Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments

Autoři: Alba Noël aff001;  Gwendoline Van Soen aff001;  Isabelle Rouaud aff001;  Eric Hitti aff002;  Sophie Tomasi aff001
Působiště autorů: Univ Rennes, CNRS, ISCR–UMR 6226, Rennes, France aff001;  LTSI, UMR_S 1099, UFR Sciences Pharmaceutiques et Biologiques, Rennes, France aff002
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227816


In the context of research for new cytotoxic compounds, obtaining bioactive molecules from renewable sources remain a big challenge. Microorganisms and more specifically Actinobacteria from original sources are well known for their biotechnological potential and are hotspots for the discovery of new bioactive compounds. The strain DP94 studied here had shown an interesting cytotoxic activity of its culture broth (HaCaT: IC50 = 8.0 ± 1.5 μg/mL; B16: IC50 = 4.6 ± 1.8 μg/mL), which could not been explained by the compounds isolated in a previous work. The increase of the cytotoxic activity of extracts was investigated, based on a Taguchi L9 orthogonal array design, after DP94 culture in TY medium using two different vessels (bioreactor or Erlenmeyer flasks). Various culture parameters such as temperature, pH and inoculum ratio (%) were studied. For experiments conducted in a bioreactor, stirring speed was included as an additional parameter. Significant differences in the cytotoxic activities of different extracts on B16 melanoma cancer cell lines, highlighted the influence of culture temperature on the production of cytotoxic compound(s) using a bioreactor. A culture in Erlenmeyer flasks was also performed and afforded an increase of the production of the active compounds. The best conditions for the highest cytotoxicity (IC50 on B16: 6 ± 0.5 μg/mL) and the highest yield (202.0 mg/L) were identified as: pH 6, temperature 37°C and 5% inoculum.

Klíčová slova:

Analysis of variance – Bacterial growth – Cytotoxicity – Experimental design – High performance liquid chromatography – Lichenology – B16 cells – Nocardia


1. Barka EA, Vatsa P, Sanchez L, Gaveau-Vaillant N, Jacquard C, Klenk H-P, et al. Taxonomy, Physiology, and Natural Products of Actinobacteria. Microbiol Mol Biol Rev. 2016;80: 1–43. doi: 10.1128/MMBR.00019-15 26609051

2. Burg RW, Miller BM, Baker EE, Birnbaum J, Currie SA, Hartman R, et al. Avermectins, New Family of Potent Anthelmintic Agents: Producing Organism and Fermentation. Antimicrob Agents Chemother. 1979;15: 361–367. doi: 10.1128/aac.15.3.361 464561

3. Linke HA, Mechlinski W, Schaffner CP. Production of amphotericin B-14C by Streptomyces nodosus fermentation, and preparation of the amphotericin B-14C-methyl-ester. J Antibiot (Tokyo). 1974;27: 155–160.

4. Lacal JC, Vázquez D, Fernandez-Sousa JM, Carrasco L. Antibiotics that specifically block translation in virus-infected cells. J Antibiot (Tokyo). 1980;33: 441–446.

5. Umezawa H, Ishizuka M, Maeda K, Takeuchi T. Studies on bleomycin. Cancer. 1967;20: 891–895. doi: 10.1002/1097-0142(1967)20:5<891::aid-cncr2820200550>3.0.co;2-v 5337399

6. Nemoto A, Hoshino Y, Yazawa K, Ando A, Mikami Y, Komaki H, et al. Asterobactin, a new siderophore group antibiotic from Nocardia asteroides. J Antibiot (Tokyo). 2002;55: 593–597.

7. Bilyk O, Luzhetskyy A. Metabolic engineering of natural product biosynthesis in actinobacteria. Curr Opin Biotechnol. 2016;42: 98–107. doi: 10.1016/j.copbio.2016.03.008 27058643

8. Axenov-Gibanov DV, Voytsekhovskaya IV, Tokovenko BT, Protasov ES, Gamaiunov SV, Rebets Y, et al. Actinobacteria Isolated from an Underground Lake and Moonmilk Speleothem from the Biggest Conglomeratic Karstic Cave in Siberia as Sources of Novel Biologically Active Compounds. PLOS ONE. 2016;11. doi: 10.1371/journal.pone.0149216 26901168

9. Aschenbrenner IA, Cernava T, Berg G, Grube M. Understanding Microbial Multi-Species Symbioses. Front Microbiol. 2016;7. doi: 10.3389/fmicb.2016.00180 26925047

10. Suzuki MT, Parrot D, Berg G, Grube M, Tomasi S. Lichens as natural sources of biotechnologically relevant bacteria. Appl Microbiol Biotechnol. 2016;100: 583–595. doi: 10.1007/s00253-015-7114-z 26549239

11. Selbmann L, Zucconi L, Ruisi S, Grube M, Cardinale M, Onofri S. Culturable bacteria associated with Antarctic lichens: affiliation and psychrotolerance. Polar Biol. 2010;33: 71–83. doi: 10.1007/s00300-009-0686-2

12. Parrot D, Antony-Babu S, Intertaglia L, Grube M, Tomasi S, Suzuki MT. Littoral lichens as a novel source of potentially bioactive Actinobacteria. Sci Rep. 2015;5: 15839. doi: 10.1038/srep15839 26514347

13. Bates ST, Cropsey GWG, Caporaso JG, Knight R, Fierer N. Bacterial Communities Associated with the Lichen Symbiosis. Appl Environ Microbiol. 2011;77: 1309–1314. doi: 10.1128/AEM.02257-10 21169444

14. Grube M, Cardinale M, de Castro JV, Müller H, Berg G. Species-specific structural and functional diversity of bacterial communities in lichen symbioses. ISME J. 2009;3: 1105–1115. doi: 10.1038/ismej.2009.63 19554038

15. Parrot D, Legrave N, Delmail D, Grube M, Suzuki M, Tomasi S. Review–Lichen-Associated Bacteria as a Hot Spot of Chemodiversity: Focus on Uncialamycin, a Promising Compound for Future Medicinal Applications. Planta Med. 2016;82: 1143–1152. doi: 10.1055/s-0042-105571 27220082

16. Noël A, Ferron S, Rouaud I, Gouault N, Hurvois J-P, Tomasi S. Isolation and Structure Identification of Novel Brominated Diketopiperazines from Nocardia ignorata—A Lichen-Associated Actinobacterium. Molecules. 2017;22: 371. doi: 10.3390/molecules22030371 28264516

17. Bhattacharya A, Das S, Majumder P, Batish A. Estimating the effect of cutting parameters on surface finish and power consumption during high speed machining of AISI 1045 steel using Taguchi design and ANOVA. Prod Eng. 2009;3: 31–40.

18. Kim O-S, Cho Y-J, Lee K, Yoon S-H, Kim M, Na H, et al. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol. 2012;62: 716–721. doi: 10.1099/ijs.0.038075-0 22140171

19. Shirling EB, Gottlieb D. Methods for characterization of Streptomyces species1. Int J Syst Evol Microbiol. 1966;16: 313–340. doi: 10.1099/00207713-16-3-313

20. Bézivin C, Tomasi S, Lohézic-Le Dévéhat F, Boustie J. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine. 2003;10: 499–503. doi: 10.1078/094471103322331458 13678234

21. Cho JY, Kang JY, Hong YK, Baek HH, Shin HW, Kim MS. Isolation and Structural Determination of the Antifouling Diketopiperazines from Marine-Derived Streptomyces praecox 291–11. Biosci Biotechnol Biochem. 2012;76: 1116–1121. doi: 10.1271/bbb.110943 22790932

22. Perrone P, Daverio F, Valente R, Rajyaguru S, Martin JA, Lévêque V, et al. First Example of Phosphoramidate Approach Applied to a 4‘-Substituted Purine Nucleoside (4‘-Azidoadenosine): Conversion of an Inactive Nucleoside to a Submicromolar Compound versus Hepatitis C Virus. J Med Chem. 2007;50: 5463–5470. doi: 10.1021/jm070362i 17914786

23. Bode HB, Bethe B, Höfs R, Zeeck A. Big Effects from Small Changes: Possible Ways to Explore Nature’s Chemical Diversity. ChemBioChem. 2002;3: 619–627. doi: 10.1002/1439-7633(20020703)3:7<619::AID-CBIC619>3.0.CO;2-9 12324995

24. Parrot D, Intertaglia L, Jehan P, Grube M, Suzuki MT, Tomasi S. Chemical analysis of the Alphaproteobacterium strain MOLA1416 associated with the marine lichen Lichina pygmaea. Phytochemistry. 2018;145: 57–67. doi: 10.1016/j.phytochem.2017.10.005 29091816

25. Abdelmohsen UR, Cheng C, Viegelmann C, Zhang T, Grkovic T, Ahmed S, et al. Dereplication Strategies for Targeted Isolation of New Antitrypanosomal Actinosporins A and B from a Marine Sponge Associated-Actinokineospora sp. EG49. Mar Drugs. 2014;12: 1220–1244. doi: 10.3390/md12031220 24663112

26. Schrader KK, Harries MD, Page PN. Temperature effects on biomass, geosmin, and 2-methylisoborneol production and cellular activity by Nocardia spp. and Streptomyces spp. isolated from rainbow trout recirculating aquaculture systems. J Ind Microbiol Biotechnol. 2015;42: 759–767. doi: 10.1007/s10295-015-1600-2 25724337

27. Dutta S, Basak B, Bhunia B, Sinha A, Dey A. Approaches towards the enhanced production of Rapamycin by Streptomyces hygroscopicus MTCC 4003 through mutagenesis and optimization of process parameters by Taguchi orthogonal array methodology. World J Microbiol Biotechnol. 2017;33: 90. doi: 10.1007/s11274-017-2260-3 28390015

28. Jia B, Jin ZH, Mei LH. Medium Optimization Based on Statistical Methodologies for Pristinamycins Production by Streptomyces pristinaespiralis. Appl Biochem Biotechnol. 2008;144: 133. doi: 10.1007/s12010-007-8012-3 18456945

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