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.

Construction of Quintuple Protease and Double Amylase Gene Deletant for Heterologous Protein Production in <i>Aspergillus oryzae</i> KBN616

“…4), which was in accordance with the previously described results (Nemoto et al, 2012, Yoshino-Yasuda et al, 2013. Additional deletion of the taaG1 gene completely eliminated all detectable α-amylase activity.…”

“…taaG3 gene expression is approximately 4-fold higher than that of the taaG1 gene due to the substitution of the CCAAT sequence to CCAAA in the taaG1 gene promoter (Nemoto et al, 2012, Yoshino-Yasuda et al, 2013. The taaG3 gene was deleted first since the loss of taaG3 gene function can be detected easily using the starch plate assay.…”

Molecular Analysis of AsamyR Gene Encoding Transcriptional Factor for Amylolytic Gene from Shoyu Koji Mold, Aspergillus sojae KBN1340

“…Isolation and structural features of AsamyR gene Three genes, AspecA, AsrglA and AstaaG1, from A. sojae KBN1340 have been already cloned and characterized (Yoshino-Yasuda et al, 2011, Yoshino-Yasuda et al, 2012, Yoshino-Yasuda et al, 2013. Their nucleotide sequences including the 5'-and 3'-flanking regions shared significant identity (94.9%, 93.4% and 94.1%) with those from A. oryzae.…”

“…In the promoter region of the A. sojae α-amylase gene (AstaaG1 gene), a CGGAAATTTAAAGG sequence, that is the AmyR binding site of the taaG2 gene, is present at position -264 to -251, as described previously (Yoshino-Yasuda et al, 2013). Therefore, AsAmyR should also regulate the induction of amylolytic genes in A. sojae.…”

“…In order to elucidate the regulation system of the A. sojae α-amylase gene, AstaaG1, we have previously reported its structure and expression (Yoshino-Yasuda et al, 2013). Disruption of the AstaaG1 gene has shown that AsTaaG1 is responsible for α-amylase activity in A. sojae.…”