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

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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.

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Acknowledgments

We thank John Ondeyka for the preparation of Fig. 1; Thomas Fulton for use of the Nodulisporium sp. genomic cosmid library, and Maria Losada for assistance in the RNA experiments. We also thank William Strohl, Jan Tkacz, Richard Monaghan, and Thomas Fulton for critically reading the manuscript.

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  1. Merck Research Laboratories, P.O. Box 2000, RY80Y-325, Rahway, NJ, 07065, USA

    C. Ireland, N. Peekhaus, P. Lu, R. Sangari, A. Zhang, P. Masurekar & Z. An

  2. Merck Research Laboratories, P.O. Box 4, WP26A-4000, West Point, PA, 19486, USA

    Z. An

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  1. C. Ireland
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  2. N. Peekhaus
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  7. Z. An
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Correspondence to Z. An.

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Ireland, C., Peekhaus, N., Lu, P. et al. The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production. Appl Microbiol Biotechnol 79, 451–459 (2008). https://doi.org/10.1007/s00253-008-1440-3

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  • DOI: https://doi.org/10.1007/s00253-008-1440-3

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