Whole genome surveying and metabolomic profiling aided navigation of therapeutic plethora of Streptomyces melanogenes WPF1 isolated from Pinus patula rhizosphere

Abstract

Rhizosphere microbial diversity from Western Ghats remains a relatively less explored source of therapeutic agents. Targeted screening of actinomycetes from an under studied area led to the isolation of 24 actinomycetes from Pinus patula rhizosphere. Screening led to identification of Streptomyces melanogenes WPF1 with strong therapeutic potential. Delineation of the whole genome of WPF1 and metabolic profiling of the partially purified active fraction (PF10) led to confirmation of kinamycin D, oleic acid, palmitic acid and other components. These molecules in unison can be correlated to the recorded biological activities of the active fraction. AntiSMASH analysis of the WPF1 genome identified 26 biosynthetic gene clusters (BGCs) associated with secondary metabolite biosynthesis, including one cluster exhibiting over 97% similarity to known Kinamycin biosynthetic pathway. LC–MS and high-resolution mass spectrometry (HRMS) analyses revealed presence of Kinamycin D in the partially purified fraction PF10. PF10 demonstrated selective antibacterial activity against Gram-positive human pathogens and effectively inhibited their biofilm formation. Additionally, PF10 suppressed the proliferation of cancer cell lines, with IC₅₀ values ranging from 2.0 to 2.5 µg/mL, and induced apoptosis, as evidenced by Poly (ADP-ribose) polymerase (PARP) cleavage, activation of caspase 3 and caspase 9 and Bax protein accumulation. These findings highlight S. melanogenes WPF1 as a promising source of bioactive secondary metabolites, and importance of rhizosphere microbial diversity for potential therapeutic discovery.

Graphical abstract

Keywords

Streptomyces melanogenes / Antimicrobial / Anticancer / Kinamycin D / antiSMASH / GC–MS / LC–MS

References

[1] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol, 1990, 215: 403-410.

[2] Andrews S (2010) FastQC: a quality control tool for high throughput sequence data.

[3] Avire NJ, Whiley H, Ross K. A review of Streptococcus pyogenes: public health risk factors, prevention and control. Pathogens, 2021, 10(2): 248.

[4] Bavelloni A, Focaccia E, Piazzi M, Errani C, Blalock W, Faenza I. Cell cycle arrest and apoptosis induced by kinamycin F in human osteosarcoma cells. Anticancer Res, 2017, 37(8): 4103-4109.

[5] Bellotti D, Remelli M. Deferoxamine B: a natural, excellent and versatile metal chelator. Molecules, 2021, 26(11): 3255

[6] Benov L. Effect of growth media on the MTT colorimetric assay in bacteria. PLoS ONE, 2019, 14(8e0219713

[7] Benov L. Improved formazan dissolution for bacterial MTT assay. Microbiol Spectr, 2021, 9: e01637-e1721.

[8] Blin K, Shaw S, Steinke K, Villebro R, Ziemert N, Lee SY, Medema MH, Weber T. antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res, 2019, 47(W1): W81-W87.

[9] Braz VS, Melchior K, Moreira CG. Escherichia coli as a multifaceted pathogenic and versatile bacterium. Front Cell Infect Microbiol, 2020, 10548492

[10] Challinor VL, Bode HB. Bioactive natural products from novel microbial sources. Ann N Y Acad Sci, 2015, 1354(1): 82-97.

[11] Chen S, Zhou Y, Chen Y, Gu J. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018, 34(17): i884-i890.

[12] Cruz CD, Shah S, Tammela P. Defining conditions for biofilm inhibition and eradication assays for Gram-positive clinical reference strains. BMC Microbiol, 2018, 18: 173

[13] Cunha MB, Jorge AF, Nunes MJ, Sousa JR, Lança MJ, Gomes da Silva M, Gaudêncio SP. GC/MS fatty acid profile of marine-derived actinomycetes from extreme environments: chemotaxonomic insights and biotechnological potential. Mar Drugs, 2024, 23(1): 1

[14] Dan VM, Sanawar R, Mohan GM, Cheriyan SP, Kumar TRS. Urdamycin V from Streptomyces sp induces p53 independent apoptosis in cervical cancer cells inconsiderate of HPV status and inhibited growth of Gram-positive human pathogens. Nat Prod Res:, 2024

[15] Deepthi KS, Salim S, Anugraha AS, Sugathan S. Whole genome sequence data of Streptomyces californicus TBG-201, a chitinolytic actinomycete isolated from the Vandanam sacred groves of Alleppey District, Kerala, India. Data Brief, 2023, 48109228

[16] Demain AL, Sanchez S. Microbial drug discovery: 80 years of progress. J Antibiot, 2009, 621): 5-16.

[17] Deng B, Kong W, Suo H, Shen X, Newton MA, Burkett WC, Zhao Z, John C, Sun W, Zhang X, Fan Y, Hao T, Zhou C, Bae-Jump VL. Oleic acid exhibits anti-proliferative and anti-invasive activities via the PTEN/AKT/mTOR pathway in endometrial cancer. Cancers (Basel), 2023, 15(22): 5407

[18] Desbois AP, Smith VJ. Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential. Appl Microbiol Biotechnol, 2010, 85: 1629-1642.

[19] Dilika F, Bremner PD, Meyer JJM. Antibacterial activity of linoleic and oleic acids isolated from Helichrysum pedunculatum: a plant used during circumcision rites. Fitoterapia, 2000, 71(4): 450-452.

[20] Easa PS, Rajesh KP. Forests and its biodiversity in the Western Ghats of Kerala. Ecohydrology of Kerala, 2025, Amsterdam. Elsevier77-95.

[21] Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics, 2016, 32(19): 3047-3048.

[22] Gama MG, Anagha B, Ratheesh S, Vipin DM, Pradeep CK. First metabolic profiling of Indian wild edible mushroom Amanita hemibapha reveals antimicrobial compounds that infers selective activity against Gram-positive human pathogens. Nat Prod Res, 2025

[23] Giulitti F, Petrungaro S, Mandatori S, Tomaipitinca L, De Franchis V, D'Amore A, Filippini A, Gaudio E, Ziparo E, Giampietri C. Anti-tumor effect of oleic acid in hepatocellular carcinoma cell lines via autophagy reduction. Front Cell Dev Biol, 2021, 9629182

[24] Grela E, Kozłowska J, Grabowiecka A. Current methodology of MTT assay in bacteria—a review. Acta Histochem, 2018, 120(4): 303-311.

[25] Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinformatics, 2013, 29(8): 1072-1075.

[26] Harald B. The antibiotic resistance crisis and the development of new antibiotics. Microb Biotechnol, 2024, 17(7e14510

[27] Harir M, Bendif H, Bellahcene M, Fortas Z, Pogni R (2018) Streptomyces secondary metabolites. In: Basic biology and applications of actinobacteria. IntechOpen, https://doi.org/10.5772/intechopen.79890

[28] Hasinoff BB, Wu X, Yalowich JC, Goodfellow V, Laufer RS, Adedayo O, Dmitrienko GI. Kinamycins A and C, bacterial metabolites that contain an unusual diazo group, as potential new anticancer agents: antiproliferative and cell cycle effects. Anticancer Res, 2006, 17(7): 825-837.

[29] Hata T, Omura S, Iwai Y, Nakagawa A, Otani M, Ito S, Matsuya T. A new antibiotic, kinamycin: fermentation, isolation, purification and properties. J Antibiot, 1971, 24(6): 353-359.

[30] Hatim S, Elena C, Alessia S, Paul H, Jacopo G, Enrica C, et al. . The WHO bacterial priority pathogens list 2024: a prioritisation study to guide research, development, and public health strategies against antimicrobial resistance. Lancet Infect Dis, 2025, 25(9): 1033.

[31] Held T, Kutzner HJ. Genetic recombination in Streptomyces michiganensis DSM 40015 revealed three genes responsible for the formation of melanin. J Basic Microbiol, 1991, 312): 127-134.

[32] Jolley KA, Bray JE, Maiden MC. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res, 2018, 3124

[33] Khaleel K (2016) Western Ghats—A hot spot of biodiversity. In: Western Ghats – From Ecology to Economics, p 54.

[34] Khan NA, Barthes N, McCormack G, O'Gara JP, Thomas OP, Boyd A. Sponge-derived fatty acids inhibit biofilm formation of MRSA and MSSA by down-regulating biofilm-related genes specific to each pathogen. J Appl Microbiol, 2023, 134(8lxad152

[35] Khorshed A, Arpita M, Suranjana S, Yi-Ming Z, et al. . Streptomyces: the biofactory of secondary metabolites. Front Microbiol, 2022, 13968053

[36] Kongue MD, Talontsi FM, Lamshöft M, Kenla TJ, Dittrich B, Kapche GD, Spiteller M. Sonhafouonic acid, a new cytotoxic and antifungal hopene-triterpenoid from Zehneria scabra camerunensis. Fitoterapia, 2013, 85: 176-180.

[37] Kumar P, Lee JH, Beyenal H, Lee J. Fatty acids as antibiofilm and antivirulence agents. Trends Microbiol, 2020, 28(9): 753-768.

[38] Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods, 2012, 9(4): 357-359.

[39] Letizia M, Diggle SP, Whiteley M. Pseudomonas aeruginosa: ecology, evolution, pathogenesis and antimicrobial susceptibility. Nat Rev Microbiol, 2025

[40] Li JM, Lin N, Zhang Y, Chen X, Liu Z, Lu R, Bian F, Liu H, Pflugfelder SC, Li DQ. Ectoine protects corneal epithelial survival and barrier from hyperosmotic stress by promoting anti-inflammatory cytokine IL-37. Ocul Surf, 2024, 32: 182-191.

[41] McGaw LJ, Jäger AK, Van Staden J. Antibacterial effects of fatty acids and related compounds from plants. S Afr J Bot, 2002, 68(4): 417-423.

[42] Meier-Kolthoff JP, Göker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun, 2019, 10(1): 2182.

[43] Meier-Kolthoff JP, Carbasse JS, Peinado-Olarte RL, Göker M. TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Res, 2022, 50D1): D801-D807.

[44] Miranda MF, Freddie B, Isabella S. The global cancer burden and human development: a review. Scand J Public Health, 2018, 46(1): 27-36.

[45] Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res, 2007, 35(suppl_2): W182-W185.

[46] Muralikrishnan B, Dan VM, Vinodh JS, Jamsheena V, Ramachandran R, Thomas S, Kumar RA. Anti-microbial activity of chrysomycin A produced by Streptomyces sp. against Mycobacterium tuberculosis. RSC Adv, 2017, 7(58): 36335-36339.

[47] Nirwati H, Damayanti E, Sholikhah EN, Mutofa M, Widada J. Soil-derived Streptomyces sp. GMR22 producing antibiofilm activity against Candida albicans: bioassay, untargeted LC-HRMS, and gene cluster analysis. Heliyon, 2022, 8(4e09333

[48] O'Hara KA, Wu X, Patel D, Liang H, Yalowich JC, Chen N, Goodfellow V, Adedayo O, Dmitrienko GI, Hasinoff BB. Mechanism of the cytotoxicity of the diazoparaquinone antitumor antibiotic kinamycin F. Free Radic Biol Med, 2007, 43(8): 1132-1144.

[49] Omura S, Nakagawa A, Yamada H, Hata T, Furusaki A, Watanabe T. Structures and biological properties of kinamycin A, B, C, and D. Chem Pharm Bull, 1973, 21(5): 931-940.

[50] Preuss HG, Echard B, Dadgar A, Talpur N, Manohar V, Enig M, Bagchi D, Ingram C. Effects of essential oils and monolaurin on Staphylococcus aureus: in vitro and in vivo studies. Toxicol Mech Methods, 2005, 15(4): 279-285.

[51] Proksee. https://proksee.ca/projects/new.

[52] Ramadan AMAA, Zidan SAH, Shehata RM, El-Sheikh HH, Ameen F, Stephenson SL, Al-Bedak OAHM. Antioxidant, antibacterial, and molecular docking of methyl ferulate and oleic acid produced by Aspergillus pseudodeflectus AUMC 15761 utilizing wheat bran. Sci Rep, 2024, 14(1): 3183

[53] Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics, 2014, 3014): 2068-2069.

[54] She P, Chen L, Liu H, Zou Y, Luo Z, Koronfel A, Wu Y. The effects of D-tyrosine combined with amikacin on the biofilms of Pseudomonas aeruginosa. Microb Pathog, 2015, 86: 38-44.

[55] Siddharth S, Vittal RR, Wink J, Steinert M. Diversity and bioactive potential of actinobacteria from unexplored regions of Western Ghats, India. Microorganisms, 2020, 8(2225

[56] Smitka TA, Bonjouklian R, Perun TJ, Hunt AH, Foster RS, Mynderse JS, Yao RC. A83016A, a new kinamycin type antibiotic. J Antibiot, 1992, 45(4): 581-583.

[57] Song L, Wang F, Liu C, Guan Z, Wang M, Zhong R, et al. . Isolation and evaluation of Streptomyces melanogenes YBS22 with potential application for biocontrol of rice blast disease. Microorganisms, 2023, 11(12): 2988

[58] Sugawara R, Onuma M. Melanomycin, a new antitumor substance from Streptomyces. II. Description of the strain. J Antibiot Ser A, 1957

[59] Tesser R, Vitiello R, Russo V, Turco R, Di Serio M, Lin L, Li C. Oleochemistry products. Industrial Oil Plant: Application Principles and Green Technologies, 2020, Singapore. Springer201-268.

[60] Wang X, Zhang C, Bao N. Molecular mechanism of palmitic acid and its derivatives in tumor progression. Front Oncol, 2023, 13: 1224125

[61] Waterhouse RM, Seppey M, Simão FA, Manni M, Ioannidis P, Klioutchnikov G, Kriventseva EV, Zdobnov EM. Busco applications from quality assessments to gene prediction and phylogenomics. Mol Biol Evol, 2018, 35(3): 543-548.

[62] Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol, 2017, 136e1005595

[63] Wu S, Zhu Z, Fu L, Niu B, Li W. WebMGA: a customizable web server for fast metagenomic sequence analysis. BMC Genomics, 2011, 12: 444.

[64] Yoon BK, Jackman JA, Valle-González ER, Cho NJ. Antibacterial free fatty acids and monoglycerides: biological activities, experimental testing, and therapeutic applications. Int J Mol Sci, 2018, 194): 1114

[65] Zhaochao W, Juanjuan Y, Chenjie W, Hua Yi, Hong W, Jianwei C. The deep mining era: genomic, metabolomic, and integrative approaches to microbial natural products from 2018 to 2024. Mar Drugs, 2025, 23(7): 261

Funding

Manipal Academy of Higher Education, Manipal

RIGHTS & PERMISSIONS

The Author(s)