R ESEARCH ARTICLE

ScienceAsia 49 (2023): 661–669

doi: 10.2306/scienceasia1513-1874.2023.067

Genomic analysis and physicochemical screening of
ikarugamycin, fumaquinone, and a new compound, pudicinfrom Streptomyces sp. 3MP-14
Nattaporn Klykleunga,† , Hirotaka Matsuob,c,† , Masato Iwatsukic , Yōko Takahashic , Satoshi Ōmurac ,Masahiro Yukid , Takuji Kudod , Moriya Ohkumad , Rataya Luechapudiporne , Somboon Tanasupawata,∗ ,Takuji Nakashimac,fa

Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University,
Bangkok 10330 ThailandbResearch Center for Medicinal Plant Resources, National Institutes of Biomedical Innovation, Health and Nutrition,
1-2 Hachimandai, Tsukuba 305-0843 JapancŌmura Satoshi Memorial Institute, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641 Japand
Japan Collection of Microorganisms, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba,
Ibaraki 305-0074 Japane
Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University,
Bangkok 10330 ThailandfResearch Organization for Nano and Life Innovation, Waseda University, 513 Waseda, Tsurumaki-cho, Shin-juku-ku,
Tokyo 162-0041 Japan∗

Corresponding author, e-mail: Somboon.T@chula.ac.th† These authors contributed equally to this work.
Received 16 Jan 2023, Accepted 4 Jun 2023
Available online 12 Aug 2023
ABSTRACT: An endophytic actinomycete, strain 3MP-14 isolated from the root of Mimosa pudica belonged to the genus
Streptomyces and was closely related to Streptomyces mimosae JCM 33328T (100%) based on 16S rRNA gene sequences.
The strain had genome sizes of 7.2 Mb with genomic G+C contents of 73.4 mol%. The average nucleotide identity(ANI) and digital DNA-DNA hybridization (dDDH) of strain 3MP-14 were 100% when compared with S. mimosae JCM
33328T . Based on the polyphasic approach, dDDH, and ANI, strain 3MP-14 was identified as Streptomyces mimosae.
Twenty-four putative secondary metabolite biosynthetic gene clusters (BGCs) were identified from the draft genome
analysis of strain 3MP-14. In the course of chemical investigation for the products of the strain, a physicochemical (PC)screening was conducted, and a new compound, designated as pudicin, and two known antimicrobials, ikarugamycin
and fumaquinone, were identified. The structure elucidation of pudicin revealed a unique structurally new compoundwith N -acetylcysteine moiety using one-dimensional and two-dimensional nuclear magnetic resonance (1D and 2D
NMR spectroscopy) and Electrospray Ionization Mass Spectrometry (ESI-MS). Pudicin showed slight antioxidative
activity based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method with the IC50 value of 394.89 ±3.88 µg/ml.
KEYWORDS: actinomycetes, physicochemical screening, pudicin, Mimosa pudica, Streptomyces, secondary metabolite

INTRODUCTION
The genus Streptomyces is aerobic, Gram-positive filamentous bacteria that form spore chains on the extensively branched substrate and aerial mycelia. Streptomyces species have LL-diaminopimelic acid and nodiagnostic sugar in whole-cell hydrolysates. They arewidely distributed in various habitats, including terrestrial, hot springs, marine, desert, and plants [1]. Over50 percent of microbial compounds were produced
from actinomycetes, especially the genus Streptomyces
which was the largest bioactive producer among theothers [2]. Endophytic actinomycetes which resided
in the tissues of healthy host plants as endosymbionts
are the potential resources of new species and new secondary metabolites for developing new drug discovery
with many drugs and analogs successfully used in clin-

ical practice [3, 4]. Special pressure and selective environment have led to evolution of endophytes to produce new enzymes for adapting to their hosts, enabling
the biosynthesis of new secondary metabolites [5, 6].
In addition, many traditional medicinal plants havewidely local ethnobotanical use in alternative medicine
in Thailand. Thus, medicinal plants are promising
as isolation sources for discovering new endophytic
actinomycetes and novel bioactive compounds in thisstudy.
A program designed to discover new endophytic
actinomycetes and their bioactive secondary metabolites was investigated. The search for new secondary compounds was optimized by physicochemical(PC) screening [2, 7] based on the spectrum obtained
from liquid chromatography with tandem mass spectroscopy (LC/MS/MS) data and compared with thewww.scienceasia.org

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natural product databases in the Dictionary of Natural
Products (http://dnp.chemnetbase.com). Therefore,
in this study we investigated the taxonomic positionof strain 3MP-14 isolated from the root of M. pudica(Fabaceae) based on polyphasic approaches and themass and ultraviolet (UV) spectra of the potential antimicrobials identified. Additionally, the antioxidative
activity of the new compound was determined using
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method with the half maximal inhibitory concentration (IC50 ) value.
MATERIALS AND METHODS
Isolation and identification of strain 3MP-14
The root of M. pudica (Fabaceae) was collectedfrom Kanchanaburi Province, Thailand (14°310 1200 N
98°490 1500 E), and strain 3MP-14 was isolated as previously described [8]. After incubation on International Streptomyces Project (ISP) 4 medium at 30 °C for
30 days, morphological characteristics were observed
by a scanning electron microscope (model JSM-5600,
JEOL, Tokyo, Japan). Cultural characteristics were
investigated on ISP media [9]: ISP 2, ISP 3, ISP 4,
ISP 5, ISP 6, ISP 7, and nutrient agar plates. The
aerial and substrate mycelia and soluble pigments were
determined using the National Bureau of Standards(NBS)/Inter-Society Color Council (ISCC) color chart[10]. Carbon utilization was performed as describedby Shirling and Gottlieb [9]. Biochemical characteristics, temperatures, pH, and NaCl concentrations for
growth were observed as described by Klykleung et al[8]. API ZYM system (bioMérieux, Marcy-l’Étoile,
France) was used to determine enzymatic activities.
Whole-cell sugars and diaminopimelic acid were evaluated as reported by Staneck and Roberts [11]. Phospholipids were analyzed following the method of Minnikin et al [12]. Menaquinones were analyzed by themethod of Collin et al [13]. Cellular fatty acids wereanalyzed as reported by Sasser [14].
Genomic DNA was isolated and purified as reported previously [15]. The 16S rRNA gene wasamplified and sequenced by Macrogen (Seoul, Korea)
using universal primers as previously described by
Lane [16]. The Basic Local Alignment Search Tool
(BLAST) analysis was assessed using the EzBioCloud
web-based tool. The neighbor-joining phylogenetic
tree was reconstructed using Molecular Evolutionary
Genetics Analysis (MEGA) 11 [17]. Whole-genome
sequencing was carried out by an Illumina Miseq platform (Illumina) using 2 × 250 bp paired-end reads.
Assembly of the reads to contigs was accomplishedby using St. Petersburg genome assembler (SPAdes)
3.12 [18]. The draft assemblies have been submitted to GenBank and are publicly available. Averagenucleotide identity (ANI) values were calculated withpairwise genome alignment of the draft genome sequences of related type strains by JSpeciesWS web
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ScienceAsia 49 (2023)

service [19, 20]. Calculation of the digital DNA-DNA
hybridization (dDDH) values was performed by the
Genome-to-Genome Distance Calculator (GGDC 2.1)
using the BLAST+ method [21]. The G+C content wasdetermined according to the genomic DNA sequences.
Results were evaluated by the recommended formula 2(identities/HSP length), which is useful when dealing
with the draft genomes.
Genomic analysis of strain 3MP-14
The draft genome of strain 3MP-14 was annotatedby the National Center for Biotechnology Information(NCBI) Prokaryotic Genome Annotation Pipeline [22],Glimmer v3.02 [23], and Rapid Annotation using Subsystem Technology [24]. Identification of potential
secondary metabolite biosynthetic gene clusters wasachieved using antibiotics and Secondary metabolite
Analysis Shell (antiSMASH) v6.0 [25] and confirmedwith manual BLAST alignment.
Preliminary screening for antimicrobial activity
The strain was cultivated in seed medium 301 [26]at 30 °C for 5 days (on a 200 rpm shaker) and in a
production medium (ISP 2) for 7 days. The ethanol
crude extract was screened for its antimicrobial activity
against Staphylococcus aureus ATCC 25923, Kocuria
rhizophila ATCC 9341, Bacillus subtilis ATCC 6633,
Escherichia coli ATCC 25922, Pseudomonas aeruginosa
ATCC 27853, and Candida albicans ATCC 10231 using
agar well diffusion method [26, 27]. The plates werethen incubated at 37 °C for 24 h, except for the C. albicans ATCC 10231, which was incubated at 30 °C.
Fermentation and purification of a newcompound, pudicin
Briefly, strain 3MP-14 was cultivated in 200 ml of theseed medium (shaking at 200 rpm) at 30 °C for 4
days. The inoculum was transferred into a 30-liter jar
fermenter containing 20 l of the production medium(2.5% soluble starch, 1.0% dry yeast, 1.0% defatted
wheat germ, 0.5% glycerol, 0.3% meat extract, and
0.3% CaCO3 ; adjusted to pH 7.0 before sterilization)with aeration 10 l/min and agitation of 250 rpm at
30 °C for 7 days. Purification of a new compound,pudicin, was subsequently guided by its physicochemical properties such as MS and UV spectra, using liquid
chromatography/ultraviolet (LC/UV) and liquid chromatography/mass spectrometry (LC/MS) equipment.
The purification procedure of pudicin is summarized inFig. 1. Twenty liters of culture broth were centrifuged;
the supernatant was passed through a column of Diaion HP-20 (100 i.d. × 250 mm, Mitsubishi ChemicalCo., Tokyo, Japan). After washing with water, thefraction containing pudicin was eluted with methanol(MeOH) and concentrated in vacuo. This fraction
was dissolved in H2 O, and 3 volumes of ethyl acetate(EtOAc) were added. The EtOAc layer was collected

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ScienceAsia 49 (2023)

in the 13C-NMR spectra. LC/electrospray ionization(ESI)/MS spectra were measured using an MS system(TripleTOF 5600+ System; AB Sciex, Framingham,
MA, USA) coupled to a HPLC system (ExionLC AC; AB
Sciex). Infrared (IR) spectra were recorded using an IR
spectrometer (FT-710; Horiba Ltd., Kyoto, Japan). UV
spectra were obtained using a spectrophotometer (U2810; Hitachi High-Tech Science Co, Tokyo, Japan).
Optical rotation was measured on a polarimeter (DIP1000; JASCO Co, Tokyo, Japan). All solvents were purchased from Kanto Chemical Co., Inc., Tokyo, Japan.
In vitro antioxidant activity of pudicin using DPPH
radical scavenging assay

Fig. 1 Purification scheme for pudicin.

and concentrated in vacuo, yielding 30.8 g of material. This material was separated into 4 fractions bysilica gel column chromatography (60 i.d. × 200 mm,
Fuji Silysia Chemical Co., Ltd., Aichi, Japan) withhexane and EtOAc solvent system (hexane:EtOAc =8:2, 6:4, stepwise separation), followed by chloroform(CHCl3 ) and MeOH solvent system (CHCl3 :MeOH =20:1, 8:2, stepwise separation). The eluted fraction(CHCl3 :MeOH = 20:1) containing pudicin was concentrated to yield 801.3 mg. The material was applied
on an ODS column (25 i.d. × 100 mm, Fuji Silysia
Chemical Co., Ltd.) and eluting system (0–100%
gradient system with MeOH aq. in 30 min and followedby 100% MeOH for 20 min, flow rate 20 ml/min) to
give 5 fractions every 10 min. The fourth fraction(224.0 mg) containing pudicin was subjected to semipreparative High Performance Liquid Chromatography(HPLC) (10 i.d. × 250 mm; YMC-Actus Triart PFP, YMC
Co., Ltd., Kyoto, Japan) using a 70% MeOH aq. (0.1%
formic acid) isocratic solvent system to obtain pudicin(17.7 mg). The final purification was performedby preparative HPLC (10 i.d. × 250 mm; YMC-Actus
C18, YMC Co., Ltd.) using 80% MeOH aq. (0.1%
formic acid) isocratic solvent system to obtain pudicin(9.4 mg).
General experimental procedures for chemical
analyses1
H-NMR at 400 MHz and 13C-NMR at 100 MHz at
600 MHz (AVANCE III HD600; Bruker, Kanagawa,Japan) were performed. Chemical shifts are expressedin parts per million and referenced to residual CD3 OD(3.31 ppm) and CDCl3 (7.26 ppm) in the 1H-NMR
spectra and CD3 OD (49.0 ppm) and CDCl3 (77.0 ppm)

The DPPH radical scavenging activity was determinedfollowing the procedure of Chansriniyom et al [28].
The 200 µl of pudicin (0.05–1.0 mg/ml) in EtOH wasadded to 0.2 ml of a 0.002% DPPH solution in 96well microplates (NUNC, Gillingham, UK). The reaction was incubated for 30 min in the dark at room
temperature. The absorbance was measured using a
Corona Grating Microplate Reader SH-9000 (Corona
Electric, Ibaraki, Japan) at 517 nm. Ascorbic acid wasused as a positive control. The percentage of DPPH
radical scavenging activity was calculated as follows:
DPPH radical scavenging activity (%) =(Ac − As )
× 100
Ac

where Ac and As mean the absorbance of control andsample, respectively. The experiment was conductedin triplicate. The percentage of inhibition at eachconcentration was measured, and IC50 was calculated.
Ferric reducing antioxidant power (FRAP) assay
The ferric (Fe+3 ) reduction of pudicin (at the final
concentration of 1 mg/ml) was carried out according
to the method of Wong et al [29]. The ferric-reducing
ability of plasma (FRAP) reagent was prepared bymixing 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6tripyridyl-s-triazine (TPTZ), and 20 mM FeCl3 · 6 H2 O
in a ratio of 10:1:1 at 37 °C. The 20 µl of pudicin
or ascorbic acid (positive control) was added to 96well microplates (NUNC, Sigma Aldrich, UK), followedby 280 µl of FRAP reagent. The reaction mixtures
were incubated in the dark at room temperature for
15 min, and then the absorbances were measured at
593 nm. The Fe+3 reduction was calculated as follows:
The reduction of Fe+3 is expressed in µM of ascorbic
acid equivalents by calculating from the calibration
curve. FRAP calibration graphs were constructed byplotting absorbances (minus reagent blanks) versus
each concentration of the standard in the assay system(µM).
Statistical analysis
Data were calculated as means ± standard deviation(SD). Significant differences were tested by one-way
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Fig. 2 Scanning electron micrograph of strain 3MP-14 grownon ISP 4 agar medium at 30 °C for 30 days. Bar, 1 µm.

ANOVA for the separation of means (SPSS 15.0 for
windows).
RESULTS AND DISCUSSION
Isolation and identification of strain 3MP-14
Strain 3MP-14 (=JCM 33207= TISTR 2647) grew wellon ISP 4 and nutrient agar and moderately on ISP 2,
ISP 3, and nutrient agar, whereas it grew poorly on
ISP 5, ISP 6, and ISP 7 media (Table S1). White
aerial masses were formed on ISP 3 and ISP 4 media.
The soluble pigment was not produced on the tested
media. Spiral spore chains with 4 to 6 turns, circular
to elliptical shape (0.5–0.7 × 0.6–0.8 µm in size),and smooth on-surface spores were formed on ISP 4
medium (Fig. 2). The whole-cell hydrolysates of thestrain contained LL-diaminopimelic acid, glucose, andribose. The menaquinones were MK-9 (H8 ), MK-10(H6 ), and MK-10 (H8 ). The predominant fatty acid was
iso-C16:0 (50.8%) (Table S3). The phospholipids were
phosphatidylethanolamine (PE), phosphatidylinositol(PI), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), unidentified phosphoglycolipid (PGL), andunidentified phospholipid (PL) (Fig. S1). Growth of
this strain occurred at 20–37 °C (optimum 30 °C),
pH 6.0–11.0 (optimum pH 7.0–9.0), and 0–3% NaCl(w/v). The 16S rRNA gene sequence of strain 3MP-14
revealed that it was closely related to S. mimosae 3MP10T (100%), S. zhaozhouensis DSM 42101T (98.76%),
S. sedi JCM 16909T (98.45%), and S. triticirhizae DSM
107172T (98.35%). The phylogenetic tree showed it
was on the same node and shared the cluster withstrain 3MP-10T (Fig. S2). Strain 3MP-14 showed thedifference in utilization of myo-inositol and enzyme
activities of cystine arylamidase, α-chymotrypsin, and
α-fucosidase from S. triticirhizae DSM 107172T [30],
S. mimosae 3MP-10T , S. zhaozhouensis DSM 42101T ,and S. sedi JCM 16909T as shown in Table 1 andTable S2.
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ScienceAsia 49 (2023)

The draft genome sequence of strain 3MP-14 was
7,197,316 bp in size with the average in silico DNA
G+C content of 73.4 mol% (Table 2). The dDDH
value between the genomes of strain 3MP-14 and 3MP10T was 100% (C.I. model 100–100%), while thedDDH values between the genomes of these strains
and related species, including S. triticirhizae DSM
107172T (RFFJ00000000), S. zhaozhouensis DSM
42101T (OCNE00000000), and S. sedi JCM 16909T(VDGT00000000) were 55.7% (C.I. model 52.9–
58.4%), 29.9% (C.I. model 27.5–32.4%) and 29.6%(C.I. model 27.2–32.1%), respectively (Table S4).
These values were significantly lower than the threshold value of 70% commonly used to delineate separated species status [31]. The ANIb and ANIm values of the draft genomes between strain 3MP-14 andS. mimosae 3MP-10T were 100%. ANIb values between
this strain and related species, S. triticirhizae DSM
107172T , S. zhaozhouensis DSM 42101T , and S. sedi
JCM 16909T were 94.49, 84.88 and 84.74%, respectively. ANIm values between this strain and related
species, S. zhaozhouensis DSM 42101T and S. sedi JCM
16909T were 94.62, 88.01 and 87.92%, respectively(Table S4). Both dDDH and ANI values of strain
3MP-14 and S. mimosae 3MP-10T were 100% whichindicated it was identified as Streptomyces mimosae[8, 19].
The GenBank/EMBL/DDBJ accession number for
16S rRNA gene sequences of strain 3MP-14 isLC379255. The Whole Genome Shotgun project ofstrain 3MP-14 has been deposited at DDBJ/ENA/GenBank, and the accession number is VDLZ00000000.
Genomic analysis of strain 3MP-14
A total of 6,077 coding genes from the draft genome ofstrain 3MP-14 were predicted by RAST, of which 34%
were hypothetical. Genome sequence analysis of strain
3MP-14 revealed the presence of at least 24 candidate BGCs. Twenty-four putative secondary metabolite
BGCs, predicted in the draft genome of strain 3MP14, comprised 10 different types of BGCs, including
non-ribosomal peptide synthetase (NRPS), polyketide
synthetase (PKS), thiopeptide, lanthipeptide, terpenes,
butyrolactone, bacteriocin, siderophores, ectoine, andtRNA-dependent cyclodipeptide synthases. The BGCs
potentially involved in the biosynthesis of secondary
metabolites are presented in Table 3.
The investigation of BGCs in strain 3MP-14 withthe known gene clusters was equal to 100% and over
50%. This finding might possibly indicate an ability
for the biosynthesis of naringenin, geosmin, ectoine,polyketomycin, and desferrioxamine. As an instanceof below 20% gene similarity to the known BGCs,
strain 3MP-14 showed hits with different metabolites:
3% similarity to murayaquinone and lactonamycin;
7% similarity to marinophenazine; 8% similarity toazicemicin, cahuitamycins, and chlortetracycline; 12%

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Table 1 Differential characteristics of strain 3MP-14 and related type strains. Strains: 1, 3MP-14; 2, S. mimosae 3MP-10T ;3, S. triticirhizae DSM 107172T ; 4. S. zhaozhouensis DSM 42101T ; and 5, S. sedi JCM 16909T . +, positive; w, weakly positive;−, negative. Data are from this study, except strain DSM 107172T [30].
Characteristic
Soluble pigment on ISP 3
NaCl tolerance (%, w/v)
pH range for growth
Growth temperature ( °C)
Nitrate reduction
Starch hydrolysis
Gelatin liquefaction
Utilization of
L-Arabinose
D-Fructose
D-Galactose
myo-Inositol
Lactose
D-Maltose
D-Mannitol
D-Mannose
L-Rhamnose
D-Ribose

1

2

3

4

5

None
0–3
6–11
20–37
−
+
−

None
0–3
6–11
20–37
−
+
−

None
0–6
4–6
15–45
−
+
+

None
0–7
6–10
15–37
+
+
+

Dark green
0–5
7–8
15–37
−
−
−

+
+
+
+
+
w
+
+
+
+

+
+
+
w
+
w
+
+
+
+

−
−
−
−
−
−
−
+
−
+

−
−
+
−
+
+
+
−
−
−

−
+
−
−
−
+
−
−
−
−

Table 2 Genome features of strain 3MP-14 and related type strains.
FeatureAccession no.
Genome size (bp)Genome coverage
Number of contig
Total gene
The no. of CDS
DNA GC content (%)
a3MP-14

S. mimosae
3MP-10T

VDLZ00000000
7,197,316
300X
38
6,145
6,077
73.4

VDLY00000000
7,198,341
300X
45
6,149
6,081
73.4

a

S. triticirhizae
DSM 107172TaS. zhaozhouensis
DSM 42101T

S. sediJCM 16909T

RFFJ00000000
6,563,286
252X
428
6,029
5,696
73.3

OCNE00000000
6,532,426
143X
64
5,625
5,559
73.4

VDGT00000000
6,603,797
300X
51
5,598
5,534
72.9

Data from GenBank.

similarity to ikarugamycin; 13% similarity to maklamicin; and 14% similarity to versipelostatin. Nevertheless, several key genes in the metabolic pathways
were absent; it is ambiguous to investigate the eventual
function of each biosynthetic gene cluster in synthesizing each secondary metabolite. The BGCs with alow percentage of similarity to known clusters may
not represent functional gene clusters, or they may
be complemented by genes in other regions. Several
genes exhibited minimal similarity to known BGCs;
some may even be part of other gene clusters dueto their composition of one or two genes. Further
investigation is required to explain the role of thesepathways.
PC screening for secondary metabolites of strain
3MP-14The culture broth of strain 3MP-14 showed antibacterial activity against S. aureus ATCC 25923, K. rhizophila
ATCC 9341, and B. subtilis ATCC 6633. Therefore, it
is suggested that antimicrobial agents were includedin the culture broth of strain 3MP-14. The culture
broth of strain 3MP-14 was added to equal volumesof ethanol and analyzed by LC/UV and LC/MS. The
Mass and UV spectra of each peak were compared withthose of known compounds using existing databases
such as the Dictionary of Natural Products (http://dnp.
chemnetbase.com) as well as in-house databases. One
new polycyclic aromatic compound, named pudicin,
together with two known compounds, fumaquinone
and ikarugamycin were found (Fig. S3). Fumaquinone(m/z 303.1223 [M+H]+ calculated value for C17 H19 O5
303.1227) exhibited maximal UV absorbance at 220,
265, 304, and 428 nm. Ikarugamycin (m/z 479.2912
calculated value for C29 H39 O4 N2 479.2910) exhibited
maximal UV at 224 and 326 nm. Ikarugamycin andfumaquinone are known for their wide range of antimicrobials [31, 32].
Structure elucidation of a new compound, pudicin
Pudicin was isolated as a pale yellow amorphoussolid; [α]30
D −114.1° (c = 0.1, MeOH), IR (KBr) νmax
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Table 3 Potential BGCs in the genome of strain 3MP-14.
No.

Type

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

T1PKS
hgIE-KST2PKS, T1PKS, PKS-like
TerpeneBacteriocin
Siderophore, T1PKSLAP, thiopeptide
Terpene
T3PKS
CDPS
TerpeneLanthipeptide
Butyrolactone
Butyrolactone, PKS-like
NRPS, Butyrolactone
T1PKS, NRPS
TerpeneTerpene
T1PKS
T3PKS, otherSiderophore
Ectoine
T1PKS
T1PKS

a

Length (bp)36,422
46,13872,486
21,218
10,84990,23424,706
21,841
29,74020,822
22,40637,024
9,942
31,64660,372
49,372
19,277
21,22133,73543,4408,791
10,396
12,066
10,531

Similar BGC (gene no.)Similarity (%)aMaklamicin–
Polyketomycin
Chlortetracycline–
Tirandamycin–
HopeneNaringenin
Marinophenazine
Geosmin
Cahuitamycins
Murayaquinone
Lactonamycin
Azicemicin
Ikarugamycin
Isorenieratene
Isorenieratene
Meoabyssomicin/Abyssomicin
Furaquinocin
Desferrioxamine
EctoineLaidlomycin
Versipelostatin

13
–
83
8
–
33
–
30
100
7
100
8
3
3
8
12
42
25
25
34
50
100
28
14

The percentage of genes in reference BGC showing similarity to the BGC, which is calculated by antiSMASH.

Fig. 3 1H−1 H COSY (bold) and selected HMBC (arrow)correlations of pudicin.

cm−1 : 3350, 2908, 2873, 2850, 1715, 1647, 1614,
1566, 1454, 1376, 1342, 1308, 1239, 1214, 1123;
UV λmax nm ("): 204 (680,000), 246 (1,190,000),
344 (640,000); 1H and 13C NMR data are shownin Table 3. The HRESI-TOF-MS of pudicin produced
at m/z 600.2576 [M+Na]+ (calculated value for
C30 H43 NO8 SNa: m/z 600.2607) indicated the molecular formula was C30 H43 NO8 S. The absorption maxima
of the UV spectrum were observed at 211, 248, and342 nm. Analysis of the 1H NMR (Table 4 and Fig. S4)
and HSQC data (Fig. S5) revealed 4 methyls at δH
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0.90 (H-20), 1.13 (H-19), 1.18 (H-17), and 1.50 (H18), an acetyl group at δH 2.00 (H-50 ), 6 methylenesat δH 0.89, 1.81 (H-7), 0.90, 1.76 (H-5), 1.29, 1.87
(H-4), 1.32, 1.79 (H-15), 2.90, 3.08 (H-30 ), and 2.96
(H-14), 5 methyne protons at δH 1.46 (H-6), 1.83
(H-7a), 1.95 (H-9a), 2.39 (H-3), and 2.81 (H-12),
an oxymethyne proton at 3.81 (H-16), a heteroatom
bonded proton at δH 4.59 (H-20 ), and 3 olefinic protons
at δH 5.72 (H-8), 5.74 (H-9), 5.86 (H-11). The 13C
NMR (Table 4 and Fig. S6) and HSQC data showed
the presence of 30 carbons classified into a ketone
group at δC 205.2 (C-1), 2 carbonyl carbons at δC
173.3 (C-40 ) and 173.8 (C-10 ), 3 sp2 carbons including
an oxygenated carbon at δC 104.8 (C-1a), 137.8 (C10a), and 183.1 (C-2), 3 sp2 methine carbons at δC
117.8 (C-11), δC 123.5 (C-9), and δC 136.4 (C-8), 6
sp3 methylene carbons at δC 42.7 (C-7), 41.8 (C-14),
40.1 (C-15), 37.1 (C-5), 36.0 (C-30 ), and 29.1 (C-4),
3 sp3 quaternary carbons at 79.0 (C-13), 71.2 (C-10),
and 43.2 (C-2a), 7 sp3 methyne carbons including 2
heteroatom bonded methyne carbons at δC 67.7 (C16), 54.3 (C-20 ), 52.3 (C-9a), 42.6 (C-3), 40.6 (C-12),
39.9 (C-7a), and 34.2 (C-6), and 5 sp3 methyl carbons
including an acetyl carbon atδC 25.0 (C-18), 22.9 (C20), 22.5 (C-50 ), 23.3 (C-17), and 19.3 (C-19). The
gross structure of pudicin was confirmed by analysesof two-dimensional (2D) NMR data, including 1H−1 H
Correlation Spectroscopy (COSY) and Heteronuclear
Multiple Bond Correlation (HMBC) spectra in CD3 OD(Fig. S7 and Fig. S8). The 1H−1 H COSY data indicated667

ScienceAsia 49 (2023)
Table 4 1H and 13C NMR chemical shifts of pudicin.

partial structure d and the HMBC correlations from H20 to C-40 , from H-30 to C-10 , and from H-50 to C-40
revealed N -acetylcysteine moiety. The connectivity ofthis moiety at C-13 through C-14 was revealed by the
HMBC correlations from H-30 to C-14, from H-14 to C30 , C-1, C-12, and C-13, and from H-12 to C-14. The
HMBC correlations from H-11 to C-10, from H-11 andH-9 to C-2 (4 J HMBC), from H-18 to C-9a, C-10, andC-10a, from H-3 to C-19, and from H-19 to C-2, C-2a,and C-3 revealed the connectivity of 2 partial structures, 5-(2-hydroxypropyl)cyclohex-3-en-1-one frameand cyclohexene, through 4,6-dimethylcyclohex-1-en1-ol frame. Finally, 4 hydroxy groups were attachedat C-2, C-10, C-13, and C-16 by accounting for themolecular formula and chemical shifts. The central
part of this structure was also supported by the dataof 1D and 2D NMR measured in CDCl3 (Fig. S10 toFig. S13). As shown in Fig. S10, 1H NMR showed a
downfield proton signal of 2-OH (14.9 ppm). The
HMBC correlations (Fig. S14) from 2-OH to C-1a, C2, and C2a supported this structure. Thus, the planar
structure was determined, as shown in Fig. 3.
Screening for antimicrobial activity
Pudicin showed no antimicrobial activity against S. aureus ATCC 25923, K. rhizophila ATCC 9341, B. subtilis
ATCC 6633, E. coli ATCC 25922, P. aeruginosa ATCC
27853, and C. albicans ATCC 10231. The culture brothof strain 3MP-14 showed antibacterial activity against
S. aureus ATCC 25923, K. rhizophila ATCC 9341, andB. subtilis ATCC 6633.
In vitro antioxidant activity of pudicin

Recorded for 1H NMR at 600 MHz and for 13C NMR at
150 MHz in methanol-d4 (CD3 OD).

partial structures a (C-3–C-6 and C3–C-7a), b (C-8–C9a), c (C-11–C-12, C-12–C-15 and C-15–C-17), and d(C-20 –C-30 ) as shown in Fig. 3. The partial structurea and the HMBC correlations from H-3 to C-7, from
H-7 to C-3 and C-5, and from H-20 to C-5, C-6, andC-7 revealed methylcyclohexane frame. The partial
structure b and the HMBC correlations from H-8 to
C-3, C-7a, and C-9a, from H-9 to C-2a and C-7a,
and from H-9a to C-8 revealed cyclohexene frame,
confirming the connectivity of cyclohexene frame andmethylcyclohexane frame at C-3 and C-7a. This bicyclic structure was also supported by the TOCSY
correlations (Fig. S9). The partial structure c and the
HMBC correlations from H-11 to C-1a and C-13, from
H-12 to C-1, C-10a, and C-13, and from H-15 to C13 revealed 5-propylcyclohex-3-en-1-one frame. The

For the DPPH assay, 500 µg/ml of pudicin showed
57.38 ± 3.88% DPPH free radical scavenging activity
compared to standard ascorbic acid, which showed
89.65 ± 0.05% at 16 µg/ml concentration. The IC50
value of pudicin was 394.89 ± 3.88 µg/ml. For the
FRAP assay, the amount of reduced iron was correlatedwith the antioxidant capacity of pudicin. The activity
was reported in terms of ascorbic acid equivalents(µM). The 100 µg/ml of pudicin showed slight antioxidant activity as 0.02 µM of ascorbic acid equivalents.
Based on polyphasic studies, dDDH, and ANI, thisstrain was identified as Streptomyces mimosae [8, 19].
Twenty-four putative secondary metabolite BGCs werealso predicted in the draft genome of strain 3MP14 [25]. The related biosynthetic secondary metabolite genes have been emphasized as PKS type I, II,
III, and NRPS. The result of PC screening suggested
that strain 3MP-14 produced ikarugamycin and fumaquinone (Fig. S4). Despite no fumaquinone in
the BGCs, the BGCs of ikarugamycin were detected
as 12% similarity in the genome of strain 3MP-14.
These data strongly support the identification of aknown compound. Ikarugamycin has been reported
in Streptomyces sp. No. 8603 [32], an endophyticwww.scienceasia.org

668
Streptomyces harbinensis NEAU-Da3T [33], and Streptomyces zhaozhouensis CA-185989 [34]. It was apolycyclic tetramate macrolactam (PTMs) that exhibited antitumor, antibacterial, and antifungal activities[33–36], while fumaquinone, a prenylated naphthoquinone antibiotic, was produced by Streptomyces fumanus LL-F42248 and showed antimicrobial activity
against Gram-positive bacteria [37].
Upon analyzing the genome of S. mimosae
3MP-14 and 3MP-10T , it was discovered that theikarugamycin BGC was present. Similarly, the genomeof S. triticirhizae DSM 107172T also contained theikarugamycin BGC. Thus, by comparing the genome
analyses of strains 3MP-14, 3MP-10T , and DSM
107172T , it can be concluded that the ikarugamycin
BGCs are present in all 3 strains. This indicates that theikarugamycin biosynthetic gene cluster is conserved
within this phylogenetic cluster. As previously reported, the known piericidin A1 could be estimatedfrom BCGs of S. chumphonensis KK1-2T [38] but wasabsent in closely related strains unrelated to the species
within the phylogenetic cluster. Therefore, combining
chemical and in silico analysis of attractive compounds
is a potential approach.
In addition to antioxidant activity, the DPPH assay of pudicin showed slight free radical scavenging
activity at 50–500 µg/ml compared to ascorbic acid.
Therefore, our strain could be a source of antioxidants, as reported in Streptomyces sp. AM-S1 [39]and a mangrove-derived Streptomyces sp. MUM212[40]. Based on secondary metabolite BGC analysis, ectoine (C6 H10 N2 O2 ) and naringenin (C15 H12 O5 )showed 100% BGC similarity; however, these secondary metabolites were not produced by strain 3MP14 (Table 4). Nevertheless, some peaks could not
be identified as known compounds in the LC chart(Fig. S4) from the PC properties and genome. Therefore, some new compounds may be left in the culture
broth of strain 3MP-14. Combination of PC screening
and BGC analysis could effectively search for newcompounds.

CONCLUSION
Streptomyces mimosae 3MP-14 isolated from the rootof Mimosa pudica was characterized and identified
based on polyphasic approaches, the 16S rRNA gene,and genome sequence analysis. Two known compounds, ikarugamycin and fumaquinone, and a newcompound, pudicin, were discovered from the culture
broth of strain 3MP-14 by PC screening. Pudicin
has a unique structure with a 4-membered ring and
N -acetylcysteine moiety. It showed the presence ofantioxidants in the DPPH assay. Twenty-four putative
secondary metabolite BGCs were identified from thedraft genome analysis of the strain.
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ScienceAsia 49 (2023)

Appendix A. Supplementary data
Supplementary data associated with this article can befound at http://dx.doi.org/10.2306/scienceasia1513-1874.
2023.067.

Acknowledgements: This study was supported by the Thailand Research Fund for a 2017 Royal Golden Jubilee Ph.D.
Program as a scholarship to NK, the Institute for Fermentation, Osaka (IFO), Japan, and the Grant for International
Research Integration, Research Pyramid, Ratchadaphiseksomphot Endowment Fund (CUGRP_61_01_33_01), Chulalongkorn University. We thank the Pharmaceutical ResearchInstrument Center, Faculty of Pharmaceutical Sciences, Chulalongkorn University for providing research facilities.

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Appendix A. Supplementary data
Table S1 Cultural characteristics of strain 3MP-14.
MediumCharacteristic

3MP-143MP-10T

ISP 2

GrowthArial mycelium
Substrate mycelium
Soluble pigment

Moderate
Pale yellow
Vivid yellowish green
None

Moderate
Pale yellow
Vivid yellowish green
None

ISP 3

GrowthArial mycelium
Substrate mycelium
Soluble pigment

Moderate
White
Pale yellowish
None

Moderate
White
Pale yellowish
None

ISP 4

GrowthArial mycelium
Substrate mycelium
Soluble pigment

Good
White
Pale yellowish
None

Good
White
Pale yellowish
None

ISP 5

GrowthArial mycelium
Substrate mycelium
Soluble pigment

Poor
Pale yellow
Light yellow green
None

Poor
Pale yellow
Light yellow green
None

ISP 6

GrowthArial mycelium
Substrate mycelium
Soluble pigment

Poor
Pale yellowish
yellow
None

Poor
Pale yellowish
Pale yellow
None

ISP 7

GrowthArial mycelium
Substrate mycelium
Soluble pigment

Poor
Pale yellow
yellow
None

Poor
Pale yellow
yellow
None

NA

GrowthArial mycelium
Substrate mycelium
Soluble pigment

Good
Pale yellow
Pale yellowish
None

Good
Pale yellow
Pale yellowish
None

Table S2 Differential characteristics of strain 3MP-14 and related type strains. Strains: 1, 3MP-14; 2, S. mimosae 3MP-10T ; 3,S. triticirhizae DSM 107172T ; 4, S. zhaozhouensis DSM 42101T ; and 5, S. sedi JCM 16909T . +, positive; w, weakly positive;−, negative. Data are from this study, except DSM 107172T was from https://bacdive.dsmz.de/strain/159640 (accessed date:
March 30,2023).
Characteristic

1

2

3

4

5

Enzyme activity
Esterase (C4)
Leucine arylamidase
Valine arylamidase
Cystine arylamidase
Trypsinα-Chymotrypsin
Alkaline phosphatase
Acid phosphatase
α-Fucosidase
α-Galactosidase
β-Galactosidase
α-Glucosidase
β-Glucosidase
α-Glucosaminidase
α-Mannosidase

+
+
+
w
+
w
w
+
+
+
+
+
+
+
+

+
+
+
+
+
+
w
+
w
+
+
+
+
+
+

+
+
+
+
+
w
+
+
+
+
+
+
+
+
+

+
w
+
+
−
+
−
+
−
−
−
+
+
−
−

−
+
+
w
−
−
−
−
−
w
+
−
−
w
w

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Table S3 Cellular fatty acid compositions (%) of strain 3MP-14 and related type strains. Strains: 1, 3MP-14; 2, S. mimosae
3MP-10T ; 3, S. triticirhizae DSM 107172T ; 4, S. zhaozhouensis DSM 42101T ; and 5, S. sedi JCM 16909T . Data are from thisstudy, except strain DSM 107172T [30].
Fatty acid

1

2

3

4

5

Saturated fatty acid
C16:0
C17:0
C17:0 cyclo
C18:0

2.0
2.0
1.4
–

5.8
–
–
2.0

14.2
8.1
7.6
–

6.7
1.2
–
1.7

4.3
2.2
1.7
–

Unsaturated fatty acidC17:1 ω8c
C18:1 ω9c

–
–

–
–

–
2.4

3.4
1.8

3.7
–

Branched fatty acidi-C10:0
i-C14:0
i-C15:0
a-C15:0
i-C16:0
i-C17:0
i-C17:0 3OH
a-C17:0
i-C18:0
3OH-C8:0

4.5
–
2.6
2.8
50.8
1.7
1.8
7.2
1.2
1.7

–
1.9
4.1
21.8
36.2
3.5
–
13.0
1.0
–

–
–
–
7.3
22.0
5.2
–
17.4
–
–

–
–
–
3.1
31.6
–
–
14.4
–
–

–
–
1.3
2.4
45.9
1.8
–
14.6
1.9
–

Unsaturated Brancheda-C15:1 A
i-C16:1 G
a-C17:1 ω9c
i-C18:1 H
Summed feature 3a
Summed feature 5b
Summed feature 8c
Summed feature 9d

–
7.7
5.3
–
–
–
–
3.2

–
–
3.4
–
–
1.3
–
1.9

–
–
–
–
5.1
–
0.6
–

1.2
10.0
8.9
1.2
6.6
–
1.5
1.2

–
6.1
5.0
–
1.8
–
–
2.5

–, not detected. a C16:1 ω7c/C16:1 ω6c; b anteiso-C18:0 /C18:2 ω6,9c; c C18:1 ω7c; d 10Me-C16:0 . Strain DSM 107172T
contained other C17:1 ω7c (5.6%), C15:0 (2.2%), and C18:1 ω8c (1.1%).

Table S4 ANIb and ANIm values (%) and the digital (in silico) DNA-DNA hybridization (dDDH) values between the draft
genome of strain 3MP-14 and related type strains. Draft genomes: 1, Strain 3MP-14; 2, S. mimosae 3MP-10T ; 3, S. triticirhizae
DSM 107172T ; 4, S. zhaozhouensis DSM 42101T ; and 5, S. sedi JCM 16909T .
QuerygenomeReference
genomeANIb

ANIm% dDDH(Formula 2)*

Model C.I.

Distance

Prob.
DDH ¾70%

G+C
difference

2
3
4
5

100
94.49
84.88
84.74

100
94.62
88.01
87.92

100
55.7
29.9
29.6

100–100%
52.9–58.4%
27.5–32.4%
27.2–32.1%

0.00000.05950.14290.1443

98.30
37.41
0.10
0.09

0.00
0.10
0.02
0.48

1
1
1
1
*

Recommended formula (identities/HSP length), which is liberated of genome length and is thus prosperous against theuse of incomplete draft genome.

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S3

Fig. S1 Polar lipid profiles of strain 3MP-14 based on two-dimensional thin layer chromatograms detected with anisaldehyde,
phosphomolybdic acid, ninhydrin, Drangendorff, and Molybdenum blue as spraying reagent. Solvent system I; Chloroform:Methanol:Water (65:25:4; %, v/v). Solvent system II; Chloroform:Acetic acid:Methanol:Water (80:15:12:4; %, v/v). PE,
phosphatidylethanolamine; DPG, diphosphatidylglycerol; PI, phosphatidylinositol; PG, phosphatidylglycerol; PL, unidentified
phospholipid; GL, unidentified glycolipid; and PGL, unidentified phosphoglycolipid.

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Fig. S2 Neighbor-joining phylogenetic tree based on almost complete 16S rRNA gene (1,457 nt) showing the relationshipbetween strain 3MP-14 and Streptomyces species. Micrococcus antarcticus JCM 11467T was used as an outgroup. The number
at node showed the level of bootstrap support for branched points, based on 1000 resampling (values ¾ 50 are shown).
Asterisk (*) and # indicated the branches were recovered in the maximum-likelihood and maximum-parsimony, respectively.

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S5

Fig. S3 LC-UV/MS analysis of the culture broth extracted with MeOH of strain 3MP-14.

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S6Fig. S4 1H NMR spectrum of pudicin (600 MHz, CD3 OD).

Fig. S5 13C NMR spectrum of pudicin (150 MHz, CD3 OD).

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S7

Fig. S6 HSQC spectrum of pudicin (600 MHz, CD3 OD).

Fig. S7 1H−1 H COSY spectrum of pudicin (600 MHz, CD3 OD).

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S8Fig. S8 HMBC spectrum of pudicin (600 MHz, CD3 OD).

Fig. S9 TOCSY spectrum of pudicin (600 MHz, CD3 OD).

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S9

Fig. S10 1H NMR spectrum of pudicin (600 MHz, CDCl3 ).

Fig. S11 13C NMR spectrum of pudicin (150 MHz, CDCl3 ).

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S10Fig. S12 HSQC spectrum of pudicin (600 MHz, CDCl3 ).

Fig. S13 HMBC spectrum of pudicin (600 MHz, CDCl3 ).

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S11

Fig. S14 Partial 1H and 13C NMR chemical shifts and HMBC correlations of pudicin (600 MHz, CDCl3 ).

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