Molecular Analysis of the α-Amylase Gene, AstaaG1, from Shoyu Koji Mold, Aspergillus sojae KBN1340

Abstract

Aspergillus sojae generally has only one ortholog of the Aspergillus oryzae taa (α-amylase) gene. The AstaaG1 gene from a shoyu koji mold, A. sojae KBN1340, comprised 2,063 bp with eight introns. AsTaaG1 consisted of 498 amino acid residues possessing high identity to other Aspergilli α-amylase sequences. Disruption of the AstaaG1 gene resulted in no detectable α-amylase production in starch medium. Promoter activity of the AstaaG1 gene, monitored by xylanase activity, was upregulated with replacement of the CCAAT-like sequence. Site-directed mutation of the CCAAT-like sequence increased xylanase production approximately four times higher than that of the wild type. These results clearly demonstrate that the decreased copy number of the taa gene and the low affinity binding sequence to the Hap complex lead to the lower amylolytic activity of A. sojae compared to that of A. oryzae.