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Cloning, characterization and expression of an α-amylase gene from Streptomyces griseus IMRU3570

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Summary

A gene, amy, encoding an α-amylase, was cloned on a 4.8 kb Sau3A fragment from the DNA of Streptomyces griseus IMRU3570. The gene was localized to a 2.27 kb fragment by subcloning and deletion mapping experiments. The gene contained an open reading frame (ORF) of 1698 nucleotides that encoded a protein of 566 amino acids with a deduced Mr of 59713 Da. Dot-blot analysis revealed that the copy number of the transcript in S. lividans transformed with the amy gene was 2.8-fold higher than in the donor S. griseus strain in good agreement with the proportionally higher secretion of amylase in S. lividans. A transcription initiation site was found approximately 64 by upstream from the ATG translation start codon. The promoter of the amy gene was subcloned on a 290 by HindIII-EcoRI fragment. Expression of a neomycin resistance gene from the amy promoter was negatively regulated by glucose. A 219 nucleotide fragment extending from the single BstEII site to the end of the amy gene was dispensable since active α-amylase was secreted after deletion of this region and coupling of a TGA translation stop codon.

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Authors and Affiliations

  1. Area de Microbiología, Facultad de Biología, Universidad de León, E-24071, Lehn, Spain

    Tomás Vigal, José A. Gil, Antonio Daza, María Dolores García-González & Juan F. Martín

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  1. Tomás Vigal
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  2. José A. Gil
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  3. Antonio Daza
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  4. María Dolores García-González
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  5. Juan F. Martín
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Communicated by H. Hennecke

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Vigal, T., Gil, J.A., Daza, A. et al. Cloning, characterization and expression of an α-amylase gene from Streptomyces griseus IMRU3570. Molec. Gen. Genet. 225, 278–288 (1991). https://doi.org/10.1007/BF00269860

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  • DOI: https://doi.org/10.1007/BF00269860

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