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World Journal of Microbiology and Biotechnology

, Volume 31, Issue 10, pp 1543–1554 | Cite as

Production and secretion of naphthoquinones is mediated by the MFS transporter MFS1 in the entomopathogenic fungus Ophiocordyceps sp. BCC1869

  • Pratoomporn Khaokhajorn
  • Sompid Samipak
  • Sutichai Nithithanasilp
  • Morakot Tanticharoen
  • Alongkorn AmnuaykanjanasinEmail author
Original Paper

Abstract

Naphthoquinones are deep red polyketide pigments produced by the ant-pathogenic fungus Ophiocordyceps sp. BCC1869. In culture, biosynthesis of these naphthoquinones remains at a low level during the first 20 days and reaches its maximum production level at approximately 50 days. The MFS transporter gene MFS1 was previously identified in Ophiocordyceps sp. BCC1869 from a subtractive EST library between the fungus grown under naphthoquinone-inductive and naphthoquinone-repressive conditions. We cloned and sequenced this transporter gene, which has an open reading frame of 1505 bp and three introns (48, 52, and 58 bp). Phylogenetic analysis showed this MFS transporter was tightly clustered with fungal riboflavin transporters. Functional analysis of this gene was performed by overexpression of MFS1 under the control of a strong, constitutive promoter. We successfully transformed the fungus with this overexpression plasmid using PEG-protoplast transformation, which generated nine transformants per µg of plasmid. RT-PCR indicated that the MFS1 expression level in the overexpressing strains increased 3- to 10-fold compared to the wild type. HPLC analysis of crude extracts of mutants and wild type demonstrated that four naphthoquinone derivatives, erythrostominone, epierythrostominol, deoxyerythrostominone, and deoxyerythrostominol, were the major naphthoquinones produced and excreted in staggering quantities (20- to 2300-fold) in 7-day old liquid cultures by the mutant C7, compared to the wild type. High resolution electrospray ionization mass spectrometry verified mass spectra of these purified metabolites. Three other naphthoquinone derivatives, whose structures have not been identified, were also detected in high amount in the mutant liquid cultures.

Keywords

Naphthoquinone MFS transporter Overexpression Ophiocordyceps Secretion Erythrostominone 

Notes

Acknowledgments

We are indebted to Chakapong Intaraudom and Drs. Juntira Panya and Taridaporn Bunyapaiboonsri for great help and advice in chemical analysis. We thank Wiwat Somyong for help in compound extraction. We are also grateful to Dr. Chanikul Chutrakul and Sarocha Panchanawaporn for advice in fungal transformation. This work was supported by the National Center for Genetic Engineering and Biotechnology’s Platform Program.

Supplementary material

11274_2015_1903_MOESM1_ESM.pdf (98 kb)
Supplementary material 1 (PDF 97 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Pratoomporn Khaokhajorn
    • 1
    • 2
  • Sompid Samipak
    • 2
  • Sutichai Nithithanasilp
    • 1
  • Morakot Tanticharoen
    • 1
    • 3
  • Alongkorn Amnuaykanjanasin
    • 1
  1. 1.Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC)National Science and Technology Development Agency (NSTDA)PathumthaniThailand
  2. 2.Department of Genetics, Faculty of ScienceKasetsart UniversityBangkokThailand
  3. 3.School of Bioresources and TechnologyKing Mongkut’s University of Technology ThonburiBangkokThailand

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