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FacultyDepartment of Environmental Management / Graduate School of Agriculture
DegreePh.D. in Agriculture
Commentator Guide
Last Updated :2020/09/02

Research Activities

Research Areas

  • Life sciences, Applied microbiology

Published Papers

  • Isobutanol production in Corynebacterium glutamicum: Suppressed succinate by-production by pckA inactivation and enhanced productivity via the Entner-Doudoroff pathway., Hasegawa S, Jojima T, Suda M, Inui M, Metabolic engineering, Metabolic engineering, Jan. 2020 , Refereed
  • Evaluation of the influence of sprinkling powdered slaked lime on microorganisms for the prevention of domestic animal infectious diseases., Mori M, Sakagami Y, Hamazaki Y, Jojima T, Environmental technology, Environmental technology, 1 - 11, Apr. 2018 , Refereed
  • Improving Process Yield in Succinic Acid Production by Cell Recycling of Recombinant Corynebacterium glutamicum, Jojima, Toru, Noburyu, Ryoji, Suda, Masako, Okino, Shohei, Yukawa, Hideaki, Inui, Maysayuki, Fermentation, Fermentation, 2(1), 5, 2016 , Refereed
  • Identification of a HAD superfamily phosphatase, HdpA, involved in 1,3-dihydroxyacetone production during sugar catabolism in Corynebacterium glutamicum, Toru Jojima, Takafumi Igari, Wataru Gunji, Masako Suda, Masayuki Inui, Hideaki Yukawa, FEBS LETTERS, FEBS LETTERS, 586(23), 4228 - 4232, Nov. 2012 , Refereed
    Summary:Corynebacterium glutamicum produces 1,3-dihydroxyacetone (DHA) as metabolite of sugar catabolism but the responsible enzyme is yet to be identified. Using a transposon mutant library, the gene hdpA (cgR_2128) was shown to encode a haloacid dehalogenase superfamily member that catalyzes dephosphorylation of dihydroxyacetone phosphate to produce DHA. Inactivation of hdpA led to a drastic decrease in DHA production from each of glucose, fructose, and sucrose, indicating that HdpA is the main enzyme responsible for DHA production from sugars in C. glutamicum. Confirmation of DHA production via dihydroxyacetone phosphatase finally confirms a long-speculated route through which bacteria produce DHA. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • Metabolic engineering of Corynebacterium glutamicum for hyperproduction of polymer-grade L- and D-lactic acid., Yota Tsuge, Naoto Kato, Shogo Yamamoto, Masako Suda, Toru Jojima, Masayuki Inui, Applied microbiology and biotechnology, Applied microbiology and biotechnology, 103(8), 3381 - 3391, Apr. 2019 , Refereed
    Summary:Strain development is critical for microbial production of bio-based chemicals. The stereo-complex form of polylactic acid, a complex of poly-L- and poly-D-lactic acid, is a promising polymer candidate due to its high thermotolerance. Here, we developed Corynebacterium glutamicum strains producing high amounts of L- and D-lactic acid through intensive metabolic engineering. Chromosomal overexpression of genes encoding the glycolytic enzymes, glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, triosephosphate isomerase, and enolase, increased L- and D-lactic acid concentration by 146% and 56%, respectively. Chromosomal integration of two genes involved in the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-dehydro-3-deoxyphosphogluconate aldolase), together with a gene encoding glucose-6-phosphate dehydrogenase from Zymomonas mobilis, to bypass the carbon flow from glucose, further increased L- and D-lactic acid concentration by 11% and 44%, respectively. Finally, additional chromosomal overexpression of a gene encoding NADH dehydrogenase to modulate the redox balance resulted in the production of 212 g/L L-lactic acid with a 97.9% yield and 264 g/L D-lactic acid with a 95.0% yield. The optical purity of both L- and D-lactic acid was 99.9%. Because the constructed metabolically engineered strains were devoid of plasmids and antibiotic resistance genes and were cultivated in mineral salts medium, these strains could contribute to the cost-effective production of the stereo-complex form of polylactic acid in practical scale.
  • Efficient construction of xenogeneic genomic libraries by circumventing restriction-modification systems that restrict methylated DNA, Satoshi Hasegawa, Toru Jojima, Masayuki Inui, Journal of Microbiological Methods, Journal of Microbiological Methods, 146, 13 - 15, Mar. 01 2018 , Refereed
    Summary:An efficient method to construct xenogeneic genomic libraries with low errors and bias by circumventing restriction-modification systems that restrict methylated DNA was developed. Un-methylated genomic DNA of Escherichia coli prepared by ϕ29 DNA polymerase was introduced to Corynebacterium glutamicum R after ligation with un-methylated vector plasmids.
  • Enhanced Glucose Consumption and Organic Acid Production by Engineered Corynebacterium glutamicum Based on Analysis of a pfkB1 Deletion Mutant, Satoshi Hasegawa, Yuya Tanaka, Masako Suda, Toru Jojima, Masayuki Inui, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 83(3), Feb. 2017 , Refereed
    Summary:In the analysis of a carbohydrate metabolite pathway, we found interesting phenotypes in a mutant strain of Corynebacterium glutamicum deficient in pfkB1, which encodes fructose-1-phosphate kinase. After being aerobically cultivated with fructose as a carbon source, this mutant consumed glucose and produced organic acid, predominantly L-lactate, at a level more than 2-fold higher than that of the wild-type grown with glucose under conditions of oxygen deprivation. This considerably higher fermentation capacity was unique for the combination of pfkB1 deletion and cultivation with fructose. In the metabolome and transcriptome analyses of this strain, marked intracellular accumulation of fructose-1-phosphate and significant upregulation of several genes related to the phosphoenolpyruvate: carbohydrate phosphotransferase system, glycolysis, and organic acid synthesis were identified. We then examined strains overexpressing several of the identified genes and demonstrated enhanced glucose consumption and organic acid production by these engineered strains, whose values were found to be comparable to those of the model pfkB1 deletion mutant grown with fructose. L-Lactate production by the ppc deletion mutant of the engineered strain was 2,390 mM (i.e., 215 g/liter) after 48 h under oxygen deprivation, which was a 2.7-fold increase over that of the wild-type strain with a deletion of ppc. IMPORTANCE Enhancement of glycolytic flux is important for improving microbiological production of chemicals, but overexpression of glycolytic enzymes has often resulted in little positive effect. That is presumably because the central carbon metabolism is under the complex and strict regulation not only transcriptionally but also posttranscriptionally, for example, by the ATP/ADP ratio. In contrast, we studied a mutant strain of Corynebacterium glutamicum that showed markedly enhanced glucose consumption and organic acid production and, based on the findings, identified several genes whose overexpression was effective in enhancing glycolytic flux under conditions of oxygen deprivation. These results will further understanding of the regulatory mechanisms of glycolytic flux and can be widely applied to the improvement of the microbial production of useful chemicals.
  • Engineering the glycolytic pathway: A potential approach for improvement of biocatalyst performance, Toru Jojima, Masayuki Inui, BIOENGINEERED, BIOENGINEERED, 6(6), 328 - 334, Nov. 2015 , Refereed
    Summary:The glycolytic pathway is a main driving force in the fermentation process as it produces energy, cell component precursors, and fermentation products. Given its importance, the glycolytic pathway can be considered as an attractive target for the metabolic engineering of industrial microorganisms. However, many attempts to enhance glycolytic flux, by overexpressing homologous or heterologous genes encoding glycolytic enzymes, have been unsuccessful. In contrast, significant enhancement in glycolytic flux has been observed in studies with bacteria, specifically, Corynebacterium glutamicum. Although there has been a recent increase in the number of successful applications of this technology, little is known about the mechanisms leading to the enhancement of glycolytic flux. To explore the rational applications of glycolytic pathway engineering in biocatalyst development, this review summarizes recent successful studies as well as past attempts.
  • Thermal and Solvent Stress Cross-Tolerance Conferred to Corynebacterium glutamicum by Adaptive Laboratory Evolution, Shinichi Oide, Wataru Gunji, Yasuhiro Moteki, Shogo Yamamoto, Masako Suda, Toru Jojima, Hideaki Yukawa, Masayuki Inui, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 81(7), 2284 - 2298, Apr. 2015 , Refereed
    Summary:Reinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. Here, we report on adaptive laboratory evolution (ALE) of the amino acid-producing bacterium Corynebacterium glutamicum under thermal stress. After 65 days of serial passage of the transgenic strain GLY3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were isolated, and all the mutations they acquired were identified by whole-genome resequencing. Of the 21 mutations common to the three strains, one large deletion and two missense mutations were found to promote growth of the parental strain under thermal stress. Additive effects on thermotolerance were observed among these mutations, and the combination of the deletion with the missense mutation on otsA, encoding a trehalose-6-phosphate synthase, allowed the parental strain to overcome the upper limit of growth temperature. Surprisingly, the three evolved strains acquired cross-tolerance for isobutanol, which turned out to be partly attributable to the genomic deletion associated with the enhanced thermotolerance. The deletion involved loss of two transgenes, pfk and pyk, encoding the glycolytic enzymes, in addition to six native genes, and elimination of the transgenes, but not the native genes, was shown to account for the positive effects on thermal and solvent stress tolerance, implying a link between energy-producing metabolism and bacterial stress tolerance. Overall, the present study provides evidence that ALE can be a powerful tool to refine the phenotype of C. glutamicum and to investigate the molecular bases of stress tolerance.
  • Metabolic engineering for improved production of ethanol by Corynebacterium glutamicum, Toru Jojima, Ryoji Noburyu, Miho Sasaki, Takahisa Tajima, Masako Suda, Hideaki Yukawa, Masayuki Inui, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 99(3), 1165 - 1172, Feb. 2015 , Refereed
    Summary:Recombinant Corynebacterium glutamicum harboring genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) can produce ethanol under oxygen deprivation. We investigated the effects of elevating the expression levels of glycolytic genes, as well as pdc and adhB, on ethanol production. Overexpression of four glycolytic genes (pgi, pfkA, gapA, and pyk) in C. glutamicum significantly increased the rate of ethanol production. Overexpression of tpi, encoding triosephosphate isomerase, further enhanced productivity. Elevated expression of pdc and adhB increased ethanol yield, but not the rate of production. Fed-batch fermentation using an optimized strain resulted in ethanol production of 119 g/L from 245 g/L glucose with a yield of 95 % of the theoretical maximum. Further metabolic engineering, including integration of the genes for xylose and arabinose metabolism, enabled consumption of glucose, xylose, and arabinose, and ethanol production (83 g/L) at a yield of 90 %. This study demonstrated that C. glutamicum has significant potential for the production of cellulosic ethanol.
  • Promiscuous activity of (S,S)-butanediol dehydrogenase is responsible for glycerol production from 1,3-dihydroxyacetone in Corynebacterium glutamicum under oxygen-deprived conditions, Toru Jojima, Takafumi Igari, Yasuhiro Moteki, Masako Suda, Hideaki Yukawa, Masayuki Inui, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 99(3), 1427 - 1433, Feb. 2015 , Refereed
    Summary:Corynebacterium glutamicum can consume glucose to excrete glycerol under oxygen deprivation. Although glycerol synthesis from 1,3-dihydroxyacetone (DHA) has been speculated, no direct evidence has yet been provided in C. glutamicum. Enzymatic and genetic investigations here indicate that the glycerol is largely produced from DHA and, unexpectedly, the reaction is catalyzed by (S,S)-butanediol dehydrogenase (ButA) that inherently catalyzes the interconversion between S-acetoin and (S,S)-2,3-butanediol. Consequently, the following pathway for glycerol biosynthesis in the bacterium emerges: dihydroxyacetone phosphate is dephosphorylated by HdpA to DHA, which is subsequently reduced to glycerol by ButA. This study emphasizes the importance of promiscuous activity of the enzyme in vivo.
  • Development of Growth-Arrested Bioprocesses with Corynebacterium glutamicum for Cellulosic Ethanol Production from Complex Sugar Mixtures, Toru Jojima, Alain A. Vertès, Masayuki Inui, Hideaki Yukawa, Biorefineries: Integrated Biochemical Processes for Liquid Biofuels, Biorefineries: Integrated Biochemical Processes for Liquid Biofuels, 121 - 139, Aug. 18 2014 , Refereed
    Summary:In conventional fermentation processes used in industrial microbiology, product formation and biomass formation often occur in parallel. The yield and cost of goods might be optimized by separating these two phenomena into two distinct phases, however. This so-called growth-arrested bioprocess can be operated particularly efficiently using cells of Corynebacterium glutamicum as biocatalysts for the production of biofuels and other commodity chemicals. Compared to traditional fermentation processes, growth-arrested bioprocesses exhibit properties that make them particularly suited to a variety of applications in lignocellulosic biorefineries. In this chapter, we describe the fundamental attributes of growth-arrested bioprocesses and review recent advances in the genetic engineering of high-performing strains tailor-designed for the production of biofuels, such as cellulosic ethanol, and commodity chemicals.
  • Engineering of Corynebacterium glutamicum for High-Yield L-Valine Production under Oxygen Deprivation Conditions, Satoshi Hasegawa, Masako Suda, Kimio Uematsu, Yumi Natsuma, Kazumi Hiraga, Toru Jojima, Masayuki Inui, Hideaki Yukawa, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 79(4), 1250 - 1257, Feb. 2013 , Refereed
    Summary:We previously demonstrated efficient L-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of L-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the L-valine yield. Eliminating these by-products therefore was deemed key to improving the L-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and L-valine production dropped considerably due to the severely elevated intracellular NADH/NAD(+) ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher L-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM L-valine at a yield of 88% mol mol of glucose(-1) after 24 h under oxygen deprivation, a vastly improved yield over our previous best.
  • Overexpression of Genes Encoding Glycolytic Enzymes in Corynebacterium glutamicum Enhances Glucose Metabolism and Alanine Production under Oxygen Deprivation Conditions, Shogo Yamamoto, Wataru Gunji, Hiroaki Suzuki, Hiroshi Toda, Masako Suda, Toru Jojima, Masayuki Inui, Hideaki Yukawa, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 78(12), 4447 - 4457, Jun. 2012 , Refereed
    Summary:We previously reported that Corynebacterium glutamicum strain Delta ldhA Delta ppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165,2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD(+) ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.
  • Improvement of the Redox Balance Increases L-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions, Satoshi Hasegawa, Kimio Uematsu, Yumi Natsuma, Masako Suda, Kazumi Hiraga, Toru Jojima, Masayuki Inui, Hideaki Yukawa, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 78(3), 865 - 875, Feb. 2012 , Refereed
    Summary:Production of L-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the L-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to im-balanced cofactor production and consumption in the overall L-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of L-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for L-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD(+) ratio significantly decreased, and glucose consumption and L-valine production drastically improved. Moreover, L-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD(+) ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for L-valine production under oxygen deprivation conditions. The L-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM L-valine after 24 h with a yield of 0.63 mol mol of glucose(-1) and the L-valine productivity reached 1,940 mM after 48 h.
  • Metabolic engineering of bacteria for utilization of mixed sugar substrates for improved production of chemicals and fuel ethanol, Toru Jojima, Masayuki Inui, Hideaki Yukawa, Biofuels, Biofuels, 2(3), 303 - 313, May 2011 , Refereed
    Summary:Heightened concerns about dwindling cheap world oil supplies, and adverse global climate change blamed on man's unsustainable dependence on such finite fossil fuels, have led to increased urgency in the pursuit of new and effective technologies for the production of energy and chemicals from renewable feedstocks. Among several viable renewable resources, lignocellulosic biomass has long been recognized as abundant enough to potentially meet most future demands for transportation fuels and chemicals. Before this is realized, however, sizeable advances in technologies that underpin the concept of the biorefinery must be made. As newer innovations continue to be made on the way towards industrial-scale lignocellulosic biorefineries, inefficiencies in the conversion of the pentose sugar component of lignocellulosic hydrolysates have been so prioritized in numerous experimental biological production processes as to culminate in a healthy body of literature, particularly in the last decade or so. This article aims to present the current state of metabolic engineering of bacteria for utilization of mixed sugar substrates for improved production of chemicals and fuels from lignocellulosic biomass. © 2011 Future Science Ltd.
  • Engineering of sugar metabolism of Corynebacterium glutamicum for production of amino acid l-alanine under oxygen deprivation, Toru Jojima, Miho Fujii, Eiji Mori, Masayuki Inui, Hideaki Yukawa, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 87(1), 159 - 165, Jun. 2010 , Refereed
    Summary:Corynebacterium glutamicum was genetically engineered to produce l-alanine from sugar under oxygen deprivation. The genes associated with production of organic acids in C. glutamicum were inactivated and the alanine dehydrogenase gene (alaD) from Lysinibacillus sphaericus was overexpressed to direct carbon flux from organic acids to alanine. Although the alaD-expressing strain produced alanine from glucose under oxygen deprivation, its productivity was relatively low due to retarded glucose consumption. Homologous overexpression of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the alaD-expressing strain stimulated glucose consumption and consequently improved alanine productivity. In contrast gapA overexpression did not affect glucose consumption under aerobic conditions, indicating that oxygen deprivation engendered inefficient regeneration of NAD(+) resulting in impaired GAPDH activity and reduced glucose consumption in the alanine-producing strains. Inactivation of the alanine racemase gene allowed production of l-alanine with optical purity greater than 99.5%. The resulting strain produced 98 g l(-1) of l-alanine after 32 h in mineral salts medium. Our results show promise for amino acid production under oxygen deprivation.
  • Xylitol production by recombinant Corynebacterium glutamicum under oxygen deprivation, Miho Sasaki, Toru Jojima, Masayuki Inui, Hideaki Yukawa, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 86(4), 1057 - 1066, Apr. 2010 , Refereed
    Summary:Wild-type Corynebacterium glutamicum produced 0.6 g l(-1) xylitol from xylose at a productivity of 0.01 g l(-1) h(-1) under oxygen deprivation. To increase this productivity, the pentose transporter gene (araE) from C. glutamicum ATCC31831 was integrated into the C. glutamicum R chromosome. Consequent disruption of its lactate dehydrogenase gene (ldhA), and expression of single-site mutant xylose reductase from Candida tenuis (CtXR (K274R)) resulted in recombinant C. glutamicum strain CtXR4 that produced 26.5 g l(-1) xylitol at 3.1 g l(-1) h(-1). To eliminate possible formation of toxic intracellular xylitol phosphate, genes encoding xylulokinase (XylB) and phosphoenolpyruvate-dependent fructose phosphotransferase (PTS(fru)) were disrupted to yield strain CtXR7. The productivity of strain CtXR7 increased 1.6-fold over that of strain CtXR4. A fed-batch 21-h CtXR7 culture in mineral salts medium under oxygen deprivation yielded 166 g l(-1) xylitol at 7.9 g l(-1) h(-1), representing the highest bacterial xylitol productivity reported to date.
  • Sugar transporters in efficient utilization of mixed sugar substrates: current knowledge and outlook, Toru Jojima, Crispinus A. Omumasaba, Masayuki Inui, Hideaki Yukawa, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85(3), 471 - 480, Jan. 2010 , Refereed
    Summary:There is increasing interest in production of transportation fuels and commodity chemicals from lignocellulosic biomass, most desirably through biological fermentation. Considerable effort has been expended to develop efficient biocatalysts that convert sugars derived from lignocellulose directly to value-added products. Glucose, the building block of cellulose, is the most suitable fermentation substrate for industrial microorganisms such as Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae. Other sugars including xylose, arabinose, mannose, and galactose that comprise hemicellulose are generally less efficient substrates in terms of productivity and yield. Although metabolic engineering including introduction of functional pentose-metabolizing pathways into pentose-incompetent microorganisms has provided steady progress in pentose utilization, further improvements in sugar mixture utilization by microorganisms is necessary. Among a variety of issues on utilization of sugar mixtures by the microorganisms, recent studies have started to reveal the importance of sugar transporters in microbial fermentation performance. In this article, we review current knowledge on diversity and functions of sugar transporters, especially those associated with pentose uptake in microorganisms. Subsequently, we review and discuss recent studies on engineering of sugar transport as a driving force for efficient bioconversion of sugar mixtures derived from lignocellulose.
  • Engineering of pentose transport in Corynebacterium glutamicum to improve simultaneous utilization of mixed sugars, Miho Sasaki, Toru Jojima, Hideo Kawaguchi, Masayuki Inui, Hideaki Yukawa, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85(1), 105 - 115, Nov. 2009 , Refereed
    Summary:Corynebacterium glutamicum strains CRA1 and CRX2 are able to grow on l-arabinose and d-xylose, respectively, as sole carbon sources. Nevertheless, they exhibit the major shortcoming that their sugar consumption appreciably declines at lower concentrations of these substrates. To address this, the C. glutamicum ATCC31831 l-arabinose transporter gene, araE, was independently integrated into both strains. Unlike its parental strain, resultant CRA1-araE was able to aerobically grow at low (3.6 g center dot l(-1)) l-arabinose concentrations. Interestingly, strain CRX2-araE grew 2.9-fold faster than parental CRX2 at low (3.6 g center dot l(-1)) d-xylose concentrations. The corresponding substrate consumption rates of CRA1-araE and CRX2-araE under oxygen-deprived conditions were 2.8- and 2.7-fold, respectively, higher than those of their respective parental strains. Moreover, CRA1-araE and CRX2-araE utilized their respective substrates simultaneously with d-glucose under both aerobic and oxygen-deprived conditions. Based on these observations, a platform strain, ACX-araE, for C. glutamicum-based mixed sugar utilization was designed. It harbored araBAD for l-arabinose metabolism, xylAB for d-xylose metabolism, d-cellobiose permease-encoding bglF (317A) , beta-glucosidase-encoding bglA and araE in its chromosomal DNA. In mineral medium containing a sugar mixture of d-glucose, d-xylose, l-arabinose, and d-cellobiose under oxygen-deprived conditions, strain ACX-araE simultaneously and completely consumed all sugars.
  • Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions, Miho Sasaki, Toru Jojima, Masayuki Inui, Hideaki Yukawa, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 81(4), 691 - 699, Dec. 2008 , Refereed
    Summary:Corynebacterium glutamicum R was metabolically engineered to broaden its sugar utilization range to D-xylose and D-cellobiose contained in lignocellulose hydrolysates. The resultant recombinants expressed Escherichia coli xylA and xylB genes, encoding D-xylose isomerase and xylulokinase, respectively, for D-xylose utilization and expressed C. glutamicum R bglF(317A) and bglA genes, encoding phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) beta-glucoside-specific enzyme IIBCA component and phospho-beta-glucosidase, respectively, for D-cellobiose utilization. The genes were fused to the non-essential genomic regions distributed around the C. glutamicum R chromosome and were under the control of their respective constitutive promoter trc and tac that permitted their expression even in the presence of D-glucose. The enzyme activities of resulting recombinants increased with the increase in the number of respective integrated genes. Maximal sugar utilization was realized with strain X5C1 harboring five xylA-xylB clusters and one bglF(317A)-bglA cluster. In both D-cellobiose and D-xylose utilization, the sugar consumption rates by genomic DNA-integrated strain were faster than those by plasmid-bearing strain, respectively. In mineral medium containing 40 g l(-1) D-glucose, 20 g l(-1) D-xylose, and 10 g l(-1) D-cellobiose, strain X5C1 simultaneously and completely consumed these sugars within 12 h and produced predominantly lactic and succinic acids under growth-arrested conditions.
  • An efficient succinic acid production process in a metabolically engineered Corynebacterium glutamicum strain, Shohei Okino, Ryoji Noburyu, Masako Suda, Toru Jojima, Masayuki Inui, Hideaki Yukawa, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 81(3), 459 - 464, Dec. 2008 , Refereed
    Summary:A Corynebacterium glutamicum strain (Delta ldhA-pCRA717) that overexpresses the pyc gene encoding pyruvate carboxylase while simultaneously exhibiting a disrupted ldhA gene encoding <l-lactate dehydrogenase was investigated in detail for succinic acid production. Succinic acid was shown to be efficiently produced at high-cell density under oxygen deprivation with intermittent addition of sodium bicarbonate and glucose. Succinic acid concentration reached 1.24 M (146 g l-1) within 46 h. The yields of succinic acid and acetic acid from glucose were 1.40 mol mol-1(0.92 g g-1) and 0.29 mol mol-1 (0.10 g g-1), respectively. The succinic acid production rate and yield depended on medium bicarbonate concentration rather than glucose concentration. Consumption of bicarbonate accompanied with succinic acid production implied that added bicarbonate was used for succinic acid synthesis.
  • Basidiomycete Lentinula edodes CDC5 and a novel interacting protein CIPB bind to a newly isolated target gene in an unusual manner, Takehito Nakazawa, Shinya Kaneko, Yasumasa Miyazaki, Toru Jojima, Takashi Yamazaki, Shiho Katsukawa, Kazuo Shishido, FUNGAL GENETICS AND BIOLOGY, FUNGAL GENETICS AND BIOLOGY, 45(6), 818 - 828, Jun. 2008 , Refereed
    Summary:We isolated a target gene for the Lentinula edodes putative transcription factor Le.CDC5 that contains a c-Myb-type DNA-binding domain. The gene, termed ctg1, encodes a novel protein (159 amino acid residues) with a leucine zipper-like sequence and contains a 7-bp Le.CDC5-binding sequence, 5'GCAATCT3', in its transcribed region downstream of the start codon. Chromatin immunoprecipitation analysis strongly suggested that intracellular Le.CDC5 binds to this 7-bp sequence on L. edodes chromatin. Binding was most efficient on chromatin from the stipes of mature fruiting bodies. Two Le.CDC5-interaction partners were identified in L. edodes and named CIPA and CIPB. The CIPB protein (127 amino acid residues) binds to a 6-bp sequence with the consensus sequence 5'CAACAC/T/G3'. The ctg1 gene contains nine 6-bp consensus (or consensus-like) sequences, six are in the 5'-upstream region and three in the transcribed region downstream of the start codon. At least two each of the upstream and downstream sequences appear to bind CIPB in vitro. We suggest that Le.CDC5 and CIPB can cooperatively regulate the expression of ctg1. (c) 2008 Elsevier Inc. All rights reserved.
  • Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions (Applied Microbiology and Biotechnology DOI: 10.1007/s00253-007-1199-y), Kaori Yasuda, Toru Jojima, Masako Suda, Shohei Okino, Masayuki Inui, Hideaki Yukawa, Applied Microbiology and Biotechnology, Applied Microbiology and Biotechnology, 78(4), 737, Mar. 2008 , Refereed
  • Production of isopropanol by metabolically engineered Escherichia coli, Toru Jojima, Masayuki Inui, Hideaki Yukawa, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 77(6), 1219 - 1224, Jan. 2008 , Refereed
    Summary:A genetically engineered strain of Escherichia coli JM109 harboring the isopropanol-producing pathway consisting of five genes encoding four enzymes, thiolase, coenzyme A (CoA) transferase, acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824, and primary-secondary alcohol dehydrogenase from C. beijerinckii NRRL B593, produced up to 227 mM of isopropanol from glucose under aerobic fed-batch culture conditions. Acetate production by the engineered strain was approximately one sixth that produced by a control E. coli strain bearing an expression vector without the clostridial genes. These results demonstrate a functional isopropanol-producing pathway in E. coli and consequently carbon flux from acetyl-CoA directed to isopropanol instead of acetate. This is the first report on isopropanol production by genetically engineered microorganism under aerobic culture conditions.
  • Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions, Kaori Yasuda, Toru Jojima, Masako Suda, Shohei Okino, Masayuki Inui, Hideaki Yukawa, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 77(4), 853 - 860, Dec. 2007 , Refereed
    Summary:Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetylcoenzyme A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions.
  • Large-scale identification of transcripts expressed in a symbiotic fungus (Termitomyces) during plant biomass degradation, Toru Johjima, Yaovapa Taprab, Napavarn Noparatnaraporn, Toshiaki Kudo, Moriya Ohkuma, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 73(1), 195 - 203, Nov. 2006 , Refereed
    Summary:Fungus-growing termites have a symbiotic relationship with the basidiomycetes of the genus Termitomyces. This symbiotic system is able to degrade dead plant material efficiently. We conducted expressed sequence tag (EST) analysis of a symbiotic Termitomyces fungus degrading plant material in a field nest of the termite Macrotermes gilvus. A subtractive cDNA library was also investigated to facilitate the discovery of genes expressed specifically under the symbiotic conditions. A total of 2,613 ESTs were collected and resulted in 1,582 nonredundant tentative consensus sequences, of which approximately 59% showed significant similarity to known protein sequences. A number of homologous sequences to genes involved in plant cell wall degradation were identified and a majority of them encoded putative pectinolytic enzymes. Real-time quantitative reverse transcriptase polymerase chain reaction analyses confirmed significant upregulation of putative stress response genes under symbiotic conditions. The present ESTs database provides a valuable resource for molecular biological study of plant material degradation in the symbiosis between termites and fungi.
  • Diversity and abundance of termites along an altitudinal gradient in Khao Kitchagoot National Park, Thailand, Tetsushi Inoue, Yoko Takematsu, Akinori Yamada, Yuichi Hongoh, Toru Johjima, Shigeharu Moriya, Yupaporn Sornnuwat, Charunee Vongkaluang, Moriya Ohkuma, Toshiaki Kudo, JOURNAL OF TROPICAL ECOLOGY, JOURNAL OF TROPICAL ECOLOGY, 22(5), 609 - 612, Sep. 2006 , Refereed
  • Symbiotic fungi produce laccases potentially involved in phenol degradation in fungus combs of fungus-growing termites in Thailand, Y Taprab, T Johjima, Y Maeda, S Moriya, S Trakulnaleamsai, N Noparatnaraporn, M Ohkuma, T Kudo, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 71(12), 7696 - 7704, Dec. 2005 , Refereed
    Summary:Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.
  • Fungal Community Analysis of Fungus Gardens in Termite Nests, Shigeharu Moriya, Tetsushi Inoue, Moriya Ohkuma, Taprab Yaovapa, Tohru Johjima, Poonpilai Suwanarit, Utaiwan Sangwamt, Charunee Vongkaluang, Napavarn Noparatnaraporn, Toshiaki Kudo, Microbes and Environments, Microbes and Environments, 20(4), 243 - 252, 2005 , Refereed
    Summary:Fungus-growing termites cultivate fungi of the genus Termitomyces in gardens inside their nests. Despite various reports of the presence of Termitomyces in these gardens, the entire fungal community structure of the gardens has not yet been described. To clarify whether the fungal crops in the gardens are monocultures of Termitomyces, we examined 18 fungus gardens derived from 5 species of fungus-growing termites covering 3 genera. Phylogenetic analysis of DNA sequences revealed that non-Termitomyces fungi might have inhabited the gardens. However, terminal restriction fragment length polymorphism (T-RFLP) analysis clearly demonstrated that non-Termitomyces fungi made up only a minor population within the gardens. This suggests that the fungus gardens were maintained as almost complete monocultures of Termitomyces. © 2005, Japanese Society of Microbial Ecology & The Japanese Society of Soil Microbiology. All rights reserved.
  • A cDNA homologue of Schizosaccharomyces pombe cdc5(+) from the mushroom Lentinula edodes: characterization of the cDNA and its expressed product, Y Miyazaki, T Jojima, T Ono, T Yamazaki, K Shishido, BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION, BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION, 1680(2), 93 - 102, Oct. 2004 , Refereed
    Summary:A cDNA homologue of Schizosaccharomyces pombe cdc5(+) was isolated from the basidiomycete mushroom Lentinula edodes and it was named Le.cdc5 cDNA. The deduced Le.CDC5 (842 amino acid residues) possessed N-terminal amino acid sequence highly homologous to those of S. pombe cdc5(+) gene product (Sp.cdc5p) and Sp.cdc5p-related proteins (SPCDC5RPs). The N-terminal 185 amino acid peptide of Le.CDC5 (Le.CDC5(1-185) peptide) produced in Escherichia coli was subjected to random binding-site selection analysis, revealing that Le.CDC5(1-185) peptide binds to a 7-bp sequence with the consensus sequence of 5' GCAATGT3' (complementary; 5' ACATTGC3'). Genomic binding-site (GBS) cloning by using Le.CDC5(1-185) peptide resulted in an isolation of the DNA fragment that contained three sets of 7-bp consensus-like sequence and TATA box. The Le.CDC5 protein contained two putative phosphorylation sites of cAMP-dependent protein kinase (A kinase) in its C-terminus. There exists a possible leucine zipper between the two phosphorylation sites. The Le.CDC5 fragment containing the two phosphorylation sites was actually phosphorylated by commercially available A kinase. Yeast two-hybrid analysis suggested the homodimerization of Le.CDC5 protein probably through the leucine zipper. Northern blot analysis showed that Le.cdc5 gene is most actively transcribed in primordia and small immature fruiting bodies of L. edodes, implying that Le.cdc5 may play a role in the beginning and early stage of fruiting-body formation. (C) 2004 Elsevier B.V. All rights reserved.
  • Isolation of a ras gene from the basidiomycete Coriolus hirsutus and use of its promoter for the expression of Pleurotus ostreatus manganese(II) peroxidase cDNA in C. hirsutus, Takashi Yamazaki, Toshiro Kiyofuji, Toru Johjima, Susumu Kajiwara, Akira Tsukamoto, Jun Sugiura, Kazuo Shishido, Mycoscience, Mycoscience, 45(5), 317 - 323, 2004 , Refereed
    Summary:A ras gene homologue (named Ch.ras) was cloned from the basidiomycete Coriolus hirsutus. Ch.ras has a coding capacity of 215 amino acids (aa) interrupted by six small introns. The deduced Ch.Ras protein exhibited significant homology (86.5% or 86.0% identical) to the basidiomycete Coprinus cinereus Ras (215 aa) and Lentinula edodes Ras (217 aa) proteins. The 5′-upstream region of Ch.ras contains two GC boxlike sequences, one TATA box-like sequence, one CCAAT box, and three CT-rich sequences. Primer extension analysis showed the presence of three transcriptional initiation sites: one is located in the most upstream CT-sequence and the other two just after it. By using the 1.4-kb fragment containing the promoter elements and transcriptional initiation sites, we have constructed the chromosome-integrating vector pHRP, which is useful for the expression of foreign genes in C. hirsutus. The Pleurotus ostreatus manganese(II) peroxidase (MnP) cDNA (designated mnpc) was inserted into the downstream of the Ch.ras promoter elements of pHRP, yielding pHRP-mnp. We obtained, with pHRP-mnp, C. hirsutus strains that show high levels of enzymatic activity of MnP and efficiently degrade pentachlorophenol (PCP), a chlorinated aromatic toxic compound.
  • Isolation and cDNA cloning of novel hydrogen peroxide-dependent phenol oxidase from the basidiomycete Termitomyces albuminosus, T Johjima, M Ohkuma, T Kudo, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 61(3), 220 - 225, May 2003 , Refereed
    Summary:A novel hydrogen peroxide-dependent phenol oxidase (TAP) was isolated from the basidiomycete Termitomyces albuminosus. TAP is an extracellular monomeric enzyme with an estimated molecular weight of 67 kDa. The purified enzyme can oxidize various phenolic compounds in the presence of hydrogen peroxide, but cannot oxidize 3,4-dimethoxybenzyl (veratryl) alcohol. Mn-II was not required for catalysis by TAP. The optimum pH for TAP activity was 2.3, which is the lowest known optimum pH for a fungal phenol oxidase. The cDNA encoding TAP was cloned with reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers based on the N-terminal amino acid sequence of TAP and 5' rapid amplification of cDNA ends (RACE)PCR. The cDNA encodes a mature protein of 449 amino acids with a 55-amino-acid signal peptide. The deduced amino acid sequence of TAP showed 56% identity with dye-decolorizing heme peroxidase (DYP) from the ascomycete Geotrichum candidum Dec 1, but no homology with other known peroxidases from fungi.
  • Electrochemical characterization of lignin peroxidase from the white-rot basidiomycete Phanerochaete chrysosporium, M Oyadomari, H Shinohara, T Johjima, H Wariishi, H Tanaka, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 21(4-6), 291 - 297, Feb. 2003 , Refereed
    Summary:Electrochemical analysis of lignin peroxidase (LiP) was performed using a pyrolytic graphite electrode coated with peroxidase-embedded tributylmethyl phosphonium chloride membrane. The formal redox potential of ferric/ferrous couples of LiP was - 126 mV (versus SHE), which was comparable with that of manganese peroxidase (MnP) and horseradish peroxidase (HRP). Yet, only LiP is capable of oxidizing non-phenolic substrates with a high redox potential. Since with decreasing pH, the redox potential increased, an incredibly low pH optimum of LiP as peroxidase at 3.0 or lower was proposed as the clue to explain LiP mechanisms. A low pH might be the key for UP to possess a high redox potential. The pK(a) values for the distal His in peroxidases were calculated using redox data and the Nernst equation, to be 5.8 for LiP, 4.7 for MnP, and 3.8 for HRP A high pK(a) value of the distal His might be crucial for LiP compound 11 to uptake a proton from the solvent. As a result, LiP is able to complete its catalytic cycle during the oxidation of non-proton-donating substrates. In compensation, LiP has diminished its reactivity toward hydrogen peroxide. (C) 2002 Published by Elsevier Science B.V.
  • Chemical analysis of food processing by the fungus-growing termite Macrotermes gilvus, T Johjima, T Inoue, M Ohkuma, N Noparatnaraporn, T Kudo, SOCIOBIOLOGY, SOCIOBIOLOGY, 42(3), 815 - 824, 2003 , Refereed
    Summary:In order to investigate the mechanism of food processing by the fungus-growing termite Macrotermes gilvus, we analyzed chemical composition of fallen leaves, foodstore, and newer and older part of fungus comb. Proportions of extractives changed considerably during food processing, while proportions of holocellulose remained mostly unchanged. Contents of water-soluble phenolics were much lower in the old part of the fungus comb than in the fallen leaves. Possible roles of foodstore and Termitomyces for reduction of litter toxicity are discussed.
  • Molecular phylogeny of symbiotic basidiomycetes of fungus-growing termites in Thailand and their relationship with the host, Y Taprab, M Ohkuma, T Johjima, Y Maeda, S Moriya, T Inoue, P Suwanarit, N Noparatnaraporn, T Kudo, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66(5), 1159 - 1163, May 2002 , Refereed
    Summary:Termitomyces-related symbiotic basidiomycetes in the nests of fungus-growing termites (Macrotermitinae) of several genera in Thailand were cultivated and analyzed phylogenetically based on the DNA sequence of nuclear ribosomal RNA genes. The relationships of the symbiotic fungi With host termites and their locality were apparently complex, supporting intricate mechanisms for the termites to acquire the symbionts.
  • Veratryl alcohol binding sites of lignin peroxidase from Phanerochaete chrysosporium, T Johjima, H Wariishi, H Tanaka, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 17(2), 49 - 57, Apr. 2002 , Refereed
    Summary:Possible binding sites of lignin peroxidase (LiP) for veratry I alcohol (VA) were investigated by determining the reactivity of three different chemically modified LiPs against VA when acting (i) as a reducing substrate, (ii) as a rescuing reagent for the rapid conversion of LiPIII* back to native LiP, and (iii) as an enzyme-bound redox mediator. The enzyme was chemically modified to alter its surface properties by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence and the absence of 2-aminoethanesulfonic acid to introduce sulfo and N-acylurea groups instead of carboxylic groups, respectively. LiP was also modified by N-bromosuccinimide (NBS) to yield Trp-modified enzyme. The spectral characteristics and compound I formation rates of modified LiPs were identical to those of unmodified LiP. The activities for VA oxidation by modified LiPs were significantly reduced but with almost unchanged pH dependencies. Several other substrates including phenolic. anionic, and polymeric substrates were also utilized to characterize the activity of the modified enzymes. Kinetic analysis of these reactions strongly suggests that LiP has at least two substrate oxidation sites, one is Trp 171 for VA oxidation and the other is for anionic substrate oxidation. For VA-supported oxidation of ferric cytochrome c (Cc(3+)), the VA binding site was estimated to be the same as that of VA oxidation. The reactivities of chemically modified LiPs with excess H2O2 were investigated, indicating the formation of compound III* species. Compound III* species of EDC-LiP and unmodified LiP were converted back to ferric enzymes by adding VA. However, there was a reduced recovery of ferric NBS-LiP, suggesting that the VA binding site for VA-derived rapid reversion of compound III* is located at a different site from that for VA oxidation. (C) 2002 Elsevier Science B.V. All rights reserved.
  • Direct binding of hydroxylamine to the heme iron of Arthromyces ramosus peroxidase - Substrate analogue that inhibits compound I formation in a competitive manner, H Wariishi, D Nonaka, T Johjima, N Nakamura, Y Naruta, S Kubo, K Fukuyama, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 275(42), 32919 - 32924, Oct. 2000 , Refereed
    Summary:The interaction of hydroxylamine (HA) with Arthromyces ramosus peroxidase (ARP) was investigated by kinetic, spectroscopic, and x-ray crystallographic techniques. HA inhibited the reaction of native ARP with H2O2 in a competitive manner. Electron absorption and resonance Raman spectroscopic studies indicated that pentacoordinate high spin species of native ARP are converted to hexacoordinate low spin species upon the addition of HA, strongly suggesting the occurrence of a direct interaction of HA with ARP heme iron. Kinetic analysis exhibited that the apparent dissociation constant is 6.2 mM at pH 7.0 and that only one HA molecule likely binds to the vicinity of the heme. pH dependence of HA binding suggested that the nitrogen atom of HA could be involved in the interaction with the heme iron. X-ray crystallographic analysis of ARP in complex with HA at 2.0 Angstrom resolution revealed that the electron density ascribed to HA is located in the distal pocket between the heme iron and the distal His(56). HA seems to directly interact with the heme iron but is too far away to interact with Arg(52). In HA, it is likely that the nitrogen atom is coordinated to the heme iron and that hydroxyl group is hydrogen bonded to the distal His(56).
  • Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium, T Johjima, N Itoh, M Kabuto, F Tokimura, T Nakagawa, H Wariishi, H Tanaka, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 96(5), 1989 - 1994, Mar. 1999 , Refereed
    Summary:Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 mu M. The LiP-DHP interaction was controlled by the ionization group with a pK(a) of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI-DHP and LiPII-DHP interactions were calculated to be 350 and 250 mu M, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s(-1), respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His-Asp...proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.
    Summary:Binding properties of lignin peroxidase (LiP) against a synthetic lignin (DHP) were studied utilizing the resonant mirror biosensor, Among several ligninolytic enzymes, only LiP specifically binds to DHP. A one-electron transfer from DHP to LiP compound I was characterized using a stopped-flow technique, showing that a binding interaction of DHP with LiP compound I.
  • Fourier transform Raman assignment of guaiacyl and syringyl marker bands for lignin determination, M Takayama, T Johjima, T Yamanaka, H Wariishi, H Tanaka, SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY, SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY, 53(10), 1621 - 1628, Sep. 1997 , Refereed
    Summary:A near infrared fourier transform Raman (NIR-FTR) spectroscopic technique was utilized to characterize lignin in wood. The Raman bands for C=C stretching derived from 4-hydroxy-3-methoxyphenyl (guaiacyl) nuclei and from 3,5-dimethoxy-4-hydroxyphenyl (syringyl) nuclei exist independently. The NIR-FTR analysis of a series of lignin model compounds indicated that a syringyl band was shifted to a lower frequency compared to a guaiacyl band. This shift was also observed in chemically synthesized lignin (DHP). Syringyl DHP, in which all the aromatic nuclei consist of syringyl type, exhibited a C=C stretching band at 1594 cm(-1), while guaiacyl DHP exhibited the band at 1599 cm(-1). These bands were designated as syringyl and guaiacyl marker bands, respectively. Chemical and physical treatment of hardwood and softwood exhibited different characteristics. One of the reasons is the chemical structure of lignin. Softwood mainly contains only guaiacyl lignin, while hardwood contains both guaiacyl and syringyl lignin, and the syringyl/guaiacyl (S/G) ratio varies among species. Under high-resolution conditions (1 cm(-1)), the NIR-FTR spectra of 10 hardwoods (wood meal samples) revealed that both syringyl and guaiacyl marker bands existed. On the other hand, the spectra of softwoods contained only a guaiacyl marker bands existed. On the other hand, the spectra of softwoods contained only a guaiacyl marker band. The S/G ratio in hardwood calculated from the peak area intensity ratio of two marker bands shows a linear relationship with the SIG ratio obtained from conventional nitrobenzene oxidation analysis with the correlation factor>0.96. Furthermore, if peak component separation analysis was combined, low-resolution spectral data gave a similar S/G ratio. Either syringyl of guaiacyl marker bands can be assigned in the NIR-FTR spectra of wood blocks (saw-cut surface). This spectral technique may provide an easy-handling and non-destructive analytical method for lignin determination. (C) 1997 Elsevier Science B.V.
  • The effect of ligand field strength on nonresonance Raman characteristics of hemoproteins, T Johjima, H Wariishi, H Tanaka, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 226(3), 601 - 606, Sep. 1996 , Refereed
    Summary:A series of hemoproteins were characterized using Raman spectroscopic technique under non-resonant (near-infrared excited) conditions. All the proteins used in this study contain an iron-protoporphyrin IX with a coordinated histidine as a proximal ligand. Hemoproteins exhibited a near-infrared Raman shift at 1372 cm(-1), only when heme was in the ferric state, while the peak completely disappeared when heme iron was reduced. The intensity of this peak was weakened upon the coordination of electron-donating ligands to heme iron. Therefore, the characteristics of this peak are different from the oxidation marker band assigned by the resonance Raman spectroscopy, rather, the intensity is strongly related to the sixth ligand field strength. In addition, the peak intensity may also reflect the distance between heme iron and the sixth ligand. (C) 1996 Academic Press, Inc.

Books etc

  • Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, Jojima, T, Inui, T, Yukawa, H, Contributor, L-isoleucine, Wilely,   2010
  • Termitomyces/termite interactions, Rouland-Lefèvre, Corinne, Inoue, Tetsushi, Johjima, Toru, Contributor,   2006


  • リサイクル部会,バイオマス部会,ガス化部会ー三部会合同シンポジウム 廃棄物,バイオマス,石炭等利用技術の最新動向 ソフトバイオマスからのバイオエタノール製造技術開発, 湯川英明, 城島透, J Jpn Inst Energy, 87, 5, 333, 335,   2008 05 20 ,
  • ソフトバイオマスからのバイオ燃料製造技術とRITEの研究開発, 城島透, 湯川英明, 酵素工学ニュ-ス, 59, 7, 11,   2008 04 ,
  • 夢のある農林水産研究 ソフトバイオマスからのバイオ燃料製造, 城島透, 湯川英明, 農林水産技術研究ジャーナル, 31, 1, 50, 52,   2008 01 01 ,
  • 2J14-4 Production of organic acids from sugar mixture by recombinant Corynebacterium glutamicum, SASAKI Miho, JYOJIMA Toru, OKINO Shohei, INUI Masayuki, YUKAWA Hideaki, 日本生物工学会大会講演要旨集, 19,   2007 ,
  • 人工水田におけるネオニコチノイド系農薬が微生物生態系に及ぼす影響評価, 尾枝良哉, 伊藤秋実, 脇屋香, 城島透, 早坂大亮, 森美穂, 日本防菌防黴学会年次大会要旨集, 45th, 292,   2018 11 12 ,