Screening, characterization, and phylogenetic analysis of Methioninase-Producing Indigenous Streptomyces spp. Isolated from the Upper Lake of Bhopal, Madhya Pradesh, India

*Corresponding author: Pratik Yadav Email: Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Screening, characterization, and phylogenetic analysis of Methioninase-Producing Indigenous Streptomyces spp. Isolated from the Upper Lake of Bhopal, Madhya Pradesh, India Pratik Yadav 1, *, Rakesh Mehta 2 and Ragini Gothalwal 3 1 Department of Biotechnology, Barkatullah University, Bhopal. 2 Department of Botany and Biotechnology. Govt., MGM College Itarsi. 3 Department of Biotechnology, Barkatullah University, Bhopal. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(02), 290-300 Publication history: Received on 06 April 2025; revised on 11 May 2025; accepted on 13 May 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.22.2.0493 Abstract Methioninase, an enzyme with potential for cancer treatment, catalyzes the degradation of methionine, which supports tumor growth and metastasis. Streptomyces species, known for their secondary metabolite production, are prime candidates for methioninase production. This study aimed to isolate and screen indigenous Streptomyces strains from aquatic environments around the Upper Lake of Bhopal, India, to identify those with methioninase activity. Samples were collected from five locations, and Streptomyces spp. were isolated using starch casein agar and screened with a phenol red-based assay for methioninase production. Seven out of 20 isolates exhibited positive methioninase activity. Molecular identification through 16S rRNA sequencing confirmed the identity of these isolates, revealing genetic diversity among the methioninase-producing strains. Phylogenetic analysis further supported the distinctiveness of these strains. The study emphasizes the promising potential of these isolates for enzyme-based cancer therapies and contributes to bioprospecting efforts for novel therapeutic agents. The isolated Streptomyces spp. from the Upper Lake of Bhopal exhibit potent methioninase activity, highlighting their potential for enzyme therapy, particularly in cancer treatment. The molecular characterization of these isolates provides a foundation for further research into optimizing methioninase production. These findings contribute significantly to the growing interest in microbial enzymes as therapeutic agents, particularly in the context of cancer therapy. Keywords: L-Methioninase; Streptomyces spp.; Cancer Therapy; Microbial Diversity Phylogeny 1. Introduction Methioninase is a vital enzyme with significant therapeutic potential, particularly for cancer treatment, owing to its ability to degrade methionine, an amino acid that promotes tumor growth and metastasis (Zhang et al., 2017). The growing interest in methioninase for enzyme therapy in treating methionine-dependent cancers has driven research into optimizing its production (Tan et al., 2019). Streptomyces spp., known for their ability to produce a wide range of secondary metabolites, are promising candidates for such studies, especially from diverse ecological environments (Shirling & Gottlieb, 1966). This study focuses on isolating and screening indigenous Streptomyces strains from various locations around the Upper Lake of Bhopal, Madhya Pradesh, India. As one of Asia's largest artificial lakes, the Upper Lake offers a unique ecosystem with diverse microbial populations, making it a prime source for discovering novel actinobacterial species (Rai et al., 2021). Previous studies have shown that aquatic soil and sediment samples are rich in Streptomyces spp. with enzymatic activities, including methioninase production (El-Naggar & Eldin, 2020). World Journal of Biology Pharmacy and Health Sciences, 2025, 22(02), 290-300 291 The main objective of this research is to explore the phylogenetic relationships among methioninase-producing Streptomyces spp. isolated from the Upper Lake of Bhopal, M.P., India. Screening, isolation, and identification of strains with potent methioninase activity will pave the way for potential cancer therapy applications, contributing to the growing body of knowledge in microbial bioprospecting for valuable enzymatic properties. 2. Materials and Methods 2.1. Isolation & Screening of Streptomyces Species 2.1.1. Sample Collection Marshy soil mixed water samples were collected from five locations around the Upper Lake of Bhopal, India, during the sampling period from August 2022 to July 2023. Pre-sterilized glass bottles were used for sample collection, and precautions were taken to avoid contamination. Each sample was assigned a code and stored at 4-8°C in the laboratory. The different sampling points are mentioned in table 1. Table 1 The different points of soil mixed water sampling around Upper Lake of Bhopal S. No. Sampling Sites Water Sample Sample Code 1st Collection 2nd Collection 1. Van Vihar Turbid and muddy water S1 S6 2. Boat Club Turbid and muddy water S2 S7 3. Kamla Park Turbid and muddy water S3 S8 4. Kaliasot Dam Turbid and muddy water S4 S9 5. Bairagarh Turbid and muddy water S5 S10 2.2. Isolation of Streptomyces Species Starch casein agar (M801 HiMedia) was used for isolating Streptomyces spp. from the samples, followed by sub-culturing on inorganic salt starch agar (ISSA) for pure cultures. Samples were serially diluted and inoculated onto agar plates using the spread plate method, followed by incubation at 30°C for 8-10 days. The composition of the media is mentioned in table 2. Colonies were enumerated using a digital colony counter, and pure cultures were prepared through repeated sub-culturing (Shirling & Gottlieb, 1966). Table 2 Composition of starch casein agar media (M801 HiMedia) S.No. Ingredients Quantity in Grams/Litre 1. Soluble starch 10.00 2. Casein (Vitamin Free) 0.30 3. KNO3 2.00 4. MgSO4.7H2O 0.05 5. K2HPO4 2.00 6. NaCl 2.00 7. CaCO3 0.02 8. FeSO4.7H2O 0.01 9. Agar 18.00 10. Distilled water 1000 All ingredients homogenised in per litre distilled water then sterilized and poured in sterile plates World Journal of Biology Pharmacy and Health Sciences, 2025, 22(02), 290-300 292 2.3. Rapid Assay for Methioninase Streptomyces spp. Indigenous Streptomyces spp. isolates were screened for methioninase activity using a phenol red-based rapid assay. Starch agar plates (pH 7.0) with 0.07% phenol red and methionine, the composition of which is mentioned in Table 3. were inoculated and incubated at 28°C for 48-72 hours. Methioninase-positive isolates caused a pink coloration around colonies, indicating ammonia release from methionine degradation. This method, adapted from Arfi et al. (2003) and Selim et al. (2015), was confirmed using modified media per William and Hariharan (2013). Table 3 Composition of Starch agar with 0.07% phenol red and 0.5% L-methionine S.No. Ingredients Quantity in Grams/Litre 1. Meat Extract 40 2. Soluble Starch 10 3. Methionine 5 4. Phenol red indicator 0.7 5. Agar 15 6. pH 7±0.2 All ingredients homogenised in per litre distilled water then sterilized and poured in sterile plates; Later, the methioninase +ve Streptomyces spp. isolates will be then subjected to molecular studies for sequencing of 16S rRNA for identification of Streptomyces spp. species. 2.4. Molecular Identification of Streptomyces spp. 2.4.1. DNA Extraction The Streptomyces spp. isolates positive for methioninase activity were identified using 16S rRNA gene analysis. Genomic DNA was extracted using a modified CTAB method (Kieser et al., 2000; Sambrook & Russell, 2001). Isolates were cultured on starch nitrate agar at 28°C for 5-7 days, transferred to broth, and incubated with shaking for 48-72 hours. Cell pellets were harvested, washed with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and treated with 10 mg/mL lysozyme. Lysis was performed using 10% SDS and prewarmed CTAB buffer (2% CTAB, 100 mM Tris-HCl, 20 mM EDTA, 1.4 M NaCl, 0.5% β-mercaptoethanol, pH 8.0). DNA was extracted using chloroform:isoamyl alcohol (24:1, v/v), treated with RNase A (10 mg/mL), and precipitated with isopropanol. The DNA pellet was washed with 70% ethanol, air-dried, and dissolved in TE buffer. Quality was confirmed via 0.8% agarose gel, and DNA was used for 16S rRNA amplification. 2.4.2. Amplification of 16S rDNA and Phylogenetic Analysis The 16S rRNA gene from Streptomyces spp. isolates was amplified using universal primers 27F (5'- AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') procured from BioServe Biotechnologies. The PCR reaction mixture included 2 µL genomic DNA, 2 µL primers, 5 µL 10X assay buffer, 5 µL MgCl₂ (25 mM), 5 µL dNTP mix (2.5 mM), 0.5 µL Taq DNA polymerase, and 30.5 µL molecular-grade water, forming a 50 µL volume. PCR was conducted in a Prima-96 Thermal Cycler with conditions: initial denaturation at 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 45°C for 1 minute, and 72°C for 30 seconds, followed by final extension at 72°C for 10 minutes. Amplified products were confirmed on 1% agarose gel. Partial sequencing was outsourced to Biokart, Bangalore, and analyzed via NCBI BLAST. Phylogenetic analysis was performed using MEGA 11 with CLUSTAL W alignment to construct a phylogenetic tree. 3. Results and Discussion 3.1. Enumeration of Target Microbial Species The CFU count from various soil samples diluted at 10⁻⁷ (see Table 4) demonstrates significant microbial diversity, with Kamla Park (348 CFU) having the highest microbial presence, followed by Kaliasot Dam (199 CFU) and Boat Club (157 CFU). The lowest counts were observed at Bairagarh (104 CFU) and Van Vihar National Park (15 CFU). These findings suggest that nutrient-rich environments, such as parks and water-proximal areas, support a higher prevalence of potential methioninase-producing Streptomyces spp., consistent with their role in anticancer enzyme production. Such areas, often rich in organic matter, provide ideal conditions for microbial proliferation. Similar patterns have been noted in studies indicating that organic content influences microbial community structures and enzyme production potential World Journal of Biology Pharmacy and Health Sciences, 2025, 22(02), 290-300 293 (Xu et al., 2020; Arfi et al., 2003). This reinforces the suitability of these environments for isolating Streptomyces with biotechnological applications. Table 4 Culture response on primary culture plates S. No. Master Plate Code Sample Dilution Used CFU Count 1. A Van Vihar National Park 15 2. B Boat Club 36 3. C Kamla Park 348 4. D Kaliasot Dam 199 5. E Bairagarh 104 6. F Van Vihar National Park 117 7. G Boat Club 157 8. H Kamla Park 259 9. I Kaliasot Dam 11 10. J Bairagarh 53 3.2. Confirmation of Methioninase Production The screening of 20 Streptomyces spp. isolates through rapid assay for methioninase production revealed that only seven isolates (S2, S3, S6, S7, S9, S13, and S17) exhibited positive activity, while the remaining 13 isolates were negative in present study (see Table 5). This result aligns with studies by Selim et al. (2015), who noted variability in methioninase activity among Streptomyces isolates, with only 30% demonstrating extracellular enzyme production. Similarly, Peela and Porana, (2017) emphasized that methioninase production is strain-specific and often influenced by environmental and cultural factors. Furthermore, findings by El-Sayed, et al. (2010) highlight the importance of targeted isolation strategies to identify potent methioninase producers for therapeutic applications. This study underscores the potential of selected isolates for further optimization and molecular characterization to enhance methioninase production, a promising enzyme for applications such as cancer therapy. Table 5 Response of pure indigenous Streptomyces spp. isolates for methioninase production activity S. No. Isolate Code Methioninase production activity 1. S1 –Ve 2. S2 +Ve 3. S3 +Ve 4. S4 –Ve 5. S5 –Ve 6. S6 +Ve 7. S7 +Ve 8. S8 –Ve 9. S9 +Ve 10. S10 –Ve 11. S11 –Ve 12. S12 –Ve 13. S13 +Ve World Journal of Biology Pharmacy and Health Sciences, 2025, 22(02), 290-300 294 14. S14 –Ve 15. S15 –Ve 16. S16 –Ve 17. S17 +Ve 18. S18 –Ve 19. S19 –Ve 20. S20 –Ve 3.3. Molecular Identification and Phylogenetic Analysis The 16S rRNA region of indigenous Streptomyces spp. isolates (S2, S3, S6, S7, S9, S13, and S17) was successfully amplified, producing a sharp 1500 bp band on 1% agarose gel. These amplified fragments were then subjected to partial genome sequencing using Sanger’s method, with sequencing services provided by Biokart India Pvt. Ltd., Bangalore. The resulting sequences were analyzed for homology and identity using the NCBI BLAST tool, and the results are summarized in Table 6 and Table 7. Table 6 The 16S rRNA sequences of Streptomyces spp. isolates obtained after partial Sanger’s sequencing S. No. Isolate Code Sequencing ID Obtained Sequence 1. S2 >_S2_2023_27F_F01.ab 1 GGTTCGAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGC CTATCAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCG ACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACAC TGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG CAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGC AGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTA AACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTAC CTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCC GCGGTAATACGTAGGGCGCAAGCGTTGTCCAAAATTAT TGGGCGTAAAGAGCTCGTAGGCGGTTTGTCACGTCGGT TGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGA TACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAAT TCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGA ACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGA CGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTA GATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTA GGTGTGGGCAACATTCCCCGTTGTCCGTGCCGCAGCTA ACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAG GCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGC GGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAA CCTTACCAAGGCTTGACATACACCGGAAAACCCTGGAG ACAGGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCAT GGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAG TCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGC AGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCG GGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTC ATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACA ATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAG CGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTC TGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATC GCAGATCAGCATTGCTAAACT 2. S3 >_S3_2023_27F_ E02.ab1 AGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGT AGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGA GACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGA World Journal of Biology Pharmacy and Health Sciences, 2025, 22(02), 290-300 295 ATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCC GCGTGAGGGATGACGGCCTTCGGGTTCCAAACCTCTTT CAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAA GAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTA AAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAG CCCGGGTTTTAACCCCGGGTCTGCAGTCGATACGGGCA GGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGT AGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGT GGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGG AGCGAAAGCGTGGGGAGCGAACAGGATTGAGTACCCT GGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGG CAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTA AGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAAC TCCAAGGGGTTGACGGGGGCCCGCACAAGCGGCGGAGC ATGTGGCTTAATTCGACGCAACGCGAACCCCCTTACCA AGGCTTGACATACACCGGAAAGCATCAGAGATGGTGCC CCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCG TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGTCCCGTGTTGCCAGCAAGCCCTT CGCCGTGTTGGGGACTCCCGGGAGACCGCCGGGGTCAA CT 3. S6 >_S6_2023_27F_C03.ab 1 GGGGTCTAATACCGGATGACACTTTCTCTCGCATGGGA GAAGGTTGAAAGCTCCGGCGGTGCAGGATGAGCCCGCG GCCTATCAGCTAGTTGGTGAGGTAGAAGCTCACCAAGG CGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCAC ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC AGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGA TGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTT GTAAACCTCTTTCAGCAGGGAATTAGCGAAAGTGACGG TACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCA GCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAAT TATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTC GGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGT CGATACGGGCAGGCTAGAGTGTGGTAGGGGAGATCGG AATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGA GGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTAC TGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGA TTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGAA CTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAG CTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGC AAGGCTAAAACTCAAAGGAATTGACGGGCCCGGGCACA AGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAA GAACCTTACCAAGGCTTGACATCGCCCGGAAAGCCGTA GAGATACGGCCCCCCTTGTGGTCGGGTGACAGGTGGTG CATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCC AGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACT GCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAA GTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCT ACAATGGCAGGTACAATGAGCTGCGAAGCCGCGAGGCG GAGCGAATCTCAAAAAGCCTGTCTCAGTTCGGATTGGG GTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTA A 4. S7 >_S7_2023_27F_D01.ab 1 GTCTCCGTGTGGAAAGCTCCGGCGGTGCAGGATGAGCC CGCGGCCTATCAGCTTGAAGGTGGGGTGATGGCCTACC AAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGG CCACACTGGGACTGAGACACGGCCCAGACTCCTACGGG World Journal of Biology Pharmacy and Health Sciences, 2025, 22(02), 290-300 296 AGGCAGCAGTGGGGAATATTGCCCAATGGGCGCAAGCC TGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGG GTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCGAGTGA CGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCCA GCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGG AATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGC GTCGGATGTGAAAGCCCGGGGCTTAACCCCGGGTCTGC AAACGATACGGGCAGGCTAGAGTTCGGCAGGGGAGAT TAAAATTGGTGGTGTAGCGGTGAAATGCGCAGATATC AGGAGGAACACCGGTGGCGAAGGCGGATCTCTGCCCCG ATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAAC AGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTG GGCACTAGGTGTGGGCGGCATTCCACGTCGTCCGTGCC GCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGG CCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCG CACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACG CGAAGAACCTTACCAAGGCTTGACATACACCGGAAAGC CGTAGAGATACGGCCCCCCTTGTGGTCGGTGTACAGGT GGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTG GGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGT TGCCAGCAACTCCTTTCGGGGAGGTTGGGACTCACGGG AGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGAC GTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACAC GTGCTACAATGGCCGGTACAATGAGCTGCGATGCCGTG AGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGA TTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGC TAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTT CCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAG TCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGG GAGGGAGTCGTCGAAGGTTT 5. S9 >_S9_2023_27F_A04.ab 1 GTAGCCCGGCCTGAGAGGGCGACCGGCCACACTGGGAC TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGG GGAATATTGCACAATGGCGAAAGCCTGATGCAGCGACG CCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCT TTCAGCAGGGAAGAAGCCCGAGTGACGGTACCTGCAGA AGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAA TACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGT AAAGAGCTCGCAGGCGGCTTGTCACGTCGGATGTGAAA GCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGC TAGCTAGAGTGTGGTAGGGGAGATCGGAATTCCTGGT GTAGCGGTGAAATGCGCAGATATCACCCGGAACACCGG TGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAG GAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCC TGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTG GCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATT AAGTTCCCCGCTGGGGAGTNCGGGCGCAAGGCTAAAAC TCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGC ATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCA AGGCTTGACATATACCGGAAAGCATCAGAGATGGTGCC CCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCG TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTT CGGGGTGATGGGGACTCACAGGAGACTGCCGGGGTCAA CTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCC CCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGG TACAATGAGCTGCGATGCCGCGAGGCGGAGCGAATCTC AAAAAGCCGGTCTCAGTTCGGATTGGGGT World Journal of Biology Pharmacy and Health Sciences, 2025, 22(02), 290-300 297 6. S13 >_S13_2023_27F_B01.a b1 GGGACGGGGTTAAAAGCTCCGGCGGTGAAGGATGAGCC CGCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACC AAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGG CCACACTGGGACTGAGACACGGCCCAGACTCCTACGGG AGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGC CTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCG GGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGT GACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGC CAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCC GGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTC ACGTCGGATGTGAAAGCCCGGGGCTTAACCCCGGGTCT GCATTCGATACGGGCTAGCTAGAGTGTGGTAGGGGAG ATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATAT CAAAAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCC ATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAA CAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTT GGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGC CGCAGCTAACGCATTAAGAACCCCGCCTGGCCAGTACG GCCGCAAGGCTAAAACTCAAAGGAATTGACGGGCCCCC GCACAAGCAGCGGAGCATGTGGCAAAATTCGACGCAAC GCGAAGAACCTTACCAAGGCAAGACATATACCGGTTTG CATCAGAGATGGTGCCCCCCTTGTGGTCGGTATACAGG TGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTT GGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTG TTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGG AGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGAC GTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACAC GTGCTACAATGGCCGGTACAATGAGCTGCGATGTCGCA AGGCGGAGCGAATCTCAAAAAGCCGG 7. S17 >_S17_2023_27F_H01.a b1 CTCATGGGGGACGGTTGAAAGCTCCGGCGGTGCAGGAT GAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGC CTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCG ACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCT ACGGGAGGCAGCAGTGGGGAAAATTGCACAATGGGCG AAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGC CTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGA AAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTAC GTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTT GTCCGGAATTATTGCCTGTAAAGAGCTCGTAGGCGGTC TGTCACGTCGGGTGTGAAAGCCCGGGGCTTAACCCCGG GTCTGCATTCGATACGGGCAGACTAGAGTGTGGTAGGG GAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGA TATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGG GCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCG AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACG TTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGT GCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTA CGGCCGCAAGGCTAAAACTCAAAGGAATTGACGCGGGC CCGCACAAGCAGCCCAGCATGTGGCTTAATTCGACGCA ACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAA AGCATCAGAGATGGTGCCCGGGTTGTGGTCGGTGTACA GGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATG TTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTG TGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACA GGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACG ACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCAC ACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCG CAAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAG World Journal of Biology Pharmacy and Health Sciences, 2025, 22(02), 290-300 298 Table 7 Closest resemblance of indigenous Streptomyces spp. isolates with reference to 16S rRNA region sequence analysis using NCBI BLAST tool. S. No. Streptomyces spp. Isolate Code Closest Neighbour upon Alignment Accession Percentage Resemblance 1. S2 Streptomyces mutabilis strain NBRC 12800 NR_112281.1 99.65% 2. S3 Streptomyces thinghirensis strain S10 NR_116901.1 98.36% 3. S6 Streptomyces rectiviolaceus strain NBRC 100765 NR_112590.1 99.49% 4. S7 Streptomyces albus strain DSM 40313 NR_025615.1 99.13% 5. S9 Streptomyces griseus strain KACC 20084 NR_042791.1 99.51% 6. S13 Streptomyces griseolus strain NBRC 3719 NR_112493.1 98.62% 7. S17 Streptomyces niveus strain NRRL 2466 NR_115784.1 99.00% Table 7 shows the closest relatives of indigenous Streptomyces spp. isolates based on 16S rRNA sequence analysis using the NCBI BLAST tool. The isolates, including S2 (Streptomyces mutabilis), S3 (Streptomyces thinghirensis), and others, exhibit high sequence similarity, ranging from 98.36% to 99.65%, with the highest similarity observed for isolate S2. These results confirm the close genetic relationship of the isolates with known Streptomyces strains. Figure 1 Phylogenetic tree generated among 7 different indigenous Streptomyces spp. isolates from pooled data of nucleotides by Test Neighbor-Joining statistical method through MEGA 11 software version 11.0.13 / CLUSTAL W The phylogenetic analysis of seven indigenous Streptomyces spp. isolates was conducted using MEGA 11 software, employing the neighbor-joining method with 1000 bootstrap replications. The isolates, identified as Streptomyces griseus, S. griseolus, S. niveus, S. rectiviolaceus, S. albus, S. mitabilis, and S. thinghirensis, formed two major clades. The first clade included S. griseus and S. griseolus, which exhibited a close evolutionary relationship, supported by high bootstrap values (91). S. griseus is known for its methioninase production, which has biotechnological relevance, especially in cancer therapy (Tan et al., 2010). The second clade consisted of S. albus, S. mitabilis, and S. thinghirensis,