Department of Environmental Management Professor/Manager
Last Updated :2024/05/29

Researcher Information

Research funding number

  • 20754229

J-Global ID

Research Interests

  • バイオリファイナリー   代謝工学   

Research Areas

  • Life sciences / Applied microbiology

Association Memberships


Published Papers

  • Toru Jojima; Yuki Ioku; Yasuhisa Fukuta; Norifumi Shirasaka; Yoshinobu Matsumura; Miho Mori
    International journal of systematic and evolutionary microbiology 73 (5) 2023/05
  • Toru Jojima; Takafumi Igari; Ryoji Noburyu; Akira Watanabe; Masako Suda; Masayuki Inui
    Biotechnology for Biofuels Springer Science and Business Media LLC 14 (1) 45 - 45 2021/12 [Refereed]
    Abstract Background It is interesting to modify sugar metabolic pathways to improve the productivity of biocatalysts that convert sugars to value-added products. However, this attempt often fails due to the tight control of the sugar metabolic pathways. Recently, activation of the Entner–Doudoroff (ED) pathway in Escherichia coli has been shown to enhance glucose consumption, though the mechanism underlying this phenomenon is poorly understood. In the present study, we investigated the effect of a functional ED pathway in metabolically engineered Corynebacterium glutamicum that metabolizes glucose via the Embden–Meyerhof–Parnas (EMP) pathway to produce ethanol under oxygen deprivation. This study aims to provide further information on metabolic engineering strategies that allow the Entner–Doudoroff and Embden–Meyerhof–Parnas pathways to coexist. Results Three genes (zwf, edd, and eda) encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis were expressed in a genetically modified strain, C. glutamicum CRZ2e, which produces pyruvate decarboxylase and alcohol dehydrogenase from Z. mobilis. A 13C-labeling experiment using [1-13C] glucose indicated a distinctive 13C distribution of ethanol between the parental and the ED-introduced strains, which suggested an alteration of carbon flux as a consequence of ED pathway introduction. The ED-introduced strain, CRZ2e-ED, consumed glucose 1.5-fold faster than the parental strain. A pfkA deletion mutant of CRZ2e-ED (CRZ2e-EDΔpfkA) was also constructed to evaluate the effects of EMP pathway inactivation, which showed an almost identical rate of glucose consumption compared to that of the parental CRZ2e strain. The introduction of the ED pathway did not alter the intracellular NADH/NAD+ ratio, whereas it resulted in a slight increase in the ATP/ADP ratio. The recombinant strains with simultaneous overexpression of the genes for the EMP and ED pathways exhibited the highest ethanol productivity among all C. glutamicum strains ever constructed. Conclusions The increased sugar consumption observed in ED-introduced strains was not a consequence of cofactor balance alterations, but rather the crucial coexistence of two active glycolytic pathways for enhanced glucose consumption. Coexistence of the ED and EMP pathways is a good strategy for improving biocatalyst productivity even when NADPH supply is not a limiting factor for fermentation.
  • Miho Mori; Yoshikazu Sakagami; Megumi Tanaka; Ryo Inoue; Toru Jojima
    Journal of food protection 83 (7) 1234 - 1240 2020/07 
    ABSTRACT: Hygiene management of domestic refrigerators is an important aspect of food poisoning prevention. The aim of the present study was to confirm the relationship between microbial contamination and hygiene management by measuring microbial levels and investigating temperature and cleaning frequency and method of domestic refrigerators in Japan. We analyzed three internal sections (the egg compartment, bottom shelf, and vegetable drawer) of 100 domestic refrigerators in Japan. Salmonella, Listeria monocytogenes, and Yersinia enterocolitica were not found in any of the refrigerators, but coliforms and Escherichia coli were detected in more than one household, and Staphylococcus aureus was the most frequently isolated pathogen. The prevalences of these microorganisms had similar tendencies in all three sections sampled and were highest in the vegetable drawer. The temperature distribution in the refrigerators was also investigated, and a temperature >6.1°C (improper temperature) was found in 46.2% of the areas surveyed. Only 17% of the respondents cleaned their refrigerators monthly or more often, and this frequency was lower than that reported in other countries. Fifty percent of the respondents used only water to clean the refrigerator, 10% used only an alcohol or disinfecting wipe, and 8% used only a dry cloth. Although no significant correlations were found between microbial contamination and temperatures in refrigerators, correlations were found between microbial contamination and refrigerator cleaning frequency and/or method. To our knowledge, this is the first detailed survey concerning relationships between microbial contamination and hygiene management in domestic refrigerators in Japan. The data obtained can be used to promote food poisoning management in Japanese households.
  • Hasegawa S; Jojima T; Suda M; Inui M
    Metabolic engineering 59 24 - 35 1096-7176 2020/01 [Refereed]
    On the basis of our previous studies of microbial L-valine production under oxygen deprivation, we developed isobutanol-producing Corynebacterium glutamicum strains. The artificial isobutanol synthesis pathway was composed of the first three steps of the L-valine synthesis pathway; and the subsequent Ehrlich Pathway: pyruvate was converted to 2-ketoisovalerate in the former reactions; and the 2-keto acid was decarboxylated into isobutyraldehyde, and subsequently reduced into isobutanol in the latter reactions. Although there exists redox cofactor imbalance in the overall reactions, i.e., NADH is generated via glycolysis whereas NADPH is required to synthesize isobutanol, it was resolved by taking advantage of the NAD-preferring mutant acetohydroxy acid isomeroreductase encoded by ilvCTM and the NAD-specific alcohol dehydrogenase encoded by adhA. Each enzyme activity to synthesize isobutanol was finely tuned by using two kinds of lac promoter derivatives. Efficient suppression of succinate by-production and improvement of isobutanol yield resulted from inactivation of pckA, which encodes phosphoenolpyruvate carboxykinase, whereas glucose consumption and isobutanol production rates decreased because of the elevated intracellular NADH/NAD+ ratio. On the other hand, introduction of the exogenous Entner-Doudoroff pathway effectively enhanced glucose consumption and productivity. Overexpression of phosphoenolpyruvate:carbohydrate phosphotransferase system specific to glucose and deletion of ilvE, which encodes branched-chain amino acid transaminase, further suppressed by-products and improved isobutanol productivity. Finally, the produced isobutanol concentration reached 280 mM at a yield of 84% (mol/mol glucose) in 24 h.
  • Yota Tsuge; Naoto Kato; Shogo Yamamoto; Masako Suda; Toru Jojima; Masayuki Inui
    Applied microbiology and biotechnology 103 (8) 3381 - 3391 0175-7598 2019/04 [Refereed]
    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.
  • Mori M; Sakagami Y; Hamazaki Y; Jojima T
    Environmental technology 1 - 11 0959-3330 2018/04 [Refereed]
  • Satoshi Hasegawa; Toru Jojima; Masayuki Inui
    Journal of Microbiological Methods Elsevier B.V. 146 13 - 15 1872-8359 2018/03 [Refereed]
    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.
  • Satoshi Hasegawa; Yuya Tanaka; Masako Suda; Toru Jojima; Masayuki Inui
    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.
  • Jojima, Toru; Noburyu, Ryoji; Suda, Masako; Okino, Shohei; Yukawa, Hideaki; Inui, Maysayuki
    Fermentation 2 (1) 5  2016 [Refereed]
  • Toru Jojima; Masayuki Inui
    BIOENGINEERED TAYLOR & FRANCIS INC 6 (6) 328 - 334 2165-5979 2015/11 [Refereed]
    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.
  • Shinichi Oide; Wataru Gunji; Yasuhiro Moteki; Shogo Yamamoto; Masako Suda; Toru Jojima; Hideaki Yukawa; Masayuki Inui
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY AMER SOC MICROBIOLOGY 81 (7) 2284 - 2298 0099-2240 2015/04 [Refereed]
    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.
  • Toru Jojima; Ryoji Noburyu; Miho Sasaki; Takahisa Tajima; Masako Suda; Hideaki Yukawa; Masayuki Inui
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 99 (3) 1165 - 1172 0175-7598 2015/02 [Refereed]
    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.
  • Toru Jojima; Takafumi Igari; Yasuhiro Moteki; Masako Suda; Hideaki Yukawa; Masayuki Inui
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 99 (3) 1427 - 1433 0175-7598 2015/02 [Refereed]
    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.
  • Toru Jojima; Alain A. Vertès; Masayuki Inui; Hideaki Yukawa
    Biorefineries: Integrated Biochemical Processes for Liquid Biofuels Elsevier Inc. 121 - 139 2014/08 [Refereed]
    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.
  • Satoshi Hasegawa; Masako Suda; Kimio Uematsu; Yumi Natsuma; Kazumi Hiraga; Toru Jojima; Masayuki Inui; Hideaki Yukawa
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY AMER SOC MICROBIOLOGY 79 (4) 1250 - 1257 0099-2240 2013/02 [Refereed]
    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.
  • Toru Jojima; Takafumi Igari; Wataru Gunji; Masako Suda; Masayuki Inui; Hideaki Yukawa
    FEBS LETTERS WILEY 586 (23) 4228 - 4232 1873-3468 2012/11 [Refereed]
    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.
  • Shogo Yamamoto; Wataru Gunji; Hiroaki Suzuki; Hiroshi Toda; Masako Suda; Toru Jojima; Masayuki Inui; Hideaki Yukawa
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY AMER SOC MICROBIOLOGY 78 (12) 4447 - 4457 0099-2240 2012/06 [Refereed]
    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.
  • Satoshi Hasegawa; Kimio Uematsu; Yumi Natsuma; Masako Suda; Kazumi Hiraga; Toru Jojima; Masayuki Inui; Hideaki Yukawa
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY AMER SOC MICROBIOLOGY 78 (3) 865 - 875 0099-2240 2012/02 [Refereed]
    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.
  • Toru Jojima; Masayuki Inui; Hideaki Yukawa
    Biofuels 2 (3) 303 - 313 1759-7269 2011/05 [Refereed]
    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.
  • Toru Jojima; Miho Fujii; Eiji Mori; Masayuki Inui; Hideaki Yukawa
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 87 (1) 159 - 165 0175-7598 2010/06 [Refereed]
    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.
  • Miho Sasaki; Toru Jojima; Masayuki Inui; Hideaki Yukawa
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 86 (4) 1057 - 1066 0175-7598 2010/04 [Refereed]
    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.
  • Toru Jojima; Crispinus A. Omumasaba; Masayuki Inui; Hideaki Yukawa
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 85 (3) 471 - 480 0175-7598 2010/01 [Refereed]
    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.
  • Miho Sasaki; Toru Jojima; Hideo Kawaguchi; Masayuki Inui; Hideaki Yukawa
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 85 (1) 105 - 115 0175-7598 2009/11 [Refereed]
    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.
  • Miho Sasaki; Toru Jojima; Masayuki Inui; Hideaki Yukawa
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 81 (4) 691 - 699 0175-7598 2008/12 [Refereed]
    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.
  • Shohei Okino; Ryoji Noburyu; Masako Suda; Toru Jojima; Masayuki Inui; Hideaki Yukawa
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 81 (3) 459 - 464 0175-7598 2008/12 [Refereed]
    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.
  • Takehito Nakazawa; Shinya Kaneko; Yasumasa Miyazaki; Toru Jojima; Takashi Yamazaki; Shiho Katsukawa; Kazuo Shishido
    FUNGAL GENETICS AND BIOLOGY ACADEMIC PRESS INC ELSEVIER SCIENCE 45 (6) 818 - 828 1087-1845 2008/06 [Refereed]
    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.
  • Toru Jojima; Masayuki Inui; Hideaki Yukawa
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 77 (6) 1219 - 1224 0175-7598 2008/01 [Refereed]
    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.
  • Kaori Yasuda; Toru Jojima; Masako Suda; Shohei Okino; Masayuki Inui; Hideaki Yukawa
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 77 (4) 853 - 860 0175-7598 2007/12 [Refereed]
    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.
  • Toru Johjima; Yaovapa Taprab; Napavarn Noparatnaraporn; Toshiaki Kudo; Moriya Ohkuma
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 73 (1) 195 - 203 0175-7598 2006/11 [Refereed]
    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.
  • Tetsushi Inoue; Yoko Takematsu; Akinori Yamada; Yuichi Hongoh; Toru Johjima; Shigeharu Moriya; Yupaporn Sornnuwat; Charunee Vongkaluang; Moriya Ohkuma; Toshiaki Kudo
    JOURNAL OF TROPICAL ECOLOGY CAMBRIDGE UNIV PRESS 22 (5) 609 - 612 0266-4674 2006/09 [Refereed]
  • Y Taprab; T Johjima; Y Maeda; S Moriya; S Trakulnaleamsai; N Noparatnaraporn; M Ohkuma; T Kudo
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY AMER SOC MICROBIOLOGY 71 (12) 7696 - 7704 0099-2240 2005/12 [Refereed]
    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.
  • Shigeharu Moriya; Tetsushi Inoue; Moriya Ohkuma; Taprab Yaovapa; Tohru Johjima; Poonpilai Suwanarit; Utaiwan Sangwamt; Charunee Vongkaluang; Napavarn Noparatnaraporn; Toshiaki Kudo
    Microbes and Environments 20 (4) 243 - 252 1347-4405 2005 [Refereed]
    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.
  • Y Miyazaki; T Jojima; T Ono; T Yamazaki; K Shishido
    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.
  • Takashi Yamazaki; Toshiro Kiyofuji; Toru Johjima; Susumu Kajiwara; Akira Tsukamoto; Jun Sugiura; Kazuo Shishido
    Mycoscience Springer Japan 45 (5) 317 - 323 1340-3540 2004 [Refereed]
    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.
  • T Johjima; M Ohkuma; T Kudo
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY SPRINGER 61 (3) 220 - 225 0175-7598 2003/05 [Refereed]
    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.
  • M Oyadomari; H Shinohara; T Johjima; H Wariishi; H Tanaka
    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC ELSEVIER SCIENCE BV 21 (4-6) 291 - 297 1381-1177 2003/02 [Refereed]
    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.
  • T Johjima; T Inoue; M Ohkuma; N Noparatnaraporn; T Kudo
    SOCIOBIOLOGY UNIV ESTADUAL FEIRA SANTANA 42 (3) 815 - 824 0361-6525 2003 [Refereed]
    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.
  • Y Taprab; M Ohkuma; T Johjima; Y Maeda; S Moriya; T Inoue; P Suwanarit; N Noparatnaraporn; T Kudo
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 66 (5) 1159 - 1163 0916-8451 2002/05 [Refereed]
    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.
  • T Johjima; H Wariishi; H Tanaka
    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC ELSEVIER SCIENCE BV 17 (2) 49 - 57 1381-1177 2002/04 [Refereed]
    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.
  • H Wariishi; D Nonaka; T Johjima; N Nakamura; Y Naruta; S Kubo; K Fukuyama
    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).
  • T Johjima; N Itoh; M Kabuto; F Tokimura; T Nakagawa; H Wariishi; H Tanaka
    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.
  • T Jojima; N Itoh; H Wariishi; H Tanaka
    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.
  • M Takayama; T Johjima; T Yamanaka; H Wariishi; H Tanaka
    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.
  • T Johjima; H Wariishi; H Tanaka
    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

  • Corynebacterium glutamicum: From Systems Biology to Biotechnological Applications
    Jojima, T; Inui, T; Yukawa, H (Biotechnological Application of Corynebacterium glutamicum Under Oxygen Deprivation)Caister Academic Press 2015 151-160
  • Biorefineries, Integrated Biochemical Process for Liquid Biofuels
    Jojima, T; Inui, T; Yukawa, H (Development of growth-arrested bioprocesses with Corynebacterium glutamicum for cellulosic ethanol production from complex sugar mixtures)Elsevier 2014 121-139
  • Microbiology Monograph series, Corynebacterium glutamicum.
    Jojima, T; Inui, T; Yukawa, H (Biorefinery applications of Corynebacterium glutamicum)Springer Berlin Heidelberg 2012 149-172
  • セルロース系バイオエタノール製造技術-食料クライシス回避のために
    城島透; 乾将之; 湯川英明 (ソフトバイオマスを原料にしたコリネ型細菌による混合糖同時変換エタノール製造技術)エヌティーエス出版 2010 277-290
  • 未利用バイオマスの活用技術と事業性評価
    城島透; 湯川英明 (Contributor非可食バイオマスからのエタノール製造技術と化学品生産)サイエンス&テクノロジー株式会社 2010 235-247
  • 次世代バイオエタノール生産の技術革新と事業展開
    城島透; 湯川英明 (Contributor海外におけるセルロースエタノール導入・研究開発動向)フロンティア出版 2010 35-49
  • Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology
    Jojima, T; Inui, T; Yukawa, H (ContributorL-isoleucine)Wilely 2010 209-214
  • Termitomyces/termite interactions
    Rouland-Lefèvre, Corinne; Inoue, Tetsushi; Johjima, Toru (Contributor)2006 335-350


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