Naoto Okazaki

Molecular Cloning and Characterization of a Transcriptional Activator Gene, amyR, Involved in the Amylolytic Gene Expression in Aspergillus oryzae

A gene, designated amyR, coding for a transcriptional activator involved in amylolytic gene expression has been cloned from Aspergillus oryzae by screening for a clone that enabled to reverse the reduced expression of the α-amylase gene (amyB) promoter. amyR encodes 604 amino acid residues of a putative DNA-binding protein carrying a zinc binuclear cluster motif (Zn(II)2Cys6) belonging to the GAL4 family of transcription factors. The amyR gene disruptants showed a significant restricted growth on starch medium and produced little of the amylolytic enzymes including α-amylase and glucoamylase compared with a non-disruptant, indicating that amyR is a transcriptional activator gene involved in starch/maltose-induced efficient expression of the amylolytic genes in A. oryzae. In addition, sequencing analysis found that amyR, agdA (encoding α-glucosidase), and amyA (encoding α-amylase), are clustered on a 12-kb DNA fragment of the largest chromosome in A. oryzae, and that amyR is about 1.5 kb upstream of agdA and transcribed in the opposite direction. Furthermore, transcriptional analysis revealed that the amyR gene was expressed in the presence of glucose comparable to the level in the presence of maltose, while the amylolytic genes were transcribed at high levels only in the presence of maltose.

Effects of Aldehyde Dehydrogenase and Acetyl-CoA Synthetase on Acetate Formation in Sake Mash

To reveal the mechanism of the production of acetate by sake yeast (Saccharomyces cerevisiae), the expression of genes encoding aldehyde dehydrogenase (ALD), acetyl-CoA synthetase (ACS) and acetyl-CoA hydrolase (ACH), which are related to acetate production, was investigated. Northern blot analysis using total RNA of sake yeast isolated from sake mash revealed that all of the tested genes, ACS1, ACS2, ALD2/3, ALD4, ALD6 and ACH1, were transcribed during sake fermentation. Transcription of ALD2/3 was detected only in the early stage of sake fermentation. A static culture of sake yeast in hyperosmotic media including 1 M sorbitol or 20% glucose resulted in high acetate production and increased transcription of ALD2/3. This is the same result as reported in an aerobic condition, and induction of ALD2/3 seemed to be one reason for high acetate production at high glucose concentration during fermentation. Overexpression of ACS2 resulted in low acetate production both during small-scale sake fermentation and in a static liquid culture. On the other hand, over-expression of ACS1 did not change acetate productivity significantly in a static culture. These results indicate that ALD2/3 and ACS2 play important roles for acetate production during sake fermentation.

Mycelial distribution in rice Koji

Abstract Mycelial distribution of Aspergillus oryzae in rice koji was measured to elucidate the steric growth mode in the solid-state culture. Mycelial density showed a exponential distribution against the distance from the surface of rice koji . On the basis of this phenomenon, mycelial distribution was expressed by a mathematical model and total mycelia were shown by the following equation: Y t = ac ∫e b ( R − r ) r 2 d r + Y s where Y t is the total mycelia in koji , the first term in right side is the mycelia in the inner part of rice grain and Y s is the surface mycelia. The variable r is the distance from the center of the koji , R is the radius of the koji , and a , b , and c are parameters. The values of each parameter were obtained by the least squares method using the experimental data and the distribution of mycelia both in the surface and in the matrix of rice grain was shown quantitatively. The steric growth mode of fungi in rice grain was shown using these techniques. The mycelial distribution and enzyme composition in rice koji made in sake factories were examined and the results were analyzed by a principal component analysis. The results suggested that when the degree of mycelial penetration is low the activities of acid protease and acid carboxypeptidase are high.

Identification of a mutation causing a defective spindle assembly checkpoint in high ethyl caproate-producing sake yeast strain K1801

In high-quality sake brewing, the cerulenin-resistant sake yeast K1801 with high ethyl caproate-producing ability has been used widely; however, K1801 has a defective spindle assembly checkpoint (SAC). To identify the mutation causing this defect, we first searched for sake yeasts with a SAC-defect like K1801 and found that K13 had such a defect. Then, we searched for a common SNP in only K1801 and K13 by examining 15 checkpoint-related genes in 23 sake yeasts, and found 1 mutation, R48P of Cdc55, the PP2A regulatory B subunit that is important for the SAC. Furthermore, we confirmed that the Cdc55-R48P mutation was responsible for the SAC-defect in K1801 by molecular genetic analyses. Morphological analysis indicated that this mutation caused a high cell morphological variation. But this mutation did not affect the excellent brewing properties of K1801. Thus, this mutation is a target for breeding of a new risk-free K1801 with normal checkpoint integrity. Graphical abstract K1801 (triploid) having 2 copies of R48P-type cdc55 and 1 copy of R48-type cdc55 showed a defective spindle assembly checkpoint (SAC). Cdc55-R48P is semidominant mutation in the SAC function.

Phenotypic Diagnosis of Lineage and Differentiation During Sake Yeast Breeding

Sake yeast was developed exclusively in Japan. Its diversification during breeding remains largely uncharacterized. To evaluate the breeding processes of the sake lineage, we thoroughly investigated the phenotypes and differentiation of 27 sake yeast strains using high-dimensional, single-cell, morphological phenotyping. Although the genetic diversity of the sake yeast lineage is relatively low, its morphological diversity has expanded substantially compared to that of the Saccharomyces cerevisiae species as a whole. Evaluation of the different types of breeding processes showed that the generation of hybrids (crossbreeding) has more profound effects on cell morphology than the isolation of mutants (mutation breeding). Analysis of phenotypic robustness revealed that some sake yeast strains are more morphologically heterogeneous, possibly due to impairment of cellular network hubs. This study provides a new perspective for studying yeast breeding genetics and micro-organism breeding strategies.