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Streptomyces monashensis sp.
nov., a novel mangrove soil
actinobacterium from East
Malaysia with antioxidative
potential
Jodi Woan-Fei Law1, Hooi-Leng Ser1,2,3, Nurul-Syakima Ab Mutalib
4, Surasak Saokaew
1,5,6,
Acharaporn Duangjai1,5,7, Tahir Mehmood Khan2,8, Kok-Gan Chan
9,10, Bey-Hing Goh2,3,5 &
Learn-Han Lee1,3,5
A new Streptomyces species discovered from Sarawak mangrove soil is described, with the proposed
name – Streptomyces monashensis sp. nov. (strain MUSC 1JT). Taxonomy status of MUSC 1JT was
determined via polyphasic approach. Phylogenetic and chemotaxonomic properties of strain MUSC
1JT were in accordance with those known for genus Streptomyces. Based on phylogenetic analyses, the
strains closely related to MUSC 1JT were Streptomyces corchorusii DSM 40340T (98.7%), Streptomyces
olivaceoviridis NBRC 13066T (98.7%), Streptomyces canarius NBRC 13431T (98.6%) and Streptomyces
coacervatus AS-0823T (98.4%). Outcomes of DNA–DNA relatedness between strain MUSC 1JT and its
closely related type strains covered from 19.7 ± 2.8% to 49.1 ± 4.3%. Strain MUSC 1JT has genome size
of 10,254,857 bp with DNA G + C content of 71 mol%. MUSC 1JT extract exhibited strong antioxidative
activity up to 83.80 ± 4.80% in the SOD assay, with signicant cytotoxic eect against colon cancer cell
lines HCT-116 and SW480. Streptomyces monashensis MUSC 1JT (=DSM 103626T = MCCC 1K03219T)
could potentially be a producer of novel bioactive metabolites; hence discovery of this new species
may be highly signicant to the biopharmaceutical industry as it could lead to development of new and
useful chemo-preventive drugs.
Natural products play an important part in the development of drugs as they have been the source of many of
the active ingredients of medicines1. Microbes have been extensively explored as sources for bioactive natural
products due to their production of unique secondary metabolites which are required for defense and survival
in harsh environments2. Members of the phylum Actinobacteria have been one of the primary sources of bioac-
tive natural products, owing to their capability to produce abundant secondary metabolites comprising diverse
chemical structures and biological activities3. In particular, the genus Streptomyces has brought upon a benecial
1Novel Bacteria and Drug Discovery Research Group, Microbiome and Bioresource Research Strength, Jerey Cheah
School of Medicine and Health Sciences, Monash University Malaysia, 47500, Bandar Sunway, Selangor Darul Ehsan,
Malaysia. 2Biofunctional Molecule Exploratory Research Group, Biomedicine Research Advancement Centre, School
of Pharmacy, Monash University Malaysia, 47500, Bandar Sunway, Selangor Darul Ehsan, Malaysia. 3Institute of
Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou, 510006, P. R. China.
4UKM Medical Molecular Biology Institute (UMBI), UKM Medical Centre, University Kebangsaan Malaysia, Kuala
Lumpur, Malaysia. 5Center of Health Outcomes Research and Therapeutic Safety (Cohorts), School of Pharmaceutical
Sciences, University of Phayao, Phayao, Thailand. 6Pharmaceutical Outcomes Research Center (CPOR), Faculty of
Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand. 7Division of Physiology, School of Medical
Sciences, University of Phayao, Phayao, Thailand. 8The Institute of Pharmaceutical Sciences, University of Veterinary
and Animal Sciences, Lahore, Pakistan. 9Division of Genetics and Molecular Biology, Institute of Biological Sciences,
Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia. 10International Genome Centre, Jiangsu
University, Zhenjiang, China. Correspondence and requests for materials should be addressed to K.-G.C. (email:
kokgan@um.edu.my) or B.-H.G. (email: goh.bey.hing@monash.edu) or L.-H.L. (email: lee.learn.han@monash.edu)
Received: 13 September 2018
Accepted: 21 January 2019
Published online: 28 February 2019
OPEN
Corrected: Author Correction
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impact to the pharmaceutical industry by accounting for approximately 80% of the Actinobacteria derived natural
products3–6.
In the early 1940 s, Professor Dr. Waksman and Professor Dr. Henrici7 proposed the genus Streptomyces
comprising Gram positive lamentous bacteria that are well-known as prolic producers of numerous com-
pounds with various bioactivities including antibacterial, antifungal, antioxidant, anticancer, and immunosup-
pression5,8–10. us far, the exploration of new taxa is one of the successful approaches to uncover new chemical
scaolds or therapeutic agents11. Interest in the benecial properties of Streptomyces has led to eorts to explore
these organisms found in a variety of habitats such as terrestrial, marine, desert, and plants - resulting in about
844 validly identied species to date (http://www.bacterio.cict.fr/)12,13. Recently, there has been increasing scien-
tic interest in the discovery of novel Streptomyces from underexplored area such as the mangrove environment,
in hopes that this could lead to the extraction of new and useful compounds from these novel species14,15. In
fact, mangrove environments are currently considered one of the best marine resources for the isolation of novel
Streptomyces16.
Globally, the largest percentage distribution of mangrove forests of 42% is found to be in Asia, followed by
20% in Africa, 15% in North and Central America, 12% in Oceania, and 11% South America17. Malaysia is among
the most mangrove-rich country in Asia with the state of Sarawak being an area which has abundant mangrove
forests that are mostly remained undisturbed18. Mangrove environments are unique and dynamic as they are
mainly situated in the intertidal zones of tropical and subtropical coastal regions19. Furthermore, a variety of ter-
restrial, freshwater, and marine organisms inhabit the mangrove forests20. Mangroves are vastly rich in nutrient
and organic matter resulting from countless microbial enzymatic and metabolic activities21. In addition, man-
grove environments experience alterations in salinity and tidal gradient constantly. All these factors will eventu-
ally assist in the rapid development of species diversity which occurs as a reaction to environmental variations
and triggers metabolic pathway adaptations in living organisms which could result in generation of imperative
metabolites3,21. Hence, these reasons have essentially driven the investigation of Streptomyces population present
in Sarawak mangrove forests which then created a chance for novel species discovery.
Mangrove derived Streptomyces are a valuable source of bioactive secondary metabolites22. e production of
secondary metabolites by Streptomyces oen occurs when environmental stresses are present, such as, presence
of competing microorganisms or nutrient depletion23. Upon exposure to stressful conditions like depletion of
nutrients, Streptomyces bacteria undergo complex morphological changes, during which they initially develop a
network of branched laments known as the substrate mycelium (vegetative phase) and subsequently form aerial
multinucleated mycelium and spores (reproductive sporulation phase)23,24. During this shiing phase, many
interesting secondary metabolites are produced to ensure the survival of Streptomyces under stressful or unfa-
vorable environments24. Additionally, Streptomyces have a large genome of approximately 8–10 Mbp containing
more than 20 biosynthetic gene clusters that encode enzymes for the biosynthesis of secondary metabolites25.
Aside from ensuring the survival of the organism, this unique characteristic of Streptomyces hints at the capability
to produce novel bioactive secondary metabolites. e bioactive secondary metabolites produced by Streptomyces
are structurally diverse26; the commonly found compounds include polyketides, peptides, pyrroles, β-lactams,
and terpenes23,24. Many novel bioactive compounds have been discovered from mangrove derived Streptomyces
including: (1) chalcomycin B, a novel macrolide antibiotic isolated from Streptomyces sp. B706427; (2) xiamycin A,
a novel pentacyclic indolosesquiterpene with anti-HIV activity isolated from Streptomyces sp. GT2002150328; (3)
balomycin K, a novel antifungal macrolide isolated from Streptomyces avotricini Y12-2629; and (4) streptocar-
bazoles A and B, novel indolocarbazoles with cytotoxic activity isolated from Streptomyces sp. FMA30.
Also, there is increasing evidence that novel Streptomyces from the mangrove are valuable sources of antioxi-
dant and anticancer compounds. A study conducted by Hong et al.31 found that new species Streptomyces isolate
162227 and 0614149 isolated from mangrove sites in China were capable of inhibiting Human Colon Tumor 116
cells. In Malaysia, a number of novel Streptomyces strains have been identied from mangrove environments.
For instance, Streptomyces pluripotens20, Streptomyces mangrovisoli32, Streptomyces humi33, Streptomyces antioxi-
dans15, Streptomyces malaysiense14, and Streptomyces colonosanans5. Some of these novel mangrove Streptomyces
have been associated with potential antioxidant and anticancer activities, for example, Streptomyces mangrovisoli
exhibited strong antioxidant activity and the antioxidant agent was identied as Pyrrolo[1,2-a]pyrazine-1,4-dione,
hexahydro-32. Streptomyces malaysiense and Streptomyces colonosanans were reported to exhibit strong antioxi-
dant activity as well as demonstrating cytotoxicity against colon cancer cell lines5,14.
Oxidative stress is a condition where there is a cumulative production of oxygen free radicals through either
endogenous or exogenous insults along with insucient antioxidant defense, and has been associated with car-
cinogenesis8,34. e accumulation of free radicals may cause modication or damage to vital biological macromol-
ecules such as lipids, proteins, and DNA. As a result, DNA mutations might occur which could increase cancer
risk32,34. Antioxidants play a vital role in biological systems by scavenging the excessive free radicals in order to
prevent the harmful eects caused by oxidative stress5. Given that cancer is a major public health issue, scientists
are actively searching for eective cancer treatment options which include the discovery of potent natural antiox-
idant and anticancer agents from microbial sources5,35,36. Streptomyces is proven to be a good source of anticancer
drugs; a number of anticancer drugs currently in use have been derived from Streptomyces such as bleomycin,
dactinomycin, mitomycin C, and doxorubicin37–40. Hence, this triggered our interest to look into the potential
antioxidant and anticancer activities of Sarawak mangrove-derived Streptomyces.
is study was conducted to investigate novel Streptomyces strains isolated from mangrove soil sampled at
Sarawak, East Malaysia. Strain MUSC 1JT was recovered from one of the soil samples and polyphasic approach
based on genotypic, chemotaxonomic and phenotypic features veried that it is a novel Streptomyces species.
Whole genome of strain MUSC 1JT was analyzed via next generation sequencing technique. is study further
explored the antioxidant and cytotoxic potentials of the extract of this bacterium. With the application of gas
chromatography-mass spectrometry (GC-MS), the active compounds present in the extract that were accountable
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for the observed bioactivities were identied. e outcome of current research provides an in depth understand-
ing of Streptomyces monashensis sp. nov. MUSC 1JT from dierent perspectives and also demonstrates the poten-
tial of this strain in producing bioactive compounds with antioxidant and cytotoxic activities.
Results
Genotypic, phylogenetic, and genomic analyses of strain MUSC 1JT. e nearly full-length 16S
rRNA gene sequence was attained for strain MUSC 1JT (1490 bp; GenBank/EMBL/DDBJ accession number
KP998432). Based on the 16S rRNA sequences, phylogenetic trees were reconstructed to determine the evolu-
tionary relationship of this strain with its related type strains (Figs1, S1 and S2). Results were in agreement that
the most closely related strain is S. coacervatus AS-0823T (98.4% sequence similarity) with shortest evolution-
ary distance, as they formed distinct clade at bootstrap value of ≥50% in the neighbour-joining (Fig.1), maxi-
mum-likelihood (Fig.S1), and maximum-parsimony (Fig.S2) phylogenetic trees. e 16S rRNA gene sequence
analysis for strain MUSC 1JT revealed that this strain exhibited the highest similarity to strain S. corchorusii DSM
40340T (98.7%), S. olivaceoviridis NBRC 13066T (98.7%), and S. canarius NBRC 13431T (98.6%).
Furthermore, the results of DDH revealed that the DNA–DNA relatedness levels between strain MUSC 1JT
and S. corchorusii JCM 4467T (34.8 ± 3.3%), S. olivaceoviridis JCM 4499T (49.1 ± 4.3%), S. canarius JCM 4549T
(19.7 ± 2.8%) and S. coacervatus JCM 17318T (21.1 ± 3.2%) were signicantly below 70%–recommended cut-o
point for the delineation of bacterial species41. Besides, strain MUSC 1JT yielded a distinctive BOX-PCR n-
gerprint which can be dierentiated from its closely related type strains (Supplementary Fig.S3). e results of
phylogenetic analysis, DDH, and BOX-PCR ngerprint analysis were consistent and thus supported that strain
MUSC 1JT represents a novel species of Streptomyces genus.
In addition, the whole genome sequencing showed that the genome of strain MUSC 1JT consists of
10,254,857 bp with average coverage of 170.0-fold (Table1). e whole project of strain MUSC 1JT was deposited
at DDBJ/EMBL/GenBank under accession number MLYO00000000 and the version described in this paper is the
rst version (MLYO0100000). A total of 9,310 coding genes was predicted on MUSC 1JT genome, which assigned
to 445 subsystems, along with 68 tRNA and 4 rRNA genes. Based on RAST annotation, the majority of the genes
are involved in amino acid and derivative metabolism (8.06%), carbohydrate metabolism (7.45%), followed by
cofactor, vitamin, prosthetic group, and pigment metabolism (4.19%).
Whole genome comparisons between strain MUSC 1JT and its closely related type strain S. corchorusii DSM
40340T was also performed. Analysis based on Clusters of Orthologous Groups (COG) functional categories
showed that similar distribution of genes between strain MUSC 1JT and S. corchorusii DSM 40340T; highest num-
ber of known proteins were found to be involved in essential processes like transcription (Class K) followed by
carbohydrate transport and metabolism (Class G) (aer removing uninformative classes such as R and S in the
analysis) (Table2). Further analysis using Artermis Comparison Tool (ACT)42 which uses BLAST to compare
two or more genomes revealed large amount of synteny exists between strain MUSC 1JT and S. corchorusii DSM
40340T (Fig.2). Nonetheless, the ANI value comparing strain MUSC 1JT and S. corchorusii DSM 40340T was cal-
culated to be 86.03%. ANI has become increasingly popular due to the availability of whole genome sequences.
e ANI analysis is primarily done by computation comparisons of two genome sequences to determine the
genetic relatedness between prokaryotic strains43. A report by Goris et al.44 has described that 95% ANI and 69%
conserved DNA corresponded with the cut-o point of 70% DDH for species delineation. e ANI value reected
by strain MUSC 1JT and type strain S. corchorusii DSM 40340T was found to be well below the recommended
value by Goris et al.44. is nding was also in line with the outcome of DDH analysis between strain MUSC 1JT
and S. corchorusii DSM 40340T (DNA-DNA relatedness of 34.8 ± 3.3%, <70%). Furthermore, additional analy-
ses of strain MUSC 1JT and its other closely related strains that possessed >98% 16S rRNA sequence similarity
have revealed ANI values between 82–87%, which falls signicantly below the recommended value (TableS1).
erefore, the novel status of the strain MUSC 1JT was further conrmed based on these extensive genomic
comparative analyses.
Apart from that, both of the genomes were also submitted to antiSMASH to detect presence of biosynthetic
gene clusters. From the analysis, more than 120 clusters were detected on strain MUSC 1JT genome related to var-
ious biosynthetic gene clusters including type-I polyketide synthetase, indole biosynthesis, and siderophores pro-
duction. One of the common biosynthetic gene clusters within strain MUSC 1JT and S. corchorusii was selected
for comparison – biosynthetic gene cluster related to production of desferrioxamine B. e gene clusters were
highly similar and pairwise comparison of the gene encoding for IucA/IucC family protein responsible for pro-
duction of desferrioxamine revealed that gene similarities of 88.29% (Fig.3)45. e presence of these biosynthetic
gene clusters indicates the bioactive potential of strain MUSC 1JT and suggesting its ability in producing such
valuable bioactive compounds.
Chemotaxonomic analyses of strain MUSC 1JT. e results of chemotaxonomic analyses revealed that
strain MUSC 1JT presented a type I cell-wall as it contains LL-diaminopimelic acid46, an amino acid found to be
present in many other species of the genus Streptomyces5,19,20,32,47–49. e predominant menaquinones of strain
MUSC 1JT were identied as MK-9(H8) (55%) and MK-9(H6) (16%). e detection of these predominant men-
aquinones is in agreement with the report of Kim et al.50. e whole cell sugars detected were glucose and ribose.
Strain MUSC 1JT has a G + C content of 71 mol% and it was in the range of 67.0–78.0 mol% as described for
Streptomyces50.
e fatty acid proles of strain MUSC 1JT and its closely related type strains are presented in Table3. e
major cellular fatty acids in strain MUSC 1JT were identied as anteiso-C15: 0 (19.3%), iso-C16: 0 (19.1%), iso-C15: 0
(13.0%), anteiso-C17: 0 (11.2%), and C16: 0 (10.8%). e fatty acid prole of strain MUSC 1JT displayed high levels
of similarities with those of closely related phylogenetic neighbors such as S. coacervatus JCM 17318T, S. olivace-
oviridis JCM 4499T and S. corchorusii JCM 4467T, as they also contain anteiso-C15: 0 (19.3–28.6%) as their major
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fatty acid (Table3). However, quantitative dierences can be observed in the fatty acid proles of strain MUSC
1JT and its closely related type strains; for example, anteiso-C15: 0 (19.3%) was found to be predominant in strain
MUSC 1JT (Table3), but the quantity of the same fatty acid was much higher in S. olivaceoviridis JCM 4499T
(28.6%). Polar lipids analysis revealed the presence of phospholipid, phosphatidylglycerol, phosphatidylinositol,
Figure 1. Neighbour-joining phylogenetic tree based on almost complete 16S rRNA gene sequences (1490
nucleotides) showing the relationship between Streptomyces monashensis MUSC 1JT and representatives of
some other related taxa. Numbers at nodes indicate percentages of 1000 bootstrap re-samplings, only values
above 50% are shown. Bar, 0.002 substitutions per site. Asterisks indicate that the corresponding nodes were
also recovered using the maximum-likelihood and maximum-parsimony tree-making algorithms.
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phosphoglycolipid, and diphosphatidylglycerol in strain MUSC 1JT (Fig.4). Outcomes of polar lipids analysis of
closely related type strains were included as supplementary information (Supplementary Fig.S4).
Phenotypic analyses of strain MUSC 1JT. Phenotypic analyses in this study revealed that the mangrove
forest soil-derived MUSC 1JT strain grows well on ISP 2, ISP 3, ISP 5, ISP 6, ISP 7, Streptomyces agar, and nutrient
agar aer 7–14 days at 28 °C; grows moderately on starch casein agar and actinomycetes isolation agar, and does not
grow on ISP 4. e colors of the aerial and substrate mycelium were media-dependent as shown in TableS2. Based
on the observation of 14-day-old culture grown on ISP 2 agar, the aerial and vegetative hyphae of strain MUSC 1JT
were abundant and well developed. ese morphological features of strain MUSC 1JT (Fig.5) conform to those
observed in genus Streptomyces, hence, this indicated that strain MUSC 1JT belongs to the genus Streptomyces51.
For the analysis of temperature, pH, and NaCl tolerance, the results indicated that growth was found to occur at
24–40 °C (optimum 28–32 °C), at pH 6.0–8.0 (optimum pH 7.0), and with 0–6% NaCl tolerance (optimum 0–2%).
Cells were found to be positive for catalase and hemolytic activity. Moreover, the cells were capable of hydrolyzing
soluble starch, carboxymethylcellulose, casein and tributyrin, but unable to hydrolyze chitin and xylan. In addition,
Streptomyces monashen sis MUSC 1JT
Genome size (bp) 10,254,857
Contigs 218
Contigs N50 (bp) 159,229
G + C content % 71
Protein coding genes 9,310
tRNA 68
rRNA 2 (5S), 1 (16S), 1 (23S)
Table 1. General features of Streptomyces monashensis MUSC 1JT genome.
Class
MUSC 1JTS. corchorusii
DescriptionCounts %Counts %
A 5 0.07 7 0.09 RNA processing and modication
B 1 0.01 1 0.01 Chromatin structure and dynamics
C 468 6.37 466 6.21 Energy production and conversion
D 58 0.79 51 0.68 Cell cycle control, cell division,
chromosome partitioning
E 574 7.81 591 7.88 Amino acid transport and metabolism
F 131 1.78 125 1.67 Nucleotide transport and metabolism
G 638 8.68 649 8.65 Carbohydrate transport and metabolism
H 291 3.96 287 3.83 Coenzyme transport and metabolism
I 360 4.90 371 4.95 Lipid transport and metabolism
J 229 3.12 235 3.13 Translation, ribosomal structure and
biogenesis
K 961 13.08 995 13.26 Transcription
L 252 3.43 240 3.20 Replication, recombination and repair
M 307 4.18 312 4.16 Cell wall/membrane/envelope biogenesis
N 7 0.10 11 0.15 Cell motility
O 200 2.72 201 2.68 Posttranslational modication, protein
turnover, chaperones
P 270 3.67 233 3.11 Inorganic ion transport and metabolism
Q 389 5.29 358 4.77 Secondary metabolites biosynthesis,
transport and catabolism
R 983 13.38 1100 14.66 General function prediction only
S 487 6.63 492 6.56 Function unknown
T 518 7.05 559 7.45 Signal transduction mechanisms
U 63 0.86 58 0.77 Intracellular tracking, secretion, and
vesicular transport
V 153 2.08 157 2.09 Defense mechanisms
W 1 0.01 1 0.01 Extracellular struc tures
Z 2 0.03 2 0.03 Cytoskeleton
Tot a l 7348 100 7502 100
Table 2. Comparison between MUSC 1JT and Streptomyces corchorusii DSM 40340T based on COG functional
categories.
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the phenotypic properties presented in Table4 demonstrated that strain MUSC 1JT was distinguishable from its
closely related members of the genus Streptomyces. e compounds listed are utilized as sole carbon sources by
MUSC 1JT: acetic acid, α-D-glucose, α-D-lactose, α-hydroxy-butyric acid, α-keto-butyric acid, α-keto-glutaric
acid, β-hydroxyl-D, L-butyric acid, β-methyl-D-glucoside, bromo-succinic acid, citric acid, D-arabitol, D-aspartic
acid, D-cellobiose, dextrin, D-fructose, D-fucose, D-galactose, D-galacturonic acid, D-glucose-6-phosphate,
D-gluconic acid, D-glucuronic acid, D-lactic acid methyl ester, D-malic acid, D-maltose, D-mannitol, D-mannose,
D-melibiose, D-ranose, D-saccharic acid, D-salicin, D-sorbitol, D-trehalose, D-turanose, formic acid, gelatin,
gentiobiose, glucuronamide, glycerol, glycyl-L-proline, inosine, L-fucose, L-galactonic acid lactone, L-lactic acid,
L-malic acid, L-rhamnose, methyl pyruvate, mucic acid, N-acetyl-β-D-mannosamine, N-acetyl-D-galactosamine,
N-acetyl-D-glucosamine, pectin, p-hydroxyl-phenylacetic acid, propionic acid, quinic acid, stachyose, sucrose,
Tween 40, γ-amino-butyric acid and myo-inositol. e following compounds are utilized as sole nitrogen sources
by MUSC 1JT: L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid and
L-serine. Results of chemical sensitivity assays revealed that cells are resistant to 1% sodium lactate, aztreonam,
nalidixic acid, potassium tellurite, rifamycin RV and sodium bromate. While the cells are sensitive to fusidic acid,
D-serine, guanine HCl, lincomycin, lithium chloride, minocycline, niaproof 4, sodium butyrate, tetrazolium blue,
tetrazolium violet, troleandomycin and vancomycin.
Results of genomic and phylogenetic analysis, chemotaxonomic and phenotypic analyses proven that strain
MUSC 1JT isolated from Sarawak mangrove soil is qualified to be assigned as a novel species in the genus
Streptomyces, for which the name Streptomyces monashensis sp. nov. is proposed.
Antioxidant activity of strain MUSC 1JT extract. In this study, the antioxidant potential of novel strain
MUSC 1JT was evaluated using SOD activity assay, ABTS assay, and metal chelating assay. Based on the results of
all the assays, the extract of strain MUSC 1JT exhibited signicant radical scavenging ability (Table5). e capa-
bility of strain MUSC 1JT extract to scavenge in vitro oxygen-derived species like superoxide anion (O2⋅−) was
analyzed via SOD activity assay, which utilizes the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H
-tetrazolium, monosodium salt (WST) reduction method. e superoxide anion radical in this assay is generated
through hypoxanthine-xanthine oxidase reaction, followed by the reduction of WST to WST-1 yellow formazan
by the superoxide radical5,8,52. Strain MUSC 1JT extract possesses SOD-like activity up to 83.80 ± 4.80% by vir-
tue of scavenging the superoxide anion radical and subsequently inhibiting the development of yellow WST-1
formazan. e extract exhibited signicant SOD-like activity (P < 0.05) ranging from 42.41 ± 1.58% (at 0.25 mg/
mL) to 83.80 ± 4.80% (at 2 mg/mL). In addition, antioxidant activity of strain MUSC 1JT extract was conrmed
by ABTS assay. e production of ABTS radical cation in this assay was initiated by the reaction between a strong
oxidizing agent potassium persulfate with ABTS salt53. e extract was able to scavenge the ABTS radical gener-
ated in the assay with signicant activity of 12.33 ± 3.07% at concentration of 2 mg/mL (Table5).
e ability of strain MUSC 1JT extract in exhibiting metal chelating activity further demonstrated its antioxi-
dant potential. In a metal chelating assay, the ferrozine added can quantitatively form complexes with Fe2+, result-
ing in a formation of Fe2+-ferrozine complex that can be disrupted in the presence of other chelating agents54. e
presence of strain MUSC 1JT extract exhibited a signicant metal chelating activity, with highest activity recorded
at 75.50 ± 1.44% at 2 mg/mL concentration (Table5). e antioxidative potential of MUSC 1JT extract is empha-
sized through its metal chelating ability by preventing transition metals from promoting the generation of ROS5,14.
Figure 2. Synteny map of Streptomyces monashensis MUSC 1JT (top) and Streptomyces corchorusii DSM 40340T
(bottom) genomes built using ACT.
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Cytotoxic activity of strain MUSC 1JT extract. Generally, strain MUSC 1JT extract showed promising
cytotoxic activity against the colon cancer cell lines tested. e results of strain MUSC 1JT extract tested against
the colon cancer cell lines were presented in Fig.5. Aer 72 hours of treatment with strain MUSC 1JT extract,
the results revealed that the extract had signicant cytotoxic eect against both colon cancer cell lines (P < 0.05)
(Fig.6). e extract demonstrated highest cytotoxicity against SW480, with cell viability of 81.7 ± 4.0% at the
highest tested extract concentration of 400 µg/mL. As for HCT-116 colon cancer cells, the extract exhibited cell
viability of 82.3 ± 5.3% at concentration of 400 µg/mL. Morphological studies were conducted using phase con-
trast microscopy to visualize the response of SW480 and HCT-116 cells aer treated with MUSC 1JT extract.
Figure 3. Biosynthetic gene clusters related to production of siderophore, desferrioxamine B for Streptomyces
monashensis MUSC 1JT and Streptomyces corchorusii DSM 40340T.
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Fatty acid 1234
iso-C12:0 0.2 0.1 — —
C12:0 0.2 0.1 — —
iso-C13:0 0.4 0.1 0.2 0.2
anteiso-C13:0 0.5 0.2 0.3 0.2
C13:0 0.2 — — —
iso-C14:0 5.2 5.1 1.6 2.6
C14:0 0.9 0.8 0.5 0.5
iso-C15:0 13.0 5.1 7.9 8.4
anteiso-C15:0 19.3 25.0 28.6 26.2
C15:0 3.9 1.4 1.6 1.7
iso-C16:1 H 0.2 0.2 — 0.2
iso-C16:0 19.1 22.4 11.5 16.4
C16:1 Cis 9 0.9 1.7 0.4 0.4
C16:0 10.8 13.3 11.8 10.4
C16:0 9Methyl 0.7 0.5 0.6 0.8
anteiso-C17:1 C 0.5 0.8 1.0 0.9
iso-C17:0 7.5 4.3 7.3 7.4
anteiso-C17:0 11.2 15.9 23.5 19.4
C17:1 Cis 9 — 0.2 — 0.2
C17:0 Cyclo 0.3 — 0.5 0.7
C17:0 4.1 1.8 1.6 1.6
iso-C18:0 0.5 0.7 0.5 0.4
iso-C17:0 2OH — — — 0.2
C18:0 0.5 0.4 — 0.4
Table 3. Cellular fatty acid composition of Streptomyces monashensis MUSC 1JT and its closely related
Streptomyces species. Strains: 1, Streptomyces monashensis sp. nov. MUSC 1JT; 2, Streptomyces coacervatus JCM
17318T; 3, Streptomyces olivaceoviridis JCM 4499T; 4, Streptomyces corchorusii JCM 4467T. −, <0.1% or not
detected. All data are obtained concurrently from this study.
Figure 4. Two dimensional total lipid prole of Streptomyces monashensis MUSC 1JT. DPG,
diphosphatidylglycerol; PG, phosphatidylglycerol; PGL, phosphoglycolipid; PI, phosphatidylinosotitol; PL,
phospholipid; PN*, possibility of PME, phosphatidylmonomethylethanolamine/PE, phosphatidylethanolamine/
OH-PE, hydroxyphosphatidylethanolamine.
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It can be observed that the cancer cells have shrunk and rounded-up aer treatment with MUSC 1JT extract at
400 µg/mL (Supplementary Fig.S5).
GC-MS analysis for chemical proling of strain MUSC 1JT extract. Since strain MUSC 1JT exhibited
signicant antioxidant and cytotoxic activities in the experiments, GC-MS analysis was performed to assist in
chemical proling and the identication of compounds present in the extract. e outcome of GC-MS analysis
of strain MUSC 1JT extract which revealed 14 compounds is presented in Table6: Pyrazine, 2,5-dimethyl- (1),
Pyrazine, trimethyl- (2), 2-Pyrrolidone (3), 2-Piperidinone (4), Indolizine (5), Pyrazine, 3,5-dimethyl-2-propyl-
(6), Phenol, 2,4-bis(1,1-dimethylethyl)- (7), Benzoic acid, 4-ethoxy-, ethyl ester (8), (3R,8aS)-3-Methyl-
1,2,3,4,6,7,8,8a-octahydropyrrolo[1,2-a]pyrazine-1,4-dione (9), Pyrrolo[1,2-a]pyrazine-1,4-dione,
hexahydro- (10), Phenol, 3,5-dimethoxy- (11), Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-
(12), 9H-Pyrido[3,4-b]indole (13), Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)- (14), with
chemical structures illustrated in Fig.6. e main classes of compounds found in the extract include pyrazine,
pyrrolidone, piperidone, indolizine, phenolic compound, benzoic acid ester, pyrrolopyrazine, and β-carboline
alkaloid.
Discussion
In the life cycle of Streptomyces, the development of aerial mycelium is initiated aer 2 days and it will continue to
mature into spores up to 10 days55. During this transition, it is when Streptomyces will start to produce secondary
metabolites24,55. In this study, 10-days fermentation process was performed using a complex HFM 1 medium on
strain MUSC 1JT to encourage cell growth and production of secondary metabolites. e metabolites of strain
MUSC 1JT were then extracted using methanol as extraction solvent. e extract was subjected to bioactivity
testing pertaining its antioxidant activity and cytotoxicity against cancer cells.
Oxidative stress caused by uncontrolled production of oxygen free radicals (e.g. O2·−, ·OH) has been recog-
nized as one of the key causes of health disorders including cancer, coronary heart disease, diabetes mellitus,
and neurodegenerative diseases8,56–58. Antioxidants can reduce the presence of free radicals, thereby protecting
the human body from damage caused by oxidative stress and consequently providing a positive eect on human
health by preventing or decreasing the risk of diseases such as cancer15,57. Streptomyces bacteria have been one of
the high-yielding sources of natural antioxidants. Among the new antioxidants discovered from Streptomyces are
carazostatin A isolated from Streptomyces chromofuscus DC 11859, carquinostatin A isolated from Streptomyces
exfoliates 2419-SVT260, diphenazithionin isolated from Streptomyces griseus ISP 523661, and ageloline A isolated
from Streptomyces sp. SBT34562. Results of SOD activity assay, ABTS assay, and metal chelating assay revealed the
antioxidative capability of strain MUSC 1JT, which could suggest that the strain might be capable of producing
potent antioxidant(s) that could be useful in dealing with oxidative stress.
Since the association between oxidative stress and the initiation of carcinogenesis was established, researchers
have been actively searching for potential antioxidants as well as anticancer agents that could be used for pre-
vention and/or treatment of cancer63. Among the dierent types of cancer, colorectal cancer is one of the most
common cancers- ranking as the third most commonly diagnosed cancer globally and second most commonly
diagnosed cancer in Malaysia64,65. e cytotoxic potential of strain MUSC 1JT was evaluated using the MTT assay
on human colon cancer cell lines: HCT-116 and SW480. Two dierent cancer cell lines with dierent genetic
makeup (e.g. HCT-116 cells contain wildtype p53; SW480 cells contain mutated p53) were used as panels in this
study to observe whether there is any varying ecacy in the cytotoxic activity of the extract against these cells5,66.
As a result, slight dierences in the cytotoxicity were observed in these two cancer cell lines following the expo-
sure to strain MUSC 1JT extract. is could be due to their distinctive susceptibility or resistance towards the
extract which contributed by their unique genetic makeup.
Figure 5. Scanning electron microscope of Streptomyces monashensis MUSC 1JT.
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Further analysis such as the GC-MS analysis was performed and this allowed the identication of compounds
that may account for the bioactivities exhibited by strain MUSC 1JT extract. Among the identied compounds
were the phenolic compounds that consist of an aromatic ring bearing one or more hydroxyl groups, also well
known for their antioxidant properties67. e phenolic compounds detected in strain MUSC 1JT extract were
Phenol, 2,4-bis(1,1-dimethylethyl)- (7) and Phenol, 3,5-dimethoxy- (11) (Fig.7). Both of the phenolic com-
pounds were previously detected in several Streptomyces strains, whereby Phenol, 2,4-bis(1,1-dimethylethyl)- (7)
in Streptomyces cavouresis KUV3968, Streptomyces sp. MUM2568, and Streptomyces colonosanans5, while both
Phenol, 2,4-bis(1,1-dimethylethyl)- (7) and Phenol, 3,5-dimethoxy- (11) in Streptomyces antioxidans15. Moreover,
these phenolic compounds have been associated with the antioxidant and cytotoxic activities exhibited by these
Streptomyces strains.
Heterocyclic compounds were detected in the extract of strain MUSC 1JT, such instances include the pyra-
zines and pyrrolopyrazines. Pyrazines are heterocyclic compounds that can be found in nature and are com-
monly produced by microorganisms69. Pyrazines are typical volatile and odorous metabolites produced by
Characteristic 1 2 3 4
Morphology (on ISP 2):
Color of aerial mycelium Light
Greenish
Yellow
Pale
Yellowish
Green
Pale
Yellowish
Green Yellowish White
Color of substrate mycelium Strong
Greenish
Yellow
Brilliant
Greenish
Yellow Pale Yellow Pale Yellow
Growth at:
26 °C +(+) (+) (+)
36 °C (+)+ + +
pH 8 (+)− − −
2% NaCl +(+) (+) (+)
Hemolytic + − + +
Hydrolysis of:
Tributyrin (lipase) + + + −
Carboxymethylcellulose (cellulase) + − + −
Carbon source utilization:
D-maltose + − + +
D-turanose + + − −
Stachyose + − + +
β-methyl-D-glucoside + + − −
D-salicin + + − −
N-acetyl-D-galactosamine + − − −
3-methyl glucose − + − −
D-fucose + − + −
D-fructose-6-PO4 − + + −
D-aspartic acid + + − −
D-serine − − + −
L-galactonic acid lactone + − + +
p-hydroxy-phenylacetic acid + − − −
α-hydroxy-butyric acid + − + +
α-keto-butyric acid + − + +
acetoacetic acid − + − −
Chemical sensitivity assays:
Guanidine HCl − + − −
Tetrazolium violet − + − −
Tetrazolium blue − + − −
Sodiu m bromate + + + −
Table 4. Dierentiation characteristics of Streptomyces monashensis MUSC 1JT and type strains of
phylogenetically closely related species of the genus Streptomyces. Strains: 1, Streptomyces monashensis sp. nov.
MUSC 1JT; 2, Streptomyces coacervatus JCM 17318T; 3, Streptomyces olivaceoviridis JCM 4499T; 4, Streptomyces
corchorusii JCM 4467T. All data were obtained concurrently in this study. +Positive; −negative; (+)weak. All
strains are positive for production of catalase, protease, and amylase; whilst negative for production of xylanase
and chitinase. All strains are positive for utilization of acetic acid, α-D-lactose, β-hydroxyl-D, L-butyric acid,
citric acid, dextrin, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, D-mannose, D-melibiose,
D-ranose, D-sorbitol, D-trehalose, gelatin, gentiobiose, glycyl-L-proline, L-fucose, L-malic acid, mucic acid,
pectin, Tween 40 and γ-amino-butyric acid.
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Streptomyces70,71 and they have also been detected in a number of other bacteria from various sources, for instance,
Corynebacterium glutamicum72, Chondromyces crocatus73, Serratia rubidaea, Serratia odorifera, Serratia caria as
well as Cedecea davisae74. Some of the pyrazines were reported to be associated with antioxidant, anticancer, and
antimicrobial activities15,75,76. Compounds Pyrazine, 2,5-dimethyl- (1), Pyrazine, trimethyl- (2), and Pyrazine,
3,5-dimethyl-2-propyl- (6) (Fig.7) were previously detected in other microorganisms such as Streptomyces citreus
CBS 109.60, Streptomyces antioxidans, and Corynebacterium glutamicum15,70,72. Previous studies also reported
that these compounds exhibited antitumor and antioxidant activities. For example, Wang and Tao77 reported the
detection of Pyrazine, 2,5-dimethyl- (1) in the metabolites of Stigmatella WXNXJ-B as one of the compounds
contributing to the antitumor activities on human liver carcinoma cells and human breast cancer cells. As for
pyrrolopyrazines, they can be found in or are produced by Streptomyces32. Pyrrolopyrazines are known to exert
various bioactivities including antioxidant, antitumor, antibacterial, antifungal, and anti-angiogenesis32,78,79.
As an example, the compound Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- (10) has been successfully
purified from marine sponge-associated Bacillus sp. where it exhibited significant antioxidant effect which
could assist in reducing oxygen free radical induced cellular oxidative damage58. Also, compounds (3R,8aS)-
3-Methyl-1,2,3,4,6,7,8,8a-octahydropyrrolo[1,2-a]pyrazine-1,4-dione (9) and Pyrrolo[1,2-a]pyrazine-1,4-dione,
hexahydro- (10) (Fig.7) were previously detected in crude extracts of Streptomyces pluripotens80 which were
suggested to be responsible for the potent antioxidant activity exerted by the strain. Additionally, pyrrolopyrazine
compounds have been associated with promising anticancer activity. e ndings of this study suggested that
these heterocyclic compounds could have contributed to the antioxidant activity and cytotoxic activity of strain
MUSC 1JT extract against the tested colon cancer cells.
A tricyclic indole β-carboline alkaloid, 9H-Pyrido[3,4-b]indole (13) (Fig.7) also known as norharman was
detected in strain MUSC 1JT extract and it has been reported to demonstrate antitumor and cytotoxic activities
in previous studies81,82. Compound 9H-Pyrido[3,4-b]indole (13) was previously detected in Pseudoalteromonas
Antioxidants
assays Concentration of strain
MUSC 1JT extract (mg/mL) Mean ± standard
error (%)
SOD
0.25 42.41 ± 1.58*
0.50 66.55 ± 2.10*
1.00 80.06 ± 3.38*
2.00 83.80 ± 4.80*
ABTS
0.25 5.06 ± 1.84
0.50 10.50 ± 1.04*
1.00 9.42 ± 1.33*
2.00 12.33 ± 3.07*
Metal chelating
0.25 11.82 ± 2.87*
0.50 27.32 ± 2.90*
1.00 44.84 ± 1.85*
2.00 75.50 ± 1.44*
Table 5. Radical scavenging activity of Streptomyces monashensis MUSC 1JT evaluated using ABTS, metal
chelating, and SOD assays. Symbol (*) indicates p < 0.05 signicant dierence between strain MUSC 1JT extract
and controls (without strain MUSC 1JT extract).
Figure 6. Cytotoxic activity of Streptomyces monashensis MUSC 1JT extract against human colon cancer cell
lines. e measurement of cell viability was done using MTT assay. e graphs show cytotoxicity eect of
MUSC 1JT extract against (A) SW480, and (B) HCT-116. All data are expressed as mean ± standard deviation
and signicance level are set as 0.05. Symbol (*) indicates p < 0.05 signicant dierence between the cells
treated with MUSC 1JT extract and control (without MUSC 1JT extract).
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piscicida by Zheng et al.83 and it was cytotoxic against the tested HeLa cervical-cancer cell line and the BGC-823
stomach-cancer cell line. Also, the study conducted by Tan et al.8 suggested that the presence of 9H-Pyrido[3,4-b]
indole (13) in Streptomyces sp. MUM256 could be responsible for the observed anticancer eect against colon
cancer cells (HCT 116, HT 29, and Caco-2).
Finally, the compounds 2-Pyrrolidone (3), 2-Piperidinone (4), Indolizine (5), and Benzoic acid, 4-ethoxy-,
ethyl ester (8) (Fig.7) discovered in the extract of strain MUSC 1JT were also found in other microbes.
Sathiyanarayanan et al.84 reported the detection of 2-Pyrrolidone (3) in Streptomyces sp. MAPS15 which showed
antimicrobial activity. Ser et al.15 detected 2-Piperidinone (4) and Indolizine (5) in Streptomyces antioxidans.
Benzoic acid, 4-ethoxy-, ethyl ester (8) was previously detected in Bacillus sp. and Streptomyces colonosanans5,85.
From the results of GC-MS analysis, it can be concluded that majority of the chemical compounds detected in
the extract of strain MUSC 1JT are recognized for their antioxidative and cytotoxic activities against cancer cells.
Hence, these identied compounds might be the factors contributing to the antioxidant and cytotoxic activities
demonstrated by extract from strain MUSC 1JT. However, additional studies are required to determine the exact
compound or combination of compounds that contributed to the observed activities.
No. Retention
time (min) Compound Class Molecular
formula Molecular
weight (MW) Qua lity
(%)
1 13.547 Pyrazine, 2,5-dimethyl- Pyrazine C6H8N2108 74
2 19.675 Pyrazine, trimethyl- Pyrazine C7H10N2122 80
3 23.869 2-Pyrrolidone Pyrrolidone C4H7NO 85 86
4 29.654 2-Piperidinone Piperidone C5H9NO 99 74
5 34.970 Indolizine Indolizine C8H7N 117 83
6 36.103 Pyrazine, 3,5-dimethyl-2-propyl- Pyrazine C9H14N2150 72
7 44.485 Phenol, 2,4-bis(1,1-dimethylethyl)- Phenolic compound C14H22O 206 93
8 44.897 B enzoic acid, 4-ethoxy-, ethyl ester Benzoic acid ester C11H14O3194 95
9 51.701 (3R,8aS)-3-Methyl-1,2,3,4,6,7,8,8a-octahydropyrrolo[1,2-a]pyrazine-1,4-dione Pyrrolopyrazine C8H12N2O2168 90
10 53.314 Pyr rolo[1,2-a]pyrazine-1,4-dione, hexahydro- Pyrrolopyrazine C7H10N2O2154 94
11 56.187 Phenol, 3,5-dimethoxy- Phenolic compound C8H10O3154 53
12 59.523 Pyr rolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- Pyrrolopyrazine C11H18N2O2210 78
13 60.387 9H-Pyrido[3,4-b]indole β-carboline alkaloid C11H8N2168 95
14 72.082 Pyr rolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)- Pyrrolopyrazine C14H16N2O2244 98
Table 6. Compounds identied from Streptomyces monashensis MUSC 1JT extract using GC-MS.
Figure 7. Chemical structures of constituents detected in Streptomyces monashensis MUSC 1JT extract using
GC-MS.
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Meanwhile, the genomic studies of Streptomyces provide a basis for better understanding of the secondary
metabolism and the production of target bioactive metabolites, thus creating an opportunity to obtain novel
bioactive compounds86,87. With the availability of NGS technology, the whole genome of strain MUSC 1JT was
subjected to sequencing. e availability of whole genome sequences provides a new point of view for novel strain
identication as the information allows in-depth genomic comparisons. For instance, the calculation of ANI
of conserved genes present in two sequenced strains have been suggested to be comparable to results from the
conventional DDH method44,88. Apart from that, whole genome sequences allow genome mining, which in turn
enables identication of gene clusters for natural product biosynthesis, and subsequently accelerate the discovery
of potential drug leads. In the current study, the biosynthetic gene clusters related to production of desferrioxam-
ine B was detected in MUSC 1JT. Even though desferrioxamine has long been used clinically to treat iron toxicity,
various studies suggested the potential use of this compound to manage other diseases including osteoporosis89,
neurodegenerative diseases90,91 and cancer92,93. Nonetheless, the genome potential of MUSC 1JT genome prompts
application of advanced techniques like genome editing with CRISPR-Cas9 systems to accentuate its ability in
producing these bioactive metabolites. Altogether, these ndings highlight the value of this mangrove derived
novel strain MUSC 1JT in the biopharmaceutical eld.
Description of Streptomyces monashensis sp. nov
Streptomyces monashensis sp. nov. (mo.nash.en’sis. N.L. masc. adj. referring to Monash University).
Cells stain Gram-positive and light greenish yellow aerial and strong greenish yellow substrate mycelium on
ISP 2 agar. Coloration of aerial and substrate mycelium are media-dependent (TableS2). Optimal cell growth
occurred at 28–32 °C, pH 7.0, with 0–2% NaCl. Cells are positive for catalase and hemolytic activities, as well as
capable of producing amylase, cellulase, protease, and lipase enzymes.
e cell wall peptidoglycan contains LL-diaminopimelic acid. e predominant menaquinones are MK-9(H8)
and MK-9(H6). Whole cell sugars detected include glucose and ribose. e polar lipids consist of phospholipid,
phosphatidylglycerol, phosphatidylinositol, phosphoglycolipid and diphosphatidylglycerol. e major cellular
fatty acids (>10%) are anteiso-C15: 0, iso-C16: 0, iso-C15: 0, anteiso-C17: 0 and C16: 0.
e type strain is MUSC 1JT (=DSM 103626T = MCCC 1K03219T) isolated from mangrove sediments collected
from the Sarawak mangrove forest located in East Malaysia. e 16S rRNA gene sequence of strain MUSC 1JT
has been deposited in GenBank/EMBL/DDBJ under the accession number KP998432. The genome size of
strain MUSC 1JT is 10,254,857 bp with average coverage of 170.0-fold and its G + C content is approximately
71 mol%. e whole project of strain MUSC 1JT was deposited at DDBJ/EMBL/GenBank under accession num-
ber MLYO00000000 and the version described in this paper is the rst version (MLYO0100000).
Conclusion
In summary, the strain MUSC 1JT, a novel species of the genus Streptomyces was successfully isolated and
identied from mangrove soil collected at the mangrove forest of Kuching, Sarawak, East Malaysia. e name
Streptomyces monashensis sp. nov. is proposed and the type strain is MUSC 1JT (=DSM 103626T = MCCC
1K03219T). e ndings of this study demonstrated that strain MUSC 1JT exhibits strong antioxidant activity
as high as 83.80 ± 4.80% via SOD assay as well as signicant cytotoxic activity against colon cancer cell lines SW
480 and HCT-116. is study provides a comprehensive description of the novel strain Streptomyces monashensis
MUSC 1JT and elucidates the potential of the strain in the biopharmaceutical industry. e potent antioxidative
activity of Streptomyces monashensis MUSC 1JT shows the strain to be a potentially good microbial source that
could contribute to drug discovery, especially with regard to development of potential antioxidant agents from
this strain. Hence, it is worthwhile to conduct further studies to provide in-depth understanding on the antioxi-
dative property of this strain.
Materials and Methods
Soil sampling, isolation and maintenance of strain. Soil samples were originated from a mangrove
forest in Malaysia, specically, in the area of Kuching of Sarawak. Collection of soil samples was carried out
in June 2015; the isolation and maintenance of Streptomyces isolates were conducted according to previously
described method5. Eighty-eight Streptomyces isolates were successfully recovered from the soil samples and in
vitro preliminary bioactivity screening of methanolic Streptomyces extracts was performed (data not shown).
Strain MUSC 1JT, isolated from sampling site KTTAS 5 (1°41′47.77″N 110°11′16.05″E), was discovered as one of
the putative novel isolates with potential antioxidant and cytotoxic activities.
Genotypic, phylogenetic, and genomic analyses. Methods of genomic DNA extraction of the strain
were adapted from Hong et al.31 and the methods of PCR amplication of the 16S rRNA gene were adapted
from Lee et al.20 using TurboCycler 2 (Blue-Ray Biotech, Taipei, Taiwan). The nearly-complete 16S rRNA
gene sequence of strain MUSC 1JT was obtained via molecular cloning. Multiple alignment of 16S rRNA gene
sequence of strain MUSC 1JT with representative sequences of related type strains in the genus Streptomyces
was performed using CLUSTAL-X soware94; the reference sequences were retrieved from the GenBank/EMBL/
DDBJ databases. Firstly, the sequence alignment was veried manually and adjusted. en, MEGA version 6.095
was used to construct the phylogenetic trees with neighbor-joining (Fig.1), maximum-likelihood algorithms
(Supplementary Fig.S1), and maximum-parsimony algorithms (Supplementary Fig.S2). e evolutionary dis-
tances for neighbor-joining algorithm were computed by the Kimura’s two-parameter model. Tree topologies
were assessed by bootstrap analyses based on 1000 resamplings method of Felsenstein96. e levels of sequence
similarity were assessed by EzBioCloud server (http://www.ezbiocloud.net/)97.
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Genomic DNA extraction followed by DNA-DNA hybridization (DDH)5,14 were performed on strain MUSC
1JT and its closely related type strains S. corchorusii JCM 4467T, S. olivaceoviridis JCM 4499T, S. canarius JCM
4549T, and S. coacervatus JCM 17318T. e G + C content of strain MUSC 1JT was determined and BOX-PCR
ngerprinting was performed according to previously established protocol5,98,99.
Chemotaxonomic characteristics. e chemotaxonomic analyses were performed by the Identication
Service of the DSMZ, Braunschweig5,14,15,19,20, which include evaluation of: cell wall peptidoglycan, whole cell
sugars, respiratory quinones, fatty acids, and polar lipids.
Phenotypic characteristics. Cultural morphology and Gram staining of strain MUSC 1JT was investi-
gated based on established protocol5. ISCC-NBS color charts were used for the assignment of the colony color of
strain MUSC 1JT. Cellular morphology of strain MUSC 1 was observed using Light microscopy (80i, Nikon) and
scanning electron microscopy (JEOL-JSM 6400)5,14. Temperature, pH, and NaCl tolerance of strain MUSC 1JT
growth were evaluated in this study5. Production of melanoid pigments and enzymatic activities (e.g. catalase,
hemolytic, amylolytic, cellulase, lipase etc.) of strain MUSC 1JT were investigated using established protocol5,14,100.
Carbon-source utilization and chemical sensitivity of Streptomyces strains were analyzed using Biolog GenIII
MicroPlate (Biolog, USA).
e phenotypic assays mentioned in this study were performed concurrently for strain MUSC 1JT, S. corchorusii
JCM 4467T, S. olivaceoviridis JCM 4499T and S. coacervatus JCM 17318T.
Whole genome sequencing and bioinformatics analysis of strain MUSC 1JT. Genomic DNA
extraction and whole genome sequencing of strain MUSC 1JT were conducted according to the methods described
in previous studies5,101–107. Trimmed sequences were de novo assembled with CLC Genomic Workbench version
7 (CLC bio, Denmark). Prodigal version 2.6108 was used for gene prediction, while RNAmmer and tRNAscan
SE version 1.21 were used for rRNA and tRNA prediction109,110. e genome assembly was submitted to Rapid
Annotation using Subsystem Technology (RAST) database and NCBI Prokaryotic Genomes Annotation Pipeline
(PGAP) for annotation5. e genome of closely related strains (e.g. S. corchorusii DSM 40340T) were retrieved
from NCBI database for comparison using BLAST before building synteny map using Artermis Comparison Tool
(ACT)42. e calculations of average nucleotide identity (ANI) values were performed on EzBioCloud (https://
www.ezbiocloud.net/tools/ani). AntiSMASH was used to detect presence of biosynthetic gene clusters related to
secondary metabolites111.
Preparation of strain MUSC 1JT extract. Extract of MUSC 1JT was prepared according to previously
established protocol5,31,112, using. HFM 1 (Biomerge, Malaysia) as fermentation medium and methanol as extract-
ing solvent. Final extract of strain MUSC 1JT was suspended in dimethyl sulphoxide (DMSO) before proceeding
to bioactivity tests5.
Examination of antioxidant activity of MUSC 1JT extract. Superoxide anion scavenging/superox-
ide dismutase (SOD) activity the extract was investigated using SOD assay Kit–WST (Sigma-Aldrich) according
to previously described protocol5,8. e outcome of the reaction was recorded by measuring the absorbance at
450 nm.
e 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay was carried out for the evalua-
tion of antioxidant activity the extract using established protocol113. e resultant absorbance was then measured
at 743 nm; with the reduction in absorbance value as an indication of the alteration in radical amount.
Metal Chelating activity of the extract was investigated based on the procedure derived from earlier study113.
Outcome of the reaction was determined through absorbance measured at 562 nm using a microplate reader.
Maintenance and growth condition of human derived cancer cell lines. In this study, the tested
human derived cancer cell lines were maintained in RPMI (Roswell Park Memorial Institute)-1640 (Gibco) sup-
plemented with 10% fetal bovine serum and 1x antibiotic-antimycotic (Gibco) in a humidied incubator at 37 °C
with 5% CO2 in 95% air5,8.
Examination of cytotoxicity activity of MUSC 1JT using 3-(4,5-dimethylthazol-2yl)-2,5-diphenyl
tetrazolium-bromide (MTT) assay. is study involved the evaluation of strain MUSC 1JT extract against
human derived colon cancer cell lines: SW480 and HCT-116. MTT assay was used for the investigation of cyto-
toxic activity of strain MUSC 1JT extract8,36. Microplate reader was used to analyze the cell viability at wavelength
570 nm (with 650 nm as reference wavelength). e morphology of the cells was observed using an inverted
microscope.
Gas chromatography-mass spectrometry (GC-MS) analysis. GC-MS analysis was conducted accord-
ing to the protocol previously described by Law et al.5. e instrument involved was Agilent Technologies 6980 N
(GC) equipped with 5979 Mass Selective Detector (MS), with HP-5MS (5% phenyl methyl siloxane) capillary
column of dimensions 30.0 m × 250 µm × 0.25 µm and helium as carrier gas at 1 mL/min. is study utilized NIST
05 Mass Spectral Library.
Statistical analysis. Antioxidant and cytotoxic activities assays in this study were carried out in quadru-
plicate. e collected data was analyzed using SPSS statistical analysis soware and stated as mean ± standard
deviation (SD). e signicant dierences between groups were determined through one-way analysis of variance
(ANOVA) and appropriate post hoc test (Tukey). e signicance level of p ≤ 0.05 was used for all data analyses
in this study.
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Acknowledgements
is work was nancially supported by MOSTI ScienceFund Grant (Project No. 06-02-10-SF0300) and External
Industry Grants from Biotek Abadi Sdn Bhd (Vote No. GBA-808138 and GBA-808813) awarded to L.-H.L.,
University of Malaya for Research Grant (GA001-2016, GA002-2016, and PPP Grant No. PG133-2016A) awarded
to K.-G.C. e authors are thankful to Professor Bernhard Schink for the support in the Latin etymology of the
new species name.
Author Contributions
e experiments, data analysis, and manuscript writing were performed by J.W.-F.L. and H.-L.S., while N.-S.A.M.,
S.S., A.D., T.M.K., K.-G.C., B.-H.G. and L.-H.L. provided vital guidance, insight and technical support for the
completion of the project. L.-H.L. and B.-H.G. founded the research project.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-39592-6.
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