Cloning, characterization and expression of an alpha-amylase gene from Streptomyces griseus IMRU3570.
A gene, amy, encoding an alpha-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 bp upstream from the ATG translation start codon. The promoter of the amy gene was subcloned on a 290 bp 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 alpha-amylase was secreted after deletion of this region and coupling of a TGA translation stop codon.
Vigal
T
T
Area de Microbiología, Facultad de Biología, Universidad de León, Spain.
Gil
J A
JA
Daza
A
A
García-González
M D
MD
Martín
J F
JF
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004351:Drug Resistance / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013305:Streptomyces griseus / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D014169:Transformation, Bacterial; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D000516:alpha-Amylases; D009355:Neomycin