平成17年度 研究成果

M.Tominaga, YH.Lee, R.Hayashi, Y.Suzuki, O.Yamada, K.Sakamoto, K.Gotoh, O.Akita.

To help assess the potential for aflatoxin production by Aspergillus oryzae, the structure of an aflatoxin biosynthesis gene homolog cluster in A. oryzae RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99% similarity to those of Aspergillus flavus, three genes shared 93% similarity or less. A 257-bp deletion in the aflT region, a frameshift mutation in norA, and a base pair substitution in verA were found in A. oryzae RIB 40. In the aflR promoter, two substitutions were found in one of the three putative AreA binding sites and in the FacB binding site. PCR primers were designed to amplify homologs of aflT, nor-1, aflR, norA, avnA, verB, and vbs and were used to detect these genes in 210 A. oryzae strains. Based on the PCR results, the A. oryzae RIB strains were classified into three groups, although most of them fell into two of the groups. Group 1, in which amplification of all seven genes was confirmed, contained 122 RIB strains (58.1% of examined strains), including RIB 40.

Seventy-seven strains (36.7%) belonged to group 2, characterized by having only vbs, verB, and avnA in half of the cluster. Although slight expression of aflR was detected by reverse transcription-PCR in some group 1 strains, including RIB 40, other genes (avnA, vbs, verB, and omtA) related to aflatoxin production were not detected. aflR was not detected in group 2 strains by Southern analysis.

T.Akao, M.Yamaguchi, A.Yahara, K.Yoshiuchi, H.Fujita, O.Yamada, O.Akita, T.Ohmachi, Y.Asada, T.Yoshida.

1,2-alpha-Mannosidase catalyzes the specific cleavage of 1,2-alpha-mannose residues from protein-linked N-glycan. In this study, a novel DNA sequence homologous to the authentic 1,2-alpha-mannosidase was cloned from a cDNA library prepared from solid-state cultured Aspergillus oryzae. The fmanIB cDNA consisted of 1530 nucleotides and encoded a protein of 510 amino acids in which all consensus motifs of the class I alpha-mannosidase were conserved. Expression of the full length of 1,2-alpha-mannosidase cDNA by the Aspergillus host, though it has rarely been done with other filamentous-fungal mannosidase, was successful with fmanIB and caused an increase in both intracellular and extracellular mannosidase activity. The expressed protein (FmanIBp) specifically hydrolyzed 1,2-alpha-mannobiose with maximal activity at a pH of 5.5 and a temperature of 45 degrees C. With Man(9)GlcNAc(2) as the substrate, Man(5)GlcNAc(2) finally accumulated while hydrolysis of the 1,2-alpha-mannose residue of the middle branch was rate-limiting. To examine the intracellular localization of the enzyme, a chimeric protein of FmanIBp with green fluorescent protein was constructed. It showed a dotted fluorescence pattern in the mycelia of Aspergillus, indicative of the localization in intracellular vesicles. Based on these enzymatic and microscopic results, we estimated that FmanIBp is a fungal substitute for the mammalian Golgi 1,2-alpha-mannosidase isozyme IB. This and our previous report on the presence of another ER-type mannosidase in A. oryzae (Yoshida et al., 2000) support the notion that the filamentous fungus has similar steps of N-linked glycochain trimming to those in mammalian cells.

M.Okuda, I.Aramaki, T.Koseki, N.Inouchi, and K.Hashizume

Structural and physicochemical characteristics of endosperm starch from milled rice grains of seven Japanese cultivars used in sake production were examined. Amylose content was 15.2 - 20.2%, number-average degree of polymerization (DP(n)) of amylose was 900 - 1,400, and the ratio of short-to-long chain amylopectin was 2.7 - 3.5, respectively. The degree of retrogradation of purified starch stored for seven days at 4℃ after gelatinization was 20 - 31%. The degree of retrogradation correlated negatively with the ratio of short-to-long chain amylopectin. The effect of holding time after steaming on enzyme digestibility and starch retrogradation of steamed rice grains was investigated. The longer the holding time after steaming, the greater the extent of retrogradation, and the less the degree of enzymatic digestibility. The decreased rate of enzyme digestibility correlated with amylopectin chain length distribution. Samples with short-chain amylopectin exhibited a slow decrease in enzyme digestibility. It was determined that the structure and retrogradation properties of endosperm starch in Japanese rice cultivars affect the decreasing rate of enzyme digestibility of the steamed, milled rice grains.

M.Tamaki, S.Kurita, M.Toyomaru, T.Itani, T.Tsuchiya, I.Aramaki and M.Okuda

This study was designed to determine whether or not the difference in the physical properties between white-core and non-white-core kernels of the rice varieties for sake brewing is associated with their starch properties. We used two rice cultivars for sake brewing, Senbon-nishiki and Yamada-nishiki, from three different plots in Hiroshima prefecture. Hardness values of kernels were significantly higher in non-white-core than in white-core kernels in both varieties. Vickers hardness (VH) values were lowest at the center of the kernel in both types of kernels. VH values of white-core tissues of white-core kernels were significantly lower than those of corresponding tissues of non-white-core kernels. No significant differences were observed between the two types of kernels in VH values of the surrounding translucent tissues and in the starch properties (amylose content, pasting properties analyzed using a rapid viscoanalyzer and gelatinization properties analyzed using a differential scanning calorimetry). These results suggest that the difference in physical properties between the two types of kernels of the rice varieties for sake brewing are associated with the difference in structure of endosperm cells and not in starch properties.

T.Yamane, S.T.Jeong, N.Goto-Yamamoto, Y.Koshita, and S.Kobayashi

Although coloration of grape berry skins is influenced by temperature, the details of its effects have not been reported. To find temperature sensitive stages for coloration and to clarify the mechanisms that underlie the effect of temperature on anthocyanin accumulation, two-week treatments at temperatures of 20℃ and 30℃ were carried out at four different stages of development and ripening using each of three potted vines of Aki Queen (Vitis labrusca x V. vinifera). Anthocyanin accumulation in the skins was significantly higher at 20℃ than at 30℃ after the temperature treatment, and the most sensitive stage for the temperature treatment was from one to three weeks after coloring began (stage III). Furthermore, at harvest, the grapes treated at 20℃ in stage III contained the highest concentration of anthocyanin. After temperature treatment in stage III, the concentration of abscisic acid (ABA), a plant hormone related to anthocyanin accumulation, in the berry skins was 1.6 times higher at 20℃ than at 30℃. The copy numbers of accumulated mRNA of anthocyanin biosynthetic enzyme genes and a myb-related regulate gene, VvmybA1, were also higher at 20℃ than at 30℃. These results and previous reports indicate that the high and low temperatures during ripening, especially in stage III, likely affect the production and/ or degradation of ABA in berry skins and that the endogenous ABA level affects the expression of VvmybA1; the product of VvmybA1 then controls the expression of the anthocyanin biosynthetic enzyme genes.

N.Goto-Yamamoto, H.Mouri, M.Azumi, and K.J.Edwards

Nine newly developed microsatellite markers for grapes were characterized. Using these markers and eight reported markers, seven Occidental and eight Oriental cultivars were analyzed, including Japanese and Chinese cultivars of Vitis vinifera, two cultivars of V. labrusca, and one sample each of V. riparia and V. rotundifolia. Calculated phenetic distances (1 - proportion of shared alleles) agreed well with the classification of grapes. A dendrogram based on the phenetic distances showed a clear separation of species of Vitis, as well as Oriental and Occidental cultivars. Results suggested the importance of Oriental cultivars as genetic resources of grapes.

Ardiansyah,* H.Shirakawa, T.Koseki, K.Ohinata, K.Hashizume, and M.Komai

Effect of dietary supplementation of two types of rice bran fraction on blood pressure (BP), lipid profile, and glucose metabolism in stroke-prone spontaneously hypertensive rats was studied. Male 4-week-old rats were divided into one group fed the AIN-93M-based control (C) diet and two groups fed diet supplemented with 60 g/kg of Driselase and ethanol fractions (DF and EF, respectively) of rice bran. After 8 weeks feeding, the BP decreased in the DF and EF groups in comparison with the C group (p < 0.01). Plasma ACE inhibitory activity, BUN, BUN/creatinine ratio, albumin, triglyceride, and glucose levels were lower in the DF and EF groups than in the C group (p < 0.01). Plasma nitric oxide and urinary 8-hydroxy-2'-deoxyguanosine levels were lower in the DF and EF groups than in the C group (p < 0.01). Rice bran fractions appear to have a beneficial dietary component that improves hypertension, hyperlipidemia, and hyperglycemia.

T.Koseki, Y.Miwa, T.Akao, O.Akita, K.Hashizume

We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused α-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward α-naphthylacetate (C2), lower activity toward α-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.

A.Mizuno, H.Tabei, and M.Iwahuti

We isolated a mutant with low acetic acid and high ethanol productivities from 2-deoxyglucose-resistant mutants of brewer's yeast NCYC1245 (Saccharomyces cerevisiae). To determine the mechanism for these properties in the mutant (2DGR19) during fermentation, gene expression and enzyme activity related to acetic acid and ethanol production were investigated. DNA microarray analysis revealed that the transcriptional levels of many genes involved in glycolysis were higher in 2DGR19 than in NCYC1245. Among these transcriptional levels of 2DGR19 relative to NCYC1245, the expression level of ADH4 encoding alcohol dehydrogenase (ADH) was highest, which corresponded to the high ADH activity in 2DGR19. Quantitative PCR analysis also revealed that the transcriptional level of ADH4 was the highest among ADH1 to ADH4. Although no significant differences in the transcriptional levels of ALD2 to ALD6 encoding acetaldehyde dehydrogenase (ALD) between 2DGR19 and NCYC1245 were observed, ALD activity in 2DGR19 was lower. Using quantitative PCR analysis, ALD6 was found to be the most highly expressed among the ALD2 to ALD6 genes. These results indicate that ALD6 contributes to a low ALD activity, depending on post-transcriptional regulation. A high ADH activity appeared to be the major reason for the high ethanol productivity of 2DGR19. A low ALD activity was considered to be principally responsible for a low acetic acid productivity, although a high ADH activity also might have played a role. Beer brewed using 2DGR19 in pilot-scale high-gravity brewing contained about half as much acetic acid and 1.1% more ethanol compared with that brewed using NCYC1245. The use of 2DGR19 may overcome difficulties associated with high-gravity brewing.

中村洋二郎、下飯仁、伊藤清

白麹菌から細胞表層タンパク質の候補（CwpA）を得たが、本遺伝子のコードするタンパク質はN末端とC末端の双方に疎水性領域をもち、GPIアンカータンパク質の特徴的な構造を示した。

本タンパク質は膜画分に存在し、PI-PLC処理を行わない場合には界面活性剤層に抽出され、PI-PLC処理を行いリン脂質の部分を切断除去すると水層に移行した。また、GIP付加シグナルの直前にストップコドンを人工的に導入すると、細胞に保持されず、分泌された。以上の結果より、CwpAはGPIアンカータンパク質であることが示された。

GFPを付加したCwpAは細胞表層に蛍光を示したので、細胞膜にGPIを介してアンカリングされていることが示された。

磯谷敦子、宇都宮仁、神田涼子、岩田博、中野成美

市販酒における「老香」に関与する香気成分について検討するため、全国市販酒類調査において老香を指摘された市販清酒（老香清酒）の香気成分の定量および官能評価を行った。比較のため、「老香」を指摘されなかった市販清酒（老香なし清酒）および長期熟成酒（貯蔵期間3年以上）についても分析を行った。揮発性アルデヒド類、エチルエステル類、ポリスルフィド（DMDS, DMTS）、フルフラール、ソトロンは、老香なし清酒＜老香清酒＜長期熟成酒の順に多くなった。主成分分析の結果、老香清酒はポリスルフィドが相対的に多く、長期熟成酒で貯蔵期間が特に長いものはソトロン、揮発性アルデヒド類、フルフラールおよびコハク酸ジエチルが多いことが示唆された。市販酒の官能評価の結果、老香強度とDMTS濃度の対数との間には相関がみられたが、ソトロン濃度の対数との間には相関がみられなかった。これらの結果から、DMTSは市販酒における「老香」に大きく寄与していることが示唆された。

M.Shobayashi, N.Mukai, K.Iwashita, Y.Hiraga, H.Iefuji

S-adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study.

In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolate by this method. These mutants accumulated 1.7-5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.

Y.Fujita, E.Ukena, H.Iefuji, Y.Giga-Hama, K.Takegawa.

Methionine synthase (EC2.1.1.14) catalyses the final step in methionine synthesis i.e., methylation of homocysteine. A search of the Schizosaccharomyces pombe genomic database revealed a gene designated SPAC9.09, encoding a protein with significant homology to methionine synthase. Disruption of SPAC9.09 caused methionine auxotrophy, thus the gene was identified as a methionine synthase and designated met26+. We investigated additional phenotypes of the met26 mutant and found that it exhibited a remarkable growth defect in the absence of adenine even in media supplemented with methionine. This phenotype was not observed in other methionine auxotrophs. In the budding yeast Saccharomyces cerevisiae that has been reported to utilize homocysteine in cysteine synthesis, lack of a functional methionine synthase did not cause a requirement for adenine. The introduction of genes from S. cerevisiae constituting the cystathionine pathway (CYS4 and CYS3) into S. pombe met26D cells restored growth in the absence of adenine. HPLC analysis showed that total homocysteine content in met26D cells was higher than in other methionine auxotrophs and that introduction of the S. cerevisiae cystathionine pathway decreased total homocysteine levels. These data demonstrate that accumulation of homocysteine causes a defect in adenine biosynthesis in the met26 mutant.

C.Lin, Y.Hiraga, K.Masaki, H.Iefuji and K.Ohkata

In order to elucidate the nature of a novel lipase (CSL), isolated from the yeast Cryptococcus spp. S-2, in chiral recognition by comparison with that of immobilized PPL, the desymmetrization and asymmetrization of prochiral 2-phenyl-1,3-propanediol (1a), 2-benzyl-1,3-propanediol (1b), 2-methyl-2-phenyl-1,3-propanediol (1c), 2-benzyl-2-methyl-1,3-propanediol (1d), 2-ethyl-2-phenyl-1,3-propanediol (1e), and 2-benzyl-2-ethyl-1,3-propanediol (1f) by acetylation was investigated. Acetylation of 1a with excess vinyl acetate by the CSL-enzyme catalyst gave the corresponding monoacetate 2a with high enantioselectivity (80% ee) in 46% yield. Very high levels of desymmetrization were observed in the tertiary systems of 1c-f, giving the corresponding monoacetates 2c-f, respectively, in >97%. In the desymmetrization of diols 1a, 1c, 1d, and 1f, the sense of chiral differentiation of CSL was opposite to that of immobilized porcine pancreatic lipase (PPL).

A.Fujita, N.Goto-Yamamoto, I.Aramaki, and K.Hashizume

In order to investigate the control mechanism of flavonol biosynthesis of grapevine, we obtained five genomic sequences (FLS1 to FLS5) of putative flavonol synthase genes from Vitis vinifera cv. Cabernet Sauvignon. The mRNA of five FLSs accumulated in flower buds and flowers, while the mRNA of FLS2, FLS4, and FLS5 accumulated in small berry skins and then decreased toward veraison. At the ripening stage, the mRNA of only FLS4 and FLS5 accumulated again. This change in mRNA accumulation did not contradict the flavonol accumulation in the berry skins. Shading of the berries completely inhibited the increase in flavonol content and mRNA accumulation of FLS4, but did not affect the mRNA accumulation of FLS5. The effects of light and plant hormones on flavonol accumulation were different from those on anthocyanin accumulation. Thus flavonol biosynthesis appears to be under a different control system from that of anthocyanin biosynthesis.

M.Tamaki, R.Kihara, M.Okuda, I.Aramaki, Z.Katsuba and T.Tsuchiya

By successive crossing using Hattan-type varieties originating from Hattanso" as a parent, "Hattan-type varieties" of rice suitable for brewing the original Hiroshima sake have been bred. In this study, the difference in the properties of starch and protein among the Hattan-type varieties was examined. Six Hattan-type varieties, Hattanso, Hattan No.10, Hattan No.35, Hattan No.40, Hattan-nishiki No.1 and Hattan-nishiki No.2, were used. As the properties of starch, amylose content, pasting properties and gelatinization properties were examined. The pasting and gelatinization properties were examined using a rapid viscoanalyzer (RVA) and a differential scanning calorimetry (DSC), respectively. As the properties of protein, the compositional ratio of two types of protein bodies (PB-II/PB-I) was analyzed. However, no significant differences in the above properties were observed among these Hattan-type varieties. The above properties of starch and protein in Hattanso seem to be retained in all of these varieties. In these varieties, breeding might not have been aimed at improvement of the properties of starch and protein.

S.T.Jeong, N.Goto-Yamamoto, K.Hashizume, and M.Esaka

Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are involved in the biosynthesis pathway of cyanidin- and delphinidin-based anthocyanins, as well as quercetin and myricetin (flavonols), and procyanidin and prodelphinidin (flavan-3-ols). Grape (Vitis vinifera) accumulates these three classes of flavonoids and is presumed to have both F3'h and F3'5'h. Thus, we obtained the genomic sequences of F3'h and F3'5'h from grape and determined their mRNA levels as well as the flavonoid composition of several grape organs to investigate whether the transcription of these genes controls the flavonoid composition. The flower, stem, tendril, and seed, which accumulated a higher level of mRNA of F3'h than F3'5'h, showed reasonable compositions of flavonols and/or flavan-3-ols. The organs that accumulated a high level of F3'5'h mRNA contained a high level of delphinidin-based anthocyanins (berry skin at the harvest stage) or prodelphinidin (small leaf), consistent with the mRNA levels. However, not all the classes of flavonoid showed a similar composition according to the mRNA levels of F3'h and F3'5'h. The compositions of anthocyanins and flavonols in the small leaf were inconsistent with the mRNA levels. Consequently, other mechanisms in addition to gene transcription are also expected to control the composition of each flavonoid class.

A.Fujita, N.Soma, N.Goto-Yamamoto, H.Shindo, T.Kakuta, T.Koizumi, and K.Hashizume

Proanthocyanidins, which are polymers and oligomers of flavan-3-ol units, are important components of grape for red winemaking and accumulate in the berry skins and seeds. Major flavan-3-ol units of grape proanthocyanidins are (-)-epicatechin and (-)-epigallocatechin. It was recently reported that (-)-epicatechin and (-)-epigallocatechin were biosynthesized from corresponding anthocyanidins by anthocyanidin reductase (ANR). In the current study, we obtained a genomic sequence of the ANR gene of Vitis vinifera cv. Cabernet Sauvignon (VvANR). The deduced amino acid sequence of VvANR conserved the characteristic sequence of ANR of other plants. Southern blot analysis showed that grape ANR is probably encoded by a single copy gene in its haploid genome. Real-time quantitative-PCR using berry skins and seeds showed that the mRNA of VvANR accumulates at the early stage of berry development and then decreases toward the ripening stage. Our analysis and other reports show that proanthocyanidins accumulate in the berry skins and seeds before veraison, especially during the early stage of development, and then decrease in concentration during ripening. Thus, the change of mRNA accumulation substantially coincides with the change of proanthocyanidin accumulation in the berry skins and seeds. These results indicate that at least a part of (-)-epicatechin and (-)-epigallocatechin biosynthesis is probably controlled by the transcription of VvANR.

K.Masaki, N.R.Kamini, H.Ikeda, and H.Iefuji

A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (e-caprolactone), and poly(3-hydroxybutyrate).

M.Shobayashi, S.Mitsueda, M.Ago, T.Fujii, K.Iwashita, H.Iefuji

Ergosterol is an essential component of yeast cell to maintain the integrity of membrane and investigated as an important factor of ethanol tolerance of yeast cells. We investigated the effects of brewing conditions on ergosterol contents of S. cerevisiae K-9, sake yeast, kinds of Saccharomyces cerevisiae, produce more than 20% of ethanol, and X2180-1A, laboratory yeast. K-9 has a higher total ergosterol contents under the all conditions which we examined than X2180-1A. Ethanol and hypoxia were found to have negative and synergistic effects on total ergosterol contents of both strains and significantly reduced the free ergosterol contents of X2180-1A but only slightly reduced the free ergosterol contents of K-9. The maintenance of free ergosterol contents under brewing conditions might be an important character of sake yeast strains. DNA microarray analysis also showed higher expression of ergosterol biosynthesis genes in K-9 than X2180-1A.

M.Machida, K.Asai, M.Sano, T.Tanaka, T.Kumagai, G.Terai, K.Kusumoto, T.Arima, O.Akita, Y.Kashiwagi, K.Abe, K.Gomi, H.Horiuchi, K.Kitamoto, T.Kobayashi, M.Takeuchi, D.W.Denning, J.E.Galagan, W.C.Nierman, J.Yu, D.B.Archer, J.W.Bennett, D.Bhatnagar, T.E.Cleveland, N.D.Fedorova, O.Gotoh, H.Horikawa, A.Hosoyama, M.Ichinomiya, R.Igarashi, K.Iwashita, P.R.Juvvadi, M.Kato, Y.Kato, T.Kin, A.Kokubun, H.Maeda, N.Maeyama, J.Maruyama, H.Nagasaki, T.Nakajima, K.Oda, K.Okada, I.Paulsen, K.Sakamoto, T.Sawano, M.Takahashi, K.Takase, Y.Terabayashi, J.R.Wortman, O.Yamada, Y.Yamagata, H.Anazawa, Y.Hata, Y.Koide, T.Komori, Y.Koyama, T.Minetoki, S.Suharnan, A.Tanaka, K.Isono, S.Kuhara, N. Ogasawara and H. Kikuchi

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.

H.Wu, K.Ito, and H.Shimoi

Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae.

向井伸彦、木曽邦明

著者らは、発ガン性が疑われているカルバミン酸エチル(EC)について、市販アルコール飲料（清酒、合成清酒、焼酎）での調査を行った。 調査対象の市販酒類として、清酒については通常製品（古酒あるいは長期貯蔵酒の表示のないもの）が125点、古酒（長期貯蔵酒）が32点であり、合成清酒は4点、焼酎は19点（芋焼酎：6点、麦焼酎：5点、米焼酎：4点、黒糖焼酎：1点、そば焼酎：1点、胡麻焼酎：1点、泡盛：1点）であった。清酒（通常製品（古酒以外））では、平均47μg/kg、最大210μg/kg含有していた。清酒（古酒）では、平均183μg/kg、最大1100μg/kg含有していた。合成清酒では、いずれも定量限界(20μg/kg)未満であった。焼酎では、15点が定量限界(20μg/kg)未満であり、最大37μg/kg含有していた。清酒（古酒）では、EC含有量と着色度及び紫外部吸収との間に高い正の相関がみられた。

向井伸彦、木曽邦明、家藤治幸

著者らは、バニリンの前駆物質である4-ビニルグアヤコール (4-VG) 含有量を高めた焼酎の製造を試みた。米焼酎と泡盛の小仕込み試験において、酵素剤（ヘミセルラーゼ剤）を添加することにより、もろみの遊離フェルラ酸が増加するとともに、留液の4-VGが増加した。4-VG生成能を有するワイン酵母を併用することで4-VGはさらに増加した。

K.Mori, H.Saito, N.Goto-Yamamoto, M.Kitayama, S.Kobayashi, S.Sugaya, H.Gemma and K.Hashizume

Potted Pinot noir grapevines were grown under continuous high temperature (30℃) or low night (15℃) and high day (30℃) temperatures after veraison. Half of the total number of clusters of each vine was sprayed with 250 ppm abscisic acid (ABA) at veraison. Anthocyanin accumulation in berry skins grown under high night temperatures was lower than that in berries grown under low night temperatures. HPLC analysis showed that the ratios of delphinidin-3-glucoside, cyanidin-3-glucoside and petunidin-3-glucoside to the total anthocyanin content were greatly reduced under high night temperatures. ABA treatment enhanced anthocyanin accumulation under high night temperatures to almost the same level as under low night temperatures; the ratio of each anthocyanin to the total anthocyanin was not affected by ABA treatment.

T.Ohnuki, T.Ozaki, T.Yoshida, F.Sakamoto, N.Kozai, E.Wakai, A.J.Francis, and H.Iefuji

We determined the association of uranium in yeast cells S. cerevisiae grown in medium containing high (1g･L-1) or low (0.2g･L-1) concentrations of phosphate after exposure for 96 h to a 4 10-4mol･L-1 U(VI) solution at pH 3.2 or 4.7. The analysis was made using a field emission scanning electron microscope equipped with energy dispersive spectroscopy (FESEM-EDS), transmission electron microscopy (TEM), and visible diffuse reflectance spectrometry. Cells grown in the high-phosphate medium rapidly accumulated U(VI) from solution at pH 3.2 over the first 24 h, followed by a slow uptake until 96 h, whereas in cells grown in low-phosphate medium, U(VI) accumulation reached a steady state within 24 h. FESEM-EDS analyses revealed the formation of a U(VI)-bearing precipitate on the yeast cells grown in high-phosphate medium after only 48 h exposure; no precipitate was detected on cells grown in low-phosphate medium up to 96 h. These results suggest that sorption onto the cell surfaces was the dominant process initially. Analysis of the U(VI)-bearing precipitates by all three methods demonstrated the presence of H-autunite, HUO2PO4 ･ 4H2O. Thermodynamic calculations suggest that the chemical compositions of the solutions containing yeast grow in high-phosphate medium were undersaturated with respect to H-autunite, but were supersaturated with ten times more U(VI) and P than were actually observed. Apparently, the sorbed U(VI) on the cell surfaces reacts with P released from the yeast to form H-autunite by local saturation. The U(VI) uptake by yeast cells grown in high phosphate medium at pH 4.7, along with the thermodynamic calculation, indicated that more H-autunite is precipitated in neutral pH solution than in acid solution. Thus, U(VI)-phosphate mineralization on the cells of microorganisms should be taken into account for predicting U(VI) mobility in the environment.

F.Sakamoto, T.Ohnuki, N.Kozai, E.Wakai, T.Fujii, H.Iefuji, and A.J.Francise

We have carried out the growth experiments of 3 strains of yeast in a medium containing uranium (VI) to elucidate the effect of U (VI) on the growth of microorganisms. Hansenula fabianii J640 grew in the liquid medium containing 0.1 mM U (VI) at lower rate than the control, but Saccharomyces cerevisiae did not grow under this condition. The H. fabianii J640 pre-cultured for 21 h in the liquid medium without U (VI) grew even after the exposure to 1 mM U (VI), but did not grow without pre-cultivation. For the pre-cultured H. fabianii J640, radioactivity of U in the medium was the same as the initial one for 110 h, and then gradually decreased. TEM-EDS analysis of H. fabianii J640 exposed to 1 mM U (VI) for 165 h showed accumulation of U (VI) on the cells. When H. fabianii J640 was not pre-cultured, radioactivity of U in the medium was lower than the initial one. These results indicated that U (VI) inhibits the growth of yeast, and that the adsorption of U (VI) by the cells depends on the metabolism of yeast.

M.Shimizu, K.Miyashita, H.Kitagaki, K.Ito, and H.Shimoi

Sake yeasts are used for sake brewing and have a crucial role in the quality of sake, since they produce not only ethanol but also various compounds that provide sake flavors. Therefore, the appropriate selection and monitoring of a strain used in sake mash is important. However, the identification of specific sake yeast strains have been difficult, because sake yeasts have similar characteristics in taxonomic and physiological analyses. We found amplified fragment length polymorphisms (AFLPs) in the PCR products of the AWA1 gene of sake yeast strains. The AWA1 gene encodes a cell wall protein that is responsible for foam formation in sake mash. This polymorphism of the AWA1 gene can be used for the identification of sake yeast strains.

H.Hisada, Y.Hata, A.Kawato, Y.Abe, O.Akita

Fungi contain distinct genes encoding the same class of enzyme that are differentially regulated according to conditions. We cloned two catalase genes, catA and catB, from Aspergillus oryzae. The catA gene predicts a 747-amino-acid polypeptide sharing 81% identity with Aspergillus fumigatus catalase (catA) and 77% with Aspergillus nidulans catalase (catA). The catB gene predicts a 725-amino-acid polypeptide sharing 82% identity with A. fumigatus catalase (catB) and 75% with A.nidulans catalase (catB). However, the catA and catB genes share little homology (41%) with one another, suggesting that each gene belongs to a distinct gene family. Overexpression studies demonstrated that both genes encode a functional catalase. Promoter assays indicated that the catA gene is developmentally regulated as it was preferentially expressed in solid-state cultures undergoing sporulation. However, its expression was not affected by hydrogen peroxide treatment. Conversely, the catB gene was highly expressed under all culture conditions tested, and it was induced by hydrogen peroxide treatment. These results suggest that the catB gene may be mainly used for detoxification of oxidative stress while the catA gene may have another role such as chaperoning proteins in the spore.

A.Isogai, H.Utsunomiya, R.Kanda, and H.Iwata

Changes in the aroma of sake during aging were investigated by aroma extract dilution analysis (AEDA) and quantitative analysis using the stir bar sorptive extraction (SBSE) method. In AEDA, more odor zones were detected in aged sake than in fresh. The dilution factors of aldehydes, polysulfides, and some esters were greater in the aged sake, and their increase during aging was confirmed through a quantitative analysis of sake stored for 0 to 35 years. Among these compounds, 3-methylbutanal, methional, and dimethyltrisulfide (DMTS) were present in aged sake at concentrations exceeding their odor thresholds, and the highest odor active-value was observed for DMTS. Sensory tests showed that supplementation with DMTS contributed to both the total odor intensity and the sulfury odor of aged sake aroma.

A.Miki, A.Isogai, H.Utsunomiya, and H.Iwata

2,4,6-Trichloroanisole (TCA), which has been identified as the main component responsible for the cork taint in wine, was detected in sake samples having a musty/muddy off-flavor by stir bar sorptive extraction (SBSE). We confirmed that TCA is one of the components causing this off-flavor in sake, as in other alcoholic beverages, from a sensory analysis showing the correlation between TCA concentration and the intensity of the musty/muddy off-flavor. We investigated the route of TCA production in the rice koji preparation process and in the moromi mash process for sake brewing. We found that TCA is produced mainly by the biomethylation of 2,4,6-trichlorophenol (TCP) by rice koji in brewing and that TCP originates from the wooden tools used in preparing rice koji.

M.Okuda, I.Aramaki, T.Koseki, H.Satoh, and K.Hashizume

Using rice samples derived from normal rice cultivars and endosperm starch mutant, we investigated key factors contributing to the enzyme digestibility of steamed rice grains. The chemical composition of polished rice grains, structural features of endosperm starch, and enzyme digestibility of steamed rice grains were examined. The protein content of polished rice grains was 4.6 - 9.1%, amylose content was 4 - 27%, the DPn of purified amylose was 900 - 1,600, the amylopectin short/long chain ratio was 1.2 - 5.9, and the enzyme digestibilities of steamed polished rice grains were 0.9-12.6 ^。Brix. Amylose content and RVA parameters (viscosity, breakdown, and setback) correlated significantly with enzyme digestibility of steamed rice grains. Multiple regression formulas were constructed to predict digestibility of steamed rice grain as a function of the molecular characteristics of the starch. When both amylose content and the short/long chain amylopectin ratio were used as predictor variables, they accounted for >80% of the observed variance in digestibility of steamed rice grains. Multiple regression revealed that the more digestible rice samples had starch with a lower amylose content and more shortchain amylopectin. Reassociation of amylose-lipid complex and recrystallization of amylopectin in the stored steamed rice grains was monitored by differential scanning calorimetry (DSC), and the observed retrogradation properties were related to the structural characteristics of starch and to the enzyme digestibility of steamed rice grains.

M.Tamaki, R.Kihara, T.Itani, Z.Katsuba, T.Tsuchiya, H.Matsumoto, K.Suenari and I.Aramaki

By successive crossing using Hattan-type varieties originating from "Hattanso" as a parent "Hattan-type varieties" of rice suitable for brewing the original Hiroshima sake have been bred. The varieties were improved inheriting the flavor of Hattan-type sake, and Hattan-nishiki N0.1 and No.2 were bred in 1984. However, neither variety was suitable for brewing high-grade sake such as Ginjoshu and Daiginjoshu, which require high-degree polishing of rice grains. Therefore, their parent cultivar, Hattan No. 35, is attracting attention for the production of high-grade sake. We have been trying to breed a new variety, which retains the sake-brewing suitability of Hattan-type varieties but can endure high-degree polishing. In this study, to establish a guideline for further breeding, we examined the polishing characteristics and suitability for sake brewing of six Hattan-type varieties derived from Hattanso. In the process of breeding of "Hattan-type varieties" of rice, grain size, white-core size and % of white-core grains increased resulting in an increased suitability for sake brewing, such as water absorptivity and digestibility, in Hattan No.35, Hattan-nishiki N0.1 and Hattan-nishiki N0.2. However, Hattannishiki N0.1 and N0.2 had many ellipsoidal-white-cores with large white tissue, which cause the grains to be easily broken during high-degree polishing. On the other hand, Hattan N0.35, having grains with relatively many lined-white-cores with small white tissue, was superior for high-degree polishing. In the future, breeding of a new variety, which has the superior cultivation characteristics of Hattan-nishiki N0.1 and No.2, but with improved white-core characteristics, is expected.

加藤拓、村島健司、下飯仁、伊藤清

白麹の非耐酸性α-アミラーゼ（AU-amylase）は、液体培養においてグリセロールやグルコースを炭素源に用いた場合、マルトースを用いた場合とほぼ同等に生産されたが、黄麹菌タカアミラーゼ（TAKA-amylase）のマルトース培地における生産量と比べると生産レベルは低かった。AU-amylase とTAKA-amylaseの遺伝子は、タンパク質コード領域とプロモーター領域（欠失部分を除く）ともに、相同性が非常に高かったが、AU-amylase遺伝子のプロモーター中には64bpの欠失が認められた。レポーター遺伝子を用いた解析の結果から、AU-amylaseの低生産性はプロモーター中の欠失に基づき、グリセロールやグルコース培地での生産性は菌自体の性質によることが示唆された。グルコアミラーゼも AU-amylaseと同様に、グリセロールやグルコース培地で生産された。

米原由希、小関卓也、奥田将生、荒巻功、橋爪克己

イネ登熟期の低温が原料米の酒造適性に及ぼす影響を解析した。低温で栽培された山田錦及び日本晴は20分吸水率や消化性(Brix)が上昇し、カリウム含量が低下した。粘度特性や糊化特性はそれぞれRVA及びDSCを用いて調べた。低温で栽培されたコメはRVA及びDSCによる糊化開始温度が顕著に低下した。また、低温で栽培されたコメのアミロース含有量及びアミロペクチンにおける短鎖/長鎖比が上昇した。これらのことは気象条件が原料米の酒造適性に関わることを示唆しており、消化性はアミロペクチンの短鎖/長鎖比が大きく寄与していると推察される。

向井伸彦、 木曽邦明

著者らは、CIE表色系を用いた清酒の色彩測定を行った。古酒に関して、着色度と非常に高い相関がみられたのは、L^*、b^*、C^*、Y、x及びyであった。着色度の値からCIE表色値を推定することができた。Xy座標図において、古酒の分布はメラノイジン溶液の分布と類似していた。アデニン要求性酵母の使用や紅麹の使用による赤色の清酒の色彩に関して、
CIE表色系を用いることで色彩を的確に捉えられることが示唆された。市販古酒を表色値L^*a^*b^*を用いてクラスター分析を行い、3群に分けることができた。

S.Kobayashi, N.Goto-Yamamoto, H. Hirochika

By using white-skinned cultivars ('Italia' and 'Muscat of Alexandria') and putative red-skinned sports ('Ruby Okuyama' and 'Flame Muscat') from the white cultivars, we analyzed the expression and function of a myb-related gene, VvmybA1, involved in the regulation of anthocyanin biosynthesis in grapes. A simple sequence repeat (SSR) analysis showed that 'Ruby Okuyama' and 'Flame Muscat' are true derivatives by bud mutation from 'Italia' and 'Muscat of Alexandria', respectively. The VvmybA1 transcript was detected in berry skins of 'Ruby Okuyama' after coloring had begun; it was not detectable in those of 'Italia'. Three VvmybA cDNAs (VvmybA1, VvmybA2, and VvmybA3) from 'Ruby Okuyama' under the control of the 35S promoter were introduced into somatic embryos of 'Kyoho' by particle bombardment. Upon the introduction of VvmybA1, redo cells were induced in the embryos, whereas the introduction of VvmybA2 or VvmybA3 failed to do so. The VvmybA1 cDNA was also shown to have the ability to induce the anthocyanin-producing cells in the skin tissues of 'Muscat of Alexandria', The relationship between the skin-color mutations and the detailed structure of the VvmynA1 gene is also discussed.

T.Koseki, Y.Miwa, S.Fushinobu, K.Hashizume

We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward α-naphthylacetate (C2), lower activity toward α-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser¹¹⁹, Ser¹⁴⁶, Asp¹⁶⁸ and Asp²⁰², were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser¹¹⁹ and Asp²⁰² are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased Km and decreased kcat. The catalytic efficiency of S146A was a 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.

橋爪克己、奥田将生、沼田美子代、櫻尾尚平、小関卓也、畑冨士夫、蓮尾徹夫、庄嶋修

長期貯蔵米精米時の砕米率は玄米水分に密接に関係していた。水分含量の低い（11～13%）試料は精米時に低い砕米率を示し、一方、高い水分含量（15%以上）の試料の砕米率は大変高かった。長期貯蔵米の乾燥処理は精米時の砕米発生の防止に有効であり、処理の有効性は一俵張り精米機で確認された。長期貯蔵米の剛度は水分によって変化した。この剛度と水分含量の関係は試験した2試料間で同じではなく、これが精米時の異なる試料の砕けの頻度に影響を及ぼしていると考えられた。乾燥処理は米粒のヘキサナール含量をわずかに増加させ、また、白米の吸水率を増加させた。

K.Kobayashi, K.Kusaka, T. Takahashi, and K. Sato

A simultaneous assay method for diacetyl and acetoin was developed to investigate the formation of diacetyl during the brewing of alcoholic beverages. A GC-MS analysis after the extraction from neutralized sample by ethyl acetate gave accurate assay results. The detection limit was below 0.1 mg/l and the assay was quantitative from 0.1 to 100 mg/l for both compounds. Unlike other methods, the assay results were unaffected by the presence of α-acetolactate (up to 26 mg/l), which easily decomposes to diacetyl or acetoin, because the extraction condition prevents the decomposition and extraction of this acidic compound. Since our assay is compatible with samples that contain α-acetolactate, the kinetic parameters for decomposition of α-acetolactate to diacetyl and acetoin were determined. The decomposition rate constants were affected by the ethanol concentration. Overall kinetics for the decomposition of α-acetolactate was formulated as a function of ethanol concentration, pH and temperature. The kinetics can be applied to alcoholic beverages such as sake.

福田央、中津茂生、三上重明

酵母のポリガラクツロナーゼ遺伝子(PGU1)を発現ベクター(pQE80L)に連結した組換えプラスミドを、大腸菌Origami B株に形質転換した。 形質転換した大腸菌をポリガラクツロナーゼタンパク質の生産誘導条件下で培養したところ、ポリガラクツロナーゼ活性が検出された。このポリガラクツロナーゼタンパク質を市販の簡易アフィニティカラムを用いて精製した。ポリガラクツロナーゼタンパク質は、pH3.7～5.5で活性を示し、至適pHは3.8、至適温度は40℃であった。大腸菌の発現系を用いたポリガラクツロナーゼタンパク質の生産は簡易に実施できたことから、タンパク質に関するバイオテクノロジー学習の効果的教材になりうると考えられた。

N.Tomishige, Y.Noda, H.Adachi, H.Shimoi and K.Yoda

The fungal GAS1-related genes encode GPI-anchored beta-1,3-glucanosyltransferase, and their loss causes a defect in the assembly of the cell wall. The KEX2 gene encodes a processing protease in the late Golgi compartment and its loss also results in defects in the cell wall. Simultaneous mutations of these genes are lethal in Saccharomyces cerevisiae. To understand the basis of this synthetic lethality, we screened for multicopy suppressors and identified 13 SKG (suppressor of kex2 gas1 synthetic lethality) genes. SKG1 encodes a transmembrane protein that localizes on the inner surface of the plasma membrane at the bud and in the daughter cell. The multicopy SKG1 increases the sensitivity of cells to zymolyase, and the skg1Delta null mutation increases resistance to it. This zymolyase susceptibility corresponds to an increase of alkali-soluble beta-1,3-glucan and a decrease of chitin in the cell wall. Thus SKG1 encodes a novel protein that affects the cell wall polymer composition in the growing region of the cell.

T. Yamada, H. Shimoi and K. Ito

GeneFilters(r) and Northern blot analysis revealed that the sake yeast strain Kyokai no. 7 (K7) showed a higher expression level of OLE1, which encodes a Δ-9 fatty acid desaturase gene, compared with the laboratory yeast strain X2180-1A. Other sake yeasts also showed a high expression level of OLE1. Unsaturated fatty acid concentrations in strain K7 are higher than that in strain X2180-1A, suggesting that the higher expression level of OLE1 in sake yeasts increases the unsaturated fatty acid content in the cell membrane. Experiments using OLE1 promoter:lacZ fusion reporter genes revealed that both the cis element of the OLE1 promoter and trans factors are involved in the increased expression of OLE1 in sake yeasts.