Researcher Profile and Settings

Affiliation

• Faculty of Engineering　Applied Chemistry　Biotechnology

Job Title

• Professor

Degree

• Agriculture

URL

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

Research funding number

• 70264679

J-Global ID

• 200901050726079892

Research Interests

• 生合成   天然物   Natural product biosynthesis   

Research Areas

• Life sciences / Applied microbiology
• Life sciences / Bioorganic chemistry

Educational Organization

•  Bachelor's degree program　School of Engineering
•  Master's degree program　Graduate School of Chemical Sciences and Engineering
•  Doctoral (PhD) degree program　Graduate School of Chemical Sciences and Engineering

Academic & Professional Experience

• 2010/04 - Today Hokkaido University Faculty of Engineering
• 1994/04 - 2010/03 Toyama Prefectural University
• 1994/04 - 2010/03 - Biotechnology Research Center, Toyama Prefectural University
• 1985 - 1994 協和発酵工業(株)東京研究所
• 1985 - 1994 Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd.

Education

•        - 1985  Nagoya University
•        - 1985  Nagoya University  Graduate School, Division of Agriculture
•        - 1983  Nagoya University  School of Agricultural Sciences
•        - 1983  Nagoya University  Faculty of Agriculture

Association Memberships

• 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   

Research Activities

Published Papers

• 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 1389-1723 2024/02
• 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 2024/01/10 

  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 2023/08/04 

  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 2023/05/03 

  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

  2023/01/12
• 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 2023/01 

  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) 2022/10/26 

  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) 1439-4227 2022/06/20 [Refereed]
  
• 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) 1433-7851 2022/02/07 [Refereed]
  
• Identification of pulvomycin as an inhibitor of the futalosine pathway

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

  The Journal of Antibiotics 0021-8820 2021/08/20 [Refereed]
   

  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 1367-5435 2021/06/11 [Refereed]
   

  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 0960-894X 2021/03 [Refereed]
   

  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 2041-6520 2021 [Refereed]
   

  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 0021-8561 2020/06/10 [Refereed]
   

  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 2020/05/19 [Refereed]
   

  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 2020/03/20 [Refereed]
   

  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 1554-8929 2019/11 [Refereed][Not invited]
  
• 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 0021-8820 2019/10 [Refereed][Not invited]
   

  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  2045-2322 2019/02 [Refereed][Not invited]
   

  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 [Refereed][Not invited]
  
• 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 [Refereed][Not invited]
  
• 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 [Refereed][Not invited]
  
• 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 [Refereed][Not invited]
  
• 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 [Refereed][Not invited]
  
• Searching for potent and specific antibiotics against pathogenic Helicobacter and Campylobacter strains.

  Y. Ogasawara, T. Dairi

  J. Ind. Microbiol. Biotechnol 46 409 - 414 2019 [Refereed][Invited]
  
• 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 1433-7851 2018/05 [Refereed][Not invited]
  
• Peptide epimerization machineries found in microorganisms

  Yasushi Ogasawara, Tohru Dairi

  Frontiers in Microbiology 9 156  1664-302X 2018/02/06 [Refereed][Invited]
   

  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

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

  JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY AMER CHEMICAL SOC 66 (5) 1191 - 1196 0021-8561 2018/02 [Refereed][Not invited]
   

  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 mu M) 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 1347-4421 2018/02/01 [Refereed][Not invited]
   

  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 [Refereed][Not invited]
  
• Aplasmomycin and boromycin are specific inhibitors of the futalosine pathway.

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

  J Antibiot 2018 [Refereed][Not invited]
  
• 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 [Refereed][Not invited]
  
• 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 [Refereed][Not invited]
  
• 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 [Refereed][Not invited]
  
• 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 [Refereed][Not invited]
  
• 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-INTERNATIONAL EDITION Wiley-Blackwell 56 (44) 13847 - 13851 1433-7851 2017/10 [Refereed][Not invited]
   

  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 0947-6539 2017/08 [Refereed][Not invited]
   

  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 1554-8929 2017/07 [Refereed][Not invited]
   

  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 0021-8820 2017/06 [Refereed][Not invited]
  
• 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 0002-7863 2017/03 [Refereed][Not invited]
   

  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 1433-7851 2017/02 [Refereed][Not invited]
   

  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  2045-2322 2016/10 [Refereed][Not invited]
   

  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 1523-7060 2016/10 [Refereed][Not invited]
   

  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 0960-894X 2016/08 [Refereed][Not invited]
   

  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

  Yasushi Ogasawara, Junpei Kawata, Motoyoshi Noike, Yasuharu Satoh, Kazuo Furihata, Tohru Dairi

  ACS CHEMICAL BIOLOGY 11 (6) 1686 - 1692 1554-8929 2016/06 [Refereed][Not invited]
   

  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 0175-7598 2016/05 [Refereed][Not invited]
   

  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 0021-8820 2016/02 [Refereed][Not invited]
  
• 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 1389-1723 2015/09 [Refereed][Not invited]
   

  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 0916-8451 2015 [Refereed][Not invited]
   

  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 - + 1552-4450 2015/01 [Refereed][Not invited]
   

  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 1389-1723 2014/02 [Refereed][Not invited]
   

  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 0916-8451 2014 [Refereed][Not invited]
   

  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 0175-7598 2014/01 [Refereed][Not invited]
   

  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 [Refereed][Not invited]
  
• 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 0099-2240 2013/12 [Refereed][Not invited]
   

  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 0002-7863 2013/10 [Refereed][Not invited]
   

  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 0006-2960 2013/07 [Refereed][Not invited]
   

  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 1389-1723 2013/06 [Refereed][Not invited]
   

  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 0002-7863 2013/01 [Refereed][Not invited]
   

  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 0969-0239 2012/12 [Refereed][Not invited]
   

  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  1932-6203 2012/08 [Refereed][Not invited]
   

  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 1439-4227 2012/03 [Refereed][Not invited]
   

  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 0076-6879 2012 [Refereed][Not invited]
   

  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 0175-7598 2011/11 [Refereed][Not invited]
   

  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 1364-5072 2011/10 [Refereed][Not invited]
   

  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 Analysis of biosynthetic machinery of fusicoccin produced by Phomopsis amygdali(Oral Presentation)

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

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会 (53) 115 - 120 2011/09/02 

  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 dehypoxanthine futalosine, the biosynthetic intermediate in an alternative biosynthetic pathway for menaquinones

  Arata Yajima, Saki Kouno, Tohru Dairi, Manami Mogi, Ryo Katsuta, Haruo Seto, Tomoo Nukada

  TETRAHEDRON LETTERS 52 (38) 4934 - 4937 0040-4039 2011/09 [Refereed][Not invited]
   

  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 1552-4450 2011/07 [Refereed][Not invited]
   

  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 0002-7863 2011/03 [Refereed][Not invited]
   

  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 0066-4804 2011/02 [Refereed][Not invited]
   

  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 0021-8820 2011/01 [Refereed][Not invited]
  
• Convergent strategies in biosynthesis

  Tohru Dairi, Tomohisa Kuzuyama, Makoto Nishiyama, Isao Fujii

  NATURAL PRODUCT REPORTS 28 (6) 1054 - 1086 0265-0568 2011 [Refereed][Not invited]
   

  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 [Refereed][Not invited]
  
• Analysis of the Lactobacillus Metabolic Pathway

  Masahiro Kuratsu, Yoshimitsu Hamano, Tohru Dairi

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 76 (21) 7299 - 7301 0099-2240 2010/11 [Refereed][Not invited]
   

  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 0040-4039 2010/01/06 [Refereed][Not invited]
  
• 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 0960-894X 2009/10 [Refereed][Not invited]
   

  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 0021-8820 2009/07 [Refereed][Not invited]
   

  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 0916-8451 2009/05 [Refereed][Not invited]
   

  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 0960-894X 2009/02 [Refereed][Not invited]
   

  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 0022-3263 2009/02 [Refereed][Not invited]
   

  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 0037-1017 2009 [Refereed][Not invited]
  
• 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 0036-8075 2008/09 [Refereed][Not invited]
   

  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 0916-8451 2008/04 [Refereed][Not invited]
   

  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 - + 0002-7863 2008/04 [Refereed][Not invited]
   

  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 0021-8820 2008/03 [Refereed][Not invited]
   

  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 0916-8451 2008/02 [Refereed][Not invited]
   

  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 0175-7598 2007/12 [Refereed][Not invited]
   

  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 0916-8451 2007/12 [Refereed][Not invited]
   

  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 0163-3864 2007/11 [Refereed][Not invited]
   

  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 0027-8424 2007/02 [Refereed][Not invited]
   

  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 0021-924X 2007/01 [Refereed][Not invited]
   

  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 1439-4227 2007/01 [Refereed][Not invited]
   

  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 天然有機化合物討論会 2006 "P - 355" 2006/07/23
• 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 0021-9193 2006/02 [Refereed][Not invited]
   

  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 0916-8451 2006/02 [Refereed][Not invited]
   

  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 0099-2240 2005/08 [Refereed][Not invited]
   

  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 0014-5793 2005/07 [Refereed][Not invited]
   

  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

  JOURNAL OF ANTIBIOTICS 58 (4) 227 - 243 0021-8820 2005/04 [Refereed][Not invited]
   

  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 1359-7345 2005/02 [Refereed][Not invited]
   

  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 0175-7598 2004/11 [Refereed][Not invited]
   

  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 0021-8820 2004/11 [Refereed][Not invited]
   

  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 0916-8451 2004/06 [Refereed][Not invited]
   

  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 0916-8451 2004/04 [Refereed][Not invited]
   

  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 Engineering of phenylacetaldehyde reductase for improved substrate conversion in concentrated 2-propanol

  Makino Y., Dairi T., Itoh N.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 44 S217  2004
• 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 0021-8820 2003/11 [Refereed][Not invited]
   

  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 0175-7598 2003/07 [Refereed][Not invited]
   

  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 0022-3263 2003/07 [Refereed][Not invited]
   

  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 0021-924X 2003/01 [Refereed][Not invited]
   

  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 0021-9258 2002/10 [Refereed][Not invited]
   

  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 0021-8820 2002/10 [Refereed][Not invited]
  
• 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 0014-2956 2002/05 [Refereed][Not invited]
   

  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 0916-8451 2002/04 [Refereed][Not invited]
   

  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 0003-9861 2002/02 [Refereed][Not invited]
   

  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 0002-1407 2002 [Refereed][Not invited]
  
• 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 0021-9193 2001/10 [Refereed][Not invited]
   

  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 0916-8451 2001/07 [Refereed][Not invited]
   

  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 1-2 1545 53 - 66 0006-3002 2001/02 [Refereed][Not invited]
  
• 2 Development of Methodology for Biochemical and Biosynthetic Studies of Isoprenoid Compounds using Perdeuterated Mevalonate

  Eguchi Tadashi, Dekishima Yasumasa, Matsushima Yoshitaka, Tamegai Hideyuki, Kakinuma Katsumi, Takagi Motoki, Kuzuyama Tomohisa, Seto Haruo, Misawa Norihiko, Hamano Yoshimitsu, Dairi Tohru

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会実行委員会 43 7 - 12 2001 

  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 0175-7598 2000/07 [Refereed][Not invited]
   

  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 0026-8925 2000/01 [Refereed][Not invited]
   

  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 0175-7598 1999/09 [Refereed][Not invited]
   

  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 0099-2240 1999/06 [Refereed][Not invited]
   

  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 0175-7598 1999/05 [Refereed][Not invited]
   

  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 0021-9258 1998/07 [Refereed][Not invited]
   

  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 0014-2956 1998/07 [Refereed][Not invited]
   

  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 0099-2240 1998/02 [Refereed][Not invited]
   

  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 0385-5600 1998 [Refereed][Not invited]
   

  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 4 53 124 - 126 0907-4449 1998/01 [Refereed][Not invited]
   

  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 0907-4449 1998/01/01 [Refereed][Not invited]
   

  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 0916-8451 1997/09 [Refereed][Not invited]
   

  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 0021-9193 1997/06 [Refereed][Not invited]
   

  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 0014-2956 1997/04 [Refereed][Not invited]
   

  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 0021-9258 1996/11 [Refereed][Not invited]
   

  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 0021-8820 1995/10 [Refereed][Not invited]
  
• 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 0916-8451 1995/10 [Refereed][Not invited]
   

  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 0916-8451 1995/10 [Refereed][Not invited]
   

  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 0099-2240 1995/08 [Refereed][Not invited]
   

  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 0916-8451 1995/07 [Refereed][Not invited]
   

  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 0916-8451 1995/06 [Refereed][Not invited]
   

  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 0022-1287 1993/03 [Refereed][Not invited]
   

  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 0026-8925 1992/12 [Refereed][Not invited]
   

  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 0026-8925 1992/12 [Refereed][Not invited]
   

  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 0026-8925 1992/03 [Refereed][Not invited]
   

  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 0021-9193 1991/11 [Refereed][Not invited]
   

  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 0021-8820 1989/06 [Refereed][Not invited]
  
• 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 0022-2836 1985 [Refereed][Not invited]
  

Books etc

• Isoprenoid Synthesis in Plants and Microorganisms

  DAIRI Tohru (ContributorBiosynthetic genes and enzymes of isoprenoids produced by actinomycetes)
  Springer 2013
• Comprehensive Natural Products II Chemistry and BiologyMander, L., Lui, H.-W., Eds.volume 1, pp. 789–814.

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

MISC

• Searching for Specific Inhibitors of the Alternative Pathway of Peptidoglycan Biosynthesis

  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
• Biosynthetic Studies on Antitumor Antibiotic Mitomycins

  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
• Biosynthetic studies on natural product containing α,α-disubstituted amino acid

  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
• Biosynthesis of lasso peptide RES-701

  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
• Dissecting Biosynthetic Machinery of Microalgal Polyunsaturated Fatty Acid Synthase

  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
• 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
• Biosynthesis of D-amino acid containing peptides in microorganism

  小笠原泰志, 佐藤康治, 大利徹  生化学  93-  (3)  329  -337  2021/06  [Refereed][Invited]
  
• 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
• 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
• Epimerase involved in polyglutamic acid biosynthesis

  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
• Biosynthesis of the antitumor antibiotics mitomycins

  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
• Reaction mechanism of ATP-dependent peptide epimerase MurL

  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
• 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
• Enhanced production of ergothioneine by recombinant Escherichia coli

  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
• 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  2019/11  [Not refereed][Invited]
  
• 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)  2019/03/05  [Not refereed][Not invited]
  
• 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)  2019/03/05  [Not refereed][Not invited]
  
• 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)  2019/03/05  [Not refereed][Not invited]
  
• 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)  2019/03/05  [Not refereed][Not invited]
  
• 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)  2019/03/05  [Not refereed][Not invited]
  
• 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
• 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
• 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
• 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
• 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
• 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
• 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  2018/09/22  [Not refereed][Not invited]
  
• 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  2018/09/22  [Not refereed][Not invited]
  
• 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  2018/09/10  [Not refereed][Not invited]
  
• Fermentative overproduction of ergothioneine, a sulfur-containing antioxidant by metabolic engineering of cysteine-overproducing Escherichia coli

  大津厳生, 河野祐介, 佐藤康治, 大利徹  Fragrance Journal  46-  (7)  65‐70  2018/07/15  [Not refereed][Not invited]
  
• 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
• Studies on the Biosynthesis and Heterologous Production of Fungal Terpenoids

  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
• 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
• 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
• 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
• 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  [Not refereed][Not invited]
  
• 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  2017/08/08  [Not refereed][Not invited]
  
• 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  2017/08/08  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• Bioengineering utilizing microorganisms

  大利 徹  生化学  89-  (2)  221  -229  2017  [Not refereed][Not invited]
  
• 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  2016/09/08  [Not refereed][Not invited]
  
• 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  2016/09/08  [Not refereed][Not invited]
  
• 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  2016/08/25  [Not refereed][Not invited]
  
• 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  2016/08/25  [Not refereed][Not invited]
  
• 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  2016/08/25  [Not refereed][Not invited]
  
• 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)  2016/03/05  [Not refereed][Not invited]
  
• 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)  2016/03/05  [Not refereed][Not invited]
  
• 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)  2016/03/05  [Not refereed][Not invited]
  
• 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
• New gene responsible for para-aminobenzoate biosynthesis

  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
• 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
• 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  2015/11/14  [Not refereed][Not invited]
  
• 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  2015/09/25  [Not refereed][Not invited]
  
• 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  2015/09/25  [Not refereed][Not invited]
  
• 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  2015/09/25  [Not refereed][Not invited]
  
• 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  2015/09/25  [Not refereed][Not invited]
  
• 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

  NOIKE MOTOYOSHI, MATSUI TAKASHI, OGASAWARA YASUSHI, MORITA HIROYUKI, DAIRI TOORU  日本農芸化学会大会講演要旨集(Web)  2015-  3E11P02 (WEB ONLY)  2015/03/05  [Not refereed][Not invited]
  
• C‐末にD‐Trpを持つラッソペプチドの生合成機構

  NOMURA SATOSHI, OGASAWARA YASUSHI, SATO YASUHARU, DAIRI TOORU  日本農芸化学会大会講演要旨集(Web)  2015-  3E11P05 (WEB ONLY)  2015/03/05  [Not refereed][Not invited]
  
• 3S-Ba05 Amido bond formation catalyzed by newly discovered ATP-grasp-ligases

  Dairi Tohru  日本生物工学会大会講演要旨集  67-  344  -344  2015
• Studies on the new menaquinone biosynthetic pathway found in microorganisms and application for drug discovery to pathogenic Helicobacter and Chlamydia strains

  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
• A new enzyme catalyzing amide bond formation

  大利 徹  バイオサイエンスとインダストリー  73-  (4)  274  -279  2015
• 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

  NOIKE MOTOYOSHI, MATSUI TAKASHI, SATO YASUHARU, MORITA HIROYUKI, DAIRI TOORU  日本農芸化学会北海道支部講演会講演要旨  2014-  30  2014/09/22  [Not refereed][Not invited]
  
• 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

  RYU SEII, FUJIMORI MICHIKO, MINAMI ATSUSHI, NOIKE MOTOYOSHI, SATO YASUHARU, OGURI HIROKI, OIKAWA HIDEAKI, DAIRI TOORU  イソプレノイド研究会例会講演要旨集  24th-  19  2014/09/12  [Not refereed][Not invited]
  
• バクテリアにおける新規葉酸生合成関連遺伝子の同定

  SATO YASUHARU, KOBAYASHI DAIKI, DAIRI TOORU  日本生物工学会大会講演要旨集  66th-  162  -162  2014/08/05  [Not refereed][Not invited]
  
• エルゴチオネインは酸化ストレスから放線菌を守る

  NAKAJIMA SHUNSUKE, SATO YASUHARU, DAIRI TOORU  日本生物工学会大会講演要旨集  66th-  162  -162  2014/08/05  [Not refereed][Not invited]
  
• 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

  NOIKE MOTOYOSHI, MATSUI TAKASHI, SATO YASUHARU, MORITA HIROYUKI, DAIRI TOORU  日本放線菌学会大会講演要旨集  29th-  92  2014/06/19  [Not refereed][Not invited]
  
• 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

  NOIKE MOTOYOSHI, OYA KOICHI, SASAKI IKUO, MARUYAMA CHITOSE, ISHIKAWA JUN, SATO YASUHARU, HAMANO YOSHIMITSU, ITO HAJIME, DAIRI TOORU  日本農芸化学会大会講演要旨集(Web)  2014-  2A04P08 (WEB ONLY)  2014/03/05  [Not refereed][Not invited]
  
• 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, FUJIMORI MICHIKO, MINAMI ATSUSHI, NOIKE MOTOYOSHI, SATO YASUHARU, OGURI HIROKI, OIKAWA HIDEAKI, DAIRI TOORU  日本農芸化学会大会講演要旨集(Web)  2014-  2A04P10 (WEB ONLY)  2014/03/05  [Not refereed][Not invited]
  
• エルゴチオネインはStreptomyces coelicolorを酸化ストレスから守る

  NAKAJIMA SHUNSUKE, SATO YASUHARU, YANASHIMA KENTARO, MATSUI TOMOMI, DAIRI TOORU  日本農芸化学会大会講演要旨集(Web)  2014-  2A04P11 (WEB ONLY)  2014/03/05  [Not refereed][Not invited]
  
• 2S-Da05 A novel peptide ligase catalyzing a capping of N-terminus of peptides

  Noike Motoyoshi, Ooya Koichi, Dairi Tohru  日本生物工学会大会講演要旨集  66-  98  -98  2014
• Peptide synthesis cooperatively achieved by peptide ligase and ribosomes

  Noike Motoyoshi, Morita Hiroyuki, Dairi Tohru, Matsui Takashi, Ooya Koichi, Sasaki Ikuo, Maruyama Chitose, Hamano Yoshimitsu, Ishikawa Jun, Satoh Yasuharu, Ito Hajime  Symposium on the Chemistry of Natural Products, symposium papers  56-  (0)  Oral8  2014  [Not refereed][Not invited]
   

  Peptide antibiotics are biosynthesized by two distinct machineries: (i) ribosome-based synthesis, and (ii) ribosome-independent synthesis exemplified by nonribosomal peptide synthesis. Pheganomycin (PGM) consists of the nonproteinogenic amino acid (S)-2-(3,5-dihydroxy-4-methoxyphenyl)-2-guanidinoacetic acid (1) at the N-terminus and a proteinogenic core peptide derived from NVKDGPT or NVKDR. The biosynthetic gene cluster responsible for the catalysis was identified in Streptomyces cirratus, containing a gene encoding a precursor peptide, including both core peptides, and several genes plausibly encoding enzymes for 1 biosynthesis and tailoring reactions. We searched for the gene responsible for the peptide bond formation between 1 and the peptides in the cluster, and identified pgm1, which has an ATP-grasp domain. A pgm1-deletion mutant lost PGM productivity, and recombinant PGM1 catalyzed the ATP-dependent peptide bond formation. This is the first example of cooperative peptide synthesis achieved by ribosomes and peptide ligases using a peptide as a nucleophile. PGM1 accepted a variety of peptides as the nucleophile and its flexibility was comprehended by the crystal structure of PGM1 and mutagenesis analyses.

• 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

  MATSUI TAKASHI, OTAKI SHOTA, NOIKE MOTOYOSHI, DAIRI TOORU, MORITA HIROYUKI  日本結晶学会年会講演要旨集  2014-  79  2014  [Not refereed][Not invited]
  
• ビタミンKの生合成

  大利 徹  ビタミンKと疾患  40  -46  2014  [Not refereed][Not invited]
  
• 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  2013/11/27  [Not refereed][Not invited]
  
• 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  2013/11/27  [Not refereed][Not invited]
  
• 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  2013/09/05  [Not refereed][Not invited]
  
• 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  2013/09/05  [Not refereed][Not invited]
  
• 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  2013/03/05  [Not refereed][Not invited]
  
• 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  2013/03/05  [Not refereed][Not invited]
  
• 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  2013/03/05  [Not refereed][Not invited]
  
• 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  2012/09/06  [Not refereed][Not invited]
  
• 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  2012/09/05  [Not refereed][Not invited]
  
• 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  2012/07/01  [Not refereed][Not invited]
  
• 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  2012/07/01  [Not refereed][Not invited]
  
• 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  2012/07/01  [Not refereed][Not invited]
  
• 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  2012/07/01  [Not refereed][Not invited]
  
• 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  2012/03/05  [Not refereed][Not invited]
  
• 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  2012/03/05  [Not refereed][Not invited]
  
• 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  2011/07/01  [Not refereed][Not invited]
  
• 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  2011/02/01  [Not refereed][Not invited]
  
• 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  2011/02/01  [Not refereed][Not invited]
  
• Studies on an alternative menaquinone biosynthetic pathway operating in actinomycetes

  大利 徹  日本放線菌学会誌  25-  (2)  2  -6  2011
• 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  [Not refereed][Not invited]
  
• 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  2010/09/01  [Not refereed][Not invited]
  
• 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  2010/09/01  [Not refereed][Not invited]
  
• 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  2010/07/01  [Not refereed][Not invited]
  
• 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  2010/07/01  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 33. Characterization of diterpene synthase genes in Aspergillus oryzae

  Chiba Yasutaka, Sugai Yoshinori, Shiono Yoshihito, Dairi Tohru, Mitsuhashi Wataru, Kawaide Hiroshi, Toyomasu Tomonobu  植物化学調節学会研究発表記録集  (44)  47  -47  2009/10/06  

  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  [Refereed][Invited]
  
• 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  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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
• Comparison of enzymatic properties of ent-copalyl diphosphate synthases for biosynthesis of phytoalexins and gibberellins in rice

  HAYASHI Yutaka, HIROSE Yuko, ONODERA Yu, MITSUHASHI Wataru, SASSA Takeshi, DAIRI Tohru, TOYOMASU Tomonobu  植物の生長調節 = Regulation of plant growth & development  42-  34  -34  2007/10/05  

  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
• 2C16-2 An improved procedure for selecting highly active phenylacetaldehyde reductase mutants

  MAKINO Yoshihide, DAIRI Tohru, ITOH Nobuya  日本生物工学会大会講演要旨集  19-  87  -87  2007
• 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  2006/11  [Not refereed][Not invited]
  
• 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  2006/11  [Not refereed][Not invited]
  
• 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  2006/11  [Not refereed][Not invited]
  
• Cloning and characterization of a cDNA encoding diterpene cyclase responsible for fusicoccin biosynthesis

  TSUKAHARA Mai, TOYOMASU Tomonobu, KANEKO Akane, NIIDA Rie, MITSUHASHI Wataru, DAIRI Tohru, KATO Noboru, SASSA Takeshi  植物の生長調節 = Regulation of plant growth & development  41-  61  -61  2006/10/04
• 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  [Not refereed][Not invited]
  
• Studies on biosynthetic genes and enzymes of isoprenoids produced by actinomycetes

  DAIRI TOHRU  The Japanese journal of antibiotics  58-  (1)  87  -98  2005/02/25
• Studies on eubacterial diterpene cyclases found in actinomycetes

  DAIRI Tohru, HAMANO Toshimitsu, ITOH Nobuya, KUZUYAMA Tomohisa, FURIHATA Kazuo, SETO Haruo  Nippon Nōgeikagaku Kaishi  vol. 76, No. 12, 1191-1194-  (12)  1191  -1194  2002  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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  2001/02  [Not refereed][Not invited]
   

  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  2000/09  [Not refereed][Not invited]
   

  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  2000/09  [Not refereed][Not invited]
  
• 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  2000/04/15  [Not refereed][Not invited]
   

  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  2000/04  [Not refereed][Not invited]
   

  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  2000/03/05
• 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  [Not refereed][Not invited]
  
• 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  2000/01  [Not refereed][Not invited]
   

  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.
• Alteration of stereoselectivity of L-allo-threonine aldolase by random mutation

  Kataoka Michihiko, Kawabata Hiroshi, Liu Ji-Quan, Dairi Tohru, Itoh Nobuya, Yamada Hideaki, Shimizu Sakayu  日本生物工学会大会講演要旨集  11-  160  -160  1999
• 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  1998/06  [Not refereed][Not invited]
   

  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.
• Cofactor characterization of low-specificity D-threonine aldolase

  Liu Ji-Quan, Dairi Tohru, Itoh Nobuya, Kataoka Michihiko, Shimizu Sakayu, Yamada Hideaki  日本生物工学会大会講演要旨集  10-  168  -168  1998
• Studies on Antibiotic-Biosynthetic Genes and Enzymes Catalyzing Unique Reatctions.

  Tohru Dairi  Actinomycetologica  12-  (2)  75  -83  1998  [Not refereed][Not invited]
  
• Protoplasting and regeneration of strains belonging to the genus Actinomadura.

  Actinomycetologica  11-  (1)  1  -5  1997  [Not refereed][Not invited]
  
• 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  1996/03/05
• 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  1996/03/05
• Electrophoretic fraction of Ganoderma licidium extract under non-denatured polyacrylamide gel

  Journal of Traditional Medicines  13-  504  -505  1996  [Not refereed][Not invited]
  
• 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  1995/10  [Not refereed][Not invited]
  
• 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  1995/07/05
• Potential Antifungal and Anti-HIV Agent ; Prodimicin.

  ┣DBExp. Opin. Patents. (/)-┫DB  (4)  1483  -1491  1994  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  

Industrial Property Rights

• 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  2017/10/12

  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  2014/04/25

  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  2013/07/05

  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  2012/03/22

  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  2009/05/28

  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  2008/10/16

  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  2008/01/24

  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  2004/08/26

  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  2004/07/08

  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

Awards & Honors

• 2023/03 Japan Society for Bioscience, Biotechnology, and Agrochemistry (JSBBA) JSBBA Award
   Studies on biosynthetic enzymes leading structural and functional diversities to microbial natural products.
• 2022/03 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
• 2021/03 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
• 2019/03 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
• 2017/03 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
• 2015/06 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 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 長瀬財団 Nagase Foundation Award
   

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

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

Research Grants & Projects

• Dissecting biosynthetic machineries of natural peptides leading to structural and functional diversities.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S)

  Date (from‐to) : 2022/04 -2027/03 

  Author : 大利 徹, 小笠原 泰志, 森田 洋行, 濱野 吉十
• Dissecting reaction mechanisms of novel peptide epimerases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2022/03 

  Author : Dairi Tohru

   

  (1) MurL catalyzing the epimerization of the terminus L-Glu of UDP-MurNAc-L-Ala-L-Glu, an intermediate in the alternative biosynthetic pathway of peptidoglycan, was shown to activate the substrate by adenylation of α-carbonyl of the L-Glu. By crystal structure analysis, we were unable to determine the active amino acid residues of MurL, and co-crystal of MurL with a mimic of adenylated substrate was thought to be essential for estimation of the active residues and the reaction mechanism. (2) MslH catalyzed the epimerization of C-terminal L-Trp in a ribosomal peptide MS-271. By crystal structure analysis, active amino acid residues, divalent cation trapping residues, and several substrate recognition residues were identified. (3) Polyglutamate contains D-Glu besides L-Glu. PgsA, a polyglutamate biosynthetic enzyme, was suggested to be the epimerase catalyzing isomerization of terminal L-Glu that is just ligated to the elongating polyglutamate substrate.
• Redesigning Biosynthetic Machineries (Project Team)

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/06 -2021/03 

  Author : Abe Ikuro

   

  In this project, we have made a dramatic development from simply learning biosynthetic machineries to designing new blueprints for producing desired natural products. Based on the detailed analyses of the structural diversity of natural products from the viewpoint of genes, enzymes, and reactions, we established a new world-leading technology platform for creation of complex functional molecules by rational reconstruction of biosynthetic machineries as a new academic field. In order to facilitate the research plan, the project team formulated a research policy and provided a forum to actively promote exchanges, collaborations, and joint research among individual research promoters. Researchers from various fields collaborated and complemented each other in their areas of expertise to conduct joint research and create a new academic field.
• Studies on biosynthetic machinery of biologically active natural products

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/06 -2021/03 

  Author : Dairi Tohru

   

  Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) 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 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 DHA synthase into an EPA synthase by one amino acid substitution.
• Redesigning Biosynthetic Machineries (International Activity Support Team)

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/06 -2021/03 

  Author : Abe Ikuro

   

  In this project, we have made a dramatic development from simply learning biosynthetic machineries to designing new blueprints for producing desired natural products. We established a new world-leading technology platform for creation of complex functional molecules by rational reconstruction of biosynthetic machineries as a new academic field. In order to strongly promote joint research with foreign countries, the International Activity Support Group invited outstanding foreign researchers and held lecture meetings on a regular basis. Furthermore, international symposiums were held with the aim of strengthening cooperation and exchange with the U.S., Germany, China, and other countries. We strategically promoted joint research with foreign countries, focusing on medium-term support for young researchers. Many of the results of joint research with foreign countries resulted in excellent co-authored papers.
• Biosynthesis of the Carbonylmethylene Structure Found in a Class of Pseudotripeptides, Ketomemicins

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2015/04 -2018/03 

  Author : Dairi Tohru, SATOH YASUHARU, OGASAWARA YASUSHI, MORITA HIROYUKI

   

  We recently discovered novel pseudotripeptides, ketomemicins, which possess a C-terminal pseudodipeptide connected with a carbonyl methylene instead of an amide bond. The carbonyl methylene structure is stable than amide bond and its biological significance has been shown in several natural and synthetic compounds. Despite the biological significances of these compounds, little is known about its biosynthetic machinery. We therefore examined it by in vitro studies with recombinant enzymes. Consequently, an aldolase, dehydratase, PLP-dependent glycine-C-acetyltransferase, and dehydrogenase were revealed to be involved in the formation of the pseudodipeptide with malonyl-CoA and phenylpyruvate as starter substrates.
• Biosynthetic machinery: Deciphering and regulating the system for creating structural diversity of baioactive metabolites

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2010/04 -2016/03 

  Author : OIKAWA Hideaki, EGUCHI Tadashi, ABE Ikuro, KUZUYAMA Tomohisa, DAIRI Tohru, IKEDA Haruo, GOMI Katsuya, SAITO Kazuki, KANAYA Shigehiko, ISHIKAWA Jun

   

  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。
• Peptide synthesis cooperatively achieved by peptide ligase and ribosomes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2013/04 -2015/03 

  Author : DAIRI Tohru

   

  Pheganomycin (PGM) (1) consists of a nonproteinogenic amino acid, (S)-2-(3,5-dihydroxy-4-methoxyphenyl)-2-guanidinoacetic acid (2) at the N-terminus and a proteinogenic core peptide derived from NVKDGPT or NVKDR. The biosynthetic gene cluster was identified in Streptomyces cirratus to contain a gene encoding a precursor peptide, which included both the core peptides, and several genes plausibly encoding enzymes for 2 biosynthesis. We identified a gene (pgm1) responsible for the peptide bond formation between 2 and the peptides in the cluster. A pgm1-disruptant lost 1 productivity and recombinant PGM1 catalyzed the ATP-dependent peptide bond formation. This is the first example of cooperative peptide synthesis achieved by ribosome and peptide ligase using a peptide as a nucleophile. PGM1 accepted a variety of peptides as the nucleophile and the flexibility was comprehended by the crystal structure of PGM1 and the mutagenesis analyses.
• Studies on biosynthetic machineries for cyclic terpenoids and nucleoside antibiotics

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2011/04 -2015/03 

  Author : DAIRI Tohru, KATO Nobuo, SATOH Yasuharu, ARAI Ryouichi

   

  1. Fusicoccin A is a diterpene glucoside produced by the fungus Phomopsis amygdali and its derivative lacking the OH group at the 12-position is revealed to be a potential anti-cancer drug. We therefore constructed a mutant producing the desirable intermediate by biosynthetic engineering. 2. Three fungal prenyltransferases, PaxC, PaxD, and AtmD, all of which are responsible for biosynthesis of indole diterpene compounds, were characterized. 3. Aminodeoxyfutalosine is the first intermediate in the new menaquinone biosynthetic pathway. We demonstrated that a radical S-adenosyl methionine enzyme (MqnE) catalyzed the addition of the adenosyl radical to the double bond of 3-[(1-carboxyvinyl)oxy]benzoic acid derived from chorismate by MqnA. 4. During the biosynthetic studies of pheganomycin, we identified a new ATP-grasp enzyme, which phosphorylated the non-proteogenic amino acids with ATP and the successive nucleophilic attack of the peptides.
• Study on a novel folate biosynthesis pathway on micoorganism

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2012/04 -2014/03 

  Author : SATOH Yasuharu, DAIRI Tohru, KURATSU Masahiro, OBAYASHI Daiki

   

  Folate is an essential cofactor in all living cells. para-Aminobenzoate (pABA), a building block of folate, is usually derived from chorismate in the shikimate pathway by reactions of PabABC. 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 pabABC 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 (dpabABC) as a host. A tracer experiment using [U-13C6]glucose suggested that pABA was de novo synthesized in the transformant. An E. coli dpabABCdaroB mutant carrying the NE1434 gene exhibited a prototrophic phenotype to pABA, suggesting that compounds in the shikimate pathway including chorismate were not utilized as substrates by NE1434.
• Estimation of an alternative biosynthetic pathway for co-factors by close investigation of genome databases.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2012 -2012 

  Author : DAIRI Tohru, SATOH Yasuharu

   

  The invovment of new enzymes for co-factors biosynthesis was examined. pABA; A function unknown gene, NE1434, was obtained from Nitrosomonas europaea by shotgun cloning experiment using pABA-auxotrophic E. coli mutant (△pabABC) as a host. Glutathione; Streptomyces strains do not use glutathione. However, SCO0910 was confirmed to be glutamate-cysteine ligase and was essential for ergothioneine biosynthesis. Taurine; SCO3035 in S. coelicolor A3(2) was reported to be the first enzyme in taurine biosynthesis (J. Bacteriol. 188, 5561, 2006). To investigate taurine biosynthesis in S. coelicolor A3(2), SCO3416, 2782, and 2017, putative enzymes catalyzing thesecond reaction, were used for in vitro assay. However, all enzymes did not show the expected activity.
• Screening of natural products which inhibits a new menaquinone biosynthetic pathway

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2010 -2012 

  Author : DAIRI Tohru

   

  The futalosine pathway is operating in some bacteria for the biosynthesis of menaquinone. Futalosine is converted into dehypoxanthinyl futalosine (DHFL) by MqnB.In this study, three routes to the formation of DHFL were suggested. DHFL may have been directly formed by MqnB in Thermus thermophilus. In Streptomyces coelicolor, aminodeoxy FL (AFL) was converted to FL by deaminases, then to DHFL. In contrast, MqnB of Helicobacter pylori directly converted AFL into DHFL. To identify compounds that specifically inhibit the futalosin pathway, we used B. halodurans that has FL pathway as a test strain. Finally, two candidate fungal culture broths were obtained.
• 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)

  Date (from‐to) : 2011 -2011 

  Author : 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

  Date (from‐to) : 2008 -2009 

  Author : 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。
• Studies on diterpene cyclases found in eubacteria

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2007 -2009 

  Author : 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

  Date (from‐to) : 2006 -2007 

  Author : 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。
• Studies on a new biosynthetic pathway for menaquinone (vitamin K)

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2005 -2007 

  Author : SETO Haruo, SUE Masayuki, DAIRI Toru

   

  We found that some pathogenic bacteria including Helicobacter pylori and Campylobacter jejuniutilized a new biosynthetic pathway for menaquinone. This pathway is also present in the genus Streptomyces and branches from the well known menaquinone biosynthetic pathway at chorismate. Since this pathway does not present in human, its inhibitors are expected to be useful as antibacterial substances. In order to reveal the details of this pathway, we carried out the following experiments. 1. At the onset of the experiments, we established unequivocal assignment of 13C-N1VIR spectrum of menaquinone. 2. Labeling experiments using glucoses labeled by^<13>C at different position proved that menaquinone was formed from erythrose, phosphoenolpyruvate and two different C_2 units originating from C5-C6 of glucose. 3. Several kinds of blocked mutants of Streptomyces coelicolor that required menaquinone for their growth were prepared. 4. Based on the results obtained by labeling experiments, 1,4-naphthoquinone-6-carboxylic acid was assumed to be a biosynthetic intermediate of the new pathway. Therefore, we prepared this acid by oxidation of naphthalene-2-carboxylic acid by using Ce(SO_4)_2. This compound enabled the growth of a menaquinone auxotroph of S coelicolor indicating that the synthetic compound is a true intermediate of the new pathway. 5. By detailed investigation of metabolites accumulated by mutants of S coelicolor a nucleoside compound, futalosine, was isolated. This compound consisted of inosine and a m-substituted benzoic acid derivative, and had been isolated from another strain of Streptomyces as an antitumor substance. This compound was proved to be a biosynthetic intermediate of the new pathway, since it supported the growth of some menaquinone auxotrophs of S coelicolor. 6. By utilizing other menaquinone auxotrophs of S coelicolor, several intermediates after futalosine were identified.
• Studies on production and improvement of terpenoids using biosynthetic gene cluster in fungi and plants.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

  Date (from‐to) : 2004 -2006 

  Author : SASSA Takeshi, YAMANE Hisakazu, HOSHINO Tsutomu, KATO Nobuo, OIKAWA Hideaki, DAIRI Tohou

   

  A cDNA encoding fusicoccadiene synthase (PaFS) was isolated from mycelia of fusicoccin-producing fungi. PaFS possesses two domain, terpene cyclase domain at N-termini and prenyltransferase domain at C-termini, and shows multifunctional catalyzation. We have clarified the detailed cyclization mechanism forming fusicoccane skeleton from GGDP, and have succeeded in synthesizing a fucicoccin derivative that has a differentiation inducing activity. Mechanism on construction of molecular skeleton of diterpene, phomaction was investigated by chemical methods. Heterologous expression of the genes responsible for biosynthesis of diterpene, aphidicolin using yeast was explored. A common biosynthetic intermediate of indole diterpenoide was synthesized as non-and deuterium-labeled form. The deuterium-labeled intermediate was converted into paxilline and emindole DA via epoxidation and cyclization in two different fungi in an intact manner. These results give first evidence that various indole diterpenoide are biosynthesized from a common intermediate. We have cloned three diterpene cyclase genes and one tetraterpene cyclase gene from actinomycetes. Of the four cyclases, enzymatic properties of the two diterpene cyclases were investigated with recombinant enzymes. The functional analyses were performed in respect to Rv3377c and Rv3378c involved in Mycobacterium tuberculosis H37 strain. These genes encode diterpene synthases for producing halimane skeleton. Studies on squalene cyclase were also conducted to gain more insights into the catalytic mechanism. Successful site-specific incorporation of fluorophenylalanines into the catalytic sites demonstrated the importance of cation-pi interaction. In vitro translation system revealed the function of a diterpene cyclase gene from the moss Physcomoitrella patens. This system was also available for determination of production and localization of cytochrome P450 monooxygenase proteins. The function of P450 monooxygenases responsible for biosynthesis of plant hormones, brassinosteroid and gibberellin, was revealed by heterologous expression in Pichia pastoris. We identified 6 cDNAs encoding diterpene cyclases responsible for phytoalexin bisynthesis in rice. We indicated strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis. The presence of phytocassane biosynthesis gene cluster on chromosome 2 was also suggested.
• Studies on isoprenoid biosynthetic genes isolated from prokaryotes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2004 -2006 

  Author : DAIRI Tohru, MATSUURA Nobuyasu

   

  We have 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-copalyl diphosphate(CDP) synthase and a geranylgeranyl diphosphate(GGDP) synthase, respectively, by experiments using recombinant enzymes. ORF3,that did not show similarities with any other known proteins was also expressed in E.coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into pimara-9(11),15-diene. 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 Mg^<2+> and Zn^<2+> for its activity. The optimum pH and temperature were 5.5 and 35℃. The Km value was calculated to be 13.7±1.0μM 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℃. The Km value for ent-CDP was estimated to be 2.6±0.2μM and the kcat value was 1.4 x 10^<-3>/sec.
• Studies on diterpene cydases found in actinomycetes.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2002 -2003 

  Author : DAIRI Tohru

   

  Fubacterial diterpene cyclase genes have been doned from a diterpenoid-antibiotic, terpentecin producer. Their products, ORF11 and ORF12, were confirmed to be essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same -basic skeleton as terpentecm. Functional analyses of these two enzymes were also performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a cydized intermediate (terpentedienol diphosphate, TDP), and then it was transformed into TTE by the ORF12 product. Interestingly, the ORF12 product directly reacted with GGDP and converted GGDP into three olefinic compounds. Moreover, the ORF12 product reacted even with farnesyl diphosphate (FDP) giving three olefinic compounds, which had the same structures as those formed from GGDP except for 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 reaction by an ionization-initiated reaction of substrates to an allylic carbocation followed by deprotonation to the olefin. Kinetics of the ORF12 product indicated that the affinity for TDP and GGDP were higher than that of FDP and that the relative activity of the reaction converting TDP into TTE. was highest among the reactions using TDP, GGDP, or FDP as the substrate. These results suggested that an actual reaction catalyzed by the ORF12 was the conversion of TDP into TTE in vivo.
• Scientific research on elucidation of terpene-cyclic reactions in the biosynthesis of bioactive microbial terpenoids.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

  Date (from‐to) : 2001 -2003 

  Author : SASSA Takeshi, OIKAWA Hideaki, KATO Nobuo, HOSHINO Tsutomu, TOYOMASU Tomonobu, DAIRI Tohru

   

  (1)We isolated (+)-cyatha-3,12-diene from an erinacine-producing fungus, and confirmed its stereostructure by semi-synthesis from erinacine P. The cyathane hydrocarbon could be obtained from GGDP by the cell-free system. From a fusicoccin-mass-producing fungus we isolated (+)-δ-araneosene as the presumed bicyclic hydrocarbon intermediate. (+)-Fusicocca-3(16),10(14)-diene was also isolated and its identification suggested the presence of a new fusicoccenyl cation in the biosynthetic pathway. (2)We identified two gene clusters responsible for diterpene biosynthesis by chromosome waling from GGDP synthase genes from Phomopsis amygdali. The gene clusters led us to successful isolation and characterization of two cDNAs encoding diterpene cyclases. (3)The gene cluster for aphidicolin biosynthesis has been identified by PCR-based genome walking using reported sequence of cDNA coding the diterpene cyclase. Based on non-enzymatic cyclization under various conditions and molecular orbital calculations, cyclization mechanism for construction of aphidicolane skeleton was proposed. (4)We have cloned two diterpene cyclase genes essential for biosynthesis of terpentecin. By using recombinant enzymes, it was revealed that one enzyme converted GGDP into a cyclized intermediate (terpentedienol diphosphate), and then it was transformed into terpentetriene by the other enzyme. (5)By using the site-specific mutants and the substrate analogs, we clearly demonstrated that the steric bulk size of active site residues has an important role in determining the folding conformation of squalene substrate during the polycyclization cascade.
• Studies on the biosynthesis of terpenoids produced by streptomycetes.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2001 

  Author : DAIRI Tohru

   

  Two gene clusters containing the mevalonate pathway genes and the terpentecin (TP) biosynthetic genes were cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, TP producer. In the former cluster, seven genes enecoding geranylgerairyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarbosylase (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 Ε. coli and Streptomyces Iividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate paftway were involved in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in Ε. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. In the latter cluster, two ORFs, ORF11 and ORF12 that encode proteins showing similarities to eucaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were found. The two cyclase genes were expressed in Streptomyces Iividans. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has a common basic skeleton as does TP. The two enzymes were overproduced in Ε. coli and purified to homogeneity. The recombinant ORF11 product converted GGDP into an intermediate with diphosphate, and then it was transformed into terpenteteiene by the recombinant ORF12 product. To the best of our knowledge, this is the first report about a eubacterial DC.
• Cloning and Analysis of Tautomycin Biosynthetic Genes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1996 -1998 

  Author : UBUKATA Makoto, MATSUURA Nobuyasu, DAIRI Tohru

   

  Tautomycin (TM) isolated from a culture broth of Streptomyces spiroverticillatus is an inhibitor of protein phosphatases 1 and 2A. Since TM and its analogues are expected to be useful probes for elucidating the signal trasduction of mammalian cells, we studied on the absolute structure, conformational analysis, structure-activity relationship, and biosynthesis of the polyketide compound, TM. In the present study, we have studied on the cloning and analysis of TM biosynthetic genes. Here I report the results of experiments done in 1996, 1997 and 1998. 1. Development of transformation system: Protoplast formation and regeneration of the TM producer were achieved by using the reported procedure for other Streptomyces sp. In addition to above experiments, the transformation system was established by curing the producer of the cryptic plasmid by a heat shock treatment. The satisfactory efficiency of 2 x 10ィイD15ィエD1 transformants/μgDNA was obtained using the pIJ702 plasmid, with the result that we optimized various conditions of the transformation system. 2. Cloning of the putative TM biosynthetic gene: The KS and AT primers, which were designed from the published sequences in PKS Type I, were used in PCR strategy to obtain products. The primer design strategy was successful in identifying a fragment from S. spiroverticillatus genomic DNA and a PKS on cosmid pWE15. Sequence analysis of the PCR product strongly suggests that we have cloned a PKS gene flagment in the TM producer. 3. Gene disruption of the putative TM biosynthetic gene: To determine whether the PKS gene fragment obtained from PCR strategy indeed encodes PKS involving in TM biosynthesis, the fragments were then used for gene disruption by recombinational insertion of the cloned 1.8 kb region into the corresponding region on the chromosome. The resulting 15 strains were fermented and assayed for the presence of TM. Although none of the strains produced TM, an another antibiotic xanthostatin was produced by all strains. Genomic Southern hybridization analyses showed that TM biosynthetic genes in these strains were deleted from the genomic DNA.
• Reaction Mechanisms and Application of New Bacterial Enzymes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1996 -1997 

  Author : ASANO Yasuhisa, KOMEDA Hidenobu, KATO Yasuo, DAIRI Tohru

   

  (1) Phenylalanine dehydrogenase (PheDH) : PheDHs from Bacillus sphaericus, B.badius, Sporosarcina ureae were purified from Escherichia coli transformants in large scales and sent to Prof.Engel (University College Dublin, Ireland) and Prof.Rice (University of Sheffield, U.K.) for X-ray crystallography. Based on informations of X-ray crystallography of glutamate dehydrogenase, chimeric enzymes of PheDH with altered substrate specificities were constructed. (2) Methylaspartate ammonia-lyase (MAL) : MALs from Enterobacteria such as Enterobacter, Citrobacter, Proteus were purified to homogeneities and their enxymological properties were analyzed in detail. MAL from Citrobacter amalonaticus strain YG-1002 was digested with endo peptidases and N-terminal amino acid sequences were determined. Based on the information, DNA probes were synthesized. The gene for the enzyme was cloned form the genomic library of c.amalonaticus strain YG-1002 by PCR and Southern hybridization. The sequence of the gene was compared with that of a strict anaerobe Clostridium tetanomorphum. MAL was crystallized and sent to Prof.Rice for studies of X-ray crystallography. (3) Opine dehydrogenase (ODH) : The gene for ODH from Arthrobacter sp.IC was cloned and expressed in E.coli. The enzyme was purified in a large scale and sent to Prof.Rice, and on its X-ray studies were started. The first structure of a (D,L) superfamily member, N-(1-D-Carboxylethyl)-L-norvaline dehydrogenase from Arthrobacter sp.strain 1C,has been solved to 1.8* resolution and the location of the bound coenzyme determined. (4) Results were presented in some international academic meetings.
• Development of Novel Lyases and Their Application to the Synthesis of Optically Active Compounds

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1995 -1996 

  Author : ASANO Yasuhisa, YAMADA Hideaki, DAIRI Tohru, KATO Yasuo

   

  First, we screened for microorganisms having new hydro-lyases catalyzing hydration of maleic acid and itaconic acid, respectively, to produce D-malic acid and citramalic acid which are food additives and starting materials in synthetic organic chemistry, etc. The bacterial maleate hydratase from Arthrobacter sp. was purified to homogeneity and characterized. The culture and reaction conditions for the production of optically pure D-malate from maleate were studied. Under an optimum condition, 87 grams/liter of D-malate was produced in 20 h. The yield was 72 mole %. (S)-(+)-Citramalate producing activity from itaconate was also studied. A.denitrificans produced (S)-(+)-Citramalate in a 99.9 % enantiomeric excess. Under an optimum condition, 27 g of (S)-(+)-citramalate per liter was produced in 30 h. The yield was 69 mol%. Second, a screening was carried out for new 3-methylaspartate ammonia-lyases which catalyze the synthesis of L-aspartic acid derivatives from fumaric acid derivatives. We discovered for the first time that 3-methylaspartate ammonia-lyase (MAL) producers are relatively widely distributed in the family of facultatively anaerobic Enterobacteriaceae. Crystalline MALs from Citrobacter freundii, C.amalonaticus, and Morganella morganii, were characterized. Using cell-free extracts of the isolates, optically pure (2S, 3S)-3-methylaspartic, (2S, 3S)-3-ethylaspartic, and (2R, 3S)-3-chloroaspartic acids were synthesized from corresponding fumaric acid derivatives.
• Studies on biosynthesis of terpenoids produced by microorganisms

  Date (from‐to) : 1996
• Studies on antibiotic biosynthesis

  Date (from‐to) : 1994
• Studies on biosynthesis of natural product
• Studies on biosynthetic genes and enzymes of isoprenoids produced by microorganisms

Educational Activities

Teaching Experience

Position History

• 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