Streptomyces monashensis sp. nov., a novel mangrove soil actinobacterium from East Malaysia with antioxidative potential

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 significant cytotoxic effect 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 significant to the biopharmaceutical industry as it could lead to development of new and useful chemo-preventive drugs.

www.nature.com/scientificreports www.nature.com/scientificreports/ for the observed bioactivities were identified. The outcome of current research provides an in depth understanding of Streptomyces monashensis sp. nov. MUSC 1J T from different perspectives and also demonstrates the potential of this strain in producing bioactive compounds with antioxidant and cytotoxic activities.

Results
Genotypic, phylogenetic, and genomic analyses of strain MUSC 1J t . The nearly full-length 16S rRNA gene sequence was attained for strain MUSC 1J T (1490 bp; GenBank/EMBL/DDBJ accession number KP998432). Based on the 16S rRNA sequences, phylogenetic trees were reconstructed to determine the evolutionary relationship of this strain with its related type strains (Figs 1, S1 and S2). Results were in agreement that the most closely related strain is S. coacervatus AS-0823 T (98.4% sequence similarity) with shortest evolutionary distance, as they formed distinct clade at bootstrap value of ≥50% in the neighbour-joining (Fig. 1), maximum-likelihood (Fig. S1), and maximum-parsimony (Fig. S2) phylogenetic trees. The 16S rRNA gene sequence analysis for strain MUSC 1J T revealed that this strain exhibited the highest similarity to strain S. corchorusii DSM 40340 T (98.7%), S. olivaceoviridis NBRC 13066 T (98.7%), and S. canarius NBRC 13431 T (98.6%).
Furthermore, the results of DDH revealed that the DNA-DNA relatedness levels between strain MUSC 1J T and S. corchorusii JCM 4467 T (34.8 ± 3.3%), S. olivaceoviridis JCM 4499 T (49.1 ± 4.3%), S. canarius JCM 4549 T (19.7 ± 2.8%) and S. coacervatus JCM 17318 T (21.1 ± 3.2%) were significantly below 70%-recommended cut-off point for the delineation of bacterial species 41 . Besides, strain MUSC 1J T yielded a distinctive BOX-PCR fingerprint which can be differentiated from its closely related type strains ( Supplementary Fig. S3). The results of phylogenetic analysis, DDH, and BOX-PCR fingerprint analysis were consistent and thus supported that strain MUSC 1J T represents a novel species of Streptomyces genus.
In addition, the whole genome sequencing showed that the genome of strain MUSC 1J T consists of 10,254,857 bp with average coverage of 170.0-fold ( Table 1). The whole project of strain MUSC 1J T was deposited at DDBJ/EMBL/GenBank under accession number MLYO00000000 and the version described in this paper is the first version (MLYO0100000). A total of 9,310 coding genes was predicted on MUSC 1J T 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 1J T and its closely related type strain S. corchorusii DSM 40340 T was also performed. Analysis based on Clusters of Orthologous Groups (COG) functional categories showed that similar distribution of genes between strain MUSC 1J T and S. corchorusii DSM 40340 T ; highest number of known proteins were found to be involved in essential processes like transcription (Class K) followed by carbohydrate transport and metabolism (Class G) (after removing uninformative classes such as R and S in the analysis) ( Table 2). 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 1J T and S. corchorusii DSM 40340 T (Fig. 2). Nonetheless, the ANI value comparing strain MUSC 1J T and S. corchorusii DSM 40340 T was calculated to be 86.03%. ANI has become increasingly popular due to the availability of whole genome sequences. The ANI analysis is primarily done by computation comparisons of two genome sequences to determine the genetic relatedness between prokaryotic strains 43 . A report by Goris et al. 44 has described that 95% ANI and 69% conserved DNA corresponded with the cut-off point of 70% DDH for species delineation. The ANI value reflected by strain MUSC 1J T and type strain S. corchorusii DSM 40340 T was found to be well below the recommended value by Goris et al. 44 . This finding was also in line with the outcome of DDH analysis between strain MUSC 1J T and S. corchorusii DSM 40340 T (DNA-DNA relatedness of 34.8 ± 3.3%, <70%). Furthermore, additional analyses of strain MUSC 1J T and its other closely related strains that possessed >98% 16S rRNA sequence similarity have revealed ANI values between 82-87%, which falls significantly below the recommended value (Table S1). Therefore, the novel status of the strain MUSC 1J T was further confirmed 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 1J T genome related to various biosynthetic gene clusters including type-I polyketide synthetase, indole biosynthesis, and siderophores production. One of the common biosynthetic gene clusters within strain MUSC 1J T and S. corchorusii was selected for comparison -biosynthetic gene cluster related to production of desferrioxamine B. The gene clusters were highly similar and pairwise comparison of the gene encoding for IucA/IucC family protein responsible for production of desferrioxamine revealed that gene similarities of 88.29% (Fig. 3) 45 . The presence of these biosynthetic gene clusters indicates the bioactive potential of strain MUSC 1J T and suggesting its ability in producing such valuable bioactive compounds.
Chemotaxonomic analyses of strain MUSC 1J t . The results of chemotaxonomic analyses revealed that strain MUSC 1J T presented a type I cell-wall as it contains LL-diaminopimelic acid 46 , an amino acid found to be present in many other species of the genus Streptomyces 5,19,20,32,[47][48][49] . The predominant menaquinones of strain MUSC 1J T were identified as MK-9(H 8 ) (55%) and MK-9(H 6 ) (16%). The detection of these predominant menaquinones is in agreement with the report of Kim et al. 50 . The whole cell sugars detected were glucose and ribose. Strain MUSC 1J T has a G + C content of 71 mol% and it was in the range of 67.0-78.0 mol% as described for Streptomyces 50 .
Phenotypic analyses of strain MUSC 1J t . Phenotypic analyses in this study revealed that the mangrove forest soil-derived MUSC 1J T strain grows well on ISP 2, ISP 3, ISP 5, ISP 6, ISP 7, Streptomyces agar, and nutrient agar after 7-14 days at 28 °C; grows moderately on starch casein agar and actinomycetes isolation agar, and does not grow on ISP 4. The colors of the aerial and substrate mycelium were media-dependent as shown in Table S2. Based on the observation of 14-day-old culture grown on ISP 2 agar, the aerial and vegetative hyphae of strain MUSC 1J T were abundant and well developed. These morphological features of strain MUSC 1J T (Fig. 5) conform to those observed in genus Streptomyces, hence, this indicated that strain MUSC 1J T belongs to the genus Streptomyces 51 .
Results of genomic and phylogenetic analysis, chemotaxonomic and phenotypic analyses proven that strain MUSC 1J T 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 1J t extract.
In this study, the antioxidant potential of novel strain MUSC 1J T 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 1J T exhibited significant radical scavenging ability ( Table 5). The capability of strain MUSC 1J T extract to scavenge in vitro oxygen-derived species like superoxide anion (O 2 ⋅ − ) 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. The 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 radical 5,8,52 . Strain MUSC 1J T extract possesses SOD-like activity up to 83.80 ± 4.80% by virtue of scavenging the superoxide anion radical and subsequently inhibiting the development of yellow WST-1 formazan. The extract exhibited significant 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 1J T extract was confirmed by ABTS assay. The production of ABTS radical cation in this assay was initiated by the reaction between a strong oxidizing agent potassium persulfate with ABTS salt 53 . The extract was able to scavenge the ABTS radical generated in the assay with significant activity of 12.33 ± 3.07% at concentration of 2 mg/mL ( Table 5).
The ability of strain MUSC 1J T extract in exhibiting metal chelating activity further demonstrated its antioxidant potential. In a metal chelating assay, the ferrozine added can quantitatively form complexes with Fe 2+ , resulting in a formation of Fe 2+ -ferrozine complex that can be disrupted in the presence of other chelating agents 54 . The presence of strain MUSC 1J T extract exhibited a significant metal chelating activity, with highest activity recorded at 75.50 ± 1.44% at 2 mg/mL concentration ( Table 5). The antioxidative potential of MUSC 1J T extract is emphasized through its metal chelating ability by preventing transition metals from promoting the generation of ROS 5,14 . www.nature.com/scientificreports www.nature.com/scientificreports/ Cytotoxic activity of strain MUSC 1J t extract. Generally, strain MUSC 1J T extract showed promising cytotoxic activity against the colon cancer cell lines tested. The results of strain MUSC 1J T extract tested against the colon cancer cell lines were presented in Fig. 5. After 72 hours of treatment with strain MUSC 1J T extract, the results revealed that the extract had significant cytotoxic effect against both colon cancer cell lines (P < 0.05) (Fig. 6). The 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 contrast microscopy to visualize the response of SW480 and HCT-116 cells after treated with MUSC 1J T extract.     14), with chemical structures illustrated in Fig. 6. The 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 after 2 days and it will continue to mature into spores up to 10 days 55 . During this transition, it is when Streptomyces will start to produce secondary metabolites 24,55 . In this study, 10-days fermentation process was performed using a complex HFM 1 medium on strain MUSC 1J T to encourage cell growth and production of secondary metabolites. The metabolites of strain MUSC 1J T were then extracted using methanol as extraction solvent. The 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. O 2 · − , ·OH) has been recognized as one of the key causes of health disorders including cancer, coronary heart disease, diabetes mellitus, and neurodegenerative diseases 8,[56][57][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 effect on human health by preventing or decreasing the risk of diseases such as cancer 15,57 . 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 118 59 , carquinostatin A isolated from Streptomyces exfoliates 2419-SVT2 60 , diphenazithionin isolated from Streptomyces griseus ISP 5236 61 , and ageloline A isolated from Streptomyces sp. SBT345 62 . Results of SOD activity assay, ABTS assay, and metal chelating assay revealed the antioxidative capability of strain MUSC 1J T , 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 prevention and/or treatment of cancer 63 . Among the different 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 Malaysia 64,65 . The cytotoxic potential of strain MUSC 1J T was evaluated using the MTT assay on human colon cancer cell lines: HCT-116 and SW480. Two different cancer cell lines with different 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 efficacy in the cytotoxic activity of the extract against these cells 5,66 . As a result, slight differences in the cytotoxicity were observed in these two cancer cell lines following the exposure to strain MUSC 1J T extract. This could be due to their distinctive susceptibility or resistance towards the extract which contributed by their unique genetic makeup. www.nature.com/scientificreports www.nature.com/scientificreports/ Further analysis such as the GC-MS analysis was performed and this allowed the identification of compounds that may account for the bioactivities exhibited by strain MUSC 1J T extract. Among the identified compounds were the phenolic compounds that consist of an aromatic ring bearing one or more hydroxyl groups, also well known for their antioxidant properties 67 . The phenolic compounds detected in strain MUSC 1J T extract were Phenol, 2,4-bis(1,1-dimethylethyl)-(7) and Phenol, 3,5-dimethoxy-(11) (Fig. 7). Both of the phenolic compounds were previously detected in several Streptomyces strains, whereby Phenol, 2,4-bis(1,1-dimethylethyl)- (7) in Streptomyces cavouresis KUV39 68 , Streptomyces sp. MUM256 8 , and Streptomyces colonosanans 5 , while both Phenol, 2,4-bis(1,1-dimethylethyl)-(7) and Phenol, 3,5-dimethoxy- (11) in Streptomyces antioxidans 15 . Moreover, these phenolic compounds have been associated with the antioxidant and cytotoxic activities exhibited by these Streptomyces strains.
A tricyclic indole β-carboline alkaloid, 9H-Pyrido [3,4-b]indole (13) (Fig. 7) also known as norharman was detected in strain MUSC 1J T extract and it has been reported to demonstrate antitumor and cytotoxic activities in previous studies 81  www.nature.com/scientificreports www.nature.com/scientificreports/ 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 effect against colon cancer cells (HCT 116, HT 29, and Caco-2).
From the results of GC-MS analysis, it can be concluded that majority of the chemical compounds detected in the extract of strain MUSC 1J T are recognized for their antioxidative and cytotoxic activities against cancer cells. Hence, these identified compounds might be the factors contributing to the antioxidant and cytotoxic activities demonstrated by extract from strain MUSC 1J T . However, additional studies are required to determine the exact compound or combination of compounds that contributed to the observed activities.   www.nature.com/scientificreports www.nature.com/scientificreports/ 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 compounds 86,87 . With the availability of NGS technology, the whole genome of strain MUSC 1J T was subjected to sequencing. The availability of whole genome sequences provides a new point of view for novel strain identification 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 method 44,88 . Apart from that, whole genome sequences allow genome mining, which in turn enables identification 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 desferrioxamine B was detected in MUSC 1J T . 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 osteoporosis 89 , neurodegenerative diseases 90,91 and cancer 92,93 . Nonetheless, the genome potential of MUSC 1J T genome prompts application of advanced techniques like genome editing with CRISPR-Cas9 systems to accentuate its ability in producing these bioactive metabolites. Altogether, these findings highlight the value of this mangrove derived novel strain MUSC 1J T in the biopharmaceutical field.
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 (Table S2). 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.
The type strain is MUSC 1J T (=DSM 103626 T = MCCC 1K03219 T ) isolated from mangrove sediments collected from the Sarawak mangrove forest located in East Malaysia. The 16S rRNA gene sequence of strain MUSC 1J T has been deposited in GenBank/EMBL/DDBJ under the accession number KP998432. The genome size of strain MUSC 1J T is 10,254,857 bp with average coverage of 170.0-fold and its G + C content is approximately 71 mol%. The whole project of strain MUSC 1J T was deposited at DDBJ/EMBL/GenBank under accession number MLYO00000000 and the version described in this paper is the first version (MLYO0100000).

Conclusion
In summary, the strain MUSC 1J T , a novel species of the genus Streptomyces was successfully isolated and identified from mangrove soil collected at the mangrove forest of Kuching, Sarawak, East Malaysia. The name Streptomyces monashensis sp. nov. is proposed and the type strain is MUSC 1J T (=DSM 103626 T = MCCC 1K03219 T ). The findings of this study demonstrated that strain MUSC 1J T exhibits strong antioxidant activity as high as 83.80 ± 4.80% via SOD assay as well as significant cytotoxic activity against colon cancer cell lines SW 480 and HCT-116. This study provides a comprehensive description of the novel strain Streptomyces monashensis MUSC 1J T and elucidates the potential of the strain in the biopharmaceutical industry. The potent antioxidative activity of Streptomyces monashensis MUSC 1J T 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 antioxidative property of this strain.

Materials and Methods
soil sampling, isolation and maintenance of strain. Soil samples were originated from a mangrove forest in Malaysia, specifically, 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 method 5 . 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 1J T , 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 amplification 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 1J T was obtained via molecular cloning. Multiple alignment of 16S rRNA gene sequence of strain MUSC 1J T with representative sequences of related type strains in the genus Streptomyces was performed using CLUSTAL-X software 94 ; the reference sequences were retrieved from the GenBank/EMBL/ DDBJ databases. Firstly, the sequence alignment was verified manually and adjusted. Then, MEGA version 6.0 95 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). The evolutionary distances 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 Felsenstein 96 . The levels of sequence similarity were assessed by EzBioCloud server (http://www.ezbiocloud.net/) 97 . www.nature.com/scientificreports www.nature.com/scientificreports/ Genomic DNA extraction followed by DNA-DNA hybridization (DDH) 5,14 were performed on strain MUSC 1J T and its closely related type strains S. corchorusii JCM 4467 T , S. olivaceoviridis JCM 4499 T , S. canarius JCM 4549 T , and S. coacervatus JCM 17318 T . The G + C content of strain MUSC 1J T was determined and BOX-PCR fingerprinting was performed according to previously established protocol 5,98,99 . Chemotaxonomic characteristics. The chemotaxonomic analyses were performed by the Identification Service of the DSMZ, Braunschweig 5,14,15,19,20 , 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 1J T was investigated based on established protocol 5 . ISCC-NBS color charts were used for the assignment of the colony color of strain MUSC 1J T . 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 study 5 . Production of melanoid pigments and enzymatic activities (e.g. catalase, hemolytic, amylolytic, cellulase, lipase etc.) of strain MUSC 1J T were investigated using established protocol 5,14,100 . Carbon-source utilization and chemical sensitivity of Streptomyces strains were analyzed using Biolog GenIII MicroPlate (Biolog, USA).
The phenotypic assays mentioned in this study were performed concurrently for strain MUSC 1J T , S. corchorusii JCM 4467 T , S. olivaceoviridis JCM 4499 T and S. coacervatus JCM 17318 T .
Whole genome sequencing and bioinformatics analysis of strain MUSC 1J t . Genomic DNA extraction and whole genome sequencing of strain MUSC 1J T were conducted according to the methods described in previous studies 5, [101][102][103][104][105][106][107] . Trimmed sequences were de novo assembled with CLC Genomic Workbench version 7 (CLC bio, Denmark). Prodigal version 2.6 108 was used for gene prediction, while RNAmmer and tRNAscan SE version 1.21 were used for rRNA and tRNA prediction 109,110 . The genome assembly was submitted to Rapid Annotation using Subsystem Technology (RAST) database and NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) for annotation 5 . The genome of closely related strains (e.g. S. corchorusii DSM 40340 T ) were retrieved from NCBI database for comparison using BLAST before building synteny map using Artermis Comparison Tool (ACT) 42 . The 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 metabolites 111 .
Preparation of strain MUSC 1J t extract. Extract of MUSC 1JT was prepared according to previously established protocol 5,31,112 , using. HFM 1 (Biomerge, Malaysia) as fermentation medium and methanol as extracting solvent. Final extract of strain MUSC 1J T was suspended in dimethyl sulphoxide (DMSO) before proceeding to bioactivity tests 5 .
Examination of antioxidant activity of MUSC 1J t extract. Superoxide anion scavenging/superoxide dismutase (SOD) activity the extract was investigated using SOD assay Kit-WST (Sigma-Aldrich) according to previously described protocol 5,8 . The outcome of the reaction was recorded by measuring the absorbance at 450 nm.
The 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay was carried out for the evaluation of antioxidant activity the extract using established protocol 113 . The 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 study 113 . 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) supplemented with 10% fetal bovine serum and 1x antibiotic-antimycotic (Gibco) in a humidified incubator at 37 °C with 5% CO 2 in 95% air 5,8 . Examination of cytotoxicity activity of MUSC 1J t using 3-(4,5-dimethylthazol-2yl)-2,5-diphenyl tetrazolium-bromide (Mtt) assay. This study involved the evaluation of strain MUSC 1J T extract against human derived colon cancer cell lines: SW480 and HCT-116. MTT assay was used for the investigation of cytotoxic activity of strain MUSC 1J T extract 8,36 . Microplate reader was used to analyze the cell viability at wavelength 570 nm (with 650 nm as reference wavelength). The morphology of the cells was observed using an inverted microscope.
Gas chromatography-mass spectrometry (GC-Ms) analysis. GC-MS analysis was conducted according to the protocol previously described by Law et al. 5 . The 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. This study utilized NIST 05 Mass Spectral Library. statistical analysis. Antioxidant and cytotoxic activities assays in this study were carried out in quadruplicate. The collected data was analyzed using SPSS statistical analysis software and stated as mean ± standard deviation (SD). The significant differences between groups were determined through one-way analysis of variance (ANOVA) and appropriate post hoc test (Tukey). The significance level of p ≤ 0.05 was used for all data analyses in this study.