Applied Microbiology and Biotechnology

, Volume 79, Issue 3, pp 451–459

The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production

  • C. Ireland
  • N. Peekhaus
  • P. Lu
  • R. Sangari
  • A. Zhang
  • P. Masurekar
  • Z. An
Applied Genetics and Molecular Biotechnology

Abstract

Nodulisporic acid A (NAA), an insecticidal indole diterpene, is produced by the fungus Nodulisporium sp. Since indole-3-glycerolphosphate is the precursor of the indole moiety of NAA, it is suggested that the activity of tryptophan synthetase may play a role in NAA biosynthesis. To investigate this hypothesis, the tryptophan synthetase gene TRP1 of Nodulisporium sp. was cloned and characterized. The gene consists of three introns of 146, 68, and 57 bp. The four exons encode a protein of 712 amino acids, the sequence of which is highly homologous to that of other fungal tryptophan synthetase proteins. The transcription initiation site was mapped 66 bp upstream to the ATG, and the polyA tail attachment site is 169 bp downstream to the translation stop codon. Replacement of the N-terminal half of the gene with a hygromycin selection marker yielded mutants with the tryptophan auxotroph/hygromycin-resistance (trp/hyr) phenotype. The TRP1 mutants required a high concentration of tryptophan supplement in solid medium (10 mM) to sustain minimal growth and failed to produce NAA in the production medium (FFL-CAM) supplemented with high concentrations of tryptophan.

Keywords

Trytophan synthetase Nodulisporium sp Nodulisporic acid A 

References

  1. An Z, Farman ML, Budde A, Taura S, Leong SA (1996) New cosmid vectors for library construction, chromosome walking and restriction mapping in filamentous fungi. Gene 176:93–96CrossRefGoogle Scholar
  2. Ballance DJ (1991) Transformation systems for filamentous fungi and an overview of fungal gene structure. In: Leong SA, Berka RM (eds) Molecular industrial mycology: systems and applications for filamentous fungi. Marcel Dekker, New York, pp 1–29Google Scholar
  3. Binninger DM, Skrzynia C, Pukkila PJ, Casselton LA (1987) DNA-mediated transformation of the basidiomycete Coprinus cinereus. EMBO J 6:835–840Google Scholar
  4. Bowden DW, Muller-Kahle H, Fulton TR, Gravius TC, Barker DF, Donis-Keller H (1988) Studies on locus expression, library representation, and chromosome walking using an efficient method to screen cosmid libraries. Gene 71:391–400CrossRefGoogle Scholar
  5. Bruchez JJP, Eberle J, Russo VEA (1993) Regulatory sequences involved in the translation of Neurospora crassa mRNA: Kozak sequences and stop codons. Fungal Genet Newslett 40:85–88Google Scholar
  6. Burns DM, Horn V, Paluh J, Yanofsky C (1990) Evolution of the tryptophan synthetase of fungi: analysis of experimentally fused Escherichia coli tryptophan synthetase alpha and beta chains. J Biol Chem 265:2060–2069Google Scholar
  7. Burns DM, Yanofsky C (1989) Nucleotide sequence of the Neurospora crassa trp-3 gene encoding tryptophan synthetase and comparison of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae and Escherichia coli. J Biol Chem 264:3840–3848Google Scholar
  8. Byrd AD, Schardl CL, Songlin PJ, Mogen KL, Siegel MR (1990) The b-tubulin gene of Epichloe typhina from perennial ryegrass (Lolium perenne). Curr Genet 18:347–354CrossRefGoogle Scholar
  9. Byrne KM, Smith SK, Ondeyka JG (2002) Biosynthesis of nodulisporic acid A: precusor studies. J Am Chem Soc 124:7055–7076CrossRefGoogle Scholar
  10. Eckert SE, Kubler E, Hoffmann B, Braus GH (2000) The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system. Mol Gen Genet 263:867–876CrossRefGoogle Scholar
  11. Floss HG, Mothes U (1964) Uber den Einfluss von Tryptophan und analogen Verbindungan auf die Biosynthese von Clavinalkaloiden in saprophytischer Kultur. Arch Mikrobiol 48:213–221CrossRefGoogle Scholar
  12. Fulton TR, Ibrahim N, Losada MC, Grzegorski D, Tkacz JS (1999) A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium. Mol Gen Genet 262:714–720CrossRefGoogle Scholar
  13. Krupinski VM, Robbers JE, Floss HG (1976) Physiological study of ergot: induction of alkaloid synthesis by tryptophan at the enzymatic level. J Bacteriol 125:158–165Google Scholar
  14. Marfey P (1984) Determination of D-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res Commun 49:591–596Google Scholar
  15. Monaghan RL, Polishook JD, Pecore V, Bills GF, Nallin-Omstead M, Streicher SL (1995) Discovery of novel secondary metabolites from fungi—is it really a random walk through a random forest. Can J Bot 73(S1):S923–S931CrossRefGoogle Scholar
  16. Ondeyka JG, Helms GL, Hensens OD, Goetz MA, Zink DL, Tsipouras A, Shoop WL, Slayton L, Dombrowski AW, Polishook JD, Ostlind DA, Tsou NN, Ball RG, Singh SB (1997) Nodulisporic acid A, a novel and potent insecticide from Nodulisporium sp. Isolation, structure determination, and chemical transformations. J Am Chem Soc 119:8809–8816CrossRefGoogle Scholar
  17. Ostlind DA, Felcetto T, Misura A, Ondeyka J, Smith S, Goetz M, Shoop W, Mickle W (1997) Discovery of a novel indole diterpene insecticide using first instars of Lucilia sericata. Med Vet Entomolo 11:407–408CrossRefGoogle Scholar
  18. Polishook JD, Ondeyka JG, Dombrowski AW, Pelaez F, Platas G, Teran AM (2001) Biogeography and relatedness of Nodulisporium strains producing nodulisporic acid. Mycologia 93:1125–1137CrossRefGoogle Scholar
  19. Radford A, Parish JH (1997) The genome and genes of Neurospora crassa. Fungal Genet Biol 21:258–266CrossRefGoogle Scholar
  20. Rodrigues KF, Samuels GJ (1990) Preliminary study of endophytic fungi in a tropical palm. Mycological Research 94:827–830CrossRefGoogle Scholar
  21. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  22. Shoop WL, Gregory LM, Zakson-Aiken M, Michael BF, Haines HW, Ondeyka JG, Meinke PT, Schmatz DM (2001) Systemic efficacy of nodulisporic acid against fleas on dogs. J Parasitol 87:419–423Google Scholar
  23. Skrzynia C, Binninger DM, Alspaugh JA, Pukkila PJ (1989) Molecular characterization of TRP1, a gene coding for tryptophan synthetase in the basidiomycete Coprinus cinereus. Gene 81:73–82CrossRefGoogle Scholar
  24. Smith MM, Warren VA, Thomas BS, Brochu RM, Ertel EA, Rohrer S, Schaeffer J, Schmatz D, Petuch BR, Tang YS, Meinke PT, Kaczorowski GJ, Cohen CJ (2000) Nodulisporic acid opens insect glutamate-gated chloride channels: identification of a new high affinity modulator. Biochemistry 39:5543–5554CrossRefGoogle Scholar
  25. Vining LC (1970) Effect of tryptophan on alkaloid biosynthesis in cultures of a Claviceps species. Can J Microbiol 16:473–480CrossRefGoogle Scholar
  26. Yanofsky C (1984) Comparison of regulatory and structural regions of genes of tryptophan metabolism. Mol Biol Evol 1:143–161Google Scholar
  27. Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci U S A 81:1470–1474CrossRefGoogle Scholar
  28. Zalkin H, Yanofsky C (1982) Yeast gene TRP5: structure, function, regulation. J Biol Chem 257:1491–1500Google Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • C. Ireland
    • 1
  • N. Peekhaus
    • 1
  • P. Lu
    • 1
  • R. Sangari
    • 1
  • A. Zhang
    • 1
  • P. Masurekar
    • 1
  • Z. An
    • 1
    • 2
  1. 1.Merck Research LaboratoriesRahwayUSA
  2. 2.Merck Research LaboratoriesWest PointUSA

Personalised recommendations