Utilization of the TEF1-alpha gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae.
For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translation-elongation factor 1 alpha, was cloned from the same strain and used for expression of polygalacturonase genes. The TEF1 gene comprised 1647 bp with three introns. The TEF1-alpha protein consisted of 460 amino acids possessing high identify to other fungal TEF proteins. Two nucleotide sequences homologous to the upstream activation sequence, characterized for the ribosomal protein genes in Saccharomyces cerevisiae, as well as the pyrimidine-rich sequences were present in the TEF1 gene promoter region, suggesting that the A, oryzae TEF1 gene has a strong promoter activity. Two expression vectors, pTFGA300 and pTFGB200 for production of polygalacturonases A and B respectively, were constructed by using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned from the same strain comprised 1226 bp with two introns and encoded a protein of 367 amino acids with high similarity to other fungal polygalacturonases. PGA and PGB were secreted at approximately 100 mg/l in glucose medium and purified to homogeneity. PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature optimum of 45 degrees C. PGB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 degrees C.
Food Research Institute, Aichi Prefectural Government, Nagoya, Japan.
N
N
Kitamoto
J
J
Matsui
Y
Y
Kawai
A
A
Kato
S
S
Yoshino
K
K
Ohmiya
N
N
Tsukagoshi
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000737:chemistry / Q000201:enzymology*; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004271:DNA, Fungal / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics*; D011096:Polygalacturonase / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013025:Glycine max / Q000737:chemistry; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000091262:TEA Domain Transcription Factors; C057182:TEAD1 protein, human; D014157:Transcription Factors; D011096:Polygalacturonase