Isolation and partial characterization of a cryptic polyene gene cluster in Pseudonocardia autotrophica

  • Mi-Yeon Lee
  • Ji Seon Myeong
  • Hyun-Joo Park
  • Kyuboem Han
  • Eung-Soo Kim
Original Paper

Abstract

The polyene antibiotics, a category that includes nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very promising antifungal polyketide compounds and are typically produced by soil actinomycetes. The biosynthetic gene clusters for these polyenes have been previously investigated, revealing the presence of highly similar cytochrome P450 hydroxylase (CYP) genes. Using polyene CYP-specific PCR screening with several actinomycete genomic DNAs, Pseudonocardia autotrophica was determined to contain a unique polyene-specific CYP gene. Genomic DNA library screening using the polyene-specific CYP gene probe identified a positive cosmid clone, which contained a DNA fragment of approximately 34.5 kb. The complete sequencing of this DNA fragment revealed a total of seven complete and two incomplete open reading frames, which were found to be highly similar, but still unique, when compared to previously known polyene biosynthetic genes. These results suggest that the polyene-specific screening approach may constitute an efficient method for the isolation of potentially valuable cryptic polyene biosynthetic gene clusters from various rare actinomycetes.

Keywords

Polyene Polyketide Antifungal Cryptic gene cluster Pseudonocardia Cytochrome P450 hydroxylase 

Notes

Acknowledgements

The authors are very grateful for the technical support provided by the ERC in Inha University. This work was financially supported by a 21C Frontier R&D program MG 3-1 Grant from the Korean Ministry of Science and Technology.

References

  1. 1.
    Aparicio JF, Colina AJ, Ceballos E, Martín JF (1999) The biosynthetic gene cluster for the 26-membered ring polyene macrolide pimaricin. J Biol Chem 274:10133–10139CrossRefPubMedGoogle Scholar
  2. 2.
    Aparicio JF, Fouces R, Mendes MV, Olivera N, Martín JF (2000) A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis. Chem Biol 7:895–905CrossRefPubMedGoogle Scholar
  3. 3.
    Aparicio JF, Caffrey P, Gil JA, Zotchev SB (2002) Polyene antibiotic biosynthesis gene clusters. Appl Microbiol Biotechnol 61:179–188PubMedGoogle Scholar
  4. 4.
    Bolard J (1986) How do the polyene macrolide antibiotics affect the cellular membrane properties? Biochim Biophys Acta 864:257–304PubMedGoogle Scholar
  5. 5.
    Brautaset T, Sekurova ON, Sletta H, Ellingsen TE, Strom AR, Valla S, Zotchev SB (2000) Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway. Chem Biol 7:395–403CrossRefPubMedGoogle Scholar
  6. 6.
    Brautaset T, Bruheim P, Sletta H, Hagen L, Ellingsen TE, Strom AR, Valla S, Zotchev SB (2002) Hexaene derivatives of nystatin produced as a result of an induced rearrangement within the nysC polyketide synthase gene in Streptomyces noursei ATCC 11455. Chem Biol 9:367–373CrossRefPubMedGoogle Scholar
  7. 7.
    Caffrey P, Lynch S, Flood E, Finnan S, Oliynyk M (2001) Amphotericin biosynthesis in Streptomyces nodosus: deductions from analysis of polyketide synthase and late genes. Chem Biol 8:713–723CrossRefPubMedGoogle Scholar
  8. 8.
    Campelo AB, Gil JA (2002) The candicidin gene cluster from Streptomyces griseus IMRU 3570. Microbiology 148:51–59PubMedGoogle Scholar
  9. 9.
    Gupte M, Kulkarni P, Ganguli BN (2002) Antifungal antibiotics. Appl Microbiol Biotechnol 58:46–57CrossRefPubMedGoogle Scholar
  10. 10.
    Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. A laboratory manual. John Innes Foundation, Norwich UKGoogle Scholar
  11. 11.
    Mendes MV, Recio E, Fouces R, Luiten R, Martin JF, Aparicio JF (2001) Engineered biosynthesis of novel polyenes: a pimaricin derivative produced by targeted gene disruption in Streptomyces natalensis. Chem Biol 8:635–644CrossRefPubMedGoogle Scholar
  12. 12.
    Munro AW, Lindsay JG (1996) Bacterial cytochromes P-450. Mol Microbiol 20:1115–1125PubMedCrossRefGoogle Scholar
  13. 13.
    O’Keefe DP, Harder PA (1991) Occurrence and biological function of cytochrome P450 monooxygenases in actinomycetes. Mol Microbiol 5:2099–2105PubMedCrossRefGoogle Scholar
  14. 14.
    Resat H, Sungur FA, Baginski M, Borowski E, Aviyente V (2000) Conformational properties of amphotericin B amide derivatives—impact on selective toxicity. J Comput Aided Mol Des 14:689–703CrossRefPubMedGoogle Scholar
  15. 15.
    Rodriguez E, McDaniel R (2001) Combinatorial biosynthesis of antimicrobials and other natural products. Curr Opin Microbiol 4:526–534CrossRefPubMedGoogle Scholar
  16. 16.
    Seo YW, Cho KW, Lee HS, Yoon TM, Shin JH (2000) New polyene macrolide antibiotics from Streptomyces sp. M90025. J Microbiol Biotechnol 10:176–180Google Scholar
  17. 17.
    Zazopoulos E, Huang K, Staffa A, Liu W, Bachmann BO, Nonaka K, Ahlert J, Thorson JS, Shen B, Farnet CM (2003) A genomics-guided approach for discovering and expressing cryptic metabolic pathways. Nat Biotechnol 21:187–190CrossRefPubMedGoogle Scholar
  18. 18.
    Zotchev S, Haugan K, Sekurova O, Sletta H, Ellingsen TE, Valla S (2000) Identification of a gene cluster for antibacterial polyketide-derived antibiotic biosynthesis in the nystatin producer Streptomyces noursei ATCC 11455. Microbiology 146:611–619PubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2005

Authors and Affiliations

  • Mi-Yeon Lee
    • 1
  • Ji Seon Myeong
    • 1
  • Hyun-Joo Park
    • 2
  • Kyuboem Han
    • 3
  • Eung-Soo Kim
    • 1
  1. 1.Department of BiotechnologyInha UniversityIncheonKorea
  2. 2.SongWon Envichem Inc.SeoulKorea
  3. 3.Hanson Biotech Co., Ltd 201 IACRIHan Nam UniversityDaejeonKorea

Personalised recommendations