基本情報

所属

• 工学研究院　応用化学部門　生物工学分野

職名

• 教授

学位

• 博士(農学)（東京大学）

ホームページURL

https://www.eng.hokudai.ac.jp/labo/tre/ABCLab_jp/

科研費研究者番号

• 70264679

J-Global ID

• 200901050726079892

研究キーワード

• 生合成   天然物   Natural product biosynthesis   

研究分野

• ライフサイエンス / 応用微生物学
• ライフサイエンス / 生物有機化学

担当教育組織

•  学士課程　工学部
•  修士課程　総合化学院
•  博士課程　総合化学院

職歴

• 2010年04月 - 現在 北海道大学大学院工学研究院
• 1994年04月 - 2010年03月 富山県立大学生物工学研究センター
• 1994年04月 - 2010年03月 - Biotechnology Research Center, Toyama Prefectural University
• 1985年 - 1994年 協和発酵工業(株)東京研究所
• 1985年 - 1994年 Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd.

学歴

•         - 1985年   名古屋大学   農学研究科   農芸化学(発酵)
•         - 1985年   名古屋大学
•         - 1983年   名古屋大学   農学部   農芸化学(発酵)
•         - 1983年   名古屋大学

所属学協会

• 日本生物工学会   日本放線菌学会   日本農芸化学会   The Society for Biotechnology, Japan   The Japanese Biochemical Society   The Society for Actinomycetes Japan   and Agrochemistry   Biotechnology   Japan Society for Bioscience   

研究活動情報

論文

• Optimization of tyrosol-producing pathway with tyrosine decarboxylase and tyramine oxidase in high-tyrosine-producing Escherichia coli

  Ning Shen, Yasuharu Satoh, Daisuke Koma, Hiroyuki Ohashi, Yasushi Ogasawara, Tohru Dairi

  Journal of Bioscience and Bioengineering 137 2 115 - 123 2024年02月
• Identification of a new oligomycin derivative as a specific inhibitor of the alternative peptidoglycan biosynthetic pathway.

  Shuhei Umetsu, Takeshi Tsunoda, Haruka Kiyanagi, Yuki Inahashi, Kenichi Nonaka, Tohru Dairi, Yasushi Ogasawara

  The Journal of antibiotics 2024年01月10日 

  Peptidoglycan is an important macromolecule in bacterial cell walls to maintain cell integrity, and its biosynthetic pathway has been well studied. Recently, we demonstrated that some bacteria such as Xanthomonas oryzae, a pathogen causing bacterial blight of rice, used an alternative pathway for peptidoglycan biosynthesis. In this pathway, MurD2, a MurD homolog, catalyzed the attachment of L-Glu to UDP-MurNAc-L-Ala and MurL, which did not show homology to any known protein, catalyzed epimerization of the terminal L-Glu of the MurD2 product to generate UDP-MurNAc-L-Ala-D-Glu. Because the alternative pathway also operates in some other plant pathogens and opportunistic pathogens, specific inhibitors of the alternative pathway could function as pesticides and antibiotics for these pathogens. In this study, we searched for specific inhibitors of the alternative pathway from metabolites produced by actinomycetes and identified a new oligomycin-class polyketide, which was revealed to inhibit the MurD2 reaction, in culture broth of Micromonospora sp. K18-0097.
• Peptide epimerase-dehydratase complex responsible for biosynthesis of the linaridin class ribosomal peptides.

  Wanlu Xiao, Takeshi Tsunoda, Chitose Maruyama, Yoshimitsu Hamano, Yasushi Ogasawara, Tohru Dairi

  Bioscience, biotechnology, and biochemistry 87 11 1316 - 1322 2023年08月04日 

  Grisemycin, salinipeptin, and cypemycin belong to the linaridin class of ribosomally synthesized and posttranslationally modified peptides that contain multiple dehydrobutyrine and D-amino acid residues. The biosynthetic gene clusters of these linaridins lack obvious candidate genes for the dehydratase and epimerase required to introduce dehydrobutyrine and D-amino acid residues, respectively. However, we previously demonstrated that the grisemycin (grm) cluster contained cryptic dehydratase and epimerase genes by heterologous expression of this biosynthetic gene cluster in Streptomyces lividans and proposed that two genes (grmH and grmL) with unknown functions catalyze dehydration and epimerization reactions. In this study, we confirmed that both GrmH and GrmL, which were shown to constitute a protein complex by a co-purification experiment, were required to catalyze the dehydration, epimerization, and proteolytic cleavage of a precursor peptide GrmA by in vivo experiments. Furthermore, we demonstrated that GrmH/GrmL complex accepted salinipeptin and cypemycin precursor peptides, which possess three additional amino acids.
• N-Formimidoylation/-iminoacetylation modification in aminoglycosides requires FAD-dependent and ligand-protein NOS bridge dual chemistry.

  Yung-Lin Wang, Chin-Yuan Chang, Ning-Shian Hsu, I-Wen Lo, Kuan-Hung Lin, Chun-Liang Chen, Chi-Fon Chang, Zhe-Chong Wang, Yasushi Ogasawara, Tohru Dairi, Chitose Maruyama, Yoshimitsu Hamano, Tsung-Lin Li

  Nature communications 14 1 2528 - 2528 2023年05月03日 

  Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N-O or O-S accounting for N-formimidoylation or N-iminoacetylation. The resultant product is no longer sensitive to aminoglycoside-modifying enzymes, a strategy that antibiotic-producing species employ to counter drug resistance in competing species.
• Structure of lasso peptide epimerase MslH reveals metal-dependent acid/base catalytic mechanism

  Yu Nakashima, Atsushi Kawakami, Yasushi Ogasawara, Masatoshi Maeki, Manabu Tokeshi, Tohru Dairi, Hiroyuki Morita

  2023年01月12日
• Ergothioneine production by Corynebacterium glutamicum harboring heterologous biosynthesis pathways.

  Takashi Hirasawa, Yuki Shimoyamada, Yukio Tachikawa, Yasuharu Satoh, Yusuke Kawano, Tohru Dairi, Iwao Ohtsu

  Journal of bioscience and bioengineering 135 1 25 - 33 2023年01月 

  In this study, Corynebacterium glutamicum was engineered to produce ergothioneine, an amino acid derivative with high antioxidant activity. The ergothioneine biosynthesis genes, egtABCDE, from Mycolicibacterium smegmatis were introduced into wild-type and l-cysteine-producing strains of C. glutamicum to evaluate their ergothioneine production. In the l-cysteine-producing strain, ergothioneine production reached approximately 40 mg L-1 after 2 weeks, and the amount was higher than that in the wild-type strain. As C. glutamicum possesses an ortholog of M. smegmatis egtA, which encodes an enzyme responsible for γ-glutamyl-l-cysteine synthesis, the effect of introducing egtBCDE genes on ergothioneine production in the l-cysteine-producing strain was evaluated, revealing that a further increase to more than 70 mg L-1 was achieved. As EgtBs from Methylobacterium bacteria are reported to use l-cysteine as a sulfur donor in ergothioneine biosynthesis, egtB from Methylobacterium was expressed with M. smegmatis egtDE in the l-cysteine-producing strain. As a result, ergothioneine production was further improved to approximately 100 mg L-1. These results indicate that utilization of the l-cysteine-producing strain and introduction of heterologous biosynthesis pathways from M. smegmatis and Methylobacterium bacteria are effective for improved ergothioneine production by C. glutamicum.
• First direct evidence for direct cell-membrane penetrations of polycationic homopoly(amino acid)s produced by bacteria

  Yamato Takeuchi, Kazunori Ushimaru, Kohei Kaneda, Chitose Maruyama, Takashi Ito, Kazuya Yamanaka, Yasushi Ogasawara, Hajime Katano, Yasuo Kato, Tohru Dairi, Yoshimitsu Hamano

  Communications Biology 5 1 2022年10月26日 

  Abstract Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. Although the biological significance of polycationic homopoly(amino acid)s remains unclear, increasing attention has recently been focused on their potential use to achieve cellular internalization. Here, for the first time, we provide direct evidence that two representative bacterial polycationic isopeptides, ε-poly-l-α-lysine (ε-PαL) and ε-oligo-l-β-lysine (ε-OβL), were internalized into mammalian cells by direct cell-membrane penetration and then diffused throughout the cytosol. In this study, we used clickable ε-PαL and ε-OβL derivatives carrying a C-terminal azide group, which were enzymatically produced and then conjugated with a fluorescent dye to analyze subcellular localization. Interestingly, fluorescent proteins conjugated with the clickable ε-PαL or ε-OβL were also internalized into cells and diffused throughout the cytosol. Notably, a Cre recombinase conjugate with ε-PαL entered cells and mediated the Cre/loxP recombination, and ε-PαL was found to deliver a full-length IgG antibody to the cytosol and nucleus.
• Biosynthetic Gene Cluster of Linaridin Peptides Contains Epimerase Gene

  Wanlu Xiao, Yasuharu Satoh, Yasushi Ogasawara, Tohru Dairi

  ChemBioChem 23 12 2022年06月20日 [査読有り]
  
• Identification of Cyclopropane Formation in the Biosyntheses of Hormaomycins and Belactosins: Sequential Nitration and Cyclopropanation by Metalloenzymes

  Xiaojun Li, Ryo Shimaya, Tohru Dairi, Wei‐chen Chang, Yasushi Ogasawara

  Angewandte Chemie International Edition 61 7 2022年02月07日 [査読有り]
  
• Identification of pulvomycin as an inhibitor of the futalosine pathway

  Yasushi Ogasawara, Shuhei Umetsu, Yuki Inahashi, Kenichi Nonaka, Tohru Dairi

  The Journal of Antibiotics 2021年08月20日 [査読有り]
   

  Menaquinone is an essential cofactor in the electron-transfer pathway for bacteria. Menaquinone is biosynthesized from chorismate using either the well-known canonical pathway established by pioneering studies in model microorganisms or the futalosine pathway, which we discovered in Streptomyces. Because Helicobacter pylori, which causes stomach cancer, uses the futalosine pathway and most beneficial intestinal bacteria including lactobacilli use the canonical pathway, the futalosine pathway will be a great target to develop antibiotics specific for H. pylori. Here, we searched for such compounds from metabolites produced by actinomycetes and identified pulvomycin from culture broth of Streptomyces sp. K18-0194 as a specific inhibitor of the futalosine pathway.
• Discovery of an alternative pathway of peptidoglycan biosynthesis: A new target for pathway specific inhibitors

  Yasushi Ogasawara, Tohru Dairi

  Journal of Industrial Microbiology and Biotechnology 2021年06月11日 [査読有り]
   

  Peptidoglycan in bacterial cell walls is a biopolymer consisting of sugars and amino acids and plays important role in maintaining cell integrity from the environment. Its biosynthesis is a major target for antibiotics and the genes and enzymes involved in the biosynthetic pathway have been well studied. However, we recently identified an alternative pathway in the early stage of peptidoglycan biosynthesis in Xanthomonas oryzae, a plant pathogen causing bacterial blight disease of rice. The distribution of the alternative pathway is limited to relatively few bacterial genera that contain many pathogenic species, including Xylella and Stenotrophomonas, besides Xanthomonas. Thus, the alternative pathway is an attractive target for the development of narrow-spectrum antibiotics specific to pathogens. In this minireview, we summarize the discovery of the alternative pathway and identification of its specific inhibitors.
• Flavonoids from Woodfordia fruticosa as potential SmltD inhibitors in the alternative biosynthetic pathway of peptidoglycan

  Yuan-E Lee, Takeshi Kodama, Nwet Nwet Win, Dae-Won Ki, Nhat Nam Hoang, Chin Piow Wong, Khine Zar Wynn Lae, Hla Ngwe, Tohru Dairi, Hiroyuki Morita

  Bioorganic & Medicinal Chemistry Letters 36 127787 - 127787 2021年03月 [査読有り]
   

  SmltD is an ATP-dependent ligase that catalyzes the condensation of UDP-MurNAc-L-Ala and L-Glu to form UDP-MurNAc-L-Ala-L-Glu, in the newly discovered peptidoglycan biosynthesis pathway of a Gram-negative multiple-drug-resistant pathogen, Stenotrophomonas maltophilia. Phytochemical investigation of the 70% ethanol extract from Woodfordia fruticosa flowers collected in Myanmar led to the identification of anti-SmltD active flavonoids, kaempferol 3-O-(6''-galloyl)-β-D-glucopyranoside (1), astragalin (2), and juglalin (3). Among them, 1 showed the most potent SmltD inhibitory activity. An enzyme steady-state kinetic study revealed that 1 exerted competitive inhibition with respect to ATP. The results of this study provided an attractive foundation for the further development of novel inhibitors of SmltD.
• Identification of the peptide epimerase MslH responsible for d-amino acid introduction at the C-terminus of ribosomal peptides

  Zhi Feng, Yasushi Ogasawara, Tohru Dairi

  Chemical Science 12 7 2567 - 2574 2021年 [査読有り]
   

  The biosynthesis of d-tryptophan containing lasso peptide MS-271 involves the epimerization of a ribosomal peptide MslA catalyzed by a novel class of metal- and cofactor-independent peptide epimerase MslH.

• High Production of Ergothioneine in Escherichia coli using the Sulfoxide Synthase from Methylobacterium strains

  Tomoyuki Kamide, Shun Takusagawa, Naoyuki Tanaka, Yasushi Ogasawara, Yusuke Kawano, Iwao Ohtsu, Yasuharu Satoh, Tohru Dairi

  Journal of Agricultural and Food Chemistry 68 23 6390 - 6394 2020年06月10日 [査読有り]
   

  We previously constructed a heterologous production system for ergothioneine (ERG) in Escherichia coli using five ERG biosynthesis genes (egtABCDE) from Mycobacterium smegmatis. However, significant amounts of hercynine (HER), an intermediate of ERG, as ERG were accumulated, suggesting that the reaction of EgtB catalyzing the attachment of γ-glutamylcysteine (γGC) to HER to yield hercynyl-γ-glutamylcysteine sulfoxide was a bottleneck. In this study, we searched for other EgtBs and found many egtB orthologs in diverse microorganisms. Among these, Methylobacterium strains possessed EgtBs that catalyze the direct conversion of HER into hercynylcysteine sulfoxide with l-cysteine (l-Cys) as a sulfur donor, in a manner similar to those of acidobacterial CthEgtB and fungal Egt1. An in vitro study with recombinant EgtBs from Methylobacterium brachiatum and Methylobacterium pseudosasicola clearly showed that both enzymes accepted l-Cys but not γGC. We reconstituted the ERG production system in E. coli with egtB from M. pseudosasicola; ERG productivity reached 657 mg L-1.
• Recent advances in functional analysis of polyunsaturated fatty acid synthases.

  Shohei Hayashi, Yasuharu Satoh, Yasushi Ogasawara, Tohru Dairi

  Current opinion in chemical biology 59 30 - 36 2020年05月19日 [査読有り]
   

  Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid, eicosapentaenoic acid, and arachidonic acid are essential fatty acids for humans. PUFAs are biosynthesized by either desaturases/elongases from oleic acid or PUFA synthases from acetyl units. PUFA synthases are composed of three or four subunits, and each creates a specific PUFA even though the multiple catalytic domains in each subunit are very similar. We recently dissected these PUFA synthases by in vivo and in vitro experiments and elucidated how the enzymes control PUFA profiles. Moreover, for the first time, we converted a practical microalgal docosahexaenoic acid synthase into an eicosapentaenoic acid synthase based on the results.
• Off-Loading Mechanism of Products in Polyunsaturated Fatty Acid Synthases.

  Shohei Hayashi, Yasushi Ogasawara, Yasuharu Satoh, Chitose Maruyama, Yoshimitsu Hamano, Tohru Dairi

  ACS chemical biology 15 3 651 - 656 2020年03月20日 [査読有り]
   

  Marine microorganisms de novo biosynthesize polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid by polyunsaturated fatty acid (PUFA) synthases composed of three or four polypeptides in a manner similar to fatty acid synthases (FASs). FASs usually possess thioesterase (TE) domains to release free fatty acids from acyl carrier protein (ACP)-tethered intermediates. Here, we investigated the off-loading mechanism with microalgal and bacterial PUFA synthases through in vivo and in vitro experiments. The in vitro experiments with acyltransferase (AT)-like domains and acyl-ACP substrates clearly demonstrated that the AT-like domains catalyzed the hydrolysis of acyl-ACPs to yield free fatty acids.
• Subtle Control of Carbon Chain Length in Polyunsaturated Fatty Acid Synthases.

  Naka M, Ikeuchi K, Hayashi S, Satoh Y, Ogasawara Y, Dairi T

  ACS chemical biology 2019年11月 [査読有り][通常論文]
  
• Identification of actinomycin D as a specific inhibitor of the alternative pathway of peptidoglycan biosynthesis.

  Ogasawara Y, Shimizu Y, Sato Y, Yoneda T, Inokuma Y, Dairi T

  The Journal of antibiotics 73 2 125 - 127 2019年10月 [査読有り][通常論文]
   

  Peptidoglycan is an indispensable component of bacterial cell walls. We recently discovered an alternative peptidoglycan biosynthetic pathway, which involves two enzymes, MurD2 and MurL, catalyzing the ligation of l-Glu to UDP-MurNAc-l-Ala and epimerization of the terminal l-Glu of the MurD2 product, respectively. Because the pathway operates in Xanthomonas oryze, a pathogen causing bacterial blight of rice, we searched for specific inhibitors from metabolites produced by actinomycetes to obtain a lead compound to function as an agrochemical. Actinomycin D was isolated from Streptomyces parvulus NBRC 13193 as a specific inhibitor of the pathway. In vitro analysis indicated that actinomycin D inhibited the MurD2 reaction.
• Gram-scale fermentative production of ergothioneine driven by overproduction of cysteine in Escherichia coli

  Tanaka, Naoyuki, Kawano, Yusuke, Satoh, Yasuharu, Dairi, Tohru, Ohtsu, Iwao

  SCIENTIFIC REPORTS 9 1895  2019年02月 [査読有り][通常論文]
   

  Ergothioneine (ERG), a unique thiol compound, is suggested to function as an antioxidant and cytoprotectant. Despite several recent attempts to produce ERG using various organisms, its yield was still very low and the costs remained high. Since the level of ERG produced depends strictly on the availability of three distinct precursor amino acids (L-cysteine (Cys), L-histidine, and L-methionine (Met)), metabolic engineering for enhancement of the flux toward ERG biosynthesis is required. Herein, we took advantage of a high-Cys production system using Escherichia coli cells, in which Cys biosynthesis and excretion were activated, and applied it to the fermentative production of ERG from glucose. The Cys overproduction in E. coli cells carrying the egtBCDE genes from Mycobacterium smegmatis was effective for ERG production. Furthermore, coexpression of the egtA gene, which encodes gamma-glutamylcysteine synthetase that synthesizes the gamma-glutamylcysteine used as a sulfur source of ERG biosynthesis, enhanced ERG production even though E. coli intrinsically has gamma-glutamylcysteine synthetase. Additionally, disruption of the metJ gene that encodes the transcriptional repressor involved in Met metabolism was effective in further increasing the production of ERG. Finally, we succeeded in the high-level production of 1.31 g/L ERG in a fed-batch culture process using a jar fermenter.
• In vitro characterization of MitE and MitB: formation of N-acetylglucosaminyl-3-amino-5-hydroxybenzoyl-MmcB as a key intermediate in the biosynthesis of antitumor antibiotic mitomycins.

  Y. Ogasawara, Y. Nakagawa, C. Maruyama, Y. Hamano, T. Dairi

  Bioorg. Med. Chem. Lett. 29 2076 - 2078 2019年 [査読有り][通常論文]
  
• Involvement of Peptide Epimerization in Poly-γ-glutamic Acid Biosynthesis.

  Y. Ogasawara, M. Shigematsu, S. Sato, H. Kato, T. Dairi

  Org. Lett. 21 3972 - 3975 2019年 [査読有り][通常論文]
  
• Amino Acid Residues Recognizing Isomeric Glutamate Substrates in UDP-N-acetylmuramic acid-L-alanine-glutamate Synthetases.

  R. Feng, Y. Satoh, H. Morita, Y. Ogasawara, T. Dairi

  ACS Chem. Biol 14 975 - 978 2019年 [査読有り][通常論文]
  
• Control mechanism for carbon chain length in polyunsaturated fatty acid synthases.

  S. Hayashi, M. Naka, K. Ikeuchi, M. Otsuka, K. Kobayashi, Y. Satoh, Y. Ogasawara, C. Maruyama, Y. Hamano, T. Ujihara, T. Dairi

  Angew. Chem. Int. Ed. 58 6605 - 6610 2019年 [査読有り][通常論文]
  
• Control mechanism for cis-double bond formation by polyunsaturated fatty acid synthases.

  S. Hayashi, Y. Satoh, Y. Ogasawara, C. Maruyama, Y. Hamano, T. Ujihara, T. Dairi

  Angew. Chem. Int. Ed. 58 2326 - 2330 2019年 [査読有り][通常論文]
  
• Searching for potent and specific antibiotics against pathogenic Helicobacter and Campylobacter strains.

  Y. Ogasawara, T. Dairi

  J. Ind. Microbiol. Biotechnol 46 409 - 414 2019年 [査読有り][招待有り]
  
• Enzymatic Formation of a Skipped Methyl-Substituted Octaprenyl Side Chain of Longestin (KS-505a): Involvement of Homo-IPP as a Common Extender Unit.

  Ozaki T, Shinde SS, Gao L, Okuizumi R, Liu C, Ogasawara Y, Lei X, Dairi T, Minami A, Oikawa H

  Angewandte Chemie (International ed. in English) 57 22 6629 - 6632 2018年05月 [査読有り][通常論文]
  
• Peptide epimerization machineries found in microorganisms

  Yasushi Ogasawara, Tohru Dairi

  Frontiers in Microbiology 9 156  2018年02月06日 [査読有り][招待有り]
   

  D-Amino acid residues have been identified in peptides from a variety of eukaryotes and prokaryotes. In microorganisms, UDP-N-acetylmuramic acid pentapeptide (UDP-MurNAc-L-Ala-D-Glu-meso-diaminopimelate-D-Ala-D-Ala), a unit of peptidoglycan, is a representative. During its biosynthesis, D-Ala and D-Glu are generally supplied by racemases from the corresponding isomers. However, we recently identified a unique unidirectional L-Glu epimerase catalyzing the epimerization of the terminal L-Glu of UDP-MurNAc-L-Ala-L-Glu. Several such enzymes, introducing D-amino acid resides into peptides via epimerization, have been reported to date. This includes a L-Ala-D/L-Glu epimerase, which is possibly used during peptidoglycan degradation. In bacterial primary metabolisms, to the best of our knowledge, these two machineries are the only examples of peptide epimerization. However, a variety of peptides containing D-amino acid residues have been isolated from microorganisms as secondary metabolites. Their biosynthetic mechanisms have been studied and three different peptide epimerization machineries have been reported. The first is non-ribosomal peptide synthetase (NRPS). Excellent studies with dissected modules of gramicidin synthetase and tyrocidine synthetase revealed the reactions of the epimerization domains embedded in the enzymes. The obtained information is still utilized to predict epimerization domains in uncharacterized NRPSs. The second includes the biosynthetic enzymes of lantibiotics, which are ribosome-dependently supplied peptide antibiotics containing polycyclic thioether amino acids (lanthionines). A mechanism for the formation of the D-Ala moiety in lanthionine by two enzymes, dehydratases catalyzing the conversion of L-Ser into dehydroalanine and enzymes catalyzing nucleophilic attack of the thiol of cysteine into dehydroalanine, was clarified. Similarly, the formation of a D-Ala residue by reduction of the dehydroalanine residue was also reported. The last type of machinery includes radical-S-adenosylmethionine (rSAM)-dependent enzymes, which catalyze a variety of radical-mediated chemical transformations. In the biosynthesis of polytheonamide, a marine sponge-derived and ribosome-dependently supplied peptide composed of 48 amino acids, a rSAM enzyme (PoyD) is responsible for unidirectional epimerizations of multiple different amino acids in the precursor peptide. In this review, we briefly summarize the discovery and current mechanistic understanding of these peptide epimerization enzymes.
• Heterologous and High Production of Ergothioneine in Escherichia coli

  Ryo, Osawa, Tomoyuki, Kamide, Yasuharu, Satoh, Yusuke, Kawano, Iwao, Ohtsu, Tohru, Dairi

  Journal of agricultural and food chemistry 66 5 1191 - 1196 AMER CHEMICAL SOC 2018年02月 [査読有り][通常論文]
   

  Ergothioneine (ERG) is a histidine-derived thiol compound suggested to function as an antioxidant and cytoprotectant in humans. Therefore, experimental trials have been conducted applying ERG from mushrooms in dietary supplements and as a cosmetic additive. However, this method of producing ERG is expensive; therefore, alternative methods for ERG supply are required. Five Mycobacterium smegmatis genes, egtABCDE, have been confirmed to be responsible for ERG biosynthesis. This enabled us to develop practical fermentative ERG production by microorganisms. In this study, we carried out heterologous and high-level production of ERG in Escherichia coli using the egt genes from M. smegmatis. By high production of each of the Egt enzymes and elimination of bottlenecks in the substrate supply, we succeeded in constructing a production system that yielded 24 mg/L (104 μM) secreted ERG.
• Functional analysis of methyltransferases participating in streptothricin-related antibiotic biosynthesis

  Haruka Niikura, Chitose Maruyama, Yasushi Ogasawara, Kazuo Shin-ya, Tohru Dairi, Yoshimitsu Hamano

  Journal of Bioscience and Bioengineering 125 2 148 - 154 2018年02月01日 [査読有り][通常論文]
   

  Streptothricin (ST) and its related compounds produced by Streptomyces strains are broad-spectrum antibiotics that consist of carbamoylated D-gulosamine, amino-acid side chain, and streptolidine lactam moieties. BD-12, a streptothricin-related antibiotic, has a glycine-derived side chain and two N-methyl groups, whereas ST-F carrying the L-β-lysine side chain has no methyl group. In our previous studies, we identified and characterized the BD-12 and ST biosynthetic gene clusters. Here we report the functional analysis of two methyltransferase genes (orf 6 and orf 13) in the BD-12 biosynthetic gene cluster. Combinatorial biosynthesis using these two methyltransferase genes and the ST biosynthetic gene cluster resulted in the production of three methylated forms of ST-F. Among them, N,N′-dimethyl-ST-F, a novel compound generated in the present study, showed bacteria-specific antibiotic activities, although ST-F exhibits antibiotic activities against both prokaryotes and eukaryotes. Our findings also demonstrated that the orf 6 and orf 13 genes are responsible for the N-methylations of the amide bonds in the streptolidine lactam and in the amino-acid side chain linkage, respectively, and that N-methyl modification of the streptolidine lactam confers resistance in part against an ST hydrolase, SttH.
• Ergothioneine production with Aspergillus oryzae.

  S. Takusagawa, Y. Satoh, I. Ohtsu, T. Dairi

  Biosci Biotechnol Biochem. 2018年 [査読有り][通常論文]
  
• Aplasmomycin and boromycin are specific inhibitors of the futalosine pathway.

  Y. Shimizu, Y. Ogasawara, A. Matsumoto, T. Dairi

  J Antibiot 2018年 [査読有り][通常論文]
  
• Enzymatic formation of a skipped methyl-substituted octaprenyl side chain of longestin (KS-505a): Involvement of homo-IPP as a common extender unit.

  T. Ozaki, S.S. Shinde, L. Gao, R. Okuizumi, C. Liu, Y. Ogasawara, X Lei, T. Dairi, A. Minami, H. Oikawa

  Angew. Chem. Int. Ed. 20 6178 - 6182 2018年 [査読有り][通常論文]
  
• Biosynthetic Gene Cluster of a D‐Tryptophan‐Containing Lasso Peptide, MS‐271.

  Z. Feng, Y. Ogasawara, S. Nomura, T. Dairi

  ChemBioChem 19 2045 - 2048 2018年 [査読有り][通常論文]
  
• Total Biosynthesis of Brassicicenes: Identification of a Key Enzyme for Skeletal Diversification.

  A. Tazawa, Y. Ye, T. Ozaki, C. Liu, Y. Ogasawara, T. Dairi, Y. Higuchi, N. Kato, K. Gomi, A. Minami, H. Oikawa

  Org. Lett. 20 6178 - 6182 2018年 [査読有り][通常論文]
  
• Novel enzymology in futalosine-dependent menaquinone biosynthesis.

  S. Joshi, D. Fedoseyenko, N. Mahanta, H. Manion, S. Naseem, T. Dairi, TP. Begley

  Curr Opin Chem Biol 47 134 - 141 2018年 [査読有り][通常論文]
  
• Synthesis of Acylborons by Ozonolysis of Alkenylboronates: Preparation of an Enantioenriched Amino Acid Acylboronate

  Jumpei Taguchi, Toshiki Ikeda, Rina Takahashi, Ikuo Sasaki, Yasushi Ogasawara, Tohru Dairi, Naoya Kato, Yasunori Yamamoto, Jeffrey W. Bode, Hajime Ito

  Angewandte Chemie 56 44 13847 - 13851 Wiley-Blackwell 2017年10月 [査読有り][通常論文]
   

  A concise synthesis of acylborons was achieved by ozonolysis of alkenyl MIDA (N-methyliminodiacetic acid) boronates. This reaction exhibits excellent functional-group tolerance and is applicable to various acyl MIDA boronates and potassium acyltrifluroborates (KATs) which could not be synthesized by previous methods. In addition, alpha-amino acylborons, which would be essential for peptide ligations, were prepared for the first time. The acylboron of l-alanine was obtained in high enantiopurity and found to be configurationally stable. Oligopeptide synthesis between the alpha-amino KATs and amino acid in dilute aqueous media was studied.
• Biosynthesis of Oligopeptides Using ATP-Grasp Enzymes

  Yasushi Ogasawara, Tohru Dairi

  CHEMISTRY-A EUROPEAN JOURNAL 23 45 10714 - 10724 2017年08月 [査読有り][通常論文]
   

  Peptides are biologically occurring oligomers of amino acids linked by amide bonds and are indispensable for all living organisms. Many bioactive peptides are used as antibiotics, antivirus agents, insecticides, pheromones, and food preservatives. Nature employs several different strategies to form amide bonds. ATP-grasp enzymes that catalyze amide bond formation (ATP-dependent carboxylate-amine ligases) utilize a strategy of activating carboxylic acid as an acylphosphate intermediate to form amide bonds and are involved in many different biological processes in both primary and secondary metabolisms. The recent discovery of several new ATP-dependent carboxylate-amine ligases has expanded the diversity of this group of enzymes and showed their usefulness for generating oligopeptides. In this review, an overview of findings on amide bond formation catalyzed by ATP-grasp enzymes in the past decade is presented.
• N-Phenylacetylation and Nonribosomal Peptide Synthetases with Substrate Promiscuity for Biosynthesis of Heptapeptide Variants, JBIR-78 and JBIR-95

  Kunpei Takeda, Kohei Kemmoku, Yasuharu Satoh, Yasushi Ogasawara, Kazuo Shin-ya, Tohru Dairi

  ACS CHEMICAL BIOLOGY 12 7 1813 - 1819 2017年07月 [査読有り][通常論文]
   

  JBIR-78 (1) and JBIR-95 (2), both of which are heptapeptide derivatives isolated from Kibdelosporangium sp, AK-AA56, have the same amino acid sequences except for the second amino acid phenylacetic acid (Paa)L-Val-D-Asp (1)/D-cysteic acid(2)-D-Ala-(3S)-3-hydroxy-D-Leu-Gly-D-Ala-L-Phe. Heterologous expression of the biosynthetic gene cluster including genes encoding nonribosomal peptide synthetases, (NRPS) and in vitro assays. with recombinant OrfA, an L-cysteic acid synthase homologue, suggested the single A domain in module 2 activates both L-Asp and 1-cysteic acid to yield 1 and 2 respectively although the substrate specificities of the A domains. of NRPSs are Usually, strict Biosynthetic mechanism of introduction of N-terminal Paa was also investigated. Recombinant Ora and Orf2 similar to subunits of pyruvate dehydrogenase complex catalyzed the conversion of phenylpyruvate into phenylacetyl-CoA together with dihydrolipoyl, dehydrogenase whose encoding gene is located outside of the gene cluster. Moreover, we showed that phenylacetyl-CoA was directly condensed with L-Val, which was tethered to a peptidyl carrier protein, at the first condensation domain in the NRPS.
• Identification of tirandamycins as specific inhibitors of the futalosine pathway

  Yasushi Ogasawara, Kensuke Kondo, Ayumi Ikeda, Rikako Harada, Tohru Dairi

  JOURNAL OF ANTIBIOTICS 70 6 798 - 800 2017年06月 [査読有り][通常論文]
  
• A Glycopeptidyl-Glutamate Epimerase for Bacterial Peptidoglycan Biosynthesis

  Ruoyin Feng, Yasuharu Satoh, Yasushi Ogasawara, Tohru Yoshimura, Tohru Dairi

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 139 12 4243 - 4245 2017年03月 [査読有り][通常論文]
   

  D-Glutamate (Glu) supplied by Glu racemases or D-amino acid transaminase is utilized for peptidoglycan biosynthesis in microorganisms. Comparative genomics has shown that some microorganisms, including Xanthomonas oryzae, perhaps have no orthologues of these genes. We performed shotgun cloning experiments with a D-Glu auxotrophic Escherichia coli mutant as the host and X. oryzae as the DNA donor. We obtained complementary genes, XOO_1319 and XOO_1320, which are annotated as a hypothetical protein and MurD (UDP-MurNAc-L-Ala-D-Glu synthetase), respectively. By detailed in vitro analysis, we revealed that XOO_1320 is an enzyme to ligate L-Glu to UDP-MurNAc-L-Ala, providing the first example of MurD utilizing L-Glu, and that XOO_1319 is a novel enzyme catalyzing epimerization of the terminal L-Glu of the product in the presence of ATP and Mg2+. We investigated the occurrence of XOO_1319 orthologues and found that it exists in some categories of microorganisms, including pathogenic ones.
• Biosynthesis of the Carbonylmethylene Structure Found in the Ketomemicin Class of Pseudotripeptides

  Junpei Kawata, Taiki Naoe, Yasushi Ogasawara, Tohru Dairi

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 56 8 2026 - 2029 2017年02月 [査読有り][通常論文]
   

  We recently discovered novel pseudotripeptides, the ketomemicins, which possess a C-terminal pseudodipeptide connected with a carbonylmethylene instead of an amide bond, through heterologous expression of gene clusters identified in actinobacteria. The carbonylmethylene structure is a stable isostere of the amide bond and its biological significance has been shown in several natural and synthetic products. Despite the biological importance of these compounds, little is known about how the carbonylmethylene structure is biosynthesized. In this work, we fully characterized the biosynthetic machinery of the pseudodipeptide. An aldolase, dehydratase, PLP-dependent glycine-C-acetyltransferase, and dehydrogenase were involved in the formation of the pseudodipeptide, with malonyl-CoA and phenylpyruvate as starter substrates.
• Enhanced production of polyunsaturated fatty acids by enzyme engineering of tandem acyl carrier proteins

  Shohei Hayashi, Yasuharu Satoh, Tetsuro Ujihara, Yusuke Takata, Tohru Dairi

  SCIENTIFIC REPORTS 6 35441  2016年10月 [査読有り][通常論文]
   

  In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A. These ACPs were previously shown to be important for PUFA productivity. In this study, we examined their function in more detail. PUFA productivities increased depending on the number of ACPs without profile changes in each subunit A of eukaryotic and prokaryotic PUFA synthases. We also constructed derivative enzymes from subunit A with 5 x ACPs. Enzymes possessing one inactive ACP at any position produced similar to 30% PUFAs compared with the parental enzyme but unexpectedly had similar to 250% productivity compared with subunit A with 4 x ACPs. Enzymes constructed by replacing the 3rd ACP with an inactive ACP from another subunit A or ACP-unrelated sequences produced similar to 100% and similar to 3% PUFAs compared with the parental 3rd ACP-inactive enzyme, respectively. These results suggest that both the structure and number of ACP domains are important for PUFA productivity.
• Biosynthesis of Shearinine: Diversification of a Tandem Prenyl Moiety of Fungal Indole Diterpenes

  Chengwei Liu, Atsushi Minami, Tohru Dairi, Katsuya Gomi, Barry Scott, Hideaki Oikawa

  ORGANIC LETTERS 18 19 5026 - 5029 2016年10月 [査読有り][通常論文]
   

  The late-stage biosynthetic pathway of the indole diterpene shearinine involving four enzymatic reactions (JanQDOJ) was elucidated by an efficient heterologous expression system using Aspergillus oryzae. Key oxidative cyclization, forming a characteristic A/B bicyclic shearinine core by flavoprotein oxidase, was studied using a substrate analogue and a buffer containing (H2O)-O-18. These experimental data provided evidence that JanO catalyzes two-step oxidation via a hydroxylated product and that the JanO reaction involves the hydride-transfer mechanism.
• Characterization of three amidinotransferases involved in the biosynthesis of ketomemicins

  Yasushi Ogasawara, Michiko Fujimori, Junpei Kawata, Tohru Dairi

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 26 15 3662 - 3664 2016年08月 [査読有り][通常論文]
   

  We recently reported a novel class of amide bond forming enzymes (peptide ligases) involved in the biosynthesis of pheganomycins, resorcinomycins and ketomemicins. This class of enzymes exclusively utilizes N-alpha-amidino amino acids as the N-terminal substrate. In this Letter, we characterized three new amidinotransferases involved in the biosynthesis of ketomemicins and showed that L-arginine was the amidino-acceptor of amidinotransferases in both the Micromonospora sp. and Streptomyces mobaraensis clusters, while the Salinispora tropica enzyme recognized L-valine. Unexpectedly, the S. tropica enzyme accepted several different amino acids as amidino acceptors in addition to L-valine. Accordingly, we re-investigated the specific metabolites governed by the gene cluster of S. tropica and identified several minor congeners of ketomemicin C with different N-terminal amidino-amino acids. These results indicate that the amidinotransferase of S. tropica is promiscuous and could be useful to generate new ketomemicin-type natural products. (C) 2016 Elsevier Ltd. All rights reserved.
• Exploring Peptide Ligase Orthologs in Actinobacteria—Discovery of Pseudopeptide Natural Products, Ketomemicins.

  Y. Ogasawara, J. Kawata, M. Noike, Y. Satoh, K. Furihata, T. Dairi

  ACS Chem. Biol. 11 6 1686 - 1692 2016年06月 [査読有り][通常論文]
   

  We recently identified a novel peptide ligase (PGM1), an ATP-grasp-ligase, that catalyzes amide bond formation between (5)-2-(3,5-dihydroxy-4-methoxypheny1)-2guanidinoacetic acid and ribosomally supplied oligopeptides in pheganomycin biosynthesis. This was the first example of an ATP-grasp-ligase utilizing peptides as nucleophiles. To explore the potential of this type of enzyme, we performed a BLAST search and identified many orthologs. The orthologs of Streptomyces mobaraensis, Salinispora tropica, and Micromonospora sp. were found in similar gene clusters consisting of six genes. To probe the functions of these genes, we heterologously expressed each of the dusters in Streptomyces lividans and detected novel and structurally similar pseudotripeptides in the broth of all transformants. Moreover, a recombinant PGM1 ortholog of Micromonospora sp. was demonstrated to be a novel dipeptide ligase catalyzing amide bond formation between amidino-arginine and dipeptides to yield tripeptides; this is the first report of a peptide ligase utilizing dipeptides as nudeophiles.
• Advanced functionalization of polyhydroxyalkanoate via the UV-initiated thiol-ene click reaction

  Kenji Tajima, Kosuke Iwamoto, Yasuharu Satoh, Ryosuke Sakai, Toshifumi Satoh, Tohru Dairi

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 100 10 4375 - 4383 2016年05月 [査読有り][通常論文]
   

  Polyhydroxyalkanoates (PHAs) incorporating vinyl-bearing 3-hydroxyalkanoates were prepared in 8.5-12.9 g L-1 yield. The molar ratios (0-16 mol%) of the vinyl-bearing 3-hydroxyalkanoate derivatives were controlled by the continuous feeding of undecylenate at various concentrations. Subsequently, the PHAs were functionalized by UV-initiated thiol-ene click reaction and chemical modification. H-1 NMR spectra suggested that 3-mercaptopropionic acid and 2-aminoethanethiol were successfully introduced into the vinyl-bearing PHA. Subsequently, chemical modification using fluorescein or a fibronectin active fragment (GRGDS) was attempted. The former yielded a PHA derivative capable of emitting fluorescence under UV irradiation, which was useful for determining the miscibility of PHA in a composite film comprising poly-EY-lactic acid (PLLA) and PHA. In the latter case, PHA bearing GRGDS peptides exhibited cell adhesiveness, suggesting that its biocompatibility was improved upon peptide introduction. Taken together, the UV-initiated thiol-ene click reaction was demonstrated to be useful in PHA modification.
• Structure and activity relationships of the anti-Mycobacterium antibiotics resorcinomycin and pheganomycin

  Yasushi Ogasawara, Koichi Ooya, Michiko Fujimori, Motoyoshi Noike, Tohru Dairi

  JOURNAL OF ANTIBIOTICS 69 2 119 - 120 2016年02月 [査読有り][通常論文]
  
• Ergothioneine protects Streptomyces coelicolor A3(2) from oxidative stresses

  Shunsuke Nakajima, Yasuharu Satoh, Kentaro Yanashima, Tomomi Matsui, Tohru Dairi

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 120 3 294 - 298 2015年09月 [査読有り][通常論文]
   

  Thiol compounds with low-molecular weight, such as glutathione, mycothiol (MSH), bacillithiol, and ergothioneine (ERG), are known to protect microorganisms from oxidative stresses. Mycobacteria and actinobacteria utilize both MSH and ERG. The biological functions of MSH in mycobacteria have been extensively studied by genetic and biochemical studies, which have suggested it has critical roles for detoxification in cells. In contrast, the biological functions of ERG remain ambiguous because its biosynthetic genes were only recently identified in Mycobacterium avium. In this study, we constructed mutants of Streptomyces coelicolor A3(2), in which either the MSH or ERG biosynthetic gene was disrupted, and examined their phenotypes. A mshC (SC01663)-disruptant completely lost MSH productivity. In contrast, a disruptant of the egtA gene (SC00910) encoding gamma-glutamyl-cysteine synthetase unexpectedly retained reduced productivity of ERG, probably because of the use of L-cysteine instead of gamma-glutamyl-cysteine. Both disruptants showed delayed growth at the late logarithmic phase and were more susceptible to hydrogen peroxide and cumene hydroperoxide than the parental strain. Interestingly, the ERG-disruptant, which still kept reduced ERG productivity, was more susceptible. Furthermore, the ERG-disruptant accumulated 5-fold more MSH than the parental strain. In contrast, the amount of ERG was almost the same between the MSH-disruptant and the parental strain. Taken together, our results suggest that ERG is more important than MSH in S. coelicolor A3(2). (c) 2015, The Society for Biotechnology, Japan. All rights reserved.
• Identification and analysis of the resorcinomycin biosynthetic gene cluster

  Koichi Ooya, Yasushi Ogasawara, Motoyoshi Noike, Tohru Dairi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 79 11 1833 - 1837 2015年 [査読有り][通常論文]
   

  Resorcinomycin (1) is composed of a nonproteinogenic amino acid, (S)-2-(3,5-dihydroxy-4-isopropylphenyl)-2-guanidinoacetic acid (2), and glycine. A biosynthetic gene cluster was identified in a genome database of Streptoverticillium roseoverticillatum by searching for orthologs of the genes responsible for biosynthesis of pheganomycin (3), which possesses a (2)-derivative at its N-terminus. The cluster contained a gene encoding an ATP-grasp-ligase (res5), which was suggested to catalyze the peptide bond formation between 2 and glycine. A res5-deletion mutant lost 1 productivity but accumulated 2 in the culture broth. However, recombinant RES5 did not show catalytic activity to form 1 with 2 and glycine as substrates. Moreover, heterologous expression of the cluster resulted in accumulation of only 2 and no production of 1 was observed. These results suggested that a peptide with glycine at its N-terminus may be used as a nucleophile and then maturated by a peptidase encoded by a gene outside of the cluster.
• A peptide ligase and the ribosome cooperate to synthesize the peptide pheganomycin

  Motoyoshi Noike, Takashi Matsui, Koichi Ooya, Ikuo Sasaki, Shouta Ohtaki, Yoshimitsu Hamano, Chitose Maruyama, Jun Ishikawa, Yasuharu Satoh, Hajime Ito, Hiroyuki Morita, Tohru Dairi

  NATURE CHEMICAL BIOLOGY 11 1 71 - + 2015年01月 [査読有り][通常論文]
   

  Peptide antibiotics are typically biosynthesized by one of two distinct machineries in a ribosome-dependent or ribosome-independent manner. Pheganomycin (PGM (1)) and related analogs consist of the nonproteinogenic amino acid (S)-2-(3,5-dihydroxy-4-hydroxymethyl)phenyl-2-guanidinoacetic acid (2) and a proteinogenic core peptide, making their origin uncertain. We report the identification of the biosynthetic gene cluster from Streptomyces cirratus responsible for PGM production. Unexpectedly, the cluster contains a gene encoding multiple precursor peptides along with several genes plausibly encoding enzymes for the synthesis of amino acid 2. We identified PGM1, which has an ATP-grasp domain, as potentially capable of linking the precursor peptides with 2, and validate this hypothesis using deletion mutants and in vitro reconstitution. We document PGM1's substrate permissivity, which could be rationalized by a large binding pocket as confirmed via structural and mutagenesis experiments. This is to our knowledge the first example of cooperative peptide synthesis achieved by ribosomes and peptide ligases using a peptide nucleophile.
• New gene responsible for para-aminobenzoate biosynthesis

  Yasuharu Satoh, Masahiro Kuratsu, Daiki Kobayashi, Tohru Dairi

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 117 2 178 - 183 2014年02月 [査読有り][通常論文]
   

  Folate is an essential cofactor in all living cells for one-carbon transfer reactions. para-Aminobenzoate (pABA), a building block of folate, is usually derived from chorismate in the shikimate pathway by reactions of aminodeoxychorismate synthase (PabA and -B) and 4-amino-4-deoxychorismate lyase (PabC). We previously suggested that an alternative pathway for pABA biosynthesis would operate in some microorganisms such as Lactobacillus fermentum and Nitrosomonas europaea since these bacteria showed a prototrophic phenotype to pABA despite the fact that there are no orthologs of pabA, -B, and -C in their genome databases. In this study, a gene of unknown function, NE1434, was obtained from N. europaea by shotgun cloning using a pABA-auxotrophic Escherichia coli mutant (Delta pabABC) as a host. A tracer experiment using [U-C-13(6)]glucose suggested that pABA was de novo synthesized in the transformant An E. coli Delta pabABC Delta aroB mutant carrying the NE1434 gene exhibited a prototrophic phenotype to pABA, suggesting that compounds in the shilrimate pathway including chorismate were not utilized as substrates by NE1434. Moreover, the CT610 gene, an ortholog of NE1434 located in the folate biosynthetic gene cluster in Chlamydia trachomatis, also complemented pABA-auxotrophic E. toll mutants. Taken together, these results suggest that NE1434 and CT610 participate in pABA biosynthesis. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.
• A fungal prenyltransferase catalyzes the regular di-prenylation at positions 20 and 21 of paxilline

  Chengwei Liu, Motoyoshi Noike, Atsushi Minami, Hideaki Oikawa, Tohru Dairi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 3 448 - 454 2014年 [査読有り][通常論文]
   

  A putative indole diterpene biosynthetic gene cluster composed of eight genes was identified in a genome database of Phomopsis amygdali, and from it, biosynthetic genes of fusicoccin A were cloned and characterized. The six genes showed significant similarities to pax genes, which are essential to paxilline biosynthesis in Penicillium paxilli. Recombinants of the three putative prenyltransferase genes in the cluster were overexpressed in Escherichia coli and characterized by means of in vitro experiments. AmyG is perhaps a GGDP synthase. AmyC and AmyD were confirmed to be prenyltransferases catalyzing the transfer of GGDP to IGP and a regular di-prenylation at positions 20 and 21 of paxilline, respectively. AmyD is the first know example of an enzyme with this function. The K-m values for AmyD were calculated to be 7.6 +/- 0.5 M for paxilline and 17.9 +/- 1.7 M for DMAPP at a k(cat) of 0.12 +/- 0.003/s.
• Functional analysis of a prenyltransferase gene (paxD) in the paxilline biosynthetic gene cluster

  Chengwei Liu, Motoyoshi Noike, Atsushi Minami, Hideaki Oikawa, Tohru Dairi

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 98 1 199 - 206 2014年01月 [査読有り][通常論文]
   

  Paxilline is an indole-diterpene produced by Penicillium paxilli. Six genes (paxB, C, G, M, P, and Q) in paxilline biosynthetic gene cluster were previously shown to be responsible for paxilline biosynthesis. In this study, we have characterized paxD, which is located next to paxQ and has weak similarities to fungal dimethylallyl tryptophan synthase genes. PaxD was overexpressed in Escherichia coli and the purified enzyme was used for in vitro analysis. When paxilline and dimethylallyl diphosphate were used as substrates, one major and one minor product, which were identified as di-prenyl paxilline and mono-prenyl paxilline by liquid chromatography-mass spectrometry analysis, were formed. The structure of the major product was determined to be 21,22-diprenylated paxilline, showing that PaxD catalyzed the successive di-prenylation. Traces of both products were detected in culture broth of P. paxilli by liquid chromatography-mass spectrometry analysis. The enzyme is likely to be a dimer and required no divalent cations. The optimum pH and temperature were 8.0 and 37 A degrees C, respectively. The Km values were calculated as 106.4 A +/- 5.4 mu M for paxilline and 0.57 A +/- 0.02 mu M for DMAPP with a kcat of 0.97 A +/- 0.01/s.
• Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

  Koichi Tagami, Atsushi Minami, Ryuya Fujii, Chengwei Liu, Mizuki Tanaka, Katsuya Gomi, Tohru Dairi, Hideaki Oikawa

  Chembiochem 15 14 2076 - 2080 2014年 [査読有り][通常論文]
  
• Regiospecificities and prenylation mode specificities of the fungal indole diterpene prenyltransferases AtmD and PaxD

  Chengwei Liu, Atsushi Minami, Motoyoshi Noike, Hiroaki Toshima, Hideaki Oikawa, Tohru Dairi

  Applied and Environmental Microbiology 79 23 7298 - 7304 2013年12月 [査読有り][通常論文]
   

  We recently reported the function of paxD, which is involved in the paxilline (compound 1) biosynthetic gene cluster in Penicillium paxilli. Recombinant PaxD catalyzed a stepwise regular-type diprenylation at the 21 and 22 positions of compound 1 with dimethylallyl diphosphate (DMAPP) as the prenyl donor. In this study, atmD, which is located in the aflatrem (compound 2) biosynthetic gene cluster in Aspergillus flavus and encodes an enzyme with 32% amino acid identity to PaxD, was characterized using recombinant enzyme. When compound 1 and DMAPP were used as substrates, two major products and a trace of minor product were formed. The structures of the two major products were determined to be reversely monoprenylated compound 1 at either the 20 or 21 position. Because compound 2 and β-aflatrem (compound 3), both of which are compound 1-related compounds produced by A. flavus, have the same prenyl moiety at the 20 and 21 position, respectively, AtmD should catalyze the prenylation in compound 2 and 3 biosynthesis. More importantly and surprisingly, AtmD accepted paspaline (compound 4), which is an intermediate of compound 1 biosynthesis that has a structure similar to that of compound 1, and catalyzed a regular monoprenylation of compound 4 at either the 21 or 22 position, though the reverse prenylation was observed with compound 1. This suggests that fungal indole diterpene prenyltransferases have the potential to alter their position and regular/reverse specificities for prenylation and could be applicable for the synthesis of industrially useful compounds. © 2013, American Society for Microbiology.
• Menaquinone Biosynthesis: Formation of Aminofutalosine Requires a Unique Radical SAM Enzyme

  Nilkamal Mahanta, Dmytro Fedoseyenko, Tohru Dairi, Tadhg P. Begley

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 135 41 15318 - 15321 2013年10月 [査読有り][通常論文]
   

  Menaquinone (MK, vitamin K-2) is a lipid-soluble molecule that participates in the bacterial electron transport chain. In mammalian cells, MK functions as an essential vitamin for the activation of various proteins involved in blood clotting and bone metabolism. Recently, a new pathway for the biosynthesis of this cofactor was discovered in Streptomyces coelicolor A3(2) in which chorismate is converted to aminofutalosine in a reaction catalyzed by MqnA and an unidentified enzyme. Here, we reconstitute the biosynthesis of aminofutalosine and demonstrate that the missing enzyme (aminofutalosine synthase, MqnE) is a radical SAM enzyme that catalyzes the addition of the adenosyl radical to the double bond of 3-[(1-carboxyvinyl)oxy]benzoic acid. This is a new reaction type in the radical SAM superfamily.
• In Vitro Reconstitution of the Radical S-Adenosylmethionine Enzyme MqnC Involved in the Biosynthesis of Futalosine-Derived Menaquinone

  Lisa E. Cooper, Dmytro Fedoseyenko, Sameh H. Abdelwahed, Soong-Hyun Kim, Tohru Dairi, Tadhg P. Begley

  BIOCHEMISTRY 52 27 4592 - 4594 2013年07月 [査読有り][通常論文]
   

  The radical S-adenosylmethionine enzyme MqnC catalyzes conversion of dehypoxanthine futalosine (DHFL) to the unique Spiro compound cyclic DHFL in the futalosine pathway for menaquinone biosynthesis. This study describes the in vitro reconstitution of [4Fe-4S] cluster-dependent MqnC activity and identifies the site of abstraction of a hydrogen atom from DHFL by the adenosyl radical.
• Cellulose complementing factor (Ccp) is a new member of the cellulose synthase complex (terminal complex) in Acetobacter xylinum

  Naoki Sunagawa, Takaaki Fujiwara, Takanori Yoda, Shin Kawano, Yasuharu Satoh, Min Yao, Kenji Tajima, Tohru Dairi

  JOURNAL OF BIOSCIENCE AND BIOENGINEERING 115 6 607 - 612 2013年06月 [査読有り][通常論文]
   

  The cellulose complementing factor (Ccp) is known to be involved in cellulose production in the Acetobacter species. However, its precise functions remain unclear. In the current study, we identified the coding region of the ccpAx gene (ccp gene from Acetobacter xylinum) and the localization of the CcpAx in cells by generating fusion proteins tagged to an enhanced green fluorescent protein (EGFP). From the results of N-terminal sequencing of CcpAx-EGFP-fusion protein, which recovered 65% of cellulose-producing abilities of the wild-type to the ccpAx gene-knockout mutant, the ccpAx gene was determined to encode a protein with the molecular weight of 8 kDa. The amino acid sequence deduced had high similarities with the C-terminal regions of Ccp proteins from other Acetobacter species. Fluorescence microscopy analysis showed that CcpAx was longitudinally localized along with one side of the cell membrane. Additionally, the localization of AxCeSD, which is thought to be a member of the cellulose synthase complex [terminal complex (TC)] in A. xylinum, was determined in the same manner as CcpAx. Fluorescence microscopy analysis showed that AxCeSD had a localization pattern similar to that of CcpAx. Pulldown assays and isothermal titration calorimetry analysis clearly showed a significant interaction between CcpAx and AxCeSD. Taken together, these data strongly suggest that CcpAx functions as a member of the TC in A. xylinum. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.
• Reconstitution of Biosynthetic Machinery for Indole-Diterpene Paxilline in Aspergillus oryzae

  Koichi Tagami, Chengwei Liu, Atsushi Minami, Motoyoshi Noike, Tetsuya Isaka, Shuhei Fueki, Yoshihiro Shichijo, Hiroaki Toshima, Katsuya Gomi, Tohru Dairi, Hideaki Oikawa

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 135 4 1260 - 1263 2013年01月 [査読有り][通常論文]
   

  Indole-diterpenes represented by paxilline share a common pentacyclic core skeleton derived from indole and geranylgeranyl diphosphate. To shed light on the detailed biosynthetic mechanism of the paspaline-type hexacyclic skeleton, we examined the reconstitution of paxilline biosynthetic machinery in Aspergillus oryzae NSAR1 Stepwise introduction of the six pax genes enabled us to isolate all biosynthetic intermediates and to synthesize paxilline. In vitro and in vivo studies on the key enzymes, prenyltransferase PaxC and cyclase PaxB, allowed us to elucidate actual substrates of these enzymes. Using the isolated and the synthesized epoxide substrates, the highly intriguing stepwide epoxidation/cyclization mechanism for the construction of core structure has been confirmed. In addition, we also demonstrated "tandem transformation" to simultaneously introduce two genes using a single vector (paxG/paxB, pAdeA; paxP/paxQ pUNA). This may provide further option for the reconstitution strategy to synthesize more complex fungal metabolites.
• Cellulose production by Enterobacter sp CJF-002 and identification of genes for cellulose biosynthesis

  Naoki Sunagawa, Kenji Tajima, Mariko Hosoda, Shin Kawano, Ryota Kose, Yasuharu Satoh, Min Yao, Tohru Dairi

  CELLULOSE 19 6 1989 - 2001 2012年12月 [査読有り][通常論文]
   

  Enterobacter sp. CJF-002, which had been isolated as a cellulose producer with saccharides as a carbon source, was shown to efficiently produce cellulose from beet molasses (B-Mol) and biodiesel fuel by-product (BDF-B), renewable non-edible and inexpensive biomasses. The cellulose production rates of Enterobacter sp. CJF-002 using B-Mol and BDF-B as carbon sources were faster than those of Acetobacter xylinum (A. xylinum) ATCC23769, a representative cellulose producing bacterium. To clarify the biosynthetic machinery of cellulose in the strain, genes responsible for cellulose biosynthesis were cloned. Six open reading frames (ORFs) were suggested to be clustered and their amino acid sequences had high similarities with those of BcsA, BcsB, BcsZ (endoglucanase), BcsC, YhjQ, and YhjK from Escherichia coli, respectively. Of these, the former four genes showed low similarities to corresponding orthologs in a cellulose biosynthetic gene cluster of A. xylinum. A bcsC-knockout mutant produced no cellulose, confirming that the gene is essential for cellulose production of Enterobacter sp. CJF-002. The predicted three-dimensional structure of BcsZ(En) from Enterobacter sp. CJF-002 had high similarity with that of CMCax (endoglucanase) from A. xylinum ATCC23769 in spite of the low similarity in their amino acid sequences. Taken together, A. xylinum and Enterobacter sp. CJF-002 might produce cellulose via a similar synthetic mechanism.
• Molecular Breeding of a Fungus Producing a Precursor Diterpene Suitable for Semi-Synthesis by Dissection of the Biosynthetic Machinery

  Motoyoshi Noike, Yusuke Ono, Yuji Araki, Ryo Tanio, Yusuke Higuchi, Hajime Nitta, Yoshimitsu Hamano, Tomonobu Toyomasu, Takeshi Sassa, Nobuo Kato, Tohru Dairi

  PLOS ONE 7 8 e42090  2012年08月 [査読有り][通常論文]
   

  Many clinically useful pharmaceuticals are semi-synthesized from natural products produced by actinobacteria and fungi. The synthetic protocols usually contain many complicated reaction steps and thereby result in low yields and high costs. It is therefore important to breed microorganisms that produce a compound most suitable for chemical synthesis. For a long time, desirable mutants have been obtained by random mutagenesis and mass screening. However, these mutants sometimes show unfavorable phenotypes such as low viability and low productivity of the desired compound. Fusicoccin (FC) A is a diterpene glucoside produced by the fungus Phomopsis amygdali. Both FC and the structurally-related cotylenin A (CN) have phytohormone-like activity. However, only CN exhibits anti-cancer activity. Since the CN producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out, and elimination of the hydroxyl group at position 12 of FC was essential to mimic the CN-like activity. Based on detailed dissection of the biosynthetic machinery, we constructed a mutant producing a compound without a hydroxyl group at position 12 by gene-disruption. The mutant produced this compound as a sole metabolite, which can be easily and efficiently converted into an anti-cancer drug, and its productivity was equivalent to the sum of FC-related compounds produced by the parental strain. Our strategy would be applicable to development of pharmaceuticals that are semisynthesized from fungal metabolites.
• An Enzyme Catalyzing O-Prenylation of the Glucose Moiety of Fusicoccin A, a Diterpene Glucoside Produced by the Fungus Phomopsis amygdali

  Motoyoshi Noike, Chengwei Liu, Yusuke Ono, Yoshimitsu Hamano, Tomonobu Toyomasu, Takeshi Sassa, Nobuo Kato, Tohru Dairi

  CHEMBIOCHEM 13 4 566 - 573 2012年03月 [査読有り][通常論文]
   

  Isoprenoids form the largest family of compounds found in nature. Isoprenoids are often attached to other moieties such as aromatic compounds, indoles/tryptophan, and flavonoids. These reactions are catalyzed by three phylogenetically distinct prenyltransferases: soluble aromatic prenyltransferases identified mainly in actinobacteria, soluble indole prenyltransferases mostly in fungi, and membrane-bound prenyltransferases in various organisms. Fusicoccin A (FC A) is a diterpene glycoside produced by the plant-pathogenic fungus Phomopsis amygdali and has a unique O-prenylated glucose moiety. In this study, we identified for the first time, from a genome database of P. amygdali, a gene (papt) encoding a prenyltransferase that reversibly transfers dimethylallyl diphosphate (DMAPP) to the 6'-hydroxy group of the glucose moiety of FC A to yield an O-prenylated sugar. An in vitro assay with a recombinant enzyme was also developed. Detailed analyses with recombinant PAPT showed that the enzyme is likely to be a monomer and requires no divalent cations. The optimum pH and temperature were 8.0 and 50 degrees C, respectively. Km values were calculated as 0.49 +/- 0.037 mu M for FC P (a plausible intermediate of FC A biosynthesis) and 8.3 +/- 0.63 mu M for DMAPP, with a kcat of 55.3 +/- 3.3x10-3 s. The enzyme did not act on representative substrates of the above-mentioned three types of prenyltransferase, but showed a weak transfer activity of geranyl diphosphate to FC P.
• Menaquinone Biosyntheses in Microorganisms

  Tohru Dairi

  NATURAL PRODUCT BIOSYNTHESIS BY MICROORGANISMS AND PLANT, PT A 515 107 - 122 2012年 [査読有り][通常論文]
   

  In prokaryotes, menaquinone (MK) is involved in an electron-transfer pathway. Its biosynthesis was established in the 1970s and 1980s with Escherichia coli. However, a bioinformatic analysis of whole genome sequences has suggested the presence of an alternative pathway. We investigated a novel pathway in a Streptomyces strain. The C-13-labeling pattern of MK purified from a Streptomyces strain grown on [U-C-13]glucose was quite different from that of E. coli. We searched for candidate genes participating in the pathway by in silico screening, and the involvement of these genes in the pathway was confirmed by gene-disruption experiments. We also employed mutagenesis to isolate auxotrophic mutants and used these mutants as hosts for shotgun cloning experiments. Metabolites that accumulated in the culture broth of the mutants were isolated and their structures were determined. Taken together, the results indicated an alternative pathway (futalosine (FL) pathway). Moreover, there were three possible routes in the early part of the FL pathway. FL was directly formed by MqnA in Thermus thermophilus and converted into dehypoxanthinyl FL (DHFL). In Acidothermus cellulolyticus, Streptomyces coelicolor, and Helicobacter pylori, aminodeoxyfutalosine (AFL) was formed by MqnA. In the case of the former two strains, AFL was converted to FL by deaminases then to DHFL. In contrast, AFL was directly converted to DHFL in H. pylori.
• Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from Ralstonia eutropha

  Xuerong Han, Yasuharu Satoh, Toshifumi Satoh, Ken&apos, ichiro Matsumoto, Toyoji Kakuchi, Seiichi Taguchi, Tohru Dairi, Masanobu Munekata, Kenji Tajima

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 92 3 509 - 517 2011年11月 [査読有り][通常論文]
   

  A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 x 10(5), and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T (g), T (m), and T (d) (10%) were observed at -1.1A degrees C, 158.8A degrees C, and 252.7A degrees C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.
• Isolation of a thermotolerant bacterium producing medium-chain-length polyhydroxyalkanoate

  Y. Satoh, K. Tajima, S. Nakamoto, H. Xuerong, T. Matsushima, T. Ohshima, S. Kawano, T. Erata, T. Dairi, M. Munekata

  JOURNAL OF APPLIED MICROBIOLOGY 111 4 811 - 817 2011年10月 [査読有り][通常論文]
   

  Aims: The aim of this study was to isolate a thermotolerant micro-organism that produces polyhydroxyalkanoates (PHAs) composed of medium-chain-length (mcl) HA units from a biodiesel fuel (BDF) by-product as a carbon source. Methods and Results: We successfully isolated a thermotolerant micro-organism, strain SG4502, capable to accumulate mcl-PHA from a BDF by-product as a carbon source at a cultivation temperature of 45 degrees C. The strain could also produce mcl-PHA from acetate, octanoate and dodecanoate as sole carbon sources at cultivation temperatures up to 55 degrees C. Taxonomic studies and 16S rRNA gene sequence analysis revealed that strain SG4502 was phylogenetically affiliated with species of the genus Pseudomonas. This study is the first report of PHA synthesis by a thermotolerant Pseudomonas. Conclusions: A novel thermotolerant bacterium capable to accumulate mcl-PHA from a BDF by-product was successfully isolated. Significance and Impact of the Study: A major issue regarding industrial production of microbial PHAs is their much higher production cost compared with conventional petrochemical-based plastic materials. Especially significant are the cost of a fermentative substrate and the running cost to maintain a temperature suitable for microbial growth. Thus, strain SG4502, isolated in this study, which assimilates BDF by-product and produces PHA at high temperature, would be very useful for practical application in industry.
• 20 糸状菌Phomopsis amygdaliが生産するフシコクシン生合成マシナリーの全容解明(口頭発表の部)

  野池 基義, 小野 裕介, 荒木 祐治, 谷尾 亮, 濱野 吉十, 樋口 雄介, 豊増 知伸, 佐々 武史, 加藤 修雄, 大利 徹

  天然有機化合物討論会講演要旨集 53 115 - 120 天然有機化合物討論会 2011年09月02日 

  Both fusicoccin A (FC) and structurally related cotylenin A (CN) are diterpene glucosides and show a phytohormone-like activity. However, only CN induces the differentiation of human myeloid leukemia cells. Since the CN producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out and an elimination of hydroxyl group at 12-position of FC was essential to have the CN-like activity. Moreover, modified FC with hydroxyl group at 3-position was recently shown to be more effective. Therefore, we tried to identify a gene catalyzing 12-hydroxylation and to breed a mutant producing a modified FC without hydroxyl group at 12-position by a disruption of the gene. Previous identification of fusicocca-2,10(14)-diene synthase gene in Phomopsis amygdali, a FC producer, enabled us to identify a partial gene cluster for bisoynthesis of FC. However, other biosynthetic genes still remained unknown. In this study, we identified another gene cluster containing nine genes by draft genome sequencing. Of these, two cytochrome P450s catalyzing 813 and 9-hydrozylations, glycosylation, methylation, prenylation, and acetylation genes were confirmed to encode enzymes with the expected activities. We also identified a cytochrome P450 catalyzing 12-hydroxylations by a gene disruption experiment. The remaining one cytochrome P450 gene therefore probably catalyzes hydroxylation at the 19-position.
• Synthesis of (±)-Cyclic De-hypoxanthine Futalosine, the Biosynthetic Intermediate in an Alternative Biosynthetic Pathway for Menaquinones

  A. Yajima, S. Kouno, M. Mogi, R. Katsuta, T. Dairi, H. Seto, T. Nukada

  Tetrahedron Lett 52 38 4934 - 4937 2011年09月 [査読有り][通常論文]
   

  The first synthesis of (+/-)-cyclic dehypoxanthine futalosine (cyclic DHFL), a biosynthetic intermediate in the futalosine pathway for menaquinones operating in microorganisms, has been achieved. Efficient growth of the Streptomyces coelicolor mutant, which lacks the cyclic DHFL synthetase gene (mqnC gene) was observed in the presence of synthetic (+/-)-cyclic DHFL. (C) 2011 Elsevier Ltd. All rights reserved.
• Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal formation

  Shunji Takahashi, Atsushi Toyoda, Yasuyo Sekiyama, Hiroshi Takagi, Toshihiko Nogawa, Masakazu Uramoto, Ryuichiro Suzuki, Hiroyuki Koshino, Takuto Kumano, Suresh Panthee, Tohru Dairi, Jun Ishikawa, Haruo Ikeda, Yoshiyuki Sakaki, Hiroyuki Osada

  NATURE CHEMICAL BIOLOGY 7 7 461 - 468 2011年07月 [査読有り][通常論文]
   

  Spiroacetal compounds are ubiquitous in nature, and their stereospecific structures are responsible for diverse pharmaceutical activities. Elucidation of the biosynthetic mechanisms that are involved in spiroacetal formation will open the door to efficient generation of stereospecific structures that are otherwise hard to synthesize chemically. However, the biosynthesis of these compounds is poorly understood, owing to difficulties in identifying the responsible enzymes and analyzing unstable intermediates. Here we comprehensively describe the spiroacetal formation involved in the biosynthesis of reveromycin A, which inhibits bone resorption and bone metastases of tumor cells by inducing apoptosis in osteoclasts. We performed gene disruption, systematic metabolite analysis, feeding of labeled precursors and conversion studies with recombinant enzymes. We identified two key enzymes, dihydroxy ketone synthase and spiroacetal synthase, and showed in vitro reconstruction of the stereospecific spiroacetal structure from a stable acyclic precursor. Our findings provide insights into the creation of a variety of biologically active spiroacetal compounds for drug leads.
• Dioxygenases, Key Enzymes to Determine the Aglycon Structures of Fusicoccin and Brassicicene, Diterpene Compounds Produced by Fungi

  Yusuke Ono, Atsushi Minami, Motoyoshi Noike, Yusuke Higuchi, Tomonobu Toyomasu, Takeshi Sassa, Nobuo Kato, Tohru Dairi

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 133 8 2548 - 2555 2011年03月 [査読有り][通常論文]
   

  Fusicoccin A and cotylenin A are structurally related diterpene glucosides and show a phytohormone-like activity. However, only cotylenin A induces the differentiation of human myeloid leukemia cells. Since the cotylenin A producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out and a modified fusicoccin A with hydroxyl group at the 3-position showed a similar biological activity with that of cotylenin A. We then searched for an enzyme source that catalyzes the introduction of a hydroxyl group into the 3-position and found that brassicicene C, which is structurally related to fusicoccin A with hydroxyl group at the 3-position, was produced by Alternaria brassicicola ATCC96836. We recently cloned a brassicicene C biosynthetic gene cluster including the genes encoding fusicocca-2,10(14)-diene synthase and two cytochrome P450s, which were responsible for the formation of fusicocca-2,10(14)-diene-8 beta,16-diol. In this study, we report that a alpha-ketoglutarate dependent dioxygenase, the gene coding for which was located in the cluster, catalyzed a hydroxylation at the 3-position of fusicocca-2,10(14)-diene-8 beta,16-diol. On the other hand, a alpha-ketoglutarate-dependent dioxygenase, which had been identified in a fusicoccin A biosynthetic gene cluster, catalyzed the 16-oxidation of fusicocca-2,10(14)-diene-8 beta,16-diol to yield an aldehyde (8 beta-hydroxyfusicocca-1,10(14)-dien-16-al), although both dioxygenases had 51% amino acid sequence identity. These findings suggested that the dioxygenases played critical roles for the formation of the fusicoccin A-type and cotylenin A-/brassicicene C-type aglycons. Moreover, we showed that short-chain dehydrogenase/reductase located in the fusicoccin A biosynthetic gene cluster catalyzed the reduction of the aldehyde to yield fusicocca-1,10(14)-diene-8 beta,16-diol.
• Diversity of the Early Step of the Futalosine Pathway

  Chisato Arakawa, Masahiro Kuratsu, Kazuo Furihata, Tomoshige Hiratsuka, Nobuya Itoh, Haruo Seto, Tohru Dairi

  ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 55 2 913 - 916 2011年02月 [査読有り][通常論文]
   

  We recently demonstrated that the futalosine pathway was operating in some bacteria for the biosynthesis of menaquinone and that futalosine was converted into dehypoxanthinyl futalosine (DHFL) by an MqnB of Thermus thermophilus. In this study, we found that aminodeoxyfutalosine, which has adenine instead of hypoxanthine in futalosine, was directly converted into DHFL by an MqnB of Helicobacter pylori. Therefore, this step is potentially an attractive target for the development of specific anti-H. pylori drugs.
• Branched fatty acids inhibit the biosynthesis of menaquinone in Helicobacter pylori

  Runi Tanaka, Takao Kunisada, Nobuaki Kushida, Keiko Yamada, Shunsuke Ikeda, Motoyoshi Noike, Yuusuke Ono, Nobuya Itoh, Hideto Takami, Haruo Seto, Tohru Dairi

  JOURNAL OF ANTIBIOTICS 64 1 151 - 153 2011年01月 [査読有り][通常論文]
  
• Convergent strategies in biosynthesis

  Tohru Dairi, Tomohisa Kuzuyama, Makoto Nishiyama, Isao Fujii

  NATURAL PRODUCT REPORTS 28 6 1054 - 1086 2011年 [査読有り][通常論文]
   

  This review article focuses on how nature sometimes solves the same problem in the biosynthesis of small molecules but using very different approaches. Four examples, involving isopentenyl diphosphate, menaquinone, lysine, and aromatic polyketides, are highlighted that represent different strategies in convergent metabolism.
• A NEW MENAQUINONE BIOSYNTHETIC PATHWAY, DIVERSITY OF THE EARLY STEP OF THE PATHWAY

  Tohru Dairi

  PROGRESS ON POST-GENOME TECHNOLOGIES AND MODERN NATURAL PRODUCTS, 2011 9 - 9 2011年 [査読有り][通常論文]
  
• Analysis of the Lactobacillus Metabolic Pathway

  Masahiro Kuratsu, Yoshimitsu Hamano, Tohru Dairi

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 76 21 7299 - 7301 2010年11月 [査読有り][通常論文]
   

  We performed analyses of the phenotypic and genotypic relationships focusing on biosyntheses of amino acids, purine/pyrimidines, and cofactors in three Lactobacillus strains. We found that Lactobacillus fermentum IFO 3956 perhaps synthesized para-aminobenzoate (PABA), an intermediate of folic acid biosynthesis, by an alternative pathway.
• Substrate specificity of the CYC2 enzyme from Kitasatospora griseola: production of sclarene, biformene and novel bicyclic diterpenes by the enzymatic reactions of labdane- and halimane-type diterpene diphosphates

  Nakano Chiaki, Hoshino Tsutomu, Sato Tsutomu, Toyomasu Tomonobu, Dairi Tohru, Sassa Takeshi

  TETRAHEDRON LETTERS 51 1 125 - 128 2010年01月06日 [査読有り][通常論文]
  
• Functional analyses of cytochrome P450 genes responsible for the early steps of brassicicene C biosynthesis

  Makoto Hashimoto, Yusuke Higuchi, Shunji Takahashi, Hiroyuki Osada, Toshiyuki Sakaki, Tomonobu Toyomasu, Takeshi Sassa, Nobuo Kato, Tohru Dairi

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 19 19 5640 - 5643 2009年10月 [査読有り][通常論文]
   

  We previously revealed that Orf8 and Orf6, which were identified in the brassicicene C biosynthetic gene cluster in Alternaria brassicicola strain ATCC96836, were fusicoccadiene (FD) synthase and 16-O-methyltransferase, respectively. In the present Letter, the early biosynthetic steps after the formation of FD were investigated. Plasmids carrying the FD synthase gene, one (or two) of five cytochrome P450 genes (orf1, orf2, orf5, orf7, and orf11) identified in the cluster and a cytochrome P450 reductase gene cloned from strain ATCC96836 were constructed and introduced into Saccharomyces cerevisiae. Based on the structures of the compounds produced by the transformants, Orf1 is suggested to be an 8 beta-hydroxylation enzyme that yields FD 8 beta-ol ( 4), followed by 16-hydroxylation by Orf7 to produce FD 8 beta 16-diol ( 5). (C) 2009 Elsevier Ltd. All rights reserved.
• An alternative menaquinone biosynthetic pathway operating in microorganisms: an attractive target for drug discovery to pathogenic Helicobacter and Chlamydia strains

  Tohru Dairi

  JOURNAL OF ANTIBIOTICS 62 7 347 - 352 2009年07月 [査読有り][通常論文]
   

  Menaquinone is an essential vitamin as an obligatory component of the electron transfer pathway in microorganisms. Menaquinone has been shown to be derived from chorismate by eight enzymes, designated MenA to -H in Escherichia coli. However, bioinformatic analyses of whole-genome sequences have suggested that some microorganisms, such as Helicobacter pylori and Campylobacter jejuni, which are known to cause gastric carcinoma and diarrhea, respectively, do not have orthologs of most of the men genes, although they synthesize menaquinone. The (13)C-labeling pattern of menaquinone purified from Streptomyces coelicolor A3(2) grown on [U-(13)C]glucose was quite different from that of E. coli, suggesting that an alternative pathway was operating in the strain. We searched for candidate genes participating in the alternative pathway by in silico screening, and the involvement of these genes in the pathway was confirmed by gene-disruption experiments. We also used multagenesis to isolate mutants that required menaquinone for their growth and used these mutants as hosts for shotgun cloning experiments. Metabolites that accumulated in the culture broth of mutants were isolated and their structures were determined. Taking these results together, we deduced the outline of the alternative pathway, which branched at chorismate in a similar manner to the known pathway but then followed a completely different pathway. As humans and some useful intestinal bacteria, such as lactobacilli, lack the alternative pathway, it would be an attractive target for the development of chemotherapeutics. The Journal of Antibiotics (2009) 62, 347-352; doi:10.1038/ja.2009.46; published online 26 June 2009
• Enzymatic Properties of Futalosine Hydrolase, an Enzyme Essential to a Newly Identified Menaquinone Biosynthetic Pathway

  Tomoshige Hiratsuka, Nobuya Itoh, Haruo Seto, Tohru Dairi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 73 5 1137 - 1141 2009年05月 [査読有り][通常論文]
   

  In prokaryotes, menaquinone is used for respiration. In Escherichia coli, menaquinone is biosynthesized from chorismate by seven enzymes. However, very recently, we identified an alternative pathway (the futalosine pathway), which operates in some bacteria, including Streptomyces coelicolor, Helicobacter pylori, Campylobacter jejuni, and Thermus thermophilus. We describe the steps of this pathway, which branches at chorismate in a manner similar to the known pathway, but then follows a different route. This new pathway includes futalosine, an unusual nucleoside derivative consisting of inosine and o-substituted benzoate moieties, as a biosynthetic intermediate. In this study, a recombinant futalosine hydrolase (TTHA0556) of T. thermophilus, which participates in the second step of the pathway and catalyzes the reaction releasing hypoxanthine from futalosine, was prepared and used in functional analyses. Recombinant TTHA0556 formed a homotetramer and reacted only with futalosine; other structurally related nucleotides and nucleosides were not accepted. Recombinant TTHA0556 required no cofactors, and the optimum pH and temperature were 4.5 and 80 degrees C. The Km value was calculated to be 154.0 +/- 5.3 Km and the mu M value was 1.02/s. Recombinant TTHA0556 was slightly inhibited by hypoxanthine, with a Ki value of 1.1 mm.
• Identification and functional analysis of brassicicene C biosynthetic gene cluster in Alternaria brassicicola

  Atsushi Minami, Naoto Tajima, Yusuke Higuchi, Tomonobu Toyomasu, Takeshi Sassa, Nobuo Kato, Tohru Dairi

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 19 3 870 - 874 2009年02月 [査読有り][通常論文]
   

  The biosynthetic gene cluster of brassicicene C was identified in Alternaria brassicicola strain ATCC 96836 from genome database search. In vivo and in vitro study clearly revealed the function of Orf8 and Orf6 as a fusicoccadiene synthase and methyltransferase, respectively. The understanding toward the biosynthetic pathway promises construction of this type of diterpene compounds with genetic engineering. (C) 2008 Elsevier Ltd. All rights reserved.
• Biosynthetic Gene-Based Secondary Metabolite Screening: A New Diterpene, Methyl Phomopsenonate, from the Fungus Phomopsis amygdali

  Tomonobu Toyomasu, Akane Kaneko, Tetsuo Tokiwano, Yuya Kanno, Yuri Kanno, Rie Niida, Shigeyoshi Miura, Taiki Nishioka, Chiho Ikeda, Wataru Mitsuhashi, Tohru Dairi, Tomikazu Kawano, Hideaki Oikawa, Nobuo Kato, Takeshi Sassa

  JOURNAL OF ORGANIC CHEMISTRY 74 4 1541 - 1548 2009年02月 [査読有り][通常論文]
   

  The presence of the geranylgeranyl diphosphate synthase (GGS) gene is a common feature of gene clusters for diterpene biosynthesis. We demonstrated identification of a diterpene gene cluster using homology-based PCR of GGS genes and the subsequent genome walking in the fungus Phomopsis amygdali N2. Structure determination of a novel diterpene hydrocarbon phomopsene provided by enzymatic synthesis with the recombinant terpene synthase PaPS and screening of fungal broth extracts with reference to characteristic NMR signals of phomopsene allowed us to isolate a new diterpene, methyl phomopsenonate. The versatility of the gene-based screening of unidentified diterpenes is discussed in regard to fungal genomic data.
• An alternative menaquinone biosynthetic pathway operating in microorganisms

  Tohru Dairi

  Seikagaku 81 2 95 - 99 2009年 [査読有り][通常論文]
  
• An alternative menaquinone biosynthetic pathway operating in microorganisms

  Tomoshige Hiratsuka, Kazuo Furihata, Jun Ishikawa, Haruyuki Yamashita, Nobuya Itoh, Haruo Seto, Tohru Dairi

  SCIENCE 321 5896 1670 - 1673 2008年09月 [査読有り][通常論文]
   

  In microorganisms, menaquinone is an obligatory component of the electron- transfer pathway. It is derived from chorismate by seven enzymes in Escherichia coli. However, a bioinformatic analysis of whole genome sequences has suggested that some microorganisms, including pathogenic species such as Helicobacter pylori and Campylobacter jejuni, do not have orthologs of the men genes, even though they synthesize menaquinone. We deduced the outline of this alternative pathway in a nonpathogenic strain of Streptomyces by bioinformatic screening, gene knockouts, shotgun cloning with isolated mutants, and in vitro studies with recombinant enzymes. As humans and commensal intestinal bacteria, including lactobacilli, lack this pathway, it represents an attractive target for the development of chemotherapeutics.
• Identification of diterpene biosynthetic gene clusters and functional analysis of labdane-related diterpene cyclases in Phomopsis amygdali

  Tomonobu Toyomasu, Rie Nuda, Hiromichi Kenmoku, Yuri Kanno, Shigeyoshi Miura, Chiaki Nakano, Yoshihito Shiono, Wataru Mitsuhashi, Hiroaki Toshima, Hideaki Oikawa, Tsutomu Hoshino, Tohru Dairi, Nobuo Kato, Takeshi Sassa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 72 4 1038 - 1047 2008年04月 [査読有り][通常論文]
   

  Two diterpene biosynthesis gene clusters in the fusicoccin-producing fungus, Phomopsis amygdali, were identified by genome walking from PaGGS1 and PaGGS4 which encode the geranylgeranyl diphosphate (GGDP) synthases. The diterpene cyclase-like genes, PaDC1 and PaDC2, were respectively located proximal to PaGGS1 and PaGGS4. The amino acid sequences of these two enzymes were similar to those of fungal labdane-related diterpene cyclases. Recombinant PaDC1 converted GGDP mainly into phyllocladan-16 alpha-ol via (+)-copalyl diphosphate (CDP) and trace amounts of several labdane-related hydrocarbons which had been identified from the P. amygdali F6 mycelia. Since phyllocladan-16 alpha-ol had not been identified in P. amygdali F6 mycelia, we isolated phyllocladan-16 alpha-ol from the mycelia. Recombinant PaDC2 converted GGDP into (+)-CDP. Furthermore, we isolated the novel diterpenoid, phyllocladan-11 alpha,16 alpha,18-triol, which is a possible metabolite of phyllocladan-16 alpha-ol in the mycelia. We propose that genome walking offers a useful strategy for the discovery of novel natural products in fungi.
• Studies on a new biosynthetic pathway for menaquinone

  Haruo Seto, Yuusuke Jinnai, Tomoshige Hiratsuka, Miwako Fukawa, Kazuo Furihata, Nobuya Itoh, Tohru Dairi

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 130 17 5614 - + 2008年04月 [査読有り][通常論文]
   

  Menaquinone is biosynthesized from chorismate via a new pathway involving 1,4-dihydroxy-6-naphthoate in Streptomyces and presumably in pathogenic bacteria Helicobacter and Campylobacter.
• Cloning of the gene cluster responsible for the biosynthesis of brasilicardin A, a unique diterpenoid

  Yutaka Hayashi, Nobuyasu Matsuura, Hiroaki Toshima, Nobuya Itoh, Jun Ishikawa, Yuzuru Mikami, Tohru Dairi

  JOURNAL OF ANTIBIOTICS 61 3 164 - 174 2008年03月 [査読有り][通常論文]
   

  Brasilicardin A (BCA), produced by Nocardia brasiliensis IFM 0406 (currently referred to as N. terpenica), has a unique structure consisting of a diterpene skeleton with L-rhamnose, N-acetylglucosamine, amino acid, and 3-hydroxybenzoate moieties, and exhibits potent biological activities. To understand the biosynthetic machinery of this unique compound, we have cloned the corresponding gene cluster. Firstly, we cloned a gene by PCR that encodes geranylgeranyl diphosphate synthase (GGPPS), which produces a direct precursor of diterpene compounds. We obtained four candidate genes and one of the genes was confirmed to encode a GGPPS. By sequence analysis of regions flanking the GGPPS gene, we identified eleven genes (bra1-11), all oriented in the same direction. We did not, however, detect any genes related to L-rhamnose and N-acetylglucosamine biosyntheses in the flanking regions. A gene disruption experiment did indeed show that this gene cluster was responsible for BCA biosynthesis.
• Comparison of the enzymatic properties of ent-copalyl diphosphate synthases in the biosynthesis of phytoalexins and gibberellins in rice

  Yutaka Hayashi, Tomonobu Toyomasu, Yuko Hirose, Yu Onodera, Wataru Mitsuhashi, Hisakazu Yamane, Takeshi Sassa, Tohru Dairi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 72 2 523 - 530 2008年02月 [査読有り][通常論文]
   

  The rice genome contains two ent-copalyl diphosphate synthase genes: OsCPS1 acts in gibberellin (phytohormone) biosynthesis, and OsCPS2/OsCyc2 acts in the synthesis of oryzalexins A-F and phytocassanes A-E (phytoalexins). We characterized the enzymatic properties of recombinant OsCPS2/OsCyc2 fused with a tag-protein at the N-terminus, and compared them to those of OsCPS1. Several enzymatic properties of OsCPS2/OsCyc2, including the optimal pH, optimal temperature, divalent cation requirement, and kinetic values for the geranylgeranyl diphosphate (GGDP) substrate, were almost the same as those of OsCPS1. However, OsCPS2/OsCyc2 activity was not inhibited by 50-60 mu m GGDP substrate, by which the OsCPS1 activity was inhibited. Furthermore, the OsCPS1 activity exhibited approximately 70% inhibition by 100 mu m Amo-1618 (a gibberellin biosynthetic inhibitor), whereas the OsCPS2/OsCyc2 activity exhibited approximately 10% inhibition. These results indicate that the properties of OsCPS2/OsCyc2 were partially different from those of OsCPS1, although OsCPS2/OsCyc2 catalyzes the same reaction step as OsCPS1.
• Engineering the phenylacetaldehyde reductase mutant for improved substrate conversion in the presence of concentrated 2-propanol

  Yoshihide Makino, Tohru Dairi, Nobuya Itoh

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 77 4 833 - 843 2007年12月 [査読有り][通常論文]
   

  Phenylacetaldehyde reductase (PAR) from Rhodococcus sp. ST-10 is useful for chiral alcohol production because of its broad substrate specificity and high stereo-selectivity. The conversion of ketones into alcohols by PAR requires the coenzyme NADH. PAR can regenerate NADH by oxidizing additional alcohols, especially 2-propanol. However, substrate conversion by wild-type PAR is suppressed in concentrated 2-propanol. Previously, we developed the Sar268 mutant of PAR, which can convert several substrates in the presence of concentrated 2-propanol. In this paper, further mutational engineering of Sar268 was performed to achieve higher process yield. Each of nine amino acid positions that had been examined for generating Sar268 was subjected to saturation mutagenesis. Two novel substitutions at the 42nd amino acid position increased m-chlorophenacyl chloride (m-CPC) conversion. Moreover, several nucleotide substitutions identified from libraries of random mutations around the start codon also improved the PAR activity. E. coli cells harboring plasmid pHAR1, which has the integrated sequence of the top clones from the above selections, provided greater conversion of m-CPC and ethyl 4-chloro-3-oxobutanoate than the Sar268 mutant, with very high optical purity of products. This mutant is a promising novel biocatalyst for efficient chiral alcohol production.
• Cloning of the gene cluster responsible for biosynthesis of KS-505a (longestin), a unique tetraterpenoid

  Yutaka Hayashi, Hiroyasu Onaka, Nobuya Itoh, Haruo Seto, Tohru Dairi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 12 3072 - 3081 2007年12月 [査読有り][通常論文]
   

  KS-505a (longestin), produced by Streptomyces argenteolus, has a unique structure that consists of a tetraterpene (C40) skeleton, to which a 2-O-methylglucuronic acid and an o-succinyl benzoate moiety are attached. It is a novel inhibitor of calmodulin-dependent cyclic-nucleotide phosphodiesterase, which is representative of a potent anti-amnesia drug. As a first step to understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we cloned a KS505a biosynthetic gene cluster. First we searched for a gene encoding octaprenyl diphosphates, which yielded a C40 precursor by PCR, and four candidate genes were obtained. Among these, one was confirmed to have the expected enzyme activity by recombinant enzyme assay. On the basis of an analysis of the flanking regions of the gene, a putative KS-505a biosynthetic gene cluster consisting of 24 ORFs was judged perhaps to be present on a 28-kb DNA fragment. A gene disruption experiment was also employed to confirm that the cluster indeed participated in KS-505a biosynthesis. This is believed to be the first report detailing the gene cluster of a cyclized tetraterpenoid.
• Studies on terpenoids produced by actinomycetes: Oxaloterpins A, B, C, D, and E, Diterpenes from Streptomyces sp KO-3988

  Keiichiro Motohashi, Ryoko Ueno, Masayuki Sue, Kazuo Furihata, Takuo Matsumoto, Tohru Dairi, Satoshi Omura, Haruo Seto

  JOURNAL OF NATURAL PRODUCTS 70 11 1712 - 1717 2007年11月 [査読有り][通常論文]
   

  Following screening for terpenoids produced by Streptomyces sp. KO-3988, five new diterpenes named oxaloterpins A (1), B (2), C (3), D (4), and E (5) together with the known viguiepinone (6) were isolated from culture broth, and their structures were established on the basis of extensive NMR and MS analyses. The absolute configuration of oxaloterpin A was determined by the modified Mosher's method as 3R, 5S, 8S, I OR, 13S.
• Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi

  Tomonobu Toyomasu, Mai Tsukahara, Akane Kaneko, Rie Niida, Wataru Mitsuhashi, Tohru Dairi, Nobuo Kato, Takeshi Sassa

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 104 9 3084 - 3088 2007年02月 [査読有り][通常論文]
   

  Fusicoccins are a class of diterpene glucosides produced by the plant-pathogenic fungus Phomopsis amygdali. As modulators of 14-3-3 proteins, fusicoccins function as potent activators of plasma membrane H+-ATPase in plants and also exhibit unique biological activity in animal cells. Despite their well studied biological activities, no genes encoding fusicoccin biosynthetic enzymes have been identified. Cyclic diterpenes are commonly synthesized via cyclization of a C-20 precursor, geranylgeranyl diphosphate (GGDP), which is produced through condensation of the universal C-5 isoprene units dimethylallyl diphosphate and isopentenyl diphosphate by prenyltransferases. We found that (+)-fusicocca-2,10 (14)-diene, a tricyclic hydrocarbon precursor for fusicoccins, is biosynthesized from the C-5 isoprene units by an unusual multifunctional enzyme, A amygdali fusicoccadiene synthase (PaFS), which shows both prenyltransferase and terpene cyclase activities. The functional analysis of truncated mutants and site-directed mutagenesis demonstrated that PaFS consists of two domains: a terpene cyclase domain at the N terminus and a prenyltransferase domain at the C terminus. These findings suggest that fusicoccadiene can be produced efficiently in the fungus by using the C5 precursors, irrespective of GGDP availability. In fact, heterologous expression of PaFS alone resulted in the accumulation of fusicocca 2,10 (14)-diene in Escherichia cofi cells, whereas no product was detected in E. coli cells expressing Gibberella fujikuroi ent-kaurene synthase, another fungal diterpene cyclase that also uses GGDP as a substrate but does not contain a prenyltransferase domain. Genome walking suggested that fusicoccin biosynthetic enzymes are encoded as a gene cluster near the PaFS gene.
• Functional analysis of eubacterial ent-copalyl diphosphate synthase and pimara-9(11),15-diene synthase with unique primary sequences

  Chiho Ikeda, Yutaka Hayashi, Nobuya Itoh, Haruo Seto, Tohru Dairi

  JOURNAL OF BIOCHEMISTRY 141 1 37 - 45 2007年01月 [査読有り][通常論文]
   

  We have previously cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15-diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp. strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-CDP synthase and a GGDP synthase, respectively, by experiments using recombinant enzymes. In this study, ORF3, that did not show similarities with any other known proteins was expressed in Escherichia coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into PAID. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg2+ and Zn2+ for its activity. The optimum pH and temperature were 5.5 and 35 degrees C. The Km value was calculated to be 13.7 +/- 1.0 mu M for GGDP and the kcat value was 3.3 x 10(-2)/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30 degrees C. The Kin value for ent-CDP was estimated to be 2.6 +/- 0.2 mu M and the kcat value was 1.4 x 10(-3)/sec.
• A comparative analysis of the sugar phosphate cyclase superfamily involved in primary and secondary metabolism

  Xiumei Wu, Patricia M. Flatt, Oliver Schloerke, Axel Zeeck, Tohru Dairi, Taifo Mahmud

  CHEMBIOCHEM 8 2 239 - 248 2007年01月 [査読有り][通常論文]
   

  Sugar phosphate cyclases (SPCs) catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites for example, aromatic amino acids, and clinically relevant secondary metabolites, for example, aminocyclitol/aminoglycoside and ansamyclin antibiotics. Feeding experiments with isotopically labeled cyclitois revealed that cetonicacytone A, a unique C-N-aminocyclitol antibiotic isolated from an insect endophytic Actinomyces sp., is derived form 2-epi-5-valioline, a product of SPC. By using heterologous probes from the 2-epi-5-epi valiolone synthase class of SPCs, an SPC homologue, gene, cetA, was isolated from the cetoniacytone producer, CetA is closely relted to BE-orf9 found in the BE-40644 biosynthetic gene cluster from Actionoplanes sp. strain A40644. Recombinant expression of cetA and Be-orf9 and biochemical characterization of the gene products confirmed their function as 2-epi-valinolone synthases. Further phylogenetic analysis of SPC sequences revealed a new clade of SPCs that might regulate the biosynthesis of a novel set of secondary metabolites.
• P-355 Biosynthesis of a Natural Polyketide-Isoprenoid Hybrid Compound Furaquinocin A : Identification and Heterologous Expression of the Gene Cluster

  Hayashi Yutaka, Kawasaki Takashi, Kuzuyama Tomohisa, Furihata Kazuo, Itoh Nobuya, Seto Haruo, Dairi Tohru

  International Symposium on the Chemistry of Natural Products 2006 "P - 355" 天然有機化合物討論会 2006年07月23日
• Biosynthesis of a natural polyketide-isoprenoid hybrid compound, furaquinocin A: Identification and heterologous expression of the gene cluster

  T Kawasaki, Y Hayashi, T Kuzuyama, K Furihata, N Itoh, H Seto, T Dairi

  JOURNAL OF BACTERIOLOGY 188 4 1236 - 1244 2006年02月 [査読有り][通常論文]
   

  Furaquinocin (FQ) A, produced by Streptomyces sp. strain KO-3988, is a natural polyketide-isoprenoid hybrid compound that exhibits a potent antitumor activity. As a first step toward understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we have cloned an FQ A biosynthetic gene cluster by taking advantage of the fact that an isoprenoid biosynthetic gene cluster generally exists in flanking regions of the mevalonate (NW) pathway gene cluster in actinomycetes. Interestingly, Streptomyces sp. strain KO-3988 was the first example of a microorganism equipped with two distinct mevalonate pathway gene clusters. We were able to localize a 25-kb DNA region that harbored FQ A biosynthetic genes (fur genes) in both the upstream and downstream regions of one of the MV pathway gene clusters (MV2) by using heterologous expression in Streptomyces lividans TK23. This was the first example of a gene cluster responsible for the biosynthesis of a polyketide-isoprenoid hybrid compound. We have also confirmed that four genes responsible for viguiepinol [3-hydroxypimara-9(11),15-diene] biosynthesis exist in the upstream region of the other MV pathway gene cluster (MV1), which had previously been cloned from strain KO-3988. This was the first example of prokaryotic enzymes with these biosynthetic functions. By phylogenetic analysis, these two MV pathway clusters were identified as probably being independently distributed in strain KO-3988 (orthologs), rather than one cluster being generated by the duplication of the other cluster (paralogs).
• Gene cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols

  K Inoue, Y Makino, T Dairi, N Itoh

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 70 2 418 - 426 2006年02月 [査読有り][通常論文]
   

  The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed Isadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).
• Engineering of phenylacetaldehyde reductase for efficient substrate conversion in concentrated 2-propanol

  Y Makino, K Inoue, T Dairi, N Itoh

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 71 8 4713 - 4720 2005年08月 [査読有り][通常論文]
   

  Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanol acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates catalyzed by PAR. To improve the conversion efficiency in high concentrations of substrate and 2-propanol, selection of a PAR mutant library and the subsequent rearrangement of mutations were attempted. With only a single selection round and following the manual combination of advantageous mutations, PAR was successfully adapted for the conversion of high concentrations of substrate with concentrated 2-propanol. This method will be widely applicable for the engineering of enzymes potentially valuable for industry.
• fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis

  KJ Puan, H Wang, T Dairi, T Kuzuyama, CT Morita

  FEBS LETTERS 579 17 3802 - 3806 2005年07月 [査読有り][通常論文]
   

  Although flavodoxin I is indispensable for Escherichia coli growth, the exact pathway(s) where flavodoxin I is essential has not been identified. We performed transposon mutagenesis of the flavodoxin I gene, fldA, in an E. coli strain that expressed mevalonate pathway enzymes and that had a point mutation in the lytB gene of the MEP pathway resulting in the accumulation of (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP). Disruption of fldA abrogated mevalonate-independent growth and dramatically decreased HMBPP levels. The fldA(-) mutant grew with mevalonate indicating that the essential role of flavodoxin I under aerobic conditions is in the MEP pathway. Growth was restored by fldA complementation. Since GcpE (which synthesizes HMBPP) and LytB are iron-sulfur enzymes that require a reducing system for their activity, we propose that flavodoxin is essential for GcpE and possibly LytB activity. Thus, the essential role for flavodoxin I in E. coli is in the MEP pathway for isoprenoid biosynthesis. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• Studies on biosynthetic genes and enzymes of isoprenoids produced by actinomycetes

  T. Dairi

  Japanese J. Antibiot 58 4 227 - 243 2005年04月 [査読有り][通常論文]
   

  Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate, a common precursor of isoprenoids. In addition to this pathway, some Streptomyces strains possess the mevalonate (MV) pathway via which isoprenoid antibiotics are produced. We have recently cloned and analyzed the MV pathway gene clusters and their flanking regions from terpentecin, BE-40644, and furaquinocin A producers. All these clusters contained genes coding for mevalonate kinase, mevalonate diphosphate decarboxylase, phosphomevalonate kinase, type 2 IPP isomerase, HMG-CoA reductase, and HMG-CoA synthase. The order of each of the open reading frames (ORFs) is also the same, and the respective homologous ORFs show more than 70% amino acid identity with each other. In contrast to these conservative gene organizations, the biosynthetic genes of terpentecin, BE-40644, and furaquinocin A were located just upstream and/or downstream of the MV pathway gene cluster. These facts suggested that all the actinomycete strains possessing both the MV and MEP pathways produce isoprenoid compounds and the biosynthetic genes of one of these isoprenoids usually exist adjacent to the MV pathway gene cluster. Therefore, when the presence of the MV cluster is detected by molecular genetic techniques, isoprenoids may be produced by the cultivation of these actinomycete strains. During the course of these studies, we identified diterpene cyclases possessing unique primary structures that differ from those of eukaryotes and catalyze unique reactions.
• Mycobacterium tuberculosis H37Rv3377c encodes the diterpene cyclase for producing the halimane skeleton

  C Nakano, T Okamura, T Sato, T Dairi, T Hoshino

  CHEMICAL COMMUNICATIONS Issue 8: 1016–1018 8 1016 - 1018 2005年02月 [査読有り][通常論文]
   

  The cloning and functional expression of Mycobacterium tuberculosis Rv3377c in Escherichia coli revealed that this gene encodes the diterpene cyclase for producing (+)-5(6),13-halimadiene-15-ol, which accepts geranylgeranyldiphosphate as the intrinsic substrate.
• Purification and characterization of NADPH-dependent aldo-keto reductase specific for beta-keto esters from Penicillium citrinum, and production of methyl (S)-4-bromo-3-hydroxybutyrate

  N Itoh, H Asako, K Banno, Y Makino, M Shinohara, T Dairi, R Wakita, M Shimizu

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 66 1 53 - 62 2004年11月 [査読有り][通常論文]
   

  A novel beta-keto ester reductase (KER) was purified to homogeneity from recombinant Escherichia coli (pTrcKER) cells, which efficiently expressed the ker gene cloned from Penicillium citrinum IFO4631. The enzyme was monomeric and had a molecular mass of 37 kDa. It catalyzed the reduction of some beta-keto esters, especially alkyl 4-halo-3-oxobutyrates. However, it did not catalyze the reverse reaction, the dehydrogenation of alkyl 4-halo-3-hydroxybutyrates and other alcohols. The enzyme required NADPH as a cofactor and showed no activity with NADH. Therefore, it was defined as a NADPH-dependent aldo-keto reductase (AKR3E1), belonging to the AKR superfamily. The enzyme stereospecifically produced methyl (S)-4-bromo-3-hydroxybutyrate from its keto derivative with high stereospecificity (97.9% enantiomer excess). E. coli cells expressing KER and glucose dehydrogenase in the water/butyl acetate two-phase system achieved a high productivity of (S)-4-bromo-3-hydroxybutyrate (277 mM, 54 mg/ml) in the organic solvent layer.
• Presence of copalyl diphosphate synthase gene in an actinomycete possessing the mevalonate pathway

  T Kawasaki, T Kuzuyama, Y Kuwamori, N Matsuura, N Itoh, K Furihata, H Seto, T Dairi

  JOURNAL OF ANTIBIOTICS 57 11 739 - 747 2004年11月 [査読有り][通常論文]
   

  We have previously shown that gene clusters for biosyntheses of terpentecin and BE-40644, a diterpene antibiotic and a sesquiterpene antibiotic, respectively, were located in the adjacent mevalonate pathway gene clusters. In this study, a mevalonate pathway gene cluster was cloned from Streptomyces sp. strain KO-3988, which was known to produce furaquinocin A, employing a hybridization experiment using a 3-hydroxy-3-methyl glutaryl CoA (HMG-CoA) reductase gene, which had been previously cloned from the strain KO-3988, as a probe. By sequencing flanking regions, we found four open reading frames that could encode a putative cytochrome P450 (ORF1), an isoprenoid cyclase (ORF2), an unknown protein (ORF3), and a polyprenyl diphosphate synthase gene (ORF4) in the upstream region of the mevalonate pathway gene cluster, though we did not find any genes related to furaquinocin A biosynthesis. The two ORFs (ORF2 and 4) were expressed as recombinant enzymes in E. coli and used for studies to investigate functions of these products. The ORF4 product was confirmed to be a geranylgeranyl diphosphate (GGDP, C-20) synthase. The ORF2 product proved to catalyze a conversion of GGDP into copalyl diphosphate, the first example of an enzyme with this function of prokaryotic origin. These results again showed that actinomycetes possessing the mevalonate pathway usually produce an isoprenoid and that its biosynthetic gene cluster exists in adjacent the mevalonate pathway gene cluster.
• Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens

  T Nakano, K Miyake, H Endo, T Dairi, T Mizukami, R Katsumata

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 6 1345 - 1352 2004年06月 [査読有り][通常論文]
   

  For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.
• Heterologous mevalonate production in Streptawyces lividans TK23

  T Kuzuyama, T Dairi, H Yamashita, Y Shoji, H Seto

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 4 931 - 934 2004年04月 [査読有り][通常論文]
   

  Mevalonate is a ubiquitous biosynthetic intermediate of terpenoids and is used as a moisturizer in cosmetics and a chemical for biochemical research. In this study, we have achieved a heterologous production of this useful compound by expression in Streptomyces lividans TK23 of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase genes, which were cloned from Streptomyces sp. strain CL190.
• 3P110 フェニルアセトアルデヒド還元酵素の高濃度2-propanol存在下での基質変換能の改良(蛋白質 F) 蛋白質工学/進化工学)

  牧野 祥嗣, 大利 徹, 伊藤 伸哉

  生物物理 44 S217  一般社団法人 日本生物物理学会 2004年
• A relationship between the mevalonate pathway and isoprenoid production in actinomycetes

  T Kawasaki, T Kuzuyama, K Furihata, N Itoh, H Seto, T Dairi

  JOURNAL OF ANTIBIOTICS 56 11 957 - 966 2003年11月 [査読有り][通常論文]
   

  Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate. In addition to this pathway, some Streptomyces strains have the mevalonate pathway to produce terpenoid antibiotics. We have previously shown that a gene cluster for biosynthesis of terpentecin, a diterpene antibiotic, was located in adjacent the mevalonate pathway gene cluster. In this study, a mevalonate pathway gene cluster was cloned from Actinoplanes sp. strain A40644, an isoprenoid antibiotic BE-40644 producer, to examine whether the mevalonate pathway genes and isoprenoid biosynthetic genes are clustered in genomic DNA. By sequencing flanking regions a probable. BE-40644 biosynthetic gene cluster was found in the downstream region of the mevalonate pathway gene cluster. Heterologous expression of a 9-kb fragment confirmed that a set of the BE-40644 biosynthetic genes was involved in the fragment. This result suggested that the presence of the mevalonate pathway might be a good landmark to detect the production of isoprenoid compounds by actinomycetes.
• A novel enzyme, D-3-hydroxyaspartate aldolase from Paracoccus denitrificans IFO 13301: purification, characterization, and gene cloning

  JQ Liu, T Dairi, N Itoh, M Kataoka, S Shimizu

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 62 1 53 - 60 2003年07月 [査読有り][通常論文]
   

  A novel enzyme, D-3-hydroxyaspartate aldolase (D-HAA), catalyzing the conversion of D-3-hydroxyaspartate to glyoxylate plus glycine, was purified to homogeneity from Paracoccus denitrificans IFO 13301. D-HAA is strictly D-specific as to the alpha-position, whereas the enzyme does not distinguish between threo and erythro forms at the beta-position. In addition to D-3-hydroxyaspartate, the enzyme also acts on D-threonine, D-3-3,4-dihydroxyphenylserine, D-3-3,4-methylenedioxyphenylserine, and D-3-phenylserine. The D-HAA gene was cloned and sequenced. The gene contains an open reading frame consisting of 1,161 nucleotides corresponding to 387 amino acid residues. The predicted amino acid sequence displayed 35% and 22% identity with that of the D-threonine aldolase of Arthrobacer sp. DK-38 and Alcaligenes xylosoxidan IFO 12669, respectively. This is the first paper reporting both a purified enzyme with D-3-hydroxyaspartate aldolase activity and also its gene cloning.
• A new approach for the investigation of isoprenoid biosynthesis featuring pathway switching, deuterium hyperlabeling, and H-1 NMR spectroscopy. The reaction mechanism of a novel Streptomyces diterpene cyclase

  T Eguchi, Y Dekishima, Y Hamano, T Dairi, H Seto, K Kakinuma

  JOURNAL OF ORGANIC CHEMISTRY 68 14 5433 - 5438 2003年07月 [査読有り][通常論文]
   

  Recent methodology for the investigation of isoprenoid biosynthesis featuring pathway switching and hyperdeuteration has shown significant advantages in elucidating the reaction mechanism of a novel Streptomyces diterpene cyclase with use of precise atom-level analysis. Insight into the cyclization mechanism involved in the conversion of geranylgeranyl diphosphate (GGPP) into a clerodane hydrocarbon terpentetriene was obtained by heterologous expression in doubly engineered Streptomyces lividans of a diterpene cyclase gene derived from Streptomyces griseolosporeus, a producer of an unique diterpenoid cytotoxic antibiotic terpentecin, and by in vivo labeling with mevalonate-d(9). The cyclization involved electrophilic protonation, cationic ring closure, Wagner-Meer-wein-type rearrangements, and deprotonation. A key feature was that the labeled metabolite as a mixture of predominantly deuterated mosaic molecules provided sufficient information that close analysis of the labeling pattern for each individual isoprene unit was achieved primarily by H-1 NMR spectroscopy. The cyclization of GGPP into the clerodane skeleton catalyzed by the cyclase appears to involve Si-face specific protonation, intermediates with A/B chair-boat conformation, and specific methyl and hydride migrations to give an intermediary C-4 carbocation. Subsequent collapse of the cation through specific removal of the initiating proton and final elimination of diphosphate gives rise to the terpentetriene hydrocarbon.
• Interconversion of the product specificity of type I eubacterial farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase through one amino acid substitution

  T Kawasaki, Y Hamano, T Kuzuyama, N Itoh, H Seto, T Dairi

  JOURNAL OF BIOCHEMISTRY 133 1 83 - 91 2003年01月 [査読有り][通常論文]
   

  Prenyltransferases catalyze the sequential condensation of isopentenyl diphosphate into prenyl diphosphates with specific chain lengths. Pioneering studies demonstrated that the product specificities of type I prenyltransferases were mainly determined by the amino acid residues at the 4th and 5th positions before the first aspartate-rich motif (FARM) of the prenyltransferases. We previously cloned a type I geranylgeranyl diphosphate synthase (GGDPSase) gene from Streptomyces griseo-losporeus MF730-N6 [Hamano, Y., Dairi, T., Yamamoto, M., Kawasaki, T., Kaneda, K., Kuzuyama, T., Itoh, N., and Seto, H. (2001) Biosci. Biotechnol. Biochem. 65,1627-1635]. In this study, a prenyltransferase gene was cloned from Streptomyces argenteolus A-2 and was confirmed to encode a type I farnesyl diphosphate synthase (FDPSase). Interestingly, the amino acid residues at the 4th and 5th positions before the FARM were the same in these two enzymes. To identify the amino acid that determines the product chain length, mutated enzymes, GGDPSase (L-50S), FDPSase (S-50L), GGDPSase (V-8A), FDPSase (A-8V), GGDPSase (A+57L), and FDPSase (L+58A), in which the amino acid residue at the -50th, -8th, and +57th (58th) position before or after the FARM was substituted with the corresponding amino acid of the other enzyme, were constructed. The GGDPSase (A+57L) and FDPSase (L+58A) produced farnesyl diphosphate and geranylgeranyl diphosphate, respectively. On the other hand, the other mutated enzymes produced prenyl diphosphates with the same chain lengths as the wild type enzymes did. These results showed that the amino acid residue at the 57th (58th) position after the FARM also played an important role in determination of the product specificity.
• Functional analysis of eubacterial diterpene cyclases responsible for biosynthesis of a diterpene antibiotic, terpentecin

  Y Hamano, T Kuzuyama, N Itoh, K Furihata, H Seto, T Dairi

  JOURNAL OF BIOLOGICAL CHEMISTRY 277 40 37098 - 37104 2002年10月 [査読有り][通常論文]
   

  Eubacterial diterpene cyclase genes had previously been cloned from a diterpenoid antibiotic terpentecin producer (Dairi, T., Hamano, Y., Kuzuyama, T., Itoh, N., Furihata, K., and Seto, H. (2001) J. BacterioL 183, 60856094). Their products, open reading frame 11 (ORF11) and ORF12, were essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same basic skeleton as terpentecin. In this study, functional analyses of these two enzymes were performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a cyclized intermediate, terpentedienol diphosphate (TDP), which was then transformed into TTE by the ORF12 product. Interestingly, the ORF12 product directly catalyzed the conversion of GGDP into three olefinic compounds. Moreover, the ORF12 product utilized farnesyl diphosphate as a substrate to give three olefinic compounds, which had the same structures as those formed from GGDP with the exception of the chain lengths. These results suggested that the ORF11 product with a DXDD motif converted GGDP into TDP by a protonation-initiated cyclization and that the ORF12 product with a DDXXD motif completed the transformation of TDP to the olefin, terpentetriene by an ionization-initiated reaction followed by deprotonation. The kinetics of the ORF12 product indicated that the affinity for TDP and GGDP were higher than that of farnesyl diphosphate and that the relative activity of the reaction converting TDP into TTE was highest among the reactions using TDP, GGDP, or farnesyl diphosphate as the substrate. These results suggested that an actual reaction catalyzed by the ORF12 was the conversion of TDP into TTE in vivo.
• Detection of the mevalonate pathway in streptomyces species using the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene

  T Kuzuyama, S Takahashi, T Dairi, H Seto

  JOURNAL OF ANTIBIOTICS 55 10 919 - 923 2002年10月 [査読有り][通常論文]
  
• Chiral alcohol production by NADH-dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH

  N Itoh, M Matsuda, M Mabuchi, T Dairi, J Wang

  EUROPEAN JOURNAL OF BIOCHEMISTRY 269 9 2394 - 2402 2002年05月 [査読有り][通常論文]
   

  Phenylacetaldehyde reductase (PAR) produced by styrene-assimilating Corynebacterium strain ST-10 was used to synthesize chiral alcohols. This enzyme with a broad substrate range reduced various prochiral aromatic ketones and beta-ketoesters to yield optically active secondary alcohols with an enantiomeric purity of more than 98% enantiomeric excess (e.e.). The Escherichia coli recombinant cells which expressed the par gene could efficiently produce important pharmaceutical intermediates; (R)-2-chloro-1-(3-chlorophenyl)ethanol (28 mg.mL(-1)) from m -chlorophenacyl chloride, ethyl (R)-4-chloro-3-hydroxy butanoate) (28 mg.mL(-1)) from ethyl 4-chloro-3-oxobutanoate and (S)-N-tert -butoxycarbonyl(Boc)-3-pyrrolidinol from N -Boc-3-pyrrolidinone (51 mg.mL(-1)), with more than 86% yields. The high yields were due to the fact that PAR could concomitantly reproduce NADH in the presence of 3-7% (v/v) 2-propanol in the reaction mixture. This biocatalytic process provided one of the best asymmetric reductions ever reported.
• Growth-phase dependent expression of the mevalonate pathway in a terpenoid antibiotic-producing Streptomyces strain

  Y Hamano, T Dairi, M Yamamoto, T Kuzuyama, N Itoh, H Seto

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 4 808 - 819 2002年04月 [査読有り][通常論文]
   

  Streptomyces griseolosporeus MF730-N6, a terpenoid antibiotic-terpentecin (Tp) producer, has both the non-mevalonate and mevalonate pathways for the formation of IPP. The Tp biosynthetic gene (ter) and the mevalonate pathway gene cluster (mev) including an HMG-CoA reductase gene (hmgr) had previously been cloned from strain MF730-N6. In this study, two distinct dxs genes (dxs I and dxs 2) and a dxr gene, which encode DXP synthases and DXP reductoisomerase, and participate in the initial and the second step of the nonmevalonate pathway, respectively, were cloned. These gene products were expressed in E. coli and confirmed to have the expected activities. The dxs 1, dxs 2, dxr, mev, and ter genes were used for Northern blot and primer extension analyses to examine temporal expression of these genes together with a gap gene coding for GAP dehydrogenase, which was also cloned in this study and used as an internal control. Transcripts of the dxs 1, dxs 2, dxr, and gap genes were detected throughout the cultivation. On the other hand, messages of the mev and ter genes were not detected at early growth phase but appeared when Tp production started. These results suggested that the nonmevalonate pathway and the mevalonate pathway were mainly used for the primary metabolism and the secondary metabolism, respectively, and that both of the two dxs genes were actually transcribed in this strain.
• Reaction mechanism of the Co2+-activated multifunctional bromoperoxidase-esterase from Pseudomonas putida IF-3

  T Kawanami, M Miyakoshi, T Dairi, N Itoh

  ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 398 1 94 - 100 2002年02月 [査読有り][通常論文]
   

  The reaction mechanism of the Co2+-activated bromoperoxidase-esterase of Pseudomonas putida IF-3 was studied. Site-directed mutagenesis suggested that the serine residue of the catalytic triad conserved in serine hydrolases participates in the bromination and ester hydrolysis reactions. The enzyme released a trace amount of free peracetic acid depending on the concentration of H2O2, which had been considered the intermediate in the reaction of nonmetal haloperoxidases to oxidize halide ions to hypohalous acid. However, the formation of free peracetic acid could not explain the enzyme activation effect by Co2+ ions which completely depleted the free peracetic acid. In addition, the k(cat) value of the enzymatic bromination was 900-fold higher than the rate constant of free peracetic acid-mediated bromination. Those results strongly suggested that the peracetic acid-like intermediate formed at the catalytic site is the true intermediate and that the formation of free peracetic acid is only a minor reaction involving the enzyme. We propose the possible reaction mechanism of this multifunctional enzyme based on these findings. (C) 2002 Elsevier Science.
• Studies on eubacterial diterpene cyclases found in actinomycetes

  T Dairi, T Hamano, N Itoh, T Kuzuyama, K Furihata, H Seto

  NIPPON NOGEIKAGAKU KAISHI-JOURNAL OF THE JAPAN SOCIETY FOR BIOSCIENCE BIOTECHNOLOGY AND AGROCHEMISTRY 76 12 1191 - 1194 2002年 [査読有り][通常論文]
  
• Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin

  T Dairi, Y Hamano, T Kuzuyama, N Itoh, K Furihata, H Seto

  JOURNAL OF BACTERIOLOGY 183 20 6085 - 6094 2001年10月 [査読有り][通常論文]
   

  A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627-1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.
• Cloning of a gene cluster encoding enzymes responsible for the mevalonate pathway from a terpenoid-antibiotic-producing Streptomyces strain

  Y Hamano, T Dairi, M Yamamoto, T Kawasaki, K Kaneda, T Kuzuyama, N Itoh, H Seto

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 7 1627 - 1635 2001年07月 [査読有り][通常論文]
   

  A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.
• Cloning and biochemical characterization of Co(2+)-activated bromoperoxidase-esterase (perhydrolase) from Pseudomonas putida IF-3 strain.

  Itoh N, Kawanami T, Liu JQ, Dairi T, Miyakoshi M, Nitta C, Kimoto Y

  Biochimica et biophysica acta 1545 53 - 66 1-2 2001年02月 [査読有り][通常論文]
  
• 2 完全重水素化メバロン酸を用いるイソプレノイド類の代謝反応機構化学への応用(口頭発表の部)

  江口 正, 出来島 康方, 松島 芳隆, 為我井 秀行, 柿沼 勝己, 高木 基樹, 葛山 智久, 瀬戸 治男, 三沢 典彦, 濱野 吉十, 大利 徹

  天然有機化合物討論会講演要旨集 43 7 - 12 天然有機化合物討論会実行委員会 2001年 

  Isoprenoids are chemically diverse in nature, ubiquitous in living organisms, and crucial in biological processes. The biosynthesis of such isoprenoids proceeds through mevalonate and nonmevalonate pathways depending upon organisms and cellular organella, isopentenyl diphosphate (IPP) being a key intermediate in both cases. Metabolic engineering and control of these pathways should thus provide new opportunity to study intriguing chemistry and biochemistry involved and to develop selective chemotherapeutic agents and isoprenoid-related materials. We describe a new practical approach to the preparation of highly- and multiply deuterated isoprenoids, zeaxanthin and diterpene anitibiotic terpentecin being as examples, and its potential for analyzing the biosynthetic mechanism of isoprenoids, based on the metabolic engineering of microorganisms. Obviously, deuterium-labeled compounds are invaluable in biochemical, bioorganic as well as physicochemical research. Metabolically engineered E. coli DK223 (pTMV20, pACCAR25ΔcrtX) produces zeaxanthin under the presence of mevalonate. Fully deuterated mevalonolactone-d_9 (MVL-d_9), which had been synthesized, was supplemented to the culture of the above triply-engineered E. coli, and the biosynthesized zeaxanthin was extracted and purified by repeated chromatography. All the zeaxanthin formed was proved to be derived only from the supplemented MVL-d_9. This was the first example of such highly and multiply deuterated zeaxanthin, and clearly demonstrated significant potential of the present approach for the preparation of various isotope-labeled isoprenoids. Additional example of this approach was also demonstrated in the mechanistic study of cyclization reaction in the biosynthesis of diterpene anitibiotic terpentecin. Straightforward stereochemical analysis of isoprenoid biosynthesis was demonstrated by one-shot labeling of MVL-d_9 and ^1H NMR spectroscopy. Precise analysis of the simplified proton spectra of highly deuterated isoprenoids, especially under the deuterium decoupled conditions, appeared to be beneficial for mechanistic enzymology, particularly, for the key transformation involving proton attack and proton quench as observed in the terpene cyclase reactions.
• Gene cloning and overproduction of low-specificity D-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug

  JQ Liu, M Odani, T Yasuoka, T Dairi, N Itoh, M Kataoka, S Shimizu, H Yamada

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 54 1 44 - 51 2000年07月 [査読有り][通常論文]
   

  The dtaAX gene encoding a pyridoxal 5'-phosphate (pyridoxal-P)-dependent low-specificity D-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The predicted amino acid sequence displayed 54% identity with that of D-threonine aldolase from gram-positive bacteria Ar-throbacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherickia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low-specificity D-threonine aldolase was shown to be an efficient biocatalyst for resolution of L-beta-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease.
• Cloning of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from terpenoid antibiotic-producing Streptomyces strains

  T Dairi, Y Motohira, T Kuzuyama, S Takahashi, N Itoh, H Seto

  MOLECULAR AND GENERAL GENETICS 262 6 957 - 964 2000年01月 [査読有り][通常論文]
   

  We have isolated a mutant lacking 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity from a terpenoid antibiotic (terpentecin) producer, Streptomyces griseolosporeus MF730-N6, which uses both the mevalonate and nonmevalonate pathways for the formation of isopentenyl diphosphate, by screening terpentecin non-producing mutants. Terpentecin is known to be synthesized via the mevalonate pathway. The gene encoding HMG-CoA reductase (hmgg) was cloned and identified by complementation of the mutant, using a self-cloning system developed in this study for strain MF730-N6, The corresponding hmgs gene for HMG-CoA reductase was also cloned from Streptomyces sp. KO-3988, which produces the terpenoid antibiotic furaquinocin. Sequence analysis of hmgg and hmgs showed that both genes encode polypeptides of 353 amino acids which are 84% identical to each other. A search of protein sequence databases revealed that both gene products were also similar to HMG-CoA reductases from a variety of other organisms, including Streptomyces sp. CL190 (hmgg is 89% and hmgs 85%, identical to its CL190 homolog), sea urchin (40.3 and 40.5%), German cockroach (37.6 and 38.4%), and Camptotheca acuminata (39.7 and 40.8%).
• Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp strain ST-10

  JC Wang, M Sakakibara, JQ Liu, T Dairi, N Itoh

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 52 3 386 - 392 1999年09月 [査読有り][通常論文]
   

  The gene encoding phenylacetaldehyde reductase (PAR), a useful biocatalyst for producing chiral alcohols, was cloned from the genomic DNA Of the styrene-assimilating Corynebacterium sp. strain ST-10. The gene contained an opening reading frame consisting of 1,158 nucleotides corresponding to 385 amino acid residues. The subunit molecular weight was calculated to be 40,299, which was in agreement with that determined by polyacrylamide gel electrophoresis, The enzyme was sufficiently expressed in recombinant Escherichia coli cells for practical use and purified to homogeneity by three-column chromatography steps. The predicted amino acid sequence displayed only 20-29% identity with zinc-containing, NAD(+)-dependent, long-chain alcohol dehydrogenases. Nevertheless, the probable NAD(+)- and zinc-binding sites are conserved although one of the three catalytic zinc-binding residues of the zinc-containing, long-chain alcohol dehydrogenases was substituted by Asp in PAR. The protein contains 7.6 mol zinc/mol tetramer. Therefore, the enzyme was considered as a new member of zinc-containing, long-chain alcohol dehydrogenases with a particular and broad substrate specificity.
• Development of a self-cloning system for Actinomadura verrucosospora and identification of polyketide synthase genes essential for production of the angucyclic antibiotic pradimicin

  T Dairi, Y Hamano, T Furumai, T Oki

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 65 6 2703 - 2709 1999年06月 [査読有り][通常論文]
   

  A self-cloning system for Actinomadura verrucosospora, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a replicon from a newly found Actinomadura plasmid and a selectable marker cloned from the Actinomadura strain. The system has an efficiency of more than 10(5) CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from A. verrucosospora. Nucleotide sequence analysis of the 3.5-kb SalI-SphI fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis.
• A new route to L-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity D-threonine aldolase-catalyzed stereospecific resolution

  JQ Liu, M Odani, T Dairi, N Itoh, S Shimizu, H Yamada

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 51 5 586 - 591 1999年05月 [査読有り][通常論文]
   

  A new enzymatic resolution process was established for the production of L-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity D-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized DL-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichia coli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l(-1)) L-threo-MTPS was obtained from 200 mM (45.5 g l(-1)) DL-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess.
• A novel metal-activated pyridoxal enzyme with a unique primary structure, low specificity D-threonine aldolase from Arthrobacter sp. strain DK-38 - Molecular cloning and cofactor characterization

  JQ Liu, T Dairi, N Itoh, M Kataoka, S Shimizu, H Yamada

  JOURNAL OF BIOLOGICAL CHEMISTRY 273 27 16678 - 16685 1998年07月 [査読有り][通常論文]
   

  The gene encoding low specificity D-threonine aldolase, catalyzing the interconversion of D-threonine/D-allo-threonine and glycine plus acetaldehyde, was cloned from the chromosomal DNA of Arthrobacter sp, strain DK-38, The gene contains an open reading frame consisting of 1,140 nucleotides corresponding to 379 amino acid residues. The enzyme was overproduced in recombinant Escherichia coli cells and purified to homogeneity by ammonium sulfate fractionation and three-column chromatography steps. The recombinant aldolase was identified as a pyridoxal enzyme with the capacity of binding 1 mol of pyridoxal 5'-phosphate per mol of subunit, and Lys(59) Of the enzyme was determined to be the cofactor binding site by chemical modification with NaBH4. In addition, Mn2+ ion was demonstrated to be an activator of the enzyme, although the purified enzyme contained no detectable metal ions. Equilibrium dialysis and atomic absorption studies revealed that the recombinant enzyme could bind 1 mol of Mn2+ ion per mol of subunit, Remarkably, the predicted amino acid sequence of the enzyme showed no significant similarity to those of the currently known pyridoxal 5'-phosphate-dependent enzymes, indicating that low specificity D-threonine aldolase is a new pyridoxal enzyme with a unique primary structure. Taken together, low specificity D-threonine aldolase from Arthrobacter sp. strain DK-38, with a unique primary structure, is a novel metal-activated pyridoxal enzyme.
• Gene cloning, biochemical characterization and physiological role of a thermostable low-specificity L-threonine aldolase from Escherichia coli

  JQ Liu, T Dairi, N Itoh, M Kataoka, S Shimizu, H Yamada

  EUROPEAN JOURNAL OF BIOCHEMISTRY 255 1 220 - 226 1998年07月 [査読有り][通常論文]
   

  The ItaE gene encoding for a thermostable low-specificity L-threonine aldolase, which catalyzes the cleavage of L-threonine/L-allo-threonine to glycine and acetaldehyde, was cloned from Escherichia coli GS245 by the polymerase chain reaction. Construction and expression of the plasmid pLTAE, which contained the ItaE gene under the control of the lac promoter, resulted in a 227-fold increase in the specific activity above the level detected in E. coli cells containing the control vector The enzyme is thermostable: it retained its full activity upon heating at 60 degrees C for 1 h. The enzyme was thus feasibly purified to homogeneity by heat treatment and butyl-Toyopearl column chromatography, and characterized. To reveal the physiological role of the enzyme, gene disruption was performed, Knockout of the ItaE gene of wild-type E. coli did not effect the cellular growth rate, while disruption of the ItaE gene of E. coli GS245, whose serine hydroxymethyltransferase gene was knocked out, caused a significant decrease in the cellular growth rate, suggesting that the threonine aldolase is not a major source of cellular glycine in wild-type E. coli but catalyzes an alternative pathway for cellular glycine when serine hydroxymethyltransferase is inert.
• Gene cloning, nucleotide sequencing, and purification and characterization of the low-specificity L-threonine aldolase from Pseudomonas sp. strain NCIMB 10558

  JQ Liu, S Ito, T Dairi, N Itoh, M Kataoka, S Shimizu, H Yamada

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 64 2 549 - 554 1998年02月 [査読有り][通常論文]
   

  A low-specificity L-threonine aldolase (L-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5'-phosphate as a coenzyme, is strictly L specific at the alpha position, whereas it cannot distinguish between three and erythro forms at the beta position. In addition to threonine, the enzyme also acts on various other L-beta-hydroxy-alpha-amino acids, including L-beta-3,4-dihhydroxy-phenylserine, L-beta-3,4-methylenedioxyphenylserine, and L-beta-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity L-TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms, However, lysine 207 of low-specificity L-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 120 nm, Thus, Lys207 of the L-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5'-phosphate of the enzyme to catalyze the reversible aldol reaction.
• Shotgun cloning and characterization of the thymidylate synthase-encoding gene from Mycobacterium bovis BCG

  S Matsumoto, H Yukitake, N Ohara, T Dairi, H Kanbara, T Yamada

  MICROBIOLOGY AND IMMUNOLOGY 42 1 15 - 21 1998年 [査読有り][通常論文]
   

  The shotgun cloning of a Mycobacterium bovis BCG (BCG) genome into pBluescript SK (+) successfully yielded a 0.9 kbp fragment, confirming the ability of Escherichia coli thyA mutant MH2702 to grow in a thymine-depleted medium. This DNA fragment contained a gene homologous to the thymidylate synthase (TS)-encoding genes (thyA) of other organisms. An inverted repeat sequence and open reading frame (ORF) were observed at the upstream region of the thyA. A computer analysis revealed that the protein encoded by this ORF possessed a structure unique for a DNA binding protein.
• Crystallization of Arthrobacter sp. strain 1C N-(1-D-carboxyethyl)-L-norvaline dehydrogenase and its complex with NAD+

  Britton KL, Rogers HF, Asano Y, Dairi T, Kato Y, Stillman TJ, Rice DW

  Acta crystallographica. Section D, Biological crystallography 53 124 - 126 4 1998年01月 [査読有り][通常論文]
   

  The novel NAD(+)-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 Angstrom resolution. X-ray precession photographs have established that the crystals belong to space group P2(1)2(1)2, with cell parameters a = 104.9, b = 80.0, c = 45.5 Angstrom and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.
• Crystallization of Arthrobacter sp. strain 1C N-(1-D-carboxyethyl)-L-norvaline dehydrogenase and its complex with NAD+

  K. Linda Britton, H. Fiona Rogers, Yasuhisa Asano, Tohru Dairi, Yasuo Kato, Timothy J. Stillman, David W. Rice

  Acta Crystallographica Section D: Biological Crystallography 54 1 124 - 126 1998年01月01日 [査読有り][通常論文]
   

  The novel NAD+-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 Å resolution. X-ray precession photographs have established that the crystals belong to space group P21212, with cell parameters a = 104.9, b = 80.0, c = 45.5 Å and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.
• Cloning and nucleotide sequence of the putative polyketide synthase genes for pradimicin biosynthesis from Actinomadura hibisca

  T Dairi, Y Hamano, Y Igarashi, T Furumai, T Oki

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 61 9 1445 - 1453 1997年09月 [査読有り][通常論文]
   

  We cloned the putative polyketide synthase genes (pms genes) for pradimicin A biosynthesis from Actinomadura hibisca using an oligonucleotide probe designed on the basis of conserved amino acid sequences of other polyketide synthases (PKSs). By DNA sequencing of an 8.2-kb SacI fragment that hybridized with the oligonucleotide probe, 11 open reading frames (ORFs) were found. All of the ORFs except for ORF10 were predicted to be translated in the same direction. Each of the deduced ORFs has significant sequence similarity to the protein responsible for polyketide biosynthesis or spore pigmentation. In particular, ORF1, ORF2, and ORF3 were 50-70% identical with genes coding for PKSs for actinorhodin biosynthesis. Specific DNA regions similar in sequence to pms genes were found with genomic Southern hybridization in all of the pradimicin producers examined, but were not found in pradimicin nonproducers, suggesting that the genes cloned in this study encode polyketide synthase for pradimicin biosynthesis.
• L-allo-threonine aldolase from Aeromonas jandaei DK-39: Gene cloning, nucleotide sequencing, and identification of the pyridoxal 5'-phosphate-binding lysine residue by site-directed mutagenesis

  JQ Liu, T Dairi, M Kataoka, S Shimizu, H Yamada

  JOURNAL OF BACTERIOLOGY 179 11 3555 - 3560 1997年06月 [査読有り][通常論文]
   

  we have isolated the gene encoding L-all-threonine aldolase (L-allo-TA) from Aeromonas jandaei DK-39, a pyridoxal 5'-phosphate (PLP)-dependent enzyme that stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The gene contains an open reading frame consisting of 1,014 nucleotides corresponding to 338 amino acid residues. The protein molecular weight was estimated to be 36,294, which is in good agreement with the subunit molecular weight of the enzyme determined by polyacrylamide gel electrophoresis. The enzyme was overexpressed in recombinant Escherichia coli cells and purified to homogeneity by one hydrophobic column chromatography step. The predicted amino acid sequence showed no significant similarity to those of the currently known PLP-dependent enzymes but displayed 40 and 41% identity with those of the hypothetical GLY1 protein of Saccharomyces cerevisiae and the GLY1-like protein of Caenorhabditis elegans, respectively. Accordingly, L-allo-TA might represent a new type of PLP-dependent enzyme. To determine the PLP-binding site of the enzyme, all of the three conserved lysine residues of L-allo-TA were replaced by alanine by site-directed mutagenesis. The purified mutant enzymes, K51A and K224A, showed properties similar to those of the wild type, while the mutant enzyme K199A was catalytically inactive, with corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys199 of L-allo-TA probably functions as an essential catalytic residue forming an internal Schiff base with PLP of the enzyme to catalyze the reversible aldol reaction.
• The GLY1 gene of Saccharomyces cerevisiae encodes a low-specific L-threonine aldolase that catalyzes cleavage of L-allo-threonine and L-threonine to glycine - Expression of the gene in Escherichia coli and purification and characterization of the enzyme

  JQ Liu, S Nagata, T Dairi, H Misono, S Shimizu, H Yamada

  EUROPEAN JOURNAL OF BIOCHEMISTRY 245 2 289 - 293 1997年04月 [査読有り][通常論文]
   

  The GLY1 gene of Saccharomyces cerevisiae is required for the biosynthesis of glycine for cell growth [McNeil, J. B., McIntosh, E. V., Taylor, B. V., Zhang, F.-R., Tang, S. & Bognar, A. L. (1994) J. Biol. Chem. 269, 9155-9165], but its gene product has not been identified. We have found that the GLY1 protein is similar in primary structure to L-allo-threonine aldolase of Aeromonas jandiae DK-39, which stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The GLY1 gene was amplified by PCR, with a designed ribosome-binding site, cloned into pUC118, and expressed in Escherichia coli cells. The enzyme was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of about 170 kDa and consists of four subunits identical in molecular mass. The enzyme contains 2 mol pyridoxal 5'-phosphate/4 mol of subunit as a cofactor, and its absorption spectrum exhibits maxims at 280 nm and 420 nm. The enzyme catalyzes the cleavage of not only L-allo-threonine to glycine but also L-threonine. We have termed the enzyme a low-specific L-threonine aldolase to distinguish it from L-allo-threonine aldolase.
• An alkaline D-stereospecific endopeptidase with beta-lactamase activity from Bacillus cereus

  Y Asano, H Ito, T Dairi, Y Kato

  JOURNAL OF BIOLOGICAL CHEMISTRY 271 47 30256 - 30262 1996年11月 [査読有り][通常論文]
   

  We purified a novel extracellular D-stereospecific endopeptidase, alkaline D-peptidase (D-stereospecific peptide hydrolase, EC 3.4.11.-), to homogeneity from the culture broth of the soil bacterium Bacillus cereus strain DF4-B. The M(r) of the enzyme was 37,952, and it was composed of a single polypeptide chain. The optimal pH for activity was similar to 10.3, The enzyme was strictly D-stereospecific toward oligopeptides composed of D-phenylalanine such as (D-Phe)(3) and (D-Phe)(4). The enzyme also acted to a lesser extent on (D-Phe)(6), Boc-(D-Phe)(4) (where Boc is tert-butoxycarbonyl), Boc-(D-Phe)(4) methyl ester, Boc-(D-Phe)(3) methyl ester, Boc (D-Phe)(2), (D-Phe)(2), and others, but not upon their corresponding peptides composed of L-Phe, (D-Ala)(n) (n = 2-5), (D-Val)(3), and (D-Leu)(2), The mode of action of the enzyme was clarified with synthetic substrates ((D-Phe)(2)-D-Tyr and D-Tyr-(D-Phe)(2)) and eight stereoisomers of (Phe)(3). The enzyme had beta-lactamase activity toward ampicillin and penicillin G, although carboxypeptidase DD and D-aminopeptidase activities were undetectable. The gene coding for alkaline D-peptidase (adp) was cloned into plasmid pUC118, and a 1164-base pair open reading frame consisting of 388 codons was identified as the adp gene, The predicted polypeptide was similar to carboxypeptidase DD from Streptomyces R61, penicillin-binding proteins from Streptomyces lactamdurans and Bacillus subtilis, and class C beta-lactamases, Thus, the enzyme was categorized as a new ''penicillin-recognizing enzyme.''
• NUCLEOTIDE-SEQUENCE OF FORTIMICIN KL1 METHYLTRANSFERASE GENE ISOLATED FROM MICROMONOSPORA-OLIVASTEROSPORA, AND COMPARISON OF ITS DEDUCED AMINO-ACID-SEQUENCE WITH THOSE OF METHYLTRANSFERASES INVOLVED IN THE BIOSYNTHESIS OF BIALAPHOS AND FOSFOMYCIN

  T KUZUYAMA, T SEKI, T DAIRI, T HIDAKA, H SETO

  JOURNAL OF ANTIBIOTICS 48 10 1191 - 1193 1995年10月 [査読有り][通常論文]
  
• A SELF-DEFENSE GENE HOMOLOGOUS TO TETRACYCLINE EFFLUXING GENE ESSENTIAL FOR ANTIBIOTIC PRODUCTION IN STREPTOMYCES-AUREOFACIENS

  T DAIRI, K AISAKA, R KATSUMATA, M HASEGAWA

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 59 10 1835 - 1841 1995年10月 [査読有り][通常論文]
   

  By Northern blot analyses with DNA probes carrying 6-demethylchlortetracycline (6-DCT) biosynthetic genes from Streptomyces aureofaciens NRRL3203, a highly expressed gene (tcrC) was detected in a high titer producing mutant derived from the parental strain NRRL3203 by NTG mutagenesis, The analysis of the nucleotide sequence of the 2.8-kb BamHI fragment containing tcrC gene showed that the predicted tcrC gene product is a protein consisting of 512 amino acids. The deduced amino acid sequence had a high level identity with that of the self-defense gene (tet347) of Streptomyces rimosus, known to mediate oxytetracycline efflux. The tcrC gene-inactivated strains generated from strain NRRL3203 by gene replacement had a 90% decrease in the level of resistance to tetracycline and the antibiotic productivity when compared with the parental strain.
• NUCLEOTIDE SEQUENCING OF PHENYLALANINE DEHYDROGENASE GENE FROM BACILLUS-BADIUS-IAM-11059

  A YAMADA, T DAIRI, Y OHNO, XL HUANG, Y ASANO

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 59 10 1994 - 1995 1995年10月 [査読有り][通常論文]
   

  The nucleotide sequence of the pdh gene coding for PheDH from Bacillus badius IAM 11059 was analyzed. The gene consists of an ORF of 1140 nucleotides,which specifies a protein of 380 codons. The primary structure of PheDH is similar to PheDH from B. sphaericus, PheDH from Themoactinomyces intermedius, and leucine dehydrogenase from B. stearothemophilus, etc.
• CLONING, NUCLEOTIDE SEQUENCING, AND EXPRESSION OF AN OPINE DEHYDROGENASE GENE FROM ARTHROBACTER SP STRAIN 1C

  T DAIRI, Y ASANO

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 61 8 3169 - 3171 1995年08月 [査読有り][通常論文]
   

  The gene coding for opine dehydrogenase from Arthrobacter sp. strain 1C was cloned onto plasmid pBluescript KS(-), and the nucleotide sequence of the 1,077-bp open reading frame consisting of 359 codons was identified as the odh gene. Transformed Escherichia coli cells overproduced NAD(+)-dependent opine dehydrogenase under control of the promoter of the lac gene on pBluescript KS(-).
• CONSERVED ORGANIZATION OF GENES FOR BIOSYNTHESIS OF CHLORTETRACYCLINE IN STREPTOMYCES STRAINS

  T DAIRI, T NAKANO, T MIZUKAMI, K AISAKA, M HASEGAWA, R KATSUMATA

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 59 7 1360 - 1361 1995年07月 [査読有り][通常論文]
   

  By genomic Southern blot analysis, the DNA sequences homologous to the gene cluster responsible for biosynthesis of 6-demethyl-chlortetracycline in Streptomyces aureofaciens NRRL3203 mere shown to be highly conserved in independent chlortetracycline- or tetracycline producing Streptomyces strains. By contrast, oxytetracycline-producing Streptomyces strains had no hybridization with the cluster DNA.
• CLONING AND NUCLEOTIDE-SEQUENCE OF THE GENE RESPONSIBLE FOR CHLORINATION OF TETRACYCLINE

  T DAIRI, T NAKANO, K AISAKA, R KATSUMATA, M HASEGAWA

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 59 6 1099 - 1106 1995年06月 [査読有り][通常論文]
   

  Two cosmid clones containing distinct types of self-defense gene of a 6-demethylchlortetracycline producer, Streptomyces aureofaciens NRRL3203, were isolated. The gene responsible for chlorination of tetracycline (chl gene) was subcloned from one of the cosmid clones by complementation of a chlorination-deficient mutant, using a gene cloning system for strain NRRL3203 developed in this study. The nucleotide sequence analysis of a 4.4-kb SacI-BamHI fragment containing the chi gene showed that the predicted product of the chl gene is a polypeptide of 452 amino acids, and that the chl gene was preceded by two open reading frames, which could endode polypeptides of 50 kDa and 32 kDa, respectively. A search for sequences homologous to these ORFs found that the former product strongly resembles that of the 6-hydroxylation enzyme for oxytetracycline biosynthesis, and that the latter product has a weak but significant similarity to the hydroxyindole O-methyltransferase of bovine pineal gland. By Northern blot analysis, these three genes were suggested to be polycistronically transcribed.
• CHARACTERIZATION OF 2 DIFFERENT TYPES OF RESISTANCE GENES AMONG PRODUCERS OF FORTIMICIN-GROUP ANTIBIOTICS

  T OHTA, T DAIRI, M HASEGAWA

  JOURNAL OF GENERAL MICROBIOLOGY 139 139 591 - 599 1993年03月 [査読有り][通常論文]
   

  Fortimicin-A (FTM-A; astromicin)-resistance genes (fmr genes) isolated from six producers of the FTM-group of antibiotics were analysed. These genes could be classified into two types by the resistance profiles to aminoglycoside antibiotics and by their DNA homologies. Three genes, fmrT from the istamycin producer Streptomyces tenjimariensis ATCC 31603, fmrS from the sannamycin producer Streptomyces sannanensis IFO 14239 and fmrH from the sporaricin producer Saccharopolyspora hirsuta ATCC 20501, conferred resistance to FTM-A, kanamycin (Km) and neomycin B (Nm-B), but not to gentamicin (Gm). The other three genes, fmrO from the FTM-A producer Micromonospora olivasterospora ATCC 21819, fmrM from the antibiotic S F-2052 producer Micromonospora sp. SF-2098 (ATCC 31580) and fmrD from the dactimicin producer Dactylosporangium matsuzakiense ATCC 31570, conferred resistance to FTM-A, Km and Gm, but not to Nm-B. No DNA homology was detected between the two types of the resistance genes in Southern-blot analysis. The present results revealed that, in spite of the similarity of their biosynthesis genes, there are at least two different types of resistance genes among the FTM-group antibiotic producers.
• ORGANIZATION AND NATURE OF FORTIMICIN-A (ASTROMICIN) BIOSYNTHETIC GENES STUDIED USING A COSMID LIBRARY OF MICROMONOSPORA-OLIVASTEROSPORA DNA

  T DAIRI, T OHTA, E HASHIMOTO, M HASEGAWA

  MOLECULAR & GENERAL GENETICS 236 1 39 - 48 1992年12月 [査読有り][通常論文]
   

  The cloning of five DNA segments carrying at least seven genes (fms1, fms3, fms4, fms5, fms7, fms11, and fms12) that participate in fortimicin A (astromicin) biosynthesis was described previously. These DNA fragments were used to screen a cosmid library of genomic DNA in order to examine if these biosynthetic genes are clustered in Micromonospora olivasterospora. One cosmid clone (pGLM990) was obtained, which hybridized to all the probes. Complementation analysis, using mutants blocked at various steps and chimeric plasmids subcloned from pGLM990, showed that three additional genes (fms8, fms10, and fms13) are present in pGLM990. A gene conferring self-resistance to the antibiotic, which was independently cloned in Streptomyces lividans, using the plasmid vector pIJ702 was also found to be linked to the cluster of biosynthetic genes. Thus, at least ten biosynthetic genes and a self-defense gene are clustered in a chromosomal region of about 27 kb in M. olivasterospora. Interestingly, the fms8 gene which participates in the dehydroxylation step of fortimicin A biosynthesis was found to have homology with a neomycin resistance gene nmrA from the neomycin-producing Micromonospora sp. MK50. Studies using a cell-free extract of the fms8 mutant and its parent strain showed that the enzyme encoded by fms8 phosphorylates a biosynthetic precursor, fortimicin KK1, in the presence of ATP. Thus the dehydroxylation reaction is suggested to occur via the phosphorylation of the target hydroxyl group. DNA regions homologous to fms genes were found in Micromonospora sp. SF-2098 and Dactylosporangium matsuzakiense, both producers of fortimicin group antibiotics.
• N-FORMIMIDOYL FORTIMICIN-A SYNTHASE, A UNIQUE OXIDASE INVOLVED IN FORTIMICIN A BIOSYNTHESIS - PURIFICATION, CHARACTERIZATION AND GENE CLONING

  T DAIRI, K YAMAGUCHI, M HASEGAWA

  MOLECULAR & GENERAL GENETICS 236 1 49 - 59 1992年12月 [査読有り][通常論文]
   

  Micromonospora olivasterospora, a fortimicin A (FTM A, astromicin) producer, was found to carry an enzyme that converts FTM A to N-formimidoyl FTM A (FI-FTM A). This enzyme (FI-FTMase) was purified to homogeneity and shown to be a flavin adenine dinucleotide (FAD) enzyme. Tracer experiments proved that the formimidoyl group was derived from C-2 of glycine via oxidation of the amino acid in the presence of FTM A and oxygen. The gene encoding this enzyme, fms 14, was cloned using a 26-mer oligonucleotide probe, designed according to the N-terminal amino acid sequence of purified FI-FTMase, from a cosmid clone pGLM990, which has been shown to contain a cluster of FTM A biosynthetic genes. The nucleotide sequence, and biochemical and genetic analysis revealed that FI-FTMase is composed of four identical subunits of mol. wt. 52000, and contains at least one FAD per subunit. DNA regions homologous to fms14 were found in two other producers of the fortimicin group of antibiotics, Dactylosporangium matsuzakiense ATCC31570 and Micromonospora sp. SF-2098.
• SELF CLONING IN MICROMONOSPORA-OLIVASTEROSPORA OF FMS GENES FOR FORTIMICIN-A (ASTROMICIN) BIOSYNTHESIS

  T DAIRI, T OHTA, E HASHIMOTO, M HASEGAWA

  MOLECULAR & GENERAL GENETICS 232 2 262 - 270 1992年03月 [査読有り][通常論文]
   

  We have cloned the seven genes that are responsible for biosynthesis of the antibiotic fortimicin A (FTM A) using a recently developed self-cloning system that employs the plasmid vector pMO116 for Micromonospora olivasterospora. Five chimeric plasmids that restored FTM A production in M. olivasterospora mutants blocked at different biosynthetic steps were isolated by shotgun cloning. Secondary transformation using other non-producing mutants showed that two additional FTM A biosynthetic genes were included on these plasmids, and that at least four of the genes were clustered. Interestingly AN38-1, a non-producing mutant that had a defect in dehydroxylation of a precursor of FTM A, was complemented by the DNA fragment containing a neomycin resistance gene that had been cloned from a neomycin-producing strain (Micromonospora sp. FTM A non-producing strain) in the course of constructing the plasmid vector pMO116. These results clearly show that this novel gene cloning system in Micromonospora is of practical use.
• A NOVEL, HIGHLY EFFICIENT GENE-CLONING SYSTEM FOR MICROMONOSPORA STRAINS

  M HASEGAWA, T DAIRI, T OHTA, E HASHIMOTO

  JOURNAL OF BACTERIOLOGY 173 21 7004 - 7011 1991年11月 [査読有り][通常論文]
   

  A highly efficient gene-cloning system for Micromonospora olivasterospora, a producer of the antibiotic fortimicin A (astromicin), suited to shotgun cloning has been developed. The system is supported by two new advancements accomplished in this study. One is the construction of novel plasmid vectors pMO116, pMO126, pMO133, pMO136, and pMO217, all consisting of replicons from newly found Micromonospora plasmids and selectable markers cloned from a neomycin-producing Micromonospora strain. The other advancement is the establishment of a new protocol for bacterial protoplasting in which some kinds of sugar alcohols are added in precultures. Such sugar alcohols were found to sensitize a wide taxonomical range of bacteria to lysozyme. The system is reproducible and reliable and has a high efficiency of more than 10(6) CFU/mu-g of DNA.
• COMMON BIOSYNTHETIC FEATURE OF FORTIMICIN-GROUP ANTIBIOTICS

  T DAIRI, M HASEGAWA

  JOURNAL OF ANTIBIOTICS 42 6 934 - 943 1989年06月 [査読有り][通常論文]
  
• POSITIVE CONTROL OF TRANSCRIPTION INITIATION IN ESCHERICHIA-COLI - A BASE SUBSTITUTION AT THE PRIBNOW BOX RENDERS OMPF EXPRESSION INDEPENDENT OF A POSITIVE REGULATOR

  T DAIRI, K INOKUCHI, T MIZUNO, S MIZUSHIMA

  JOURNAL OF MOLECULAR BIOLOGY 184 1 1 - 6 1985年 [査読有り][通常論文]
  

書籍

• Isoprenoid Synthesis in Plants and Microorganisms

  DAIRI Tohru (担当:分担執筆範囲:Biosynthetic genes and enzymes of isoprenoids produced by actinomycetes)
  Springer 2013年
• Comprehensive Natural Products II Chemistry and BiologyMander, L., Lui, H.-W., Eds.volume 1, pp. 789–814.

  (範囲:Comprehensive Natural Products II Chemistry and BiologyMander, L., Lui, H.-W., Eds.volume 1, pp. 789–814.)
  Elsevier: Oxford, 2010年

その他活動・業績

• ペプチドグリカン新規生合成経路阻害剤の探索

  梅津秀平, 角田毅, 稲橋佑起, 野中健一, 小笠原泰志, 大利徹 日本放線菌学会大会講演要旨集 37th 2023年
• 抗腫瘍抗生物質マイトマイシンの生合成解析

  高橋佑大, 中川陽, 角田毅, 大利徹, 小笠原泰志 日本放線菌学会大会講演要旨集 37th 2023年
• α,α-二置換アミノ酸を有する天然物の生合成研究

  角田毅, 古村淳吉, 山崎陽香, 小笠原泰志, 大利徹 日本放線菌学会大会講演要旨集 37th 2023年
• ラッソペプチドRES-701の生合成研究

  山谷優花, 小笠原泰志, 佐藤康治, 大利徹 日本農芸化学会大会講演要旨集(Web) 2022 2022年
• 微細藻類由来DHA合成酵素の炭素鎖伸長反応の解析

  仲間陸, 小林飛悠, 大塚慎, 佐藤康治, 小笠原泰志, 大利徹 日本農芸化学会大会講演要旨集(Web) 2022 2022年
• 微細藻類由来DHA合成酵素のdehydrataseドメインの機能解析

  小林飛悠, 佐藤康治, 小笠原泰志, 大利徹 日本農芸化学会東北支部大会プログラム・講演要旨集 2022 2022年
• 細菌における」D-アミノ酸含有ペプチドの生合成

  小笠原泰志, 佐藤康治, 大利 徹 生化学 93 (3) 329 -337 2021年06月 [査読有り][招待有り]
  
• 微細藻類由来DHA合成酵素の炭素鎖長制御機構の解明

  仲間陸, 佐藤康治, 小笠原泰志, 大利徹 日本農芸化学会北海道支部学術講演会講演要旨集(Web) 2021 2021年
• 微細藻類由来DHA合成酵素のdehydrataseドメインの機能解析

  小林飛悠, 佐藤康治, 小笠原泰志, 大利徹 日本農芸化学会北海道支部学術講演会講演要旨集(Web) 2021 2021年
• ポリグルタミン酸生合成におけるエピメリ化酵素の同定

  加藤陽菜多, 小笠原泰志, 大利徹 日本農芸化学会大会講演要旨集(Web) 2021 2021年
• 抗腫瘍抗生物質マイトマイシンの生合成解析

  中川陽, 小笠原泰志, 大利徹 日本農芸化学会大会講演要旨集(Web) 2021 2021年
• 新規ATP依存ペプチドエピメラーゼMurLの反応機構解析

  中尾優文, 小笠原泰志, 佐藤康治, 大利徹 日本農芸化学会大会講演要旨集(Web) 2021 2021年
• 新規ATP依存ペプチドエピメラーゼMurLの反応機構解析

  中尾優文, 小笠原泰志, 佐藤康治, 大利徹 日本農芸化学会北海道支部学術講演会講演要旨集(Web) 2020 2020年
• 大腸菌によるErgothioneine発酵生産の効率化

  田草川隼, 上出倫敬, 佐藤康治, 小笠原泰志, 大津厳生, 大利徹 日本農芸化学会大会講演要旨集(Web) 2020 2020年
• 海洋微生物における多価不飽和脂肪酸生合成酵素の解析と応用

  林 祥平, 小笠原泰志, 佐藤康治, 大利 徹 B&I 77 (6) 448 -452 2019年11月 [査読無し][招待有り]
  
• 多価不飽和脂肪酸生合成酵素の論理的機能改変

  池内健心, 中真以, 林祥平, 大塚慎, 小林洸太, 佐藤康治, 小笠原泰志, 氏原哲朗, 大利徹 日本農芸化学会大会講演要旨集(Web) 2019 ROMBUNNO.1D2a12 (WEB ONLY) 2019年03月05日 [査読無し][通常論文]
  
• in vitro解析による多価不飽和脂肪酸生合成酵素の炭素鎖長制御機構の解明

  林祥平, 小笠原泰志, 佐藤康治, 丸山千登勢, 濱野吉十, 氏原哲朗, 大利徹 日本農芸化学会大会講演要旨集(Web) 2019 ROMBUNNO.1D2a10 (WEB ONLY) 2019年03月05日 [査読無し][通常論文]
  
• in vitro解析による多価不飽和脂肪酸生合成酵素のcis二重結合形成機構の解明

  林祥平, 小笠原泰志, 佐藤康治, 丸山千登勢, 濱野吉十, 氏原哲朗, 大利徹 日本農芸化学会大会講演要旨集(Web) 2019 ROMBUNNO.1D2a09 (WEB ONLY) 2019年03月05日 [査読無し][通常論文]
  
• 精密解析に基づく多価不飽和脂肪酸生合成酵素の機能改変

  林祥平, 佐藤康治, 小笠原泰志, 大利徹 日本農芸化学会大会講演要旨集(Web) 2019 ROMBUNNO.3S13D6a5 (WEB ONLY) 2019年03月05日 [査読無し][通常論文]
  
• in vivo解析による多価不飽和脂肪酸生合成酵素の生成物制御ドメインの同定

  林祥平, 中真以, 佐藤康治, 氏原哲朗, 大利徹 日本農芸化学会大会講演要旨集(Web) 2019 ROMBUNNO.1D2a08 (WEB ONLY) 2019年03月05日 [査読無し][通常論文]
  
• ロンゲスチン生合成における特異なメチル基導入機構の解明

  尾崎太郎, SHINDE Sandip S., GAO Lei, 奥泉諒, LIU Chengwei, 小笠原泰志, 大利徹, 南篤志, 及川英秋 日本農芸化学会大会講演要旨集(Web) 2019 2019年
• 抗腫瘍抗生物質マイトマイシンの生合成解析

  中川陽, 小笠原泰志, 大利徹 日本生物工学会大会講演要旨集 71st 2019年
• 抗腫瘍抗生物質マイトマイシンの初期生合成機構のin vitro解析

  中川陽, 小笠原泰志, 丸山千登勢, 濱野吉十, 大利徹 日本放線菌学会大会講演要旨集 34th 2019年
• ポリグルタミン酸生合成における新規エピメリ化反応

  佐藤将太, 重松真由子, 加藤陽菜多, 小笠原泰志, 大利徹 日本生物工学会大会講演要旨集 71st 2019年
• 精密解析に基づく多価不飽和脂肪酸生合成酵素の機能改変

  林祥平, 佐藤康治, 小笠原泰志, 大利徹 日本生物工学会大会講演要旨集 71st 2019年
• 天然抗酸化剤~エルゴチオネイン~の高効率微生物生産

  大利徹 アサヒグループ学術振興財団研究紀要(Web) 2019 2019年
• 多価不飽和脂肪酸合成酵素の不飽和度制御機構の解明

  林祥平, 佐藤康治, 小笠原泰志, 大利徹 日本農芸化学会東北支部大会プログラム・講演要旨集 153rd 71 2018年09月22日 [査読無し][通常論文]
  
• 多価不飽和脂肪酸合成酵素の機能改変

  池内健心, 林祥平, 大塚慎, 小林洸太, 佐藤康治, 大利徹 日本農芸化学会東北支部大会プログラム・講演要旨集 153rd 71 2018年09月22日 [査読無し][通常論文]
  
• 抗酸化物質エルゴチオネインの発酵生産

  佐藤康治, 河野祐介, 大津厳生, 大利徹 バイオサイエンスとインダストリー 76 (5) 402‐403 2018年09月10日 [査読無し][通常論文]
  
• 活性酸素の皮膚への影響と抗酸化剤による制御 抗酸化機能を有するエルゴチオネインのシステイン生産大腸菌による発酵生産

  大津厳生, 河野祐介, 佐藤康治, 大利徹 Fragrance Journal 46 (7) 65‐70 2018年07月15日 [査読無し][通常論文]
  
• 異種発現によるバナナ病原菌由来ジテルペン類の生合成研究

  田澤聡大, YE Ying, 尾崎太郎, LIU Chengwei, 南篤志, 小笠原泰志, 大利徹, 及川英秋 日本農芸化学会大会講演要旨集(Web) 2018 2018年
• 真菌由来希少テルペノイドの生合成研究と異種生産

  尾崎太郎, 山根桃華, 田澤聡大, YE Ying, LIU Chengwei, 小笠原泰志, 大利徹, 南篤志, 及川英秋 天然有機化合物討論会講演要旨集(Web) 60th 2018年
• カルボニルメチレン構造を有するシュードトリペプチド(Ketomemicin)の生合成機構

  大利徹 日本農芸化学会大会講演要旨集(Web) 2018 2018年
• バナナ病原菌が感染時に生産するジテルペン類の異種生産

  田澤聡大, YE Ying, 尾崎太郎, LIU Chengwei, 南篤志, 小笠原泰志, 大利徹, 及川英秋 日本化学会春季年会講演予稿集(CD-ROM) 98th 2018年
• ペプチドグリカンの新しい生成機構

  大利徹 日本農芸化学会大会講演要旨集(Web) 2018 2018年
• 放線菌が生み出した疑似ペプチド化合物-疑似ペプチド（ケトメミシン）がもつカルボニルメチレンの生合成を解明-

  小笠原 泰志, 大利 徹 化学と生物 56 76 -78 2018年 [査読無し][通常論文]
  
• 多価不飽和脂肪酸合成酵素における3‐ケトアシルリダクターゼとデヒドラターゼの機能解析

  林祥平, 佐藤康治, 小笠原泰志, 氏原哲朗, 大利徹 日本生物工学会大会講演要旨集 69th 322 2017年08月08日 [査読無し][通常論文]
  
• 大腸菌によるErgothioneineの発酵生産

  大澤怜, 上出倫敬, 佐藤康治, 河野祐介, 大津厳生, 大利徹 日本生物工学会大会講演要旨集 69th 323 2017年08月08日 [査読無し][通常論文]
  
• 細菌ペプチドグリカンの新規生合成機構

  佐藤康治, 馮 若茵, 大利 徹 B & I 75 422 -423 2017年 [査読無し][通常論文]
  
• 創薬を志向した微生物の生合成工学

  大利 徹 The Japanese Journal of Antibiotics 71 (1) 1 -11 2017年 [査読無し][通常論文]
  
• ペプチド結合を触媒する新たな酵素

  小笠原 泰志, 大利 徹 ペプチド医薬品のスクリーニング・安定化・製剤化技術 (技術情報協会) 2017年 [査読無し][通常論文]
  
• 微生物を用いた生合成工学

  大利 徹 生化学 89 (2) 221 -229 2017年 [査読無し][通常論文]
  
• ペプチド系天然化合物JBIR‐78およびJBIR‐95の生合成研究

  竹田薫平, 佐藤康治, 小笠原泰志, 新家一男, 大利徹 日本放線菌学会大会講演要旨集 31st 80 2016年09月08日 [査読無し][通常論文]
  
• 抗酸化物質Ergothioneineの酵素合成法の開発

  大澤怜, 上出倫敬, 佐藤康治, 大津厳生, 大利徹 日本放線菌学会大会講演要旨集 31st 141 2016年09月08日 [査読無し][通常論文]
  
• Photobacterium profundum由来エイコサペンタエン酸合成酵素の異種宿主発現

  中真以, 林祥平, 佐藤康治, 氏原哲朗, 大利徹 日本生物工学会大会講演要旨集 68th 253 2016年08月25日 [査読無し][通常論文]
  
• 多価不飽和脂肪酸合成酵素におけるタンデムアシルキャリアプロテインの機能解明

  林祥平, 佐藤康治, 氏原哲朗, 大利徹 日本生物工学会大会講演要旨集 68th 253 2016年08月25日 [査読無し][通常論文]
  
• 多価不飽和脂肪酸合成酵素の機能解析

  林祥平, 佐藤康治, 氏原哲朗, 大利徹 日本生物工学会大会講演要旨集 68th 168 2016年08月25日 [査読無し][通常論文]
  
• 放線菌に見出されたペプチドライゲースオルソログの機能解析

  小笠原泰志, 川田純平, 佐藤康治, 降旗一夫, 大利徹 日本農芸化学会大会講演要旨集(Web) 2016 4SY05‐3 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  
• ペプチド系天然化合物JBIR‐78および95の生合成研究

  竹田薫平, 佐藤康治, 小笠原泰志, 新家一男, 大利徹 日本農芸化学会大会講演要旨集(Web) 2016 2B038 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  
• Xanthomonas oryzaeに見出した新規アミノ酸異性化酵素

  FENG Ruoyin, 佐藤康治, 大利徹 日本農芸化学会大会講演要旨集(Web) 2016 2B040 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  
• 放線菌におけるエルゴチオネイン特異的輸送体の探索

  上出倫敬, 佐藤康治, 河野祐介, 大津厳生, 大利徹 日本農芸化学会北海道支部講演会講演要旨 2016 2016年
• パラ-アミノ安息香酸生合成に関与する新規遺伝子(2015生物工学論文賞)

  佐藤 康治, 倉都 将宏, 小林 大毅, 大利 徹 生物工学会誌 : seibutsu-kogaku kaishi 94 (2) 72 -72 2016年
• 微生物が生産する生理活性物質研究 : 生物工学的観点から (大村智先生ノーベル賞受賞記念特集 微生物由来天然物の実用化と未来)

  大利 徹 生物工学会誌 94 (7) 390 -392 2016年
• ペプチド系天然化合物JBIR‐95の生合成研究

  竹田薫平, 佐藤康治, 小笠原泰志, 新家一男, 大利徹 日本農芸化学会北海道支部講演会講演要旨 2015 12 2015年11月14日 [査読無し][通常論文]
  
• 多価不飽和脂肪酸生産能に対するacyl carrier proteinドメイン数の影響

  林祥平, 佐藤康治, 氏原哲朗, 大利徹 日本生物工学会大会講演要旨集 67th 337 -337 2015年09月25日 [査読無し][通常論文]
  
• 芳香族アミノ酸水酸化酵素の物質生産への応用

  佐藤康治, 大利徹 日本生物工学会大会講演要旨集 67th 352 -352 2015年09月25日 [査読無し][通常論文]
  
• シアノバクテリアにおけるエルゴチオネイン生合成遺伝子の探索

  大澤怜, 佐藤康治, 中嶋駿介, 大津厳生, 高木博史, 大利徹 日本生物工学会大会講演要旨集 67th 336 -336 2015年09月25日 [査読無し][通常論文]
  
• バクテリア由来フェニルアラニン水酸化酵素を利用したチロシン生産

  佐藤康治, 大利徹 日本生物工学会大会講演要旨集 67th 337 -337 2015年09月25日 [査読無し][通常論文]
  
• 緩い基質特異性を示すペプチドリガーゼの結晶構造からの考察と応用

  野池基義, 松井崇, 小笠原泰志, 森田洋行, 大利徹 日本農芸化学会大会講演要旨集(Web) 2015 3E11P02 (WEB ONLY) 2015年03月05日 [査読無し][通常論文]
  
• C‐末にD‐Trpを持つラッソペプチドの生合成機構

  野村智, 小笠原泰志, 佐藤康治, 大利徹 日本農芸化学会大会講演要旨集(Web) 2015 3E11P05 (WEB ONLY) 2015年03月05日 [査読無し][通常論文]
  
• 3S-Ba05 放線菌に見出したATP-grasp-ligaseが触媒する多様なアミド形成反応(二次代謝産物の生合成工学が切り拓く合成生物学の世界,シンポジウム)

  大利 徹 日本生物工学会大会講演要旨集 67 344 -344 2015年
• 微生物に見出されたメナキノン新規生合成経路の全容解明と抗ピロリ菌剤開発への展開

  大利 徹 旭硝子財団助成研究成果報告 Reports of research assisted by the Asahi Glass Foundation 1 -4 2015年
• アミド結合形成を触媒する新規酵素

  大利 徹 バイオサイエンスとインダストリー 73 (4) 274 -279 2015年
• 放線菌に見いだされた新規ペプチドライゲースは幅広い基質特異性を持つ

  野池基義, 松井崇, 佐藤康治, 森田洋行, 大利徹 日本農芸化学会北海道支部講演会講演要旨 2014 30 2014年09月22日 [査読無し][通常論文]
  
• インドールジテルペンの生合成に関与するプレニル基転移酵素に関する研究

  劉成偉, 藤盛道子, 南篤志, 野池基義, 佐藤康治, 大栗博毅, 及川英秋, 大利徹 イソプレノイド研究会例会講演要旨集 24th 19 2014年09月12日 [査読無し][通常論文]
  
• バクテリアにおける新規葉酸生合成関連遺伝子の同定

  佐藤康治, 小林大毅, 大利徹 日本生物工学会大会講演要旨集 66th 162 -162 2014年08月05日 [査読無し][通常論文]
  
• エルゴチオネインは酸化ストレスから放線菌を守る

  中嶋駿介, 佐藤康治, 大利徹 日本生物工学会大会講演要旨集 66th 162 -162 2014年08月05日 [査読無し][通常論文]
  
• 放線菌に見いだされた新規ペプチドライゲースは幅広い基質特異性を持つ

  野池基義, 松井崇, 佐藤康治, 森田洋行, 大利徹 日本放線菌学会大会講演要旨集 29th 92 2014年06月19日 [査読無し][通常論文]
  
• ペプチドライゲースとリボソームとの協同によるペプチド新奇生合成機構の解明

  野池基義, 雄谷洸一, 佐々木郁雄, 丸山千登勢, 石川淳, 佐藤康治, 濱野吉十, 伊藤肇, 大利徹 日本農芸化学会大会講演要旨集(Web) 2014 2A04P08 (WEB ONLY) 2014年03月05日 [査読無し][通常論文]
  
• インドールジテルペンの生合成に関与するプレニル基転移酵素に関する研究―2

  LIU Chengwei, 藤盛道子, 南篤志, 野池基義, 佐藤康治, 大栗博毅, 及川英秋, 大利徹 日本農芸化学会大会講演要旨集(Web) 2014 2A04P10 (WEB ONLY) 2014年03月05日 [査読無し][通常論文]
  
• エルゴチオネインはStreptomyces coelicolorを酸化ストレスから守る

  中嶋駿介, 佐藤康治, 簗島謙太郎, 松井智未, 大利徹 日本農芸化学会大会講演要旨集(Web) 2014 2A04P11 (WEB ONLY) 2014年03月05日 [査読無し][通常論文]
  
• 2S-Da05 ペプチドのN-末をキャッピングする新奇ペプチドライゲース(天然物生合成研究の最前線,シンポジウム)

  野池 基義, 雄谷 洸一, 大利 徹 日本生物工学会大会講演要旨集 66 98 -98 2014年
• リボソームと新規ペプチドライゲースによる協同的ペプチド生成機構

  野池 基義, 森田 洋行, 大利 徹, 松井 崇, 雄谷 洸一, 佐々木 郁雄, 丸山 千登勢, 濱野 吉十, 石川 淳, 佐藤 康治, 伊藤 肇 天然有機化合物討論会講演要旨集 56 (0) Oral8 2014年 [査読無し][通常論文]
   

  【目的】 ペプチド系抗生物質の生合成では、リボソーム関与の生合成に加え、Non Ribosomal Peptide Synthase（NRPS）に代表されるリボソーム非関与の生合成機構も用いられる。また、近年、ATP依存のアミノ酸リガーゼやtRNA依存のアミノ酸転移酵素によるペプチド結合生成機構も報告されている。このようにペプチド系抗生物質の生合成には多様性があるが、ペプチドの基本骨格そのものは上記の何れかの機構で生合成される。

  放線菌が生産するペプチド系抗生物質、pheganomycin（PGM、下図）は、N-末のL-3,5-dihydroxy-amidino-phenylglycine（DHPG）誘導体に、何れもL-体からなるAsn-tertiary Leu-Lys-Asp-Arg、あるいはAsn-tertiary Leu-Lys-Asp-Gly-Pro-Thrが結合した2種類が知られている¹。tert-Leuは、最近bottromycinの生合成研究で明らかにされたように^2,3、radical S-adenosyl methionine （SAM）酵素によるVal側鎖のメチル化により生成すると推定されることから、DHPG のみが非タンパク性のアミノ酸と考えられる。PGMの生合成を考察すると、非タンパク性のDHPGを含むことからNRPSで生成すると推定されるが、一般にアミノ酸をアデニル化するNRPS のA-ドメインの基質認識は厳密であり、どのように2種類の配列からなるペプチドが生合成されるか興味が持たれたことから、その生合成機構の解明を試みた。

  【方法および結果】 バンコマイシンに含まれるDHPGの基本骨格は、DpgAからDpgCにより生合成されることが報告されている（下図）⁴。そこでこれら遺伝子のオーソログをPGM生産菌のドラフトゲノム配列中に探索した結果、生合成遺伝子クラスターを見出した（次ページ図）⁵。本クラスターには、DHPG誘導体の合成に関与すると推定される、DpgC反応に続くアミノ基転移酵素、アミジノ基転移酵素、4位メチル化と水酸化酵素遺伝子（P450）、また、Val側鎖のメチル化に関与すると推定されるradical SAM酵素遺伝子が存在した。しかし、最長8つのアミノ酸からなるNVKDGPTを合成し得る巨大なNRPSを近傍に見出せなかった。そこで、クラスター内にペプチダーゼが存在したことも考慮し、ペプチド部分がリボソーム関与で合成される可能性を考え塩基配列を精査した結果、38アミノ酸からなる遺伝子を見出した。このペプチドはNVKDRとNVKDGPTの両方の配列を含んでおり、C-末の多様性を矛盾なく説明できた。

  そこで次に、本クラスターがPGMの生合成に関与することを証明するために、プレカーサーペプチドをコードする構造遺伝子の破壊を行った。相同組換えにより構造遺伝子のみを欠失させた株を構築した結果、PGMの生産性が消失したことから、PGMのペプチド性アミノ酸からなるC-末部分は、リボソームにより合成されたプレカーサーペプチドに由来すると考えられた⁵。

  しかし、N-末のDHPG誘導体とプレカーサーペプチドから供給されたペプチドの結合を触媒する酵素に関しては候補遺伝子を見出せなかった。クラスター内には、A-ドメイン1つを持つNRPS-A（649アミノ酸）、T-ドメインを持つNRPS-T（89アミノ酸）、C-ドメインを持つNRPS-C（441アミノ酸）が存在した（上図）。これまでNRPSのA-ドメインで活性化されたアミノ酸類に対し、ペプチドが求

  (View PDFfor the rest of the abstract.)

• ペプチド系抗生初質フェガノマイシンの生合成に見いだされた新規ペプチドライゲースPGM1の結晶構造解析

  松井崇, 大瀧翔太, 野池基義, 大利徹, 森田洋行 日本結晶学会年会講演要旨集 2014 79 2014年 [査読無し][通常論文]
  
• ビタミンKの生合成

  大利 徹 ビタミンKと疾患 40 -46 2014年 [査読無し][通常論文]
  
• ペプチド系抗生物質フェガノマイシンの生合成に関与する新規ペプチドライゲース

  野池基義, 雄谷洸一, 佐々木郁雄, 丸山千登勢, 石川淳, 佐藤康治, 濱野吉十, 伊藤肇, 大利徹 日本農芸化学会北海道支部講演会講演要旨 2013 24 2013年11月27日 [査読無し][通常論文]
  
• エルゴチオネインはStreptomyces coelicolorを酸化ストレスから守る

  中嶋駿介, 佐藤康治, 簗島謙太郎, 松井智未, 大利徹 日本農芸化学会北海道支部講演会講演要旨 2013 18 2013年11月27日 [査読無し][通常論文]
  
• 放線菌に見出した新規ペプチドライゲース

  野池基義, 雄谷洸一, 佐々木郁雄, 丸山千登勢, 石川淳, 佐藤康治, 濱野吉十, 伊藤肇, 大利徹 日本放線菌学会大会講演要旨集 28th 62 2013年09月05日 [査読無し][通常論文]
  
• エルゴチオネインはStreptomyces coelicolorを酸化ストレスから守る

  中嶋駿介, 佐藤康治, 簗島謙太郎, 松井智未, 大利徹 日本放線菌学会大会講演要旨集 28th 63 2013年09月05日 [査読無し][通常論文]
  
• ペプチド系抗生物質,ボトロマイシン生合成機構の解明

  佐藤康治, 大門美保, 簗島謙太郎, 尾仲宏康, 石川淳, 大利徹 日本農芸化学会大会講演要旨集(Web) 2013 WEB ONLY 3C22A12 2013年03月05日 [査読無し][通常論文]
  
• バクテリアにおける新規葉酸生合成経路の解明

  佐藤康治, 小林大毅, 大利徹 日本農芸化学会大会講演要旨集(Web) 2013 WEB ONLY 3C22A11 2013年03月05日 [査読無し][通常論文]
  
• ペプチド系抗生物質,フェガノマイシン生合成機構の解明

  野池基義, 雄谷洸一, 佐藤康治, 丸山千登勢, 濱野吉十, 石川淳, 大利徹 日本農芸化学会大会講演要旨集(Web) 2013 WEB ONLY 3C22A13 2013年03月05日 [査読無し][通常論文]
  
• 放線菌が持つメナキノン新規生合成経路の解明と特異的阻害剤の探索

  池田駿介, 池田安由美, 佐藤康治, 野池基義, 瀬戸治男, 大利徹 日本放線菌学会大会講演要旨集 27th 104 2012年09月06日 [査読無し][通常論文]
  
• 熱安定性酵素を用いたポリヒドロキシアルカン酸のin vitro合成

  橋本佳季, HAN Xuerong, 佐藤康治, 田島健次, 田口精一, 大利徹 高分子学会予稿集(CD-ROM) 61 (2) ROMBUNNO.1PB118 2012年09月05日 [査読無し][通常論文]
  
• バイオディーゼル燃料副生成物からのバクテリアセルロースの生産

  小瀬亮太, 吉田誠, 砂川直輝, 田島健次, 大利徹 セルロース学会年次大会講演要旨集 19th 122 2012年07月01日 [査読無し][通常論文]
  
• 遺伝子組換え酢酸菌によるセルロース誘導体の合成

  佐藤智章, 砂川直輝, 小瀬亮太, 田島健次, 大利徹 セルロース学会年次大会講演要旨集 19th 61 2012年07月01日 [査読無し][通常論文]
  
• 遺伝子組換え酢酸菌によるGlcNAc含有バクテリアセルロースの合成

  砂川直輝, 今井友也, YAO Min, 小瀬亮太, 田島健次, 大利徹 セルロース学会年次大会講演要旨集 19th 15 -16 2012年07月01日 [査読無し][通常論文]
  
• 酢酸菌のセルロース合成におけるbglxAおよび下流遺伝子の機能

  砂川直輝, 松田恵利子, 小瀬亮太, YAO Min, 田島健次, 大利徹 セルロース学会年次大会講演要旨集 19th 133 2012年07月01日 [査読無し][通常論文]
  
• バクテリアにおける新規葉酸生合成経路の解明

  佐藤康治, 大利徹 日本農芸化学会大会講演要旨集(Web) 2012 WEB ONLY 2A06A03 2012年03月05日 [査読無し][通常論文]
  
• フタロシン(FL)経路の解析

  池田駿介, 柏崎恭平, 小野裕介, 池田安由美, 佐藤康治, 矢島新, 瀬戸治男, 大利徹 日本農芸化学会大会講演要旨集(Web) 2012 WEB ONLY 2A06A02 2012年03月05日 [査読無し][通常論文]
  
• Enterobacter sp.CJF002株におけるバクテリアセルロース合成の解析

  砂川直輝, 細田真梨子, 田島健次, 河野信, YAO Min, 佐藤康治, 大利徹 セルロース学会年次大会講演要旨集 18th 137 2011年07月01日 [査読無し][通常論文]
  
• 海藻由来ポリヒドロキシアルカン酸合成菌の単離・同定

  栗城夢実, 田島健次, 佐藤康治, 尾島孝男, 小林孝紀, 大利徹 高分子学会北海道支部研究発表会講演要旨集 45th 47 2011年02月01日 [査読無し][通常論文]
  
• 皮膚における脂肪細胞の効果—皮膚を構成する他細胞との相互作用

  大木菜津美, 金山寿之, 佐藤康治, 田島健次, 大利徹, 棟方正信 高分子学会北海道支部研究発表会講演要旨集 45th 46 2011年02月01日 [査読無し][通常論文]
  
• 2010年度学会賞受賞論文 放線菌が持つメナキノン新規生合成経路の解明

  大利 徹 日本放線菌学会誌 25 (2) 2 -6 2011年
• 微生物におけるビタミンＫ生合成の分子機構と生物学的意義

  大利 徹 腎と代謝 24 179 -186 2011年 [査読無し][通常論文]
  
• 官能基修飾によるポリヒドロキシアルカン酸の機能化

  岩本浩介, 谷尾亮, 佐藤康治, 田島健次, 堺井亮介, 佐藤敏文, 鳥谷部哲也, 松島得雄, 覚知豊次, 大利徹, 棟方正信, 棟方正信 高分子学会予稿集(CD−ROM) 59 (2 Disk1) ROMBUNNO.1PD114 2010年09月01日 [査読無し][通常論文]
  
• 改良水‐有機溶媒二相反応系による新規ポリヒドロキシアルカン酸の合成

  HAN Xuerong, 佐藤康治, 田島健次, 鳥谷部哲也, 松島得雄, 大利徹, 棟方正信, 棟方正信 高分子学会予稿集(CD−ROM) 59 (2 Disk1) ROMBUNNO.1PC113 2010年09月01日 [査読無し][通常論文]
  
• Acetobacter xylinumにおけるbglxA下流遺伝子のクローニングと解析

  松田恵利子, 砂川直輝, 吉田誠, 田島健次, 佐藤康治, 棟方正信, 大利徹 セルロース学会年次大会講演要旨集 17th 104 2010年07月01日 [査読無し][通常論文]
  
• 酢酸菌セルロース合成におけるAxCesD N末端の機能解析

  砂川直輝, 藤原孝彰, 田島健次, YAO Min, 佐藤康治, 棟方正信, 田中勲, 大利徹 セルロース学会年次大会講演要旨集 17th 110 2010年07月01日 [査読無し][通常論文]
  
• バイオディーゼル副生成物を原料としたバクテリアセルロース合成

  砂川直輝, 吉田誠, 松田恵利子, 細田真梨子, 佐藤康治, 松島得雄, 田島健次, 棟方正信, 大利徹 日本生物工学会シンポジウム講演要旨集 2010 33 2010年 [査読無し][通常論文]
  
• 33.麹菌のジテルペン合成酵素(口頭発表)

  千葉 康隆, 菅井 佳宣, 塩野 義人, 大利 徹, 三橋 渉, 川出 洋, 豊増 知伸 植物化学調節学会研究発表記録集 (44) 47 -47 2009年10月06日 

  Fungi produce diterpenes exhibiting phytohormone-related activities, such as gibberellin and fusicoccin. We identified a diterpene synthase gene PaFS, which is responsible for fusicoccin biosynthesis, in the mycelium of fusicoccin-producing fungus Phomopsis amygdali (Toyomasu et al., PNAS, 104: 3084-3088, 2007). PaFS (719 amino acids) possesses the diterpene cyclase domain at N-terminus and prenyltransferase domain at C-terminus. Our database search suggested that PaFS homologues exist in genome DNA of several fungal species. Among them, we found two PaFS homologous genes in Aspergillus oryzae genome database. It has been believed that A. oryzae produces few secondary metabolites including terpenoids. In this study, we characterize the two homologues, A. oryzae diterpene synthase like 1(AoDSL1) and AoDSL2. AoDSL1 contained a stop codon in region encoding prenyltransferase domain probably because of naturally occurred nonsense mutation, and is perhaps expressed as a truncated enzyme composed of N-terminal 412 amino acid residues. AoDSL2 encoded 728 amino acid residues, in which a successive Asp of DDxxD motif in the prenyltransferase domain was substituted by AN (position 482-483). These possible naturally occurred mutations perhaps resulted in loss of prenyltransferase activities. Recombinant AoDSL1 converted GGDP into two unknown diterpene cyclic hydrocarbons. The effects of site directed mutagenesis of possible naturally occurred mutation on enzyme activity will be presented.
• 微生物に見出されたメナキノン新規生合成経路

  大利 徹 日本生化学会誌 81 (2) 95 -99 2009年 [査読有り][招待有り]
  
• 細菌に見出されたメナキノン新規生合成経路

  大利 徹 バイオサイエンスとインダストリィー 67 (3) 102 -106 2009年 [査読無し][通常論文]
  
• 創薬標的としての新規メナキノン生合成系

  大利 徹 細胞工学 28 (4) 366 -371 2009年 [査読無し][通常論文]
  
• 創薬ターゲットとしてのメナキノン新規生合成経路

  大利 徹 ファルマシア 45 (9) 870 -874 2009年 [査読無し][通常論文]
  
• 糸状菌が生産するジテルペン化合物の生合成に関する研究

  南篤志, 田島直人, 橋元誠, 加藤雅士, 小林哲夫, 豊増知伸, 佐々武史, 加藤修雄, 大利徹 日本農芸化学会大会講演要旨集 2008 2008年
• イネのフィトアレキシンとジベレリン生合成に関与するent-コパリル2リン酸合成酵素の酵素的性質の比較

  林 豊, 廣瀬 祐子, 小野寺 悠, 三橋 渉, 佐々 武史, 大利 徹, 豊増 知伸 植物の生長調節 = Regulation of plant growth & development 42 34 -34 2007年10月05日 

  Gibberellins, a phytohormone, are biosynthesized from geranylgeranyl diphosphate (GGDP) via ent-kaurene. In higher plants, entkaurene was formed from GGDP via ent-copalyl diphsphate (ent-CDP) by two distinct diterpene cyclases, ent-CDP synthase and ent-kaurene synthase. Rice (Oryza sativa L.) produces diterpene phytoalexins, such as oryzalexins, momilactones and phytocassanes, of which biosynthetic hydrocarbon intermediates are converted from GGDP by the similar manner as ent-kaurene synthesis. We have shown that rice has two ent-CDP synthase genes; OsCPSl is for gibberellin biosynthesis and OsCPS2/OsCyc2 is for oryzalexins A-F and phytocassanes A-E. Although ent-CDP synthases for gibberellin biosynthesis from other plant species were well characterized, we have known little about properties of rice ent-CDP synthase for phytoalexin biosynthesis.Here, we enzymatically characterized recombinant putative mature OsCPS2/OsCyc2 as well as OsCPS4/OsCycl, syn-CDP synthase for mimilactones A and B and oryzalexin S, and compared to OsCPS1. The optima of pH (around neutral), temperature (around 30 oC), and divalent ion (0.1 mM Mg^<2+>) on OsCPS2/OsCyc2 and OsCPS4/OsCyc1 were almost the same as OsCPS1. Interestingly, the inhibition rate of activities of OsCPS2/OsCyc2 and OsCPS4/OsCyc1 by substrate, GGDP (50-60μM), and Amo-1618 (10^<-5>-10^<-4>M), a gibberellin biosynthetic inhibitor which suppresses ent-CDP synthase activity, was less than that of OsCPS1.
• Nocardia属細菌が持つイソプレノイド生合成遺伝子資源の探索(平成18年度共同利用研究報告)

  大利 徹, 三上 襄 千葉大学真菌医学研究センター報告 11 97 -97 2007年
• 2C16-2 高活性なフェニルアセトアルデヒド還元酵素変異体を選択する系の改良(酵素学・酵素工学・タンパク質工学,一般講演)

  牧野 祥嗣, 大利 徹, 伊藤 伸哉 日本生物工学会大会講演要旨集 19 87 -87 2007年
• Further improvement of engineered phenylacetaldehyde reductase (PAR) on activity under concentrated 2-propanol - Abstracts

  Yoshihide Makino, Tohru Dairi, Nobuya Itoh JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 42 (3-4) 116 -117 2006年11月 [査読無し][通常論文]
  
• Cloning and expression of cyclo(Leu-Phe) oxidase gene from an actinomycete Streptomyces albulus K023

  Machiko Nagao, Atsushi Morimoto, Takashi Tamura, Tohru Dairi, Hiroshi Kanzaki JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 42 (3-4) 126 -126 2006年11月 [査読無し][通常論文]
  
• Chiral alcohol production by enantioselective reduction with immobilized recombinant E-coli cells expressing PAR and LSADH - Abstracts

  Masatoshi Nakamura, Kousuke Inoue, Yoshihide Makino, Tohru Dairi, Nobuya Itoh JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 42 (3-4) 116 -116 2006年11月 [査読無し][通常論文]
  
• フシコクシン生合成に関与する環化酵素遺伝子のクローニングと機能解析

  塚原 麻伊, 豊増 知伸, 金子 あかね, 新井田 理絵, 三橋 渉, 大利 徹, 加藤 修雄, 佐々 武史 植物の生長調節 = Regulation of plant growth & development 41 61 -61 2006年10月04日
• 放線菌の多様なイソプレノイド生合成遺伝子

  大利 徹 バイオサイエンスとインダストリー 64 (6) 2006年 [査読無し][通常論文]
  
• 放線菌が生産するイソプレノイド抗生物質の生合成研究

  大利 徹 The Japanese journal of antibiotics 58 (1) 87 -98 2005年02月25日
• 原核生物に見いだされたジテルペンサイクラーゼの機能解析

  大利 徹, 濱野 吉十, 伊藤 伸哉, 葛山 智久, 降旗 一夫, 瀬戸 治男 日本農芸化学会誌 vol. 76, No. 12, 1191-1194 (12) 1191 -1194 2002年 [査読無し][通常論文]
  
• テルペノイド抗生物質生産放線菌からのメバロン酸経路遺伝子クラスターのクローニング

  濱野 吉十, 大利 徹, 山本 正浩, 川崎 崇, 金田 一秀, 葛山 智久, 伊藤 伸哉, 瀬戸 治男 日本農芸化学会誌 vol. 76, No. 11, 1092-1093 (11) 1092 -1093 2002年 [査読無し][通常論文]
  
• Cloning and biochemical characterization of Co2+-activated bromoperoxidase-esterase (perhydrolase) from Pseudomonas putida IF-3 strain

  N Itoh, T Kawanami, JQ Liu, T Dairi, M Miyakoshi, C Nitta, Y Kimoto BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1545 (1-2) 53 -66 2001年02月 [査読無し][通常論文]
   

  The gene encoding Co2+-activated bromoperoxidase (BPO)-esterase (EST), catalyzing the organic acid-assisted bromination of some organic compounds with H2O2 and Br- and quite specific hydrolysis of (R)-acetylthioisobutyric acid methyl ester, was cloned from the chromosomal DNA of the Pseudomonas putida IF-3 strain. The bpo-est gene comprises 831 bp and encoded a protein of 30181 Da. The enzyme was expressed at a high level in Escherichia coli and purified to homogeneity by ammonium sulfate fractionation and two-step column chromatographies. The recombinant enzyme required acetic acid, propionic acid, isobutyric acid or n-butyric acid in addition to H2O2 and Br- for the brominating reaction and was activated by Co2+ ions. It catalyzed the bromination of styrene and indene to give the corresponding racemic bromohydrin. Although the enzyme did not release free peracetic acid in the reaction mixture, chemical reaction with peracetic acid could well explain such enzymatic reactions via a peracetic acid intermediate. The results indicated that the enzyme was a novel Co2+ -activated organic acid-dependent BPO (perhydrolase)-EST, belonging to the non-metal haloperoxidase-hydrolase family. (C) 2001 Elsevier Science B.V. All rights reserved.
• Diversity of microbial threonine aldolases and their application

  JQ Liu, T Dairi, N Itoh, M Kataoka, S Shimizu, H Yamada JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 10 (1-3) 107 -115 2000年09月 [査読無し][通常論文]
   

  Threonine aldolase catalyzes the reversible interconversion of certain beta-hydroxy-alpha-amino acids and glycine plus the corresponding aldehydes. Various microbial threonine aldolases with different stereospecificities were found on extensive screening, and the genes encoding the proteins were cloned and heterogeneously overexpressed in Escherichia coli. By using recombinant threonine aldolases, an enzymatic resolution process was established for the production of optically pure P-hydroxy-a-amino acids. In addition, the threonine aldolase-catalyzed direct synthesis of beta-hydroxy-alpha-amino acid from aldehyde and glycine is discussed. (C) 2000 Elsevier Science B.V. Al rights reserved.
• Studies on antibiotic biosynthetic genes and enzymes catalyzing unique reactions

  T Dairi NIPPON NOGEIKAGAKU KAISHI-JOURNAL OF THE JAPAN SOCIETY FOR BIOSCIENCE BIOTECHNOLOGY AND AGROCHEMISTRY 74 (9) 975 -983 2000年09月 [査読無し][通常論文]
  
• Studies on the nonmevalonate pathway: Conversion of 4-(cytidine 5'- diphospho)-2-C-methyl-D-erythritol to its 2-phospho derivative by 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase

  Tomohisa Kuzuyama, Motoki Takagi, Kazuhide Kaneda, Hiroyuki Watanabe, Tohru Dairi, Haruo Seto Tetrahedron Letters 41 (16) 2925 -2928 2000年04月15日 [査読無し][通常論文]
   

  A nonmevalonate pathway intermediate, 4-(cytidine 5'-diphospho)-2-C- methyl-D-erythritol, is transformed to its 2-phospho-derivative in the presence of ATP by a novel Escherichia coli enzyme, 4-(cytidine 5'- diphospho)-2-C-methyl-D-erythritol kinase. (C) 2000 Elsevier Science Ltd.
• Studies on the nonmevalonate pathway: formation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate from 2-phospho-4-(cytidine 5 '-diphospho)-2-C-methyl-D-erythritol

  M Takagi, T Kuzuyama, K Kaneda, H Watanabe, T Dairi, H Seto TETRAHEDRON LETTERS 41 (18) 3395 -3398 2000年04月 [査読無し][通常論文]
   

  2-Phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol was transformed to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate by a novel Escherichia coli enzyme involved in the nonmevalonate pathway. (C) 2000 Elsevier Science Ltd. All rights reserved.
• ユニークな反応を触媒する抗生物質生合成酵素・遺伝子群の解析

  大利 徹 日本農芸化学会誌 74 420 -422 2000年03月05日
• ユニークな反応を触媒する抗生物質生合成酵素・遺伝子の解析

  大利 徹 日本農芸化学会誌 74 (9) 975 -983 2000年 [査読無し][通常論文]
  
• Formation of 4-(cytidine 5 '-diphospho)-2-C-methyl-D-erythritol from 2-C-methyl-D-erythritol 4-phosphate by 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, a new enzyme in the nonmevalonate pathway

  T Kuzuyama, M Takagi, K Kaneda, T Dairi, H Seto TETRAHEDRON LETTERS 41 (5) 703 -706 2000年01月 [査読無し][通常論文]
   

  2-C-Methyl- D-erythritol-4-phosphate is transformed to 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol in the presence of cytidine 5'-triphosphate by a novel Escherichia coli enzyme, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, involved in the nonmevalonate pathway. (C) 2000 Elsevier Science Ltd. All rights reserved.
• 803 ランダム変異によるL-allo-threonine aldolaseの立体選択性の改変

  片岡 道彦, 川端 潤, 劉 吉泉, 大利 徹, 伊藤 伸哉, 山田 秀明, 清水 昌 日本生物工学会大会講演要旨集 11 160 -160 1999年
• Low-specificity L-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to beta-hydroxy-alpha-amino acid synthesis

  JQ Liu, S Ito, T Dairi, N Itoh, S Shimizu, H Yamada APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 49 (6) 702 -708 1998年06月 [査読無し][通常論文]
   

  Low-specificity L-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between L-threonine/L-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits with a molecular mass of 35 kDa. The enzyme, requiring pyridoxal-5'-phosphate as a coenzyme, is strictly L-specific at the alpha position, whereas it can not distinguish between threo and erythro forms at the beta position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible interconversion between glycine plus various aldehydes and L-beta-hydroxy-alpha-amino acids, including L-beta-(3,4-dihydroxyphenyl)serine, L-beta-(3,4-methylenedioxyphenyl)serine and L-beta-phenylserine, providing a new route for the industrial production of these important amino acids.
• 836 低立体選択性D-スレオニンアルドラーゼ活性の発現に必要な補酵素の同定

  劉 吉泉, 大利 徹, 伊藤 伸哉, 片岡 道彦, 清水 昌, 山田 秀明 日本生物工学会大会講演要旨集 10 168 -168 1998年
• Studies on Antibiotic-Biosynthetic Genes and Enzymes Catalyzing Unique Reactions

  Dairi Tohru 日本放線菌学会誌 12 (2) 75 -83 1998年 [査読無し][通常論文]
  
• Protoplasting and regeneration of strains belonging to the genus Actinomadura.

  Actinomycetologica 11 (1) 1 -5 1997年 [査読無し][通常論文]
  
• 新しいペニシリン認識酵素、アルカリD-ペプチダーゼの構造と機能 : 酵素

  浅野 泰久, 伊藤 元, 大野 泰世, 大利 徹, 加藤 康夫 日本農藝化學會誌 70 301 -301 1996年03月05日
• 抗真菌抗生物質Pradimicin生合成遺伝子のクローニングと一次構造解析 : 第一報 : 微生物

  大利 徹, 古米 保, 沖 俊一 日本農藝化學會誌 70 101 -101 1996年03月05日
• Electrophoretic fraction of Ganoderma licidium extract under non-denatured polyacrylamide gel

  Journal of Traditional Medicines 13 504 -505 1996年 [査読無し][通常論文]
  
• NUCLEOTIDE-SEQUENCE OF FORTIMICIN KL1 METHYLTRANSFERASE GENE ISOLATED FROM MICROMONOSPORA-OLIVASTEROSPORA, AND COMPARISON OF ITS DEDUCED AMINO-ACID-SEQUENCE WITH THOSE OF METHYLTRANSFERASES INVOLVED IN THE BIOSYNTHESIS OF BIALAPHOS AND FOSFOMYCIN

  T KUZUYAMA, T SEKI, T DAIRI, T HIDAKA, H SETO JOURNAL OF ANTIBIOTICS 48 (10) 1191 -1193 1995年10月 [査読無し][通常論文]
  
• Arthrobacter sp. 1Cのオピン脱水素酵素遺伝子 : 微生物

  大利 徹, 浅野 泰久 日本農藝化學會誌 69 270 -270 1995年07月05日
• Potential Antifungal and Anti-HIV Agent ; Prodimicin.

  ┣DBExp. Opin. Patents. (/)-┫DB (4) 1483 -1491 1994年 [査読無し][通常論文]
  
• Use of a Heterologous Gene for Molecular Breeding of Actinomycetes Producing Structurally Related Antibiotics : Self-defense Genes of Producers of Fortimicin-A(Astromicin)-group Antibiotics.

  Toshio Ohata, Tohru Dairi, Eri Hashimoto, Mamoru Hasegawa ┣DBActinomycetol. (/)-┫DB 7 (2) 145 -155 1993年 [査読無し][通常論文]
  
• Construction and Application of Gene Cloning System for ┣DBStreptomyces sannanensis. (/)-┫DB

  in "Trends in Actinomycetology in Japan", ed. by Yasumasa Koyama, Society for Actinomycetes Japan, Tokyo 101 -102 1989年 [査読無し][通常論文]
  
• Common Feature beyond the Generic Difference of the Biosynthesis of Fortimicins and Sannamycins.

  in "Trends in Actinomycetology in Japan", ed. by Yasumasa Koyama, Society for Actinomycetes Japan, Tokyo 65 -68 1989年 [査読無し][通常論文]
  
• Cloning and nucleotide sequence of the putative polyketide synthase gene for pradimicin biosynthesis from Actinomadura hibisca

  Tohru DAIRI, Yoshimitsu HAMANO, Yasuhiro IGARASHI, Tamotsu FURUMAI, Toshikazu OKI Biosci. Biotech. Biochem. 61 (9) 1445 -1453 1977年 [査読無し][通常論文]
  

特許

• 特開2017-184690:多価不飽和脂肪酸ポリケチドシンターゼ及びその利用  2017年10月12日

  大利 徹, 佐藤 康治, 林 祥平, 氏原 哲朗  協和発酵バイオ株式会社  201703017522783208
• 特許第5528666号:新規化合物デヒポキサンチニルフタロシン及びその製造方法  2014年04月25日

  大利 徹, 瀬戸 治男, 降旗 一夫, 山下 治之  株式会社ADEKA  201403020155294651
• 特許第5305615号:メナキノンの新規生合成経路に関与するタンパク質の遺伝子群を利用したメナキノンの製造方法  2013年07月05日

  大利 徹, 瀬戸 治男, 山下 治之  株式会社ADEKA  201303000741674708
• 特開2012-056899:抗ピロリ菌剤の探索方法  2012年03月22日

  大利 徹, 荒川 知里, 倉都 将宏  富山県, 協和発酵バイオ株式会社  201203046444324822
• 特開2009-114124:新規化合物、メナキノンの新規生合成経路を利用した新規化合物の製造方法、メナキノンの新規生合成経路の中間体を利用したメナキノンの生産量を増加させる製造方法、及びメナキノンの新規生合成経路の中間体を利用したメナキノンの新規生合成経路に特異的な阻害剤の探索方法  2009年05月28日

  大利 徹, 瀬戸 治男, 降旗 一夫, 山下 治之  株式会社ADEKA  200903071133439897
• 特開2008-245628:フシコッカン合成キメラ型酵素およびその遺伝子  2008年10月16日

  豊増 知伸, 佐々 武史, 大利 徹, 加藤 修雄  国立大学法人大阪大学  200903034275224151
• 特開2008-011854:メナキノンの新規生合成経路に関与するタンパク質の遺伝子群  2008年01月24日

  大利 徹, 瀬戸 治男, 山下 治之  株式会社ADEKA  200903004619160895
• 特開2004-236618:メバロン酸の製造方法及びそれにかかわる酵素遺伝子  2004年08月26日

  大利 徹, 葛山 智久, 山下 治之  旭電化工業株式会社  200903055547211647
• 特開2004-187531:メバロン酸の製造方法  2004年07月08日

  瀬戸 治男, 葛山 智久, 大利 徹, 山下 治之  旭電化工業株式会社  200903027980052215

受賞

• 2023年03月 公益社団法人 日本農芸化学会 日本農芸化学会賞
   微生物天然化合物の構造・機能多様性を創出する新規生合成酵素・機構に関する研究
• 2022年03月 日本農芸化学会 トピックス賞
   微細藻類由来DHA合成酵素の炭素鎖伸長反応の解析 

  受賞者: 仲間 陸;小林 飛悠;大塚 慎;佐藤 康治;小笠原 泰志;大利 徹
• 2021年03月 日本農芸化学会 トピックス賞
   ポリグルタミン酸生合成におけるエピメリ化酵素の同定 

  受賞者: 加藤 陽菜多、小笠原 泰志、大利 徹
• 2019年03月 日本農芸化学会 トピックス賞
   in vitro解析による多価不飽和脂肪酸生合成酵素の炭素鎖長制御機構の解明 

  受賞者: 林 祥平;小笠原 泰志;佐藤 康治;丸山 千登勢;濱野 吉十;氏原 哲朗;大利 徹
• 2017年03月 日本農芸化学会 トピックス賞
   An unprecedented glutamate epimerase for bacterial peptidoglycan biosynthesis 

  受賞者: Ruoyin FENG;Yasuharu Satoh;Yasushi Ogasawara;Tohru Yoshimura;Tohru Dairi
• 2015年06月 日本生物工学会 第23回生物工学論文賞
   New gene responsible for para-aminobenzoate biosynthesis 

  受賞者: Y. Satoh;M. Kuratsu;D. Kobayashi;T. Dairi
• 2012年 酵素応用シンポジウム運営委員会 酵素応用シンポジウム奨励賞
   

  受賞者: 大利 徹
• 2011年 長瀬財団 Nagase Foundation Award
   

  受賞者: 大利 徹
• 2010年 日本放線菌学会 日本放線菌学会 学会賞
   

  受賞者: 大利 徹
• 2004年 住木・梅澤記念賞
  
• 2004年 Sumiki & Umezawa Award from Japan Antibiotic Research Association
  
• 2002年 日本農芸化学会論文賞
  
• 2000年 日本農芸化学会奨励賞
  
• 2000年 Encouragement Award from Japan Society for Bioscience, Biotechnology, and Agrochemistry
  
• 1999年 とやま賞
  
• 1999年 TOYAMA Award from Toyama Hitodukuri Foundation
  
• 1998年 日本放線菌学会浜田賞
  
• 1998年 Encouragement Award from The Society for Actinomycetes Japan
  

共同研究・競争的資金等の研究課題

• 天然ペプチド系化合物に構造・機能多様性をもたらす新規酵素・生合成機構の解明と応用

  日本学術振興会：科学研究費助成事業 基盤研究(S)

  研究期間 : 2022年04月 -2027年03月 

  代表者 : 大利 徹, 小笠原 泰志, 森田 洋行, 濱野 吉十
• 新規ペプチドエピメラーゼ類の反応基盤解明

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2018年04月 -2022年03月 

  代表者 : 大利 徹, 森田 洋行, 濱野 吉十

   

  以下の2つのペプチド新規異性化酵素について詳細な解析を行った。 ①微生物のペプチドグリカンの生合成に関与する新規エピメラーゼ（XOO_1319）； UDP-MurNAc-L-Ala-L-Gluの末端のL-Gluのエピメリ化を触媒する酵素（MurL）は、ATPを要求する新規エピメラーゼである。反応液中にAMPが検出されることから基質はアデニル化により活性化されると予想される。そこでヒドロキシルアミンによる中間体のトラップ実験を行った結果、ヒドロキサム酸誘導体が検出されたことから、アデニル化による活性化が確認できた。また昨年度、MurLのアポ体のX線結晶構造解析を行ったが、基質結合部位と推定される領域がオープン構造とっており、反応機構を推定するまでには至らなかった。そこで、放線菌Micromonospora属由来とSalinospora属由来のMurLについてもアポ体の結晶構造解析を行った。その結果、同様の構造が得られ、オープン構造が本酵素群の共通の構造であると判断された。そこで反応機構解明に必須である、基質類縁体との共結晶を得るためにアデニル化反応中間体ミミックの合成を行った。現在、化学合成と酵素合成を組み合わせ、少量スケールでの目的化合物の合成に成功している。
  ②リボソームにより合成されるラッソペプチド（MS-271）；昨年度、MS-271のC末端のL-Trpのエピメリ化に関与する遺伝子を同定した。今年度、大腸菌異種宿主発現系を用い詳細に解析した結果、プレカーサーペプチド、エピメリ化酵素、プロテアーゼ遺伝子を発現させた結果、C末端のTrp の異性化反応が進行した。現在、in vitro実験での再現を試みている。
• 生合成リデザイン・研究総括班

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2016年06月 -2021年03月 

  代表者 : 阿部 郁朗, 菅 裕明, 梅野 太輔, 葛山 智久, 南 篤志, 渡辺 賢二, 濱野 吉十, 江口 正, 山崎 真巳, 大利 徹, 脇本 敏幸, 池田 治生

   

  本領域では、生合成の「設計図を読み解く」から、「新しい設計図を書く」方向に飛躍的な展開を図る。天然物構造多様性の遺伝子・酵素・反応の視点からの精密解析に基づき、新たに生合成工学や合成生物学の世界最先端の技術基盤を確立することで、生合成システムの合理的再構築による複雑骨格機能分子の革新的創成科学を新たな学術領域として強力に推進する。生合成リデザインの達成に向けて３つのグループを計画班に設けた。総勢１２名が計画研究代表者として参画する。限られた数の班員で、他分野との連携を強化する目的で、各計画研究には、結晶構造解析、進化分子工学などの異分野の研究者が、多数、連携研究者として含まれている。総括班では円滑な情報共有と連携の強化を進める。 領域開始の４年目（2019年度）は総括班に加え、第２期公募班が研究に従事した。領域代表者のリーダーシップにより、領域の意義がより明確になるように、各班の計画的共同研究の推進と異分野の融合に配慮した。年２回の公開シンポジウムを含む研究集会を開催し、領域の方針や計画を確認した。班員の相互の密接な情報交換、解析データの共有および共同研究の推進を図る。異分野研究者のコミュニケーションの円滑化、国内外の当該研究分野との連携を図り、本研究領域を適切な方向へ進展させた。広報担当（濱野吉十、南篤志、脇本敏幸、梅野大輔）、を中心に本学術領域ホームページを運営した。逐次研究成果を公表した。また、若手育成担当（葛山智久、山崎真巳）を中心に、若手シンポジウムなどを企画し、当該分野の若手研究者が成果報告や意見交換を行い、切磋琢磨する場を設けた。さらにシンポジウムの告知や報告、研究成果を掲載したニュースレターを定期的に発行し、関係各所に送付した。
• 高機能性生体分子の創成をめざした生合成マシナリーの基盤解明

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2016年06月 -2021年03月 

  代表者 : 大利 徹

   

  ①多価不飽和脂肪酸（PUFA）生合成酵素の精密機能解析；昨年度、海洋原核微生物由来のエイコサペンタエン酸（EPA; C20,ω3）とドコサヘキサエン酸（DHA; C22,ω3）生合成酵素内に存在するketo-synthase (KS)/chain length factor (CLF)ドメインが、生産物の炭素鎖長制御に関与することを明らかにした。両酵素のKS/CLFドメインのアミノ酸配列は高い相同性を有するが、両酵素間で互いに異なり、かつ各々の酵素群で高度に保存されているアミノ酸残基が存在する。そこで今年度は、これらが炭素鎖長制御に関与するか検討するため、DHAのアミノ酸残基を対応するEPAの残基に置換した改変酵素を20作成した。その結果、2つの改変酵素がEPAのみを生産したことから、極限られた残基が鎖長制御に関与することが明らかになった。 昨年度、得られた知見を基に真核藻類由来のDHA生合成酵素のKSドメインに変異導入した結果、EPAをDHAと等量生合成する改変酵素を得た。今年度はCLF領域に変異導入した結果、よりEPAを多く生産する改変酵素（EPA/DHA比が1.7）を得た。 さらに、生合成の最終反応であるACP (acyl carrier protein)からのDHA/EPAの切り離しに関与する新規なacyltransferase-likeドメインを同定した。 ②新規アミド結合形成酵素（MurNAc-L-Ala-L-Glu synthetase）；本生合成機構は、イネ白葉枯病菌が利用している。したがって、本酵素の阻害剤は特異的農薬になると考えられることから放線菌と糸状菌の培養液に候補化合物を探索した。その結果、アクチノマイシンDが本酵素を阻害することがわかった。
• 生合成リデザイン・国際活動支援班

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2016年06月 -2021年03月 

  代表者 : 阿部 郁朗, 菅 裕明, 濱野 吉十, 南 篤志, 池田 治生, 脇本 敏幸, 渡辺 賢二, 梅野 太輔, 江口 正, 大利 徹, 葛山 智久, 山崎 真巳

   

  本領域は、日本のお家芸とも言える天然物化学研究を基盤に発展、展開しており、我が国が国際的にリードしている研究領域である。こうした基盤研究の質の高さに加え、天然物に対する国民一人一人の理解度や認知度が高い点が、我が国の強みであると言えるが、若手研究者の国際的なネットワーク形成や、海外研究者との共同研究の推進に対しては、積極的な支援が必要である。このような国際活動に対しては、領域全体で支援することで、各研究者が個人個人で形成した海外ネットワークが強固なものとなり有効である。また、本領域研究者間で有用な情報を共有し、利用していくことは、今後も本領域が国際的にリードしていくためには必要不可欠である。特に、若手研究者に対する支援は人的なネットワーク形成に加え、最先端技術、手法またはそのシーズの輸入という点からみても効果が高いと考えられ、領域全体としての支援姿勢を明確にしていきたい。 ４年目（2019年度）も、昨年に引き続き、これまでに行った国際セミナーや国際シンポジウムの講演者や、国際活動支援班と人的つながりのある研究者を中心として国際活動支援班で協議の上、海外の優れた研究者を招待して講演会を開催した。計画研究班の中で、数ヶ月単位の海外研究者との共同研究を必要とする研究者に対する支援を行い、海外との共同研究を戦略的に推進した。コロナウイルス感染拡大により、第２回日独天然物生合成セミナーは中止となった。
• シュードペプチド新規形成機構の解明と応用

  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 : 2015年04月 -2018年03月 

  代表者 : 大利 徹, 濱野 吉十, 佐藤 康治, 小笠原 泰志, 森田 洋行

   

  当研究室で見出した、シュードトリペプチド（ケトメミシン）が持つカルボニルメチレン構造の生合成機構の解明を行った。ケトメミシン生合成遺伝子クラスターに存在する4つの遺伝子の関与が考えられたため組換え酵素を用いて検討した。その結果、アルドラーゼがマロニルCoAとフェニルピルビン酸からベンジルリンゴ酸CoAの生成を触媒し、次いで脱水酵素によりベンジルフマリルCoAへと変換され、ピリドキサールリン酸依存酵素であるグリシン-C-アセチルトランスフェラーゼが2回目の炭素-炭素結合形成を触媒し、最後に還元酵素により二重結合が還元されカルボニルメチレン構造を持つシュードジペプチドが生成することを証明した。
• 生合成マシナリー研究の総括

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2010年04月 -2016年03月 

  代表者 : 及川 英秋, 江口 正, 阿部 郁朗, 葛山 智久, 大利 徹, 池田 治生, 五味 勝也, 斉藤 和季, 金谷 重彦, 石川 淳, 齊藤 和季, 石川 淳, 阿部 郁朗, 江口 正, 五味 勝也, 池田 治生, 金谷 重彦, 大利 徹

   

  新学術領域領域代表者と8名の計画研究代表者(江口、大利、阿部、池田、五味、斉藤、金谷、石川)、5名の評価委員の意見を取りまとめ、新学術領域研究「生合成マシナリー」の成果報告シンポジウムを、聴衆が期待できる週末のアクセスが良い場所という観点から、東京都港区のコクヨホールに決定した。6月21日の日曜日には、100名近い企業からの出席者や一般参加者に対し、5年間の研究成果を、A01,A02,A03班の班長および指名された顕著な成果を挙げた合計７名の講演者が、判りやすく説明した。
  最終の文部科学省へ提出する成果報告書は、３班構成の各班長が、班員個人より事務局に提出された5年間の領域活動で得られた成果に関する資料をもとにデータを整理し、論文発表、学会発表、シンポジウムや講演会での依頼講演、新聞・テレビなどのメディアでの公表状況、各賞の受賞状況、さらには領域内での共同研究など各班の成果を取りまとめた後、事後評価ヒアリング、さらには一般公開用資料としてデータファイルを作成した。これをベースに冊子体も作成し、公的な研究機関や関連研究者に送付した。
  6月から10月オープンキャンパスや夏休み期間中には、領域代表が所属する北海道大学や班員が所属する研究機関で、中高生を対象にオープンラボなどの企画で、研究活動を疑似体験してもらうために、実験を撮影したビデオの上映、さらに実験室の見学を行なった。さらに11月14－15日（土・日）、東京都江東区にある日本科学未来館で開かれた大規模なサイエンスコミュニケーションのイベント、サイエンスアゴラに出展し、総括班でアウトリーチ用に作成した領域活動の広報用アニメーションやクイズ形式の説明用プレゼンファイルを利用して、来場者に研究成果を説明した。
• ペプチド化合物のリボソームとNRPSによる協同的新規生成機構の解明

  日本学術振興会：科学研究費助成事業 挑戦的萌芽研究

  研究期間 : 2013年04月 -2015年03月 

  代表者 : 大利 徹

   

  放線菌が生産するペプチド系抗生物質、フェガノマイシン（PM）は、N-末のフェニルグリシン誘導体（PheGly）に長さと配列の異なるペプチド（NVKDRとNVKDGPT）が結合している2種類が知られている。その生合成研究を行った結果、2つのペプチド部分がリボソームにより、38アミノ酸からなるプレカーサーペプチドとして供給されること、さらにATP-graspモチーフを持つPGM1がPheGlyをATP依存的にリン酸化し、次いで、2つのペプチドが求核剤として働くことを明らかにした。本酵素は幅広い基質特異性を有したことから結晶構造解析も行った。
• 環状テルペノイドおよびヌクレオシド系抗生物質生合成マシナリーの解明と再構築

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2011年04月 -2015年03月 

  代表者 : 大利 徹, 加藤 修雄, 佐藤 康治, 新井 亮一

   

  ①糸状菌が生産するFusicoccinの12位水酸基を除去した誘導体が新規抗がん剤のリード化合物として期待されている。そこで生合成工学により、目的中間体を蓄積する菌株を育種した。②カビ由来のインドールジテルペンの生合成に関与するプレニル基転移酵素PaxC、PaxD、AtmDの機能解析を行った。③メナキノン新規生合成経路中間体であるフタロシンの生成機構の解明を行い、S-アデノシルメチオニン由来のアデノシルラジカルを直接基質に用いる新規酵素の機能解明に成功した。④非タンパク性のアミノ酸をリン酸化し、次いでペプチドのアミノ基を求核剤に用いてアミドを形成する新規ペプチドリガーゼを見出した。
• 微生物ゲノム情報を活用した新規葉酸生合成経路の探索とその全容解明

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2012年04月 -2014年03月 

  代表者 : 佐藤 康治, 大利 徹, 倉都 将宏, 小林 大毅

   

  葉酸は全ての生物に必須の補酵素である。葉酸の部分骨格であるパラアミノ安息香酸（pABA）はシキミ酸経路中間体より生合成されることが知られている。申請者らはバクテリアゲノムの詳細な解析の結果、pABA生合成に関与する既知遺伝子が欠落したバクテリアを見出した。そのうちpABA原栄養性と考えられたLactobacillus fermentumやNitrosomonas europaeaには通常経路とは異なる新規pABA生合成経路の存在が予想された。本研究では、N. europaea由来NE1434遺伝子産物がシキミ酸経路中間体を基質せずにde novo合成することを明らかとした。
• ゲノムデータベースの精査による補酵素類の新規生合成経路の予測と検証

  日本学術振興会：科学研究費助成事業 挑戦的萌芽研究

  研究期間 : 2012年 -2012年 

  代表者 : 大利 徹, 佐藤 康治

   

  ゲノムデータベースの精査により新規経路・酵素の存在を予測し、その検証を行った。具体的には、Nitrosomonas eutrophaでは4-アミノ安息香酸の合成に機能未知のNE1434が関与すること、Streptomyces coelicolorのglutamate-cysteine ligase様遺伝子SCO0910はergothioneineの生合成に関与すること、S.coelicolorでは、真核生物でタウリン生合成経路に関与する2つのオルソログSCO3035、およびSCO3416,/2782/2017を持つ。しかし組換え酵素を用いて検討した結果、これらはタウリンの生合成には関与しないと推定された。
• メナキノン新規生合成経路をターゲットとした抗ピロリ菌剤の開発

  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 : 2010年 -2012年 

  代表者 : 大利 徹

   

  微生物のメナキノン新規生合成経路(フタロシン(FL)経路)の詳細な解析を行った。その結果、FL経路の初発反応は3タイプあることが解った。(1)FLを生成後、デヒポキサチニルフタロシン(DHFL)へ変換、(2)アミノデオキシフタロシン(AFL、FLが持つイノシンの代わりにアデノシンを持つ化合物)を生成後、脱アミノ化し、DHFLへ変換、(3)AFL生成後、直接DHFLへ変換。また、天然物からの抗ピロリ菌リード化合物の探索も行い、2つの候補化合物を得た。
• 微生物が生産する環状テルペノイド生合成マシナリーの解明と再構築

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2011年 -2011年 

  代表者 : 大利 徹
• 病原菌が持つメナキノン新規生合成経路の全容解明と経路特異的阻害剤の探索

  日本学術振興会：科学研究費助成事業 特定領域研究

  研究期間 : 2008年 -2009年 

  代表者 : 大利 徹

   

  メナキノン(ビタミンK)は、微生物にとって電子伝達系成分として生育に必須である。筆者は、Helicobacter属(ピロリ菌)、Campylobacter属、Wolinella属などの病原微生物や、放線菌Streptomyces属などの微生物では、今まで知られていた経路とは全く異なるフタロシン経路で生合成されることを見出した。今年度、本経路の2番目の反応を触媒するfutalosine hydrolase(MqnB、最近EC3.2.2.26が付与された)の諸性質を高度好熱菌Thermus thermophilusの組換え酵素を用いて検討した。その結果、以下の性質を示した。(1)Futalosineのみが基質となり他の核酸類縁体は基質にならないこと、(2)4量体を形成、(3)至的pHは4.5、(4)至的温度は80度、(5)Km値154.0±5.3μM、kcat1.02/s、(6)hypoxanthineにより弱く阻害されること(Ki値1.1mM)。本酵素がhypoxanthineにより弱く阻害されたことから、hypoxanthine誘導体を合成し、本酵素特異的阻害剤を探索することにより、抗ピロリ菌剤の開発が可能になると期待された。 また天然物からの抗ピロリ菌リード化合物の探索も行った。大学設備ではピロリ菌を培養できないため、2種類のBacillus属細菌を用いた系で一次スクリーニングを行った。同じBacillus属に属しながら、Bacillus haloduransがメナキノン生合成の際、新規経路を使うのに対し、Bacillus subtilisは既知経路を使う。そこで、(1)B.haloduransに対し抗菌作用を示すが、B.subtilisには影響を及ぼさない化合物を放線菌・カビの培養液中に探索した。次に、(2)B.haloduransに対する生育阻害が、外からメナキノンを添加した際、回復するか検討した。その結果、放線菌の培養中に1つの候補化合物を見出した。現在、本化合物の精製と構造解析を行っている。
• 原核生物に見出された新規ジテルペン環化酵素の機能解析と応用

  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 : 2007年 -2009年 

  代表者 : 大利 徹

   

  ジテルペン化合物は(炭素数20)、香料、医薬・医薬中間体、植物ホルモンなど重要な化合物を含んでいる。これら化合物の生合成の第一段階は、環化酵素が直鎖状の基質であるゲラニルゲラニル2 リン酸の末端オレフィンをプロトン化、または2 リン酸を脱離することによってカルボカチオンを生成することから始まる。最終的にカルボカチオンが捕捉中性化されるまで、各環化酵素特有のカチオン中間体を経る反応が順次進行し、多種多様なイソプレノイド骨格へと導かれる。反応の第一段階が共通であるにもかかわらず、最終的に多様な骨格を持つ化合物が生成する事実は、環化酵素の特定アミノ酸残基のみが反応に関与するのではなく、酵素全体のアミノ酸残基が種々のカルボカチオン中間体の生成に関与することを示唆する。従って、これら環化酵素の構造機能相関解析を行うことにより、任意の段階でカルボカチオンが捕捉中性化された化合物の生成を制御できる可能性がある。しかし環化酵素に関しては、真核生物を材料に用いた場合、酵素調製が難しいことなどから一部の酵素を除いて殆ど解析が行われていない。このような背景下、筆者は原核生物と真核生物起源のイソプレノイド生合成遺伝子を多数取得しており、今回、(1)お米由来の2 つのent-copalyl diphosphate 生合成酵素、(2)原核生物のNocardia 属放線菌が生産するジテルペン化合物、brasilicardin A 生合成遺伝子クラスターの取得、(3)原核生物のStreptomyces 属放線菌により生産されるテトラテルペン化合物(KS-505a)生合成遺伝子クラスターの取得とこれらの機能解析を行った。
• 病原菌が持つメナキノン新規生合成経路の全容解明と経路特異的阻害剤の探索

  日本学術振興会：科学研究費助成事業 特定領域研究

  研究期間 : 2006年 -2007年 

  代表者 : 大利 徹

   

  メナキノン(ビタミンK)は,人間にとっては血液凝固に必要なビタミンであり,また微生物では電子伝達系成分として生育に必須である。申請者は,胃潰瘍・胃がんの原因菌として知られているHelicobacter属細菌、食中毒原因菌として知られているCampylobacter属細菌,グラム陽性の土壌細菌であるStreptomyces属細菌などの微生物では,既知の生合成経路として知られているコリスミ酸からメナキノンに至る5ステップの生合成遺伝子群が全く存在しないことに気づいた。そこで,この新規経路の全容解明を行っている。 1.これまでにStreptomyces属放線菌を材料に用いて,バイオインフォマティクスにより絞り込んだ遺伝子を破壊することにより,7つの遺伝子群が新規経路に関与することを明らかにした(これらの成果については未発表のため,これら遺伝子群を以下men06,men26,men27,men50,men90,men92,men94と略号で記載する)。 2.また新規経路遺伝子の網羅的取得を目指し,変異剤によるメナキノン生合成欠損株の誘導と相補遺伝子の取得も行った結果,新規経路は既知経路同様コリスミ酸を出発基質とするが,その後全く別経路を経ることも分かった。 3.上述の破壊株を2つ組み合わせてメナキノン非存在下で混合培養を行った結果,幾つかの菌株の組み合わせで生育が認められた。本現象を利用し,破壊株が関与する生合成上の相対的な位置を(men06)→(men26,men27,men50)→(men90,men92,men94)→メナキノンのように明らかにすることができた。 4,上述したアッセイ系を用い,破壊株が蓄積する中間体の精製・構造決定を行い,3つの化合物の構造を決定した。
• 新規メナキノン(ビタミンK)生合成経路に関する研究

  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 : 2005年 -2007年 

  代表者 : 瀬戸 治男, 須恵 雅之, 大利 徹

   

  我々は従来とは全く異なる新規メナキノン(MK)生合成経路が、ある種の細菌、例えばHelicobacter pyloriなどに存在することを見出した。これらの細菌には病原菌が含まれ、この経路の阻害剤は新しい作用機作を有する感染症治療薬となる可能性が高いと考えられる。過去3年度の研究により、我々は以下の成果をあげた。 1.取込実験の準備段階として、MKの13C-NMRスペクトルの厳密な帰属に成功した。 2.種々の位置を13Cで標識したグルコースを用いての標識実験により、MKがコリスミ酸を出発物質として、従来知られているのとは別の経路で生合成されることを示した。 3.種々の方法でStreptomyces coelicolorのMK要求性の数種の突然変異株を調製した。 4.標識実験の結果より推定された予想中間体である1,4-ナフトキノン-6-カルボン酸をナフタレン-2-カルボン酸のCe(SO4)2酸化により調製した。この化合物をS.coelieolorのMK要求株へ添加実験することにより、生合成中間体であることを証明した。 4.S.coelicolorのメナキノン要求株の蓄積物を精査した結果、ヌクレオシド化合物であるフタロシンを単離・構造を決定した。この化合物はイノシンと置換安息香酸が結合した構造からなるが、既に抗腫瘍性を抗生物質として他の放線菌から分離されている。 5.さらに他の変異株を用いる実験によりフタロシン以降の数種の生合成中間体の同定にも成功した。
• 微生物・植物テルペノイド生合成遺伝子クラスターの取得と利用による物質生産と開発

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2004年 -2006年 

  代表者 : 佐々 武史, 山根 久和, 星野 力, 加藤 修雄, 及川 英秋, 大利 徹

   

  フシコクシンの生合成に関与する環化酵素遺伝子のcDNAクローニングに成功した。その環化酵素PaFSはGDP合成酵素と一次構造及び機能が連結したキメラ酵素だった。 PaFSによるGGDPからフシコッカン骨格への環化過程を解明するとともに、類縁のコチレニンと同等の細胞分化誘導活性を有するフシコクシン誘導体の創出に成功した。PAF拮抗剤フォマクチンの骨格構築機構を化学的手法により検討した。酵母低温発現系を用いてDNAポリメラーゼα阻害剤アフィディコリン生合成遺伝子の発現を検討した。インドールジテルペノイドの共通生合成中間体(非標識体と重水素標識体)を合成し、菌体中での酸化と環化によりパキシリンとエミンドールDAに変換されることを証明した。放線菌から新たに3つのジテルペン、1つのテトラテルペン環化酵素遺伝子をクローニングした。前者の内の2つの酵素学的諸性質を組換え酵素を用いて詳細に解析した。結核菌のジテルペン合成酵素の機能解析行い、その抗マクロファージ活性を発見した。スクアレン環化酵素のカチオン/π相互作用を実証し、トリテルペンのワンポット合成も行った。試験管内合成系によるコケジテルペン環化酵素遺伝子の迅速機能解析で成果を挙げた。メタノール資化性酵母による複数の植物ホルモン生合成P450の機能解析に成功した。イネのジテルペン系ファイトアレキシン生合成に関与する環化酵素6種を全て特定した。イネの4番染色体にモミラクトン生合成遺伝子クラスターが存在していることを発見した。また、2番染色体にファイトカサン生合成遺伝子クラスターが存在する可能性を示した。
• 微生物イソプレノイド生合成遺伝子資源の機能開発と応用

  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 : 2004年 -2006年 

  代表者 : 大利 徹, 松浦 信康

   

  1.筆者はこれまでに、イソプレノイド化合物の共通出発原料であるイソペンテニル2リン酸の生合成経路としてMEP経路とメバロン酸経路を併せ持つ放線菌は、例外なくイソプレノイド化合物を生産し、更にそれらの生合成遺伝子群は、メバロン酸経路遺伝子群近傍に存在することを見出した。本方法論を駆使し、Streptomyces sp.KO-3988株からイソプレノイド-ポリケタイド融合化合物として初めての例となるfuraquinocin A生合成遺伝子群、および原核生物起源として初めて3-hydroxypimara-9(11),15-diene生合成遺伝子群を取得した。さらに、後者の生合成に関与する2つのジテルペン環化酵素{ent-copalyl diphosphate生合成酵素(CDPS)とpimaradiene生合成酵素(PMDS)}を組換え酵素として発現させ酵素学的諸性質を詳細に検討した。その結果、CDPSは基質であるgeranylgeranyl diphosphate(GGDP)を環化しent-copalyl diphosphateへと変換し、次いでPMDSがpimara-9(11),15-dieneへと変換することを明らかにした。また、両酵素の詳細な酵素学的諸性質を検討し以下の結果を得た。CDPS:至適温度35℃、至適pH 5.5、Km 13.7μM±1.0、Vmax 36.4±0.9、金属要求性、Mgで高活性。PMDS:至適温度30℃、至適pH 7.0、Km 2.57μM±0.2、Vmax 0.96±0.014、金属要求性、Mg、Coで高活性。 2.放線菌Nocardia brasiliensis IFM 0406株が生産するbrasilicardin A(BC)は、ジテルペン、アミノ酸、糖、芳香環からなるユニークな天然生理活性物質である。その生合成を明らかにする第一歩として、生合成遺伝子クラスターの取得・解析を行った。IFM 0406株はメバロン酸経路を有さないため上記方法論は使えない。そこで、BCがジテルペン骨格を有することを利用し、最初にGGDP生合成遺伝子を取得した後、周辺領域を探索することにより11の遺伝子群からなるBCの推定生合成遺伝子クラスターを取得した。これらが実際にBCの生合成に関与することを証明するため、異種宿主であるS.lividansに導入した結果、2つの特異的化合物が生産された。LC-MSで解析した結果、これら化合物はBC関連化合物と推定されたことから、取得した遺伝子群はBC生合成遺伝子群と推定された。
• 原核生物起源のジテルペンサイクラーゼに関する研究

  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 : 2002年 -2003年 

  代表者 : 大利 徹

   

  テルペンテシン(Tp)生産菌からクローニングした2つのテルペノイドサイクラーゼ様遺伝子産物(Cyc1とCyc2)は、geranylgeranyl diphosphate (GGDP)をTpの基本骨格を有するterpentetriene (TTF)へと変換する。両酵素の詳細な反応機構をin vitroで解析した結果、(i)Cyc1はmonomerで、Cyc2はdimerで存在すること、(ii)Cyc1は、GGDPを環化し、terpentedienol diphosphate (TDP)へと変換し、次いで、TDPは、Cyc2によりTTEへと変換されること、(iii)両反応ともに、Mg^<2+>今を要求し(至適濃度1mM)、反応の至適pHは中性付近であり、至適温度は、30度前後であること、(iv)Cyc1のGGDPに対するKmは、64.2±5.7μMであり、またV/maxは、94.7±6.9U/mgであること、(v)Cyc2は、GGDPと炭素数が15のfarnesyl diphosphate (FDP)にも直接作用し、各々から、TDPやTTEとは異なる3つのオレフィン化合物の混合物を生成する反応を触媒すること、(vi)Cyc2が触媒するこれら3つの反応の速度定数を求めた結果、Cyc2のTDP、GGDP、FDPに対するKmは、それぞれ7.6±0.6μM、7.9±0.6μM、61.7±3.0μMであり、炭素数20の基質に対する親和性が高かく、また、Vmax/Km値も、本来の反応か、GGDP、FDPを用いた場合に比べ、それぞれ15倍、58倍高いことなどを明らかにした。 また、一部の放線菌は、isopentenyl diphosphate (IPP)の生合成経路としてメバロン酸経路とMEP経路を合わせ持っている。これら放線菌におけるメバロン酸経路の存在意義を調べた結果、(i)主に二次代謝産物としてのテルペノイド化合物の生産に用いられるIPPを供給していること、(ii)メバロン酸経路遺伝子群は、テルペノイド化合物生合成遺伝子群とクラスターを成していることなどを明らかにした。 さらに、prenyl diphosphate synthaseの反応産物の鎖長決定に重要なアミノ酸残基を同定し、FDP synthaseとGGDPしsynthaseの1アミノ酸を相互置換することにより、反応産物の鎖長が人れ換ることを明らかにした。
• 生理活性環状微生物テルペン類における生合成環化反応の全容解明と開発基盤研究

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2001年 -2003年 

  代表者 : 佐々 武史, 及川 英秋, 加藤 修雄, 星野 力, 豊増 知伸, 大利 徹

   

  1.エリナシン生産菌から、生合成炭化水素(+)-cyatha-3,12-dieneを分離し、エリナシンPからの半合成により立体構造を確定した。また、セルフリー系でGGDPからの変換を確認した。フシコクシン高生産株から、生合成炭化水素の推定中間体である(+)-δ-araneoseneを分離・同定した。また、fusicocca-3(16),10(14)-dieneを分離し、生合成経路上の新たなフシコッカンカチオン中間体の存在を推定した。2.フシコクシン生産菌から,保存性が高いGGDP合成酵素遺伝子を取得し,それより染色体歩行を行い,ジテルペン生合成遺伝子クラスターを同定した。このクラスター上に2種のジテルペン環化酵素遺伝子を見出したのでそれらについてcDNAクローニングし、機能を解析した。3.アフィディコリン生産菌から取得した環化酵素をコードするcDNAの塩基配列を利用して、PCRによる染色体歩行を行い、アフィディコリン生合成遺伝子クラスターを同定した。また種々の条件下で行った非酵素的環化および分子軌道計算に基づいて、詳細なアフィディコラン骨格の構築機構を推定した。4.ジテルペン化合物テルペンテシン生産放線菌から、テルペンテシンの生産に関与する2つのジテルペンサイクラーゼ遺伝子をクローニングした。組換え酵素を用いた解析により、1つの酵素は基質であるGGDPをterpentedienol diphosphate (TDP)へと変換し、次いでもう一つの酵素が、TDPをterpentetrieneへと変換することが分かった。5.スクアレン環化酵素の部位特異的変酵素の機能解析及び基質アナログの酵素反応実験を通して、基質の環化フォールデングは活性部位の立体的なサイズによって決定されることを明らかにした。
• 放線菌に於けるテルペノイド化合物の生合成研究

  日本学術振興会：科学研究費助成事業

  研究期間 : 2000年 -2001年 

  代表者 : 大利 徹

   

  ジテルペン系抗生物質Terpentecin(Tp)生産菌Streptomyces griseolosporeusを用いて、放線菌に於けるテルペノイド化合物の生合成研究を行った。本菌株は、テルペノイド化合物の共通出発物質であるisopentenyl diphosphateの生合成経路と.して、従来から知られていたメバロン酸経路に加え、近年微生物や植物の色素体に於いてその存在が明らかになった非メバロン酸経路の両経路を併せ持っている。そこで、何故本菌株が2つの経路を有するのか?それらの生理学的意義は何か?を明らかにするため、本菌株から、3-hydroxy-3-methylgrutaryl CoA synthaseからmevalonate diphosphate decarboxylaseに至るメバロン酸経路の全遺伝子群、非メバロン酸経路の初発の反応を触媒する1-deoxy-D-xylulose 5-phosphate(Dxp)synthase遺伝子を2つ、2番目の反応を触媒するDxp reductoisomerase(Dxr)遣伝子、TPの直接前駆体となるgeranylgeranyl dip hosphate(GGDP)の生合成遺伝子、およびTPの生合成遺伝子群を取得した。これらの遺伝子群を用いて種々の解析を行った結果、(i)メバロン酸経路遺伝子群、GGDP生合成遺伝子、TPの生合成遺伝子群が染色体上でクラスターを成しており、これらがTP生産時に連動して発現していること、(ii)非メバロン酸経路は生育初期から後期まで定常的に発現しているが、非メバロン酸経路のみでは、TP生合成に必要な十分量のIPPを供給できず、不足分をメバロン酸経路により補っていること、(iii)2つのDxpsynthaseは、何れも菌体内で発現していること、(iv)TPの生合成遺伝子クラスター内に、原核生物起源としては始めてのジテルペンサイクラーゼ遺伝子を見出した。本酵素遺伝子は、2つの酵素Cyc1とCyc2からなり、両酵素を大腸菌で発現させ、in vitroで解析した結果、CyclがGGDPを環化した2リン酸中間体へと変換し、次いでCyc2が中間体をTPの基本骨格を有するTerpentetrieneへと変換することを明らかにした。興味深いことに、Cyc2は上記反応に加え、単独でGGDPから3つのシャントプロダクトを生成する反応も触媒すること、さらに炭素数15のfarn esyl diphosphateにも作用し、GGDPを基質に用いたときと同様に、3つのシャントプロダクトを生成する反応も触媒することが判明した。
• トウトマイシン生合成遺伝子のクローニングと解析

  日本学術振興会：科学研究費助成事業

  研究期間 : 1996年 -1998年 

  代表者 : 生方 信, 松浦 信康, 大利 徹

   

  Streptomyces spiroverticillatusにより生産されるトウトマイシン(TM)は、タンパク質脱リン酸化酵素(PP1及びPP2A)阻害剤である。TMは無水マレイン酸骨格と直鎖状ポリケチドが結合したユニークな構造を持つことから、TM生合成遺伝子の発現と調節を、遺伝子組換え手法を用いた生合成遺伝子群の取得と一次構造の解析により明かにすることを目的とする。まず、既知のType I PKSで高度に保存されている塩基配列を基にプライマーを合成し、TM生産菌染色体DNAとのPCRを行ったところ特異的断片が増幅された。PCR産物により特異的に認識される、TM生産菌染色体DNA由来の約1.8kbの遺伝子断片が、TM生合成遺伝子由来のものであることを証明するため、pIJ702を用いてトウトマイシン生産菌の形質転換効率の向上を目指し、2x10^4transformants/μgDNAの転換効率を得た。次に、上記遺伝子断片をチオストレプトン耐性遺伝子を組み込んだpUC19を用いてサブクローニングを行いプラスミド(pTM201)を構築した。TM生産菌プロトプラストに対して相同組換えによる形質転換を行い遺伝子破壊を試みた。得られた15株の形質転換体は、全てTM生産能を失っていたが、親株が生産するもう一つの抗生物質キサントスタチンの生産能を保持していた。サザンブロット法により、これらの閉鎖株のTM生合成遺伝子は欠失していることが明らかとなった。
• 新規微生物酵素の反応機構および応用に関する研究

  日本学術振興会：科学研究費助成事業

  研究期間 : 1996年 -1997年 

  代表者 : 浅野 泰久, ヘイデン ブロナ, アーテミュイク ピーター, ライス デイビット・W, 米田 英伸, 加藤 康夫, 大利 徹, ライス デイビッド・W, ウォーラー デイビッド・, ブリットン K・リンダ

   

  (1)フェニルアラニン脱水素酵素:Bacillus sphaericus,B.badius,Sporosarcina ureae由来のフェニルアラニン脱水素酵素を大腸菌形質転換株より大量に得た。本酵素をアイルランド、ダブリン大学のエンゲル教授および英国シェフィールド大学のライス教授に送付した。グルタミン酸脱水素酵素のX線構造解析の結果のフィードバックを受けて、フェニルアラニン脱水素酵素の活性中心と目される残基周辺に部位特異的変異をかけ、キメラ酵素を作成した。 (2)メチルアスパラギン酸アンモニアリアーゼ:細菌Enterobacter,Citrobacter,Proteus等由来の腸内細菌群のメチルアスパラギン酸アンモニアリアーゼを高度に精製し、酵素化学的特性を詳細に調べた。腸内細菌群中におけるメチルアスパラギン酸アンモニアリアーゼは、お互いに高い類似点を有しているのみならず、偏性嫌気性菌由来の同酵素とも相同性を有していた。結晶化を行うとともに、シェフィールド大学のライス教授に送付し、X線構造解析を開始した。 (3)オピン脱水素酵素:Arthrobacter由来のオピン脱水素酵素遺伝子をクロニーングし、大腸菌形質転換株より本酵素を大量に高度に精製した。本酵素をシェフィールド大学のライス教授に送付し、X線構造解析に成功した。NADX^+存在下、非存在下において得られた結晶から、いずれも1.8Åの分解能のデータを得た。 (4)国際会議等において、これまでの研究成果をまとめて発表した。
• 新規リアーゼ類の開発と光学活性有機酸合成への利用

  日本学術振興会：科学研究費助成事業

  研究期間 : 1995年 -1996年 

  代表者 : 浅野 泰久, 山田 秀明, 大利 徹, 加藤 康夫

   

  フマル酸を異性化しマフラーゼによってLーリンゴ酸に至るマレイン酸の代謝系が一般的に知られているが、マレイン酸を水和しDーリンゴ酸を生成するマレートヒドラターゼについては余り知られていない。そこで、強いマレートヒドラターゼを有する細菌Arthrobacter sp.の休止菌体反応によるマレイン酸からDーリンゴ酸の製造検討を行った。さらに本酵素を精製しその諸性質を明らかにした。また安価なイタコン酸を基質とし、土壌由来の細菌Alcaligenes denitrificansの菌体反応による(S)-(+)シトラマル酸の製造を検討した。光学純度は99.9%であった。反応30時間で28g/literの(S)-(+)シトラマル酸(交換率は80.0%、モル収率は68.8%)が蓄積した。 安価なフマル酸誘導体へのアンモニアの付加反応を触媒するメチルアスパラギン酸アンモニアリアーゼを検索し、非天然型のL-アスパラギン酸誘導体の選択的な合成を検討した。(S)-グルタミン酸を含む培地で集積培養を行い、顕著な3-メチルアスパルターゼ活性を有する通性嫌気性菌Citrobacter freundii、Morganella morganii、Citrobacter amalonaticus、およびEnterobacter sp.を得た。各菌の無細胞抽出液を用いて、それぞれ光学的に純粋なthreo-(2S,3S)-3-メチル-アスパラギン酸、threo-(2S,3S)-3-エチル-アスパラギン酸、threo-(2S,3S)-3-クロロ-アスパラギン酸を好収率で合成した。上記の菌の無細胞抽出液中より、本酵素を結晶状に精製した。各酵素は偏性嫌気性菌であるClostridium tetanomorphum由来の本酵素と酵素学的諸性質において類似性を示したが、基質特異性は異なっていた。
• Studies on biosynthesis of terpenoids produced by microorganisms

  研究期間 : 1996年
• Studies on antibiotic biosynthesis

  研究期間 : 1994年
• Studies on biosynthesis of natural product
• Studies on biosynthetic genes and enzymes of isoprenoids produced by microorganisms

教育活動情報

主要な担当授業

大学運営

学内役職歴

• 2020年4月1日 - 2022年3月31日 教育研究評議会評議員
• 2020年4月1日 - 2022年3月31日 大学院総合化学院長

委員歴

• 2020年04月 - 現在   日本放線菌学会   学会長
• 2017年 - 現在   日本農芸化学会   創立100周年記念事業組織委員会特別委員会 委員
• 2016年04月 - 現在   バイオインダストリィー協会   B&I 編集幹事
• 2019年05月 - 2021年04月   日本生物工学会   理事
• 2019年02月 - 2021年02月   日本農芸化学会   広報委員長
• 2017年 - 2018年   日本放線菌学会   副会長
• 2015年 - 2018年   日本農芸化学会   理事