Lab. of Biocatalyst and Bioprocessing,Department of Biotechnology

Research Field

The use of enzymes and/or microorganisms (biocatalysts) for the efficient production of useful compounds, such as pharmaceutical and agrochemical intermediates, especially chiral compounds; Asymmetric bioreduction/biooxidation; Metagenomics for biocatalysis; Functional food materials production; Protein engineering, Biohalogenation and son.

Recent Research Themes

  1. Asymmetric hydrogen-transfer bioreduction producing chiral alcohols.
  2. Asymmetric biooxidation using styrene monooxygenase producing chiral epoxides.
  3. Screening of gene-specific amplicons from metagenomes (S-GAM method) for biocatalysis.
  4. Biohalogenation: mechanisms and applications.
  5. Protein engineering and directed evolution of oxidoreducatases to increase their activity in polar/non-polar organic solvents.
  6. Invention of a protein with an advanced function.

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Research Intererst

Itoh PhotoProf.
Nobuya Itoh
(born in 1955)
Makino PhotoLecturer.
Yoshihide Makino
(born in 1970)
Toda PhotoAssistant Prof.
Hiroshi Toda
(born in 1977)
Screening and finding applications for enzymes; mainly oxidoreductases for bioprocessing

Metagenomics for biocatalysis

Biohalogenation
Protein engineering of oxidoreductases with the aim of expanding the range of applications for these enzymes

Protein structure and function
Development of bioprocess for production of fine chemicals by enzymes

Bioconversion and metabolic engineering

address:
Lab. Biocatalysis and Bioprocessing,
Department of Biotechnology, Toyama Prefectural University,
Kurokawa 5180, Imizu, Toyama 939-0398, JAPAN
TEL: +81-766-56-7500
FAX: +81-766-56-2498
E-mail: nbito_AT_pu-toyama.ac.jp (Nobuya Itoh), makino_AT_pu-toyama.ac.jp (Yoshihide Makino)
htoda_AT_pu-toyama.ac.jp (Hiroshi Toda)
PLEASE REPLACE '_AT_' WITH '@'

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Report

(1) Production of chiral alcohols by asymmetric hydrogen-transfer bioreduction

PAR and LSADH indicate the opposite stereoselectivitySeveral examples of ketone reduction with reductases and dehydrogenases have been described, since these processes theoretically produce alcohol with 100% conversion from ketone. However, these enzymes have a number of disadvantages, including narrow substrate spectrum and low productivity due to the need for a cofactor regeneration system, such as NADH and NADPH. Therefore, these bioreduction processes are economical only when the cofactor can be regenerated in situ in a second catalytic cycle (for example, formate/formate dehydrogenase (FDH) or glucose/glucose dehydrogenase (GDH)).

Another suitable hydrogen donor for bioreduction is 2-propanol, which is used for the regeneration of NADP(H) because of its chemical properties and low cost. Recently, we reported that phenylacetaldehyde reductase (PAR) from the styrene-assimilating Rhodococcus (former Corynebacterium) sp. strain ST-10 and a novel ADH (LSADH) from Leifsoniasp. strain S749 are unique NADH-dependent alcohol dehydrogenases (ADH) with a broad substrate range of more than 40 ketones. These enzymes display high enantioselectivity to generate (S)- and (R)-form alcohols, respectively, without an additional coenzyme regeneration system, as both are able to regenerate NADH in the presence of 2-propanol. PAR or its mutated enzyme (Sar268) and LSADH are superior asymmetric hydrogen-transfer biocatalysts used to produce (S)- and (R)-alcohols.

Recombinant E. coli cells overexpressing par and lsadh produced the highest levels of chiral alcohols from ketones ever reported (50-300 g/L reaction mixture) and enantiomeric excess (e.e.).

(2) Bioproduction of aromatic and aliphatic (S)-epoxyalkanes by styrene monooxygenase from Rhodococcus sp. ST-10

Bioproduction of epoxyalkanes by RhSMOEnantiopure epoxides are important compounds for the synthesis of chiral materials including pharmaceuticals and agrochemicals. Enantioselective epoxidation of prochiral alkenes is a straightforward strategy for producing enantiopure epoxides, and many chemical approaches, such as Sharpless epoxidation, metal-salen catalyst, and fructose-derivatives, have been studied to achieve this aim. However, further improvements are required for the direct asymmetric epoxidation of straight-chain terminal alkenes due to the difficulty of their enantioselective epoxidation. Recently, we reported the isolation and characterization of styrene monooxygenase (RhSMO) of Rhodococcus sp. ST-10. RhSMO catalyzes the epoxidation of styrene to (S)-styrene oxide with excellent enantiomeric excess (ee > 99%), and can catalyze the epoxidation of styrene derivatives and straight-chain aliphatic alkenes (e.g., 1-hexene, 1-octene) to the corresponding (S)-epoxyalkanes with a good ee (>80%). We have developed a novel biocatalysis for the efficient synthesis of enantioenriched (S)-epoxyalkanes using RhSMO.

We constructed an Escherichia coli biocatalyst expressing RhSMO and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH) for the regeneration of NADH. The constructed biocatalyst could catalyze the asymmetric epoxidation of various styrene derivatives using 2-propanol as a hydrogen donor with an excellent ee (>90%). Furthermore, this biocatalyst exhibited broad substrate specificities for functional/nonfunctional, terminal/internal, and di-/tri-substituted aliphatic alkenes. Biocatalysis using RhSMO is suitable for the direct asymmetric epoxidation of various alkenes.

(3) Screening of gene-specific amplicons from metagenomes (S-GAM method) for biocatalysis

Scheme of S-GAM methodMetagenomics is an emerging tool for the isolation of genes. However, most previous approaches have featured metagenomic DNA extraction and Escherichia coli library construction, followed by sequence- or function/molecule-based screens of the library. Such approaches are very time-consuming, especially in terms of detection. Although PCR amplification of truncated genes from metagenomes would facilitate the identification of genes, the available methods are not very efficient in many cases and often fail to produce complete functional genes.

We constructed a metagenomic library of enzyme genes that focused on the Leifsonia sp. adh gene (lsadh) and Rhodococcous sp. styrene monooxygenase gene (Rhsmo). Our approach, which involved PCR amplification of nearly full-length genes from metagenomes fused with the terminal region of an each expressing vector, enabled the isolation of many novel and diverse target genes and their homologs (Fig. 1). In the case of lsadh, 1200 clones of the 2000 colonies obtained were identified as adh-positive (~60%). The genes obtained via this approach encoded a wide variety of amino acid sequences: 8-99% for Hladh (for homologous Leifsonia adh) and 55-99% for Hsmo. Thus, the screening of gene-specific amplicons from metagenomes (S-GAM) approach showed high efficiency for obtaining gene resources from metagenomes. We also confirmed the potential use of novel enzymes as biocatalysts for converting ketones to various anti-Prelog chiral alcohols by HLADHs.

(4) Functional food production by laccase reaction

Fig.1 Reaction of EGC (3) and EGCG (4) with gallic acid by laccase-catalyzed oxidation.We recently found that laccase (EC 1.10.3.2) was able to synthesize two benzotropolone derivatives, epitheaflagallin (5), and epitheaflagallin 3-O-gallate (6) (Fig. 1), from crude tea catechins in the presence of oxygen and gallic acid.1) These compounds have been previously synthesized from EGC (3)/EGCg (4) in the presence of catechol/pyrogallol by Takino and Imagawa.2) Using purified compounds including (1), (2), (3), (4), we confirmed that (5) and (6) were synthesized from (3) and (4), respectively, through laccase oxidation reaction with gallic acid. The reaction involves the preferential oxidation of gallic acid and the pyrogalloyl group (B ring) in (3) and (4) to the quinone intermediate galloquinone, followed by the Michael addition of the oxidized pyrogalloyl group in (3)/(4) and subsequent carbonyl addition across the ring and decarboxylation to yield benzotropolone skeletone. Recently, Sang et al. reported the enzymatic synthesis of tea theaflavin derivatives from catechin derivatives in the presence of hydrogen peroxide and peroxidase (EC 1.11.1.7).3) In peroxidase oxidation, the catechoyl/pyrogalloyl/galloyl groups in EC (1)/ECg (2)/EGC (3)/EGCg (4) are easily oxidized to form the quinone intermediates that subsequently yield the various theaflavin-related compounds. In our oxidation system, preferential conversion of (3) and (4) is possible, and very little EC (1) and ECG (2) reacted with the galloquinone intermediate in the reaction mixture. Laccase-dependent preferential oxidation of gallic acid and the pyrogalloyl group in (3) and (4) to the galloquinone intermediate makes it possible to convert (3) and (4) in crude tea catechins to (5) and (6).

Outline of Research Themes

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Publications

| 1997 | 1998 | 1999 | 2000 | 2001 | 2002 | 2003 | 2004 | 2005 | 2006 |
| 2007 | 2008 | 2009 | 2010 | 2011 |2012|2013|2014|2015|

[1997]

  1. N. Itoh, Volatile Haligenated Compounds from Marine Algaem: Their Formation Mechanisms and Geochemical Aspects, Recent. Res. Devel. in Phytochem., 1, 309-327 (1997).
  2. N. Itoh, M. Tujita, T. Ando, G. Hisatomi and T. Higashi, Formation and Emission of Monohalomethanes from Marine Algae, Phytochemistry, 45, 67-73 (1997).
  3. N. Itoh, R. Morihama, J.-C. Wang, K. Okada and N. Mizuguchi, Purification and Characterization of Phenylacetaldehyde Reductase from a Styrene-assimilating Corynebacterium Strain ST-10, Appl. Environ. Microbiol., 63, 3783-3788 (1997).
  4. N. Itoh, K. Hayashi, T. Ito, N. Mizuguchi and K. Okada, Characterization of Styrene Oxide Isomerase, a Key Enzyme of Styrene and Styrene Oxide Metabolism in Corynebacterium sp., Biosci. Biotech. Biochem., 61, 2058-2062 (1997).
  5. J. Miura and N. Itoh, Determination of Vanadium, Cobalt, Nickel and Iron in Bromoperoxidaes from Corallina pilulifera and Pseudomonas putida by High Performance Liquid Chromatography with Spectrophotometric Detection, J. Liq. Chromato., 20, 2367-2376 (1997).
  6. T. Dairi, Y. Hamano, Y. Igarashi, T. Furumai and T. Oki, Protoplasting and Regeneration of Strains Belonging to the Genus Actinomadura, Actinomycetol., 11, 1-5 (1997).
  7. J. Q. Liu, T. Dairi, M. Kataoka, S. Shimizu and H. Yamada, 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, J. Bacteriol., 179, 3555-3560 (1997).
  8. J. Q. Liu, S. Nagata, T. Dairi, H. Misono, S. Shimizu and H. Yamada, The GLY1 Gene of Saccharomyces cerevisiae Encodes a Low Specific L-Threonine Aldolase that Catalyzes Cleavage of L-Threonine and L-allo-Threonine to Glycine: Expression of the Gene in Escherichia coli and Purification and Characterization of the Enzyme, Eur. J. Biochem., 245, 289-293 (1997).
  9. T. Dairi, Y. Hamano, Y. Igarashi, T. Furumai and T. Oki, Cloning and Nucleotide Sequence of the Putative Polyketide Synthase Genes for Pradimicin Biosynthesis from Actinomadura hibisca, Biosci. Biotech. Biochem., 61, 1445-1453 (1997).

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[1998]

  1. J. Q. Liu, S. Ito, T. Dairi, N. Itoh, M. Kataoka, S. Shimizu, and H. Yamada, Gene Cloning, Nucleotide Sequencing, and Purification and Characterization of the Low-specificity L-Threonine Aldolase from Pseudomonas sp. Strain NCIMB10558. Appl. Environ. Microbiol., 64, 549-554 (1998).
  2. J. Q. Liu, S. Ito, T. Dairi, N. Itoh, S. Shimizu and H. Yamada, Low-specificity L-Threonine Aldolase of Pseudomonas sp. NCIMB 10558: Purification, Characterization and its Application to Beta-hydroxy-alpha-amino Acid Synthesis. Appl. Microbiol. Biotechnol., 49, 702-708 (1998).
  3. J. Q. Liu, T. Dairi, N. Itoh, M. Kataoka, S. Shimizu and H. Yamada, Gene Cloning, Biochemical Characterization and Physiological Role of a Thermostable Low-specificity L-Threonine Aldolase from Escherichia coli. Eur. J. Biochem., 254, 220-226 (1998).
  4. J. Q. Liu, T. Dairi, N. Itoh, M. Kataoka, S. Shimizu and H. Yamada, 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. J. Biol. Chem., 273, 16678-16685 (1998).
  5. S. Matsumoto, H. Yukitake, N. Ohara, T. Dairi, H. Kanbara and T. Yamada, Shotgun Cloning and Characterization of the Thymidylate Synthase-Encoding Gene from Mycobacterium bovis BCG, Microbiol. Immunol., 42, 15-21 (1998).
  6. K. L. Britton, H. F. Rogers, Y. Asano, T. Dairi, Y. Kato, T. J. Stillman and D. W. Rice, Crystallization of Arthrobacter sp. Strain 1C N-(1-D-Carboxyethyl)-L-norvaline Dehydrogenase and its Complex with NAD+, Acta Cryst., D54, 124-126 (1998).

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[1999]

  1. M. Wada, T. Matsumoto, S. Nakamori, M. Sakamoto, M. Kataoka, J.-Q. Liu, N. Itoh, H. Yamada and S. Shimizu, Purification and Characterization of a Novel Enzyme, L-Threo-3-hydroxyaspartate Dehydratase from Pseudomonas sp. T62, FEMS MIcrobiol. Lett., 179, 147-151 (1999).
  2. J. C. Wang, M. Sakakibara, J.Q. Liu, T. Dairi and N. Itoh, Cloning, Sequence Analysis, and Expression in Escerichia coli of the Gene Encoding Phenylacetaldehyde Reductase from Styrene-assimilating Corynebacterium sp. Strain ST-10. Appl. Microbiol. Biotechnol., 52, 386-392 (1999).
  3. J. Q. Liu, M. Odani, T. Dairi, N. Itoh, S. Shimizu and H. Yamada, 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. Appl. Microbiol. Biotechnol., 51, 586-591 (1999).
  4. N. Itoh, Y. Tujibata and J.-Q. Liu, Cloning and Overexpression in Escherichia coli of the Gene Encoding Dihydroxyacetone Kinase Isoenzyme I from Schizosaccharomyces pombe, and its Application to Dihysroxyacetone Phosphate Production, Appl. Microbiol. Biotechnol., 51, 193-200 (1999).
  5. N. Itoh, N. Mizuguchi and M. Mabuchi, Production of Chiral Alcohols by Enantioselective Reduction with NADH-dependendt Phenylacetaldehyde Reducatse from Corynebacterium strain, ST-10, J. Mol. Catal. :B, 6, 41-50 (1999).
  6. J.C.-Wang, M. Sakakibara, M. Matsuda and N. Itoh, Site-directed Mutagenesis of Two Zinc-binding Centers of the NADH-dependent Phenylacetaldehyde Reductase from Styrene-assimilating Corynebacterium sp. strain ST-10, Biosci. Biotechnol. Biochem., 63, 2216-2218 (1999).
  7. T. Dairi, Y. Hamano, T. Furumai and Toshikazu Oki, Development of a Self-cloning System for Actinomadura verrucosospora and Identification of Polyketide Synthase Genes Essential for Production of the Angucyclic Antibiotic Pradimicin, Appl. Environ. Microbiol., 65, 2703-2709 (1999).

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[2000]

  1. T. Dairi, Y. Motohira, T. Kuzuyama, S. Takahashi, N. Itoh and H. Seto, Cloning of the Gene Encoding 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase from Terpenoid Antibiotics-Producing Stereptomyces Strains, Mol. Gen. Genet., 262, 957-964 (2000).
  2. J. Q. Liu, M. Odani, T. Yasuoka, T. Dairi, N. Itoh, M. Kataoka, S. Shimizu and H. Yamada, 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, Appl. Microbiol. Biotechnol., 54, 44-51 (2000).
  3. N. Itoh, C. Yachi and T. Kudome, Determining a Novel NAD+-dependent Amine Dehydrogenase with a Broad Substrate Range from Streptomyces virginiae IFO 12827: Purification and Characterization, J. Mol. Cat. :B, 10, 281-290 (2000).
  4. T. Kuzuyama, M. Takagi, K. Kaneda, T. Dairi and H. Seto, Formation of 4-(Cytidine 5'-iphospho)-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, Tetrahedron Lett., 41, 703-706 (2000).
  5. T. Kuzuyama, M. Takagi, K. Kaneda, H. Watanabe, T. Dairi and H. Seto, 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, Tetrahedron Lett., 41, 2925-2928 (2000).
  6. M. Takagi, T. Kuzuyama, K. Kaneda, H. Watanabe, T. Dairi and H. Seto, 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, Tetrahedron Lett., 41, 3395-3398 (2000).

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[2001]

  1. Y. Hamano, T. Dairi, M. Yamamoto, T., Kawasaki, K., Kaneda, T. Kuzuyama, N. Itoh and H. Seto, Cloning of a Gene Cluster Encoding Enzymes Responsible for the Mevalonate Pathway from a Terpenoid-antibiotic-producing Streptomyces strain, Biosci. Biotech. Biochem., 65, 1627-1635 (2001).
  2. N. Itoh, T. Kawanami, J. Q. Liu, T. Dairi, M. Miyakoshi, C. Nitta and Y. Kimoto, Cloning and Biochemical Characterization of Co2+-Activated Bromoperoxidase-esterase (Perhydrolase) from Pseudomonas putida, Biochimica. Biophysica. Acta, 1545, 53-66 (2001).
  3. N. Ohsawa, Y. Ogata, N. Okada and N. Itoh, Physiological Function of Bromoperoxidase in the Red Marine Alga, Corallina pilulifera: Production of Bromoform as an Allelochemical and the Simultaneous Elimination of Hydrogen Peroxidase, Phytochemistry, 58, 683-692 (2001).
  4. N. Ohsawa, M. Tsujita, S. Morikawa and N. Itoh, Purification and Characterization of a Monohalomethane-producing Enzyme S-Adenosyl-L-methionine: Halide Ion Methyltransferase from a Marine Microalga, Pavlova pinguis, Biosci. Biotechnol. Biochem., 65, 2397-2404 (2001).
  5. T. Dairi, Y. Hamano, T. Kuzuyama, N. Itoh, K. Furihata and H. Seto, Eubacterial Diterpene Cyclase Genes Essential for Production of Isoprenoid Antibiotic Terpentecin, J. Bacteriol., 183, No. 20, 6085-6094 (2001).

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[2002]

  1. T. Kawanami, M. Miyakoshi, T. Dairi and N. Itoh, Reaction Mechanism of the Co2+-Activated Multifunctional Bromoperoxidase-Esterase from Pseudomonas putida IF-3, Arch. Biochem. Biophys., 398, 94-100 (2002).
  2. N. Itoh, M. Matuda, M. Mabuchi, T. Dairi and J. Wang, Chiral Alcohol Production by NADH-dependent Phenylacetaldehyde Reductase Coupled with in situ Regeneration of NADH, Eur. J. Biochem., 269, 2394-2402 (2002).
  3. Y. Hamano, T. Dairi, M. Yamamoto, T. Kuzuyama, N. Itoh, H. Seto, Growth-phase dependent expression of the mevalonate pathway in a terpenoid antibiotic-producing Streptomyces strain, Biosci. Biotechnol. Biochem., 66, 808-819 (2002).
  4. Y. Hamano, T. Kuzuyama, N. Itoh, K. Furihata, H. Seto, and T. Dairi, Functional Analysis of Eubacterial Diterpene Cyclases Responsible for Biosynthesis of a Diterpene Antibiotic Terpentecin, J. Biol. Chem., 277, 37098-37104 (2002).
  5. 5. T. Kuzuyama, S. Takahashi, T. Dairi, and H. Seto, Detection of the mevalonate pathway in Streptomyces species using the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene, J. Antibiot., 55, 919-923 (2002).

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[2003]

  1. T. Kawasaki, Y. Hamano, T. Kuzuyama, N. Itoh, H. Seto, and T. Dairi, Interconversion of the Product Specificity of Type I Eubacterial Farnesyl Diphosphate Synthase and Geranylgeranyl Diphosphate Synthase through One Amino Acid Substitution, J. Biochem., 133, 83-91 (2003).
  2. T. Eguchi, Y. Dekishima, Y. Hamano, T. Dairi, H. Seto, and K. Kakinuma, Application of New Biosynthetic Approach Featuring Pathway Switching, Deuterium Hyperlabeling and 1H NMR Spectroscopy. The Reaction Mechanism of Novel Streptomyces Diterpene Cyclase., in press, J. Org. Chem. (2003).
  3. N. Itoh, T. Kawanami, C. Nitta, N. Iwata, S. Usami, Y. Abe, and Y. Koide, Characterization of pNI10 Plasmid in Pseudomonas and The Construction of an Improved Escherichia and Pseudomonas Shuttle Vector, pNUK73, Appl. Microbiol. Biotechnol., 61, 240-246 (2003).
  4. J. Q. Liu, T. Dairi, N. Itoh, M. Kataoka, and S. Shimizu, A Novel Enzyme, D-3-hydroxyaspartate Aldolase from Paracoccus dentrificans IFO 13301: Purification, Caracterization, and Gene Cloning. Appl. Microbiol. Biotechnol., 62, 53-60 (2003).

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[2004]

  1. N. Itoh, H. Asako, K. Banno, Y. Makino, R. Wakita, M. Shimizu, Purification and characterization of NADPH-dependent aldo-keto reductase specific for β-keto esters from Penicillium citrinum, Appl. Microbiol. Biotechnol., 66, 53-62 (2004).
  2. T. Kawasaki, T. Kuzuyama, Y. Kuwamori, N. Matsuura, N. Itoh, K. Furihata, H. Seto, T. Dairi, Presence of copslyl diphosphate synthase gene in an antinomycete possessing the MV pathways, J. Antibiotics, 57, 739-747 (2004).
  3. T. Kuzuyama, T. Dairi, H. Yamashita, Y. Shoji, and H. Seto, Heterologous mevalonate production in Streptomyces lividans TK23, Biosci. Biotech. Biochem., 68, 931-934 (2004).
  4. T. Nakano, K. Miyake, H. Endo, T. Dairi, T. Mizukami, and R. Katsumata, Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens. Biosci. Biotech. Biochem., 68, 1345-1352 (2004).

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[2005]

  1. H. Asako, R. Wakita, K. Matsumura, M. Shimizu, J. Sakai, N. Itoh, Purification and cDNA cloning of NADPH-dependent aldoketoreductase, involved in asymmetric reduction of methyl 4-bromo-3-oxobutyrate, from Penicillium citrinum IFO463, Appl. Environ. Microbiol., 71, 1101-1104 (2005).
  2. K. Inoue, Y. Makino, N. Itoh, Production of (R)-chiral alcohols by a hydrogen-transfer bioreduction with NADH-dependent Leifsonia alcohol dehydrogenase (LSADH), Tetrahedron: Asymmetry, 16, 2539-2549 (2005).
  3. K. Inoue, Y. Makino, N. Itoh, Purification and characterization of a novel alcohol dehydrogenase from Leifsonia sp. strain S749: a promising biocatalyst for an asymmetric hydrogen transfer bioreduction, Appl. Environ. Microbiol., 71, 3633-3641 (2005).
  4. Y. Makino, K. Inoue, T. Dairi, N. Itoh, Engineering of phenylacetaldehyde reductase for efficient substrate conversion in concentrated 2-propanol, Appl. Environ. Microbiol., 71, 4713-4720 (2005).
  5. C. Nakano, T. Okamura, T. Sato, T. Dairi, and T. Hoshino, Mycobacterium tuberculosis H37Rv 3377c encodes the diterpene cyclase for producing halimane skeleton. Chem. Commun., Issue 8: 1016-1018 (2005).
  6. K. J. Puan, H. Wang, T. Dairi, T. Kuzuyama, and C. T. Morita, fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis. FEBS Lett. 579, 3802-3806, (200).
  7. T. Dairi, Studies on biosynthetic genes and enzymes of isoprenoids produced by actinomycetes, J. Antibiot. 58, 227-243 (2005).

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[2006]

  1. K. Inoue, Y. Makino, T. Dairi, N. Itoh, Gene cloning and expression of Leifsonia alcohol dehydrogenased (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols. Biosci. Biotechnol. Biochem., 70, 418-426 (2006).
  2. T. Kawasaki, Y. Hayashi, T. Kuzuyama, K. Furihata, N. Itoh, H. Seto, and T. Dairi, Biosynthesis of a natural polyketide-isoprenoid hybrid compound furaquinocin A: Identification and heterologous expression of the gene cluster. J. Bacteriol., 188, 1236-1244 (2006).
  3. Z.Kamenarska, M.Hiraoka, N.Itoh, Preliminary study on the bromoperoxidase activity of some macroalgae from Japan, Compt. Rend. Acad. Bulg. Sci., 59, 71-74(2006).

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[2007]

  1. N. Itoh, M. Nakamura, K. Inoue, Y. Makino, Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction, Appl. Microbiol. Biotechnol., 75, 1249-1256 (2007).
  2. Y. Makino, T. Dairi, N. Itoh, Engineering the phenylacetaldehyde reductase mutant for improved substrate conversion in the presence of concentrated 2-propanol, Appl. Microbiol. Biotechnol., 77, 833-843 (2007).
  3. Z. Kamenarska, T. Taniguchi, N. Ohsawa, M. Hiraoka, N. Itoh, A vanadium-dependent bromoperoxidase in the marine red alga Kappaphycus alvarezii (Doty) Doty displays clear substrate specificity, Phytochemistry, 68, 1358-1366 (2007).
  4. N. Itoh, Y. Katsube, K. Yamamoto, N. Nakajima, K. Yoshida, Laccase-catalyzed conversion of green tea catechins in the presence of gallic acid to epitheaflagallin and epitheaflagallin 3-O-gallate, Tetrahedron, 63, 9488-9492 (2007).
  5. Y. Hayashi, H. Onaka, N. Itoh, H. Seto, T. Dairi, Cloning of the gene cluster responsible for biosynthesis of KS-505a (longestin), a unique tetraterpenoid, Biosci. Biotech. Biochem., 71, No.12 (2007).
  6. T. Toyomasu, M. Tsukahara, A. Kaneko, R. Niida, W. Mitsuhashi, T. Dairi, N. Kato,T. Sassa, Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi, Proc. Natl. Acad. Sci. U S A, 104, 3084-3088 (2007).
  7. K. Motohashi, R. Ueno, M. Sue, K. Furihata, T. Matsumoto, T. Dairi, S. Omura, H. Seto, Studies on terpenoids produced by actinomycetes: oxaloterpins A, B, C, D, and E, diterpenes from Streptomyces sp. KO-3988, J. Nat. Prod. 70, 712-1717 (2007).

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[2008]

  1. H. Asako, M. Shimizu, N. Itoh, Engineering of NADPH-dependent aldo-keto reductase from Penicillium citrinum by directed evolution to improve thermostability and enantioselectivity, Appl. Microbiol. Biotechnol., 80, 803-812, (2008).
  2. T. Hiratsuka, K. Furihata, J. Ishikawa, H. Yamashita, N. Itoh, T. Dairi, An alternativemenaquinone biosynthetic pathway operating in microorganisms, Science, 19, 1670-1673 (2008).
  3. Y. Makino, N. Itoh, A knowledge-based structure-discriminating function that requires only main-chain atom coorcinates, BMC Structural Biology, 8, 46 (2008).
  4. Y. Hayashi, N. Matsuura, H. Toshima, N. Itoh, J. Ishikawa, Y. Mikami, T. Dairi, Cloning of the gene cluster responsible for the biosynthesis of brasilicardin A, a unique diterpenoide. J. Antibiot., 61, 164-174 (2008).
  5. H. Seto, Y. Jinnai, T. Hiratsuka, M. Fukawa, K. Furihata, N. Itoh, T. Dairi, Studies on a new biosynthetic pathway for menaquinone, J. Am. Chem. Soc., 130, 5614-5615, (2008).

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[2009]

  1. N. Itoh, H. Toda, M. Matsuda, T. Negishi, T. Taniguchi, N. Ohsawa, Involvement of S-adenosylmethionine-dependent halide/thiol methyltransferase (HTMT) in methyl halide emissions from agricultural plants: isolation and characterization of an HTMT-coding gene from Raphanus sativus (daikon radish), BMC Plant Biology, 9:116, (2009).
  2. H. Asako, M. Shimizu, N. Itoh, Biocatalytic production of (S)-4-bromo-3-hydroxybutyrate and structurally related chemicals and their application, Appl. Microbiol. Biotechnol., 84, 397-405, 2009.
  3. N. Itoh, Y. Katsube, Biocatalytic conversion of green tea catechins to epitheaflagallin, epitheaflagallin, 3-O-gallate, and theaflavins: production of promising functional foods, In Handbook of Green Tea and Health Research (H. McKinley and M. Jamieson, eds.), pp. 419-427, Nova Science Publishers, Inc., (2009).
  4. T. Hiratsuka, N. Itoh, H. Seto, T. Dairi, Enzymatic properties of futalosine hydrolase, an enzyme essential to a new identified menaquinone biosynthetic pathway, Biosci. Biotechnol. Biochem., 73, 1137-1141 (2009).
  5. A. Minami, N. Tajima, Y. Higuchi, T. Toyomasu, T. Sassa, N. Kato, T. Dairi, Identification and Functional Analysis of Brassicicene C Biosynthetic Gene Cluster in Alternaria brassicicola., Bioorg. Med. Chem. Lett., 19, 870-874 (2009).
  6. C. Nakano, T. Hoshino, T. Sato, T. Toyomasu, T. Dairi, T. Sassa, 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, Tetrahedron Lett., 51, 125-128 (2009).
  7. T. Dairi, An alternative menaquinone biosynthetic pathway operating in microorganisms: an attractive target for drug discovery to pathogenic Helicobacter and Chlamydia strains, J. Antibiot., 62, 347-352 (2009).

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[2010]

  1. J. Kurokawa, M. Asano, S. Nomoto, Y. Makino, N. Itoh, Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum: A useful enzymatic regeneration system of NAD+ from NADH, J. Biosci. Bioeng., 109,218-223, (2010).
  2. H. Asako, M. Shimizu, Y. Makino, N. Itoh, Biocatalytic reduction system for the production of chiral methyl (R)/(S)-4-bromo-3-hydroxybutyrate, Tetrahedron Lett., 51, 2664-2666 (2010).

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[2011]

  1. H. Toda, N. Ityoh, Isolation and characterization of a gene encoding a S-adenosyl-L-methionine-dependent halide/thiol methyltransferase (HTMT) from the marine diatom Phaeodactylum trichornutum: Biogenic mechanism of CH3I emission in oceans, Phytochemistry, 72, 337-343 (2011).
  2. C. Arakawa, M. Kuratsu, K. Furihata, T. Hiratsuka, N. Itoh, H. Seto, T. Dairi, Diversity of the early step of the futalosine pathway, Antimicrob. Agents Chemother., 55, 913-916 (2011).
  3. R. Tanaka, T. Kunisada, N. Kushida, K. Yamada, S. Ikeda, M. Noike, Y. Ono, N. Itoh, H. Takami, H. Seto, T. Dairi, Branched fatty acids inhibit the biosynthesis of menaquinone in Helicobacter pylori, J. Antibiotics, 64, 151-153 (2011).

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[2012]

  1. N. Itoh, K. Isotani, M. Nakamura, K. Inoue, Y. Isogai, Y. Makino, Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol-water medium, Appl. Microbiol. Biotechnol., 93, 1075-1085, (2012).
  2. K. Isotani, J. Kurokawa, N. Itoh, Production of (R)-3-quinuclidiol by E. coli biocatalysts possessing NADH-dependent 3-quinuclidinone reductase (QNR or bacC) from Microbacterium luteolum and Leifsonia alcohol dehydrogenase (LSADH), Int. J. Mol. Sci., 13, 13542-13553 (2012).
  3. H. Toda, N. Itoh, Isolation and characterization of styrene metabolic genes from styrene-assimilating soil bacteria Rhodococcus sp. ST-5 and ST-10, J. Biosci. Bioeng., 113, 12-19 (2012).
  4. H. Toda, R. Imae, T. Komio, N. Itoh, Expression and characterization of styrene monooxygenases of Rhodococcus sp. ST-5 and ST-10 for synthesizing enantiopure (S)-epoxides, Appl. Microbiol. Biotechnol., 96, 407-418 (2012)
  5. H. Toda, R. Imae, N. Itoh, Efficient biocatalysis for the production of enantiopure (S)-epoxides using styrene monooxygenase (SMO) and Leifsonia alcohol dehydrogenase (LSADH) system, Tetrahedron: Asymmetry, 23, 1542-1549 (2013)
  6. S. Yamamoto, W. Gunji, H. Suzuki, H. Toda, M. Suda, T. Jojima, M. Inui, H. Yukawa, Overexpression of glycolytic genes enhances Corynebacterium glutamicum glucose metabolism and alanine production under oxygen-deprived conditions. Appl. Environ. Microbiol., 78, 4447-4457 (2012).

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[2013]

  1. K. Isotani, J. Kurokawa, F. Suzuki, S. Nomoto, T. Negishi, M. Matsuda, N. Itoh, Gene cloning and characterization of two NADH-dependent 3-quinuclidinone reductases from Microbacterium luteolum JCM 9174, Appl. Environ. Microbiol., 79, 1378-1384 (2013).
  2. Y. Makino, N. Itoh, Development of an improved phenylacetaldehyde reductase mutant by an efficient selection procedure, Appl. Microbiol. Biotechnol., 79, 1378-1384 (2013).
  3. N. Itoh, Y. Makino, Protein engineering: development of novel enzymes for the improved reduction of C=O double bonds, In: E. Brenna (ed), Synthetic methods for biologically active molecules, pp. 139-185, Wiley-VCH, Germany (2013).

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[2014]

  1. N. Itoh, S. Kariya, J. Kurokawa, Efficient PCR-based amplification of diverse alcohol dehydrogenase genes from metagenomes for improving biocatalysis: Screening of gene-specific amplicons from metagenomes, Appl. Environ. Microbiol., 80, 6280-6289 (2014).
  2. N. Itoh, Use of anti-Prelog stereospecific alcohol dehydrogenase from Leifsonia and Pseudomonas for producing chiral alcohols, Appl. Microbiol. Biotechnol., 98, 3889-3904 (2014).
  3. N. Itoh, K. Isotani, Y. Makino, M. Kato, K. Kitayama, T. Ishimota, PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenomes for biocatalysis, Enzyme Microb. Technol., 55, 140-150 (2014).
  4. Y. Yokouchi, A. Ooki, S. Hahimoto, N. Itoh, A study on the production and emission of marin-derived volatile halocarbons, In: M. Uematsu et al. (ed), Western Pacific Air-Sea Interaction Study, pp. 1-25, TERRAPUB (2014).
  5. H. Toda, R. Imae, N. Itoh, Bioproduction of chiral epoxyalkanes using styrene monooxygensae from Rhodococcus sp. strain ST-10 (RhSMO), Adv. Synth. Catal., 356, 3443-3450 (2014).

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[2015]

  1. H. Toda, T. Ohuchi, R. Imae, N. Itoh, Microbial production of aliphatic (S)-epoxyalkanes by using Rhodococcus sp. strain ST-10 styrene monooxygenase expressed in organic-solvent-tolerant Kocuria rhizophila DC2201, Appl. Environ. Microbiol., 81:1919-1925 (2015).
  2. C. E. Paul, D. Tischler, A. Riedel, T. Heine, N. Itoh, F. Hollmann, Nonenzymatic regeneration of styrene monooxygenase for catalysis, ACS Catal., 5:2961-2965 (2015).

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Ph.D Theses

  • (N. Ohsawa)
    "Studies on the halogenating enzymes from marine algae and their physiological function" (2001)
  • (Y. Hamano)
    "Studies on the biosynthesis of terpenoids produced by streptomyces" (2002)
  • (T. Kawanami)
    "Studies on the multifunctional bromoperxidase from Pseudomonas putida IF-3 and its application" (2003)
  • (T. Kawasaki)
    "Studies on the biosynthetic genes and enzymes responsible for the formation of basic skeletons of isoprenoids produced by actinomycetes" (2005)
  • (K. Inoue)
    "Studies on the production of chiral alcohols using alcohol dehydrogenase (LSADH) from Leifsonia sp. S749" (2006)
  • (Y. Hayashi)
    "Studies on the key enzymes leading to the diversity of isoprenoids found in actinomycetes" (2007)
  • (T. Hiratsuka)
    "An alternative menaquinone biosynthetic pathway operating in microorganisms" (2009)
  • (H. Asako)
    "Studies on the production of chiral methyl (S)-4-bromo-3-hydroxybutyrate using a novel NADPH-dependent beta-keto ester reductase from Penicillium citrinum"(2011)
  • (K. Isotani)
    "Studies on the efficient biocatalysis for producing optically pure alcohols including (R)-(-)-quinuclidinol"(2013)

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