Cloning of the alpha-amylase cDNA of Aspergillus shirousamii and its expression in Saccharomyces cerevisiae.
alpha-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3' non-coding regions, respectively, so far determined. The alpha-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active alpha-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.
Research Institute of Brewing Resources Co., Ltd., Tokyo, Japan.
I
I
Shibuya
G
G
Tamura
T
T
Ishikawa
S
S
Hara
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics*; D010582:Bacteriophage lambda; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003470:Culture Media; D004247:DNA / Q000032:analysis*; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D000516:alpha-Amylases / Q000235:genetics*
D003470:Culture Media; D004247:DNA; D000516:alpha-Amylases; D005087:Glucan 1,4-alpha-Glucosidase