平成24年度 研究成果

Muneyoshi Kanai, Mitsunori Masuda, Yasumichi Takaoka, Hiroko Ikeda, Kazuo Masaki, Tsutomu Fujii and Haruyuki Iefuji

To isolate an S-adenosylmethionine (SAM)-accumulating yeast strain and to develop a more efficient method of producing SAM, we screened methionine-resistant strains using the yeast deletion library of budding yeast and isolated 123 strains. The SAM content in 81 of the 123 strains was higher than that in the parental strain BY4742. We identified ADO1 encoding adenosine kinase as one of the factors participating in high SAM accumulation. The X_△ado1 strain that was constructed from the X2180-1A strain (MAT a, ATCC 26786) could accumulate approximately 30-fold (18 mg/g dry cell weight) more SAM than the X2180-1A strain in yeast extract peptone dextrose medium. Furthermore, we attempted to identify the molecular basis underlying the differences in SAM accumulation betweenX_△ado1 and X2180-1A strains. DNA microarray analysis revealed that the genes involved in the methionine biosynthesis pathway, phosphate metabolism, and hexose transport were mainly overexpressed in theX_△ado1strain compared with the X2180-1A strain. We also determined the levels of various metabolites involved in the methionine biosynthesis pathway and found increased content of SAM, tetrahydrofolate (THF), inorganic phosphate, polyphosphoric acid, and S-adenosylhomocysteine in theX_△ado1 strain. In contrast, the content of 5-methyl-THF, homocysteine, glutathione, and adenosine was decreased. These results indicated that the △ado1 strain could accumulate SAM because of preferential activation of the methionine biosynthesis pathway.

Akira Yoshimi, Motoaki Sano, Azusa Inaba, Yuko Kokubun, Tomonori Fujioka, Osamu Mizutani, Daisuke Hagiwara, Takashi Fujikawa, Marie Nishimura, Shigekazu Yano, Shin Kasahara, Kiminori Shimizu, Masashi Yamaguchi, Kazuyoshi Kawakami, Keietsu Abe

Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and ¹³C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species.

T. Hashimoto, T. Ishihara, Y. Kakudo, K. Kaneko, T. Kenjo, Y. Masumura, M. Nakahara, W. Nakamura, S. Oshima, M. Takahashi, T. Yamamoto

1. Relative repeatability standard deviation (RSDr) and repeat-ability limit (r₉₅) for determination of total purine content using the HPLC-UV method ranged from 2.2 to 8.5% and from 2.9 to 20.6 mg/L, respectively, and were judged acceptable.
2. Relative reproducibility standard deviation (RSD_R) and repro-ducibility limit (R₉₅) for determination of total purine content using the HPLC-UV method ranged from 15.6 to 31.4 % and from 9.1 to 131.2 mg/L, respectively, and were judged unacceptable.

Okuda, M., Joyo, M., Tokuoka, M, Hashiguchi, T., Goto-Yamamoto, N., Yamaoka, H. and Shimoi, H.

Possible contamination by radioactive cesium (Cs), released by the Fukushima Daiichi Nuclear Plant Accident in Japan on March 2011, has been a matter of concern with respect to Japanese sake made from rice grains cultivated in affected fields. In this study, the behavior of stable ¹³³Cs, which is a useful analogue for predicting the behavior of radioactive Cs, was investigated in the production of sake using rice grains harvested in Japan in 2010. The concentration of stable ¹³³Cs in the polished rice grains decreased gradually with decreasing milling ratios until a ratio of 70% was reached, and below that point, it did not change significantly. The ¹³³Cs concentration in the 70% polished rice was approximately 20% of that found in brown rice. The sake was brewed on a small scale using 70% polished rice, and the transfer of ¹³³Cs from rice to sake was examined. Approximately 30-40% of ¹³³Cs in the 70% polished rice was removed during the washing and the steeping of the rice grains, and approximately 40% of the ¹³³Cs in the 70% polished rice was transferred to the sake. If the radioactive Cs species behaves similarly, these results suggest that brown rice containing 100 Bq/kg radioactivity of Cs would generate 70% polished rice grains containing 20 Bq/kg and that the sake brewed from these grains would contain 3-5 Bq/kg.

Yoshiyuki Toyoshima,, Akemi Takahashi, Hisaki Tanaka, Jun Watanabe, Yoshinobu Mogi, Tatsuo Yamazaki, Ryoko Hamada, Kazuhiro Iwashita, Katsuya Satoh, Issay Narumi

Aspergillus oryzae is a fungus that is used widely in traditional Japanese fermentation industries. In this study, the lethal and mutagenic effects of different linear energy transfer (LET) radiation in freeze-dried conidia of A. oryzae were investigated. The lethal effect, which was evaluated by a 90% lethal dose, was dependent on the LET value of the ionizing radiation. The most lethal ionizing radiation among that tested was ¹²C⁵⁺ ion beams with an LET of 121 keV/μm. The ¹²C⁵⁺ ion beams had a 3.6-times higher lethal effect than low-LET (0.2 keV/μm) γ-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. ¹²C⁶⁺ ion beams with an LET of 86 keV/μm were the most effective in inducing selenate resistance. The mutant frequency following exposure to ¹²C⁶⁺ ion beams increased with an increase in dose and reached 3.47 × 10^-3 at 700 Gy. In the dose range from 0-700 Gy, ¹²C⁵⁺ ion beams were the second most effective in inducing selenate resistance, the mutant frequency of which reached a maximum peak (1.67 × 10^-3) at 400 Gy. To elucidate the characteristics of mutation induced by ionizing radiation, mutations in the sulphate permease gene (sB) and ATP sulfurylase gene (sC) loci, the loss of function of which results in a selenate resistant phenotype, were compared between ¹²C⁵⁺ ion beams and γ-rays. We detected all types of transversions and transitions. For frameshifts, the frequency of a +1 frameshift was the highest in all cases. Although the incidence of deletions > 2 bp was generally low, deletions > 20 bp were characteristic for ¹²C⁵⁺ ion beams. γ-rays had a tendency to generate mutants carrying a multitude of mutations in the same locus. Both forms of radiation also induced genome-wide large-scale mutations including chromosome rearrangements and large deletions. These results provide new basic insights into the mutation breeding of A. oryzae using ionizing radiation.

松丸克己、磯谷敦子、藤田晃子、須藤茂俊、木崎康造

平成22酒造年度全国新酒鑑評会（第99回鑑評会）は、当該年度生産清酒を全国的に調査研究することにより、製造技術と酒質の現状及び動向を明らかにし、もって清酒の品質及び酒造技術の向上に資するとともに、国民の清酒に対する認識を高めることを目的として、日本酒造組合中央会との共催により実施した。

審査は、酒類総合研究所広島事務所において、平成23年4月20日（水）から22日（金）の3日間に予審を行い、5月10日（火）及び11日（水）に決審を行った。また、5月25日（水）に東広島運動公園体育館で製造技術研究会を、6月15日（水）に東京池袋のサンシャインシティ・ワールドインポートマート展示ホールで公開きき酒会を開催した。

出品は、875点であった（今年度より、山田錦の使用割合によるⅠ部・Ⅱ部の区分分けは廃止された）。審査の結果、優秀と認められた437点を入賞酒とし、さらに、決審において特に優秀と認められた244点に金賞を授与した。また、出品酒を公開する製造技術研究会及び公開きき酒会には、全国からそれぞれ、1,361人及び2,759人が来場した。

出品酒については、審査に加え酒質の現状及び動向を調査研究するため、出品者の記載による「全国新酒鑑評会出品酒調査表」（以下、「調査表」という。）の内容を集計するとともに成分分析を行った。

福田央、水谷治、金井宗良、高橋正之、木崎康造

単式蒸留しょうちゅうの品質を全国的に調査研究することにより、製造技術と酒質の現状及び動向を把握するとともに製造業者の参考とするため、第34回本格焼酎鑑評会を日本酒造組合中央会と共催で開催した。出品資格として、単式蒸留しょうちゅう製造免許を有する製造者で、日本酒造組合中央会の組合員に限定した。官能審査は平成23年6月2日（木）及び3日(金)に行い、製造技術研究会は6月24日（金）に当所で開催し、出品関係者の参考に供した。

出品酒の官能審査と成分分析を行ったので、以下、その結果の概要について報告する。

橋口知一、伊豆英恵、松丸克己

国内で市販されている核果蒸留酒及びリキュール（外国産、国内産）のカルバミン酸エチルを分析した。外国産の核果蒸留酒では16点全点から、核果リキュールでは35点中22点から、国内産核果蒸留酒では7点中6点から、マルメロリキュールでは3点中2点からカルバミン酸エチルが検出された。外国産の核果蒸留酒の中央値は0.265 mg/l、核果リキュールの中央値は0.01 mg/lであった。国内産の核果蒸留酒の平均値は0.03 mg/l、最大でも0.10 mg/lであり、マルメロリキュールの平均値は0.02 mg/lであった。国内産の核果蒸留酒及びマルメロリキュールの健康への懸念は比較的小さいものと考えられる。

伊豆英恵、橋口知一、堀井幸江、須藤茂俊、松丸克己

本研究では、本格焼酎市販品（麦焼酎、芋焼酎、米焼酎、蕎麦焼酎、黒糖焼酎、酒粕焼酎、泡盛）のアルコールの炭素安定同位体比（δ¹³C）と水の酸素安定同位体比（δ¹⁸O）を分析し、安定同位体比の産地や原料判別の指標としての可能性を調べた。米焼酎の小仕込試験により、仕込水のδ¹⁸Oが米焼酎（割水して約アルコール25%にしたもの）に含まれる水のδ¹⁸Oと近い値を示すことがわかった。原料に関わらず、本格焼酎に含まれる水のδ¹⁸Oと産地の緯度に相関がみられた（R²=0.67）。黒糖を原料とする黒糖焼酎のアルコールのδ¹³C（-17.6 ± 0.8‰）は米、麦、芋、蕎麦、酒粕を原料とする本格焼酎よりも高い値を示した。国産麦使用の表示がある麦焼酎のアルコールのδ¹³C（-26.2 ± 0.8‰）は麦の産地表示のない麦焼酎（-24.8 ± 1.4‰）に比べ、低い値を示した。芋麹使用の表示がある芋焼酎のアルコールのδ¹³C（-28.4 ± 0.2‰）は米麹使用の表示がある芋焼酎（-27.8 ± 0.4‰）に比べ、低い値を示した。以上のように本格焼酎のアルコールのδ¹³Cと水のδ¹⁸Oは産地や原料判別の指標として有用であると示唆された。

Hashizume, K., Ito, T., Shimonohashi, M., Kokita, A., Tokiwano, T., Okuda, M.

Taste-active hydrophobic compounds in a charcoal-untreated sake sample were subjected to a taste dilution analysis (TDA) method. All of the high TD factor fractions showed a bitter or astringent taste in common, but their taste character were different. The taste-active compounds of the high-TDA factor fractions were purified by taste-guided fractionations, using RP-HPLC and instrumental analysis. From each of the seven fractions, ferulic acid, ethyl ferulate, tryptophol, three previously reported bitter-tasting peptides, and two novel ethyl esters of peptide of 10 amino acid residues were identified. All the identified compounds had a similar taste character to that of the TDA fractions analyzed. Ethyl ferulate and the ethyl esters of the peptides showed a moderately bitter taste. The concentration of the identified compounds in seven jyunmai-type sake samples was determined. This concentration was decreased dose dependently by a charcoal treatment which is commonly applied in the final step of sake manufacture, notably in the compounds of high hydrophobicity.

Kazuhiko Imamura, Yoshihito Tsuyama, Terukage Hirata, Sumihiro Shiraishi, Kazutoshi Sakamoto, Osamu Yamada, Osamu Akita, Hitoshi Shimoi

WYK-1 is a dipeptidyl peptidase IV inhibitor produced by Aspergillus oryzae strain AO-1. Because WYK-1 is an isoquinoline derivative consisting of three l-amino acids, we hypothesized that a nonribosomal peptide synthetase was involved in its biosynthesis. We identified 28 nonribosomal peptide synthetase genes in the sequenced genome of A. oryzae RIB40. These genes were also identified in AO-1. Among them, AO090001000009 (wykN) was specifically expressed under WYK-1-producing conditions in AO-1. Therefore, we constructed wykN gene disruptants of AO-1 after nonhomologous recombination was suppressed by RNA interference to promote homologous recombination. Our results demonstrated that the disruptants did not produce WYK-1. Furthermore, the expression patterns of 10 genes downstream of wykN were similar to the expression pattern of wykN under several conditions. Additionally, homology searches revealed that some of these genes were predicted to be involved in WYK-1 biosynthesis. Therefore, we propose that wykN and the 10 genes identified in this study constitute the WYK-1 biosynthetic gene cluster.

磯谷敦子、神田涼子、岩田博、須藤茂俊

老香の主要成分DMTSの生成に対する製成・ろ過資材の抑制効果を調べたところ、活性炭には効果がみられなかったが、2種類のセライトに顕著な抑制効果がみられた。セライト中の有効成分を探索した結果、バナジウムであることが明らかとなった。清酒にバナジウムを添加して貯蔵すると、DMTSおよびDMDSの生成は抑制されたが、アルデヒド類が増加した。官能評価の結果、バナジウムの添加により硫黄様のにおいは抑制されたが、カラメル様・甘いにおいの強度が増加した。老香成分すべてを抑制する目的でバナジウムを使用するのは難しいが、長期熟成酒製造の観点からは有効利用できる可能性があると思われた。

Kei Takahashi, Masafumi Tokuoka, Hiromi Kohno, Nobuko Sawamura, Yuka Myoken, and Akihiro Mizuno

Fermented foods and beverages contain several different types of dipeptides, which are believed to be important components for taste. To date, however, a method for the comprehensive analysis of dipeptides in these products has not yet been established. In this study, the comprehensive analysis of dipeptides in alcoholic beverages was performed by a high separation method based on the structural characteristics of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized dipeptides as well as dipeptide quantification and structural estimation using ultra-high-pressure liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) and UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS), respectively. Dipeptide content was found to differ considerably among Japanese sake, beer, and wine; UHPLC-MS/MS analysis revealed that many types of dipeptides are present in sake. Dipeptide quantification analysis identified 32 types of dipeptides within the concentration range of 1.1-97.2 μM in sake. The analysis was validated by dipeptide recovery of 64.0%-107.2% (2.5 μM of standard) with a relative standard deviation of ≤33.2% from an actual alcoholic sample. Furthermore, UHPLC-Q-TOFMS analysis suggested the existence of more than 40 types of dipeptides in sake. Thus, by the combined analysis methods, we discovered that more than 60 dipeptides are present in sake. This research is the first report of dipeptide profiling of fermented alcoholic beverages by comprehensive analysis.

Kazuya Koyama, Hiroko Ikeda, Puspa Raj Poudel, Nami Goto-Yamamoto

Biosynthesis of phenolic compounds is known to be sensitive to light environments, which reflects the possible role of these compounds for photoprotection in plants. Herein, the effects of UV and visible light on biosynthesis of flavonoids was investigated, i.e., proanthocyanidins (PAs) and flavonols, in young berry skins of a red-wine grape, Vitis vinifera cv. Cabernet Sauvignon. Shading with light-proof boxes from the flowering stage until 49 days after treatment (DAT) partially decreased PA concentrations, and completely decreased flavonol concentrations in the berry skins. Shading decreased the transcript abundance of a flavonol-related genes more remarkably than those of PA-related genes. In addition, light exclusion influenced the composition of PAs, such as the decrease in the proportion of trihydroxylated subunits and the mean degree of polymerization (mDP) within PAs. However, solar UV exclusion did not affect the concentration and composition of PAs, whereas this exclusion remarkably decreased the flavonol concentration. Consistently, UV exclusion did not influence the transcript levels of PA-related genes, whereas it dramatically decreased that of flavonol-related genes. These findings indicated a different light regulation of the biosynthesis of these flavonoids in young berry skins of wine grape. Visible light primarily induces biosynthesis of PAs and affects their composition, whereas UV light specifically induces biosynthesis of flavonols. Distinct roles of members of a MYB transcription factor family for light regulation of flavonoid biosynthesis were proposed.

Daisuke Watanabe, Yuya Araki, Yan Zhou, Naoki Maeya, Takeshi Akao, and Hitoshi Shimoi

Sake yeast cells have defective entry into the quiescent state, allowing them to sustain high fermentation rates. To reveal the underlying mechanism, we investigated the PAS kinase Rim15p, which orchestrates initiation of the quiescence program in Saccharomyces cerevisiae. We found that Rim15p is truncated at the carboxyl terminus in modern sake yeast strains as a result of a frameshift mutation. Introduction of this mutation or deletion of the full-length RIM15 gene in a laboratory strain led to a defective stress response, decreased synthesis of the storage carbohydrates trehalose and glycogen, and impaired G1 arrest, which together closely resemble the characteristic phenotypes of sake yeast. Notably, expression of a functional RIM15 gene in a modern sake strain suppressed all of these phenotypes, demonstrating that dysfunction of Rim15p prevents sake yeast cells from entering quiescence. Moreover, loss of Rim15p or its downstream targets Igo1p and Igo2p remarkably improved the fermentation rate in a laboratory strain. This finding verified that Rim15p-mediated entry into quiescence plays pivotal roles in the inhibition of ethanol fermentation. Taken together, our results suggest that the loss-of-function mutation in the RIM15 gene may be the key genetic determinant of the increased ethanol production rates in modern sake yeast strains.

Takeshi Akao, Akinori Yahara, Kazutoshi Sakamoto, Osamu Yamada, Osamu Akita and Takashi Yoshida

The gene manE, encoding a probable class I endoplasmic reticulum 1,2-α-mannosidases (ER-Man), was identified from the filamentous fungus Aspergillus oryzae due to similarity to orthologs. It removes a single mannose residue from Man₉GlcNAc₂, generating Man₈GlcNAc₂ isomer B. Disruption of manE caused drastic decreases in ER-Man activity in A. oryzae microsomes.

Osamu Mizutani, Kazuo Masaki, Katsuya Gomi and Haruyuki Iefuji

Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination system has been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the transformant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced into Aspergillus oryzae protoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as a carrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced into A. oryzae protoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flanked by loxP sites. By using this method, we readily constructed a marker gene-rescued strain lacking ligD to optimize homologous recombination. Furthermore, we succeeded in integrative recombination at a loxP site in A. oryzae. Thus, we developed a simple method to use the Cre-loxP recombination system in A. oryzae by direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme.

Yu Sasano, Daisuke Watanabe, Ken Ukibe, Tomomi Inai, Hitoshi Shimoi, Hiroshi Takagi

Lignocellulosic biomass is a promising source for bioethanol production, because it is abundant worldwide and has few competing uses. However, the treatment of lignocelllulosic biomass with weak acid to release cellulose and hemicellulose generates many kinds of byproducts including furfural and 5-hydroxymethylfurfural, which inhibit fermentation by yeast, because they generate reactive oxygen species (ROS) in cells. In order to acquire high tolerance to oxidative stress in bioethanol yeast strains, we focused on the transcription activator Msn2 of Saccharomyces cerevisiae, which regulates numerous genes involved in antioxidative stress responses, and constructed bioethanol yeast strains that overexpress Msn2 constitutively. The Msn2-overexpressing bioethanol strains showed tolerance to oxidative stress, probably due to the high-level expression of various antioxidant enzyme genes. Unexpectedly, these strains showed ethanol sensitivity compared with the control strain, probably due to imbalance of the expression level between Msn2 and Msn4. In the presence of furfural, the engineered strains exhibited reduced intracellular ROS levels, and showed rapid growth compared with the control strain. The fermentation test in the presence of furfural revealed that the Msn2-overexpressing strains showed improvement of the initial rate of fermentation. Our results indicate that overexpression of the transcription activator Msn2 in bioethanol yeast strains confers furfural tolerance by reducing the intracellular ROS levels and enhances the initial rate of fermentation in the presence of furfural, suggesting that these strains are capable of adapting rapidly to various compounds that inhibit fermentation by inducing ROS accumulation. Our results not only promise to improve bioethanol production from lignocellulosic biomass, but also provide novel insights for molecular breeding of industrial yeast strains