N. Kitamoto et al., UTILIZATION OF THE TEF1-ALPHA GENE [TEF1) PROMOTER FOR EXPRESSION OF POLYGALACTURONASE GENES, PGAA AND PGAB, IN ASPERGILLUS-ORYZAE, Applied microbiology and biotechnology, 50(1), 1998, pp. 85-92
For the development of an efficient gene expression system in a shoyu
koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translati
on-elongation factor la, 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 aci
ds possessing high identity to other fungal TEF proteins. Two nucleoti
de sequences homologous to the upstream activation sequence, character
ized for the ribosomal protein genes in Saccharomyces cerevisiae, as w
ell as the pyrimidine-rich sequences were present in the TEF1 gene pro
moter region, suggesting that the A. oryzae TEF1 gene has a strong pro
moter activity. Two expression vectors, pTFGA300 and pTFGB200 for prod
uction of polygalacturonases A and B respectively, were constructed by
using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned f
rom the same strain comprised 1226 bp with two introns and encoded a p
rotein of 367 amino acids with high similarity to other fungal polygal
acturonases. PGA and PGB were secreted at approximately 100 mg/l in gl
ucose 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. P
GB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature
optimum of 55 degrees C.