Eiji Ichikawa

Regulation of the glucoamylase-encoding gene (glaB), expressed in solid-state culture (Koji) of Aspergillus oryzae

Abstract Aspergillus oryzae has two glucoamylase-encoding genes, glaA and glaB, the patterns of expression of which are different. Expression of the glaB gene is marked in solid-state culture (koji), but low in submerged culture. To elucidate the induction mechanism of the glaB promoter in solid-state culture (koji), we employed a fusion gene system using the glaA or glaB promoter and the Escherichia coli uidA gene encoding β-glucuronidase (GUS). The expression of glaB-GUS was induced by starch or maltooligosaccharides in a similar manner to that glaA-GUS, but other physical factors were found to be required for the maximal expression of the glaB gene in solid-state culture (koji). The time-course of glaB-GUS expression in solid-state culture (rice-koji making) suggested that its expression is induced by low water activity (Aw) of the medium and high temperature. When mycelia grown on a membrane under standard conditions were transferred to low-Aw and high-temperature conditions (membrane-transfer culture, MTC), glaB expression was markedly induced, but that of glaA was not. Additionally, glaB-GUS production was induced in MTC using a membrane with smaller pore size, suggesting that a physical barrier against hyphal extension could regulate glaB expression. Under conditions found to induce glaB expression, namely, starch, low-Aw, high-temperature and physical barriers, approximately 6400 U/mg-protein was obtained, equivalent to that in solid-state culture (koji). In conclusion, glucoamylase production under these induction conditions achieved in MTC reached 274 U/ml-broth, which was equivalent to the level observed in solid-state culture (koji). Northern blot analysis indicated that glaB expression was induced at the level of transcription 4 h after the transfer to the inducible conditions described above.

Comparison of two glucoamylases produced by Aspergillus oryzae in solid-state culture (koji) and in submerged culture

Abstract Two extracellular glucoamylases (EC 3.2.1.3) of Aspergillus oryzae were purified from solid-state culture (S-GA) and from submerged culture (L-GA). The two glucoamylases have different molecular masses, 65 kDa for L-GA; 63–99 kDa for S-GA, and different isoelectric points, 4.2 for L-GA; 3.9 for S-GA. Almost all of the enzymatic characteristics of the two glucoamylases were similar, except for thermal stability, initial reaction velocity on pullulan and K m value with soluble starch. Although L-GA could digest raw starch, S-GA demonstrated little activity with raw starch. Peptide mapping and amino acid composition showed that L-GA must be encoded by the glaA gene previously cloned as the glucoamylase-encoding gene from A. oryzae , but S-GA had a different primary structure than the deduced glaA product. Introduction of multiple copies of the glaA gene to A. oryzae caused on elevation of glucoamylase productivity of transformant in submerged culture but not in solid-state culture. These results suggested that the two forms of glucoamylases arise from different genes rather than result from proteolytic processing after polypeptide synthesis of a single protein.

Introduction of a feed back resistant α-isopropylmalate synthase gene of Saccharomyces cerevisiae into sake yeast

Abstract A mutant LEU4 gene (LEU4fbr-2), responsible for both the overproduction of iso-amyl alcohol in yeast and the phenotype of yeast resistant to 5,5,5-trifluoro- dl -leucine (TFL), was isolated from a TFL-resistant mutant of Saccharomyces cerevisiae F-7. The single copy number of LEU4fbr-2 complemented the leucine auxotrophy of S. cerevisiae HB190 (a, leu4, leu5), and also transformed it to TFL-resistant. Leucine-insensitive α-isopropylmalate synthase activity was detected in the crude extract of the Leu+ transformant. Also sake yeast Kyokai no. 7 (K-7) was transformed by the LEU4fbr-2 gene to TFL-resistant. The resulting transformants produced 3∼30-fold higher levels of iso-amyl alcohol (approx. 50∼475 ppm) in shaking cultures, while in static cultures the increase in productivity was only 2.5-fold compared with that of recipient strain K-7. The isolated LEU4fbr-2 gene may be useful as a positive selectable marker for the transformation of industrial yeast.