詳細

松鹿　昭則 （マツシカ　アキノリ）

  • 工学部 化学生命工学科　教授/化学生命工学科長
Last Updated :2024/04/25

コミュニケーション情報 byコメンテータガイド

  • コメント

    バイオマスなどの再生可能資源を利活用し、微生物発酵により各種のバイオ燃料、化学品、機能性食品を高効率かつ低環境負荷で生産するための基盤的な研究開発を実施しています。

研究者情報

学位

  • 博士（農学）

ホームページURL

J-Global ID

研究キーワード

  • 環境ストレス耐性   食品変敗   代謝改変   有用遺伝子のスクリーニング   発酵阻害耐性   高温発酵   耐熱性   キシロース発酵   五炭糖資化性   実用酵母   

現在の研究分野（キーワード）

    バイオマスなどの再生可能資源を利活用し、微生物発酵により各種のバイオ燃料、化学品、機能性食品を高効率かつ低環境負荷で生産するための基盤的な研究開発を実施しています。

研究分野

  • ライフサイエンス / 食品科学 / 食品工学、食品化学、食品プロセス工学
  • ライフサイエンス / 分子生物学 / 遺伝子工学
  • ものづくり技術（機械・電気電子・化学工学） / バイオ機能応用、バイオプロセス工学
  • ライフサイエンス / 応用微生物学 / 発酵食品、酵母

経歴

  • 2022年04月 - 現在  近畿大学大学院システム工学研究科 生物化学コース教授（兼務）
  • 2022年04月 - 現在  近畿大学次世代基盤技術研究所 先端化学生命工学研究センター教授（兼務）
  • 2021年04月 - 現在  近畿大学 工学部 化学生命工学科 教授
  • 2017年04月 - 2021年03月  産業技術総合研究所機能化学研究部門研究グループ長
  • 2015年04月 - 2017年03月  産業技術総合研究所 機能化学研究部門 主任研究員
  • 2011年05月 - 2012年04月  米国立再生可能エネルギー研究所 (NREL) 在外研究員

所属学協会

  • 日本分子生物学会   日本生物工学会   日本農芸化学会   中国地域バイオマス利用研究会   酵母遺伝学フォーラム   

研究活動情報

論文

  • Isabela de Oliveira Pereira; Ângela A. dos Santos; Nick C. Guimarães; Cleilton S. Lima; Eduardo Zanella; Akinori Matsushika; Sarita C. Rabelo; Boris U. Stambuk; Jaciane L. Ienczak
    Biotechnology and Bioengineering 2024年01月 [査読有り]
     
    Abstract The integration of first‐ (1G) and second‐generation (2G) ethanol production by adding sugarcane juice or molasses to lignocellulosic hydrolysates offers the possibility to overcome the problem of inhibitors (acetic acid, furfural, hydroxymethylfurfural and phenolic compounds), and add nutrients (such as salts, sugars and nitrogen sources) to the fermentation medium, allowing the production of higher ethanol titers. In this work, an 1G2G production process was developed with hemicellulosic hydrolysate (HH) from a diluted sulfuric acid pretreatment of sugarcane bagasse and sugarcane molasses. The industrial Saccharomyces cerevisiae CAT‐1 was genetically modified for xylose consumption and used for co‐fermentation of sucrose, fructose, glucose, and xylose. The fed‐batch fermentation with high cell density that mimics an industrial fermentation was performed at bench scale fermenter, achieved high volumetric ethanol productivity of 1.59 g L−1 h−1, 0.39 g g−1 of ethanol yield, and 44.5 g L−1 ethanol titer, and shown that the yeast was able to consume all the sugars present in must simultaneously. With the results, it was possible to establish a mass balance for the global process: from pretreatment to the co‐fermentation of molasses and HH, and it was possible to establish an effective integrated process (1G2G) with sugarcane molasses and HH co‐fermentation employing a recombinant yeast.
  • Viviani Tadioto; Junior Romeo Deoti; Caroline Müller; Bruna Raquel de Souza; Odinei Fogolari; Marcela Purificação; Anderson Giehl; Letícia Deoti; Ana Carolina Lucaroni; Akinori Matsushika; Helen Treichel; Boris Ugarte Stambuk; Sergio Luiz Alves Junior
    Bioprocess and Biosystems Engineering 2023年08月 [査読有り]
  • Kaisei Takemura; Junya Kato; Setsu Kato; Tatsuya Fujii; Keisuke Wada; Yuki Iwasaki; Yoshiteru Aoi; Akinori Matsushika; Tomotake Morita; Katsuji Murakami; Yutaka Nakashimada
    Journal of bioscience and bioengineering 136 1 13 - 19 2023年07月 [査読有り]
     
    Acetogens grow autotrophically and use hydrogen (H2) as the energy source to fix carbon dioxide (CO2). This feature can be applied to gas fermentation, contributing to a circular economy. A challenge is the gain of cellular energy from H2 oxidation, which is substantially low, especially when acetate formation coupled with ATP production is diverted to other chemicals in engineered strains. Indeed, an engineered strain of the thermophilic acetogen Moorella thermoacetica that produces acetone lost autotrophic growth on H2 and CO2. We aimed to recover autotrophic growth and enhance acetone production, in which ATP production was assumed to be a limiting factor, by supplementing with electron acceptors. Among the four selected electron acceptors, thiosulfate and dimethyl sulfoxide (DMSO) enhanced both bacterial growth and acetone titers. DMSO was the most effective and was further analyzed. We showed that DMSO supplementation enhanced intracellular ATP levels, leading to increased acetone production. Although DMSO is an organic compound, it functions as an electron acceptor, not a carbon source. Thus, supplying electron acceptors is a potential strategy to complement the low ATP production caused by metabolic engineering and to improve chemical production from H2 and CO2.
  • Hironaga Akita; Akinori Matsushika
    Fermentation 9 6 559 - 559 2023年06月 [査読有り]
     
    Simultaneous saccharification and fermentation (SSF) has been investigated for the efficient production of ethanol because it has several advantages such as simplifying the manufacturing process, operating easily, and reducing energy input. Previously, using lignocellulosic biomass as source materials, we succeeded in producing ethanol by SSF with Pichia kudriavzevii NBRC1279 and NBRC1664. However, various acids that fermentation inhibitors are also produced by the hydrolysis of lignocellulosic biomass, and the extent to which these acids affect the growth and ethanol productivity of the two strains has not yet been investigated. In this study, to better understand the acid tolerance mechanism of the two strains, a spot assay, growth experiment, and transcriptome analysis were carried out using Saccharomyces cerevisiae BY4742 as a control. When the three strains were cultured in SCD medium containing 15 mM formic acid, 35 mM sulfuric acid, 60 mM hydrochloric acid, 100 mM acetic acid, or 550 mM lactic acid, only P. kudriavzevii NBRC1664 could grow well under all conditions, and it showed the fastest growth rates. The transcriptome analysis showed that “MAPK signaling pathway-yeast” was significantly enriched in P. kudriavzevii NBRC1664 cultured with 60 mM hydrochloric acid, and most genes involved in the high osmolarity glycerol (HOG) pathway were up-regulated. Therefore, the up-regulation of the HOG pathway may be important for adapting to acid stress in P. kudriavzevii. Moreover, the log2-transformed fold change value in the expression level of Gpd1 was 1.3-fold higher in P. kudriavzevii NBRC1664 than in P. kudriavzevii NBRC1279, indicating that high Gpd1 expression may be accountable for the higher acid tolerance of P. kudriavzevii NBRC1664. The transcriptome analysis performed in this study provides preliminary knowledge of the molecular mechanism of acid stress tolerance in P. kudriavzevii. Our data may be useful for future studies on methods to improve the tolerance of P. kudriavzevii to acids produced from lignocellulose hydrolysis.
  • Shunsuke Kobayashi; Junya Kato; Keisuke Wada; Kaisei Takemura; Setsu Kato; Tatsuya Fujii; Yuki Iwasaki; Yoshiteru Aoi; Tomotake Morita; Akinori Matsushika; Katsuji Murakami; Yutaka Nakashimada
    Frontiers in microbiology 13 897066 - 897066 2022年05月 [査読有り]
     
    Hydrogen (H2) converted to reducing equivalents is used by acetogens to fix and metabolize carbon dioxide (CO2) to acetate. The utilization of H2 enables not only autotrophic growth, but also mixotrophic metabolism in acetogens, enhancing carbon utilization. This feature seems useful, especially when the carbon utilization efficiency of organic carbon sources is lowered by metabolic engineering to produce reduced chemicals, such as ethanol. The potential advantage was tested using engineered strains of Moorella thermoacetica that produce ethanol. By adding H2 to the fructose-supplied culture, the engineered strains produced increased levels of acetate, and a slight increase in ethanol was observed. The utilization of a knockout strain of the major acetate production pathway, aimed at increasing the carbon flux to ethanol, was unexpectedly hindered by H2-mediated growth inhibition in a dose-dependent manner. Metabolomic analysis showed a significant increase in intracellular NADH levels due to H2 in the ethanol-producing strain. Higher NADH level was shown to be the cause of growth inhibition because the decrease in NADH level by dimethyl sulfoxide (DMSO) reduction recovered the growth. When H2 was not supplemented, the intracellular NADH level was balanced by the reversible electron transfer from NADH oxidation to H2 production in the ethanol-producing strain. Therefore, reversible hydrogenase activity confers the ability and flexibility to balance the intracellular redox state of M. thermoacetica. Tuning of the redox balance is required in order to benefit from H2-supplemented mixotrophy, which was confirmed by engineering to produce acetone.
  • Kaisei Takemura; Junya Kato; Setsu Kato; Tatsuya Fujii; Keisuke Wada; Yuki Iwasaki; Yoshiteru Aoi; Akinori Matsushika; Katsuji Murakami; Yutaka Nakashimada
    Journal of bioscience and bioengineering 132 6 569 - 574 2021年09月 [査読有り]
     
    Gas fermentation is a promising biological process for the conversion of CO2 or syngas into valuable chemicals. Homoacetogens are microorganisms growing autotrophically using CO2 and H2 or CO and metabolizing them to form acetate coupled with energy conservation. The challenge in the metabolic engineering of the homoacetogens is divergence of the acetate formation, whose intermediate is acetyl-CoA, to a targeted chemical with sufficient production of adenosine triphosphate (ATP). In this study, we report that an engineered strain of the thermophilic homoacetogen Moorella thermoacetica, in which a pool of acetyl-CoA is diverted to ethanol without ATP production, can maintain autotrophic growth on syngas. We estimated the ATP production in the engineered strains under different gaseous compositions by considering redox-balanced metabolism for ethanol and acetate formation. The culture test showed that the combination of retaining a level of acetate production and supplying the energy-rich CO allowed maintenance of the autotrophic growth during ethanol production. In contrast, autotrophy was collapsed by complete elimination of the acetate pathway or supplementation of H2-CO2. We showed that the intracellular level of ATP was significantly lowered on H2-CO2 in consistent with the incompetence. In the meantime, the complete disruption of the acetate pathway resulted in the redox imbalance to produce ethanol from CO, albeit a small loss in the ATP production. Thus, preservation of a fraction of acetate formation is required to maintain sufficient ATP and balanced redox in CO-containing gases for ethanol production.
  • Isabela de Oliveira Pereira; Ângela Alves Dos Santos; Davi L Gonçalves; Marcela Purificação; Nick Candiotto Guimarães; Robson Tramontina; Natalia Coutouné; Eduardo Zanella; Akinori Matsushika; Boris U Stambuk; Jaciane Lutz Ienczak
    FEMS yeast research 21 6 foab048 - foab048 2021年09月 [査読有り]
     
    First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G-E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2, XKS1 genes and a mutant ([4-59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G-E2G production process.
  • Hironaga Akita; Yuya Itoiri; Noriyo Takeda; Zen-Ichiro Kimura; Hiroyuki Inoue; Akinori Matsushika
    Microbiology resource announcements 10 25 e0034321  2021年06月 [査読有り]
     
    Klebsiella pneumoniae subsp. pneumoniae CCI2 was isolated from leaf soil collected in Hiroshima Prefecture, Japan. The draft genome sequence comprises 78 contigs and contains 5,075,115 bp with a G+C content of 57.7%.
  • Tatsuya Fujii; Hiroyuki Inoue; Akinori Matsushika
    Applied biochemistry and biotechnology 193 10 3163 - 3172 2021年06月 [査読有り]
     
    The filamentous fungus Talaromyces cellulolyticus is a well-characterized cellulolytic and hemicellulolytic enzyme producer. In this study, the function of the tclB2 gene, which is a homolog of the manR/clrB/clr-2 gene in other filamentous fungi, in mannanolytic enzyme production by T. cellulolyticus was investigated. When a tclB2-disrupted strain (YDTclB) was grown in the presence of glucomannan, the production of β-mannanase, β-mannosidase, and α-galactosidase was decreased at the protein and transcriptional levels when compared to the control strain. In addition, a tclB2-overexpressing strain (YHTclB) showed higher β-mannanase and β-mannosidase production. When cellulose was used as a carbon source, the expression of genes encoding mannanolytic enzymes also decreased in YDTclB. These results suggested that TclB2 contributes to mannanolytic enzyme production in T. cellulolyticus. This work is the first study to identify a transcriptional regulator of mannanolytic enzyme genes in T. cellulolyticus.
  • Hironaga Akita; Tetsuya Goshima; Toshihiro Suzuki; Yuya Itoiri; Zen-ichiro Kimura; Akinori Matsushika
    Fermentation 7 2 83 - 83 2021年05月 [査読有り]
     
    Simultaneous saccharification and fermentation (SSF) is capable of performing enzymatic saccharification and fermentation for biofuel production in a single vessel. Thus, SSF has several advantages such as simplifying the manufacturing process, operating easily, and reducing energy input. Here, we describe the application of Pichia kudriavzevii NBRC1279 and NBRC1664 to SSF for bioethanol production. When each strain was incubated for 144 h at 35 °C with Japanese cedar particles, the highest ethanol concentrations were reached 21.9 ± 0.50 g/L and 23.8 ± 3.9 g/L, respectively. In addition, 21.6 ± 0.29 g/L and 21.3 ± 0.21 g/L of bioethanol were produced from Japanese eucalyptus particles when each strain was incubated for 144 h at 30 °C. Although previous methods require pretreatment of the source material, our method does not require pretreatment, which is an advantage for industrial use. To elucidate the different characteristics of the strains, we performed genome sequencing and genome comparison. Based on the results of the eggNOG categories and the resulting Venn diagram, the functional abilities of both strains were similar. However, strain NBRC1279 showed five retrotransposon protein genes in the draft genome sequence, which indicated that the stress tolerance of both strains is slightly different.
  • Hironaga Akita; Yuya Itoiri; Noriyo Takeda; Akinori Matsushika; Zen-Ichiro Kimura
    Archives of microbiology 203 4 1787 - 1793 2021年05月 [査読有り]
     
    Strain CCI5, an oligotrophic bacterium, was isolated from leaf soil collected in Japan. Strain CCI5 grew at temperatures between 25 °C and 43 °C (optimum temperature, 40 °C) and at pHs between 6.0 and 10.0 (optimum pH, 9.0). Its major fatty acids were anteiso-C15:0 and iso-C16:0, and menaquinone 7 was the only detected quinone system. In a phylogenetic analysis based on 16S rRNA gene sequences, strain CCI5 presented as a member of the genus Paenibacillus. Moreover, multilocus sequence analysis based on partial sequences of the atpD, dnaA, gmk, and infB genes showed that strain CCI5 tightly clustered with P. glycanilyticus DS-1T. The draft genome of strain CCI5 consisted of 6,864,972 bp with a G+C content of 50.7% and comprised 6,189 predicted coding sequences. The genome average nucleotide identity value (97.8%) between strain CCI5 and P. glycanilyticus DS-1T was below the cut-off value for prokaryotic subspecies delineation. Based on its phenotypic, chemotaxonomic, and phylogenetic features, strain CCI5 (= HUT-8145T = KCTC 43270T) can be considered as a novel subspecies within the genus Paenibacillus with the proposed name Paenibacillus glycanilyticus subsp. hiroshimensis subsp. nov.
  • Junya Kato; Kaisei Takemura; Setsu Kato; Tatsuya Fujii; Keisuke Wada; Yuki Iwasaki; Yoshiteru Aoi; Akinori Matsushika; Katsuji Murakami; Yutaka Nakashimada
    AMB Express 11 1 59 - 59 2021年04月 [査読有り]
     
    Gas fermentation is one of the promising bioprocesses to convert CO2 or syngas to important chemicals. Thermophilic gas fermentation of volatile chemicals has the potential for the development of consolidated bioprocesses that can simultaneously separate products during fermentation. This study reports the production of acetone from CO2 and H2, CO, or syngas by introducing the acetone production pathway using acetyl-coenzyme A (Ac-CoA) and acetate produced via the Wood-Ljungdahl pathway in Moorella thermoacetica. Reducing the carbon flux from Ac-CoA to acetate through genetic engineering successfully enhanced acetone productivity, which varied on the basis of the gas composition. The highest acetone productivity was obtained with CO-H2, while autotrophic growth collapsed with CO2-H2. By adding H2 to CO, the acetone productivity from the same amount of carbon source increased compared to CO gas only, and the maximum specific acetone production rate also increased from 0.04 to 0.09 g-acetone/g-dry cell/h. Our development of the engineered thermophilic acetogen M. thermoacetica, which grows at a temperature higher than the boiling point of acetone (58 °C), would pave the way for developing a consolidated process with simplified and cost-effective recovery via condensation following gas fermentation.
  • Hironaga Akita; Yuya Itoiri; Noriyo Takeda; Zen-Ichiro Kimura; Hiroyuki Inoue; Akinori Matsushika
    Microbiology resource announcements 10 4 2021年01月 [査読有り]
     
    Enterobacter oligotrophicus CCA3 was isolated from leaf soil collected in Hiroshima Prefecture, Japan. Here, we report the draft genome sequence of E. oligotrophicus CCA3. The draft genome sequence of E. oligotrophicus CCA3 consists of 29 contigs of 4,425,100 bp, with a GC content of 54.2%.
  • Hironaga Akita; Yuya Itoiri; Noriyo Takeda; Zen-Ichiro Kimura; Hiroyuki Inoue; Akinori Matsushika
    Journal of genomics 9 1 - 5 2021年01月 [査読有り]
     
    Strain CCI9, which was isolated from leaf soil collected in Japan, was capable of growth on poor-nutrient medium, at temperatures of 10°C to 45°C, at pHs of 4.5 to 10, and in the presence of 7.0% NaCl. We determined a draft genome sequence of strain CCI9, which consists of a total of 28 contigs containing 4,644,734 bp with a GC content of 56.1%. This assembly yielded 4,154 predicted coding sequences. Multilocus sequence analysis (MLSA) based on atpD, gyrB, infB, and rpoB gene sequences were performed to further identify strain CCI9. The MLSA revealed that strain CCI9 clustered tightly with Enterobacter roggenkampii EN-117T. Moreover, the average nucleotide identity value (98.6%) between genome sequences of strain CCI9 and E. roggenkampii EN-117T exceeds the cutoff value for prokaryotic subspecies delineation. Therefore, strain CCI9 was identified as E. roggenkampii CCI9. To clarify differences between E. roggenkampii EN-117T and CCI9, the coding proteins were compared against the eggNOG database.
  • Hironaga Akita; Yusuke Nakamichi; Tomotake Morita; Akinori Matsushika
    Biochimica et biophysica acta. Proteins and proteomics 1868 10 140476 - 140476 2020年10月 [査読有り]
     
    meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP+-dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l-2-amino-6-oxopimelate. meso-DAPDH is divided into two major clusters, types I and II, based on substrate specificity and structural characteristic. Here, we describe a novel type II meso-DAPDH from Thermosyntropha lipolytica (TlDAPDH). The gene encoding a putative TlDAPDH was expressed in Escherichia coli cells, and then the enzyme was purified 7.3-fold to homogeneity from the crude cell extract. The molecule of TlDAPDH seemed to form a hexamer, which is the typical structural characteristic of type II meso-DAPDHs. The purified enzyme exhibited oxidative deamination activity toward meso-DAP with both NADP+ and NAD+ as coenzymes. TlDAPDH exhibited reductive amination activity of corresponding 2-oxo acid to produce d-amino acid. In particular, the productivities for d-aspartate and d-glutamate have not been reported in the type II enzymes. The optimum pH and temperature for oxidative deamination of meso-DAP were 10.5 and 55°C, respectively. TlDAPDH retained more than 80% of its activity after incubation for 30 min at temperatures between 50°C and 65°C and in the pH range of 4.5-9.5. Moreover, the coenzyme and substrate recognition mechanisms of TlDAPDH were elucidated based on a multiple sequence alignment and the homology model. The results of these analyses suggested that the molecular mechanisms for coenzyme and substrate recognition of TlDAPDH were similar to those of meso-DAPDH from S. thermophilum, which is the representative type II enzyme. Based on the kinetic characteristics and structural comparison, TlDAPDH was considered to be a novel type II meso-DAPDH.
  • Hironaga Akita; Shinji Fujimoto; Keisuke Wada; Noriyo Takeda; Yuki Iwasaki; Tatsuya Fujii; Akinori Matsushika
    The Journal of general and applied microbiology 66 4 220 - 227 2020年09月 [査読有り]
     
    The discharge of industrial dyes and their breakdown products are often environmentally harmful. Here, we describe a biodegradation method using Burkholderia multivorans CCA53, which exhibits a capacity to degrade azo dyes, particularly ethyl red. Under the optimized culture conditions, 100 μM ethyl red was degraded more than 99% after incubation for 8 h. Real-time PCR analysis of azoR1 and azoR2, encoding two azoreductases, revealed that transcription level of these genes is enhanced at early phase under the optimized conditions. For a more practical approach, hydrolysates were prepared from eucalyptus or Japanese cedar chips or rice straw, and rice straw hydrolysate was used as the best medium for ethyl red biodegradation. Under those conditions, ethyl red was also degraded with high efficiency (>91%). We have thus constructed a potentially economical method for the biodegradation of ethyl red.
  • Yusuke Nakamichi; Tatsuya Fujii; Masahiro Watanabe; Akinori Matsushika; Hiroyuki Inoue
    Acta crystallographica. Section F, Structural biology communications 76 Pt 8 341 - 349 2020年08月 [査読有り]
     
    GH30-7 endoxylanase C from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C) belongs to glycoside hydrolase family 30 subfamily 7, and specifically releases 22-(4-O-methyl-α-D-glucuronosyl)-xylobiose from glucuronoxylan, as well as various arabino-xylooligosaccharides from arabinoxylan. TcXyn30C has a modular structure consisting of a catalytic domain and a C-terminal cellulose-binding module 1 (CBM1). In this study, the crystal structure of a TcXyn30C mutant which lacks the CBM1 domain was determined at 1.65 Å resolution. The structure of the active site of TcXyn30C was compared with that of the bifunctional GH30-7 xylanase B from T. cellulolyticus (TcXyn30B), which exhibits glucuronoxylanase and xylobiohydrolase activities. The results revealed that TcXyn30C has a conserved structural feature for recognizing the 4-O-methyl-α-D-glucuronic acid (MeGlcA) substituent in subsite -2b. Additionally, the results demonstrated that Phe47 contributes significantly to catalysis by TcXyn30C. Phe47 is located in subsite -2b and also near the C-3 hydroxyl group of a xylose residue in subsite -2a. Substitution of Phe47 with an arginine residue caused a remarkable decrease in the catalytic efficiency towards arabinoxylan, suggesting the importance of Phe47 in arabinoxylan hydrolysis. These findings indicate that subsite -2b of TcXyn30C has unique structural features that interact with arabinofuranose and MeGlcA substituents.
  • Hironaga Akita; Yusuke Nakamichi; Tomotake Morita; Akinori Matsushika
    MicrobiologyOpen 9 8 e1059 - e1059 2020年08月 [査読有り]
  • Keisuke Wada; Tatsuya Fujii; Hiroyuki Inoue; Hironaga Akita; Tomotake Morita; Akinori Matsushika
    Fermentation 6 3 70 - 70 2020年07月 [査読有り]
     
    Pyruvate, a potential precursor of various chemicals, is one of the fundamental chemicals produced by the fermentation process. We previously reported a pyruvate-producing Escherichia coli strain LAFCPCPt-accBC-aceE (PYR) that has the potential to be applied to the industrial production of pyruvate. In this study, the availability of the PYR strain for the production of pyruvate-derivative chemicals was evaluated using a d-lactate-producing strain (LAC) based on the PYR strain. The LAC strain expresses a d-lactate dehydrogenase-encoding gene from Lactobacillus bulgaricus under the control of a T7 expression system. The d-lactate productivity of the LAC strain was further improved by limiting aeration and changing the induction period for the expression of d-lactate dehydrogenase-encoding gene expression. Under combined conditions, the LAC strain produced d-lactate at 21.7 ± 1.4 g·L−1, which was compatible with the pyruvate production by the PYR strain (26.1 ± 0.9 g·L−1). These results suggest that we have succeeded in the effective conversion of pyruvate to d-lactate in the LAC strain, demonstrating the wide versatility of the parental PYR strain as basal strain for various chemicals production.
  • Yusuke Nakamichi; Masahiro Watanabe; Akinori Matsushika; Hiroyuki Inoue
    FEBS open bio 10 6 1180 - 1189 2020年06月 [査読有り]
     
    Xylanase B, a member of subfamily 7 of the GH30 (glycoside hydrolase family 30) from Talaromyces cellulolyticus (TcXyn30B), is a bifunctional enzyme with glucuronoxylanase and xylobiohydrolase activities. In the present study, crystal structures of the native enzyme and the enzyme-product complex of TcXyn30B expressed in Pichia pastoris were determined at resolutions of 1.60 and 1.65 Å, respectively. The enzyme complexed with 22 -(4-O-methyl-α-d-glucuronyl)-xylobiose (U4m2 X) revealed that TcXyn30B strictly recognizes both the C-6 carboxyl group and the 4-O-methyl group of the 4-O-methyl-α-d-glucuronyl side chain by the conserved residues in GH30-7 endoxylanases. The crystal structure and site-directed mutagenesis indicated that Asn-93 on the β2-α2-loop interacts with the non-reducing end of the xylose residue at subsite-2 and is likely to be involved in xylobiohydrolase activity. These findings provide structural insight into the mechanisms of substrate recognition of GH30-7 glucuronoxylanase and xylobiohydrolase.
  • Tatsuya Fujii; Akinori Matsushika
    Applied biochemistry and biotechnology 190 4 1360 - 1370 2020年04月 [査読有り]
     
    Talaromyces cellulolyticus is a promising strain for industrial cellulase production. In this study, the thaB gene, which is a homologue of the hap2/B gene in other filamentous fungi, was isolated and characterized. When grown in the presence of cellulose, culture supernatants of a thaB-disrupted strain (YDTha) exhibited decreased cellulase and xylanase enzymatic activities compared to the control strain. Furthermore, YDTha exhibited lower expression of the genes encoding cellulases and xylanases compared to the control strain. When cellobiose and lactose (soluble carbon sources) were used as carbon sources, the expression of the genes encoding cellulases and xylanases was decreased in both the YDTha and the control strains, though the expression levels in YDTha remained lower than those in the control strain. These results suggested that thaB has a positive role in cellulase and xylanase production in T. cellulolyticus.
  • Akita H; Itoiri Y; Ihara S; Takeda N; Matsushika A; Kimura Z
    Arch Microbiol 202 7 1757 - 1762 2020年02月 [査読有り]
  • Akita H; Itoiri Y; Kumagai A; Takeda N; Matsushika A; Oshiki M; Kimura Z
    J Genomic 8 21 - 24 2020年01月 [査読有り]
  • Yusuke Nakamichi; Tatsuya Fujii; Thierry Fouquet; Akinori Matsushika; Hiroyuki Inoue
    Applied and environmental microbiology 85 22 2019年11月 [査読有り]
     
    Glycoside hydrolase family 30 subfamily 7 (GH30-7) enzymes include various types of xylanases, such as glucuronoxylanase, endoxylanase, xylobiohydrolase, and reducing-end xylose-releasing exoxylanase. Here, we characterized the mode of action and gene expression of the GH30-7 endoxylanase from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C). TcXyn30C has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal cellulose binding module 1, whose cellulose-binding ability has been confirmed. Sequence alignment of GH30-7 xylanases exhibited that TcXyn30C has a conserved Phe residue at the position corresponding to a conserved Arg residue in GH30-7 glucuronoxylanases, which is required for the recognition of the 4-O-methyl-α-d-glucuronic acid (MeGlcA) substituent. TcXyn30C degraded both glucuronoxylan and arabinoxylan with similar kinetic constants and mainly produced linear xylooligosaccharides (XOSs) with 2 to 3 degrees of polymerization, in an endo manner. Notably, the hydrolysis of glucuronoxylan caused an accumulation of 22-(MeGlcA)-xylobiose (U4m2X). The production of this acidic XOS is likely to proceed via multistep reactions by putative glucuronoxylanase activity that produces 22-(MeGlcA)-XOSs (X n U4m2X, n ≥ 0) in the initial stages of the hydrolysis and by specific release of U4m2X from a mixture containing X n U4m2X. Our results suggest that the unique endoxylanase activity of TcXyn30C may be applicable to the production of linear and acidic XOSs. The gene xyn30C was located adjacent to the putative GH62 arabinofuranosidase gene (abf62C) in the T. cellulolyticus genome. The expression of both genes was induced by cellulose. The results suggest that TcXyn30C may be involved in xylan removal in the hydrolysis of lignocellulose by the T. cellulolyticus cellulolytic system.IMPORTANCE Xylooligosaccharides (XOSs), which are composed of xylose units with a β-1,4 linkage, have recently gained interest as prebiotics in the food and feed industry. Apart from linear XOSs, branched XOSs decorated with a substituent such as methyl glucuronic acid and arabinose also have potential applications. Endoxylanase is a promising tool in producing XOSs from xylan. The structural variety of XOSs generated depends on the substrate specificity of the enzyme as well as the distribution of the substituents in xylan. Thus, the exploration of endoxylanases with novel specificities is expected to be useful in the provision of a series of XOSs. In this study, the endoxylanase TcXyn30C from Talaromyces cellulolyticus was characterized as a unique glycoside hydrolase belonging to the family GH30-7, which specifically releases 22-(4-O-methyl-α-d-glucuronosyl)-xylobiose from hardwood xylan. This study provides new insights into the production of linear and branched XOSs by GH30-7 endoxylanase.
  • Wada K; Fujii T; Akita H; Matsushika A
    Appl Biochem Biotechnol 190 4 1349 - 1359 2019年11月 [査読有り]
     
    Construction of acid-tolerant strains of Saccharomyces cerevisiae is required for various bioproduction processes. We previously isolated the gene IoGAS1 from multiple stress-tolerant Issatchenkia orientalis as a gene conferring sulfuric acid resistance in S. cerevisiae, but its acid tolerance was only investigated using sulfuric acid. Here, we evaluated the growth and ethanol fermentation ability of the IoGAS1-expressing S. cerevisiae strain, B4-IoGAS1, by using various acidic reagents. B4-IoGAS1 exhibited faster growth than the control strain, B4-CON, when cultured aerobically with sulfuric, hydrochloric, formic, acetic, and lactic acids at pH below 2.4. However, the growth of B4-IoGAS1 was suppressed at pH above 2.48, irrespective of the type of acid reagents. Furthermore, B4-IoGAS1 exhibited higher performance of ethanol fermentation than B4-CON under 250 mM lactic acid condition at pH 2.37. These results demonstrate that IoGAS1 could facilitate the aerobic growth and anaerobic ethanol production under different acidic stressed conditions.
  • Toshihiro Suzuki; Tamotsu Hoshino; Akinori Matsushika
    Enzyme and Microbial Technology 129 109359 - 109359 2019年10月 [査読有り]
  • Akita H; Matsushika A; Kimura ZI
    MicrobiologyOpen 8 9 e00843  2019年09月 [査読有り]
  • Yusuke Nakamichi; Thierry Fouquet; Shotaro Ito; Akinori Matsushika; Hiroyuki Inoue
    Applied and environmental microbiology 85 13 e00552-19  2019年07月 [査読有り]
     
    In this study, we characterized the mode of action of reducing-end xylose-releasing exoxylanase (Rex), which belongs to the glycoside hydrolase family 30-7 (GH30-7). GH30-7 Rex, isolated from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), exists as a dimer. The purified Xyn30A released xylose from linear xylooligosaccharides (XOSs) 3 to 6 xylose units in length with similar kinetic constants. Hydrolysis of branched, borohydride-reduced, and p-nitrophenyl XOSs clarified that Xyn30A possesses a Rex activity. 1H nuclear magnetic resonance (1H NMR) analysis of xylotriose hydrolysate indicated that Xyn30A degraded XOSs via a retaining mechanism and without recognizing an anomeric structure at the reducing end. Hydrolysis of xylan by Xyn30A revealed that the enzyme continuously liberated both xylose and two types of acidic XOSs: 22-(4-O-methyl-α-d-glucuronyl)-xylotriose (MeGlcA2Xyl3) and 22-(MeGlcA)-xylobiose (MeGlcA2Xyl2). These acidic products were also detected during hydrolysis using a mixture of MeGlcA2Xyl n (n = 2 to 14) as the substrate. This indicates that Xyn30A can release MeGlcA2Xyl n (n = 2 and 3) in an exo manner. Comparison of subsites in Xyn30A and GH30-7 glucuronoxylanase using homology modeling suggested that the binding of the reducing-end residue at subsite +2 was partially prevented by a Gln residue conserved in GH30-7 Rex; additionally, the Arg residue at subsite -2b, which is conserved in glucuronoxylanase, was not found in Xyn30A. Our results lead us to propose that GH30-7 Rex plays a complementary role in hydrolysis of xylan by fungal cellulolytic systems.IMPORTANCE Endo- and exo-type xylanases depolymerize xylan and play crucial roles in the assimilation of xylan in bacteria and fungi. Exoxylanases release xylose from the reducing or nonreducing ends of xylooligosaccharides; this is generated by the activity of endoxylanases. β-Xylosidase, which hydrolyzes xylose residues on the nonreducing end of a substrate, is well studied. However, the function of reducing-end xylose-releasing exoxylanases (Rex), especially in fungal cellulolytic systems, remains unclear. This study revealed the mode of xylan hydrolysis by Rex from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), which belongs to the glycoside hydrolase family 30-7 (GH30-7). A conserved residue related to Rex activity is found in the substrate-binding site of Xyn30A. These findings will enhance our understanding of the function of GH30-7 Rex in the cooperative hydrolysis of xylan by fungal enzymes.
  • Akinori Matsushika
    Journal of Biological Chemistry 294 11 4065 - 4078 2019年03月 [査読有り]
  • Akinori Matsushika
    Journal of Mass Spectrometry 54 3 213 - 221 2019年03月 [査読有り]
  • 井上宏之; 中道優介; FOUQUET Thierry; 藤井達也; 渡邊真宏; 松鹿昭則
    バイオマス科学会議発表論文集(Web) 14 2019年01月
  • Akinori Matsushika
    Genome Announcements 5 38 e00910-17  2017年09月 [査読有り]
     
    ABSTRACT Ureibacillus thermosphaericus A1 was isolated from compost collected in Munakata City, Fukuoka Prefecture, Japan. Here, we report the first complete genome sequence of U. thermosphaericus . The complete genome of this strain consists of 3,488,104 bp with a GC content of 36.3% and comprises 3,362 predicted coding sequences.
  • Akinori Matsushika; Toshihiro Suzuki; Tetsuya Goshima; Tamotsu Hoshino
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 124 2 164 - 170 2017年08月 [査読有り]
     
    We previously showed that overexpression of IoGAS1, which was isolated from the multiple stress-tolerant yeast Issatchenkia orientalis, endows Saccharomyces cerevisiae cells with the ability to grow and ferment under acidic and high-salt conditions. The deduced amino acid sequence of the IoGAS1 gene product exhibits 60% identity with the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol-anchored protein essential for maintaining cell wall integrity. However, the functional roles of ScGAS1 in stress tolerance and pH regulation remain unclear. In the present study, we characterized ScGAS1 regarding its roles in tolerance to low pH and high salt concentrations. Transcriptional analysis indicated that, as for the IoGAS1 gene, ScGAS1 expression was pH dependent, with maximum expression at pH 3.0; the presence of salt increased endogenous expression of both GAS1 genes at almost all pH levels. These results suggested that ScGAS1, like IoGAS1, is involved in a novel acid- and salt-stress adaptation mechanism in S. cerevisiae. Overexpression of ScGAS1 in S. cerevisiae improved growth and ethanol production from glucose under acid stress without added salt, although the stress tolerance of the ScGAS1-overexpressing strain was inferior to that of the IoGAS1-overexpressing strain. However, overexpression of ScGAS1 did not result in increased tolerance of S. cerevisiae to combined acid and salt stress, even though ScGAS1 appears to be a salt-responsive gene. Thus, ScGAS1 is directly implicated in tolerance to low pH but does not confer salinity tolerance, supporting the view that ScGAS1 and IoGAS1 have overlapping yet distinct roles in stress tolerance in yeast. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Yosuke Kobayashi; Takehiko Sahara; Toshihiro Suzuki; Saori Kamachi; Akinori Matsushika; Tamotsu Hoshino; Satoru Ohgiya; Yoichi Kamagata; Kazuhiro E. Fujimori
    JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY 44 6 879 - 891 2017年06月 [査読有り]
     
    The pentose phosphate pathway (PPP) plays an important role in the efficiency of xylose fermentation during cellulosic ethanol production. In simultaneous saccharification and co-fermentation (SSCF), the optimal temperature for cellulase hydrolysis of lignocellulose is much higher than that of fermentation. Successful use of SSCF requires optimization of the expression of PPP genes at elevated temperatures. This study examined the combinatorial expression of PPP genes at high temperature. The results revealed that over-expression of TAL1 and TKL1 in Saccharomyces cerevisiae (S. cerevisiae) at 30 A degrees C and over-expression of all PPP genes at 36 A degrees C resulted in the highest ethanol productivities. Furthermore, combinatorial over-expression of PPP genes derived from S. cerevisiae and a thermostable yeast Kluyveromyces marxianus allowed the strain to ferment xylose with ethanol productivity of 0.51 g/L/h, even at 38 A degrees C. These results clearly demonstrate that xylose metabolism can be improved by the utilization of appropriate combinations of thermostable PPP genes in high-temperature production of ethanol.
  • Akinori Matsushika; Kanako Negi; Toshihiro Suzuki; Tetsuya Goshima; Tamotsu Hoshino
    PLOS ONE 11 9 25  2016年09月 [査読有り]
     
    The use of yeasts tolerant to acid (low pH) and salt stress is of industrial importance for several bioproduction processes. To identify new candidate genes having potential roles in low-pH tolerance, we screened an expression genomic DNA library of a multiple-stress-tolerant yeast, Issatchenkia orientalis (Pichia kudriavzevii), for clones that allowed Saccharomyces cerevisiae cells to grow under highly acidic conditions (pH 2.0). A genomic DNA clone containing two putative open reading frames was obtained, of which the putative protein-coding gene comprising 1629 bp was retransformed into the host. This transformant grew significantly at pH 2.0, and at pH 2.5 in the presence of 7.5% Na2SO4. The predicted amino acid sequence of this new gene, named I. orientalis GAS1 (IoGAS1), was 60% identical to the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol-anchored protein essential for maintaining cell wall integrity, and 58-59% identical to Candida albicans Phr1 and Phr2, pH-responsive proteins implicated in cell wall assembly and virulence. Northern hybridization analyses indicated that, as for the C. albicans homologs, IoGAS1 expression was pH-dependent, with expression increasing with decreasing pH (from 4.0 to 2.0) of the medium. These results suggest that IoGAS1 represents a novel pH-regulated system required for the adaptation of I. orientalis to environments of diverse pH. Heterologous expression of IoGAS1 complemented the growth and morphological defects of a S. cerevisiae gas1 Delta mutant, demonstrating that IoGAS1 and the corresponding S. cerevisiae gene play similar roles in cell wall biosynthesis. Site-directed mutagenesis experiments revealed that two conserved glutamate residues (E161 and E262) in the IoGas1 protein play a crucial role in yeast morphogenesis and tolerance to low pH and salt stress. Furthermore, overexpression of IoGAS1 in S. cerevisiae remarkably improved the ethanol fermentation ability at pH 2.5, and at pH 2.0 in the presence of salt (5% Na2SO4), compared to that of a reference strain. Our results strongly suggest that constitutive expression of the IoGAS1 gene in S. cerevisiae could be advantageous for several fermentation processes under these stress conditions.
  • Akinori Matsushika; Tamotsu Hoshino
    JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY 42 12 1623 - 1631 2015年12月 [査読有り]
     
    The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes.
  • Belisa B. de Sales; Bruna Scheid; Davi L. Goncalves; Marilia M. Knychala; Akinori Matsushika; Elba P. S. Bon; Boris U. Stambuk
    BIOTECHNOLOGY LETTERS 37 10 1973 - 1982 2015年10月 [査読有り]
     
    An hxt-null S. cerevisiae strain, lacking the major hexose transporters (hxt1 Delta-hxt7 Delta and gal2 Delta) but having high xylose reductase, xylitol dehydrogenase and xylulokinase activities, was transformed with a genomic DNA library from S. stipitis. Four plasmids allowing growth on xylose contained three genes encoding sugar transporters: the previously characterized XUT1 permease, and two new genes (HXT2.6 and QUP2) not previously identified as xylose transporters. High cell density fermentations with the recombinant strains showed that the XUT1 gene allowed ethanol production from xylose or xylose plus glucose as carbon sources, while the HXT2.6 permease produced both ethanol and xylitol, and the strain expressing the QUP2 gene produced mainly xylitol during xylose consumption. Cloning novel sugar transporters not previously identified in the S. stipitis genome using an hxt-null S. cerevisiae strain with a high xylose-utilizing pathway provides novel promising target genes for improved lignocellulosic ethanol production by yeasts.
  • Akinori Matsushika
    Fermentation Technology 4 1 114  2015年05月 [査読有り]
  • Hiroyuki Inoue; Seitaro Hashimoto; Akinori Matsushika; Seiya Watanabe; Shigeki Sawayama
    JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY 41 12 1773 - 1781 2014年12月 [査読有り]
     
    The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD(+)-dependent XDH or engineered NADP(+)-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD(+)-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.
  • Davi L. Goncalves; Akinori Matsushika; Belisa B. de Sales; Tetsuya Goshima; Elba P. S. Bon; Boris U. Stambuk
    ENZYME AND MICROBIAL TECHNOLOGY 63 13 - 20 2014年09月 [査読有り]
     
    Since the uptake of xylose is believed to be one of the rate-limiting steps for xylose ethanol fermentation by recombinant Saccharomyces cerevisiae strains, we transformed a hxt-null strain lacking the major hexose transporters (hxt1 Delta-hxt7 Delta, and gal2 Delta) with an integrative plasmid to overexpress the genes for xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and xylulokinase (XKS1), and analyzed the impact that overexpression of the HXT1, HXT2, HXT5 or HXT7 permeases have in anaerobic batch fermentations using xylose, glucose, or xylose plus glucose as carbon sources. Our results revealed that the low-affinity HXT1 permease allowed the maximal consumption of sugars and ethanol production rates during xylose/glucose co-fermentations, but was incapable to allow xylose uptake when this sugar was the only carbon source. The moderately high-affinity HXT5 permease was a poor glucose transporter, and it also did not allow significant xylose uptake by the cells. The moderately high-affinity HXT2 permease allowed xylose uptake with the same rates as those observed during glucose consumption, even under co-fermentation conditions, but had the drawback of producing incomplete fermentations. Finally, the high-affinity HXT7 permease allowed efficient xylose fermentation, but during xylose/glucose co-fermentations this permease showed a clear preference for glucose. Thus, our results indicate that approaches to engineer S. cerevisiae HXT transporters to improve second generation bioethanol production need to consider the composition of the biomass sugar syrup, whereby the HXT1 transporter seems more suitable for hydrolysates containing xylose/glucose blends, whereas the HXT7 permease would be a better choice for xylose-enriched sugar streams. (C) 2014 Elsevier Inc. All rights reserved.
  • Akinori Matsushika; Hiroyo Morikawa; Tetsuya Goshima; Tamotsu Hoshino
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 174 2 623 - 631 2014年09月 [査読有り]
     
    Flocculation is a desirable property in industrial yeasts and is particularly important in the fuel ethanol industry because it provides a simple and cost-free way to separate yeast cells from fermentation products. In the present study, the effect of pH and lignocellulose-derived sugars on yeast flocculation was investigated using a flocculent Saccharomyces cerevisiae, MA-R4, which has been recombinantly engineered to simultaneously co-ferment glucose and xylose to ethanol with high productivity. The flocculation level of MA-R4 dramatically decreased at pH values below 3.0 during co-fermentation of glucose and xylose. Sedimentation and microscopic observation revealed that flocculation was induced in MA-R4 when it fermented glucose, a glucose/xylose mixture, or mannose, whereas attempts to ferment xylose, galactose, and arabinose led to the loss of flocculation. MA-R4 fermented xylose and galactose more slowly than glucose and mannose. Therefore, the various flocculation behaviors shown by MA-R4 should be useful in the control of ethanol fermentation processes.
  • Akinori Matsushika
    Genome Announcements 2 4 e00733-14  2014年07月 [査読有り]
  • Tatsuya Fujii; Katsuji Murakami; Takashi Endo; Shinji Fujimoto; Tomoaki Minowa; Akinori Matsushika; Shinichi Yano; Shigeki Sawayama
    BIOPROCESS AND BIOSYSTEMS ENGINEERING 37 4 749 - 754 2014年04月 [査読有り]
     
    In the bioethanol production process, high solid saccharification and glucose/xylose co-fermentation are important technologies for obtaining increased ethanol concentrations; however, bench-scale studies using combinations of these methods are limited. In this study, we hydrolyzed high solid concentration of milled eucalyptus using commercial enzymes and obtained 138.4 g/L total monomeric sugar concentration. These sugars were fermented to 53.5 g/L of ethanol by a xylose-utilizing recombinant Saccharomyces cerevisiae strain, MA-R4. These experiments were performed in bench scale (using 50 L scale solid mixer and 70 L scale fermenter). The results obtained in this study were comparable to our previous results in laboratory scale, indicating that we successfully achieved an efficient high solid saccharification and glucose/xylose co-fermentation system in bench scale.
  • Akinori Matsushika; Tetsuya Goshima; Tamotsu Hoshino
    MICROBIAL CELL FACTORIES 13 1 18  2014年01月 [査読有り]
     
    Background: There has been much research on the bioconversion of xylose found in lignocellulosic biomass to ethanol by genetically engineered Saccharomyces cerevisiae. However, the rate of ethanol production from xylose in these xylose-utilizing yeast strains is quite low compared to their glucose fermentation. In this study, two diploid xylose-utilizing S. cerevisiae strains, the industrial strain MA-R4 and the laboratory strain MA-B4, were employed to investigate the differences between anaerobic fermentation of xylose and glucose, and general differences between recombinant yeast strains, through genome-wide transcription analysis. Results: In MA-R4, many genes related to ergosterol biosynthesis were expressed more highly with glucose than with xylose. Additionally, these ergosterol-related genes had higher transcript levels in MA-R4 than in MA-B4 during glucose fermentation. During xylose fermentation, several genes related to central metabolic pathways that typically increase during growth on non-fermentable carbon sources were expressed at higher levels in both strains. Xylose did not fully repress the genes encoding enzymes of the tricarboxylic acid and respiratory pathways, even under anaerobic conditions. In addition, several genes involved in spore wall metabolism and the uptake of ammonium, which are closely related to the starvation response, and many stress-responsive genes mediated by Msn2/4p, as well as trehalose synthase genes, increased in expression when fermenting with xylose, irrespective of the yeast strain. We further observed that transcript levels of genes involved in xylose metabolism, membrane transport functions, and ATP synthesis were higher in MA-R4 than in MA-B4 when strains were fermented with glucose or xylose. Conclusions: Our transcriptomic approach revealed the molecular events underlying the response to xylose or glucose and differences between MA-R4 and MA-B4. Xylose-utilizing S. cerevisiae strains may recognize xylose as a non-fermentable carbon source, which induces a starvation response and adaptation to oxidative stress, resulting in the increased expression of stress-response genes.
  • Tetsuya Goshima; Kanako Negi; Masaharu Tsuji; Hiroyuki Inoue; Shinichi Yano; Tamotsu Hoshino; Akinori Matsushika
    Journal of Bioscience and Bioengineering 116 5 551 - 554 2013年11月 [査読有り]
     
    We constructed a xylose-fermenting recombinant strain of thermotolerant yeast Kluyveromyces marxianus, DMB3-7. Both xylose consumption and ethanol production were remarkably increased in DMB3-7 compared to the control strain at 30°C. Furthermore, DMB3-7 produced ethanol from xylose at both 42°C and 45°C, above which xylose metabolic activity decreased. © 2013 The Society for Biotechnology, Japan.
  • Masaharu Tsuji; Tetsuya Goshima; Akinori Matsushika; Sakae Kudoh; Tamotsu Hoshino
    CRYOBIOLOGY 67 2 241 - 243 2013年10月 [査読有り]
     
    Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10 degrees C. Maximally, 48.2 g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2 g/l, 12.5 g/l and 7.2 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1-1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in told environments. (C) 2013 Elsevier Inc. All rights reserved.
  • Akinori Matsushika; Atsushi Nagashima; Tetsuya Goshima; Tamotsu Hoshino
    PLOS ONE 8 7 11  2013年07月 [査読有り]
     
    In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7-phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose.
  • Tatsuya Fujii; Akinori Matsushika; Tetsuya Goshima; Katsuji Murakami; Shinichi Yano
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 7 1579 - 1582 2013年07月 [査読有り]
     
    We prepared eight recombinant Saccharomyces cerevisae strains, including three strains generated in this study that were produced by chromosomal integration of xylose utilization pathway enzymes genes. Among these strains, MA-R4 was the most efficient at producing ethanol from rice straw enzymatic hydrolysate, indicating that it is a superior strain for bioethanol production.
  • Tetsuya Goshima; Masaharu Tsuji; Hiroyuki Inoue; Shinichi Yano; Tamotsu Hoshino; Akinori Matsushika
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 7 1505 - 1510 2013年07月 [査読有り]
     
    The yeast Kluyveromyces marxianus is considered as a potential alternative to Saccharomyces cerevisiae in producing ethanol as a biofuel. In this study, we investigated the ethanol fermentation properties of novel K marxianus strain DMB1, isolated from bagasse hydrolysates. This strain utilized sorbitol as well as various pentoses and hexoses as single carbon sources under aerobic conditions and produced ethanol from glucose in hydrolysates of the Japanese cedar at 42 degrees C. Reference strains K marxianus NBRC1777 and S. cerevisiae BY4743 did not assimilate sorbitol or ferment lignocellulosic hydrolysates to ethanol at this temperature. Thus strain DMB1 appears to be optimal for producing bioethanol at high temperatures, and might provide a valuable means of increasing the efficiency of ethanol fermentation.
  • Akinori Matsushika; Shigeki Sawayama
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 169 3 712 - 721 2013年02月 [査読有り]
     
    Glucose/xylose mixtures (90 g/L total sugar) were evaluated for their effect on ethanol fermentation by a recombinant flocculent Saccharomyces cerevisiae, MA-R4. Glucose was utilized faster than xylose at any ratio of glucose/xylose, although MA-R4 can simultaneously co-ferment both sugars. A high percentage of glucose can increase cell biomass production and therefore increase the rate of glucose utilization (1.224 g glucose/g biomass/h maximum) and ethanol formation (0.493 g ethanol/g biomass/h maximum). However, the best ratio of glucose/xylose for the highest xylose consumption rate (0.209 g xylose/g biomass/h) was 2:3. Ethanol concentration and yield increased and by-product (xylitol, glycerol, and acetic acid) concentration decreased as the proportion of glucose increased. The maximum ethanol concentration was 41.6 and 21.9 g/L after 72 h of fermentation with 90 g/L glucose and 90 g/L xylose, respectively, while the ethanol yield was 0.454 and 0.335 g/g in 90 g/L glucose and 90 g/L xylose media, respectively. High ethanol yield when a high percentage of glucose is available is likely due to decreased production of by-products, such as glycerol and acetic acid. These results suggest that ethanol selectivity is increased when a higher proportion of glucose is available and reduced when a higher proportion of xylose is available.
  • Akinori Matsushika; Shigeki Sawayama
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 168 8 2094 - 2104 2012年12月 [査読有り]
     
    The inhibitory effects of pH and acetic acid on the co-fermentation of glucose and xylose in complex medium by recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated. In the absence of acetic acid, the fermentation performance of strain MA-R4 was similar between pH 4.0-6.0, but was negatively affected at pH 2.5. The addition of acetic acid to batch cultures resulted in negligible inhibition of several fermentation parameters at pH 6.0, whereas the interactive inhibition of pH and acetic acid on the maximum cell and ethanol concentrations, and rates of sugar consumption and ethanol production were observed at pH levels below 5.4. The inhibitory effect of acetic acid was particularly marked for the consumption rate of xylose, as compared with that of glucose. With increasing initial acetic acid concentration, the ethanol yield slightly increased at pH 5.4 and 6.0, but decreased at pH values lower than 4.7. Notably, ethanol production was nearly completely inhibited under low pH (4.0) and high acetic acid (150-200 mM) conditions. Together, these results indicate that the inhibitory effects of acetic acid and pH on ethanol fermentation by MA-R4 are highly synergistic, although the inhibition can be reduced by increasing the medium pH.
  • Akinori Matsushika; Tetsuya Goshima; Tatsuya Fujii; Hiroyuki Inoue; Shigeki Sawayama; Shinichi Yano
    ENZYME AND MICROBIAL TECHNOLOGY 51 1 16 - 25 2012年06月 [査読有り]
     
    The activity of transaldolase and transketolase, key enzymes in the non-oxidative pentose phosphate pathway, is rate-limiting for xylose utilization in recombinant Saccharomyces cerevisiae. Overexpression of TAL1 and TKL1, the major transaldolase and transketolase genes, increases the flux from the pentose phosphate pathway into the glycolytic pathway. However, the functional roles of NQM1 and TKL2, the secondary transaldolase and transketolase genes, especially in xylose utilization, remain unclear. This study focused on characterization of NQM1 and TKL2, together with TAL1 and TKL1, regarding their roles in xylose utilization and fermentation. Knockout or overexpression of these four genes on the phenotype of xylose-utilizing S. cerevisiae strains was also examined. Transcriptional analysis indicated that the expression of TAL1, NQM1, and TKL1 was up-regulated in the presence of xylose. A significant decrease in both growth on xylose and xylose-fermenting ability in tal1 Delta and tkl1 Delta mutants confirmed that TALI and TKL1 are essential for xylose assimilation and fermentation. Gene disruption analysis using a tkl1 Delta mutant revealed that TKL1 is also required for utilization of glucose. Growth on xylose and xylose-fermenting ability were slightly influenced by deletion of NQM1 or TKL2 when xylose was used as the sole carbon source. Moreover, the rate of xylose consumption and ethanol production was slightly impaired in TKL1- and TKL2-overexpressing strains. NQM1 and TKL2 may thus play a physiological role via an effect on the non-oxidative pentose phosphate pathway in the xylose metabolic pathway, although their roles in xylose utilization and fermentation are less important than those of TAL1 and TKL1. (C) 2012 Elsevier Inc. All rights reserved.
  • Emiko Oguri; Osamu Takimura; Akinori Matsushika; Hiroyuki Inoue; Shigeki Sawayama
    FOOD SCIENCE AND TECHNOLOGY RESEARCH 17 4 267 - 272 2011年07月 [査読有り]
     
    The potential of spent mushroom substrate after cultivation of Pleurotus eryngii as a biomass resource for bioethanol production was investigated. Materials were pretreated by ball milling for 1 h, and enzymatic hydrolysis was then carried out. Glucose, xylose, arabinose and galactose were detected in enzymatic hydrolysates, and a > 59.0% yield of total sugars was obtained, even at a substrate concentration of 30% (w/v). Enzymatic hydrolysates were fermented using Pichia stipitis. When enzymatic hydrolysates obtained from a 20% (w/v) substrate concentration were fermented, the maximum ethanol concentration was 17.7 g and ethanol yield was 67.0%. These results indicate that corncob-based spent mushroom substrate can be used as a raw material for bioethanol production.
  • Fujii T; Yu G; Matsushika A; Kurita A; Yano S; Murakami K; Sawayama S
    Bioscience, biotechnology, and biochemistry 75 6 1140 - 1146 6 2011年06月 [査読有り]
     
    Construction of xylose- and xylo-oligosaccharide-fermenting Saccharomyces cerevisiae strains is important, because hydrolysates derived from lignocellulosic biomass contain significant amounts of these sugars. We have obtained recombinant S. cerevisiae strain MA-D4 (D-XKXDHXR), expressing xylose reductase, xylitol dehydrogenase and xylulokinase. In the present study, we generated recombinant strain D-XSD/XKXDHXR by transforming MA-D4 with a beta-xylosidase gene cloned from the filamentous fungus Trichoderma reesei. The intracellular beta-xylosidase-specific activity of D-XSD/XKXDHXR was high, while that of the control strain was under the limit of detection. D-XSD/XKXDHXR produced ethanol, and xylose accumulated in the culture supernatant under fermentation in a medium containing xylo-oligosaccharides as sole carbon source. beta-Xylosidase-specific activity in D-XSD/XKXDHXR declined due to xylose both in vivo and in vitro. D-XSD/XKXDHXR converted xylo-oligosaccharides in an enzymatic hydrolysate of eucalyptus to ethanol. These results indicate that D-XSD/XKXDHXR efficiently converted xylo-oligosaccharides to xylose and subsequently to ethanol.
  • Akinori Matsushika; Shigeki Sawayama
    ENZYME AND MICROBIAL TECHNOLOGY 48 6-7 466 - 471 2011年05月 [査読有り]
     
    Ethanol production from xylose is important for the utilization of lignocellulosic biomass as raw materials. Recently, we reported the development of an industrial xylose-fermenting Saccharomyces cerevisiae strain. MA-R4, which was engineered by chromosomal integration to express the genes encoding xylose reductase and xylitol dehydrogenase from Pichia sripitis along with S. cerevisiae xylulokinase gene constitutively using the alcohol-fermenting flocculent yeast strain, IR-2. IR-2 has the highest xylulose-fermenting ability of the industrial diploid strains, making it a useful host strain for genetically engineering xylose-utilizing S. cerevisiae. To optimize the activities of xylose metabolizing enzymes in the metabolic engineering of IR-2 for further improvement of ethanol production from xylose, we constructed a set of recombinant isogenic strains harboring different combinations of genetic modifications present in MA-R4, and investigated the effect of constitutive expression of xylulokinase and of different levels of xylulokinase and xylose reductase activity on xylose fermentation. This strain comparison showed that constitutive expression of xylulokinase increased ethanol production from xylose at the expense of xylitol excretion, and that high activity of xylose reductase resulted in an increased rate of xylose consumption and an increased glycerol yield. Moreover, strain MA-R6, which has moderate xylulokinase activity, grew slightly better but accumulated more xylitol than strain MA-R4. These results suggest that fine-tuning of introduced enzyme activity in S. cerevisiae is important for improving xylose fermentation to ethanol. (C) 2011 Elsevier Inc. All rights reserved.
  • Akinori Matsushika; Shigeki Sawayama
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 162 7 1952 - 1960 2010年11月 [査読有り]
     
    Different initial cell concentrations of a recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated for their effects on xylose fermentation and glucose-xylose cofermentation. A high initial cell concentration greatly increased both the substrate utilization and ethanol production rates. During xylose fermentation, the highest rates of xylose consumption (2.58 g/L h) and ethanol production (0.83 g/L h) were obtained at an initial cell concentration of 13.1 g/L. During cofermentation, the highest rates of glucose consumption (14.4 g/L h), xylose consumption (2.79 g/L h), and ethanol production (6.68 g/L h) were obtained at an initial cell concentration of 12.7 g/L. However, a high initial cell density had no positive effect on the maximum ethanol concentration and ethanol yield mainly due to the increased amount of by-products including xylitol. The ethanol yield remained almost constant (0.34 g/g) throughout xylose fermentation (initial cell concentration range, 1.81-13.1 g/L), while it was slightly lower at high initial cell concentrations (9.87 and 12.7 g/L) during cofermentation. The determination of the appropriate initial cell concentration is necessary for the improvement of substrate utilization and ethanol yield.
  • Akinori Matsushika; Emiko Oguri; Shigeki Sawayama
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 110 1 102 - 105 2010年07月 [査読有り]
     
    We examined the evolutionary adaptation of recombinant shochu yeast by serial anaerobic cultivation in xylose-based minimal medium. Compared with the parental strain, the adapted strain MA-S4-M1 (M1) markedly improved the growth on xylose and the anaerobic xylose consumption rate. M1 gained improved xylose utilization properties by optimizing the metabolic pathway enzymes and enhancing the uptake of xylose. (C) 2010, The Society for Biotechnology, japan. All rights reserved.
  • Akinori Matsushika; Hiroyuki Inoue; Tsutomu Kodaki; Shigeki Sawayama
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 84 1 37 - 53 2009年08月 [査読有り][招待有り]
     
    Bioethanol production from xylose is important for utilization of lignocellulosic biomass as raw materials. The research on yeast conversion of xylose to ethanol has been intensively studied especially for genetically engineered Saccharomyces cerevisiae during the last 20 years. S. cerevisiae, which is a very safe microorganism that plays a traditional and major role in industrial bioethanol production, has several advantages due to its high ethanol productivity, as well as its high ethanol and inhibitor tolerance. However, this yeast cannot ferment xylose, which is the dominant pentose sugar in hydrolysates of lignocellulosic biomass. A number of different strategies have been applied to engineer yeasts capable of efficiently producing ethanol from xylose, including the introduction of initial xylose metabolism and xylose transport, changing the intracellular redox balance, and overexpression of xylulokinase and pentose phosphate pathways. In this review, recent progress with regard to these studies is discussed, focusing particularly on xylose-fermenting strains of S. cerevisiae. Recent studies using several promising approaches such as host strain selection and adaptation to obtain further improved xylose-utilizing S. cerevisiae are also addressed.
  • Akinori Matsushika; Hiroyuki Inoue; Seiya Watanabe; Tsutomu Kodaki; Keisuke Makino; Shigeki Sawayama
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 75 11 3818 - 3822 2009年06月 [査読有り]
     
    The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP(+)-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.
  • Akinori Matsushika; Hiroyuki Inoue; Katsuji Murakami; Osamu Takimura; Shigeki Sawayama
    BIORESOURCE TECHNOLOGY 100 8 2392 - 2398 2009年04月 [査読有り]
     
    In this study, five recombinant Saccharomyces cerevisiae strains were compared for their xylose-fermenting ability. The most efficient xylose-to-ethanol fermentation was found by using the industrial strain MA-R4, in which the genes for xylose reductase and xylitol dehydrogenase from Pichia stipitis along with an endogenous xylulokinase gene were expressed by chromosomal integration of the flocculent yeast strain IR-2. The MA-R4 strain rapidly converted xylose to ethanol with a low xylitol yield. Furthermore, the MA-R4 strain had the highest ethanol production when fermenting not only a mixture of glucose and xylose, but also mixed sugars in the detoxified hydrolysate of wood chips. These results collectively suggest that MA-R4 may be a suitable recombinant strain for further study into large-scale ethanol production from mixed sugars present in lignocellulosic hydrolysates. (c) 2008 Elsevier Ltd. All rights reserved.
  • Akinori Matsushika; Seiya Watanabe; Tsutomu Kodaki; Keisuke Makino; Hiroyuki Inoue; Katsuji Murakami; Osamu Takimura; Shigeki Sawayama
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 81 2 243 - 255 2008年11月 [査読有り]
     
    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD+-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP+. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP+-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP+-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.
  • Akinori Matsushika; Shigeki Sawayama
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 106 3 306 - 309 2008年09月 [査読有り]
     
    We varied the promoter strength of xylose reductase (XR) gene and the copy number of xylulokinase (XK) gene to determine how XR and XK activities affect the xylose-fermenting abilities of recombinant Saccharomyces cerevisiae expressing xylitol dehydrogenase (XDH). The most enhanced ethanol yield and lowered xylitol yield occurred in strain I-PGK/AUR, which has high activity of both XR and XDH and moderate XK activity.
  • 藤本真司; 松鹿昭則; 秀野晃大; 柳田高志; 佐賀清崇; 井上宏之; 美濃輪智朗
    日本エネルギー学会大会講演要旨集 17th 2008年08月
  • Akinori Matsushika; Seiya Watanabe; Tsutomu Kodaki; Keisuke Makino; Shigeki Sawayama
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 105 3 296 - 299 2008年03月 [査読有り]
     
    We constructed a set of recombinant Saccharomyces cerevisiae strains with xylose-fermenting ability. A recombinant S. cerevisiae strain D-XR/ARSdR/XK, in which protein engineered NADP(+)-dependent XDH was expressed, showed 40% increased ethanol production and 23% decrease in xylitol excretion as compared with the reference strain D-XR/XDH/XK expressing the wild-type XDH.
  • Nobuhiro Azuma; Kyoko Kanamaru; Akinori Matsushika; Takafumi Yamashino; Takeshi Mizuno; Masashi Kato; Tetsuo Kobayashi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 10 2493 - 2502 2007年10月 [査読有り]
     
    His-Asp phosphorelays are widespread signal transduction mechanisms in bacteria, fungi, and higher plants. In order to investigate a His-Asp phosphorelay network in filamentous fungi, which has been genetically characterized in part, we attempted to construct an in vitro phosphotransfer network in Aspergillus nidulans comprising all the necessary components. As a first step, we established an in vitro phosphotransfer system with a histidine-containing phosphotransmitter YpdA, a response regulator SrrA, and a bacterial histidine kinase ArcB as a phosphate donor. We demonstrated the phosphotransfer from ArcB to A. nidulans YpdA and the subsequent transfer from YpdA to SrrA. This is the first direct biochemical evidence for the presence of the phosphotransfer system in filamentous fungi. Furthermore, a retrograde phosphorylation from YpdA to FphA, a histidine kinase similar to bacterial phytochrome, was found. The overall picture of the His-Asp phosphorelays in A. nidulans is discussed based on the results of the in vitro study.
  • Akinori Matsushika; Masakazu Kawamura; Yuko Nakamura; Takahiko Kato; Masaya Murakami; Takafumi Yamashino; Takeshi Mizuno
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 2 535 - 544 2007年02月 [査読有り]
     
    Together with PRR1/TOC1, PRR5 belongs to the small family of PSEUDO-RESPONSE REGULATORs (PRRs), which function as clock components of Arabidopsis thaliana. We employed a set of transgenic lines, each of which was designed to misexpress a truncated form of the PRR5 molecule, together with the original transgenic line (named PRR5-ox) that misexpresses the entire PRR5 polypeptide. The results of genetic analysis suggested that PRR5-ox seedlings showed a phenotype of hypersensitivity to red light during early photomorphogenesis in a manner dependent on red light photoreceptors (PhyA and PhyB), but independent of PRR1/TOC1. The set of newly constructed transgenic lines (named PRR5-N-ox and PRR5-C-ox) were also characterized in terms of cireadian-associated phenotypes. The results suggest that the N-terminal pseudoreceiver domain of the PRR5 molecule seems to be dispensable for the misexpressed PRR5 molecule to bring about the phenotype of red light sensitivity. However, PRR5-N-ox plants, misexpressing only the pseudo-receiver domain, showed a phenotype of long period of free-running circadian rhythms of certain clock-controlled genes. Considering these and other results, we discuss the structure and function of PRR5 in the context of current views of the circadian clock in higher plants.
  • Akinori Matsushika; Masaya Murakami; Shogo Ito; Norihito Nakamichi; Takafumi Yamashino; Takeshi Mizuno
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 2 527 - 534 2007年02月 [査読有り]
     
    Every member of a small family of PSEUDO-RESPONSE REGULATOR (PRR) genes, including TIMING OF CAB EXPRESSION 1 (TOC1 [or PRR1]), are believed to play roles close to the circadian clock in the model higher plant Arabidopsis thaliana. In this study we established a transgenic line that misexpresses (or overexpresses) the PRR7 gene. As compared with wildtype plants, the resulting PRR7-misexpressing plants (designated PRR7-ox) showed characteristic phenotypes as to hallmarked circadian-associated biological events: (i) early flowering in a manner independent of photoperiodicity, (ii) hypersensitive response to red light during early photomorphogenesis, and (iii) altered free-running rhythms with long period of clock-associated genes. Finally, a series of all transgenic lines (PRR1-ox, PRR3-ox, PRR5-ox, PRR7-ox, and PRR9-ox) were characterized comparatively with regard to their clock-associated roles. The results suggested that the five homologous PRR factors play coordinate roles, distinctively from one another, and closely to the circadian clock in higher plants.
  • Shogo Ito; Norihito Nakamichi; Takatoshi Kiba; Akinori Matsushika; Toru Fujimori; Takafami Yamashino; Takeshi Mizuno
    PLANT AND CELL PHYSIOLOGY 48 S151 - S151 2007年 [査読有り]
  • Masakazu Kawamura; Akinori Matsushika; Takafumi Yamashino; Takeshi Mizuno
    PLANT AND CELL PHYSIOLOGY 48 S188 - S188 2007年 [査読有り]
  • A Matsushika; M Kawamura; T Yamashino; T Mizuno
    PLANT AND CELL PHYSIOLOGY 47 S66 - S66 2006年 [査読有り]
  • S Ito; N Nakamichi; A Matsushika; T Fujimori; T Yamashino; T Mizuno
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 2 382 - 390 2005年02月 [査読有り]
     
    In the model higher plant Arabidopsis thaliana, a number of circadian clock-associated protein components have recently been identified. Among them, a small family of ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9) is interesting because the most probable clock component TIMING OF CAB EXPRESSION I (TOC1) belongs to this family. Several lines of evidence have already been accumulated to support the view that not only APRR1/TOC1 but also other APRR family members are crucial for a better understanding of the molecular link between circadian rhythm and light-signal transduction. Among the APRR1/TOC1 family members, the circadian-controlled APRR9 gene is unique in that its expression is rapidly induced by light at the level of transcription. In this study we dissected the regulatory cis-elements of the light-induced and/or circadian-controlled APRR9 promoter by employing not only a mutant plant carrying a T-DNA insertion in the APRR9 promoter, but also a series of APRR9-promoter::LUC (luciferase) reporters that were introduced into an Arabidopsis cultured cell line (T87 cells). Taking the results of these approaches together, we provide several lines of evidence that the APRR9 promoter contains at least two distinctive and separable regulatory cis-elements: an "L element" responsible for the light-induced expression, followed by an "R element" necessary for the fundamental rhythmic expression of APRR9. Furthermore, APRR1/ TOC1 was implicated in the L-element-mediated light response of APRR9, directly or indirectly.
  • M Murakami; A Matsushika; M Ashikari; T Yamashino; T Mizuno
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 2 410 - 414 2005年02月 [査読有り]
     
    A small family of plant proteins, designated PSEUDO RESPONSE REGULATORS (PRRs), is crucial for a better understanding of the molecular link between circadian rhythm and photoperiodic control of flowering time in the dicotyledonous model plant Arabidopsis thaliana. Recently, we showed that the monocotyledonous model plant Oryza sativa also has homologous members of the OsPRR family (Oryza sativa PRR). In the previous experiments with rice, we mainly characterized a japonica variety (Nipponbare). By employing an indica variety (Kasalath), in this study we further characterized OsPRRs with reference to the photoperiod sensitivity Hd (Heading date) QTL (quantitative trait loci) implicated in the control of flowering time in rice. The circadian-controlled and sequential expression profiles of the five OsPRR genes were observed not only for Nipponbare but also for Kasalath. Then each of these OsPRR genes was mapped on the rice chromosomes. Among these OsPRR genes, OsPRR37 was mapped very closely to Hd2-QTL, which was identified as the major locus that enhances the photoperiod sensitivity of flowering in Nipponbare. Furthermore, we found that Kasalath has a severe mutational lesion in the OsPRR37 coding sequence.
  • S Ito; N Norihito; A Matsushika; T Fujimori; T Yamashino; T Mizuno
    PLANT AND CELL PHYSIOLOGY 69 2 382 - 390 2005年02月 [査読有り]
  • A Matsushika; T Yamashino; T Mizuno
    PLANT AND CELL PHYSIOLOGY 46 S98 - S98 2005年 [査読有り]
  • S Ito; A Matsushika; H Yamada; S Sato; T Kato; S Tabata; T Yamashino; T Mizuno
    PLANT AND CELL PHYSIOLOGY 44 11 1237 - 1245 2003年11月 [査読有り]
     
    In Arabidopsis thaliana, a number of cireadian-associated factors have been identified, including TOC1 (TIMING OF CAB EXPRESSION1) that is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). As demonstrated previously, these APRR1/TOC1 quintet members are crucial for a better understanding of the molecular links between circadian rhythms and photosensory signal transduction. Here we focused on the light-induced quintet member, APRR9, and three critical issues with regard to this member were simultaneously addressed: (i) clarification of the mechanism underlying the light-dependent acute response of APRR9, (ii) clarification of the phenotype of a null mutant of APRR9, (iii) identification of protein(s) that interacts with APRR9. In this study, we present the results that support the following views. (i) A phytochrome-mediated signaling pathway(s) activates the transcription of APRR9, leading to the acute light response of APRR9. (ii) The severe mutational lesion of APRR9 singly, if not directly, affects the period (and/or phase) of free-running rhythms, in continuous light, of every circadian-controlled gene tested, including the clock genes, APRR1/TOC1, CCA1, and LHY. (iii) The APRR9 protein is capable of interacting with APRR1/TOC1, suggesting a hetrodimer formation between these cognate family members. These results are discussed within the context of a current consistent model of the Arabidopsis circadian oscillator.
  • T Yamashino; A Matsushika; T Fujimori; S Sato; T Kato; S Tabata; T Mizuno
    PLANT AND CELL PHYSIOLOGY 44 6 619 - 629 2003年06月 [査読有り]
     
    APRR1 (ARABIDPSIS PSUEDO-RESPONSE REGULATOR 1) (or TOC1, TIMING OF CAB EXPRESSION 1) is believed to be a crucial component of biological clocks of Arabidopsis thaliana. Nevertheless, its molecular function remains to be fully elucidated. Based on the results of yeast two-hybrid and in vitro binding assays, we previously showed that APRR1/TOC1 interacts with certain bHLH factors (i.e. P1F3 and PIL1, which are PHYTOCHROME INTERACTING FACTOR 3 and its homolog (PIF3-LIKE 1), respectively). To critically examine the relevance of PILI with reference to the function of APRR1/TOC1, T-DNA insertion mutants were isolated for PILL No phenotype was observed for such homozygous pill mutants, in terms of circadian-associated events in plants. We then examined more extensively a certain set of bHLH factors, which are considerably similar to PILI in their structural designs. The results of extensive analyses of such bHLH factors (namely, HFR1, PIL2, PIF4, PIL5 and PIL6) in wild-type and APRR1-overexressing (APRR1-ox) transgenic lines provided us with several new insights into a link between APRR1/TOC1 and these bHLH factors. In yeast two-hybrid assays, APRR1/TOC1 showed the ability to interact with these proteins (except for HFR1), as well as PIL1 and PIF3. Among them, it was found that the expressions of PIF4 and PIL6 were regulated in a circadian-dependent manner, exhibiting free-running robust rhythms. The expressions of PIF4 and PIL6 were regulated also by light in a manner that their transcripts were rapidly accumulated upon exposure of etiolated seedlings to light. The light-induced expressions of PIF4 and PIL6 were severely impaired in APRR1-ox transgenic lines. Taken together, here we propose the novel view that these bHLH factors (PIF4 and PIL6) might play roles, in concert with APRR1/TOC1, in the integration of light-signals to control both circadian and photomorphogenic processes.
  • N Nakamichi; A Matsushika; T Yamashino; T Mizuno
    PLANT AND CELL PHYSIOLOGY 44 3 360 - 365 2003年03月 [査読有り]
     
    A small family of genes, named ARABIDOPSIS PSEUDO RESPONSE REGULATOR (APRR), are intriguing with special reference to circadian rhythms in plants, based on the fact that one of the members (APRR1) is identical to TOC1 (TIMING OF CAB EXPRESSION 1) that is believed to encode a clock component. In Arabidopsis plants, each transcript of the APRR1/TOC1 quintet genes starts accumulating after dawn rhythmically and one after another at intervals in the order of APRR9 --> APRR7 --> APRR5 --> APRR3 --> APRR1/TOC1. To characterize such intriguing circadian-associated events, we employed an established Arabidopsis cell line (named T87). When T87 cells were grown in an appropriate light and dark cycle, cell autonomous diurnal oscillations of the APRR1/TOC1 quintet genes were observed at the level of transcription, as seen in intact plants. After transfer to the conditions without any environmental time cues, particularly in constant dark, we further showed that free-running circadian rhythms persisted in the cultured cells, not only for the APRR1/TOC1 quintet genes, but also other typical circadian-controlled genes including CCA1 (CIRCADIAN CLOCK ASSOCIATED 1), LHY (LATE ELONGATED HYPOCOTYL) and CCR2 (COLD CIRCADIAN RHYTHM RNA BINDING 2). To our knowledge, this is the first indication of cell autonomous circadian rhythms in cultured cells in Arabidopsis thaliana, which will provide us with an alternative and advantageous means to characterize the plant biological clock.
  • A Matsushika; S Ito; T Yamashino; T Kato; S Sato; S Tabata; T Mizuno
    PLANT AND CELL PHYSIOLOGY 44 S34 - S34 2003年 [査読有り]
  • A Matsushika; A Imamura; T Yamashino; T Mizuno
    PLANT AND CELL PHYSIOLOGY 43 8 833 - 843 2002年08月 [査読有り]
     
    Several Arabidopsis genes have been proposed to encode potential clock-associated components, including the Myb-related CCA1 and LHY transcription factors and a member (APRR1/TOC1) of the family of pseudo-response regulators. We previously showed that transcripts of the APRR1/TOC1 family genes each start accumulating after dawn rhythmically and sequentially at intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1/TOC1, under the conditions of continuous light. Nevertheless, no evidence has been provided that each member of the APRR1/TOC1 quintet, except for APRR1/TOC1, is indeed relevant to the mechanisms underlying circadian rhythms. Here we attempt to provide such evidence by characterizing transgenic plants that aberrantly (or constitutively) express the APRR9 gene in a manner independent of circadian rhythms. The resulting APRR9-ox plants showed intriguing phenotypes with regard to circadian rhythms, in two aspects. First, the aberrant expression of APRR9 resulted in a characteristic phenotype with regard to transcriptional events, in which short-period rhythms were commonly observed for certain circadian-regulated genes, including CCA1, LHY, APRR1/TOC1, other APRR1/TOC1 members, ELF3, and CAB2. With regard to biological consequences, such APRR9-ox plants flowered much earlier than wildtype plants, in a manner independent of photoperiodicity (or under short-day conditions). These results suggest that APRR9 (and perhaps other members of the APRR1/TOC1 quintet) must also be taken into consideration for a better understanding of the molecular mechanisms underlying circadian rhythms, and also underlying control of the flowering time through the photoperiodic long-day pathway.
  • A Matsushika; S Makino; M Kojima; T Yamashino; T Mizuno
    PLANT AND CELL PHYSIOLOGY 43 1 118 - 122 2002年01月 [査読有り]
     
    We previously identified a novel class of proteins, named pseudo-response regulators (APRRs) in Arabidopsis thaliana, each of which (APRR1, APRR3, APRR5, APRR7, and APRR9) has an intriguing structural design containing an N-terminal pseudo receiver domain and a C-terminal CONSTANS motif. Among them, APRR1 is identical to TOC1, previously proposed to be a candidate component of an Arabidopsis circadian clock. Intriguingly, expressions of the APRR1/TOC1 family of genes are under control of coordinate circadian rhythms at the level of transcription, in the manner that each APRR-transcript starts accumulating sequentially after dawn with 2 to 3 It intervals in the order: APRR9-->4PRR7-->4PRR5-->APRR3-->APRR1/TOC1. Here we examined this circadian-related event, "circadian waves of the APRR1/TOC1 quintet", by employing CCA1-overexpression (CCA1-ox) transgenic plants, based on the fact that CCA1 is a well-characterized and the most plausible oscillator component. It was found that aberrant overexpression of the CCA1 gene severely perturbed free-running and sequential rhythms of the APRR1/TOC1 family of genes. In the accompanying paper, it was shown that overexpression of APRR1 also results in a marked alteration of the CCA1 circadian rhythm, and vice versa. Taken together, it was suggested that there are intimate and mutual links between these two types of circadian-associated components (APRRs and CCA1).
  • A Matsushika; T Yamashino; T Mizuno
    PLANT AND CELL PHYSIOLOGY 43 S76 - S76 2002年 [査読有り]
  • S Makino; A Matsushika; M Kojima; T Yamashino; T Mizuno
    PLANT AND CELL PHYSIOLOGY 43 1 58 - 69 2002年01月 [査読有り]
     
    Several Arabidopsis genes have been proposed to encode potential clock-associated components, including the Myb-related CCA1 and LHY transcription factors and a member of the novel family of pseudo response regulators (APRR1/TOC1). We previously showed that mRNAs of the APRR1/TOC1 family of genes start accumulating after dawn rhythmically and sequentially at approximately 2 h intervals in the order: APRR9-->APRR7-->APRR5-->APRR3 -->APRR1/TOC1. Here we constructed APRR1-overexpressing (APRR1-ox) plants, and examined certain circadian profiles for APRRs, CCA1, LHY, GI, CCR2, and CAB2. The free-running circadian rhythms of the APRR1/TOC1 family of genes, including APRR1, were dampened in APRR1-ox plants. In particular, the light-inducible expression of APRR9 was severely repressed in APRR1-ox plants, suggesting that there is a negative APRR1-->APRR9 regulation. The free-running robust rhythm of CAB2 was also dampened in APRR1-ox. The circadian profiles of potential clock-associated genes, CCA1, LHY, GI, and CCR2 were all markedly altered in APRR1-ox, each in characteristic fashion. To gain further insight into the molecular function of APRR1, we then identified a novel Myc-related bHLH transcription factor, which physically associated with APRR1. This protein (named PIL1) is similar in its amino acid sequence to PIF3, which has been identified as a phytochrome-interacting transcription factor. These results are discussed in relation to the current idea that APRR1 (TOC1) plays a role within, or close to, the Arabidopsis central oscillator.
  • T Mizuno; A Matsushika; S Makino; M Kojima; T Kiba; A Imamura; N Hanaki; A Nakamura; T Suzuki; M Taniguchi; C Ueguchi; T Sugiyama; T Mizuno
    PLANT AND CELL PHYSIOLOGY 43 S22 - S22 2002年01月 [査読有り]
  • S Makino; A Matsushika; M Kojima; Y Oda; T Mizuno
    PLANT AND CELL PHYSIOLOGY 42 3 334 - 339 2001年03月 [査読有り]
     
    We previously identified a novel class of proteins, named Arabidopsis pseudo-response regulators (APRRs), each of which (APRR1/TOC1, APRR3, APRR5, APRR7, APRR9) has an intriguing structural design containing an N-terminal pseudo-receiver domain and a C-terminal CONSTANS motif, Expression of these APRR1/TOC1 family members is under the control of a coordinate circadian rhythm at the level of transcription such that the APRR-mRNAs start accumulating sequentially after dawn with 2 to 3h intervals in the order of APRR9 --> APRR7 --> APRR5 --> APRR3 --> APRR1/TOC1 in a given 24 h photo-period. Based on these data, we previously proposed that these sequential and rhythmic events of transcription, termed 'circadian waves of APRR1/TOC1 quintet', may be a basis of a presumed Arabidopsis biological clock (named 'bar code clock') [Matsushika et al, (2000) Plant and Cell Physiol. 41: 1002], Here we further characterized the event of circadian waves, by demonstrating that certain light stimuli ape crucial determinants to induce the robust circadian waves, and accordingly, the first-boosted and light-induced APRR9 appears to be primarily responsible for this light response of the circadian waves. Also, as such a light stimulus, a red Light pulse that is presumably perceived by phytochromes appears to be sufficient to induce (or synchronize) the APRR1/TOC1 circadian waves.
  • A Matsushika; S Makino; M Kojima; T Mizuno
    PLANT AND CELL PHYSIOLOGY 41 9 1002 - 1012 2000年09月 [査読有り]
     
    The Arabidopsis pseudo-response regulator, APRR1, has a unique structural design containing a pseudo-receiver domain and a C-terminal CONSTANS motif. This protein was originally characterized as a presumed component of the His-to-Asp phosphorelay systems in Arabidopsis thaliana, Recently, it was reported that APRR1 is identical to the TOC1 gene product, a mutational lesion of which affects the periods of many circadian rhythms in Arabidopsis plants. TOC1 is believed to be a component of the presumed circadian clock (or central oscillator). Based on these facts, in this study four more genes, each encoding a member of the APRR1/TOC1 family of pseudo-response regulators were identified and characterized with special reference to circadian rhythms. It was found that all these members of the APRR1/TOC1 family (APRR1, APRR3, APRR5, APRR7, and APRR9) are subjected to a circadian rhythm at the level of transcription. Furthermore, in a given 24 h period, the APRR-mRNAs started accumulating sequentially after dawn with 2-3 h intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1. These sequential events of transcription, termed 'circadian waves of APRR1/TOC1', were not significantly affected by the photoperiod conditions, if any (e.g. both long and short days), and the expression of APRR9 was first boosted always after dawn. Among these APRRs, in fact, only the expression of APRR9 was rapidly and transiently induced also by white light, whereas such light responses of others were very dull, if any, These results collectively support the view that these members of the APRR1/TOC1 family are together all involved in an as yet unknown mechanism underlying the Arabidopsis circadian clock, Here we propose that the circadian waves of the APRR1/TOC1 family members are most likely a molecular basis of such a biological clock in higher plants.
  • A Matsushika; T Mizuno
    JOURNAL OF BIOCHEMISTRY 127 5 855 - 860 2000年05月 [査読有り]
     
    The ArcB sensor plays a crucial role in the histidine to aspartate (His-to-Asp) phosphorelay signal transduction, which is involved in the transcriptional regulatory network that allows Escherichia coli cells to sense various respiratory growth conditions. ArcB is one of the best-studied hybrid His-kinases involved in the multi-step His-to-Asp phosphorelay. However, a major question that remains to be elucidated is how does ArcB sense an anoxic signal? The N-terminal region of ArcB is considered to be! a signal-input domain, which probably plays a role in such signal-perception. In this study, this N-terminal region of ArcB was dissected into three putative sub-domains, a "transmembrane domain," a "leucine-zipper-like domain," and a "PAS-like domain." The importance of these structural domains was assessed in vivo and in vitro by systematically analyzing a number of arcB mutants, each of which encodes a mutant ArcB protein having an amino acid substitution or a deletion within one of these sub-domains. The results are discussed with special reference to the nature of the ArcB anaerobic sensor.
  • S Takeda; A Matsushika; T Mizuno
    JOURNAL OF BIOCHEMISTRY 126 2 354 - 360 1999年08月 [査読有り]
     
    The Escherichia coli sdhCDAB operon encodes succinate dehydrogenase, an enzyme complex involved in the tricarboxylic acid (TCA) cycle. Expression of this operon is under complex transcriptional regulation in response to growth conditions, such as anaerobiosis and carbon sources. Typically, the expression of sdhCDAB is known to be subjected to "an aerobic repression" and "a glucose repression." The molecular mechanism underlying the anaerobic repression has been well documented, involving both the ArcB-ArcA two-component system and the Fnr global anaerobic regulator. However, the mechanism underlying the glucose repression is not yet clear, because the involvment of the general catabolite regulators such as CRP and CRA has been dismissed. In this study, we conducted a series of genetic analyses to identify the regulator gene(s) involved in the glucose repression of sdh. The results demonstrate that the EIICBGlc protein (the ptsG gene product), a component of the major glucose transporter, acts as a crucial mediator in glucose repression. These results support the view that the EIICBGlc protein functions not only as a glucose transporter, but also as a glucose-sensing signal transducer that modulates the glucose repression of the sdhCDAB operon.
  • A Matsushika; T Mizuno
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 62 11 2236 - 2238 1998年11月 [査読有り]
     
    The Escherichia coli ArcB sensor is involved in anaerobic phosphotransfer signal transduction. ArcB is a hybrid sensor that contains three types of phosphotransfer signaling domains in its primary amino acid sequence, namely, transmitter (or His-kinase), receiver, and histidine-containing phosphotransfer (HPt) domains. However, examination of the function of the newly-discovered HPt domain (named ArcB(c)) is still at a very early stage. To gain a general insight into the structure and function of the widespread HPt domains, on the basis of ifs three-dimensional crystal structure, in this study we constructed a certain set of mutants each having a single amino acid substitution in the HPt domain of ArcB. These ArcB(c) mutants were characterized and evaluated, based on the in vivo ability to signal the OmpR receiver via trans-phosphorylation.
  • A Matsushika; T Mizuno
    JOURNAL OF BIOCHEMISTRY 124 2 440 - 445 1998年08月 [査読有り]
     
    The Escherichia coli ArcB sensor is involved in anaerobic signal transduction. ArcB is an unorthodox His-kinase, in that it contains three types of phosphotransfer signaling domains in its primary amino acid sequence, namely, transmitter (or His-kinase), receiver, and histidine-containing phosphotransfer (HPt) domains. In this study, we first conducted an in. vivo experiment to determine whether or not the phosphorylation of the HPt domain is crucial for ArcB/ArcA-mediated anaerobic signal transduction. The results are best interpreted as meaning that the HPt domain of ArcB is important for the anaerobic signal transduction, as far as the expression of the succinate dehydrogenase (sdh) operon is concerned. We then isolated a set of ArcB mutant each with a single amino acid substitution in the HPt domain, which has lost the ability to function as a phospho-transmitter. The results of such mutational analyses, together with the three-dimensional crystal structure of the HPt domain, provided an insight into the structure and function of the HPt domain of ArcB.
  • A Matsushika; T Mizuno
    JOURNAL OF BACTERIOLOGY 180 15 3973 - 3977 1998年08月 [査読有り]
     
    The two components ArcB and ArcA play a crucial role in the signal transduction implicated in the complex transcriptional regulatory network that allows Escherichia coli to sense various respiratory growth conditions. ArcB is a hybrid sensor kinase having multiple phosphorylation sites in its primary amino acid sequence, including a transmitter, a receiver, and a histidine-containing phosphotransfer (HPt) domain. ArcA is a DNA-binding transcriptional regulator with a receiver domain. Results of recent in vitro studies revealed multistep His-to-Asp phosphotransfer circuitry in the ArcB-ArcA signaling system. For this report we conducted a series of in vivo experiments using a set of crucial ArcB mutants to evaluate the regulation of the sdh operon, The results suggested that the phosphorylated His-717 site in the HPt domain of ArcB is essential for anaerobic repression of sdh, Nonetheless, the ArcB mutant lacking this crucial His-717 site does not necessarily exhibit a null phenotype,vith respect to ArcB-ArcA signaling, The HPt mutant appears to maintain an ability to signal ArcA, particularly under aerobic conditions, which results in a significant repression of sdh. Based on these and other in vivo results, we propose a model in which ArcB functions in its own right as a dual-signaling sensor that is capable of propagating two types of stimuli through two distinct phosphotransfer pathways.

書籍

  • 公益財団法人 飯島藤十郎記念食品科学振興財団 2022年度年報（第38巻）
    松鹿昭則 (担当:分担執筆範囲:食品変敗微生物のゲノムDNAライブラリーの構築と有用遺伝子のスクリーニング)公益財団法人 飯島藤十郎記念食品科学振興財団 2023年09月
  • 酵母菌・麹菌・乳酸菌の産業応用展開
    松鹿 昭則 (担当:分担執筆範囲:木質系バイオマスからの有用物質生産に向けた酵母の育種開発)シーエムシー出版 2018年01月
  • Microbial Stress Tolerance for Biofuels
    松鹿 昭則 (担当:共著範囲:Improving biomass sugar utilization by engineered Saccharomyces csrevisiae)Springer 2012年01月
  • バイオエタノール通信 第2号
    松鹿昭則 (担当:分担執筆範囲:新規キシロース発酵性凝集性酵母の育種)地域環境資源センター（JARUS） 2009年01月
  • 微生物を使った木質バイオマスからのエタノール生産
    村上克治; 松鹿昭則; 澤山茂樹 (担当:共著範囲:)燃料電池 2008年04月
  • 木質バイオマスからのエタノール生産技術について
    澤山茂樹; 矢野伸一; 松鹿昭則; 井上宏之 (担当:共著範囲:)はかる 2007年08月

講演・口頭発表等

  • 森口 大輔; 大西 寛登; 重本 歩睦; 松鹿 昭則
    日本農芸化学会2024年度大会 2024年03月 口頭発表（一般）
  • 森口 大輔; 松鹿 昭則
    近畿大学大学院第11回院生サミット in Hiroshima 2023年08月 口頭発表（一般）
  • 生物触媒を用いたバイオプロセスに係る基盤技術開発  [招待講演]
    松鹿昭則
    近畿大学工学部 研究公開フォーラム 2022 2022年10月 公開講演，セミナー，チュートリアル，講習，講義等
  • 好熱性ホモ酢酸菌Moorella thermoaceticaの代謝改変エタノール生産において可逆性ヒドロゲナーゼ活性が細胞内酸化還元バランスを補正する  [通常講演]
    加藤淳也; 小林駿介; 和田圭介; 竹村海生; 加藤節; 藤井達也; 岩崎祐樹; 青井議輝; 森田友岳; 松鹿昭則; 村上克治; 中島田豊
    第74回日本生物工学会大会 2022年10月 口頭発表（一般）
  • 嫌気呼吸を利用した好熱性ホモ酢酸菌Moorella thermoacetica代謝改変株によるH2/CO2からのアセトン生産性の増強
    竹村海生; 加藤淳也; 加藤節; 藤井達也; 和田圭介; 岩崎祐樹; 青井議輝; 松鹿昭則; 森田友岳; 村上克治; 中島田豊
    日本農芸化学会2022年度大会 2022年03月 口頭発表（一般）
  • 好熱性ホモ酢酸菌Moorella thermoacetica代謝改変によるアセトンの高温ガス発酵
    加藤淳也; 竹村海生; 加藤節; 藤井達也; 和田圭介; 岩崎祐樹; 青井議輝; 森田友岳; 松鹿昭則; 村上克治; 中島田豊
    第73回日本生物工学会大会 2021年10月 口頭発表（一般）
  • 木質系バイオマス由来発酵物質の効率的生産に向けた酵母の育種開発  [招待講演]
    松鹿 昭則
    第96回広島大学バイオマスイブニングセミナー 2021年10月 公開講演，セミナー，チュートリアル，講習，講義等
  • Moorella thermoaceticaの H2/CO2培養において嫌気呼吸が与える増殖と物質生産への効果
    小林駿介; 加藤淳也; 竹村海生; 加藤節; 青井議輝; 中島田豊; 和田圭介; 松鹿昭則; 村上克治
    日本生物工学会西日本支部大会2020（第5回講演会） 2020年11月 口頭発表（一般）
  • 好熱性ホモ酢酸菌 Moorella thermoacetica 遺伝子工学ツール拡張に向けた試み
    加藤淳也; 小林駿介; 加藤節; 藤井達也; 和田圭介; 岩崎祐樹; 青井議輝; 松鹿昭則; 村上克治; 中島田豊
    日本生物工学会西日本支部大会2020（第5回講演会） 2020年11月 口頭発表（一般）
  • 好熱性ホモ酢酸菌Moorella thermoacetica のエタノール生産株におけるH2による増殖阻害の機構解明
    小林駿介; 加藤淳也; 和田圭介; 竹村海生; 加藤節; 青井議輝; 松鹿昭則; 村上克治; 中島田豊
    化学工学会第51回秋季大会 2020年09月 口頭発表（一般）
  • 遺伝子組換えMoorella thermoacetica による合成ガスからのエタノール生産高速化
    竹村海生; 加藤淳也; 加藤節; 藤井達也; 和田圭介; 青井議輝; 松鹿昭則; 村上 克治; 中島田豊
    日本農芸化学会2020年度中四国支部大会（第57回講演会） 2020年09月 口頭発表（一般）
  • バイオ変換グループの保有技術：微生物および酵素の改良技術
    松鹿昭則
    令和元年度 産総研 材料・化学シンポジウム「21世紀の化学反応とプロセス」 2020年02月 ポスター発表
  • Talaromyces cellulolyticus由来グルクロノキシラナーゼの機能解析
    中道優介; Fouquet Nicolas Jean Thierry; 伊藤祥太郎; 渡邊真宏; 松鹿昭則; 井上宏之
    第71回日本生物工学会大会 2019年09月 口頭発表（一般）
  • 好熱性ホモ酢酸菌Moorella thermoacetica代謝改変株を用いたエタノール生産増強に向けた培養工学的検討
    加藤淳也; 竹村海正; 岩崎祐樹; 加藤節; 青井議輝; 和田圭介; 村上克治; 松鹿昭則; 中島田豊
    第71回日本生物工学会大会 2019年09月 口頭発表（一般）
  • 糖質加水分解酵素ファミリー30-7に属するキシラナーゼの機能解析
    井上宏之; 中道優介; Fouquet Nicolas Jean Thierry; 伊藤祥太郎; 渡邊真宏; 松鹿昭則
    日本応用糖質科学会2019年度大会 2019年09月 口頭発表（一般）
  • Burkholderia multivorans CCA53株の色素分解への応用
    秋田紘長; 和田圭介; 藤井達也; 松鹿昭則
    環境バイオテクノロジー学会2019年度大会 2019年06月 ポスター発表
  • バイオテクノロジー（遺伝子解析、インフォマティクス、タンパク質構造解析）
    松鹿昭則
    技術交流会 2019年03月
  • 産総研中国センターにおける酵素開発の取り組み
    渡邊真宏; 井上宏之; 中道優介; 松鹿昭則
    産総研中国センター国際シンポジウム 2019年02月 口頭発表（一般）
  • 糸状菌由来の糖化関連酵素ライブラリーの構築と利用による植物バイオマスの限定分解技術の開発
    井上宏之; 中道優介; Fouquet Nicolas Jean Thierry; 藤井達也; 松鹿昭則
    第14回バイオマス科学会議 2019年01月 ポスター発表
  • Kluyveromyces marxianus DMB1株由来のNADPH依存性アルデヒド還元酵素の機能解析
    秋田紘長; 星野保; 松鹿昭則
    酵素工学研究会第80回講演会 2018年11月 ポスター発表
  • Talaromyces cellulolyticus由来新規GH30エンドキシラナーゼの同定
    中道優介; 藤井達也; 松鹿昭則; 井上宏之
    第70回日本生物工学会大会 2018年09月 口頭発表（一般）
  • ファミリーGH30に属するエンド型キシラナーゼの同定
    中道優介; Fouquet Nicolas Jean Thierry; 藤井達也; 渡邊真宏; 松鹿昭則; 井上宏之
    セルラーゼ研究会 第32回大会 2018年07月 ポスター発表
  • Burkholderia multivorans CCA53株の単離とリグニン分解能の解析
    秋田紘長; 木村善一郎; 星野保; 松鹿昭則
    環境バイオテクノロジー学会2018年度大会 2018年06月 ポスター発表
  • バイオエタノール  [招待講演]
    松鹿 昭則
    シンポジウム「講習会：基礎からわかるバイオ燃料」 2018年03月 公開講演，セミナー，チュートリアル，講習，講義等
  • バイオマスから機能化学品をつくるための微生物
    松鹿昭則
    平成27年度テクノブリッジフェアin北海道 2017年07月 ポスター発表
  • バイオマスからの有用物質発酵生産に向けた酵母の育種開発  [招待講演]
    松鹿 昭則
    第3回関西バイオ医療研究会 2017年06月 口頭発表（招待・特別）
  • Identification of a novel Issatchenkia orientalis GPI-anchored protein involved in tolerance to acid and salt stress
    松鹿昭則; 鈴木俊宏; 五島徹也; 星野保
    39th Symposium on Biotechnology for Fuels and Chemicals 2017年05月 ポスター発表
  • バイオマスからの有用物質生産に向けた微生物育種開発
    松鹿昭則
    平成28年度 （国研）産総研中国センター材料・化学領域機能化学研究部門／ 近畿大学次世代基盤技術研究所 先端化学生命工学研究センター[aCYBER] ジョイントミーティング 2017年03月 公開講演，セミナー，チュートリアル，講習，講義等
  • 酵母由来GPIアンカー型グルカノシルトランスフェラーゼによる耐酸性・耐塩性の強化
    松鹿昭則; 鈴木俊宏; 五島徹也; 星野保
    日本農芸化学会2017年度大会 2017年03月 口頭発表（一般）
  • バイオ燃料製造の有用要素技術開発事業/有用微生物を用いた発酵生産技術の研究開発/C5C6糖同時発酵微生物の開発
    松鹿昭則
    NEDO有用要素技術開発事業 第7回研究開発推進委員会 2017年01月 口頭発表（一般）
  • バイオマス利活用に向けたバイオ変換技術論  [招待講演]
    松鹿 昭則
    グリーンケミストリー特別講演会 2017年 公開講演，セミナー，チュートリアル，講習，講義等
  • バイオマスからの効率的発酵生産に向けた酵母の育種開発
    松鹿昭則
    技術交流会 2016年12月 公開講演，セミナー，チュートリアル，講習，講義等
  • 酵母の酸耐性・塩耐性に関与する新規GPIアンカー型タンパク質の機能解析
    松鹿昭則; 鈴木俊宏; 根冝香奈子; 五島徹也; 星野保
    第39回日本分子生物学会年会 2016年11月 ポスター発表
  • バイオ燃料製造の有用要素技術開発事業/有用微生物を用いた発酵生産技術の研究開発/C5C6糖同時発酵微生物の開発
    松鹿昭則
    NEDO有用要素技術開発事業 第6回研究開発推進委員会 2016年11月 口頭発表（一般）
  • 酵母における酸耐性・塩耐性付与遺伝子の解析と分子育種による耐性強化
    松鹿昭則; 鈴木俊宏; 根冝香奈子; 橋本智代; 五島徹也; 星野保
    第68回日本生物工学会大会 2016年09月 ポスター発表
  • Kluyveromyces marxianusにおけるキシリトール脱水素酵素の補酵素特異性改変による効果
    鈴木俊宏; 星野保; 松鹿昭則
    第68回日本生物工学会大会 2016年09月 ポスター発表
  • ペントースリン酸経路遺伝子発現の最適化によるC5C6糖同時発酵酵母のethanol生産性の向上
    小林洋介; 藤森一浩; 佐原健彦; 鈴木俊宏; 蒲池沙織; 松鹿昭則; 星野保; 扇谷悟; 鎌形洋一
    第68回日本生物工学会大会 2016年09月 ポスター発表
  • タンパク質工学によるTalaromyces cellulolyticus由来セロビオハイドロラーゼの耐熱化
    蒲池沙織; 井上宏之; 松鹿昭則; 星野保; 石川一彦
    第68回日本生物工学会大会 2016年09月 ポスター発表
  • Metabolite analysis using Kluyveromyces marxianus during xylose fermentation
    鈴木俊宏; 星野保; 松鹿昭則
    14th International Congress on Yeasts (ICY14) 2016年09月 ポスター発表
  • Ethanol production from xylose by metabolically engineered yeast strains
    松鹿昭則
    Seminar for Dr. Elvi Restiawaty, S.T., June 9, 2016 2016年06月 公開講演，セミナー，チュートリアル，講習，講義等
  • バイオ燃料製造の有用要素技術開発事業/有用微生物を用いた発酵生産技術の研究開発/C5C6糖同時発酵微生物の開発
    松鹿昭則
    NEDO有用要素技術開発事業 第5回研究開発推進委員会 2016年03月 口頭発表（一般）
  • 五炭糖発酵性改良を目指した 酵母の分子育種
    松鹿昭則
    産総研・理研交流会 2016年02月 公開講演，セミナー，チュートリアル，講習，講義等
  • HAP4遺伝子を破壊したキシロース資化性酵母による高効率エタノール生産
    松鹿昭則
    産総研中国センターシンポジウム －材料・化学研究が切り拓く産業競争力強化への道筋－ 2016年01月 ポスター発表
  • 酵母Issatchenkia orientalisにおける耐酸性・耐塩性遺伝子の単離と解析
    松鹿昭則; 根冝香奈子; 鈴木俊宏; 五島徹也; 星野保
    日本農芸化学会2016年度大会 2015年10月 ポスター発表
  • セルロース系バイオエタノール生産実用酵母Saccharomyces cerevisiae IR-2におけるペントースリン酸経路の最適化と耐熱化
    小林洋介; 藤森一浩; 佐原健彦; 鈴木俊宏; 蒲池沙織; 松鹿昭則; 星野保; 扇谷悟; 鎌形洋一
    日本農芸化学会2016年度大会 2015年10月 ポスター発表
  • キシロース資化性酵母を用いたエタノール生産に対するHAP4遺伝子破壊の効果
    松鹿昭則; 鈴木俊宏; 星野保
    第67回日本生物工学会大会 2015年10月 ポスター発表
  • Kluyveromyces marxianus由来キシロース代謝遺伝子の過剰発現による高温条件下でのキシロース発酵
    鈴木俊宏; 星野保; 松鹿昭則
    第67回日本生物工学会大会 2015年10月 ポスター発表
  • Ethanol production from xylose by metabolically engineered yeast strains
    松鹿昭則
    Seminar for Dr. Todd Pray, ABPDU, September 9, 2015 2015年09月 公開講演，セミナー，チュートリアル，講習，講義等
  • Kluyveromyces marxianusのキシロース代謝遺伝子の単離と評価
    鈴木俊宏; 星野保; 松鹿昭則
    酵母遺伝学フォーラム第48回研究報告会 2015年09月 ポスター発表
  • 木質系バイオマスからのエタノール生産のためのキシロース発酵性・耐熱性酵母の分子育種
    松鹿昭則
    機能化学研究部門 平成27年度研究交流会 2015年07月 ポスター発表
  • 農林水産省におけるバイオマス利用の研究開発について  [招待講演]
    松鹿 昭則
    平成26年度 事業化を加速する産学連携支援事業 「バイオエタノールとバイオエネルギーの将来を考える」 2014年10月 口頭発表（招待・特別）
  • 耐熱性酵母Kluyveromyces marxianus DMB1のキシロース代謝時のメタボローム解析
    鈴木俊宏; 星野保; 松鹿昭則
    第66回日本生物工学会大会 2014年09月 ポスター発表
  • Improvement of saccharification and fermentation by removal of endogenious chemicals from pretreatment lignocellulosic biomass
    星野保; 辻雅晴; 松鹿昭則
    36th Symposium on Biotechnology for Fuels and Chemicals 2014年04月 ポスター発表
  • セルロース系エタノール革新的生産システム開発事業/早生樹からのメカノケミカルパルピング前処理によるエタノール一貫生産システムの開発/酵素糖化・発酵技術の研究開発
    星野保; 松鹿昭則; 藤井達也; 小瀧努
    NEDOセルロース革新事業 平成25年度第2回研究開発推進委員会 2014年01月 口頭発表（一般）
  • 網羅的代謝変動解析によるキシロース資化性酵母の発酵特性の解明
    松鹿昭則; 星野保
    第19回E&Eフォーラム（環境・エネルギー分野研究交流会） 2013年12月
  • CE-TOFMS法を用いた炭素源変化によるキシロース発酵性実用酵母のメタボローム解析
    松鹿昭則
    第2回バイオマスリファイナリーシンポジウム（研究成果報告会） 2013年11月 ポスター発表
  • 五炭糖発酵性改良を目指した酵母の分子育種
    松鹿昭則
    第65回日本生物工学会大会 2013年09月 シンポジウム・ワークショップパネル（指名）
  • セルロース系エタノール革新的生産システム開発事業/早生樹からのメカノケミカルパルピング前処理によるエタノール一貫生産システムの開発/酵素糖化・発酵技術の研究開発
    星野保; 松鹿昭則; 藤井達也; 小瀧努
    NEDOセルロース革新事業 平成25年度第1回研究開発推進委員会 2013年08月 口頭発表（一般）
  • Metabolome analysis of recombinant industrial Saccharomyces cerevisiae with xylose-fermenting ability
    松鹿昭則; 五島徹也; 星野保
    2013 SIMB Annual Meeting 2013年08月 ポスター発表
  • Development of cellulase-producing fungi and yeast suitable for biomass conversion
    井上宏之; 松鹿昭則
    Japan - U.S. Collaboration on Clean Energy Technology Workshop 2012 2013年03月 口頭発表（一般）
  • 木質系バイオマスからの物質生産のためのキシロース発酵性・耐熱性酵母の分子育種
    五島徹也; 根冝香奈子; 松鹿昭則
    第1回バイオマスリファイナリーシンポジウム（研究成果報告会） 2013年02月 ポスター発表
  • 木質系バイオマス由来キシロース発酵性改良に向けた凝集性実用酵母の分子育種開発
    松鹿昭則; 五島徹也; 星野保
    第1回バイオマスリファイナリーシンポジウム（研究成果報告会） 2013年02月 ポスター発表
  • Ethanol production by recombinant flocculent Saccharomyces cerevisiae that can effectively co-ferment glucose and xylose
    松鹿昭則
    Seminar 2013年01月 公開講演，セミナー，チュートリアル，講習，講義等
  • セルロース系エタノール革新的生産システム開発事業/早生樹からのメカノケミカルパルピング前処理によるエタノール一貫生産システムの開発/酵素糖化・発酵技術の研究開発
    星野保; 松鹿昭則; 藤井達也; 小瀧努
    NEDOセルロース革新事業推 平成24年度第2回研究開発推進委員会 2012年12月 口頭発表（一般）
  • バイオエタノール生産用キシロース発酵性酵母の分子育種研究
    松鹿昭則
    技術交流会 2012年12月 公開講演，セミナー，チュートリアル，講習，講義等
  • 接合を利用したSaccharomyces cerevisiaeのキシロース代謝能向上
    橋本征太郎; 井上宏之; 松鹿昭則; 澤山茂樹
    第19回日本生物工学会九州支部大分大会 2012年12月 口頭発表（一般）
  • 分子育種による耐熱性酵母Kluyveromyces marxianusのキシロース発酵能の付与
    五島徹也; 松鹿昭則; 井上宏之; 矢野伸一; 星野保
    第64回日本生物工学会大会 2012年10月 口頭発表（一般）
  • Ethanol production by recombinant flocculent Saccharomyces cerevisiae that can effectively co-ferment glucose and xylose
    松鹿昭則; 澤山茂樹; 星野保
    2012 Pacific Rim Summit 2012年10月 口頭発表（一般）
  • セルロース系エタノール革新的生産システム開発事業/早生樹からのメカノケミカルパルピング前処理によるエタノール一貫生産システムの開発/酵素糖化・発酵技術の基盤研究
    星野保; 松鹿昭則; 藤井達也; 小瀧努
    NEDOセルロース革新事業 平成24年度第1回研究開発推進委員会 2012年07月 口頭発表（一般）
  • 補酵素特異性改変酵母によるキシロースからのエタノール生産
    松鹿昭則; 澤山茂樹; 小瀧努; 星野保
    環境化学技術研究部門 平成24年度研究交流会 2012年06月 ポスター発表
  • 耐熱性酵母Kluyveromyces marxianus DMB1のエタノール発酵性
    五島徹也; 井上宏之; 松鹿昭則; 矢野伸一
    日本農芸化学会2012年度大会 2012年03月 口頭発表（一般）
  • Engineering of Saccharomyces cerevisiae for efficient bioethanol production from xylose
    松鹿昭則
    NREL Seminar 2012年02月 口頭発表（一般）
  • バイオマス由来キシロース発酵性改良に向けた酵母の育種開発  [招待講演]
    松鹿 昭則
    広島大学 酵母細胞プロジェクト研究センター 春期シンポジウム 2011年03月 口頭発表（招待・特別）
  • Engineering of Saccharomyces cerevisiae for efficient bioethanol production from xylose
    松鹿昭則
    NREL and AIST meeting 2010年12月 口頭発表（一般）
  • バイオエタノール生産用キシロース発酵性酵母の分子育種
    松鹿昭則
    第5回広島大学・産総研バイオマスオープンセミナー 2010年11月 公開講演，セミナー，チュートリアル，講習，講義等
  • キシロース発酵性を付与した凝集性実用酵母のオミックス解析
    松鹿昭則; 村上克治; 永嶋淳; 澤山茂樹
    第62回日本生物工学会大会 2010年10月 ポスター発表
  • Engineering of Saccharomyces cerevisiae for efficient bioethanol production from xylose
    松鹿昭則
    NREL and AIST meeting 2010年10月 公開講演，セミナー，チュートリアル，講習，講義等
  • キシロース発酵性凝集性酵母のメタボローム解析
    松鹿昭則; 永嶋淳; 澤山茂樹
    酵母遺伝学フォーラム第43回研究報告会 2010年09月 ポスター発表
  • キシロオリゴ糖の資化性を付与した組み換え酵母によるエタノール生産
    藤井達也; 松鹿昭則; 栗田麻未; 矢野伸一; 村上克治; 澤山茂樹
    日本農芸化学会大会2010年度大会 2010年03月 ポスター発表
  • パルプ産業用機械技術と同時発酵によるバイオエタノール生産システム/ワンバッチ式バイオエタノール製造技術の研究開発 -糖化・発酵微生物-
    澤山茂樹; 矢野伸一; 井上宏之; 松鹿昭則
    NEDO先導技術開発推進委員会 2010年03月 口頭発表（一般）
  • Development of recombinant xylose-fermentable yeast suitable for biomass conversion -The progress of AIST-NREL collaboration-
    松鹿昭則
    NREL Seminar 2010年02月 口頭発表（一般）
  • 分子育種により開発したキシロース発酵性酵母によるエタノール生産
    松鹿昭則
    第6回E&Eフォーラム（環境・エネルギー分野研究交流会） 2010年02月 口頭発表（一般）
  • キシロース発酵性を付与した凝集性酵母の遺伝子発現解析
    松鹿昭則; 井上宏之; 澤山茂樹
    第32回日本分子生物学会年会 2009年12月 ポスター発表
  • 「メカノケミカルパルピング前処理によるエタノール生産技術開発」発酵技術の進捗について
    松鹿昭則
    バイオマスエネルギー先導技術開発 加速的先導技術に関するワークショップ 2009年12月 口頭発表（一般）
  • セルロース系エタノール革新的生産システム開発事業/バイオマスエタノール一貫生産システムに関する研究開発/早生樹からのメカノケミカルパルピング前処理によるエタノール一貫生産システムの開発
    坂西欣也; 澤山茂樹; 遠藤貴士; 矢野伸一; 松鹿昭則
    第1回NEDO革新事業（エタノール）推進委員会 2009年10月 口頭発表（一般）
  • Expression of the Trichoderma reesei β-xylosidase gene in Saccharomyces cerevisiae
    Guoce Yu; 矢野伸一; 井上宏之; 松鹿昭則; Xu Fang; 澤山茂樹
    International Conference on Biorefinery (IBC 09) 2009年10月 口頭発表（一般）
  • 改変型酵素を導入したキシロース発酵性実用酵母によるエタノール生産
    松鹿昭則; 井上宏之; 渡邉誠也; 小瀧努; 牧野圭祐; 澤山茂樹
    第61回日本生物工学会大会 2009年09月 口頭発表（一般）
  • Engineering of Saccharomyces cerevisiae for efficient bioethanol production from xylose
    松鹿昭則
    ブラジル・リオデジャネイロ連邦大学(UFRJ)・サンタカタリーナ連邦大学(UFSC)とのワークショップ 2009年08月 公開講演，セミナー，チュートリアル，講習，講義等
  • 出芽酵母におけるタンパク質の複数同時発現の応用
    菅野陽平; 合田孝子; 松鹿昭則; 澤山茂樹; 扇谷悟
    酵母遺伝学フォーラム第42回研究報告会 2009年07月 ポスター発表
  • Bioethanol production from nano-fibrillated lignocellulosic biomass
    井上宏之; 松鹿昭則; 澤山茂樹
    Society for Industrial Microbiology 2009 Annual Meeting and Exhibition 2009年07月 口頭発表（一般）
  • 新規キシロース発酵性酵母の分子育種
    松鹿昭則
    BTRC研究発表会 2009年06月 公開講演，セミナー，チュートリアル，講習，講義等
  • Metabolic engineering of flocculent Saccharomyces cerevisiae with genome-integrated NADP+-dependent xylitol dehydrogenase gene for ethanol production from xylose
    松鹿昭則; 井上宏之; 渡邉誠也; 小瀧努; 牧野圭祐; 澤山茂樹
    31th Symposium on Biotechnology for Fuels and Chemicals 2009年05月 ポスター発表
  • 新規キシロース発酵性賦与凝集性酵母によるエタノール生産
    松鹿昭則; 井上宏之; 村上克治; 澤山茂樹
    日本農芸化学会大会2009年度大会 2009年03月 ポスター発表
  • 新規キシロース発酵性酵母の分子育種 - バイオマス由来C5糖発酵性改良に向けた開発の現状と展望、戦略 -  [招待講演]
    松鹿昭則、井上宏之、澤山茂樹
    アルコール・バイオマス研究会講演会 2009年02月 口頭発表（招待・特別）
  • 新規キシロース発酵性凝集性酵母の育種  [招待講演]
    松鹿 昭則
    ソフトセルロース利活用技術確立事業 第2回モデル地区概要発表会 2009年01月 口頭発表（招待・特別）
  • バイオマスエネルギー高効率転換技術開発/バイオマスエネルギー先導技術研究開発/ワンバッチ式バイオエタノール製造技術の研究開発
    坂西欣也; 澤山茂樹; 遠藤貴士; 矢野伸一; 村上克治; 滝村修; 井上宏之; 寺本好邦; 松鹿昭則; 牧野圭祐; 小瀧努; Pack Seung Pil; 渡邊誠也
    平成19年度「バイオマスエネルギー高効率転換技術開発」成果報告会 2008年12月 ポスター発表
  • パルプ産業用機械技術と同時発酵によるバイオエタノール生産システム/ワンバッチ式バイオエタノール製造技術の研究開発
    澤山茂樹; 矢野伸一; 井上宏之; 松鹿昭則
    第4回NEDO先導研究（エタノール）推進委員会 2008年12月 口頭発表（一般）
  • キシロース発酵性を付与した凝集性酵母によるエタノール生産
    松鹿昭則; 井上宏之; 村上克治; 澤山茂樹
    第31回日本分子生物学会年会・第81回日本生化学会大会合同大会 2008年12月 ポスター発表
  • タンパク質工学を用いたキシロース発酵性サッカロミセス酵母の育種
    渡邉誠也; 小瀧努; 牧野圭祐; 松鹿昭則; 澤山茂樹
    第60回日本生物工学会大会 2008年08月 シンポジウム・ワークショップパネル（指名）
  • キシロースからのエタノール生産に適した実用酵母宿主株の発酵特性検討
    井上宏之; 松鹿昭則; 澤山茂樹
    第60回日本生物工学会大会 2008年08月 口頭発表（一般）
  • Engineering of Saccharomyces cerevisiae for efficient bioethanol production from xylose
    松鹿昭則
    中国・清華大学核エネルギー・新エネルギー研究所(INET)とのワークショップ 2008年08月 公開講演，セミナー，チュートリアル，講習，講義等
  • 非硫酸前処理によるバイオエタノール製造プロセスの高効率化と経済性の検討
    藤本真司; 松鹿昭則; 秀野晃大; 柳田高志; 佐賀清崇; 井上宏之; 美濃輪智朗
    第17回日本エネルギー学会大会 2008年08月 口頭発表（一般）
  • タンパク質工学的手法により改変したキシリトール脱水素酵素遺伝子導入酵母によるバイオエタノール生産
    松鹿昭則; 渡邉誠也; 小瀧努; 牧野圭祐; 澤山茂樹
    産総研・酒総研ジョイントシンポジウム - 醸造とバイオ燃料 2008年06月 ポスター発表
  • Bioethanol production from xylose using recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase
    松鹿昭則; 渡邉誠也; 小瀧努; 牧野圭祐; 澤山茂樹
    30th Symposium on Biotechnology for Fuels and Chemicals 2008年05月 ポスター発表
  • Engineering of Saccharomyces cerevisiae for efficient bioethanol production from xylose
    松鹿昭則
    中国・清華大学核エネルギー・新エネルギー研究所(INET)とのワークショップ 2008年05月 口頭発表（一般）
  • ポリメラーゼ校正機能欠損酵母株を用いたバイオエタノール生産技術
    小栗恵美子; 松鹿昭則; 村上克治; 澤山茂樹
    日本農芸化学会大会2008年度大会 2008年03月 口頭発表（一般）
  • 改変型キシロース代謝遺伝子導入酵母によるエタノール生産
    松鹿昭則; 渡邉誠也; 小瀧努; 牧野圭祐; 井上宏之; 村上克治; 澤山茂樹
    日本農芸化学会大会2008年度大会 2008年03月 口頭発表（一般）
  • ワンバッチ式バイオエタノール製造技術（発酵）の研究開発
    松鹿昭則
    第3回NEDO先導研究（エタノール）推進委員会 2008年01月 口頭発表（一般）
  • バイオマスエネルギー高効率転換技術開発/バイオマスエネルギー先導技術研究開発/ワンバッチ式バイオエタノール製造技術の研究開発
    坂西欣也; 澤山茂樹; 遠藤貴士; 矢野伸一; 村上克治; 滝村修; 井上宏之; 松鹿昭則; 寺本好邦; 牧野圭祐; 小瀧努; 渡邊誠也
    平成18年度「バイオマスエネルギー高効率転換技術開発」成果報告会 2007年10月 その他
  • 二酸化炭素とEDTA鉄による微好気性アンモニア酸化反応
    澤山茂樹; 松鹿昭則; 井上宏之
    第59回日本生物工学会大会 2007年09月 口頭発表（一般）
  • 改変型酵素を導入した酵母によるキシロースからのエタノール生産
    松鹿昭則; 渡邉誠也; 小瀧努; 牧野圭祐; 井上宏之; 村上克治; 澤山茂樹
    第59回日本生物工学会大会 2007年09月 口頭発表（一般）
  • ワンバッチ式バイオエタノール製造技術（発酵）の研究開発
    松鹿昭則
    第2回NEDO先導研究（エタノール）推進委員会 2007年08月 口頭発表（一般）
  • Effect of supplemental hemicellulase on enzymatic hydrolysis and ethanol fermentation of softwood pretreated by ball milling
    井上宏之; 矢野伸一; 滝村修; 村上克治; 松鹿昭則; 塚原建一郎; 澤山茂樹
    29th Symposium on Biotechnology for Fuels and Chemicals 2007年04月 ポスター発表
  • シロイヌナズナにおける光誘導性、時計関連遺伝子PRR9のプロ モーター解析とタンパク質解析： PRR9タンパク質の存在量も概日 変動し、暗条件で速やかに分解さ れる
    伊藤照悟; 中道範人; 木羽隆敏; 松鹿昭則; 藤森徹; 山篠貴史; 水野猛
    植物生理学会2007年度年会および第48回シンポジウム 2007年03月 口頭発表（一般）
  • シロイヌナズナの時計関連 PRR ファミリー因子の機能解析
    河村正和; 松鹿昭則; 山篠貴史; 水野猛
    植物生理学会2007年度年会および第48回シンポジウム 2007年03月 ポスター発表
  • 糸状菌Aspergillus nidulansにおけるHis-Aspリン酸リレーネットワークのin vitro解析
    東信宏; 金丸京子; 松鹿昭則; 山篠貴史; 水野猛; 加藤雅士; 小林哲夫
    日本農芸化学会中部支部第147回例会 2006年10月
  • EDTA鉄を利用した新しい嫌気性アンモニア酸化反応
    澤山茂樹; 松鹿昭則; 井上宏之
    第58回日本生物工学会大会 2006年09月 口頭発表（一般）
  • 糸状菌Aspergillus nidulansにおける His-Asp リン酸リレー情報伝達系のin vitro解析
    東信宏; 金丸京子; 松鹿昭則; 山篠貴史; 水野猛; 加藤雅士; 小林哲夫
    日本農芸化学会大会2006年度大会 2006年03月 口頭発表（一般）
  • シロイヌナズナの時計関連 PRR ファミリー因子の分子解剖による構造と機能解析
    松鹿昭則; 河村正和; 山篠貴史; 水野猛
    植物生理学会2006年度年会および第47回シンポジウム 2006年03月 口頭発表（一般）
  • シロイヌナズナの時計関連PRRファミリー因子の構造と機能
    松鹿昭則; 山篠貴史; 水野猛
    植物生理学会2005年度年会および第46回シンポジウム 2005年03月 口頭発表（一般）
  • 時計関連因子をコードするシロイヌナズナAPRR9遺伝子：光シグナル及び時計により制御されるプロモーター構造の解析
    伊藤照悟; 中道範人; 松鹿昭則; 藤森徹; 山篠貴史; 水野猛
    植物生理学会2005年度年会および第46回シンポジウム 2005年03月 ポスター発表
  • シロイヌナズナにおける時計関連APRR1/TOC1ファミリーの五重奏 : APRR9の機能解析
    伊藤照悟; 松鹿昭則; 山田寿美; 佐藤修正; 加藤友彦; 田畑哲之; 山篠貴史; 水野猛
    第26回日本分子生物学会年会 2003年12月 ポスター発表
  • The circadian-associated APRR1/TOC1 quintet of Arabidopsis thaliana, (II) : Characterization of the light-induced member, APRR9
    松鹿昭則; 伊藤照悟; 山篠貴史; 佐藤修生; 加藤友彦; 田畑哲之; 水野猛
    American Society of Plant Biologists Annual Meeting 2003 2003年07月
  • シロイヌナズナ時計関連因子 APRR9の機能解析
    松鹿昭則; 伊藤照悟; 山篠貴史; 加藤友彦; 佐藤修正; 田畑哲彦; 水野猛
    植物生理学会2003年度年会および第44回シンポジウム 2003年03月 口頭発表（一般）
  • シロイヌナズナにおける時計関連APRR1/TOC1ファミリーの五重奏 : APRR9の機能解析
    松鹿昭則; 山篠貴史; 水野猛
    第25回日本分子生物学会年会 2002年12月 ポスター発表
  • The APRR1/TOC1 quintet implicated in circadian rhythms of Arabidopsis thaliana : [I], Characterization in APRR1-ox and CCA1-ox plants
    松鹿昭則; 山篠貴史; 水野猛
    植物生理学会2002年度年会および第43回シンポジウム 2002年03月 口頭発表（一般）
  • シロイヌナズナにおける時計関連遺伝子群PRR1/TOC1ファミリー : PRR1過剰発現系を用いた解析
    松鹿昭則; 山篠貴史; 牧野聖也; 水野猛
    第24回日本分子生物学会年会 2001年12月 ポスター発表
  • シロイヌナズナにおけるサーカディアンリズム関連遺伝子群APRR1/TOC1ファミリーの解析 (II) : 何時どのようにしてサーカディアンウェイブは開始するのか？
    松鹿昭則; 牧野聖也; 小島正也; 水野猛
    植物生理学会2001年度年会および第41回シンポジウム 2001年03月 ポスター発表
  • シロイヌナズナにおけるサーカディアンリズム関連遺伝子群APRR1/TOC1ファミリーの解析 (I) : 擬似レスポンスギュレーターの発現とサーカディアンウェイブ
    牧野聖也; 松鹿昭則; 小島正也; 水野猛
    植物生理学会2001年度年会および第41回シンポジウム 2001年03月 ポスター発表
  • シロイヌナズナの擬似レギュレーター APRR1/TOC1 ファミリーの解析
    松鹿昭則; 牧野聖也; 小島正也; 水野猛
    第23回日本分子生物学会年会 2000年12月 ポスター発表
  • シロイヌナズナにおける概日性時計構成因子としての擬似レスポンスレギュレｰタｰ因子群の解析
    牧野聖也; 松鹿昭則; 小島正也; 木羽隆敏; 今村綾; 谷口光隆; 杉山達夫; 水野猛
    日本時間生物学会 2000年11月 口頭発表（一般）
  • 大腸菌の多機能センサーArcBの分子機能及び構造解析
    松鹿昭則; 水野猛
    第22回日本分子生物学会年会 1999年12月 ポスター発表
  • 大腸菌の多機能センサーArcBの分子機能及び構造解析
    松鹿昭則; 水野猛
    日本農芸化学会1999年度大会 1999年04月 口頭発表（一般）
  • 大腸菌の多機能センサーArcBの分子機能及び構造解析
    松鹿昭則; 武田真一郎; 水野猛
    第21回日本分子生物学会年会 1998年12月
  • 大腸菌ArcBセンサーの新規 His-Asp 転移 (HPt) ドメインの構造と機能解析
    松鹿昭則; 荻野智章; 山篠貴史; 水野猛
    日本農芸化学会1998年度大会 1998年04月 口頭発表（一般）
  • 好熱菌Bacillus flavocaldarius KP 1228 のgroESLのオペロンのクローニング
    松鹿昭則; 柏原真一; 鈴木讓
    日本農芸化学会1997年度大会 1997年04月 口頭発表（一般）
  • 高度好熱菌Bacillus flavocaldarius KP1228のgroESLオペロンの単離とその構造
    松鹿昭則; 柏原真一; 鈴木讓
    日本農芸化学会関西支部 第394回講演会 1996年05月 口頭発表（一般）
  • バイオ燃料製造の有用要素技術開発事業/有用微生物を用いた発酵生産技術の研究開発/C5C6糖同時発酵微生物の開発
    松鹿昭則
    NEDO有用要素技術開発事業 第4回研究開発推進委員会 口頭発表（一般）

MISC

産業財産権

受賞

  • 2016年09月 第68回日本生物工学会大会トピックス選出
     
    受賞者: 松鹿 昭則, 鈴木 俊宏, 根冝 香奈子, 橋本 智代, 五島 徹也, 星野 保
  • 2015年06月 平成27年度農林水産技術会議事務局長賞
     
    受賞者: 松鹿 昭則
  • 2007年03月 2007年（Vol.71）B.B.B.論文賞
  • 2002年03月 第9回日本植物生理学会論文賞

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2022年04月 -2025年03月 
    代表者 : 松鹿 昭則
  • 植物資源の利活用を目指した前処理およびバイオ変換技術ならびに食品中のD-アミノ酸含有量に関する研究
    国立研究開発法人 産業技術総合研究所：共同研究
    研究期間 : 2021年06月 -2025年03月 
    代表者 : 松鹿 昭則
  • 高感度分離用磁性ナノ粒子を用いた微生物、生体分子の濃縮と選択性評価
    JNC株式会社：共同研究
    研究期間 : 2023年04月 -2024年03月 
    代表者 : 白石浩平; 松鹿昭則; 櫻井伸樹
  • 高温キシロース発酵に最適な酵母の選抜および育種開発
    国立大学法人 山口大学 中高温微生物研究センター：2023年度 公募型共同研究
    研究期間 : 2023年04月 -2024年03月 
    代表者 : 松鹿 昭則 (近畿大学工学部)、山田 守 (中高温微生物研究センター)
  • 未利用柑橘資源を利用した機能性材料の開発と食品への応用
    （公財）サタケ技術振興財団：2022年度 大学研究助成金
    研究期間 : 2022年04月 -2023年03月 
    代表者 : 松鹿 昭則
  • 食品変敗微生物のゲノムDNAライブラリーの構築と有用遺伝子のスクリーニング
    （公財）飯島藤十郎記念食品科学振興財団：2022年度学術研究助成
    研究期間 : 2022年04月 -2023年03月 
    代表者 : 松鹿 昭則
  • 合成ガスからのバイオケミカル原料製造技術の開発
    新エネルギー・産業技術総合開発機構：NEDO先導研究プログラム
    研究期間 : 2020年06月 -2021年03月 
    代表者 : 中島田豊
  • 再生可能エネルギーを活用した有用物質高生産微生物デザイン
    国立研究開発法人科学技術振興機構：未来社会創造事業
    研究期間 : 2018年07月 -2021年03月 
    代表者 : 中島田豊
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 木村 善一郎; 村上 克治; 松鹿 昭則
     
    遂行予定の研究全体像は、電気を食べる微生物（電極酸化細菌）を用いたバイオリアクターで廃棄物処理・有価物生産同時プロセス（電気発酵）の構築を目指すものである。本装置は電気化学的培養装置であり、電極還元細菌と電極酸化細菌の二種類の微生物群が電極・導線を通じ電子をやり取りして物質生産を行う。本技術の特徴は最終電子受容を行う電極酸化細菌（純菌）を陽極に隔離し、エサを電流のみに制限することで廃水（廃棄物）を原料に高付加価値物質（化学品・医薬品など）を生産しうるという点であり、本研究の目的は電気をエサに様々な有価物を生産可能な微生物触媒である電極酸化細菌を育種するために、新興ゲノム編集技術であるCRISPR/Cas9を用いて当該細菌への遺伝子導入すること及び、その導入遺伝子資源を網羅的に獲得すること、さらには遺伝子導入による新規物質生産システムを構築することの3点である。 平成30年度は(1)電極酸化物質生産システムの構築、(2)固相電気培養システムによる新規遺伝子資源の獲得及び(3)CRISPR/Cas9による組換え系の構築に取り組んだ。(1)では既知電極酸化細菌である好熱菌Moorella sp. Y72株を用いて酢酸を生産する物質生産系の構築に成功した。(2)の取り組みでは平成30年度までに固体培地上で電極酸化細菌と考えられる細菌の優占的培養に成功した。今後分離株を用いたゲノムシーケンスに取り組んでいく。(3)ではY72株を用いて、当該株のゲノム内にCas9をはじめとするゲノム編集関連遺伝子の導入に取り組み、導入及び発現に成功した。今後Cas9によるゲノム切断の効率を高めるため、最適な発現・反応条件を検討していく。
  • 並行複発酵に最適なキシロース発酵性酵母創製に向けた基盤研究
    日本学術振興会：科学研究費助成事業：基盤研究 (C)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 松鹿 昭則
  • 有用微生物を用いた発酵生産技術の開発
    新エネルギー・産業技術総合開発機構：NEDO バイオ燃料製造の有用要素技術開発事業
    研究期間 : 2013年04月 -2017年03月 
    代表者 : 種田 大介
  • 早生樹からのメカノケミカルパルピング前処理によるエタノール一貫生産システムの開発
    新エネルギー・産業技術総合開発機構：NEDO セルロース系エタノール革新的生産システム開発事業
    研究期間 : 2009年04月 -2014年03月 
    代表者 : 杉浦 純
  • セルロース系バイオ燃料の生産に適合した酵素生産糸状菌およびバイオ燃料生産微生物の開発に関する研究
    経済産業省：日米エネルギー環境技術研究・標準化協力事業
    研究期間 : 2010年04月 -2013年03月 
    代表者 : 坂西 欣也
  • サトウキビ廃棄物からのエタノール生産研究
    科学技術振興機構 / 国際協力機構：地球規模課題対応国際科学協力事業
    研究期間 : 2009年04月 -2013年03月 
    代表者 : 坂西 欣也
  • 稲わら水熱・酵素糖化・エタノール発酵基盤技術の研究開発
    農林水産省：バイオ燃料変換技術研究開発（稲わら変換総合技術の開発）
    研究期間 : 2007年04月 -2012年03月 
    代表者 : 澤山 茂樹
  • メカノケミカルパルピングを利用したバイオエタノール生産技術研究開発
    新エネルギー・産業技術総合開発機構：NEDO加速的先導技術
    研究期間 : 2008年04月 -2010年03月 
    代表者 : 坂西 欣也
  • ワンバッチ式バイオエタノール製造技術の研究開発
    新エネルギー・産業技術総合開発機構：NEDOバイオマスエネルギー先導技術研究開発
    研究期間 : 2006年04月 -2009年03月 
    代表者 : 坂西 欣也
  • 大腸菌の多機能環境センサーArcBの分子機能及び構造解析
    日本学術振興会：科学研究費助成事業：特別研究員奨励費
    研究期間 : 2000年04月 -2003年03月 
    代表者 : 水野 猛

委員歴

  • 2023年11月 - 現在   Fermentation（MDPI）   Special Issue Editor (Guest Editor)
  • 2023年04月 - 現在   近畿大学工学部   動物実験小委員会 委員長
  • 2021年07月 - 現在   国立研究開発法人 産業技術総合研究所   微生物実験安全委員会委員
  • 2019年04月 - 現在   日本農芸化学会   中四国支部 参与
  • 2017年04月 - 現在   公益社団法人 日本生物工学会   西日本支部 代議員
  • 2019年07月 - 2022年03月   水産業成長産業化技術開発事業「水素細菌を原料とする純国産養魚飼料開発」   外部委員
  • 2013年06月 - 2014年03月   BMC Microbiology 編集委員

担当経験のある科目

  • 基礎ゼミ近畿大学工学部
  • 環境生命化学実験近畿大学工学部
  • 化学生命工学基礎実験近畿大学工学部
  • 卒業研究ゼミナール近畿大学工学部
  • 食品化学近畿大学 工学部
  • 食品プロセス工学特論近畿大学 大学院システム工学研究科
  • 物質化学実験近畿大学工学部
  • グリーンケミストリー近畿大学工学部
  • 卒業研究近畿大学工学部
  • 食品工学近畿大学 工学部
  • 化学概論 II近畿大学 工学部
  • フレッシュマンゼミナール近畿大学工学部
  • 化学生命工学特論近畿大学 大学院システム工学研究科
  • グリーンケミストリー近畿大学 工学部
  • フロンティア生命科学広島大学 大学院先端物質科学研究科
  • 発酵プロセス工学 Ⅱ広島大学 工学部
  • 微生物を利用したバイオ変換技術論名古屋大学 大学院生命農学研究科
  • 生物工学概論（広島大学 工学部）
  • 生命機能工学B（広島大学 大学院先端物質科学研究科）
  • 分子生命機能科学セミナー（広島大学 大学院先端物質科学研究科）
  • 基礎生命科学（広島大学 工学部）

その他のリンク

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