Biotechnology Letters

, Volume 34, Issue 7, pp 1327–1334 | Cite as

Site-directed modification of the adenylation domain of the fusaricidin nonribosomal peptide synthetase for enhanced production of fusaricidin analogs

  • Jae Woo Han
  • Eun Young Kim
  • Jung Min Lee
  • Yun Sung Kim
  • Eunjung Bang
  • Beom Seok Kim
Original Research Paper


Fusaricidins produced by Paenibacillus polymyxa DBB1709 are lipopeptide antibiotics active against fungi and Gram-positive bacteria. The cyclic hexapeptide structures of fusaricidins are synthesized by fusaricidin synthetase, a non-ribosomal peptide synthetase. The adenylation domain of the third module (FusA-A3) can recruit l-Tyr, l-Val, l-Ile, l-allo-Ile, or l-Phe, which diversifies the fusaricidin structures. Since the l-Phe-incorporated fusaricidin analog (LI-F07) exhibits more potent antimicrobial activity than other analogs, we modified a specificity-conferring sequence in the substrate binding pocket of FusA-A3 to direct the enhanced production of LI-F07. Base on comparison to the adenylation domain of gramicidin S synthetase 1 and tyrocidine synthetase 1, both of which mainly activate l-Phe, six mutant strains with altered FusA-A3 were generated using site-directed mutagenesis. M3 (I239W, I299V), M5 (I299V, G322A, V330I), and M6 (S239W, I299V, G322A, V330I) mutants produced significantly more LI-F07 than the wild-type strain.


Adenylation domain Fusaricidin analogs Non-ribosomal peptide synthetase Paenibacillus polymyxa Site-directed mutagenesis 



This study is supported by the Agenda Program of Rural Development Administration in Korea and a Korea University Grant.

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  1. Challis GL, Ravel J, Townsend CA (2000) Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chem Biol 7:211–224PubMedCrossRefGoogle Scholar
  2. Choi SK, Park SY, Kim R, Lee CH, Kim JF, Park SH (2008) Identification and functional analysis of the fusaricidin biosynthetic gene of Paenibacillus polymyxa E681. Biochem Biophys Res Commun 365:89–95PubMedCrossRefGoogle Scholar
  3. Cybulski Jr R, Sanz P, Alem F, Stibitz S, Bull R, O’Brien A (2009) Four superoxide dismutases contribute to Bacillus anthracis virulence and provide spores with redundant protection from oxidative stress. Infect Immun 77:274–285PubMedCrossRefGoogle Scholar
  4. Kajimura Y, Kaneda M (1996) Fusaricidin A, a new depsipeptide antibiotic produced by Bacillus polymyxa KT-8 taxonomy, fermentation, isolation, structure elucidation and biological activity. J Antibiot 49:129–135PubMedCrossRefGoogle Scholar
  5. Kajimura Y, Kaneda M (1997) Fusaricidins B, C and D, new depsipeptide antibiotics produced by Bacillus polymyxa KT-8: isolation, structure elucidation and biological activity. J Antibiot 50:220–228CrossRefGoogle Scholar
  6. Kurusu K, Ohba K, Arai T, Fukushima K (1987) New peptide antibiotics LI-F03, F04, F05, F07, and F08, produced by Bacillus polymyxa. I. Isolation and characterization. J Antibiot 40:1506–1514PubMedCrossRefGoogle Scholar
  7. Li J, Jensen SE (2008) Nonribosomal biosynthesis of fusaricidins by Paenibacillus polymyxa PKB1 involves direct activation of a d-amino acid. Chem Biol 15:118–127PubMedCrossRefGoogle Scholar
  8. Marahiel M, Stachelhaus T, Mootz H (1997) Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem Rev 97:2651–2674PubMedCrossRefGoogle Scholar
  9. Röttig M, Medema MH, Blin K, Weber T, Rausch C, Kohlbacher O (2011) NRPSpredictor2—a web server for predicting NRPS adenylation domain specificity. Nucleic Acids Res 39:362–367CrossRefGoogle Scholar
  10. Schwarzer D, Finking R, Marahiel MA (2003) Nonribosomal peptides: from genes to products. Nat Prod Rep 20:275–287PubMedCrossRefGoogle Scholar
  11. Stachelhaus T, Mootz HD, Marahiel MA (1999) The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem Biol 6:493–505PubMedCrossRefGoogle Scholar
  12. Stevens BW, Joska TM, Anderson AC (2005) Progress toward re engineering non ribosomal peptide synthetase proteins: a potential new source of pharmacological agents. Drug Dev Res 66:9–18CrossRefGoogle Scholar
  13. Trauger JW, Kohli RM, Mootz HD, Marahiel MA, Walsh CT (2000) Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature 407:215–218PubMedCrossRefGoogle Scholar
  14. Turgay K, Krause M, Marahiel M (1992) Four homologous domains in the primary structure of GrsB are related to domains in a superfamily of adenylate-forming enzymes. Mol Microbiol 6:529–546PubMedCrossRefGoogle Scholar
  15. Villiers BRM, Hollfelder F (2009) Mapping the limits of substrate specificity of the adenylation domain of TycA. ChemBioChem 10:671–682PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Jae Woo Han
    • 1
  • Eun Young Kim
    • 1
  • Jung Min Lee
    • 1
  • Yun Sung Kim
    • 2
  • Eunjung Bang
    • 3
  • Beom Seok Kim
    • 1
  1. 1.Division of Biotechnology, College of Life Sciences and BiotechnologyKorea UniversitySeoulRepublic of Korea
  2. 2.AgroLife Research InstituteDongbu Hitek Co., Ltd.DaejeonRepublic of Korea
  3. 3.Korea Basic Science InstituteSeoulRepublic of Korea

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