High-yield production of cytotoxic actinomycin D from endophytic Streptomyces parvulus AL036 through nutritional optimization

Thongchai Taechowisan Thanaporn Chuen-Im Phonthep Kunkasikorn Waya S. Phutdhawong   

Open Access   

Published:  Oct 30, 2025

DOI: 10.7324/JABB.2026.248963
Abstract

Endophytic actinomycetes are bacteria that live inside plant tissues without causing harm to the plant, producing various bioactive compounds. This study aimed to isolate and characterize an endophytic actinomycete from Alpinia galanga roots, and to investigate its bioactive compounds. A Streptomyces strain designated AL036 was isolated and identified as Streptomyces parvulus through morphological, biochemical, and 16S rDNA sequence analysis. Similar to S. parvulus Tc022, this strain exhibited actinomycin D as its major bioactive compound. Media optimization experiments and cytotoxic activity against non-cancerous and cancer cell lines were carried out. The starch casein medium was identified as a medium supporting significantly higher actinomycin D production (103.67 mg/L) compared to the initial medium International Streptomyces Project medium 2. Further optimization revealed that a specific carbon-to-nitrogen ratio (20 g/L soluble starch and 2 g/L casein) maximized production (199.33 mg/L). Sugar supplementation did not enhance production but influenced mycelial growth. The purified actinomycin D exhibited cytotoxicity against HeLa cells and LLC-MK2 cells, with IC50 values of 10.22 ± 2.53 μg/mL and 17.78 ± 3.97 μg/mL, respectively. This study isolated a new S. parvulus AL036, with efficient actinomycin D production similar to Tc022. Media optimization significantly improved production, highlighting the importance of carbon and nitrogen sources. Both crude extract and purified actinomycin D showed cytotoxicity and selectivity for some cancer cells.


Keyword:     Actinomycin D production Alpinia galanga Cytotoxic activity Endophytic actinomycetes Streptomyces parvulus AL036


Citation:

Taechowisan T, Chuen-im T, Kunkasikorn P, Phutdhawong WS. High-yield production of cytotoxic actinomycin D from endophytic Streptomyces parvulus AL036 through nutritional optimization. J Appl Biol Biotech 2025. Article in Press. http://doi.org/10.7324/JABB.2026.248963

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

HTML Full Text
Reference

1. Liu H, Carvalhais LC, Crawford M, Singh E, Dennis PG, Pieterse CM, et al. Inner plant values: Diversity, colonization and benefits from endophytic bacteria. Front Microbiol. 2017;8:2552. https://doi.org/10.3389/fmicb.2017.02552

2. Golinska P, Wypij M, Agarkar G, Rathod D, Dahm H, Rai M. Endophytic actinobacteria of medicinal plants: Diversity and bioactivity. Antonie Van Leeuwenhoek. 2015;108(2):267-89. https://doi.org/10.1007/s10482-015-0502-7

3. Singh R, Dubey AK. Diversity and applications of endophytic actinobacteria of plants in special and other ecological niches. Front Microbiol. 2018;9:1767. https://doi.org/10.3389/fmicb.2018.01767

4. Mauger AB, Thomas WA. NMR studies of actinomycins varying at the proline sites. Org Magn Reson. 1981;17(3):186-90. https://doi.org/10.1002/mrc.1270170311

5. Kurosawa K, Bui VP, VanEssendelft JL, Willis LB, Lessard PA, Ghiviriga I, et al. Characterization of Streptomyces MITKK-103, a newly isolated actinomycin X2-producer. Appl Microbiol Biotechnol. 2006;72(1):145-54. https://doi.org/10.1007/s00253-005-0240-2

6. Wagman GH, Marquez JA, Watkins PD, Gentile F, Murawski A, Patel M, et al. A new actinomycin complex produced by a Micromonospora species: Fermentation, isolation, and characterization. Antimicrob Agents Chemother. 1976;9:465-9. https://doi.org/10.1128/AAC.9.3.465

7. Katz E, Pugh LH. Variations on a theme. The actinomycins. Appl Microbiol. 1961;9(3):263-7. https://doi.org/10.1128/am.9.3.263- 267.1961

8. Praveen V, Tripathi CK, Bihari V. Studies on optimum fermentation conditions for actinomycin-D production by two new strains of Streptomyces spp. Med Chem Res. 2008;17:114-22. https://doi.org/10.1007/s00044-007-9042-7

9. Goldberg IH, Rabinowitz M, Reich E. Basis of actinomycin action. I. DNA binding and inhibition of RNA-polymerase synthetic reactions by actinomycin. Proc Natl Acad Sci USA. 1962;48(12):2094-101. https://doi.org/10.1073/pnas.48.12.2094

10. Koba M, Konopa J.Actinomycin D and its mechanisms of action. Postepy Hig Med Dosw. 2005;59:290-8.

11. Taechowisan T, Wanbanjob A, Tuntiwachwuttikul P, Taylor W. Identification of Streptomyces sp. Tc022, an endophyte in Alpinia galanga, and the isolation of actinomycin D. Ann Microbiol. 2006;56(2):113-7. https://doi.org/10.1007/BF03174991

12. Otoguro M, Hayakawa M, Yamazaki T, Iimura Y. An integrated method for the enrichment and selective isolation of Actinokineospora spp. in soil and plant litter. J Appl Microbiol. 2001;91(1):118-30. https://doi.org/10.1046/j.1365-2672.2001.01372.x

13. Taechowisan T, Chuen-Im T, Phutdhawong WS. Antibacterial and anticancer properties of endophenazines from Streptomyces prasinus ZO16, an endophyte in Zingiber officinale Rosc. Pak J Biol Sci. 2024;27(9):469-78. https://doi.org/10.3923/pjbs.2024.469.478

14. Meerloo JV, Kaspers GJ, Cloos J.Cell sensitivity assays: The MTT assay. Methods Mol Biol. 2011;731:237-45. https://doi.org/10.1007/978-1-61779-080-5_20

15. Williams WK, Katz E. Development of a chemically defined medium for the synthesis of actinomycin D by Streptomyces parvulus. Antimicrob Agents Chemother. 1977;11(2):281-90. https://doi.org/10.1128/AAC.11.2.281

16. Sousa MF, Lopes CE, Pereira N Jr. Development of a bioprocess for the production of actinomycin D. Braz J Chem Eng. 2002;19(3):277- 85. https://doi.org/10.1590/S0104-66322002000300002

17. Olmos E, Mehmood N, Haj Husein L, Goergen JL, Fick M, Delaunay S. Effects of bioreactor hydrodynamics on the physiology of Streptomyces. Bioprocess Biosyst Eng. 2013;36(3):259-72. https://doi.org/10.1007/s00449-012-0794-1

18. Praveen V, Tripathi D, Tripathi CK, Bihari V. Nutritional regulation of actinomycin-D production by a new isolate of Streptomyces sindenensis using statistical methods. Indian J Exp Biol. 2008;46(2):138-44.

19. Gallo M, Katz E. Regulation of secondary metabolite biosynthesis: Catabolite repression of phenoxazinone synthase and actinomycin formation by glucose. J Bacteriol. 1972;109(2):659-67. https://doi.org/10.1128/jb.109.2.659-667.1972

20. Sanchez S, Demain AL. Metabolic regulation and overproduction of primary metabolites. Microb Biotechnol. 2008;1(4):283-319. https://doi.org/10.1111/j.1751-7915.2007.00015.x

21. Barry CE, Nayar PG, Begley TP. Phenoxazinone synthase: Mechanism for the formation of the phenoxazinone chromophore of actinomycin. Biochemistry. 1989;28(15):6323-33. https://doi.org/10.1021/bi00441a026

Article Metrics
18 Views 3 Downloads 21 Total

Year

Month

Related Search

By author names