Satoshi Imayasu

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.

Identification of functional elements that regulate the glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae

Aspergillus oryzae has two glucoamylase-encoding genes, glaA and glaB, whose expressions are distinguished by the type of culture used. The glaB gene is markedly expressed in solid-state culture but is little expressed in submerged culture. In solid-state culture, glaB expression at the transcriptional level is enhanced by low-Aw (water activity), high-temperature, and physical barriers to hyphal extension, as well as by starch. To determine the cis-acting factors in the glaB promoter, deletion analysis of the promoter was done with GUS (β-glucuronidase) as the reporter. Deletion of the 27 bp from −350 to −324 (Region A) in 1.1 kb of the glaB promoter completely abolished starch, low-Aw, and high-temperature induction. Substitution of the 12-bp GC-rich motif from −335 to −324 (GC-box) resulted in significant loss of starch and low-Aw inductivities. These findings suggest that the GC-box is a cis-element essential for the high-level expression of glaB in solid-state culture.

Application of Fuzzy Control Theory to the Sake Brewing Process

Abstract Know-how of skilled operators concerning the management of the traditional sake brewing process was analyzed using fuzzy control theory, and arranged as production rules having the following four fuzzy variables: if (DB = DBi) & (DD = DDi) & (AL = ALi) then (CT = CTi). Membership functions of DB and DD were arranged in real-time using a kinetic model proposed by us previously. Based on these control rules, a fuzzy control system for regulating the moromi (sake mash) temperature was constructed. A computer simulation of sake brewing indicated that the proposed control system could be applied to control the temperature along the desired curve of traditional sake brewing.

Molecular Cloning and Overexpression of fleA Gene Encoding a Fucose-specific Lectin of Aspergillus oryzae

A protein from the cell lysate of Aspergillus oryzae was purified by column chromatography immobilized with a ferrichrysin (Fcy), which is one of the siderophores of A. oryzae. It is produced only in an iron-deficient culture and its molecular weight is estimated as 35,000 by SDS-PAGE. Two internal amino acid sequences of the protein obtained by lysylendopeptidase digestion were analyzed. Molecular cloning shows that it encodes 310 putative amino acid residues separated by 4 introns and is designated as fleA. It shows approximately 26% similarity with the gene encoding a fucose-specific lectin of Aleuria aurantia (AAL). The gene was overexpressed under control of the melO promoter in a submerged culture of A. oryzae. The fleA gene product showed hemagglutination activity against rabbit erythrocytes. A hemagglutination inhibition assay of monosaccharides showed that this lectin specifically binds to L-fucose and weakly reacts with mannose and N-acetyl-neuraminic acid.

Methods for sporulation of industrially used sake yeasts

Industrially used sake yeasts (Saccharomyces cerevisiae) such as Kyokai no. 7 and no. 9 hardly sporulate under any of the nutritional conditions examined to date. However, through acquisition of either sensitivity to ethionine or the ability to grow at 35°C in the presence of β-alanine, these sake yeasts were found to regain the ability to form asci with 4 viable spores at high frequency. These methods for the mutant-selection may facilitate the genetics of industrially used yeasts and their breeding by hybridization.

Analysis of the state characteristics of sake brewing with a neural network

The state characteristics of sake brewing were quantitatively defined as a numerical value, i.e. an SD value, expressing the time required for the alcohol concentration to reach 10 to 18 v/v%, assuming the temperature of the sake-mash (moromi) to be maintained at 15°C during brewing. A previously reported kinetic model of alcohol production was applied for this definition. The SD values varied from batch to batch, indicating that the target Baume reference pattern for the fuzzy control system reported previously should be selected in accordance with the characteristics of brewing. A procedure for estimating the brewing characteristics in the early stages was developed by applying error back-propagation learning with a neural network. This procedure enabled the fuzzy control system to generate the target Baume reference pattern according to the brewing characteristics.

Highly Efficient Sporulation Induced by Glutathione or Glutathione Thiol Esters in Sake (Kyokai No. 7) and a Wild-Type Yeast

Abstract When vegetative cells of Saccharomyces cerevisiae 4011 and a sake yeast (S. cerevisiae), Kyokai no. 7, were incubated in a sporulation medium, glutathione in the cells decreased with the increase in sporulation and was lost by the time the sporulation reached a maximum. The sporulation efficiency varied depending on the cellular glutathione content, and was lowered with a decrease of the level. Glutathione and its thiol esters significantly stimulated the sporulation of both strains when these chemicals were added to sporulation medium. The activities of glycolytic bypass enzymes, which are responsible for the synthesis of glutathione thiol ester, increased in both yeast strains during incubation in the sporulation medium. These results suggest that glutathione was converted into glutathione thiol ester and that the ester functioned as a stimulator of yeast sporulation.

On the importance of calcium and magnesium ions in yeast sporulation

Abstract Irrespective of the nutritional conditions, the sporulation frequency of wild and industrially used yeasts on agar or agarose plates has been found to vary from one experiment to another. An analysis of agar- and agarose-extracts by ion-exchange column chromatography proved that the amount of calcium and/or magnesium ions contained in the agar was a factor in the fluctuation of sporulation frequency. Furthermore, these two cations enhanced the formation of four-spored asci. When calcium or magnesium ions were added to a nutrition-deprived medium solidified with agarose containing no detectable calcium and magnesium ions, wild and industrially used sake yeasts efficiently sporulated with a frequency of 10–40%. A strictly controlled sporulation condition suitable for the analysis of meiosis and sporulation of yeast cells was constructed by using calcium and/or magnesium ions and highly purified agarose.

Induction of yeast sporulation by lysine-related compounds and glutathione in nutrition-rich conditions

Abstract In addition to dl -lysine (Kawado et al. , J. Ferment. Bioeng., 76, 391–394, 1993), dl -2-aminoadipate was found to induce sporulation in a wild-type yeast of Saccharomyces cerevisiae 4011 growing in a nutrition-rich medium without potassium acetate. In the presence of the acid at 10 mM, the sporulation frequency of the yeast cells increased in parallel with the growth and reached maximum (26%) in the stationary phase. Despite the expression of a meiosis inducer gene, IME1 , no sporulation was induced unless dl -lysine or dl -2-aminoadipate was included in the nutrition-rich medium. By using dl -2-aminoadipate in combination with glutathione, sporulation was also induced in industrially used sake yeasts growing in the nutrition-rich medium without potassium acetate.