Publications by authors named "Tomokazu Shirai"

38 Publications

Reprogramming Escherichia coli pyruvate-forming reaction towards chorismate derivatives production.

Metab Eng 2021 May 24;67:1-10. Epub 2021 May 24.

Center for Sustainable Resource Science, RIKEN, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan; Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.

Microbial metabolic pathway engineering is a potent strategy used worldwide to produce aromatic compounds. We drastically rewired the primary metabolic pathway of Escherichia coli to produce aromatics and their derivatives. The metabolic pathway of E. coli was compartmentalized into the production and energy modules. We focused on the pyruvate-forming reaction in the biosynthesis pathway of some compounds as the reaction connecting those modules. E. coli strains were engineered to show no growth unless pyruvate was synthesized along with the compounds of interest production. Production of salicylate and maleate was demonstrated to confirm our strategy's versatility. In maleate production, the production, yield against the theoretical yield, and production rate reached 12.0 g L, 67%, and up to fourfold compared to that in previous reports, respectively; these are the highest values of maleate production in microbes to our knowledge. The results reveal that our strategy strongly promotes the production of aromatics and their derivatives.
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http://dx.doi.org/10.1016/j.ymben.2021.05.005DOI Listing
May 2021

C-metabolic flux analysis in glycerol-assimilating strains of Saccharomyces cerevisiae.

J Gen Appl Microbiol 2021 May 8. Epub 2021 May 8.

School of Life Science and Technology, Tokyo Institute of Technology.

Glycerol is an attractive raw material for the production of useful chemicals using microbial cells. We previously identified metabolic engineering targets for the improvement of glycerol assimilation ability in Saccharomyces cerevisiae based on adaptive laboratory evolution (ALE) and transcriptome analysis of the evolved cells. We also successfully improved glycerol assimilation ability by the disruption of the RIM15 gene encoding a Greatwall protein kinase together with overexpression of the STL1 gene encoding the glycerol/H symporter. To understand glycerol assimilation metabolism in the evolved glycerol-assimilating strains and STL1-overexpressing RIM15 disruptant, we performed metabolic flux analysis using C-labeled glycerol. Significant differences in metabolic flux distributions between the strains obtained from the culture after 35 and 85 generations in ALE were not found, indicating that metabolic flux changes might occur in the early phase of ALE (i.e., before 35 generations at least). Similarly, metabolic flux distribution was not significantly changed by RIM15 gene disruption. However, fluxes for the lower part of glycolysis and the TCA cycle were larger and, as a result, flux for the pentose phosphate pathway was smaller in the STL1-overexpressing RIM15 disruptant than in the strain obtained from the culture after 85 generations in ALE. It could be effective to increase flux for the pentose phosphate pathway to improve the glycerol assimilation ability in S. cerevisiae.
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http://dx.doi.org/10.2323/jgam.2020.10.001DOI Listing
May 2021

Direct 1,3-butadiene biosynthesis in Escherichia coli via a tailored ferulic acid decarboxylase mutant.

Nat Commun 2021 04 13;12(1):2195. Epub 2021 Apr 13.

Center for Sustainable Resource Science, RIKEN, Yokohama, Japan.

The C4 unsaturated compound 1,3-butadiene is an important monomer in synthetic rubber and engineering plastic production. However, microorganisms cannot directly produce 1,3-butadiene when glucose is used as a renewable carbon source via biological processes. In this study, we construct an artificial metabolic pathway for 1,3-butadiene production from glucose in Escherichia coli by combining the cis,cis-muconic acid (ccMA)-producing pathway together with tailored ferulic acid decarboxylase mutations. The rational design of the substrate-binding site of the enzyme by computational simulations improves ccMA decarboxylation and thus 1,3-butadiene production. We find that changing dissolved oxygen (DO) levels and controlling the pH are important factors for 1,3-butadiene production. Using DO-stat fed-batch fermentation, we produce 2.13 ± 0.17 g L 1,3-butadiene. The results indicate that we can produce unnatural/nonbiological compounds from glucose as a renewable carbon source via a rational enzyme design strategy.
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http://dx.doi.org/10.1038/s41467-021-22504-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044207PMC
April 2021

Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803.

Metab Eng 2021 05 17;65:88-98. Epub 2021 Mar 17.

School of Agriculture, Meiji University, 1-1-1, Higashimita, Tama-ku, Kawasaki, Kanagawa, 214-8571, Japan. Electronic address:

Succinate, fumarate, and malate are valuable four-carbon (C4) dicarboxylic acids used for producing plastics and food additives. C4 dicarboxylic acid is biologically produced by heterotrophic organisms. However, current biological production requires organic carbon sources that compete with food uses. Herein, we report C4 dicarboxylic acid production from CO using metabolically engineered Synechocystis sp. PCC 6803. Overexpression of citH, encoding malate dehydrogenase (MDH), resulted in the enhanced production of succinate, fumarate, and malate. citH overexpression increased the reductive branch of the open cyanobacterial tricarboxylic acid (TCA) cycle flux. Furthermore, product stripping by medium exchanges increased the C4 dicarboxylic acid levels; product inhibition and acidification of the media were the limiting factors for succinate production. Our results demonstrate that MDH is a key regulator that activates the reductive branch of the open cyanobacterial TCA cycle. The study findings suggest that cyanobacteria can act as a biocatalyst for converting CO to carboxylic acids.
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http://dx.doi.org/10.1016/j.ymben.2021.03.007DOI Listing
May 2021

Optimal Ratio of Carbon Flux between Glycolysis and the Pentose Phosphate Pathway for Amino Acid Accumulation in .

ACS Synth Biol 2020 07 30;9(7):1615-1622. Epub 2020 Jun 30.

Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan.

Glucose is metabolized through central metabolic pathways such as glycolysis and the pentose phosphate pathway (PPP) to synthesize downstream metabolites including amino acids. However, how the split ratio of carbon flux between glycolysis and PPP specifically affects the formation of downstream metabolites remains largely unclear. Here, we conducted a comprehensive metabolomic analysis to investigate the effect of the split ratio between glycolysis and the PPP on the intracellular concentration of amino acids and their derivatives in . The split ratio was varied by exchanging the promoter of a gene encoding glucose 6-phosphate isomerase (PGI). The ratio was correlated with the transcription level and the enzyme activity. Concentrations of threonine and lysine-derivative 1,5-diaminopentane increased with an increase of the split ratio into the PPP. In contrast, concentrations of alanine, leucine, and valine were increased with an increase of the split ratio into glycolysis. These results could provide a new engineering target for improving the production of the amino acids and the derivatives.
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http://dx.doi.org/10.1021/acssynbio.0c00181DOI Listing
July 2020

Automatic Redirection of Carbon Flux between Glycolysis and Pentose Phosphate Pathway Using an Oxygen-Responsive Metabolic Switch in .

ACS Synth Biol 2020 04 23;9(4):814-826. Epub 2020 Mar 23.

Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan.

Controlling the carbon flux into a desired pathway is important for improving product yield in metabolic engineering. After entering a cell, glucose is channeled into glycolysis and the pentose phosphate pathway (PPP), which decreases the yield of target products whose synthesis relies on NADPH as a cofactor. Here, we demonstrate redirection of carbon flux into PPP under aerobic conditions in , achieved by replacing the promoter of glucose 6-phosphate isomerase gene () with an anaerobic-specific promoter of the lactate dehydrogenase gene (). The promoter replacement increased the split ratio of carbon flux into PPP from 39 to 83% under aerobic conditions. The titer, yield, and production rate of 1,5-diaminopentane, whose synthesis requires NADPH as a cofactor, were increased by 4.6-, 4.4-, and 2.6-fold, respectively. This is the largest improvement in the production of 1,5-diaminopentane or its precursor, lysine, reported to date. After aerobic cell growth, expression was automatically induced under anaerobic conditions, altering the carbon flux from PPP to glycolysis, to produce succinate in a single metabolically engineered strain. Such an automatic redirection of metabolic pathway using an oxygen-responsive switch enables two-stage fermentation for efficient production of two different compounds by a single strain, potentially reducing the production costs and time for practical applications.
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http://dx.doi.org/10.1021/acssynbio.9b00493DOI Listing
April 2020

Simultaneous increases in the levels of compatible solutes by cost-effective cultivation of Synechocystis sp. PCC 6803.

Biotechnol Bioeng 2020 06 15;117(6):1649-1660. Epub 2020 Mar 15.

Department of Agricultural Chemistrym School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan.

Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L ·day , exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.
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http://dx.doi.org/10.1002/bit.27324DOI Listing
June 2020

Reconstruction of metabolic pathway for isobutanol production in Escherichia coli.

Microb Cell Fact 2019 Jul 18;18(1):124. Epub 2019 Jul 18.

Center for Sustainable Resource Science, RIKEN, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.

Background: The microbial production of useful fuels and chemicals has been widely studied. In several cases, glucose is used as the raw material, and almost all microbes adopt the Embden-Meyerhof (EM) pathway to degrade glucose into compounds of interest. Recently, the Entner-Doudoroff (ED) pathway has been gaining attention as an alternative strategy for microbial production.

Results: In the present study, we attempted to apply the ED pathway for isobutanol production in Escherichia coli because of the complete redox balance involved. First, we generated ED pathway-dependent isobutanol-producing E. coli. Thereafter, the inactivation of the genes concerning organic acids as the byproducts was performed to improve the carbon flux to isobutanol from glucose. Finally, the expression of the genes concerning the ED pathway was modified.

Conclusions: The optimized isobutanol-producing E. coli produced 15.0 g/L of isobutanol as the final titer, and the yield from glucose was 0.37 g/g (g-glucose/g-isobutanol).
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http://dx.doi.org/10.1186/s12934-019-1171-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637570PMC
July 2019

Construction of a Model Culture System of Human Colonic Microbiota to Detect Decreased Lachnospiraceae Abundance and Butyrogenesis in the Feces of Ulcerative Colitis Patients.

Biotechnol J 2019 May 15;14(5):e1800555. Epub 2019 Apr 15.

Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan.

Compositional alteration of the gut microbiota is associated with ulcerative colitis (UC). Here, a model culture system is established for the in vitro human colonic microbiota of UC, which will be helpful for determining medical interventions. 16S ribosomal RNA sequencing confirms that UC models are successfully developed from fecal inoculum and retain the bacterial species biodiversity of UC feces. The UC models closely reproduce the microbial components and successfully preserve distinct clusters from the healthy subjects (HS), as observed in the feces. The relative abundance of bacteria belonging to the family Lachnospiraceae significantly decreases in the UC models compared to that in HS, as observed in the feces. The system detects significantly lower butyrogenesis in the UC models than that in HS, correlating with the decreased abundance of Lachnospiraceae. Interestingly, the relative abundance of Lachnospiraceae does not correlate with disease activity (defined as partial Mayo score), suggesting that Lachnospiraceae persists in UC patients at a decreased level, irrespective of the alteration in disease activity. Moreover, the system shows that administration of Clostridium butyricum MIYAIRI restores butyrogenesis in the UC model. Hence, the model detects deregulation in the intestinal environment in UC patients and may be useful for simulating the effect of probiotics.
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http://dx.doi.org/10.1002/biot.201800555DOI Listing
May 2019

Designing artificial metabolic pathways, construction of target enzymes, and analysis of their function.

Curr Opin Biotechnol 2018 12 20;54:41-44. Epub 2018 Feb 20.

Biomass Engineering Research Division, Center for Sustainable Resource Science, RIKEN, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. Electronic address:

Artificial design of metabolic pathways is essential for the production of useful compounds using microbes. Based on this design, heterogeneous genes are introduced into the host, and then various analysis and evaluation methods are conducted to ensure that the target enzyme reactions are functionalized within the cell. In this chapter, we list successful examples of useful compounds produced by designing artificial metabolic pathways, and describe the methods involved in analyzing, evaluating, and optimizing the target enzyme reaction.
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http://dx.doi.org/10.1016/j.copbio.2018.01.021DOI Listing
December 2018

Efficient 3-Hydroxybutyrate Production by Quiescent Escherichia coli Microbial Cell Factories is Facilitated by Indole-Induced Proteomic and Metabolomic Changes.

Biotechnol J 2018 May 31;13(5):e1700571. Epub 2018 Jan 31.

Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.

The authors show that quiescent (Q-Cell) Escherichia coli cultures can maintain metabolic activity in the absence of growth for up to 24 h, leading to four times greater specific productivity of a model metabolite, 3-hydroxybutyrate (3HB), than a control. Q-cells can be created by using the proton ionophore indole to halt cell division of an hns mutant strain. This uncouples metabolism from cell growth and allows for more efficient use of carbon feedstocks because less metabolic effort is diverted to surplus biomass production. However, the reason for the increased productivity of cells in the quiescent state was previously unknown. In this study, proteome expression patterns between wild-type and Q-cell cultures show that Q-cells overexpress stress response proteins, which prime them to tolerate the metabolic imbalances incurred through indole addition. Metabolomic data reveal the accumulation of acetyl-coenzyme A and phosphoenolpyruvate: excellent starting points for high-value chemical production. We demonstrate the exploitation of these accumulated metabolites by engineering a simple pathway for 3HB production from acetyl-coenzyme A. Quiescent cultures produced half the cell biomass of control cultures lacking indole, but were still able to produce 39.4 g L of 3HB compared to 18.6 g L in the control. Q-cells therefore have great potential as a platform technology for the efficient production of a wide range of commodity and high value chemicals.
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http://dx.doi.org/10.1002/biot.201700571DOI Listing
May 2018

Engineering a synthetic pathway for maleate in Escherichia coli.

Nat Commun 2017 10 27;8(1):1153. Epub 2017 Oct 27.

Center for Sustainable Resource Science, RIKEN, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.

Maleate is one of the most important dicarboxylic acids and is used to produce various polymer compounds and pharmaceuticals. Herein, microbial production of maleate is successfully achieved, to our knowledge for the first time, using genetically modified Escherichia coli. A synthetic pathway of maleate is constructed in E. coli by combining the polyketide biosynthesis pathway and benzene ring cleavage pathway. The metabolic engineering approach used to fine-tune the synthetic pathway drastically improves maleate production and demonstrates that one of the rate limiting steps exists in the conversion of chorismate to gentisate. In a batch culture of the optimised transformant, grown in a 1-L jar fermentor, the amount of produced maleate reaches 7.1 g L, and the yield is 0.221 mol mol. Our results suggest that the construction of synthetic pathways by combining a secondary metabolite pathway and the benzene ring cleavage pathway is a powerful tool for producing various valuable chemicals.
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http://dx.doi.org/10.1038/s41467-017-01233-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658364PMC
October 2017

Metabolic engineering of isopropyl alcohol-producing Escherichia coli strains with C-metabolic flux analysis.

Biotechnol Bioeng 2017 12 17;114(12):2782-2793. Epub 2017 Aug 17.

Department of Bioinfomatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan.

Metabolic engineering of isopropyl alcohol (IPA)-producing Escherichia coli strains was conducted along with C-metabolic flux analysis (MFA). A metabolically engineered E. coli strain expressing the adc gene derived from Clostridium acetobutylicum and the IPADH gene from C. beijerinckii did not produce IPA during its exponential growth phase in the aerobic batch culture. C-MFA was carried out, and revealed a deficiency in NADPH regeneration for IPA production in growth phase. Based on these findings, we used nitrogen-starved culture conditions to reduce NADPH consumption for biomass synthesis. As a result, IPA yield was increased to 20% mol/mol glucose. C-MFA revealed that the relative flux levels through the oxidative pentose phosphate (PP) pathway and the TCA cycle were elevated in nitrogen-starved condition relative to glucose uptake rate. To prevent CO release in the 6-phosphogluconate dehydrogenase (6PGDH) reaction, metabolism of this E. coli strain was further engineered to redirect glycolytic flux to the glucose 6-phosphate dehydrogenase (G6PDH) and Entner-Doudoroff (ED) pathway. IPA yield of 55% mol/mol glucose was achieved by combining the nitrogen-starved culture condition with the metabolic redirection. The C-MFA data and intracellular NADPH levels obtained under these IPA production conditions revealed linear correlations between the specific IPA production rate and NADPH concentration, as well as between IPA yield and the pyruvate dehydrogenase (PDH) flux. Our results showed that C-MFA is a helpful tool for metabolic engineering studies, and that further improvement in IPA production by E. coli may be achieved by fine-tuning the cofactor ratio and concentrations, as well as optimizing the metabolic pathways and culture conditions.
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http://dx.doi.org/10.1002/bit.26390DOI Listing
December 2017

Differences in glucose yield of residues from among varieties of rice, wheat, and sorghum after dilute acid pretreatment.

Biosci Biotechnol Biochem 2017 Aug 16;81(8):1650-1656. Epub 2017 Jun 16.

a Graduate School of Science, Technology and Innovation, Kobe University , Kobe , Japan.

Bio-refinery processes require use of the most suitable lignocellulosic biomass for enzymatic saccharification and microbial fermentation. Glucose yield from biomass solid fractions obtained after dilute sulfuric acid (1%) pretreatment (at 180 °C) was investigated using 14, 8, and 16 varieties of rice, wheat, and sorghum, respectively. Biomass solid fractions of each crop showed similar cellulose content. However, glucose yield after enzymatic hydrolysis (cellulase loading at 6.6 filter paper unit/g-biomass) was different among the varieties of each crop, indicating genotypic differences for rice, wheat, and sorghum. Nuclear magnetic resonance method revealed that the high residual level of lignin aromatic regions decreased glucose yield from solid fraction of sorghum.
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http://dx.doi.org/10.1080/09168451.2017.1336922DOI Listing
August 2017

Anionic metabolite biosynthesis enhanced by potassium under dark, anaerobic conditions in cyanobacteria.

Sci Rep 2016 08 31;6:32354. Epub 2016 Aug 31.

School of Agriculture, Meiji University, 1-1-1, Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.

Potassium (K(+)) is an essential macronutrient for all living organisms including cyanobacteria. Cyanobacteria are a group of bacteria performing oxygenic photosynthesis, widely studied in basic and applied sciences. The primary metabolism of the unicellular cyanobacterium Synechocystis sp. PCC 6803 is altered by environmental conditions, and it excretes organic acids and hydrogen under dark, anaerobic conditions. Here we demonstrated that K(+) widely changes the primary carbon metabolism of this cyanobacterium. Succinate and lactate excretion from the cells incubated under dark, anaerobic conditions was enhanced in the presence of K(+), while hydrogen production was repressed. The addition of K(+) and the genetic manipulation of acetate kinase AckA and an RNA polymerase sigma factor SigE additively increased succinate and lactate production to 141.0 and 217.6 mg/L, which are 11 and 46 times, compared to the wild-type strain without K(+), respectively. Intracellular levels of 2-oxoglutarate, succinate, fumarate, and malate increased by K(+) under dark, anaerobic conditions. This study provides the evidence of the considerable effect of K(+) on the biosynthesis of anionic metabolites in a unicellular cyanobacterium.
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http://dx.doi.org/10.1038/srep32354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006033PMC
August 2016

Toward the complete utilization of rice straw: Methane fermentation and lignin recovery by a combinational process involving mechanical milling, supporting material and nanofiltration.

Bioresour Technol 2016 Sep 11;216:830-7. Epub 2016 Jun 11.

Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan; RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. Electronic address:

Rice straw was mechanically milled using a process consuming 1.9MJ/kg-biomass, and 10g/L of unmilled or milled rice straw was used as the carbon source for methane fermentation in a digester containing carbon fiber textile as the supporting material. Milling increased methane production from 226 to 419mL/L/day at an organic loading rate of 2180mg-dichromate chemical oxygen demand/L/day, corresponding to 260mLCH4/gVS. Storage of the fermentation effluent at room temperature decreased the weight of the milled rice straw residue from 3.81 to 1.00g/L. The supernatant of the effluent was subjected to nanofiltration. The black concentrates deposited on the nanofiltration membranes contained 53.0-57.9% lignin. Solution nuclear magnetic resonance showed that lignin aromatic components such as p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) were retained primarily, and major lignin interunit structures such as the β-O-4-H/G unit were absent. This combinational process will aid the complete utilization of rice straw.
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http://dx.doi.org/10.1016/j.biortech.2016.06.029DOI Listing
September 2016

Organosolv pretreatment of sorghum bagasse using a low concentration of hydrophobic solvents such as 1-butanol or 1-pentanol.

Biotechnol Biofuels 2016 2;9:27. Epub 2016 Feb 2.

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Hyogo Kobe, 657-8501 Japan ; RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Kanagawa Yokohama, 230-0045 Japan.

Background: The primary components of lignocellulosic biomass such as sorghum bagasse are cellulose, hemicellulose, and lignin. Each component can be utilized as a sustainable resource for producing biofuels and bio-based products. However, due to their complicated structures, fractionation of lignocellulosic biomass components is required. Organosolv pretreatment is an attractive method for this purpose. However, as organosolv pretreatment uses high concentrations of organic solvents (>50 %), decreasing the concentration necessary for fractionation would help reduce processing costs. In this study, we sought to identify organic solvents capable of efficiently fractionating sorghum bagasse components at low concentrations.

Results: Five alcohols (ethanol, 1-propanol, 2-propanol, 1-butanol, and 1-pentanol) were used for organosolv pretreatment of sorghum bagasse at a concentration of 12.5 %. Sulfuric acid (1 %) was used as a catalyst. With 1-butanol and 1-pentanol, three fractions (black liquor, liquid fraction containing xylose, and cellulose-enriched solid fraction) were obtained after pretreatment. Two-dimensional nuclear magnetic resonance analysis revealed that the lignin aromatic components of raw sorghum bagasse were concentrated in the black liquor fraction, although the major lignin side-chain (β-O-4 linkage) was lost. Pretreatment with 1-butanol or 1-pentanol effectively removed p-coumarate, some guaiacyl, and syringyl. Compared with using no solvent, pretreatment with 1-butanol or 1-pentanol resulted in two-fold greater ethanol production from the solid fraction by Saccharomyces cerevisiae.

Conclusions: Our results revealed that a low concentration (12.5 %) of a highly hydrophobic solvent such as 1-butanol or 1-pentanol can be used to separate the black liquor from the solid and liquid fractions. The efficient delignification and visible separation of the lignin-rich fraction possible with this method simplify the fractionation of sorghum bagasse.
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http://dx.doi.org/10.1186/s13068-016-0427-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736640PMC
February 2016

Designing intracellular metabolism for production of target compounds by introducing a heterologous metabolic reaction based on a Synechosystis sp. 6803 genome-scale model.

Microb Cell Fact 2016 Jan 18;15:13. Epub 2016 Jan 18.

RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.

Background: Designing optimal intracellular metabolism is essential for using microorganisms to produce useful compounds. Computerized calculations for flux balance analysis utilizing a genome-scale model have been performed for such designs. Many genome-scale models have been developed for different microorganisms. However, optimal designs of intracellular metabolism aimed at producing a useful compound often utilize metabolic reactions of only the host microbial cells. In the present study, we added reactions other than the metabolic reactions with Synechosystis sp. 6803 as a host to its genome-scale model, and constructed a metabolic model of hybrid cells (SyHyMeP) using computerized analysis. Using this model provided a metabolic design that improves the theoretical yield of succinic acid, which is a useful compound.

Results: Constructing the SyHyMeP model enabled new metabolic designs for producing useful compounds. In the present study, we developed a metabolic design that allowed for improved theoretical yield in the production of succinic acid during glycogen metabolism by Synechosystis sp. 6803. The theoretical yield of succinic acid production using a genome-scale model of these cells was 1.00 mol/mol-glucose, but use of the SyHyMeP model enabled a metabolic design with which a 33 % increase in theoretical yield is expected due to the introduction of isocitrate lyase, adding activations of endogenous tree reactions via D-glycerate in Synechosystis sp. 6803.

Conclusions: The SyHyMeP model developed in this study has provided a new metabolic design that is not restricted only to the metabolic reactions of individual microbial cells. The concept of construction of this model requires only replacement of the genome-scale model of the host microbial cells and can thus be applied to various useful microorganisms for metabolic design to produce compounds.
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http://dx.doi.org/10.1186/s12934-016-0416-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717628PMC
January 2016

Metabolic design of a platform Escherichia coli strain producing various chorismate derivatives.

Metab Eng 2016 Jan 3;33:119-129. Epub 2015 Dec 3.

Center for Sustainable Resource Science, RIKEN, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan; Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan. Electronic address:

A synthetic metabolic pathway suitable for the production of chorismate derivatives was designed in Escherichia coli. An L-phenylalanine-overproducing E. coli strain was engineered to enhance the availability of phosphoenolpyruvate (PEP), which is a key precursor in the biosynthesis of aromatic compounds in microbes. Two major reactions converting PEP to pyruvate were inactivated. Using this modified E.coli as a base strain, we tested our system by carrying out the production of salicylate, a high-demand aromatic chemical. The titer of salicylate reached 11.5 g/L in batch culture after 48 h cultivation in a 2-liter jar fermentor, and the yield from glucose as the sole carbon source exceeded 40% (mol/mol). In this test case, we found that pyruvate was synthesized primarily via salicylate formation and the reaction converting oxaloacetate to pyruvate. In order to demonstrate the generality of our designed strain, we employed this platform for the production of each of 7 different chorismate derivatives. Each of these industrially important chemicals was successfully produced to levels of 1-3g/L in test tube-scale culture.
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http://dx.doi.org/10.1016/j.ymben.2015.11.007DOI Listing
January 2016

Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium.

Front Microbiol 2015 6;6:1064. Epub 2015 Oct 6.

RIKEN Center for Sustainable Resource Science Yokohama, Japan.

Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique.
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http://dx.doi.org/10.3389/fmicb.2015.01064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594341PMC
October 2015

Changes in primary metabolism under light and dark conditions in response to overproduction of a response regulator RpaA in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

Front Microbiol 2015 26;6:888. Epub 2015 Aug 26.

School of Agriculture, Meiji University, Kawasaki Japan ; RIKEN, Center for Sustainable Resource Science, Yokohama Japan.

The study of the primary metabolism of cyanobacteria in response to light conditions is important for environmental biology because cyanobacteria are widely distributed among various ecological niches. Cyanobacteria uniquely possess circadian rhythms, with central oscillators consisting from three proteins, KaiA, KaiB, and KaiC. The two-component histidine kinase SasA/Hik8 and response regulator RpaA transduce the circadian signal from KaiABC to control gene expression. Here, we generated a strain overexpressing rpaA in a unicellular cyanobacterium Synechocystis sp. PCC 6803. The rpaA-overexpressing strain showed pleiotropic phenotypes, including slower growth, aberrant degradation of an RNA polymerase sigma factor SigE after the light-to-dark transition, and higher accumulation of sugar catabolic enzyme transcripts under dark conditions. Metabolome analysis revealed delayed glycogen degradation, decreased sugar phosphates and organic acids in the tricarboxylic acid cycle, and increased amino acids under dark conditions. The current results demonstrate that in this cyanobacterium, RpaA is a regulator of primary metabolism and involved in adaptation to changes in light conditions.
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http://dx.doi.org/10.3389/fmicb.2015.00888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549654PMC
September 2015

Precipitate obtained following membrane separation of hydrothermally pretreated rice straw liquid revealed by 2D NMR to have high lignin content.

Biotechnol Biofuels 2015 18;8:88. Epub 2015 Jun 18.

RIKEN Biomass Engineering Program, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045 Japan ; Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501 Japan.

Background: Hydrothermal pretreatment of lignocellulosic biomass such as rice straw can dissolve part of the lignin and hemicellulose into a liquid fraction, thus facilitating enzyme accessibility to cellulose in bioethanol production process. Lignin is awaited to be recovered after hydrothermal pretreatment for utilization as value-added chemical, and lignin recovery also means removal of fermentation inhibitors. To recover lignin with high content from the liquid fraction, it is necessary to separate lignin and hemicellulose-derived polysaccharide. Therefore, the following processes were applied: membrane separation with nanofiltration (NF) and enzymatic hydrolysis by hemicellulase. To clarify lignin-concentrated fraction obtained during these processes, the fates of lignin and polysaccharide components were pursued by a solution NMR method and confirmed by compositional analysis of each fraction.

Results: After hydrothermal pretreatment of rice straw, the NF concentrate of the supernatant of liquid fraction was hydrolyzed by hemicellulase and the resulting black precipitate was recovered. In this black precipitate, the intensity of NMR spectra related to lignin aromatic regions increased and those related to polysaccharides decreased, compared to rice straw, the solid fraction after hydrothermal pretreatment, and the NF concentrate. The lignin content of the black precipitate was 65.8 %. Lignin in the black precipitate included 52.9 % of the acid-insoluble lignin and 19.4 % of the soluble lignin in the NF concentrate of supernatant of liquid fraction.

Conclusion: A precipitate with high lignin content was obtained from supernatants of the liquid fraction. These results suggested that precipitation of lignin was enhanced from concentrated mixtures of lignin and hemicellulosic polysaccharides by hydrolyzing the polysaccharides. Precipitation of lignin can contribute to lignin recovery from lignocellulosic biomass and, at the same time, allow more efficient ethanol production in the subsequent fermentation process.
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http://dx.doi.org/10.1186/s13068-015-0273-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476084PMC
June 2015

Changes in Lignin and Polysaccharide Components in 13 Cultivars of Rice Straw following Dilute Acid Pretreatment as Studied by Solution-State 2D 1H-13C NMR.

PLoS One 2015 17;10(6):e0128417. Epub 2015 Jun 17.

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Rokkodaicho, Nada-ku, Kobe, Hyogo, Japan; RIKEN Biomass Engineering Program, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, Japan.

A renewable raw material, rice straw is pretreated for biorefinery usage. Solution-state two-dimensional (2D) 1H-13 C hetero-nuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy, was used to analyze 13 cultivars of rice straw before and after dilute acid pretreatment, to characterize general changes in the lignin and polysaccharide components. Intensities of most (15 of 16) peaks related to lignin aromatic regions, such as p-coumarate, guaiacyl, syringyl, p-hydroxyphenyl, and cinnamyl alcohol, and methoxyl, increased or remained unchanged after pretreatment. In contrast, intensities of most (11 of 13) peaks related to lignin aliphatic linkages or ferulate decreased. Decreased heterogeneity in the intensities of three peaks related to cellulose components in acid-insoluble residues resulted in similar glucose yield (0.45-0.59 g/g-dry biomass). Starch-derived components showed positive correlations (r = 0.71 to 0.96) with glucose, 5-hydroxymethylfurfural (5-HMF), and formate concentrations in the liquid hydrolysates, and negative correlations (r = -0.95 to -0.97) with xylose concentration and acid-insoluble residue yield. These results showed the fate of lignin and polysaccharide components by pretreatment, suggesting that lignin aromatic regions and cellulose components were retained in the acid insoluble residues and starch-derived components were transformed into glucose, 5-HMF, and formate in the liquid hydrolysate.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128417PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4470627PMC
March 2016

Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

PLoS One 2015 21;10(5):e0125488. Epub 2015 May 21.

Biomass Engineering Program, RIKEN, Yokohama, Kanagawa, Japan; Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.

To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC), which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L) than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125488PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4440718PMC
February 2016

Alteration of cyanobacterial sugar and amino acid metabolism by overexpression hik8, encoding a KaiC-associated histidine kinase.

Environ Microbiol 2015 Jul 27;17(7):2430-40. Epub 2015 Jan 27.

RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.

Cyanobacteria possess circadian clocks consisting of KaiABC proteins, and circadian rhythm must closely relate to the primary metabolism. A histidine kinase, SasA, interacts with KaiC to transduce circadian signals and widely regulates transcription in Synechococcus sp. PCC 7942, although the involvement of SasA in primary metabolism has not been demonstrated at metabolite levels. Here, we generated a strain overexpressing hik8 (HOX80), an orthologue of SasA in Synechocystis sp. PCC 6803. HOX80 grew slowly under light conditions and lost viability under continuous dark conditions. Transcript levels of genes related to sugar catabolism remained higher in HOX80 under dark conditions. Metabolomic analysis revealed that under light conditions, glycogen was undetectable in HOX80, and there were decreased levels of metabolites of sugar catabolism and increased levels of amino acids, compared with those in the wild-type strain. HOX80 exhibited aberrant degradation of SigE proteins after a light-to-dark transition and immunoprecipitation analysis revealed that Hik8 directly interacts with KaiC1. The results of this study demonstrate that overexpression of hik8 widely alters sugar and amino acid metabolism, revealing the involvement of Hik8 in primary metabolism under both light and dark conditions in this cyanobacterium.
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http://dx.doi.org/10.1111/1462-2920.12715DOI Listing
July 2015

Capillary electrophoresis-mass spectrometry reveals the distribution of carbon metabolites during nitrogen starvation in Synechocystis sp. PCC 6803.

Environ Microbiol 2014 Feb 25;16(2):512-24. Epub 2013 Jun 25.

RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan; PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan.

Nitrogen availability is one of the most important factors for the survival of cyanobacteria. Previous studies on Synechocystis revealed a contradictory situation with regard to metabolism during nitrogen starvation; that is, glycogen accumulated even though the expressions of sugar catabolic genes were widely upregulated. Here, we conducted transcript and metabolomic analyses using capillary electrophoresis-mass spectrometry on Synechocystis sp. PCC 6803 under nitrogen starvation. The levels of some tricarboxylic acid cycle intermediates (succinate, malate and fumarate) were greatly increased by nitrogen deprivation. Purine and pyrimidine nucleotides were markedly downregulated under nitrogen depletion. The levels of 19 amino acids changed under nitrogen deprivation, especially those of amino acids synthesized from pyruvate and phosphoenolpyruvate, which showed marked increases. Liquid chromatography-mass spectrometry analysis demonstrated that the amount of NADPH and the NADPH/NADH ratio decreased under nitrogen depletion. These data demonstrate that there are increases in not only glycogen but also in metabolites downstream of sugar catabolism in Synechocystis sp. PCC 6803 under nitrogen starvation, resolving the contradiction between glycogen accumulation and induction of sugar catabolic gene expression in this unicellular cyanobacterium.
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http://dx.doi.org/10.1111/1462-2920.12170DOI Listing
February 2014

Cocktail δ-integration of xylose assimilation genes for efficient ethanol production from xylose in Saccharomyces cerevisiae.

J Biosci Bioeng 2013 Sep 4;116(3):333-6. Epub 2013 May 4.

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan.

Cocktail δ-integration was applied to improve ethanol production from xylose in Saccharomyces cerevisiae. Two hundred of recombinant S. cerevisiae strains possessing various copies of XYL1, XYL2, and XKS1 genes were constructed by cocktail δ-integration. Efficient strains with efficient ethanol production from xylose were successfully obtained by the fermentation test.
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http://dx.doi.org/10.1016/j.jbiosc.2013.03.020DOI Listing
September 2013

Regulation of central carbon metabolism in Saccharomyces cerevisiae by metabolic inhibitors.

J Biosci Bioeng 2013 Jul 26;116(1):59-64. Epub 2013 Feb 26.

Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan.

Metabolic inhibitors were applied for chemical regulation of central carbon metabolism in Saccharomyces cerevisiae. S. cerevisiae was treated with 10 metabolic inhibitors with various modes of action, and their activities were evaluated using a growth inhibition assay. Among the 6 active inhibitors, the effects of pyrazole (alcohol dehydrogenase inhibitor) and TTA (2-thenoyltrifluoloacetone, succinate dehydrogenase inhibitor) were analyzed in detail. The flask-scale batch-fermentation test showed that ethanol yield was reduced to 0.10 ± 0.01 g g⁻¹ and glycerol yield increased to 0.26 ± 0.01 g g⁻¹ on treatment with pyrazole at 5.0 g L⁻¹, indicating that multiple isozymes of alcohol dehydrogenase were simultaneously inhibited. The multi-targeted metabolic profiling analysis revealed that, although the TTA and pyrazole treatments affected the profiles of all central carbon metabolites in distinct manners, the level of fructose-1,6-bisphosphate commonly increased in the TTA- and pyrazole-treated S. cerevisiae by an unknown mechanism. These results demonstrate that chemical regulation of the central carbon metabolism could be used as an alternative tool to control microbial cell factories for bioproduction, or as a chemical probe to investigate the metabolic systems of useful microorganisms.
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http://dx.doi.org/10.1016/j.jbiosc.2013.01.018DOI Listing
July 2013

Evaluation of control mechanisms for Saccharomyces cerevisiae central metabolic reactions using metabolome data of eight single-gene deletion mutants.

Appl Microbiol Biotechnol 2013 Apr 7;97(8):3569-77. Epub 2012 Dec 7.

Biomass Engineering Program, RIKEN, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.

We performed metabolome and metabolite-metabolite correlation analyses for eight single-gene deletion mutants of Saccharomyces cerevisiae to evaluate the physiology of glucose metabolism. The irreversible enzyme reactions can become bottlenecks when intracellular metabolism is perturbed by direct interference from the central metabolic pathway by gene deletions or by a deletion of transcriptional regulator. Metabolome data reveal that transcriptional factor, gcr2, regulates the reaction that converts 3-phosphoglycerate into phosphoenolpyruvate. Metabolome data also suggest that the reaction catalyzed by pyruvate kinase makes one of the rate-limiting reactions throughout the glycolytic pathway.
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http://dx.doi.org/10.1007/s00253-012-4597-8DOI Listing
April 2013

Molecular mechanisms and metabolic engineering of glutamate overproduction in Corynebacterium glutamicum.

Subcell Biochem 2012 ;64:261-81

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Glutamate is a commercially important chemical. It is used as a flavor enhancer and is a major raw material for producing industrially useful chemicals. A coryneform bacterium, Corynebacterium glutamicum, was isolated in 1956 by Japanese researchers as a glutamate-overproducing bacterium and since then, remarkable progress in glutamate production has been made using this microorganism. Currently, the global market for glutamate is over 2.5 million tons per year. Glutamate overproduction by C. glutamicum is induced by specific treatments-biotin limitation, addition of fatty acid ester surfactants such as Tween 40, and addition of β-lactam antibiotics such as penicillin. Molecular biology and metabolic engineering studies on glutamate overproduction have revealed that metabolic flow is significantly altered by these treatments. These studies have also provided insight into the molecular mechanisms underlying these changes. In this chapter, we review our current understanding of the molecular mechanisms of glutamate overproduction in C. glutamicum, and we discuss the advances made by metabolic engineering of this microorganism.
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http://dx.doi.org/10.1007/978-94-007-5055-5_13DOI Listing
September 2014