283
Fig. 6 Comparison of extracts of culture broth in DMEM or
DMEM + FBS medium, a Single culture (J774.1, DMEM + FBS),
b single culture (N. tenerifensis, DMEM), c single culture (N. ten-
erifensis, DMEM + FBS), d co-culture (N. tenerifensis and J774.1,
DMEM + FBS)
Fig. 4 Comparison of optical rotation and CD spectra of 1 (natural)
and 1 (synthetic)
Fig. 5 Comparison of extracts of culture broth in mCD medium. a
Single culture (J774.1), b single culture (N. tenerifensis), c co-culture
(
N. tenerifensis and J774.1)
Fig. 7 The presumed biosynthetic pathway. a Ketosteroid isomer-
ase (an enzyme involved in reactions from 12 to 11), b 3-Ketoster-
oid-delta-1-dehydrogenase, c 17β-hydroxysteroid dehydrogenase, d
16α-hydroxylase
The biosynthetic pathway of this compound group
was proposed, as shown in Fig. 7, based on performing a
MiGAP analysis [29] and BLAST search [30] on the draft
genome of N. tenerifensis. Compounds 8–11 were pro-
posed to be biosynthesized from 12 by a group of enzymes
including ketosteroid isomerase, 3-ketosteroid-delta-1-de-
hydrogenase, 17β-hydroxysteroid dehydrogenase, and
with 10% FBS medium than in DMEM medium (Table 2).
When compounds 10 and 11 were added to J774.1 and cul-
tured for 24 h, the production of compounds 8 and 9 was
not observed (Fig. 8). These results suggested that the con-
version from compound 10 to 8 and compound 11 to 9 was
not due to J774.1. The expression of genes derived from
two enzymes, 17β-hydroxysteroid dehydrogenase (Fig. 7c)
and 16α-hydroxylase (Fig. 7d), in the strain (Table 3) may
1
6α-hydroxylase. The expression levels of the biosynthe-
sis genes in a single culture of N. tenerifensis in DMEM
medium and those in DMEM with 10% FBS medium were
then compared by RNA-seq. Two compounds, 10 and 11
were not produced in DMEM medium, but were produced
in DMEM with 10% FBS medium (Fig. 6b and c). By ana-
lyzing RNA-seq, the expression of the genes coding two
enzymes, ketosteroid isomerase and 3-ketosteroid-delta-
T
be activated by a co-culture of N. tenerifensis IFM 10554
and J774.1.
Since compounds 1, 8, and 9 were preferentially produced
by a co-culture, their cytotoxicities against J774.1 were
1
-dehydrogenase, was 4.0–4.6-fold stronger in DMEM
1
3

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Journal of Natural Medicines (2018) 72:280–289
Table 2 Presumed genes expressed in DMEM with FBS medium
Gene locationa Length aa Functiona
Contig00701 (142297..142714) 138
Similar proteinsb
%Identityb Fold changesc
Ketosteroid isomerase Ketosteroid isomerase [Nocardia brasiliensis ] 93
Ketosteroid isomerase [Nocardia altamirensis ] 92
4.6
Contig04601 (26751..28467)
571
3-Ketosteroid-delta-
3-Ketosteroid-delta-1-dehydrogenase [Nocar-
93
4.0
1
-dehydrogenase
dia brasiliensis ]
3
-Ketosteroid-delta-1-dehydrogenase [Nocar-
dia vulneris ]
93
a
Results of the MiGAP analysis
Results of the NCBI BLAST search
Results of the RNA-seq [fold change in RPKM (10- and 11-producing and non-producing conditions)]
b
c
Experimental
General experimental procedures
The following instruments were used in the present study:
a P-2200 polarimeter (JASCO) for optical rotations; a UV
mini 1240 UV–Vis spectrophotometer (Shimadzu) for
UV–Vis spectroscopy; an ECZ-600 spectrometer (JEOL)
for NMR spectroscopy (solvent chemical shifts were used
as the internal standard); and a JMS-T100LP (JEOL) for
HR-ESIMS. An HPLC system (Shimadzu) consisting of a
SCL-10AVP system controller, LC-20AD pumps, DGU-
Fig. 8 Comparison of extracts of culture broth in DMEM + FBS
medium. a Co-culture, b single culture [J774.1 cell, addition of
DMSO (control)], c single culture [J774.1 cell, addition of 10], d sin-
gle culture [J774.1 cell, addition of 11]
1
2
2A on-line degasser, CTO-10AS VP column oven, SIL-
0A autosampler, and SPD-M20A PDA detector was used
and chromatographic data were collected and processed
using Shimadzu CLASS-VP software (version 6.14 SP1,
Shimadzu). A Shimadzu LCMS system (Shimadzu) con-
examined and the results obtained revealed that they did not
exhibit cytotoxicity against J774.1.
sisting of LC-20AD pumps, a DGU-12A on-line degas-
3
ser, CTO-20A column oven, SIL-20A autosampler, SPD-
In the present study, we demonstrated a new method to
activate biosynthesis genes by culturing actinomycetes of the
genus Nocardia in the presence of animal cells and obtained
a new compound 1. By using this method, the further isola-
tion of new secondary metabolites is expected in the future.
M20A PDA detector, FCV-20AH valve unit, LCMS 2020
2
for ESIMS, and N Supplier Model 24F for the N genera-
2
2
tor was used and chromatographic data were collected and
processed using LabSolution software (version 5.42 SP4,
Shimadzu). The conditions of the LCMS analysis were as
Table 3 Genes presumed to be expressed by the co-culture
Gene locationa
Length aa Functiona
Similar proteinsb
%Identityb
69
Contig15901 (1139..1957)
272
Dehydrogenases with diꢂerent speciꢀcities Oxidoreductase [17β-HSD-like, SDR(c)]
(
related to short-chain alcohol dehydro-
[Streptomyces acidiscabies]
genases)
Oxidoreductase [17β-HSD-like, SDR(c)]
68
80
[
Streptomyces olivochromogenes ]
Contig04001 (28393..29613) 406
Cytochrome P450 monooxygenase
CYP154C5 (16α-hydroxylase) [Nocardia
farcinica ]
CYP154C3 (16α-hydroxylase) [Streptomy- 77
ces griseus subsp. griseus ]
a
Results of the MiGAP analysis
Results of the NCBI BLAST search
b
1
3

Journal of Natural Medicines (2018) 72:280–289
285
follows: 0–100% MeOH in 0.1% HCOOH, 0–30 min, lin-
Seed culture of Nocardia sp. for a co‑culture in modi‑
ear gradient, and 100% MeOH in 0.1% HCOOH, isocratic
ꢁed Czapek‑Dox medium
3
0–60 min; ꢁow rate: 0.2 mL/min; UV detection: photo-
diode array (190–600 nm); MS detection: ESI (positive
and negative) (m/z 100–2000); guard column: Develosil
ODS-HG-S (ϕ1.5 × 10 mm, Nomura chemical); col-
umn: COSMOSIL 5C -AR-II (ϕ2.0 × 150 mm, Nacalai
Each strain of Nocardia sp. was cultivated in 5 mL of
BHI + liquid medium consisting of Bacto™ brain
heart infusion (3.7 g/100 mL, Becton, Dickinson and
Company), glucose (1 g/100 mL, Wako), and glycerol
(1 mL/100 mL, Nacalai Tesque) in a 10-mL Erlenmeyer
ꢁask at 28 °C for 5 days with shaking (160 rpm). The
culture broth was added to a 50-mL tube. The super-
natant was removed after centrifugation at 3000 rpm
at 20°C for 2 min. Two milliliters of modiꢀed Czapek-
Dox medium consisting of sucrose (3 g/100 mL, Wako),
NaNO (0.3 g/100 mL, Wako), KH PO (0.1 g/100 mL,
1
8
Tesque). The following adsorbents were used for puriꢀ-
cation: Silica gel 60 F (0.25 mm, Merck) and Silica
2
54
gel 60 RP-8 F S (0.25 mm, Merck) for analytical TLC;
2
54
Silica Gel 60 N (Kanto chemical), Silica Gel PSQ 100B
Fuji Silysia chemical), and Chromatrex ODS (Fuji Sily-
sia chemical) for column chromatography; COSMOSIL
(
5
C -AR-II (ϕ 10.0 × 250 mm, Nacalai Tesque) and COS-
18
3
2
4
MOSIL Cholester column (ϕ 10.0 × 250 mm, Nacalai
Tesque) for preparative HPLC. The following instruments
were used for cultivation and extraction: GeneQuant pro
Nacalai Tesque), KCl (0.05 g/100 mL, Nacalai Tesque),
MgSO ·7H O (0.05 g/100 mL, Wako), and FeSO ·7H O
4
2
4
2
(0.001 g/100 mL, Nacalai Tesque) was added to the
tube. The supernatant was removed after centrifugation
at 3000 rpm at 20°C for 2 min. A strain suspension was
prepared by the addition of 11 mL of mCD medium to
the tube.
(
GE) for OD spectroscopy; FMC 1000 (EYELA) and
600
SOFT INCUBATOR SLI-450ND (EYELA) for an incuba-
tor; BIOLABO BL-171 (Juji Field) for a CO incubator;
2
M150-IVD (Sakuma), HIMAC CENTRIFUGE (Hitachi),
KS-5000 (Kubota), and Avanti centrifuge HP-60XP (Beck-
man Coulter) for a centrifuge.
Co‑culture in mCD medium in a cell culture ꢀask
Fifty milliliters of mCD medium were added to J774.1
in a cell culture ꢁask after the removal of DMEM with
Microbial strain
1
0% FBS medium. A suspension of Nocardia sp. was
Strains of the genus Nocardia were stored in a freeze-
dried state at the Medical Mycology Research Center,
Chiba University, Japan. These strains were reidentiꢀed
by sequencing the 16S rRNA gene.
added to the ꢁasks until the cell number ratio was reached
J774.1:Nocardia sp. = 1:10). Flasks were incubated at
8 °C for 2 weeks.
(
2
Seed culture of Nocardia sp. for a co‑culture
in DMEM with 10% FBS medium
Seed culture of a mouse macrophage‑like cell line
2
(
J774.1) in a 175‑cm cell culture ꢀask
Each strain of the Nocardia sp. culture was cultivated in
5 mL of BHI + liquid medium in a 10-mL Erlenmeyer
ꢁask at 28 °C for 5 days with shaking (160 rpm). The
culture broth was transferred to a 50-mL tube. The super-
natant was removed after centrifugation at 3000 rpm at
20 °C for 2 min. Two milliliters of DMEM were added to
the tube. The supernatant was removed after centrifugation
at 3000 rpm at 20°C for 2 min. A strain suspension was
prepared by the addition of 11 mL of DMEM to the tube.
A mouse macrophage-like cell line (J774.1) was incubated
at 37 °C in a 5% CO atmosphere in 25 mL of DMEM
2
(
Wako) with 10% fetal bovine serum (FBS, Bio West)
2
in 175-cm cell culture ꢁasks (Violamo), which reached
approximately 80–90% conꢁuency after 5–7 days. Old
culture medium was then removed and 5 mL of DMEM
with 10% FBS medium was added to the ꢁask. Cells were
scraped and collected from the bottom of the ꢁask with
a cell scraper. Cell medium was transferred to a 50-mL
tube, which was centrifuged at 2000 rpm at 20°C for
A co‑culture in DMEM with 10% FBS medium in a cell
culture ꢀask
5
min. After the removal of medium, 5 mL of DMEM
with 10% FBS medium was added to the tube. The residue
was stirred by pipetting and transferred to a hemocytom-
eter. The number of viable cells in the hemocytometer was
counted using trypan blue. Cell medium was added to each
new cell culture ꢁask with 25 mL of DMEM with 10%
FBS medium. Cells were cultured for 24 h.
Fifty milliliters of DMEM with 10% FBS medium were
added to J774.1 in a cell culture ꢁask after the removal
of medium. A suspension of Nocardia sp. was added
to the flasks until the cell number ratio was reached
1
3

286
Journal of Natural Medicines (2018) 72:280–289
(
J774.1:Nocardia sp. = 1:5). The ꢁasks were incubated at
7 °C for approximately 24 h under 5% CO .
3.55 (1H, m), 3.47 (1H, m), 2.57 (2H, m), 1.09 (3H, s),
1
3
3
and 1.07 (3H, s). C-NMR (150 MHz, CDCl ) δ: 176.5,
3
2
1
69.5, 160.2, 130.1, 127.4, 113.7, 101.2, 83.6, 78.4, 55.3,
2
0
Fermentation for mCD medium and isolation
34.1, 33.8, 33.1, 21.7, and 19.0. [α] +37.0 (c 0.1, MeOH).
D
−
1
IR (ATR) cm : 2949, 2362, 1653, 1520, 1250, 1103, and
T
+
The strain N. tenerifensis IFM 10554 was cultured at 28 °C
1032. HRESIMS m/z: 697.2991 [2 M + Na] (Calcd for
for 2 weeks on mCD medium in 63 cell culture ꢁasks in
the presence of J774.1 (cell number ratio J774.1: N. ten-
erifensis = 1:10). The culture broth (3.2 L) was centri-
fuged at 3000 rpm for 20 min to give the supernatant and
mycelial cake; the mycelial cake was extracted with MeOH
C H N O Na: 697.2948).
3
4
46
2
12
Methyl 3‑[(4R)‑2‑(4‑methoxyphenyl)‑5,5‑dimethyl‑1,3‑diox‑
ane‑4‑carboxamido] propanoate (4)
(
1.6 L × 2). The MeOH extract was combined with the
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDC·HCl, 623.4 mg, 3.25 mmol, TCI) and
4-dimethylaminopyridine (DMAP, 40 mg, 0.33 mmol, TCI)
were added to a solution of 3 (220 mg, 0.65 mmol) in dry
MeOH (13 mL, Wako), which was then stirred at room tem-
supernatant obtained above, and the combined materials
were partitioned with EtOAc (3.2 L × 3) and water. Some
of the EtOAc-soluble fraction (132.0 mg) was evaporated
and MeOH (13.2 mL) was added to the residue. A MeOH-
soluble fraction (89 mg) was subjected to ODS column
chromatography (ϕ 15 × 130 mm) eluted with 0–100%
MeOH to aꢂord 9 fractions. The fraction (4.9 mg) eluted
with 40–60% MeOH was puriꢀed by reversed-phase HPLC
perature for 22 h. After the addition of H O (30 mL), the
2
reaction mixture was extracted with CH Cl 3 times. The
2
2
organic layer was evaporated in vacuo and the residue was
separated by reversed-phase HPLC [COSMOSIL Cholester
(ϕ 10.0 × 250 mm); eluent: 80% MeOH; ꢁow rate: 4 mL/
min; UV detection: photodiode array (190–400 nm)] to give
[
COSMOSIL 5C -AR-II (ϕ 10.0 × 250 mm); eluent: 50%
18
MeOH; ꢁow rate: 5 mL/min; UV detection: photodiode array
190–400 nm)] to give compound 1 (1.5 mg, t 5.2 min).
(
4 (214 mg, 0.61 mmol, 94% yield, t 5.1 min).
R
R
1
H-NMR (600 MHz, CDCl ) δ: 7.41 (2H, d, J = 9.0 Hz),
3
Dehydropropylpantothenamide (1)
6.90 (2H, d, J = 9.0 Hz), 5.45 (1H, s), 4.06 (1H, s), 3.80
(
3H, s), 3.70 (1H, d, J = 11.0 Hz), 3.64 (1H, d, J = 11.0 Hz),
2
5
Colorless amorphous solid. [α] +14.1 (c 0.1, MeOH).
3.62 (3H, s) 3.55 (1H, m), 3.47 (1H, m), 2.54 (2H, m), 1.08
D
+
13
HRESIMS m/z: 539.3080 [2 M + Na] (calcd for
(3H, s), and 1.08 (3H, s). C-NMR (150 MHz, CDCl ) δ:
3
C H N O Na, 539.3057). UV λ (MeOH) nm (log ε):
max
172.3, 169.1, 160.2, 130.2, 127.4, 113.7, 101.2, 83.7, 78.4,
2
4
44
4
8
−
1
20
2
66 (3.8). IR (ATR) cm : 1738, 1366, and 1217. CD λ
55.3, 51.7, 34.3, 33.9, 33.0, 21.7, and 19.0. [α] +30.0 (c
nm
C
D
1
13
−1
(
MeOH) nm (∆ε): 232 (− 0.7), and 264 (0.9). H and
0.1, MeOH). IR (ATR) cm : 2956, 2364, 2348, 2310, 1733,
+
NMR data in Table 1.
and 1519. HRESIMS m/z: 374.1540 [M + Na] (Calcd for
C H NO Na: 374.1580).
1
8
25
6
N‑[[(4R)‑2‑(4‑Methoxyphenyl)‑5,5‑dimethyl‑1,3‑di‑
oxan‑4‑yl]carbonyl]‑β‑alanine (3)
Methyl (Z)‑3‑[(4R)‑2‑(4‑methoxyphenyl)‑5,5‑dime‑
thyl‑1,3‑dioxane‑4‑carboxamido] acrylate (5)
Conc. H SO (110 μL, 2.1 mmol, Wako) was added to a
2
4
solution of D-pantothenic acid calcium salt (2: 1.0 g,
Dess–Martin periodinane (154 mg, 0.36 mmol, TCI) was
added to a solution of 4 (14 mg, 0.04 mmol) in ꢁuorobenzene
(PhF, 4 mL, Wako), which was then reꢁuxed for 30 h. The
reaction mixture was quenched with saturated aqueous (aq.)
Na S O and saturated aq. NaHCO , and was then stirred
2
.1 mmol, TCI) in dry acetone (4 mL, Wako), which was
then stirred at room temperature for 2 h. p-Toluenesulfonic
acid monohydrate (p-TsOH·H O, 80 mg, 0.42 mmol, Wako).
2
p-anisaldehyde dimethyl acetal (357 μL, 2.1 mmol, Avocado
Research Chemicals) were added to the reaction mixture,
which was stirred at room temperature for 37 h. After the
2
2
4
3
until the solution became clear. The mixture was extracted
with hexane 3 times. The organic layer was evaporated in
vacuo, and the residue was separated by reversed-phase
HPLC [COSMOSIL Cholester (ϕ 10.0 × 250 mm); eluent:
80% MeOH; ꢁow rate: 4 mL/min; UV detection: photodiode
array (190–400 nm)] to give 5 (3.8 mg, 0.011 mmol, 26%
addition of H O (20 mL), the reaction mixture was extracted
2
with EtOAc 3 times. The organic layer was evaporated in
vacuo. The residue was subjected to ꢁash silica gel col-
umn chromatography (ϕ 20 × 200 mm) eluted with hexane/
acetone (1/1) to aꢂord the single diastereomer 3 (481 mg,
yield, t 8.2 min).
R
1
1
.42 mmol, 34% yield).
H-NMR (600 MHz, CDCl ) δ: 11.20 (1H, d,
3
1
H-NMR (600 MHz, CDCl ) δ: 7.40 (2H, d, J = 9.0 Hz),
J = 11.3 Hz), 7.60 (2H, d, J = 8.6 Hz), 7.46 (1H, dd,
J = 11.3, 9.0 Hz), 6.95 (2H, d, J = 8.6 Hz), 5.55 (1H, s),
5.19 (1H, d, J = 9.0 Hz), 4.23 (1H, s), 3.83 (3H, s), 3.73 (2H,
3
6
.90 (2H, d, J = 9.0 Hz), 5.45 (1H, s), 4.10 (1H, s), 3.79
(
3H, s), 3.70 (1H, d, J = 11.3 Hz), 3.64 (1H, d, J = 11.3 Hz),
1
3

Journal of Natural Medicines (2018) 72:280–289
287
1
3
m), 3.68 (3H, s), 1.17 (3H, s), and 1.11 (3H, s). C-NMR
150 MHz, CDCl ) δ: 168.4, 168.0, 160.3, 135.9, 130.0,
Dehydropropylpantothenamide (1, synthetic)
(
3
1
27.6, 113.7, 101.3, 98.0, 83.7, 78.4, 55.3, 51.2, 33.4, 21.6,
A solution of 7 (2.6 mg, 6.9 μmol) in CH COOH/H O = 4/1
3
2
2
0
−1
and 19.2. [α] − 2.3 (c 0.1, MeOH). IR (ATR) cm : 2970,
(80% CH COOH, 2 mL) was stirred at room temperature
D
3
2
3
369, 2350, 2318, 1737, 1364, and 1217. HRESIMS m/z:
for 14 h. The reaction mixture was evaporated in vacuo and
the residue was separated by reversed-phase HPLC [COS-
MOSIL Cholester (ϕ 10.0 × 250 mm); eluent: 60% MeOH;
flow rate: 4 mL/min; UV detection: photodiode array
+
72.1413 [M + Na] (Calcd for C H NO Na: 372.1423).
1
8
23
6
(
Z)‑3‑[(4R)‑2‑(4‑methoxyphenyl)‑5,5‑dimethyl‑1,3‑diox‑
ane‑4‑carboxamido] acrylic acid (6)
(190–400 nm)] to give 1 (0.7 mg, 2.7 μmol, 39% yield, t
R
6
.0 min).
2
0
−1
A solution of 5 (3.4 mg, 9.7 μmol) in 0.5 N aq. NaOH (6 mL)
was stirred at room temperature for 37 h. The solution was
neutralized by the addition of 1 N HCl, and the mixture was
extracted with EtOAc 3 times. The organic layer was evapo-
[α] +12.5 (c 0.1, MeOH). IR (ATR) cm : 2970, 2368,
D
2350, 2319, 1737, 1364, and 1217. CD λ (MeOH) nm
nm
(∆ε): 236 (-1.5), and 264 (1.3). HRESIMS m/z: 539.3060
+
[2 M + Na] (Calcd for C H N O Na: 539.3057).
2
4
44
4
8
rated in vacuo to give 6 (2.5 mg, 7.5 μmol, 77% yield).
1
H-NMR (600 MHz, CD OD) δ: 7.53 (2H, d, J = 8.6 Hz),
3
7
.44 (1H, dd, J = 9.0 Hz), 6.20 (2H, d, J = 8.6 Hz), 5.60
Fermentation for DMEM with 10% FBS medium
and isolation
(
1H, s), 5.20 (1H, d, J = 9.0 Hz), 4.32 (1H, s), 3.79 (3H, s),
.77 (1H, d, J = 11.0 Hz), 3.68 (1H, d, J = 11.0 Hz), 1.10
3
1
3
T
(
3H, s), and 1.06 (3H, s). C-NMR (150 MHz, CD OD)
The strain N. tenerifensis IFM 10554 was cultured at 37 °C
3
δ: 171.3, 169.8, 161.6, 136.6, 131.6, 128.7, 114.5, 102.5,
for 24 h under 5% CO on DMEM with 10% FBS medium in
2
2
0
1
0
1
00.0, 84.7, 79.1, 55.7, 34.3, 21.9, and 19.6. [α] -11.0 (c
cell culture ꢁasks (40 mL × 200) in the presence of J774.1
(the cell number ratio J774.1: N. tenerifensis = 1:5). The
culture broth (8.0 L) was centrifuged at 5000 rpm for 20 min
to give the supernatant and mycelial cake; the mycelial cake
was extracted with MeOH (4 L × 2). The MeOH extract was
combined with the supernatant obtained above, and the com-
bined materials were partitioned with EtOAc (8 L × 3) and
water. A part of the EtOAc-soluble fraction (319 mg) was
evaporated and MeOH (32 mL) was added to the residue.
A MeOH-soluble fraction was separated by reversed-phase
HPLC [COSMOSIL 5C -AR-II (ϕ 10.0 × 250 mm); elu-
D
−
1
.1, MeOH). IR (ATR) cm : 2970, 2369, 2350, 2318, 1738,
+
737, 1364, and 1217. HRESIMS m/z: 358.1230 [M + Na]
(
Calcd for C H NO Na: 358.1267).
17 21 6
(
4R)‑2‑(4‑methoxyphenyl)‑5,5‑dimethyl‑N‑[(Z)‑3‑oxo‑3‑(pro
pylamino)prop‑1‑en‑1‑yl]‑1,3‑dioxane‑4‑carboxamide (7)
EDC·HCl (17 mg, 0.09 mmol, TCI), 1-hydroxybenzotria-
zole monohydrate (HOBt·H O, 14 mg, 0.09 mmol, TCI),
2
and propylamine (5 mL, 0.06 mmol, Wako) were added to a
solution of 6 (10 mg, 0.03 mmol) in dry DMF (3 mL, Wako),
which was then stirred at room temperature for 24 h. The
1
8
ent: 0–100% MeOH with 0.1% HCOOH in H O, 0–30 min,
2
linear gradient; ꢁow rate: 4 mL/min; UV detection: pho-
reaction mixture was quenched with saturated aq. NaHCO
todiode array (190–400 nm)] to give 1,4-androstadine-
3
and then extracted with hexane/EtOAc = 1/4 3 times. The
organic layer was evaporated in vacuo and the residue was
separated by reversed-phase HPLC [COSMOSIL Cholester
16α, 17β-diol-3-one (8, 0.3 mg, t 19.0 min, colorless
R
2
5
amorphous solid, [α] +61.1 (c 0.05, MeOH), HRESIMS
D
+
m/z: 325.1803 [M + Na] (Calcd for C H O Na:
1
9
28
2
(
ϕ 10.0 × 250 mm); eluent: 80% MeOH; ꢁow rate: 4 mL/
325.1804)), 4-androstene-16α, 17β-diol-3-one (9, 1.1 mg,
25
min; UV detection: photodiode array (190–400 nm)] to give
t 20.5 min, colorless amorphous solid; [α] +69.7 (c 0.05,
R D
+
7
(5.0 mg, 0.013 mmol, 44% yield, t 6.8 min).
MeOH), HRESIMS m/z: 327.1951 [M + Na] (Calcd for
C H O Na: 327.1960)), 1,4-androstadine-3,17-dione (10,
R
1
H-NMR (600 MHz, CDCl ) δ: 12.05 (1H, d,
3
19 28
2
2
5
J = 11.0 Hz), 7.66 (2H, d, J = 8.9 Hz), 7.46 (1H, dd,
0.3 mg, t 22.0 min, colorless amorphous solid, [α] +84.0
R
D
+
J = 11.0, 9.0 Hz), 6.95 (2H, d, J = 8.9 Hz), 5.54 (1H, s),
(c 0.05, MeOH), HRESIMS m/z: 307.1674 [M + Na]
(Calcd for C H O Na: 307.1674)), and 4-androstene-
5
.40 (1H, t, J = 5.5 Hz) 5.00 (1H, d, J = 9.0 Hz), 4.19 (1H,
1
9
24
2
s), 3.82 (3H, s), 3.17 (2H, s), 3.28 (1H, m), 3.25 (1H, m),
3,17-dione (11, 0.2 mg, t 23.7 min, colorless amorphous
R
2
5
1
.55 (2H, m), 1.16 (3H, s), 1.10 (3H, s), and 0.94 (3H, t,
solid, [α] +96.8 (c 0.05, MeOH), HRESIMS m/z: 309.1834
D
1
3
+
J = 7.6 Hz); C-NMR (150 MHz, CDCl ) δ: 168.0, 167.7,
[M + Na] (Calcd for C H O Na: 309.1830)). A MeOH-
3
19 26
2
1
5
0
1
60.1, 133.3, 130.1, 127.7, 113.7, 101.2, 100.8, 83.9, 78.5,
insoluble compound (10.9 mg) in the EtOAc soluble fraction
2
0
5.3, 40.9, 33.4, 22.8, 21.7, 19.3, and 11.5. [α] +7.6 (c
was identiꢀed as cholesteryl oleate (12, 10.9 mg, colorless
D
−
1
25
.1, MeOH). IR (ATR) cm : 2970, 2368, 2350, 2319, 1737,
amorphous solid, [α] − 13.6 (c 0.1, CHCl ), HRESIMS
D
3
+
+
364, and 1217. HRESIMS m/z: 399.1847 [M + Na] (Calcd
m/z: 673.5939 [M + Na] (Calcd for C H O Na:
45 78 2
for C H NO Na: 399.1896).
673.5899).
2
0
28
5
1
3

288
Journal of Natural Medicines (2018) 72:280–289
6
7
8
.
.
.
Nett M, Ikeda H, Moore BS (2009) Genomic basis for natural
product biosynthetic diversity in the actinomycetes. Nat Prod Rep
Transcriptional analysis
2
6:1362–1384
RNA was extracted from the strain N. tenerifensis IFM
Bode HB, Bethe B, Höfs R, Zeeck A (2002) Big eꢂects from
small changes: possible ways to explore nature’s chemical diver-
sity. ChemBioChem 3:619–627
T
1
0554 cultured under 10- and 11-producing and non-
producing conditions. DMEM with 10% FBS medium was
used for the 10- and 11-producing conditions, and DMEM
medium without FBS was used for the non-producing condi-
Hosaka T, Ohnishi-Kameyama M, Muramatsu H, Murakami
K, Tsurumi Y, Kodani S, Yoshida M, Fujie A, Ochi K (2009)
Antibacterial discovery in actinomycetes strains with mutations
in RNA polymerase or ribosomal protein S12. Nat Biotechnol
T
tions. The strain N. tenerifensis IFM 10554 pre-cultivated
2
7:462–464
on BHI + medium at 37 °C for 5 days was inoculated into
9
.
Laureti L, Song L, Huanga S, Corre C, Leblond P, Challis GL,
Aigle B (2011) Identiꢀcation of a bioactive 51-membered mac-
rolide complex by activation of a silent polyketide synthase in
Streptomyces ambofaciens. Proc Natl Acad Sci Unit States Am
1
0- and 11-producing and non-producing media and then
incubated at 37 °C under a 5% CO atmosphere for 24 h. Fol-
2
T
lowing the incubation, the strain N. tenerifensis IFM 10554
1
08:6258–6263
was collected and suspended in ISOGEN RNA extraction
1
0. Onaka H, Mori Y, Igarashi Y, Furumai T (2011) Mycolic acid-
containing bacteria induce natural-product biosynthesis in Strep-
tomyces species. Appl Environ Microbiol 77:400–406
solution (Nippon Gene Co.). The resuspended strain N. ten-
T
erifensis IFM 10554 was homogenized using a MagNA
Lyser (Roche Diagnostics) at 7000 rpm for 20 s, and RNA
was extracted according to the ISOGEN protocol. Following
the DNase treatment, 2 μg of total RNA was treated with a
Ribo-Zero Magnetic Kit (Epicentre) to remove ribosomal
RNA. In the construction of the RNA-seq library, the mRNA
samples obtained were treated with a KAPA Standard RNA-
seq Library Preparation Kit (illumina) according to the man-
ufacturer’s protocol. Constructed libraries were sequenced
with the MiSeq system (Illumina) using Miseq Reagent Kit
v2 (Illumina). The resulting data were mapped against the
11. Hoshino S, Zhang L, Awakawa T, Wakimoto T, Onaka H, Abe I
(
2015) Arcyriaꢁavin E, a new cytotoxic indolocarbazole alkaloid
isolated by combined-culture of mycolic acid-containing bac-
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8:342–344
12. Sugiyama R, Nishimura S, Ozaki T, Asamizu S, Onaka H, Kakeya
H (2015) 5-Alkyl-1,2,3,4-tetrahydroquinolines, new membrane-
interacting lipophilic metabolites, produced by combined culture
of Streptomyces nigrescens and Tsukamurella pulmonis. Org Lett
1
7:1918–1921
13. Sakai K, Komaki H, Gonoi T (2015) Identiꢀcation and Functional
Analysis of the Nocardithiocin Gene Cluster in Nocardia pseudo-
brasiliensis. PLoS ONE 10:e0143264
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sequenced genome data of N. tenerifensis IFM10554 using
1
4. Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R,
Lee SY, Fischbach MA, Müller R, Wohlleben W, Breitling R,
Takano E, Medema MH (2015) antiSMASH 3.0-a comprehensive
resource for the genome mining of biosynthetic gene clusters.
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CLC GenomicsWorkbench.
Acknowledgments This study was supported by JSPS KAKENHI
Grant Number 17H03992 and the Strategic Priority Research Promo-
tion Program of Chiba University “Phytochemical Plant Molecular
Sciences”.
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