Skip to main content

Advertisement

SpringerLink
Log in

Bioprocesses optimization and anticancer activity of camptothecin from Aspergillus flavus, an endophyte of in vitro cultured Astragalus fruticosus

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

Emerging of endophytic fungi as potent camptothecin producers raise the hope for its commercial production, due to their rapid growth and feasibility of metabolic engineering, nevertheless, their loss of productivity with the fungal storage and subculturing is the challenge. Thus, screening for unique fungal isolate with sustainable camptothecin productivity is the objective of this work.

Results

The camptothecin productivity of the fungal endophytes of wild and in vitro cultured Astragalus fruticosus was evaluated. Aspergillus flavus ER, endophyte of A. fruticosus explant, was the potent producer (51.7 µg/l), the chemical identity of putative compound was resolved by UV, HPLC and LC-MS/MS analyses. The purified A. flavus camptothecin displayed a significant activity against HEPG-2 (IC50 0.9 mM), MCF7 and HCT29 (IC50 1.2–1.35 mM). The productivity of camptothecin by A. flavus was increased by 1.6 fold with methyljasmonate. Upon Plackett-Burman Design optimization, the yield of camptothecin was enhanced by 3 fold (150 µg/l) comparing to control. The camptothecin biosynthetic machinery of A. flavus was noticed to be attenuated with subculturing, nevertheless, this biosynthetic potency was restored upon addition of A. fruticosus methanolic extract (1%), ensuring the incidence of specific signals from plant tissues that triggers the expression of camptothecin encoding genes.

Conclusion

This is the first study deciphering the feasibility of A. flavus for sustainable production of camptothecin upon addition of A. fruticosus extracts, that could be a new platform for camptothecin scaling-up.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data Availability

All datasets generated for this study are included in the article/ Supplementary Material.

References

  1. Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT (1971) Plant Antitumor Agents. VI. The Isolation and Strcture of Taxol, a Novel Antileukemic and Antitumo Agent from Taxus bretvifolia,Journal of the American Chemical Society.2325–2327

  2. Lopez-Meyer M, Nessler CL, McKnight TD (1994) Sites of accumulation of the antitumor alkaloid camptothecin in Camptotheca acuminata. Planta Med 60:558–560

    Article  CAS  PubMed  Google Scholar 

  3. Li S, Yi Y, Wang Y, Zhang Z, Beasley RS (2002) Camptothecin accumulation and variations in Camptotheca. Planta Med 68:1010–1016

    Article  CAS  PubMed  Google Scholar 

  4. Kai G, Wu C, Gen L, Zhang L, Cui L, Ni X (2015) Biosynthesis and biotechnological production of anti-cancer drug Camptothecin. Phytochem Rev 14:525–539

    Article  CAS  Google Scholar 

  5. Uma Shaanker R, Ramesha BT, Ravikanth G, Gunaga R, Vasudeva R, Ganeshaiah KN (2008) Chemical Profiling of Nothapodytes nimmoniana for Camptothecin, an Important Anticancer Alkaloid: Towards the Development of a Sustainable Production System, in: Bioactive Molecules and Medicinal Plants. Springer, Berlin Heidelberg, pp 197–213

    Google Scholar 

  6. Hsiang YH, Hertzberg R, Hecht S, Liu LF (1985) Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260:14873–14878

    Article  CAS  PubMed  Google Scholar 

  7. Supaart Sirikantaramas T, Asano H, Sudo M, Yamazaki K, Saito (2007) Camptothecin: Therapeutic Potential and Biotechnology, Current Pharmaceutical Biotechnology. 8 196–202

  8. Demain AL, Vaishnav P (2011) Natural products for cancer chemotherapy. Microb Biotechnol 4:687–699

    Article  PubMed  PubMed Central  Google Scholar 

  9. Liu K, Ding X, Deng B, Chen W (2010) 10-Hydroxycamptothecin produced by a new endophytic Xylaria sp., M20, from Camptotheca acuminata. Biotechnol Lett 32:689–693

    Article  CAS  PubMed  Google Scholar 

  10. Lu Y, Wang H, Wang W, Qian Z, Li L, Wang J, Zhou G, Kai G (2009) Molecular characterization and expression analysis of a new cDNA encoding strictosidine synthase from Ophiorrhiza japonica. Mol Biology Rep 36:1845–1852

    Article  CAS  Google Scholar 

  11. Kutchan TM (1989) Expression of enzymatically active cloned strictosidine synthase from the higher plant Rauvolfia serpentina in Escherichia coli. FEBS Lett 257:127–130

    Article  CAS  PubMed  Google Scholar 

  12. Mcknight TD, Roessner CA, Devagupta R, Scott AI, Nessler CL (1990) Nucleotide sequence of a cdna encoding the vacuolar protein strictosidine synthase from Catharanthus roseus. Nucleic Acids Res 18:4939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yamazaki Y, Kitajima M, Arita M, Takayama H, Sudo H, Yamazaki M, Aimi N, Saito K (2004) Biosynthesis of Camptothecin. In Silico and in Vivo Tracer Study from [1-13 C]Glucose. Plant Physiol 134:161–170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Khan AA, El-Sayed AS, Akbar A, Mangravita-Novo A, Bibi S, Afzal Z, Norman DJ, Ali GS (2017) A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants. Plant Methods, 13(1):86

    Article  CAS  Google Scholar 

  15. Kusari S, Lamshöft M, Spiteller M (2009) Aspergillus fumigatus Fresenius, an endophytic fungus from Juniperus communis L. Horstmann as a novel source of the anticancer pro-drug deoxypodophyllotoxin. J Appl Microbiol 107:1019–1030

    Article  CAS  PubMed  Google Scholar 

  16. El-Sayed ASA, El-Sayed MT, Rady AM, Zein N, Enan G, Shindia A, El-Hefnawy S, Sitohy M, Sitohy B (2020) Exploiting the Biosynthetic Potency of Taxol from Fungal Endophytes of Conifers Plants; Genome Mining and Metabolic Manipulation. Molecules 25:3000

    Article  CAS  PubMed Central  Google Scholar 

  17. Karwasara VS, Dixit VK (2013) Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley. Plant Biotechnol Rep 7:357–369

    Article  Google Scholar 

  18. Bhalkar BN, Patil SM, Govindwar SP (2016) Camptothecine production by mixed fermentation of two endophytic fungi from Nothapodytes nimmoniana. Fungal Biology 120:873–883

    Article  CAS  PubMed  Google Scholar 

  19. El-Sayed ASA, Fathalla M, Yassin MA, Zein N, Morsy S, Sitohy M, Sitohy B (2020) Conjugation of Aspergillus flavipes taxol with porphyrin increases the anticancer activity of taxol and ameliorates its cytotoxic effects. Molecules, 25(2):263

  20. Puri SG, Verma V, Amna T, Qazi GN, Spiteller M (2005) An endophytic fungus from Nothapodytes foetida that produces camptothecin. J Nat Prod 68:1717–1719

    Article  CAS  PubMed  Google Scholar 

  21. Shweta S, Zuehlke S, Ramesha BT, Priti V, Mohana Kumar P, Ravikanth G, Spiteller M, Vasudeva R (2010) Uma Shaanker, Endophytic fungal strains of Fusarium solani, from Apodytes dimidiata E. Mey. ex Arn (Icacinaceae) produce camptothecin, 10-hydroxycamptothecin and 9-methoxycamptothecin. Phytochemistry 71:117–122

    Article  CAS  PubMed  Google Scholar 

  22. Pu X, Qu X, Chen F, Bao J, Zhang G, Luo Y (2013) Camptothecin-producing endophytic fungus Trichoderma atroviride LY357: isolation, identification, and fermentation conditions optimization for camptothecin production. Appl Microbiol Biotechnol 97:9365–9375

    Article  CAS  PubMed  Google Scholar 

  23. El-Sayed ASA, Hassan AEA, Shindia AA, Mohamed SG, Sitohy MZ (2016) Aspergillus flavipes methionine γ-lyase-dextran conjugates with enhanced structural, proteolytic stability and anticancer efficiency. J Mol Catal B: Enzymatic 133:S15–S24

    Article  Google Scholar 

  24. El-Sayed ASA, Khalaf SA, Azez HA, Hussein HA, EL-Moslamy SH, Sitohy B (2021) El-Baz, Production, bioprocess optimization and anticancer activity of Camptothecin from Aspergillus terreus and Aspergillus flavus, endophytes of Ficus elastica. Process Biochem 107:59–73

    Article  CAS  Google Scholar 

  25. El-Sayed ASA, Shindia AA, Ali GS, Yassin MA, Hussein H, Awad SA, Ammar HA (2021) Production and bioprocess optimization of antitumor Epothilone B analogue from Aspergillus fumigatus, endophyte of Catharanthus roseus, with response surface methodology. Enzym Microb Technol 143:109718

    Article  CAS  Google Scholar 

  26. Stierle A, Strobel G, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science 260:214–216

    Article  CAS  PubMed  Google Scholar 

  27. Pu X, Qu X, Chen F, Bao J, Zhang G, Luo Y (2013) Camptothecin-producing endophytic fungus Trichoderma atroviride LY357: Isolation, identification, and fermentation conditions optimization for camptothecin production. Appl Microbiol Biotechnol 97:9365–9375

    Article  CAS  PubMed  Google Scholar 

  28. Puri SC, Handa G, Bhat BA, Gupta VK, Amna T, Verma N, Anand R, Dhar KL, Qazi GN (2005) Separation of 9-methoxycamptothecin and camptothecin from Nothapodytes foetida by semipreparative HPLC. J Chromatogr Sci 43:348–350

    Article  CAS  PubMed  Google Scholar 

  29. Abd El-Ghani MM, El-Sayed ASA, Moubarak A, Rashad R, Nosier H, Khattab A (2021) Biosystematic study on some egyptian species of astragalus L. (fabaceae), Agriculture (Switzerland). 11:1–16

  30. El-Demerdash MM, El-Sayed AS, Georg NM, Abou-Elnour A, Nosier H (2021) Biosystematic studies of some Egyptian species of Cestrum (Solanaceae), Molecular Biology Reports.

  31. Pistelli LF (2002) Secondary metabolites of genus Astragalus: Structure and biological activity, Studies in Natural Products Chemistry. 27:443–545

  32. El-Sayed ASA, Akbar A, Iqrar I, Ali R, Norman D, Brennan M, Ali GS (2018) A glucanolytic Pseudomonas sp. associated with Smilax bona-nox L. displays strong activity against Phytophthora parasitica. Microbiol Res 207:140–152

    Article  CAS  PubMed  Google Scholar 

  33. El-Batal AI, El-Sherbiny GM, khalaf M, Abdel-Fatah SS, El-Sayed AS (2021) Production, bioprocess optimization and γ-irradiation of Penicillium polonicum, as a new Taxol producing endophyte from Ginko biloba. Biotechnol Rep 30:e00623

    Article  Google Scholar 

  34. El-Sayed AS, Shindia AA, Zaher Y (2012) L-Amino acid oxidase from filamentous fungi: Screening and optimization. Annals of Microbiology, 62(2):773–784

  35. El-Sayed ASA, Abdel-Azeim S, Ibrahim HM, Yassin MA, Abdel-Ghany SE, Esener S, Ali GS (2015) Biochemical stability and molecular dynamic characterization of Aspergillus fumigatus cystathionine γ-lyase in response to various reaction effectors. Enzym Microb Technol 81:31–46

    Article  CAS  Google Scholar 

  36. El-Sayed ASA, Ali GS (2020) Aspergillus flavipes is a novel efficient biocontrol agent of Phytophthora parasitica,Biological Control.140

  37. El-Sayed ASA, Mohamed NZ, Safan S, Yassin MA, Shaban L, Shindia AA, Shad Ali G, Sitohy MZ (2019) Restoring the Taxol biosynthetic machinery of Aspergillus terreus by Podocarpus gracilior Pilger microbiome, with retrieving the ribosome biogenesis proteins of WD40 superfamily. Sci Rep 9:11534

    Article  PubMed  PubMed Central  Google Scholar 

  38. Namdeo AG, Sharma A (2012) HPLC analysis of camptothecin content in various parts of Nothapodytes foetida collected on different periods. Asian Pac J Trop Biomed 2:389–393. doi:https://doi.org/10.1016/S2221-1691(12)60062-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Raper KB, Fennell DI (1965)The Genus Aspergillus., Williams and Wilkins,

  40. Booth C (1971) The Genus Fusarium - C. Booth - Google Books, Commonwealth Agricultural Bureaux [for the]. Commonwealth Mycological Institute, p 237

  41. Pitt JI (1979) The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces, Academic Press, London New York

  42. Frisvad JC, Samson RA (2004) Polyphasic taxonomy of Penicillium subgenus Penicillium: A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins, Studies in Mycology. (2004) 1–173

  43. El-Sayed AS, Khalaf SA, Aziz HA (2013) Characterization of homocysteine γ-lyase from submerged and solid cultures of Aspergillus fumigatus ASH (JX006238),Journal of Microbiology and Biotechnology.23

  44. El-Sayed ASA (2009) L-methioninase production by Aspergillus flavipes under solid-state fermentation.Journal of Basic Microbiology, 49:331–341

  45. El-Sayed ASA, Shindia AA, AbouZeid A, Koura A, Hassanein SE, Ahmed RM (2021) Triggering the biosynthetic machinery of Taxol by Aspergillus flavipes via cocultivation with Bacillus subtilis: proteomic analyses emphasize the chromatin remodeling upon fungal-bacterial interaction (2021) Environmental Science and Pollution Research, 28(29):39866–39881

    Article  CAS  Google Scholar 

  46. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods, Molecular Biology and Evolution. 28 2731–2739

  47. El-Sayed ASA, Shindia AA, Zeid AAA, Yassin AM, Sitohy MZ, Sitohy B (2019) Aspergillus nidulans thermostable arginine deiminase-Dextran conjugates with enhanced molecular stability, proteolytic resistance, pharmacokinetic properties and anticancer activity,Enzyme and Microbial Technology. 131

  48. El-Sayed ASA, Ali GS (2020) Aspergillus flavipes is a novel efficient biocontrol agent of Phytophthora parasitica,Biological Control.140

  49. El-Sayed ASA, Ali DMI, Yassin MA, Zayed RA, Ali GS (2019) Sterol inhibitor “Fluconazole” enhance the Taxol yield and molecular expression of its encoding genes cluster from Aspergillus flavipes. Process Biochem 76:55–67

    Article  CAS  Google Scholar 

  50. Shetaia YMH, El-Baz AF, ElMekawy A (2017) Toward Enhancing the Enzymatic Activity of a Novel Fungal Polygalacturonase for Food Industry: Optimization and Biochemical Analysis, Recent Patents on Biotechnology. 12:134–144

  51. ElMekawy A, Hegab H, El-Baz A, Hudson S (2013) Kinetic Properties and Role of Bacterial Chitin Deacetylase in the Bioconversion of Chitin to Chitosan. Recent Pat Biotechnol 7:234–241

    Article  CAS  PubMed  Google Scholar 

  52. El Sayed MT, El-Sayed ASA (2020) Biocidal activity of metal nanoparticles synthesized by fusarium solani against multidrug-resistant bacteria and mycotoxigenic fungi. Journal of Microbiology and Biotechnology, 30(2):226–236

    Article  CAS  PubMed  Google Scholar 

  53. Hassan A, Sorour NM, El-Baz A, Shetaia Y (2019) Simple synthesis of bacterial cellulose/magnetite nanoparticles composite for the removal of antimony from aqueous solution. Int J Environ Sci Technol 16:1433–1448

    Article  CAS  Google Scholar 

  54. El-Baz AF, Shetaia MY, Elkhouli RR (2011) Xylitol production by candida tropicalis under different statistically optimized growth conditions. Afr J Biotechnol 10:15353–15363

    CAS  Google Scholar 

  55. Badr H, El-Baz A, Mohamed I, Shetaia Y, El-Sayed ASA, Sorour N (2021) Bioprocess optimization of glutathione production by Saccharomyces boulardii: biochemical characterization of glutathione peroxidase. Archives of Microbiology, 203(10):6183–6196

    Article  CAS  Google Scholar 

  56. Alsaggaf MS, El-Baz AF, Badawy- SE, Moussa SH (2020)Anticancer and Antibacterial Activity of Cadmium Sulfide Nanoparticles by Aspergillus niger,

  57. El-Sayed ASA, Shindia AA, AbouZeid A, Koura A, Hassanein SE, Ahmed RM (2021) Triggering the biosynthetic machinery of Taxol by Aspergillus flavipes via cocultivation with Bacillus subtilis: proteomic analyses emphasize the chromatin remodeling upon fungal-bacterial interaction. Environmental Science and Pollution Research

  58. El-Sayed ASA, Shindia AA, Zeid AAA, Yassin AM, Sitohy MZ, Sitohy B (2019) Aspergillus nidulans thermostable arginine deiminase-Dextran conjugates with enhanced molecular stability, proteolytic resistance, pharmacokinetic properties and anticancer activity. Enzym Microb Technol 131:109432

    Article  CAS  Google Scholar 

  59. Cory AH, Owen TC, Barltrop JA, Cory JG (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 3:207–212

    Article  CAS  PubMed  Google Scholar 

  60. Patel JS, Vitoreli A, Palmateer AJ, El-Sayed A, Norman DJ, Goss EM, Brennan MS, Ali GS (2016) Characterization of Phytophthora spp. Isolated from ornamental plants in Florida. Plant Disease, 100(2):500–509

    Article  CAS  PubMed  Google Scholar 

  61. Gurudatt PS, Priti V, Shweta S, Ramesha BT, Ravikanth G, Vasudeva R, Amna T, Deepika S, Ganeshaiah KN, Uma Shaanker R, Puri S, Qazi N (2010) Attenuation of camptothecin production and negative relation between hyphal biomass and camptothecin content in endophytic fungal strains isolated from Nothapodytes nimmoniana Grahm (Icacinaceae). Curr Sci 98:1006–1010

    CAS  Google Scholar 

  62. Kusari S, Hertweck C, Spiteller M (2012) Chemical Ecology of Endophytic Fungi: Origins of Secondary Metabolites. Chem Biology 19:792–798

    Article  CAS  Google Scholar 

  63. Kusari S, Singh S, Jayabaskaran C (2014) Rethinking production of Taxol® (paclitaxel) using endophyte biotechnology. Trends in Biotechnology 32:304–311

    Article  CAS  PubMed  Google Scholar 

  64. Dief H.E-S, Hashem, E-S.A, Fawzan S, El-Sayed ASA. (2021) Alleviation of salt stress in Triticum aestivum by biopriming with Phanerochaete chrysosporium. Journal of Crop Science and Biotechnology, 24(1):103–116

    Google Scholar 

  65. Pérez-Jaramillo JE, Carrión VJ, de Hollander M, Raaijmakers JM (2018) The wild side of plant microbiomes. Microbiome 6:4–9. doi:https://doi.org/10.1186/s40168-018-0519-z

    Article  Google Scholar 

  66. Mueller UG, Sachs JL (2015) Engineering Microbiomes to Improve Plant and Animal Health. Trends Microbiol 23:606–617

    Article  CAS  PubMed  Google Scholar 

  67. Cardinale M, Grube M, Erlacher A, Quehenberger J, Berg G (2015) Bacterial networks and co-occurrence relationships in the lettuce root microbiota. Environ Microbiol 17:239–252

    Article  CAS  PubMed  Google Scholar 

  68. El-Sayed ASA, Hassan AEA, Shindia AA, Mohamed SG, Sitohy MZ (2016) Aspergillus flavipes methionine γ-lyase-dextran conjugates with enhanced structural, proteolytic stability and anticancer efficiency. Journal of Molecular Catalysis B: Enzymatic, 133:S15–S24

    Article  CAS  PubMed  Google Scholar 

  69. Bhalkar BN, Bedekar PA, Patil SM, Patil SA, Govindwar SP (2015) Production of camptothecine using whey by an endophytic fungus: Standardization using response surface methodology. https://doi.org/10.1039/c5ra12212k

  70. Kai G, Xu H, Zhou C, Liao P, Xiao J, Luo X, You L, Zhang L (2011) Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. Metabolic Eng 13:319–327. doi:https://doi.org/10.1016/j.ymben.2011.02.003

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We appreciate the financial support from Academy of Scientific Research and Technology, Egypt, to A. S.A.E.

Author information

Authors and Affiliations

  1. Enzymology and Fungal Biotechnology Lab, Botany and Microbiology Department, Faculty of Science, Zagazig University, 44519, Zagazig, Egypt

    Ashraf S. A. El-Sayed

  2. Pharmacognosy Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt

    Rawia A. Zayed & Wafaa M. Ismaeil

  3. Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, Egypt

    Ashraf F. El-Baz

Authors
  1. Ashraf S. A. El-Sayed
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rawia A. Zayed
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ashraf F. El-Baz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wafaa M. Ismaeil
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.S.E and R.Z, design the research plan and wrote the manuscript. A.B revises and edits the manuscript. W.I performs the experimental work. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Ashraf S. A. El-Sayed.

Ethics declarations

Declaration of Competing Interest

The authors declare that they have no competing interests.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

El-Sayed, A.A., Zayed, R.A., El-Baz, A.F. et al. Bioprocesses optimization and anticancer activity of camptothecin from Aspergillus flavus, an endophyte of in vitro cultured Astragalus fruticosus. Mol Biol Rep 49, 4349–4364 (2022). https://doi.org/10.1007/s11033-022-07271-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-022-07271-x

Keywords

Search

Navigation