2016 October CV

iii) Arginine/NO: Characterization of yeast NO synthase, downstream pathway and physiological roles of NO iv) Ubiquitin system: Degradation and repair...

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CURRICULUM VITAE October 10, 2016 Name: Hiroshi Takagi (高木 博史) Date of birth: July 15, 1957 Place of birth: Osaka, Japan Civic status: Married male Nationality: Japanese Present address: Office: Graduate School of Biological Sciences Nara Institute of Science and Technology 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan Phone: Office: +81-743-72-5420 Fax: Office: +81-743-72-5429 E-mail: [email protected] 1976-1980: Department of Agricultural Chemistry, Shizuoka University Awarded the degree of B.S. in Biochemistry 1980-1982: Research Institute for Biochemical Regulation, Nagoya University Awarded the degree of M.S. in Cellular Biology and Immunology for a thesis entitled “Studies on the lymphocyte circulation under the physiological conditions in rats.” under Prof. Etsuro Sugimoto 1988: Department of Agricultural Chemistry, The University of Tokyo Awarded the degree of Ph.D. in Molecular Biology and Protein Engineering for a thesis entitled “Studies on the structure and function of subtilisin E, a protease of Bacillus subtilis, by protein engineering.” under Prof. Takahisha Ohta 1982-1995: Research Scientist at Ajinomoto Co., Inc. 1986: Visiting Research Associate at Department of Biochemistry, State University of New York at Stony Brook, working under Prof. Masayori Inouye 1990-1992: Project leader (protein engineering of industrial enzymes) at Basic Research Laboratories, Ajinomoto Co., Inc. 1992-1995: Project leader (screening of new microbial enzymes and their application in food processing) at Food Research & Development Laboratories, Ajinomoto Co., Inc.

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1994-1995: Chief Biochemist at Food Research & Development Laboratories, Ajinomoto Co., Inc. 1995-2001: Associate Professor at Department of Bioscience, Fukui Prefectural University 2001-2006: Professor at Department of Bioscience, Fukui Prefectural University 2006-present: Professor at Graduate School of Biological Sciences, Nara Institute of Science and Technology Japan Society for Bioscience, Biotechnology and Agrochemistry The Japanese Biochemical Society The Molecular Biology Society of Japan The Society for Bioscience and Bioengineering Protein Science Society of Japan Yeast Genetics Society of Japan Nitric Oxide Society of Japan American Society for Microbiology American Association for the Advancement of Science Society for American Baseball Research 1. The Award of Japan Society for Bioscience, Biotechnology, and Agrochemistry for young scientists (1997) 2. The Award of Hokuriku Branch, Japanese Biochemical Society for young scientists (2001) My research area is involved in “Applied Molecular Microbiology”. The aims of my laboratory are basic studies in microbial “Bioscience” and practical applications in new “Biotechnology”. To understand in depth microbial cell functions, we search, analyze and improve various functions and mechanisms of microorganisms including yeasts and bacteria from molecular, metabolic and cellular aspects. As the best scenario, novel findings and results of our basic studies can be applied to the molecular breeding for useful microorganisms, the production of valuable biomaterials (enzymes, amino acids) and the development of promising technologies to solve environmental issues.

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1. Molecular mechanisms of stress-tolerance in yeast cells and its application for molecular breeding Yeast cells are exposed to environmental stresses during fermentations. Stress induces protein denaturation, generates abnormal proteins, and leads to growth inhibition or cell death. Therefore, stress tolerance is the key for application to industrial yeasts. We analyze various stress-tolerant mechanisms that can be applied to molecular breeding of industrial yeasts. i) Proline: Metabolic regulation, transport mechanism, physiological functions ii) N-Acetyltransferase Mpr1: Antioxidative mechanism, structural and functional analysis iii) Arginine/NO: Characterization of yeast NO synthase, downstream pathway and physiological roles of NO iv) Ubiquitin system: Degradation and repair mechanisms of stress-induced abnormal proteins, regulation of the uniqutin ligase Rsp5 activity v) Development of industrial yeast based on novel stress-tolerant mechanisms 2. Physiological roles and metabolic regulation of cysteine in Escherichia coli and their applications to cysteine fermentation Cysteine is an important amino acid both biologically and commercially. For achieving cysteine fermentation, we analyze the regulatory mechanisms regarding its biosynthesis, degradation and export, and try to construct cysteine-overproducing strains based on genomic information and metabolic engineering. 3. Folding mechanism and functional alteration of microbial proteases by new protein engineering Conventional protein engineering techniques have so far employed the introduction of mutagenesis in the protease domain to modify the enzymatic properties. A new approach, which we termed ‘pro-sequence engineering’, would be not only an important in investigations of protein folding, but also a promising technology for creating unique proteases with various beneficial properties.

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* corresponding author Original papers: 1. Folding mechanism and functional alteration of Bacillus subtilis protease, subtilisin E, by protein engineering 1) H. Ikemura, H. Takagi and M. Inouye*: Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli. J. Biol. Chem., 262, 7859-7864 (1987). 2) H. Takagi, Y. Morinaga, M. Tsuchiya, H. Ikemura and M. Inouye*: Control of folding of proteins secreted by a high expression secretion vector, pIN-III-ompA: 16-fold increase in production of active subtilisin E in Escherichia coli. Bio/Technology, 6, 948-950 (1988). 3) H. Takagi, Y. Morinaga, H. Ikemura and M. Inouye*: Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis. J. Biol. Chem., 263, 19592-19596 (1988). 4) H. Takagi*, Y. Morinaga, H. Ikemura, and M. Inouye: The role of Pro-239 in the catalysis and heat stability of subtilisin E. J. Biochem., 105, 953-956 (1989). 5) H. Takagi*, T. Takahashi, H. Momose, M. Inouye, Y. Maeda, H. Matsuzawa and T. Ohta: Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease. J. Biol. Chem., 265, 6874-6878 (1990). 6) H. Takagi*, S. Arafuka, M. Inouye and M. Yamasaki: The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis. J. Biochem., 111, 584-588 (1992). 7) H. Takagi*, T. Maeda, I. Ohtsu, Y.-C. Tsai, and S. Nakamori: Restriction of substrate specificity of subtilisin E by introduction of a side chain into a conserved glycine residue. FEBS Lett., 395, 127-132 (1996). 8) H. Takagi*, I. Ohtsu, and S. Nakamori: Construction of novel subtilisin E with high specificity, activity and productivity through multiple amino acid substitutions. Protein Engng., 10, 985-989 (1997). 9) H.-C. Mei, Y.-C. Liaw, Y.-C. Li, D.-C. Wang, H. Takagi, and Y.-C. Tsai*: Engineering subtilisin YaB: restriction of substrate specificity by the substitution of Gly124 and Gly151 with Ala. Protein Engng., 11, 109-117 (1998). 10) H. Takagi*, M. Yamamoto, I. Ohtsu, and S. Nakamori: Random mutagenesis into conserved Gly154 of subtilisin E: isolation and characterization of the revertant enzymes. Protein Engng., 11,1205-1210 (1998). 11) H. Takagi*, A. Suzumura, T. Hoshino and S. Nakamori: Gene expression of Bacillus subtilis subtilisin E in Thermus thermophilus. J. Ind. Microbiol. Biotechnol., 23, 214-

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217 (1999). 12) H. Takagi*, A. Suzumura, Y. Hasuura, T. Hoshino and S. Nakamori: Efficient selection for thermostable protease in Thermus thermophilus. Biosci. Biotech. Biochem., 64, 899-902 (2000). 13) H. Takagi*, K. Hirai, Y. Maeda, H. Matsuzawa and S. Nakamori: Engineering subtilisin E for enhanced stability and activity in polar organic solvents. J. Biochem., 127, 617-625 (2000). 14) H. Takagi*, K. Hirai, M. Wada and S. Nakamori: Enhanced thermostability of the single-Cys mutant subtilisin E under oxidizing conditions. J.Biochem., 128, 585-589 (2000). 15) M. Takahashi*, Y. Hasuura, S. Nakamori and H. Takagi: Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the proregion. J. Biochem., 130, 99-106 (2001). 16) H. Takagi*, M. Koga, S. Katsurada, Y. Yabuta, U. Shinde, M. Inouye and S. Nakamori: Functional analysis of the propeptides of subtilisin E and aqualysin I as intramolecular chaperones. FEBS Lett., 508, 210-214 (2001). 17) Y. Yabuta, H. Takagi, M. Inouye and U. Shinde*: Folding pathway mediated by an intramolecular chaperone. Propeptide-release modulates precise activation of a protease. J. Biol. Chem., 276, 44427-44434 (2001). 18) Y. Yabuta, E. Subbian, H. Takagi*, U. Shinde and M. Inouye: Folding pathway mediated by an intramolecular chaperone: dissecting conformational changes coincident with autoprocessing and the role of Ca2+ precursor maturation. J. Biochem., 131, 31-37 (2002). 19) H.-C. Mei, Y.-F. Li, C.-C. Hsu, Y.-C., Tsai* and H. Takagi*: Conversion of the cleavage specificity of subtilisin YaB on oxidized insulin chains to an elastase-like specificity by replacement of Gly124 with Ala. Biosci. Biotech. Biochem., 67, 16011604 (2003). 20) M. Takahashi, T. Sekine, N. Kuba, S. Nakamori, M. Yasuda and H. Takagi*: The production of recombinant APRP, an alkaline protease derived from Bacillus pumilus TYO-67, by in vitro-refolding of pro-enzyme fixed on a solid surface. J. Biochem., 136, 549-556 (2004). 2. Molecular mechanism of stress-tolerance in yeast cells and its application for molecular breeding 21) H. Takagi*, F. Iwamoto and S. Nakamori: Isolation of freeze-tolerant laboratory strain of Saccharomyces cerevisiae from proline-analogue-resistant mutants. Appl. Microbiol. Biotechnol., 47, 405-411 (1997).

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22) H. Takagi*, K. Sakai, K. Morida and S. Nakamori: Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae. FEMS Microbiol. Lett., 184, 103108 (2000). 23) H. Takagi*, M. Shichiri, M. Takemura, M. Mohri and S. Nakamori: Saccharomyces cerevisiae Σ1278b has novel genes of the N-acetyltransferase gene superfamily required for L-proline analogue resistance. J. Bacteriol., 182, 4249-4256 (2000). 24) Y. Kubo*, H. Takagi and S. Nakamori: Effect of gene disruption of succinate dehydrogenase on succinate production in a sake yeast strain. J. Biosci. Bioeng., 90, 619-624 (2000). 25) M. Shichiri, C. Hoshikawa, S. Nakamori and H. Takagi*: A novel acetyltranferase found in Saccharomyces cerevisiae Σ1278b that detoxifies a proline analogue, azetidine-2-carboxylic acid. J. Biol. Chem., 276, 41998-42002 (2001). 26) Y. Morita, S. Nakamori and H. Takagi*: Effect of proline and arginine metabolism on freezing stress of Saccharomyces cerevisiae. J. Biosci. Bioeng., 94, 390-394 (2002). 27) Y. Kimura, S. Nakamori and H. Takagi*: Polymorphism of the MPR1 gene required for toxic proline analogue resistance in the Saccharomyces cerevisiae complex species. Yeast, 19, 1437-1445 (2002). 28) Y. Morita, S. Nakamori and H. Takagi*: L-Proline accumulation and freeze tolerance in Saccharomyces cerevisiae are caused by a mutation in the PRO1 gene encoding γ-glutamyl kinase. Appl. Environ. Microbiol., 69, 212-219 (2003). 29) M. Nomura, S. Nakamori and H. Takagi*: Characterization of novel acetyltransferases found in the budding and fission yeasts that detoxify a proline analogue, azetidine-2-carboxylic acid. J. Biochem., 133, 67-74 (2003). 30) H. Takagi*, K. Yoshioka , N. Awano, S. Nakamori and B. Ono: Role of Saccharomyces cerevisiae serine O-acetyltransferase in cysteine biosynthesis. FEMS Microbiol. Lett., 218, 291-297 (2003). 31) M. Wada*, S. Nakamori and H. Takagi: Serine racemase homologue of Saccharomyces cerevisiae has L-threo-3-hydroxyaspartate dehydratase activity. FEMS Microbiol. Lett., 225, 189-193 (2003). 32) C. Hoshikawa, M. Shichiri, S. Nakamori and H. Takagi*: A non-conserved Ala401 in the yeast Rsp5 ubiquitin ligase is involved in degradation of Gap1 permease and stress-induced abnormal proteins. Proc. Natl. Acad. Sci. U.S.A., 100, 11505-11510 (2003). 33) Y. Terao, S. Nakamori and H. Takagi*: Gene dosage effect of L-proline biosynthetic enzymes on L-proline accumulation and freeze tolerance in Saccharomyces cerevisiae.

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Appl. Environ. Microbiol., 69, 6527-6532 (2003). 34) X.-H. Zhang, H. Takagi and J. M. Widholm*: Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination, callus and shoot formation. Plant Cell Rep., 22, 615-622 (2004). 35) T. Iwaki, N. Tanaka, H. Takagi, Y. Giga-Hama and K. Takegawa*: Characterization of end4+, a gene required for endocytosis in Schizosaccharomyces pombe. Yeast, 21, 867-881 (2004). 36) M. Nomura and H. Takagi*: Role of the yeast novel acetyltransferase Mpr1 in oxidative stress: Regulation of oxygen reactive species caused by a toxic proline catabolism intermediate. Proc. Natl. Acad. Sci. U.S.A., 101, 12616-12621 (2004). 37) X. Du and H. Takagi*: N-Acetyltransferase Mpr1 confers freeze tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species. J. Biochem., 138, 391-397 (2005). 38) K. Matsuura and H. Takagi*: Vacuolar functions are involved in stress-protective effect of intracellular proline on Saccharomyces cerevisiae. J. Biosci. Bioeng., 100, 538-544 (2005). 39) H. Takagi*, M. Takaoka, A. Kawaguchi and Y. Kubo: Effect of L-proline on sake brewing and ethanol stress in Saccharomyces cerevisiae. Appl. Environ. Microbiol., 71, 8656-8662 (2005). 40) A. Ando, F. Tanaka, Y. Murata, H. Takagi and J. Shima*: Identification and classification of genes required for tolerance to high sucrose stress revealed by genome-wide screening of Saccharomyces cerevisiae. FEMS Yeast Research, in press. 41) M. Sugiura and H. Takagi*: Yeast cell death caused by mutation of the OST2 gene encoding the ε-subunit of the Saccharomyces cerevisiae oligosaccharyltransferase. Biosci. Biotech. Biochem., 70, 1234-1241 (2006). 42) Y. Haitani, H. Shimoi and H. Takagi*: Rsp5 regulates expression of stress proteins via post-translational modification of Hsf1 and Msn4 in Saccharomyces cerevisiae. FEBS Lett., 580, 3433-3438 (2006). 43) F. Tanaka, A. Ando, T. Nakamura, H. Takagi and J. Shima*: Functional genomic analysis of commercial baker’s yeast during initial stages of model doughfermentation. Food Microbiol., 23, 717-728 (2006). 44) H. Hiraishi, M. Mochizuki and H. Takagi*: Enhancement of stress tolerance in Saccharomyces cerevisiae by overexpression of ubiquitin ligase Rsp5 and ubiquitinconjugating enzymes. Biosci. Biotech. Biochem., 70, 2762-2765 (2006). 45) H. Wu, X. Zheng, Y. Araki, H. Sahara, H. Takagi and H. Shimoi*: Global gene expression analysis of yeast cells during sake brewing. Appl. Environ. Microbiol., 72,

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7353-7358 (2006). 46) A. Ando, T. Nakamura, Y. Murata, H. Takagi and J. Shima*: Identification and classification of genes required for tolerance to freeze-thaw stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains. FEMS Yeast Res., 7, 244-253 (2007). 47) T. Nakamura, A. Ando, H. Takagi and J. Shima*: Eos1, whose deletion confers sensitivity to oxidative stress, is involved in N-glycosylation in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 353, 293-298 (2007). 48) H. Takagi*, F. Matsui, A. Kawaguchi, H. Wu, H. Simoi and Y. Kubo: Construction and analysis of self-cloning sake yeasts that accumulate proline. J. Biosci. Bioeng., 103, 377-380 (2007). 49) X. Du and H. Takagi*: N-Acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species. Appl. Microbiol. Biotech., in press. 50) T. Sekine, A. Kawaguchi, Y. Hamano and H. Takagi*: Desensitization of feedback inhibition of the Saccharomyces cerevisiae γ-glutamyl kinase enhances proline accumulation and freezing tolerance. Appl. Environ. Microbiol., 73, 4011-4019 (2007). 51) F. Tanaka-Tsuno, S. Mizukami-Murata, Y. Murata, T. Nakamura, A. Ando, H. Takagi and J. Shima*: Functional genomics of commercial baker’s yeasts that have different abilities for sugar utilization and high-sucrose tolerance under sugar conditions. Yeast, 24, 901-911 (2007). 52) M. Demae, Y. Murata, M. Hisano, Y. Haitani, J. Shima and H. Takagi*: Overexpression of two transcriptional factors, Kin28 and Pog1, suppresses the stress sensitivity caused by the rsp5 mutation in Saccharomyces cerevisiae. FEMS Microbiol. Lett., 277, 70-78 (2007). 53) Y. Haitani and H. Takagi*: Rsp5 is required for the nuclear export of mRNA of HSF1 and MSN2/4 under stress conditions in Saccharomyces cerevisiae. Genes to Cells, 13, 105-116 (2008). 54) J. Shima*, A. Ando and H. Takagi: Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains. Yeast, 25, 179-190 (2008). 55) M. Wada*, K. Okabe, M. Kataoka, S. Shimizu, A. Yokota and H. Takagi: Distribution of L-azetidine-2-carboxylate N-acetyltransferase in yeast. Biosci. Biotech. Biochem., 72, 582-586 (2008). 56) T. Kotani and H. Takagi*: Identification of amino acid residues essential for the yeast N-acetyltransferase Mpr1 activity by site-directed mutagenesis. FEMS Yeast

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Res., 8, 607-614 (2008). 57) T. Kaino and H. Takagi*: Gene expression profiles and intracellular contents of stress protectants in Saccharomyces cerevisiae under ethanol and sorbitol stresses. Appl. Microbiol. Biotech., 79, 273-283 (2008). 58) T. Nakamura, S. Mizukami-Murata, A. Ando, Y. Murata, H. Takagi and J. Shima*: Changes in gene expression of commercial baker's yeast during an air-drying process that simulates dried yeast production. J. Biosci. Bioeng., 106, 405-408 (2008). 59) T. Kaino, T. Tateiwa, S. Mizukami-Murata, J. Shima and H. Takagi*: Self-cloning baker’s yeasts that accumulate proline enhance freeze tolerance in doughs. Appl. Environ. Microbiol., 74, 5845-5849 (2008). 60) Y. Araki, H. Wu, H. Kitagaki, T. Akao, H. Takagi and H. Shimoi*: Ethanol stress stimulates the Ca2+-mediated calcineurin/Crz1 pathway in Saccharomyces cerevisiae. J. Biosci. Bioeng., 107, 1-6 (2009). 61) Y. Haitani, M. Nakata, T. Sasaki, A. Uchida and H. Takagi*: Engineering of the yeast ubiquitin ligase Rsp5: isolation of a new variant that induces constitutive inactivation of the general amino acid permease Gap1. FEMS Yeast Res., 9, 73-86 (2009). 62) T. Nakamura, H. Takagi and J. Shima*: Effects of ice-seeding temperature and intracellular trehalose contents on survival of frozen Saccharomyces cerevisiae cells. Cryobiol., 58, 170-174 (2009). 63) K. Iinoya, T. Kotani, Y. Sasano and H. Takagi*: Engineering of the yeast antioxidant enzyme Mpr1 for enhanced activity and stability. Biotechnol. Bioeng., 103, 341-352 (2009). 64) T. Hibi*, H. Yamamoto, G. Nakamura, and H. Takagi: Crystallization and preliminary crystallographic analysis on N-acetyltransferase Mpr1 from Saccharomyces cerevisiae. Acta Crystallogr., F65, 169-172 (2009). 65) H. Wu, T. Watanabe, Y. Araki, H. Kitagaki, T. Akao, H. Takagi and H. Shimoi*: Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing. J. Biosci. Bioeng., 107, 636-640 (2009). 66) T. Kaino and H. Takagi*: Proline as a stress protectant in the yeast Saccharomyces cerevisiae. Biosci. Biotech. Biochem., 73, 2131-2135 (2009). 67) H. Hiraishi, M. Okada, I. Ohtsu and H. Takagi*: Functional analysis of the yeast ubiquitin ligase Rsp5: Involvement of the ubiquitin-conjugating enzyme Ubc4 and poly-ubiquitination in ethanol-Induced down-regulation of targeted proteins. Biosci. Biotech. Biochem., 73, 2268-2273 (2009). 68) H. Hiraishi, T. Shimada, I. Ohtsu, T. Sato and H. Takagi*: The yeast ubiquitin ligase Rsp5 down-regulates the alpha subunit of nascent polypeptide-associated complex

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Egd2 under stress conditions. FEBS J., 276, 5287-5297 (2009). 69) K. Ogawa-Mitsuhashi, K. Sagane, J. Kuromitsu, H. Takagi and K. Tsukahara*: MPR1 as a novel selection marker in Saccharomyces cerevisiae. Yeast, 26, 587-593 (2009). 70) S. Takahashi*, A. Ando*, H. Takagi and J. Shima: Insufficiency of copper ion homeostasis causes freeze-thaw injury of yeast cells revealed by indirect gene expression analysis. *These authors contributed equally to this work. Appl. Environ. Microbiol., 75, 6706-6711 (2009). 71) K. Ukibe, K. Hashida, N. Yoshida and H. Takagi*: Metabolic engineering of Saccharomyces cerevisiae for astaxanthin production and oxidative stress tolerance. Appl. Environ. Microbiol., 75, 7205-7211 (2009). 72) Y. Sasano, S. Takahashi, J. Shima and H. Takagi*: Antioxidant N-acetyltransferase Mpr1/2 of industrial baker's yeast enhances fermentation ability after air-drying stress in bread dough. Int. J. Food Microbiol., 138, 181-185 (2010). 73) T. Nakamura, S. Takahashi, H. Takagi and J. Shima*: Multicopy suppression of oxidant-sensitive eos1 mutation by IZH2 in Saccharomyces cerevisiae and the involvement of Eos1 in zinc homeostasis. FEMS Yeast Res., 10, 259-269 (2010). 74) A. Nishimura, T. Kotani, Y. Sasano and H. Takagi*: An antioxidative mechanism mediated by the yeast N-acetyltransferase Mpr1: Oxidative stress-induced arginine synthesis and its physiological role. FEMS Yeast Res., 10, 687-698 (2010). 75) H. Urbanczyk, C. Noguchi, H. Wu, D. Watanabe, T. Akao, H. Takagi and H. Shimoi*: Sake yeast strains have difficulty in entering a quiescent state after cell growth cessation. J. Biosci. Bioeng., 112, 44-48 (2011). 76) Y. Sasano, Y. Haitani, I. Ohtsu, J. Shima and H. Takagi*: Proline accumulation in baker’s yeast enhances high-sucrose stress tolerance and fermentation ability in sweet dough. Int. J. Food Microbiol., 152, 40-43 (2012). 77) Y. Sasano, D. Watanabe, K. Ukibe, T. Inai, I. Ohtsu, H. Shimoi and H. Takagi*: Overexpression of the yeast transcription activator Msn2 confers furfural resistance and increases the initial fermentation rate in ethanol production. J. Biosci. Bioeng., 113, 451-455 (2012). 78) T. Kaino, Y. Tasaka, Y. Tatehashi and H. Takagi*: Functional analysis of the Cterminal region of γ-glutamyl kinase from Saccharomyces cerevisiae. Biosci. Biotech. Biochem., 76, 454-461 (2012). 79) T. Ogata*, H. Yamagishi, K. Ukibe and H. Takagi: Construction of bottomfermenting yeasts that overexpress the ubiquitin ligase RSP5 gene and their effects on high-gravity fermentation. J. Brew. Distill., 3, 1-5 (2012). 80) Y. Sasano, Y. Haitani, K. Hashida, I. Ohtsu, J. Shima and H. Takagi*:

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Overexpression of the transcription activator Msn2 enhances fermentation ability of industrial baker’s yeast in frozen dough. Biosci. Biotech. Biochem., 113, 451-455 (2012). 81) Y. Sasano, Y. Haitani, K. Hashida, I. Ohtsu, J. Shima and H. Takagi*: Simultaneous accumulation of proline and trehalose in industrial baker’s yeast enhances fermentation ability in frozen dough. J. Biosci. Bioeng., 113, 592-595 (2012). 82) Y. Sasano, Y. Haitani, K. Hashida, I. Ohtsu, J. Shima and H. Takagi*: Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker's yeast. Microb. Cell Fact., 11:40 doi:10.1186/1475-2859-11-40 (2012). 83) T. M. H. Bach, T. Hibi, R. Nasuno, G. Matsuo, Y. Sasano and H. Takagi*: Production of N-acetyl cis-4-hydroxy-L-proline by the yeast N-acetyltransferase Mpr1. J. Biosci. Bioeng., 114, 160-165 (2012). 84) A. Nishimura, R. Nasuno and H. Takagi*: The proline metabolism intermediate Δ1pyrroline-5-carboxylate directly inhibits the mitochondrial respiration in budding yeast. FEBS Lett., 586, 2411-2416 (2012). 85) S. Hasegawa, T. Ogata, K. Tanaka, A. Ando, H. Takagi and J. Shima*: Overexpression of vacuolar H+-ATPase-related genes in bottom-fermenting yeast enhances ethanol tolerance and fermentation rates during high-gravity fermentation. J. Inst. Brew., 118, 179-185 (2012). 86) T. M. H. Bach, R. Hara, K. Kino, I. Ohtsu, N. Yoshida and H. Takagi*: Microbial production of N-acetyl cis-4-hydroxy-L-proline by coexpression of the Rhizobium Lproline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1. Appl. Microbiol. Biotech., 97, 247-257 (2013). 87) A. Nishimura, N. Kawahara and H. Takagi*: The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. Biochem. Biophys. Res. Commun., 430, 137-143 (2013). 88) T. Sasaki and H. Takagi*: Phosphorylation of a conserved Thr357 in yeast Nedd4like ubiquitin ligase Rsp5 is involved in downregulation of the general amino acid permease Gap1. Genes Cells, 18, 459-475 (2013). 89) T. Inai, D. Watanabe, Y. Zhou, R. Fukada, T. Akao, J. Shima, H. Takagi and H. Shimoi*: Rim15p-mediated regulation of sucrose utilization during molasses fermentation using Saccharomyces cerevisiae strain PE-2. J. Biosci. Bioeng., 116, 591-594 (2013). 90) Y. Tatehashi and H. Takagi*: Characterization of γ-glutamyl kinase mutants from Saccharomyces cerevisiae. J. Biosci. Bioeng., 116, 576-579 (2013). 91) Y. Sasano, Y. Haitani, K. Hashida, S. Oshiro, J. Shima and H. Takagi*:

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Improvement of fermentation ability under baking-associated stress conditions by altering the POG1 gene expression in baker's yeast. Int. J. Food Microbiol., 165, 241245 (2013). 92) R. Nasuno, Y. Hirano, T. Itoh, T. Hakoshima, T. Hibi and H. Takagi*: Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism. Proc. Natl. Acad. Sci. USA, 110, 11821-11826 (2013). 93) T. Inaba, D. Watanabe, Y. Yoshiyama, K. Tanaka, J. Ogawa, H. Takagi, H. Shimoi and J. Shima*: An organic acid-tolerant HAA1-overexpression mutant of an industrial bioethanol strain of Saccharomyces cerevisiae and its application to the production of bioethanol from sugarcane molasses. AMB Express, 3:74, doi:10.1186/2191-0855-374 (2013). 94) D. Greetham*, H. Takagi and T. P. Phister: Presence of proline has a protective effect on weak acid stressed Saccharomyces cerevisiae. Antonie van Leeuwenhoek, 105, 641-652 (2014). 95) S. Uesugi, D. Watanabe, M. Kitajima, R. Watanabe, Y. Kawamura, M. Ohnishi, H. Takagi and K. Kimura*: Calcineurin inhibitors suppress the high-temperature stress sensitivity of the yeast ubiquitin ligase Rsp5 mutant: A new method of screening for calcineurin inhibitors. FEMS Yeast Res., 14, 567-574 (2014). 96) D. Watanabe, R. Kikushima, M. Aitoku, A. Nishimura, I. Ohtsu, R. Nasuno and H. Takagi*: Exogenous addition of histidine reduces copper availability in the yeast Saccharomyces cerevisiae. Microbial Cell, 1, 241-246 (2014). 97) H. Takagi*, K. Hashida, D. Watanabe, R. Nasuno, M. Ohashi, T. Iha, M. Nezuo, and M. Tsukahara: Isolation and characterization of awamori yeast mutants with l-leucine accumulation that overproduce isoamyl alcohol. J. Biosci. Bioeng., 119, 140-147 (2015). 98) T. Shiga, N. Yoshida, Y. Shimizu, E. Suzuki, T. Sasaki, D. Watanabe and H. Takagi*: Quality control of plasma membrane proteins by yeast Nedd4-like ubiquitin ligase Rsp5p under environmental stress conditions. Eukaryot. Cell, 13, 1191-1199 (2014). 99) S. Oshiro and H. Takagi*: The transcriptional activator Pog1 controls cell cycle and its phosphorylated form is downregulated by the ubiquitin ligase Dma2 in Saccharomyces cerevisiae. FEMS Yeast Res., 14, 1015-1027 (2014). 100) I. Wijayanti, D. Watanabe, S. Oshiro and H. Takagi*: Isolation and functional analysis of yeast ubiquitin ligase Rsp5 variants that alleviate the toxicity of human asynuclein. J. Biochem., 157, 251-260 (2015). 101) S. Hirayama, M. Shimizu, N. Tsuchiya, S. Furukawa, D. Watanabe, H. Shimoi, H.

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Takagi, H. Ogihara, and Y. Morinaga*: Awa1p on the cell surface of sake yeast inhibits biofilm formation and the co-aggregation between sake yeasts and Lactobacillus plantarum ML11-11. J. Biosci. Bioeng., 119, 532-537 (2015). 102) R. Nasuno, M. Aitoku, Y. Manago, A. Nishimura, Y. Sasano and H. Takagi*: Nitric oxide-mediated antioxidative mechanism in yeast through the activation of the transcription factor Mac1. PLoS One, 9(11), e113788 (2014). 103) D. Watanabe, H. Murai, R. Tanahashi, K. Nakamura, T. Sasaki and H. Takagi*: Cooperative and selective roles of the WW domains of the yeast Nedd4-like ubiquitin ligase Rsp5 in the recognition of the arrestin-like adaptors Bul1 and Bul2. Biochem. Biophys. Res. Commun., 463, 76-81 (2015). 104) R. Nasuno, S. Hirase, S. Norifune, D. Watanabe and H. Takagi*: Structure-based molecular design for thermostabilization of N-acetyltransferase Mpr1 involved in a novel pathway of L-arginine synthesis in yeast. J. Biochem., 159, 271-277 (2016). 105) D. Watanabe, Y. Zhou, A. Hirata, Y. Sugimoto, K. Takagi, T. Akao, Y. Ohya, H. Takagi and H. Shimoi*: Inhibitory role of Greatwall-like protein kinase Rim15p in alcoholic fermentation via upregulating the UDP-glucose synthesis pathway in Saccharomyces cerevisiae. Appl. Environ. Microbiol., 82, 340-351 (2016). 106) R. I. Astuti, D. Watanabe and H. Takagi*: Nitric oxide signaling and its role in oxidative stress response in Schizosaccharomyces pombe. Nitric Oxide-Biol. Chem., 52, 29-40 (2016). 107) I. Nishida, D. Watanabe, A. Tsolmonbaatar, T. Kaino, I. Ohtsu and H. Takagi*: Vacuolar amino acid transporters upregulated by exogenous proline and involved in cellular localization of proline in Saccharomyces cerevisiae. J. Gen. Appl. Microbiol., 62, 132-139 (2016). 108) H. Takagi*, J. Taguchi and T. Kaino: Proline accumulation protects Saccharomyces cerevisiae cells in the stationary phase from ethanol stress by reducing reactive oxygen species levels. Yeast, 33, 355-363 (2016). 109) Y. Yoshikawa, R. Nasuno, N. Kawahara, A. Nishimura, D. Watanabe and H. Takagi*: Regulatory mechanism of the flavoprotein Tah18-dependent nitric oxide synthesis and cell death in yeast. Nitric Oxide-Biol. Chem., 57, 85-91 (2016). 110) Y. Tatehashi, D. Watanabe and H. Takagi*: g-Glutamyl kinase is involved in selective autophagy of ribosomes in Saccharomyces cerevisiae. FEBS Lett., 590, 2906-2914 (2016). 111) D. Watanabe, A. Kaneko, Y. Sugimoto, T. Negishi, S. Ohnuki, H. Takagi and Y. Ohya*: Promoter engineering of the Saccharomyces cerevisiae RIM15 gene for improvement of alcoholic fermentation rates under stress conditions. J. Biosci. Bioeng., doi: 10.1016/j.jbiosc.2016.08.004, in press.

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112) I. Nishida, D. Watanabe and H. Takagi*: Putative mitochondrial α-ketoglutaratedependent dioxygenase Fmp12 controls utilization of proline as an energy source in Saccharomyces cerevisiae. Microbial Cell, 3, 391-397 (2016). 113) A. Tsolmonbaatar, K. Hashida, Y. Sugimoto, D. Watanabe, S. Furukawa and H. Takagi: Isolation of baker’s yeast mutants with proline accumulation that showed enhanced tolerance to baking-associated stresses. Int. J. Food Microbiol., 238, 233240 (2016). 3. Mechanism of metabolic regulation of amino acids in bacteria and molecular breeding of amino acid-overproducers 114) K. Miwa*, K. Matsui, M. Terabe, K. Ito, M. Ishida, H. Takagi, S. Nakamori and K. Sano: Construction of novel shuttle vectors and a cosmid vector for the glutamic acidproducing bacteria Brevibacterium lactofermentum and Corynebacterium glutamicum. Gene, 39, 281-286 (1985). 115) H. Takagi*, Y. Morinaga, K. Miwa, S. Nakamori and K. Sano: Versatile cloning vectors constructed with genes indigenous to a glutamic acid-producer, Brevibacterium lactofermentum. Agric. Biol. Chem., 50, 2597-2603 (1986). 116) R. Yamaguchi*, M. Terabe, K. Miwa, M. Tsuchiya, H. Takagi, Y. Morinaga, S. Nakamori, K. Sano, H. Momose and A. Yamazaki: Determination of the complete nucleotide sequence of Brevibacterium lactofermentum plasmid pAM330 and analysis of its genetic information. Agric. Biol. Chem., 50, 2771-2778 (1986). 117) S. Nakamori*, M. Ishida, H. Takagi, K. Ito, K. Miwa and K. Sano: Improved Lthreonine production by the amplification of the gene encoding homoserine dehydrogenase in Brevibacterium lactofermentum. Agric. Biol. Chem., 51, 87-91 (1987). 118) Y. Morinaga*, H. Takagi, M. Ishida, K. Miwa, T. Sato, S. Nakamori and K. Sano: Threonine production by co-existence of cloned gene coding homoserine dehydrogenase and homoserine kinase in Brevibacterium lactofermentum. Agric. Biol. Chem., 51, 93-100 (1987). 119) M. Sugimoto, A. Tanaka, T. Suzuki, H. Matsui, S. Nakamori and H. Takagi*: Sequence analysis of functional regions of homoserine dehydrogenase genes from Llysine and L-threonine-producing mutants of Brevibacterium lactofermentum. Biosci. Biotech. Biochem., 61, 1760-1762 (1997). 120) S. Nakamori*, S. Kobayashi, C. Kobayashi and H. Takagi: Overproduction of Lcysteine and L-cystine by Escherichia coli strains with a genetically altered serine acetyltransferase. Appl. Environ. Microbiol., 64, 1607-1611 (1998). 121) H. Takagi*, C. Kobayashi, S. Kobayashi and S. Nakamori: PCR random

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mutagenesis into Escherichia coli serine acetyltransferase: isolation of the mutant enzymes that cause overproduction of L-cysteine and L-cystine due to the desensitization to feedback inhibition. FEBS Lett., 452, 323-327 (1999). 122) S. Nakamori, S. Kobayashi, T. Nishimura and H. Takagi*: Mechanism of Lmethionine overproduction by Escherichia coli: the replacement of Ser54 by Asn in the MetJ protein causes the derepression of L-methionine biosynthetic enzymes. Appl. Microbiol. Biotechnol., 52,179-185 (1999). 123) H. Takagi*, N. Awano, S. Kobayashi, M. Noji, K. Saito and S. Nakamori: Overproduction of L-cysteine and L-cystine by expression of genes for feedback inhibition-sensitive serine acetyltransferase from Arabidopsis thaliana in Escherichia coli. FEMS Microbiol. Lett., 179, 453-459 (1999). 124) M. Wada*, N. Awano, K. Haisa, H. Takagi and S. Nakamori: Purification, characterization and identification of cysteine desulfhydrase of Corynebacterium glutamicum, and its relationship to cysteine production. FEMS Microbiol. Lett., 217, 103-107 (2002). 125) N. Awano, M. Wada*, A. Kohdoh, T. Oikawa, H. Takagi and S. Nakamori: Effect of cysteine desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli. Appl. Microbiol. Biotechnol., 62, 239-243 (2003). 126) M. Wada*, N. Awano, H. Yamazawa, H. Takagi and S. Nakamori: Purification and characterization of O-acetylserine sulfhydrylase of Corynebacterium glutamicum. Biosci. Biotech. Biochem., 68, 1581-1583 (2004). 127) S. Kobayashi, R. Masui, S. Yokoyama, S. Kuramitsu and H. Takagi*: A novel metal-activated L-serine O-acetyltransferase from Thermus thermophilus HB8. J. Biochem., 136, 629-634 (2004). 128) N. Awano, M. Wada, H. Mori, S. Nakamori and H. Takagi*: Identification and functional analysis of cysteine desulfhydrases in Escherichia coli. Appl. Environ. Microbiol., 71, 4149-4152 (2005). 129) Y. Haitani, N. Awano, M. Yamazaki, M. Wada, S. Nakamori and H. Takagi*: Functional analysis of L-serine O-acetyltransferase from Corynebacterium glutamicum. FEMS Microbiol. Lett., 255, 156-163 (2006). 130) Y. Kai, T. Kashiwagi, K. Ishikawa, M. K. Ziyatdinov, E. I. Redkina, M. Y. Kiriukhin, M. M. Gusyatiner, S. Kobayashi, H. Takagi and E. Suzuki*: Engineering of Escherichia coli L-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by L-cysteine. Protein Eng. Des. Sel., 19, 163167 (2006). 131) S. Yamada, N. Awano, K. Inubushi, E. Maeda, S. Nakamori, K. Nishino, A. Yamaguchi and H. Takagi*: Effect of drug transporter genes on cysteine export and

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overproduction in Escherchia coli. Appl. Environ. Microbiol., 72, 4735-4742 (2006). 132) N. Wiriyathanawudhiwong, I. Ohtsu, Z.-D. Li, H. Mori and H. Takagi*: The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli. Appl. Microbiol. Biotech., 81, 903-913 (2009). 133) I. Ohtsu*, N. Wiriyathanawudhiwong, S. Morigasaki, H. Kadokura and H. Takagi: The L-cysteine/L-cystine shuttle system provides reducing equivalents to the periplasm in Escherichia coli. J. Biol. Chem., 285, 17479-17487 (2010). 134) T. Nakatani, I. Ohtsu*, G.Nonaka, N. Wiriyathanawudhiwong, S. Morigasaki and H. Takagi: Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli. Microb. Cell Fact., 11:62 doi:10.1186/1475-2859-11-62 (2012). 135) Y. Kawano, I. Ohtsu*, K. Takumi, A. Tamakoshi, G. Nonaka, E. Funahashi, M. Ihara, and H. Takagi: Enhancement of l-cysteine production by disruption of yciW in Escherichia coli. J. Biosci. Bioeng., 119, 176-179 (2015). 136) Y. Kawano, I. Ohtsu*, A. Tamakoshi, M. Shiroyama, A. Tsuruoka, K. Saiki, K. Takumi, G. Nonaka, T. Nakanishi, T. Hishiki, M. Suematsu, and H. Takagi: Involvement of the yciW gene in l-cysteine and l-methionine metabolism in Escherichia coli. J. Biosci. Bioeng., 119, 310-313 (2015). 137) I. Ohtsu*, Y. Kawano, M. Suzuki, S. Morigasaki, K. Saiki, S. Yamazaki, G. Nonaka, and H. Takagi: Uptake of l-cystine via an ABC transporter contributes defense of oxidative stress in the l-cysteine export-dependent manner in Escherichia coli. PLoS One, 10(4), e0120619 (2015). 138) E. Funahashi, K. Saiki, K. Honda, Y. Sugiura, Y. Kawano, I. Ohtsu*, D. Watanabe, Y. Wakabayashi, T. Abe, T. Nakanishi, M. Suematsu, and H. Takagi: A finding of thiosulfate pathway for synthesis of organic sulfur compounds in Saccharomyces cerevisiae and an improvement of ethanol production. J. Biosci. Bioeng., 120, 666669 (2015). 4. Molecular analysis of microbial useful enzymes 139) H. Takagi*, M. Kondou, T. Hisatsuka, S. Nakamori, Y.-C. Tsai and M. Yamasaki: Effects of alkaline elastase from an alkalophilic Bacillus strain on the tenderization of beef meat. J. Agric. Food. Chem., 40, 2364-2368 (1992). 140) K. Washizu*, K. Ando, S. Koikeda, S. Hirose, A. Matsuura, H. Takagi, M. Motoki and K. Takeuchi: Molecular cloning of the gene for microbial transglutaminase from Streptoverticillium and its expression in Streptomyces lividans. Biosci. Biotech. Biochem., 58, 82-87 (1994). 141) S. Takehana, K. Washizu, K. Ando, S. Koikeda, K. Takeuchi, H. Matsui, M.

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Motoki and H. Takagi*: Chemical synthesis of the gene for microbial transglutaminase from Streptoverticillium and its expression in Escherichia coli. Biosci. Biotech. Biochem., 58, 88-92 (1994). 142) H. Takagi*, Y.-C. Tsai, S. Nakamori and M. Yamasaki: Improved production and recovery of alkaline elastase from alkalophilic Bacillus strain by a change of medium composition. Biosci. Biotech. Biochem., 59, 1591-1592 (1995). 143) S.-W. Cheng, H.-M. Hu, S.-W. Shen, H. Takagi, M. Asano and Y.-C. Tsai*: Production and characterization of keratinase of a feather-degrading Bacillus licheniformis PWD-1. Biosci. Biotech. Biochem., 59, 2239-2243 (1995). 144) S. Jareonkitmongkol*, M. Ohya, R. Watanabe, H. Takagi, and S. Nakamori: Partial purification of phytase from a soil isolate bacterium, Klebsiella oxytoca MO-3. J. Ferment. Bioeng., 83, 393-394 (1997). 145) M. Kawai, S. Takehana, and H. Takagi*: High-level expression of the chemically synthesized gene for microbial transglutaminase from Streptoverticillium in Escherichia coli. Biosci. Biotech. Biochem., 61, 830-835 (1997). 146) H. Takagi*, Y. Hoshino, S. Nakamori and S. Inouye: Isolation and sequence analysis of plasmid pNO33 in the ε-poly-L-lysine-producing actinomycete Streptomyces albulus IFO14147. J. Biosci. Bioeng., 89, 94-96 (2000). 147) A. Yoshizumi, M. Wada*, H. Takagi, S. Shimizu and S. Nakamori: Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding monovalent cation-activated levodione reductase from Corynebacterium aquaticum M-13. Biosci. Biotech. Biochem., 65, 830-836 (2001). 148) K. Tsujimoto, H. Takagi*, M. Takahashi, H. Yamada and S. Nakamori: Cryoprotective effect of the serine-rich repetitive sequence in silk protein sericin. J. Biochem., 129, 979-986 (2001). 149) S. Taguchi*, K. Arakawa, K. Yokoyama, S. Takehana, H. Takagi and H. Momose: Overexpression and purification of microbial pro-transglutaminase from Streptomyces cinnamoneum and in vitro processing by Streptomyces albogriseolus proteases. J. Biosci. Bioeng., 94, 478-481 (2002). 150) M. Wada*, A. Yoshizumi, Y. Noda, M. Kataoka, S. Shimuzu, H. Takagi and S. Nakamori: Production of double-chiral compound, (4R, 6R)-4-hydroxy-2,2,6trimethylcyclohexanone, by two-steps enzymatic asymmetric reduction. Appl. Environ. Microbiol., 69, 933-937 (2003). 151) S. Sogabe, A. Yoshizumi, T. A. Fukami, Y. Shiratori, S. Shimizu, H. Takagi, S. Nakamori and M. Wada*: The crystal structure and stereospecificity of levodione reductase from Corynebacterium aquaticum M-13. J. Biol. Chem., 278, 19387-19395 (2003).

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152) Y. Hoshino, S. Nakamori and H. Takagi*: Cloning and analysis of the β-lactamase gene from ε-poly-L-lysine-producing actinomycete Streptomyces albulus IFO14147. J. Biochem., 134, 473-478 (2003). 153) M. Takahashi*, K. Tsujimoto, H. Yamada, H. Takagi and S. Nakamori: The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture. Biotech. Lett., 25, 1805-1809 (2003). 154) I. Ohtsu, N. Kakuda, N. Tsukagoshi, N. Dokyu, H. Takagi, M. Wachi* and R. Aono: Transcriptional analysis of the ostA/imp gene involved in organic solvent sensitivity in Escherichia coli. Biosci. Biotech. Biochem., 68, 458-461 (2004). 155) M. Wada*, A. Yoshizumi, Y. Furukawa, H. Kawabata, M. Ueda, H. Takagi and S. Nakamori: Cloning and overexpression of the Exiguobacterium sp. F42 gene encoding a new short chain dehydrogenase, which catalyzes the stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate to ethyl (S)-3-hydroxy-3-(2thienyl)propanoate. Biosci. Biotech. Biochem., 68, 1481-1488 (2004). 156) Y. Hamano, Y. Hoshino, S. Nakamori and H. Takagi*: Overexpression and characterization of an aminoglycosides 6(acc)N-acetyltransferase with a broad specificity from ε-poly-L-lysine producer, Streptomyces albulus IFO14147. J. Biochem., 136, 517-524 (2004). 157) Y. Hamano*, I. Nicchu, Y. Hoshino, T. Kawai, S. Nakamori and H. Takagi: Development of gene delivery systems for the ε-poly-L-lysine producer, Streptomyces albulus. J. Biosci. Bioeng., 99, 636-641 (2005). 158) M. Takahashi*, K. Tsujimoto, Y. Kato, H. Yamada, H. Takagi and S. Nakamori: A sericin-derived peptide protects Sf9 insect cells from death caused by acute serum deprivation. Biotech. Lett., 27, 893-897 (2005). 159) Y. Hamano, T. Yoshida, M. Kito, S. Nakamori, T. Nagasawa* and H. Takagi*: Biological function of the pld gene product that degrades ε-poly-L-lysine selfresistance in Streptomyces albulus. Appl. Microbiol. Biotech., 72, 173-181 (2006). 160) Y. Hamano*, N. Matsuura, M. Kitamura and H. Takagi*: A novel enzyme conferring streptothricin resistance alters the toxicity of streptothricin D from broadspectrum to bacterial-specific. J. Biol. Chem., 281, 16842-16848 (2006). 161) Y. Hamano*, I. Nicchu, T. Shimizu, Y. Onji, J. Hiraki and H. Takagi*: ε-Poly-Llysine producer, Streptomyces albulus, has feedback-inhibition resistant aspartokinase, Appl. Microbiol. Biotech., 76, 873-882 (2007). 162) N. Ohhata, N. Yoshida*, H. Egami, T. Katsuragi, Y. Tani and H. Takagi: An extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4 isolated from crude oil. J. Bacteriol., 189, 6824-6831 (2007). 163) N. Yoshida*, S. Akazawa, T. Karino, H. Ishida, Y. Hata, T. Katsuragi, Y. Tani and

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H. Takagi: Functional analysis of genes encoding putative oxidoreductases in Aspergillus oryzae, which are similar to fungal fructosyl-amino acid oxidase. J. Biosci. Bioeng., 104, 424-427 (2007). 164) N. Yoshida*, N. Ohhata, Y. Yoshino, T. Katsuragi, Y. Tani and H. Takagi: Screening of carbon dioxide-requiring extreme oligotrophs from soil. Biosci. Biotech. Biochem., 71, 2830-2832 (2007). 165) K. Ukibe, T. Katsuragi*, Y. Tani and H. Takagi: Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry. FEMS Microbiol. Lett., 286, 241-248 (2008). 166) K. Yamanaka, C. Maruyama, H. Takagi and Y. Hamano*: ε-Poly-L-lysine dispersity is controlled by a highly unusual non-ribosomal peptide synthetase. Nat. Chem. Biol., 4, 766-772 (2008). 167) N. Yoshida*, T. Hayasaki and H. Takagi: Gene expression analysis of methylotrophic oxidoreductases involved in the oligotrophic growth of Rhodococcus erythropolis N9T-4. Biosci. Biotech. Biochem., 75, 123-127 (2011). 168) T. Yano, N. Yoshida* and H. Takagi: Carbon monoxide utilization of an extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4. J. Biosci. Bioeng., 114, 5355 (2012). 169) N. Yoshida*, S. Inaba and H. Takagi: Utilization of atmospheric ammonia by an extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4. J. Biosci. Bioeng, 117, 28-32 (2014). 170) A. Malik*, M. T. Hapsari, I. Ohtsu, S. Ishikawa, H. Takagi: Cloning and heterologous expression of the ftfCNC-2(1) gene from Weissella confusa MBFCNC2(1) as an extracellular active fructansucrase in Bacillus subtilis. J. Biosci. Bioeng., 119, 515-520 (2015). 171) T. Yano, N. Yoshida*, F. Yu, M. Wakamatsu and H. Takagi*: The glyoxylate shunt is essential for CO2-requiring oligotrophic growth of Rhodococcus erythropolis N9T-4. Appl. Microbiol. Biotech., 99, 5627-5637 (2015). 172) N. Yoshida*, T. Yano, K. Kedo, T. Fujiyoshi, R. Nagai, M. Iwano, E. Taguchi, T. Nishida and H. Takagi: A unique intracellular compartment formed during the oligotrophic growth of Rhodococcus erythropolis N9T-4. Appl. Microbiol. Biotech., in press. 5. Others 173) H. Takagi and K. Nakano*: The effect of vitamin A-depletion of antigen-stimulated trapping of peripheral lymphocytes in local lymph nodes of rats. Immunology, 48, 123-128 (1983).

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