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TANAKA TsutomuGraduate School of Engineering / Department of Chemical Science and EngineeringAssociate Professor
Researcher basic information
■ Research news- 25 Feb. 2020, New metabolic engineering strategy for effective sugar utilization by microbes successfully improves bioproduction of polymer raw materials
■ Research Areas
- Manufacturing technology (mechanical, electrical/electronic, chemical engineering) / Applied biofunctional and bioprocess engineering
Research activity information
■ Award- Oct. 2024 令和6年度学長表彰（財務貢献者）, 神戸大学
- Oct. 2022 神戸大学工学部 令和4年度優秀教育賞（応用化学科）
- Oct. 2022 神戸大学, 令和4年度学長表彰（財務貢献者）
- Oct. 2021 神戸大学, 令和3年度学長表彰（財務貢献者）
- Oct. 2020 神戸大学, 令和2年度学長表彰（財務貢献者）
- Oct. 2019 神戸大学工学部, 平成30年度優秀教育賞（応用化学科）
- Dec. 2014 酵素工学研究会, 酵素工学奨励賞, 微生物の細胞表層における酵素工学に関する研究Japan society
- Jan. 2014 日本農芸化学会, B.B.B.論文賞, Utilization of Lactic Acid Bacterial Genes in Synechocystis sp. PCC 6803 in the Production of Lactic AcidJapan society
- Sep. 2013 日本生物工学会, 生物工学論文賞, Direct isopropanol production from cellobiose by engineered Escherichia coli using a synthetic pathway and a cell surface display systemJapan society
- Mar. 2011 化学工学会, 研究奨励賞, 酵素を用いたタンパク質高機能化技術の開発
- Sep. 2025, Metabolic EngineeringScientific journal
- May 2025, Journal of Agricultural and Food ChemistryScientific journal
- ABSTRACT The modularization of biosynthetic pathways is a promising approach for enhancing microbial chemical production. We have developed a co-utilization method with glucose and xylose substrates to divide metabolic pathways into distinct production and energy modules to enhance the biosynthesis of para -aminobenzoic acid (pABA) in Escherichia coli . Optimizing initial glucose/xylose concentrations and eliminating carbon leakage resulted in a pABA titer of 8.22 g/L (yield: 0.23 g/g glucose). This strategy was then applied to the biosynthesis of 4APhe, a compound synthesized from chorismate without pyruvate (PYR) release. Utilizing glucose and xylose as co-substrates resulted in the production of 4.90 g/L 4APhe. Although 4APhe production did not benefit from PYR-driven energy generation as pABA production did, high titer was still achieved. This study highlights the effectiveness of modular metabolic pathway division for enhancing the production of key aromatic compounds and provides valuable insight into microbial production of chemicals that require specific biosynthetic donors such as amino groups. IMPORTANCE Microbial biosynthesis of chemicals from renewable resources offers a sustainable alternative to fossil fuel-based production. However, inefficiencies due to substrate diversion into by-products and biomass hinder optimal yields. In this study, we employed a modular metabolic engineering approach, decoupling pathways for chemical production from cell growth. Using glucose and xylose as co-substrates, we achieved the enhancement of p -aminobenzoic acid production in Escherichia coli . Additionally, we demonstrated the versatility of this approach by applying it to the biosynthesis of 4-amino-phenylalanine production. This study highlights the potential of modular metabolic pathway division for increased production of target compounds and provides valuable insight into microbial production of chemicals that require specific biosynthetic donors such as amino groups.American Society for Microbiology, Apr. 2025, Applied and Environmental MicrobiologyScientific journal
- The demand for the essential commodity chemical 1,2-propanediol (1,2-PDO) is on the rise, as its microbial production has emerged as a promising method for a sustainable chemical supply. However, the reliance of 1,2-PDO production in Escherichia coli on anaerobic conditions, as enhancing cell growth to augment precursor availability remains a substantial challenge. This study presents glucose-based aerobic production of 1,2-PDO, with xylose utilization facilitating cell growth. An engineered strain was constructed capable of exclusively producing 1,2-PDO from glucose while utilizing xylose to support cell growth. This was accomplished by deleting the gloA, eno, eda, sdaA, sdaB, and tdcG genes for 1,2-PDO production from glucose and introducing the Weimberg pathway for cell growth using xylose. Enhanced 1,2-PDO production was achieved via yagF overexpression and disruption of the ghrA gene involved in the 1,2-PDO-competing pathway. The resultant strain, PD72, produced 2.48 ± 0.15 g L-1 1,2-PDO with a 0.27 ± 0.02 g g-1-glucose yield after 72 h cultivation. Overall, this study demonstrates aerobic 1,2-PDO synthesis through the isolation of the 1,2-PDO synthetic pathway from the tricarboxylic acid cycle.Aug. 2024, Biotechnology journal, 19(8) (8), e2400210, English, International magazineScientific journal
- Short-chain esters, particularly isobutyl acetate and isoamyl acetate, hold significant industrial value due to their wide-ranging applications in flavors, fragrances, solvents, and biofuels. In this study, we demonstrated the biosynthesis of acetate esters using Yarrowia lipolytica as a host by feeding alcohols to the yeast culture. Initially, we screened for optimal alcohol acyltransferases for ester biosynthesis in Y. lipolytica. Strains of Y. lipolytica expressing atf1 from Saccharomyces cerevisiae, produced 251 or 613 mg/L of isobutyl acetate or of isoamyl acetate, respectively. We found that introducing additional copies of ATF1 enhanced ester production. Furthermore, by increasing the supply of acetyl-CoA and refining the culture conditions, we achieved high production of isoamyl acetate, reaching titers of 3404 mg/L. We expanded our study to include the synthesis of a range of acetate esters, facilitated by enriching the culture medium with various alcohols. This study underscores the versatility and potential of Y. lipolytica in the industrial production of acetate esters.Jul. 2024, Biotechnology progress, e3499, English, International magazineScientific journal
- Nitroaromatic compounds are widely used in industry, but their production is associated with issues such as the hazardousness of the process and low regioselectivity. Here, we successfully demonstrated the production of p-nitrobenzoate (PNBA) from glucose by constructing p-aminobenzoate N-oxygenase AurF-expressing E. coli. We generated this strain, which we named PN-1 by disrupting four genes involved in PNBA degradation: nfsA, nfsB, nemA, and azoR. We then expressed AurF from Streptomyces thioluteus in this strain, which resulted in the production of 945 mg/L PNBA in the presence of 1 g/L p-aminobenzoate. Direct production of PNBA from glucose was achieved by co-expressing the pabA, pabB, and pabC, as well as aurF, resulting in the production of 393 mg/L PNBA from 20 g/L glucose. To improve the PNBA titer, we disrupted genes involved in competing pathways: pheA, tyrA, trpE, pykA, and pykF. The resultant strain PN-4Ap produced 975 mg/L PNBA after 72 h of cultivation. These results highlight the potential of using microorganisms to produce other nitroaromatic compounds.Dec. 2023, Enzyme and microbial technology, 171, 110321 - 110321, English, International magazineScientific journal
- An environmentally sustainable world can be realized by using microorganisms to produce value-added materials from renewable biomass. Triacetic acid lactone (TAL) is a high-value-added compound that is used as a precursor of various organic compounds such as food additives and pharmaceuticals. In this study, we used metabolic engineering to produce TAL from glucose using an oleaginous yeast Yarrowia lipolytica. We first introduced TAL-producing gene 2-pyrone synthase into Y. lipolytica, which enabled TAL production. Next, we increased TAL production by engineering acetyl-CoA and malonyl-CoA biosynthesis pathways by redirecting carbon flux to glycolysis. Finally, we optimized the carbon and nitrogen ratios in the medium, culminating in the production of 4078 mg/L TAL. The strategy presented in this study had the potential to improve the titer and yield of polyketide biosynthesis.Oct. 2023, Journal of bioscience and bioengineering, 136(4) (4), 320 - 326, English, Domestic magazineScientific journal
- Sciscan Publishing Limited, Jul. 2023, Synthetic Biology and Engineering, 1(2) (2), 10012 - 9, English[Refereed]Scientific journal
- Elsevier BV, Mar. 2023, Enzyme and Microbial Technology, 164, 110193 - 110193, EnglishScientific journal
- The economical production of value-added chemicals from renewable biomass is a promising aspect of producing a sustainable economy. Itaconic acid (IA) is a high value-added compound that is expected to be an alternative to petroleum-based chemicals. In this study, we developed a metabolic engineering strategy for the large-scale production of IA from glucose using the fission yeast Schizosaccharomyces pombe. Heterologous expression of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus, which encodes cis-aconitate decarboxylase in the cytosol, led to the production of 0.132 g/L of IA. We demonstrated that mitochondrial localization of CAD enhanced the production of IA. To prevent the leakage of carbon flux from the TCA cycle, we generated a strain in which the endogenous malate exporter, citrate lyase, and citrate transporter genes were disrupted. A titer of 1.110 g/L of IA was obtained from a culture of this strain started with 50 g/L of glucose. By culturing the multiple mutant strain at increased cell density, we succeeded in enhancing the IA production to 1.555 g/L. The metabolic engineering strategies presented in this study have the potential to improve the titer of the biosynthesis of derivatives of intermediates of the TCA cycle.Elsevier BV, Sep. 2022, Journal of biotechnology, 358, 111 - 117, English[Refereed]Scientific journal
- Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of β-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICBGlc, and then captured by β-glucosidase.Elsevier BV, Jul. 2022, Metabolic Engineering, 72, 68 - 81, English, International magazine[Refereed]Scientific journal
- BACKGROUND: There has been an increasing demand for optically pure d-lactic and l-lactic acid for the production of stereocomplex-type polylactic acid. The d-lactic acid production from lignocellulosic biomass is important owing to its great abundance in nature. Corn steep liquor (CSL) is a cheap nitrogen source used for industrial fermentation, though it contains a significant amount of l-lactic acid, which decreases the optical purity of d-lactic acid produced. METHOD AND RESULTS: To remove l-lactic acid derived from the CSL-based medium, l-lactate oxidase (LoxL) from Enterococcus sp. NBRC 3427 was expressed in an engineered Lactiplantibacillus plantarum (formally called Lactobacillus plantarum) strain KOLP7, which exclusively produces d-lactic acid from both hexose and pentose sugars. When the resulting strain was applied for d-lactic acid fermentation from the mixed sugars consisting of the major constituent sugars of lignocellulose (35 g L-1 glucose, 10 g L-1 xylose, and 5 g L-1 arabinose) using the medium containing 10 g L-1 CSL, it completely removed l-lactic acid derived from CSL (0.52 g L-1 ) and produced 41.7 g L-1 of d-lactic acid. The l-lactic acid concentration was below the detection limit, and improvement in the optical purity of d-lactic acid was observed (from 98.2% to > 99.99%) by the overexpression of LoxL. CONCLUSION AND IMPLICATIONS: The LoxL-mediated consumption of l-lactic acid would enable the production of optically pure d-lactic acid in any medium contaminated by l-lactic acid.Wiley, Apr. 2022, Biotechnology Journal, 17(4) (4), 2100331 - 2100331, English, International magazine[Refereed]Scientific journal
- 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-1, 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.Elsevier BV, Sep. 2021, Metabolic Engineering, 67, 1 - 10, English, International magazine[Refereed]Scientific journal
- Jul. 2021, Clinical EndoscopyScientific journal
- Microbial 1,2-propanediol production using renewable feedstock is a promising method for the sustainable production of value-added fuels and chemicals. We demonstrated the metabolically engineered Escherichia coli for improvement of 1,2-propanediol production using glucose and cellobiose. The deletion of competing pathways improved 1,2-propanediol production. To reduce carbon flux toward downstream glycolysis, the phosphotransferase system (PTS) was inactivated by ptsG gene deletion. The resultant strain, GL3/PD, produced 1.48 ± 0.01 g/L of 1,2-propanediol from 20 g/L of glucose. A sugar supply was engineered by coexpression of β-glucosidase (BGL). The strain expressing BGL produced 1,2-propanediol from cellobiose at a concentration of 0.90 ± 0.11 g/L with a yield of 0.15 ± 0.01 g/g glucose (cellobiose 1 g is equal to glucose 1.1 g). As cellobiose or cellooligosaccharides a carbon source, the feasibility of producing 1,2-propanediol using an E. coli strain engineered for β-glucosidase expression are demonstrated.Elsevier Ltd, Jun. 2021, Bioresource Technology, 329, EnglishScientific journal
- We propose a novel approach to prepare affinity membranes using azido-containing surfactants and click chemistry. Porous polymeric membranes were prepared using cellulose acetate via polymer phase separation in the presence of azido-containing surfactants. Thermally induced phase separation and nonsolvent-induced phase separation were used for membrane preparation. The azido groups displayed on the membrane surfaces were conjugated with nitrilotriacetic acid via Huisgen 1,3-dipolar cycloaddition to prepare membranes that displayed affinity toward a hexahistidine-tagged protein. Two types of phase separation successfully produced porous membranes with different microstructures but showed similar separation performances. In place of nitrilotriacetic acid, D-Ala-D-Ala was conjugated to the surface of the azido-functionalized membrane. The membranes functionalized with D-Ala-D-Ala showed high affinity toward vancomycin. The present approach leads to facile surface functionalization of polymeric materials and produces affinity membranes displaying various types of ligands on their surfaces.ELSEVIER, Feb. 2021, COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS, 611, English[Refereed]Scientific journal
- Sep. 2020, Frontiers in Bioengineering and Biotechnology, 8, 569406, EnglishMetabolic engineering of shikimic acid-producing Corynebacterium glutamicum from glucose and cellobiose retaining its phosphotransferase system function and pyruvate kinase activities[Refereed]Scientific journal
- Microbial production of mevalonate from renewable feedstock is a promising and sustainable approach for the production of value-added chemicals. We describe the metabolic engineering of Escherichia coli to enhance mevalonate production from glucose and cellobiose. First, the mevalonate-producing pathway was introduced into E. coli and the expression of the gene atoB, which encodes the gene for acetoacetyl-CoA synthetase, was increased. Then, the deletion of the pgi gene, which encodes phosphoglucose isomerase, increased the NADPH/NADP+ ratio in the cells but did not improve mevalonate production. Alternatively, to reduce flux toward the tricarboxylic acid cycle, gltA, which encodes citrate synthetase, was disrupted. The resultant strain, MGΔgltA-MV, increased levels of intracellular acetyl-CoA up to sevenfold higher than the wild-type strain. This strain produced 8.0 g/L of mevalonate from 20 g/L of glucose. We also engineered the sugar supply by displaying β-glucosidase (BGL) on the cell surface. When cellobiose was used as carbon source, the strain lacking gnd displaying BGL efficiently consumed cellobiose and produced mevalonate at 5.7 g/L. The yield of mevalonate was 0.25 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing mevalonate from cellobiose or cellooligosaccharides using an engineered E. coli strain.Wiley, Jul. 2020, Biotechnology and Bioengineering, 117(7) (7), 2153 - 2164, English, International magazine[Refereed]Scientific journal
- Jan. 2020, Nature Communications, 11, 279, English[Refereed]Scientific journal
- Bioamination methods using microorganisms have attracted much attention because of the increasing demand for environmentally friendly bioprocesses. n-Butylamine production from glucose in Escherichia coli was demonstrated in this study, which has never been reported because of the absence of n-butylamine-producing pathway in nature. We focused on a transaminase-mediated cascade for bioamination from an alcohol or aldehyde. The cascade can convert an alcohol or an aldehyde to the corresponding amine with l-alanine as an amine donor. Here, n-butyraldehyde, which is a metabolic intermediate in the n-butanol producing pathway, is a potential intermediate for producing n-butylamine using this cascade. Hence, the n-butanol-producing pathway and the transaminase-mediated cascade were combined into a synthetic metabolic pathway for producing n-butylamine from glucose. Firstly, we demonstrated the conversion of n-butanol to n-butylamine using a three enzyme-mediated cascade. n-Butanol was successfully converted to n-butylamine in 92% yield in the presence of l-alanine and ammonium chloride. Then, the n-butanol-producing pathway and transaminase-mediated cascade were introduced into E. coli. Using this system, n-butylamine was successfully produced from glucose as a carbon source at a concentration of 53.2 mg L-1 after 96 h cultivation using a ppc (phosphoenolpyruvate carboxylase)-deficient strain. To the best of our knowledge, this is the first report of the direct production of n-butylamine from glucose, and may provide a starting point for the development of microbial methods to produce other bioamines.Jan. 2020, Journal of bioscience and bioengineering, 129(1) (1), 99 - 103, English, Domestic magazine[Refereed]Scientific journal
- Oct. 2019, Biotechnology and Bioengineering, 116(10) (10), 2640 - 2651[Refereed]Scientific journal
- 2019, Journal of Bioscience and Bioengineeringn-Butylamine production from glucose using a transaminase-mediated synthetic pathway in Escherichia coli[Refereed]
- BACKGROUND: Economical production of value-added chemicals from renewable biomass is a promising path to sustainability. 3-Hydroxypropionic acid (3-HP) is an important chemical for building a bio-sustainable society. Establishment of 3-HP production from renewable resources such as glucose would provide a bio-sustainable alternative to the production of acrylic acid from fossil resources. RESULTS: Here, we describe metabolic engineering of the fission yeast Schizosaccharomyces pombe to enhance 3-HP production from glucose and cellobiose via the malonyl-CoA pathway. The mcr gene, encoding the malonyl-CoA reductase of Chloroflexus aurantiacus, was dissected into two functionally distinct fragments, and the activities of the encoded protein were balanced. To increase the cellular supply of malonyl-CoA and acetyl-CoA, we introduced genes encoding endogenous aldehyde dehydrogenase, acetyl-CoA synthase from Salmonella enterica, and endogenous pantothenate kinase. The resulting strain produced 3-HP at 1.0 g/L from a culture starting at a glucose concentration of 50 g/L. We also engineered the sugar supply by displaying beta-glucosidase (BGL) on the yeast cell surface. When grown on 50 g/L cellobiose, the beta-glucosidase-displaying strain consumed cellobiose efficiently and produced 3-HP at 3.5 g/L. Under fed-batch conditions starting from cellobiose, this strain produced 3-HP at up to 11.4 g/L, corresponding to a yield of 11.2% (g-3-HP/g-glucose; given that 1 g cellobiose corresponds to 1.1 g glucose upon digestion). CONCLUSIONS: In this study, we constructed a series of S. pombe strains that produced 3-HP via the malonyl-CoA pathway. Our study also demonstrated that BGL display using cellobiose and/or cello-oligosaccharides as a carbon source has the potential to improve the titer and yield of malonyl-CoA- and acetyl-CoA-derived compounds.Nov. 2018, Microbial Cell Factories, 17(1) (1), 176 - 176, English, International magazine[Refereed]Scientific journal
- Oct. 2018, Acs Synthetic Biology, 7(11) (11)[Refereed]Scientific journal
- 化学工業社, Jul. 2018, ケミカルエンジニヤリング, 特集=革新技術が拓く新しい可能性, 63(7) (7), 7 - 10, Japanese分裂酵母を用いた有機酸生産技術の開発Scientific journal
- Fermentative production of optically pure lactic acid (LA) has attracted great interest because of the increased demand for plant-based plastics. For cost-effective LA production, an engineered Lactobacillus plantarum NCIMB 8826 strain, which enables the production of optically pure l-LA from raw starch, is constructed. The wild-type strain produces a racemic mixture of d- and l-LA from pyruvate by the action of the respective lactate dehydrogenases (LDHs). Therefore, the gene encoding D-LDH (ldhD) is deleted. Although no decrease in d-LA formation is observed in the ΔldhD mutant, additional disruption of the operon encoding lactate racemase (larA-E), which catalyzes the interconversion between d- and l-LA, completely abolished d-LA production. From 100 g L−1 glucose, the ΔldhD ΔlarA-E mutant produces 87.0 g L−1 of l-LA with an optical purity of 99.4%. Subsequently, a plasmid is introduced into the ΔldhD ΔlarA-E mutant for the secretion of α-amylase from Streptococcus bovis 148. The resulting strain could produce 50.3 g L−1 of l-LA from raw corn starch with a yield of 0.91 (g per g of consumed sugar) and an optical purity of 98.6%. The engineered L. plantarum strain would be useful in the production of l-LA from starchy materials.Wiley-VCH Verlag, May 2018, Biotechnology Journal, 13(5) (5), e1700517, English[Refereed]Scientific journal
- Cellulosomal systems are known as highly efficient biocatalysts in the degradation of lignocellulosic biomass in nature, but they remain unsuitable for industrial applications. In seeking alternatives to natural cellulosomes, casein was chosen as a scaffold for cellulase clustering. Casein is recognized as an excellent substrate for microbial transglutaminase (MTG) because it contains naturally reactive glutamine and lysine residues. A substrate peptide containing an MTG-reactive lysine residue was inserted into the C-terminus of the endoglucanase Cel5A and Cel6A from Thermobifida fusca using genetic engineering. The engineered cellulases, EG(Cel5A) and EG(Cel6A), were conjugated onto casein in different ratios by an MTG-mediated site-specific protein crosslinking reaction. Overall, a more than two-fold increase was observed when EG(Cel5A) was conjugated onto N,N-dimethylcasein, but a small or no change was observed for EG(Cel6A).Elsevier Ltd, Mar. 2018, Process Biochemistry, 66, 140 - 145, English[Refereed]Scientific journal
- A tetrameric streptavidin (SA)-appended LPETG tag was site-specifically linked to azido-containing tri-glycine via sortase A catalysis and the resulting azido-modified SA (SA-N3) was retained in the biotin-binding pocket. SA-N3 was polymerized with dibenzylcyclooctyne-modified branched poly(ethyleneglycol) (DBCO-PEG) using azido-modified branched PEG (N3-PEG) as a spacer via copper-free click chemistry. The resulting SA-based hydrogel exhibited gel-like mechanical properties and could immobilize biotin-modified molecules through biotin-SA affinity. Glucose dehydrogenase (GDH) was immobilized in the SA-based hydrogel, and the hydrogel was then coated on a glassy carbon electrode (GCE) and used for the biocatalytic oxidation of glucose. The designed GCE exhibited better performance and stability compared with GDH chemically adsorbed onto a GCE. In addition, the designed GCE anode and a Pt-carbon cathode were assembled into a glucose/O2 fuel cell that provided a maximum power density and open circuit voltage of 11.8±0.56µWcm-2 and 0.17V, respectively.Jan. 2018, Biosensors & bioelectronics, 99, 56 - 61, English, International magazine[Refereed]Scientific journal
- 2018, Basic & Clinical Pharmacology & Toxicology, 124, 17 - 17Metabolic engineering of S. pombe via CRISPR-CAS9 genome editing for 3-hydroxypropionic acid production from glucose and cellobioseScientific journal
- 2018, Basic & Clinical Pharmacology & Toxicology, 124, 18 - 18Metabolic design of Escherichia coli for production of shikimate pathway derivativesScientific journal
- 2018, Febs Open Bio, 8, 180 - 180Metabolic design of Escherichia coli for muconic acid productionScientific journal
- Metabolic engineering has been an important approach for microbial bio-production. To produce bio-chemicals with engineered microorganisms, metabolic pathways have been edited using several common strategies, including gene disruption, gene overexpression, and gene attenuation. Here, we demonstrated metabolic channeling based on enzymatic metabolic enzyme ligation as a noteworthy approach for enhancing a desired metabolic flux. To achieve metabolic channeling , the metabolic enzymes should be in close proximity in cells. In the literature, several methodologies have been recently applied to achieve metabolic channeling . Meanwhile, we have proposed a strategy for possessing metabolic enzymes in close proximity, by utilizing sortase A as a stapler to tether such enzymes in Escherichia coli. By tethering metabolic enzymes that catalyze the reactions before and after a target metabolite, the metabolic flux may be enhanced. This chapter describes the approach for enhancing acetate-producing flux by sortase-A-assisted metabolic ligation in E. coli.2018, Methods in molecular biology (Clifton, N.J.), 1772, 125 - 136, English, International magazine[Refereed]Scientific journal
- A tetrameric streptavidin (SA)-appended LPETG tag was site-specifically linked to azido-containing tri-glycine via sortase A catalysis and the resulting azido-modified SA (SA-N3) was retained in the biotin-binding pocket. SA-N3 was polymerized with dibenzylcyclooctyne-modified branched poly(ethyleneglycol) (DBCO-PEG) using azido-modified branched PEG (N3-PEG) as a spacer via copper-free click chemistry. The resulting SA-based hydrogel exhibited gel-like mechanical properties and could immobilize biotin-modified molecules through biotin-SA affinity. Glucose dehydrogenase (GDH) was immobilized in the SA-based hydrogel, and the hydrogel was then coated on a glassy carbon electrode (GCE) and used for the biocatalytic oxidation of glucose. The designed GCE exhibited better performance and stability compared with GDH chemically adsorbed onto a GCE. In addition, the designed GCE anode and a Pt-carbon cathode were assembled into a glucose/O-2 fuel cell that provided a maximum power density and open circuit voltage of 11.8 +/- 0.56 W cm(-2) and 0.17 V, respectively.ELSEVIER ADVANCED TECHNOLOGY, Jan. 2018, BIOSENSORS & BIOELECTRONICS, 99, 56 - 61, English[Refereed]Scientific journal
- Modification of the Schizosaccharomyces pombe genome is often laborious, time consuming due to the lower efficiency of homologous recombination. Here, we constructed metabolically engineered S. pombe strains using a CRISPR-Cas9 system and also demonstrated D-lactic acid (D-LA) production from glucose and cellobiose. Genes encoding two separate pyruvate decarboxylases (PDCs), an L-lactic acid dehydrogenase (L-LDH), and a minor alcohol dehydrogenase (SPBC337.11) were disrupted, thereby attenuating ethanol production. To increase the cellular supply of acetyl-CoA, an important metabolite for growth, we introduced genes encoding bacterial acetylating acetaldehyde dehydrogenase enzymes (Escherichia coli MhpF and EutE). D-LA production by the resulting strain was achieved by expressing a Lactobacillus plantarum gene encoding D-lactate dehydrogenase. The engineered strain efficiently consumed glucose and produced D-LA at 25.2 g/L from 35.5 g/L of consumed glucose with a yield of 0.71 g D-LA / g glucose. We further modified this strain by expressing beta-glucosidase by cell surface display; the resulting strain produced D-LA at 24.4 g/L from 30 g/L of cellobiose in minimal medium, with a yield of 0.68 g D-LA / g glucose. To our knowledge, this study represents the first report of a S. pombe strain that was metabolically engineered using a CRISPR-Cas9 system, and demonstrates the possibility of engineering S. pombe for the production of value-added chemicals.Dec. 2017, Metabolic engineering communications, 5, 60 - 67, English, International magazine[Refereed]Scientific journal
- The present work reviews literature describing the re-design of the metabolic pathways of a microbial host using sophisticated genetic tools, yielding strains for producing value-added chemicals including fuels, building-block chemicals, pharmaceuticals, and derivatives. This work employed Escherichia coli, a well-studied microorganism that has been successfully engineered to produce various chemicals. E. coli has several advantages compared with other microorganisms, including robustness, and handling. To achieve efficient productivities of target compounds, an engineered E. coli should accumulate metabolic precursors of target compounds. Multiple researchers have reported the use of pathway engineering to generate strains capable of accumulating various metabolic precursors, including pyruvate, acetyl-CoA, malonyl-CoA, mevalonate and shikimate. The aim of this review provides a promising guideline for designing E. coli strains capable of producing a variety of useful chemicals. Herein, the present work reviews their common and unique strategies, treating metabolically engineered E. coli as a "microbial chassis".Dec. 2017, Bioresource technology, 245(Pt B) (Pt B), 1362 - 1368, English, International magazine[Refereed]Scientific journal
- The present work reviews literature describing the re-design of the metabolic pathways of a microbial host using sophisticated genetic tools, yielding strains for producing value-added chemicals including fuels, building-block chemicals, pharmaceuticals, and derivatives. This work employed Escherichia coli, a well-studied microorganism that has been successfully engineered to produce various chemicals. E. coli has several advantages compared with other microorganisms, including robustness, and handling. To achieve efficient productivities of target compounds, an engineered E. coli should accumulate metabolic precursors of target compounds. Multiple researchers have reported the use of pathway engineering to generate strains capable of accumulating various metabolic precursors, including pyruvate, acetyl-CoA, malonyl-CoA, mevalonate and shikimate. The aim of this review provides a promising guideline for designing E. coli strains capable of producing a variety of useful chemicals. Herein, the present work reviews their common and unique strategies, treating metabolically engineered E. coli as a "microbial chassis". (C) 2017 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Dec. 2017, BIORESOURCE TECHNOLOGY, 245(Pt B) (Pt B), 1362 - 1368, English[Refereed]
- Xylooligosaccharide-assimilating Corynebacterium glutamicum strains were constructed using metabolic engineering and cell surface display techniques. First, C. glutamicum was metabolically engineered to create lysine-producing strains. Beta-xylosidase BSU17580 derived from Bacillus subtilis was then expressed on the C. glutamicum cell surface using PorH anchor protein, and enzymes involved in the xylose assimilation pathway were also expressed. Metabolic engineering had no effect on the activity of beta-xylosidase. The engineered strains efficiently consumed xylooligosaccharides and produced 12.4 mM of lysine from 11.9 g/L of xylooligosaccharides as the carbon source. Finally, co-expression of lysine decarboxylase enabled production of 11.6 mM of 1,5-diaminopentane (cadaverine) from 13 g/L of consumed xylooligosaccharides. (C) 2017 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Dec. 2017, BIORESOURCE TECHNOLOGY, 245(Pt B) (Pt B), 1684 - 1691, English[Refereed]Scientific journal
- We report a DNA-gold nanoparticle (AuNP) hybrid hydrogel in which the AuNPs crosslink enzymatically synthesized DNA to form a gel network. PCR-elongated DNA and AuNPs act as a one-dimensional polymer and cross-linkers, respectively. The DNA-AuNP hydrogel has the functional properties of both long DNA and the AuNPs.ROYAL SOC CHEMISTRY, May 2017, CHEMICAL COMMUNICATIONS, 53(43) (43), 5802 - 5805, English[Refereed]Scientific journal
- Simultaneous saccharification and fermentation (SSF) of d-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the EY-lactate dehydrogenase gene was disrupted, produced 97.7 g/L d-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8-17 h). As a result, 117.1 g/L d-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144 h of fermentation. SSF experiments were repeatedly performed for ten times and d-lactic acid was stably produced using recycled cells (118.4-129.8 g/L). On average, d-lactic acid was produced with a volumetric productivity of 2.18 g/L/h over 48 h.SPRINGER, Mar. 2017, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 101(5) (5), 1869 - 1875, English[Refereed]Scientific journal
- Simultaneous saccharification and fermentation (SSF) of d-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the EY-lactate dehydrogenase gene was disrupted, produced 97.7 g/L d-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8-17 h). As a result, 117.1 g/L d-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144 h of fermentation. SSF experiments were repeatedly performed for ten times and d-lactic acid was stably produced using recycled cells (118.4-129.8 g/L). On average, d-lactic acid was produced with a volumetric productivity of 2.18 g/L/h over 48 h.SPRINGER, Mar. 2017, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 101(5) (5), 1869 - 1875, English[Refereed]Scientific journal
- To produce styrene from a biomass-derived carbon source, Streptomyces lividans was adopted as a host strain. The gene encoding ferulic acid decarboxylase from Saccharomyces cerevisiae (FDC1) was introduced into S. lividans, and the resulting S. lividans transformant successfully expressed FDC1 and converted trans-cinnamic acid (CA) to styrene. A key factor in styrene production using microbes is the recovery of volatile styrene. In the present study, we selected polystyrene resin beads XRD-4 as the absorbent agent to recover styrene produced using S. lividans transformants, which enabled recovery of styrene from the culture broth. For styrene production from biomass-derived carbon sources, S. lividans/FDC1 was cultured together with S. lividans/p-encP, which we previously reported as a CA-producing S. lividans strain. This coculture system combined with the recovery of styrene using XAD-4 allowed the production of styrene from glucose, cellobiose, or xylo-oligosaccharide, respectively. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Dec. 2016, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 122(6) (6), 730 - 735, English[Refereed]Scientific journal
- We demonstrate metabolic enzyme ligation using a transpeptidase (Staphylococcal sortase A) in the microbial cytoplasm for the redirection of metabolic flux through metabolic channeling. Here, sortase A expression was controlled by the Inc promoter to trigger metabolic channeling by the addition of isopropyl-beta-D-thiogalactopyranoside (IPTG). We tested covalent linking of pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and evaluated the production of acetate. The time point of addition of IPTG was not critical for facilitating metabolic enzyme ligation, and acetate production increased upon expression of sortase A. These results show that sortase A-mediated enzyme ligation enhances an acetate producing flux in E. coli. We have validated that sortase A-mediated enzyme ligation offers a metabolic channeling approach to redirect a central flux to a desired flux.AMER CHEMICAL SOC, Nov. 2016, ACS SYNTHETIC BIOLOGY, 5(11) (11), 1284 - 1289, English[Refereed]Scientific journal
- To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species. The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme's k(cat)/K-m value was 0.54 mM (-1) s(-1). Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l(-1) from 10 g glucose l(-1). A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.SPRINGER, Sep. 2016, BIOTECHNOLOGY LETTERS, 38(9) (9), 1543 - 1549, English[Refereed]Scientific journal
- To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species. The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme's k(cat)/K-m value was 0.54 mM (-1) s(-1). Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l(-1) from 10 g glucose l(-1). A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.SPRINGER, Sep. 2016, BIOTECHNOLOGY LETTERS, 38(9) (9), 1543 - 1549, English[Refereed]Scientific journal
- Background: Biological applications of nanoparticles are rapidly increasing, which introduces new possibilities to improve the efficacy of radiotherapy. Here, we synthesized titanium peroxide nanoparticles (TiOxNPs) and investigated their efficacy as novel agents that can potently enhance the effects of radiation in the treatment of pancreatic cancer. Methods: TiOxNPs and polyacrylic acid-modified TiOxNPs (PAA-TiOxNPs) were synthesized from anatase-type titanium dioxide nanoparticles (TiO(2)NPs). The size and morphology of the PAA-TiOxNPs was evaluated using transmission electron microscopy and dynamic light scattering. The crystalline structures of the TiO(2)NPs and PAA-TiOxNPs with and without X-ray irradiation were analyzed using X-ray absorption. The ability of TiOxNPs and PAA-TiOxNPs to produce reactive oxygen species in response to X-ray irradiation was evaluated in a cell-free system and confirmed by flow cytometric analysis in vitro. DNA damage after X-ray exposure with or without PAA-TiOxNPs was assessed by immunohistochemical analysis of gamma-H2AX foci formation in vitro and in vivo. Cytotoxicity was evaluated by a colony forming assay in vitro. Xenografts were prepared using human pancreatic cancer MIAPaCa-2 cells and used to evaluate the inhibition of tumor growth caused by X-ray exposure, PAA-TiOxNPs, and the combination of the two. Results: The core structures of the PAA-TiOxNPs were found to be of the anatase type. The TiOxNPs and PAA-TiOxNPs showed a distinct ability to produce hydroxyl radicals in response to X-ray irradiation in a dose-and concentration-dependent manner, whereas the TiO(2)NPs did not. At the highest concentration of TiOxNPs, the amount of hydroxyl radicals increased by >8.5-fold following treatment with 30 Gy of radiation. The absorption of PAA-TiOxNPs enhanced DNA damage and resulted in higher cytotoxicity in response to X-ray irradiation in vitro. The combination of the PAA-TiOxNPs and X-ray irradiation induced significantly stronger tumor growth inhibition compared to treatment with either PAA-TiOxNPs or X-ray alone (p < 0.05). No apparent toxicity or weight loss was observed for 43 days after irradiation. Conclusions: TiOxNPs are potential agents for enhancing the effects of radiation on pancreatic cancer and act via hydroxyl radical production; owing to this ability, they can be used for pancreatic cancer therapy in the future.BIOMED CENTRAL LTD, Jul. 2016, RADIATION ONCOLOGY, 11(1) (1), 91, English[Refereed]Scientific journal
- We successfully enhanced the productivity of ectoine with Halomonas elongata by improvement of the transport of sugar. First, we carried out screening for sugar transporters capable of improving glucose and xylose consumption. We found two transporters: b3657 from Escherichia coli, which is capable of improving glucose consumption, and HEO_0208 from H. elongata, which is capable of improving xylose consumption. Using transporter-overexpressing strains, the productivity of ectoine was improved. These results indicate that sugar consumption is important for efficient ectoine production. As result of phenotypic analysis of a HEO_0208 deletion strain, we discovered that HEO_0208 is the major xylose transporter in H. elongata. This is the first report demonstrating improvement of ectoine productivity by enhancing the transport of sugar. (C) 2016 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, Jul. 2016, ENZYME AND MICROBIAL TECHNOLOGY, 89, 63 - 68, English[Refereed]Scientific journal
- We engineered efficient 2,3-butanediol (23BD) production from cellobiose using Bacillus subtilis. First, we found that B. subtilis harboring an empty vector could produce 23BD from cellobiose. However, productivity using cellobiose as a carbon source was lower than that when using glucose. This lower productivity was improved by adding purified beta-glucosidase from Thermobifida fusca YX (Tfu_0937) in the fermentation. Encouraged by these findings, we found that hydrolysis of cellobiose to glucose was an important reaction of 23BD biosynthesis in B. subtilis using cellobiose. Hence, we created efficient 23BD production from cellobiose using exogenous Tfu_0937-expressing B. subtilis. Using the engineered strain, 21.2 g L-1 of 23BD was produced after 72 h of cultivation. The productivity and yield were 0.294 g L-1 h(-1) and 0.35 g 23BD/g cellobiose, respectively. We successfully demonstrated efficient 23BD production from cellobiose by using BGL-expressing B. subtilis.SPRINGER, Jul. 2016, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 100(13) (13), 5781 - 5789, English[Refereed]Scientific journal
- Protein assemblies are an emerging tool that is finding many biological and bioengineering applications. We here propose a method for the site-specific assembly of proteins on a twigged streptavidin (SA) polymer using streptavidin as a functional scaffold. SA was genetically appended with a G tag (sortase A recognition sequence) and a Y tag (HRP recognition sequence) on its N- and C-termini, respectively, to provide G-SA-Y. G-SA-Y was polymerized using HPR-mediated tyrosine coupling, then fluorescent proteins were immobilized on the polymer by biotin-SA affinity and sortase A-mediated ligation. Fluorescence measurements showed that the proteins were immobilized in close proximity to each other. Hydrolyzing enzymes were also functionally assembled on the G-SA-Y polymer. The site-specific assembly of proteins on twigged SA polymer may find new applications in various biological and bioengineering fields. (C) 2016 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, May 2016, JOURNAL OF BIOTECHNOLOGY, 225, 61 - 66, English[Refereed]Scientific journal
- d-lactic acid is of great interest because of increasing demand for biobased poly-lactic acid (PLA). Blending poly-l-lactic acid with poly-d-lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d-lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l-lactate-deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ldhL1-pCU-PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d-lactic acid yield and productivity. d-lactic acid (27.3 g L-1) and productivity (0.75 g L-1 h(-1)) was obtained from corn stover and d-lactic acid (22.0 g L-1) and productivity (0.65 g L-1 h(-1)) was obtained from sorghum stalks using ldhL1-pCU-PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d-lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d-lactic acid. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271-278, 2016WILEY, Mar. 2016, BIOTECHNOLOGY PROGRESS, 32(2) (2), 271 - 278, English[Refereed]Scientific journal
- This journal is © The Royal Society of Chemistry. In biological systems, proteins can form well-organized, higher-order structures with unique functions that would be difficult to achieve with a single protein. These proteinaceous supramolecular structures form by self-assembly, and the spatial arrangement of the protein building blocks in them is very important. In the present study, an artificial system was developed using recombinant proteins as building blocks, which were assembled in a one-dimensional manner. The assembly of these building blocks was based on the avidin-biotin interaction. A tetrameric biotin ligand unit was designed so that the 1:4 stoichiometry of the avidin-biotin interaction was altered to a 1:2 directional interaction between the streptavidin and tetrabiotinylated protein units. In a proof-of-concept study, site-specifically tetrabiotin-labeled endoglucanase and cellulose-binding module units were prepared, and then these components were self-assembled by mixing with streptavidin to mimic a natural cellulosome. The formation of one-dimensional assemblies of the protein units depended on the stoichiometry of the avidin-biotin interaction sites in the system. Interestingly, the saccharification efficiency improved when the component ratio of protein units in the assemblies was changed. The presence of the optimal ratio of the building blocks implies the modularity of the present protein assembly system.2016, Molecular Systems Design & Engineering, 1(1) (1), 66 - 73, English[Refereed]Scientific journal
- D-lactic acid is used as a monomer in the production of poly-D-lactic acid (PDLA), which is used to form heat-resistant stereocomplex poly-lactic acid. To produce cost-effective D-lactic acid by using all sugars derived from biomass efficiently, xylose-assimilating genes encoding xylose isomerase and xylulokinase were cloned into an L-lactate-deficient strain, Lactobacillus plantarum. The resulting recombinant strain, namely L. plantarum NCIMB 8826 a triangle ldhL1-pLEM-xylAB, was able to produce D-lactic acid (at optical purity > 99 %) from xylose at a yield of 0.53 g g(-1). Simultaneous utilization of glucose and xylose to produce D-lactic acid was also achieved by this strain, and 47.2 g L-1 of D-lactic acid was produced from 37.5 g L-1 glucose and 19.7 g L-1 xylose. Corn stover and soybean meal extract (SBME) were evaluated as cost-effective medium components for D-lactic acid production. Optimization of medium composition using response surface methodology resulted in 30 % reduction in enzyme loading and 70 % reduction in peptone concentration. In addition, we successfully demonstrated D-lactic acid fermentation from corn stover and SBME in a fed-batch fermentation, which yielded 61.4 g L-1 D-lactic acid with an overall yield of 0.77 g g(-1). All these approaches are geared to attaining high D-lactic acid production from biomass sugars to produce low-cost, highly thermostable biodegradable plastics.SPRINGER, Jan. 2016, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 100(1) (1), 279 - 288, English[Refereed]Scientific journal
- In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignocellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of cell surface display using industrial microorganisms such as Escherichia coil and yeast. Cell surface display is used primarily for endowing cellulolytic activity on the host cells, and enables direct fermentation to generate useful fuels and chemicals from lignocellulosic biomass. Cell surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of surface display for industrial biotechnology are discussed. (C) 2015 Elsevier Inc. All rights reserved.PERGAMON-ELSEVIER SCIENCE LTD, Nov. 2015, BIOTECHNOLOGY ADVANCES, 33(7) (7), 1403 - 1411, English[Refereed]Scientific journal
- In the present study, sortase A-mediated immobilization of enzymes was used for the preparation of immobilized enzymes. Thermobifida fusca YX -glucosidase (BGL) or Streptococcus bovis 148 -amylase (AmyA) were produced with C-terminal sortase A recognition sequences. The resulting fusion proteins were successfully immobilized on nanoparticle surfaces using sortase A. Some properties (activity, stability, and reusability) of the immobilized fusion proteins were evaluated. Both immobilized BGL and immobilized AmyA prepared by the sortase A-mediated technique retained their catalytic activity, exhibiting activities 3.0- or 1.5-fold (respectively) of those seen with the same enzymes immobilized by chemical crosslinking. Immobilized enzymes prepared by the sortase A-mediated technique did not undergo dramatic changes in stability compared with the respective free enzymes. Thus, the sortase A-mediated technique provides a promising method for immobilization of active, stable enzymes.WILEY-V C H VERLAG GMBH, Oct. 2015, MACROMOLECULAR BIOSCIENCE, 15(10) (10), 1375 - 1380, English[Refereed]Scientific journal
- This study focused on the process development for the D-lactic acid production from cellulosic feedstocks using the Lactobacillus plantarum mutant, genetically modified to produce optically pure D-lactic acid from both glucose and xylose. The simultaneous saccharification and fermentation (SSF) using delignified hardwood pulp (5-15% loads) resulted in the lactic acid titers of 55.2-84.6 g/L after 72 h and increased productivities of 1.77-2.61 g/L/h. To facilitate the enzymatic saccharification of high-load pulp at a fermentation temperature, short-term (610 min) pulverization of pulp was conducted, leading to a significantly improved saccharification with the suppressed formation of formic acid by-product. The short-term milling followed by SSF resulted in a lactic acid titer of 102.3 g/L, an optical purity of 99.2%, and a yield of 0.879 g/g-sugars without fed-batch process control. Therefore, the process presented here shows promise for the production of high-titer D-lactic acid using the L. plantarum mutant. (C) 2015 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Jul. 2015, BIORESOURCE TECHNOLOGY, 187, 167 - 172, English[Refereed]Scientific journal
- The effect of pretreatment with peracetic acid (PAA) or an ionic liquid (1-ethyl-3-methylimidazolium acetate, [Emim][OAc]) on the synergism between endoglucanase and endoxylanase in the hydrolysis of bagasse was investigated. An endoglucanase, Cel6A, with a carbohydrate-binding module (CBM) and two endoxylanases, XynZ-C without a CBM and Xyn11A with an intrinsic xylan/cellulose binding module (XBM), were selected. The hemicellulose content, especially arabinan, and the cellulose crystallinity of bagasse were found to affect the cellulase-xylanase synergism. More specifically, higher synergism (above 3.4) was observed for glucan conversion, at low levels of arabinan (0.9%), during the hydrolysis of PAA pretreated bagasse. In contrast, [Emim][OAc] pretreated bagasse, showed lower cellulose crystallinity and achieved higher synergism (over 1.9) for xylan conversion. Ultimately, the combination of Cel6A and Xyn11A resulted in higher synergism for glucan conversion than the combination of Cel6A with XynZ-C, indicating the importance of the molecular architecture of enzymes for metabolic synergism. (C) 2015 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Jun. 2015, BIORESOURCE TECHNOLOGY, 185, 158 - 164, English[Refereed]Scientific journal
- (一社)日本放射線影響学会, May 2015, 日本放射線影響学会大会講演要旨集, 58回, 3 - 07, English新たな放射線増感剤としての過酸化チタンナノ粒子は膵癌治療における抗腫瘍効果を増強させる(Titanium Peroxide Nanoparticles, as Novel Radiosensitizers, Enhance Antitumor Efficacy in Pancreatic Cancer Therapy)
- Recently, the new trend in the second-generation ethanol industry is to use mild pretreatments, in order to reduce costs and to keep higher content of hemicellulose in the biomass. Nevertheless, a high enzyme dosage is still required in the conversion of (hemi)cellulose. The interaction between cellulases and xylanases seems to be an effective alternative to reduce enzyme loading in the saccharification process. At first, to evaluate the synergism of xylanases on bagasse degradation, we have produced two xylanases from glycoside hydrolase family 10 (GH10) and three xylanases from glycoside hydrolase family11 (GH11),from two thermophilic organisms, Thermobifida fusca and Clostridium thermocellum, and one mesophilic organism, Streptomyces lividans. Peracetic acid (PAA) pretreated bagasse was used as substrate. The combination of XynZ-C (GH10, from C. thermocellum), and XlnB (GH11, from S. lividans) presented the highest degree of synergy after 6 h (3,62). However, the combination of XynZ-C and Xyn11A (GH11, from T. fusca) resulted in the highest total yield of reducing sugars. To evaluate the synergism between xylanases and cellulases, commercial cellulase preparation from Trichoderma reesei was combined with the selected xylanases, XynZ-C and Xyn11A. About 2-fold increase was observed in the concentration of reducing sugars, when both xylanases, XynZ-C and Xyn11A, were added together with T. reesei cellulases in the reaction mixture. (C) 2015 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, May 2015, ENZYME AND MICROBIAL TECHNOLOGY, 72, 16 - 24, English[Refereed]Scientific journal
- Recently, the new trend in the second-generation ethanol industry is to use mild pretreatments, in order to reduce costs and to keep higher content of hemicellulose in the biomass. Nevertheless, a high enzyme dosage is still required in the conversion of (hemi)cellulose. The interaction between cellulases and xylanases seems to be an effective alternative to reduce enzyme loading in the saccharification process. At first, to evaluate the synergism of xylanases on bagasse degradation, we have produced two xylanases from glycoside hydrolase family 10 (GH10) and three xylanases from glycoside hydrolase family11 (GH11),from two thermophilic organisms, Thermobifida fusca and Clostridium thermocellum, and one mesophilic organism, Streptomyces lividans. Peracetic acid (PAA) pretreated bagasse was used as substrate. The combination of XynZ-C (GH10, from C. thermocellum), and XlnB (GH11, from S. lividans) presented the highest degree of synergy after 6 h (3,62). However, the combination of XynZ-C and Xyn11A (GH11, from T. fusca) resulted in the highest total yield of reducing sugars. To evaluate the synergism between xylanases and cellulases, commercial cellulase preparation from Trichoderma reesei was combined with the selected xylanases, XynZ-C and Xyn11A. About 2-fold increase was observed in the concentration of reducing sugars, when both xylanases, XynZ-C and Xyn11A, were added together with T. reesei cellulases in the reaction mixture. (C) 2015 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, May 2015, ENZYME AND MICROBIAL TECHNOLOGY, 72, 16 - 24, English[Refereed]Scientific journal
- Streptomyces lividans was adopted as a host strain for 4-vinylphenol (4VPh) production directly from cellulose. In order to obtain novel phenolic acid decarboxylase (PAD) expressed in S. lividans, PADs distributed among Streptomyces species were screened. Three novel PADs, derived from Streptomyces sviceus, Streptomyces hygroscopicus, and Streptomyces cattleya, were successfully obtained and expressed in S. lividans. S. sviceus PAD (SsPAD) could convert p-hydroxycinnamic acid (pHCA) to 4VPh more efficiently than the others both in vitro and in vivo. For 4VPh production directly from cellulose, L-tyrosine ammonia lyase derived from Rhodobacter sphaeroides and SsPAD were introduced into endoglucanase-secreting S. lividans, and the 4VPh biosynthetic pathway was constructed therein. The created transformants successfully produced 4VPh directly from cellulose. (C) 2014 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Mar. 2015, BIORESOURCE TECHNOLOGY, 180, 59 - 65, English[Refereed]Scientific journal
- In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often utilized as a cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency.OXFORD UNIV PRESS, Feb. 2015, FEMS YEAST RESEARCH, 15(1) (1), 1 - 9, English[Refereed]Scientific journal
- Background: Streptavidin is a tetrameric protein derived from Streptomyces avidinii, and has tight and specific biotin binding affinity. Applications of the streptavidin-biotin system have been widely studied. Streptavidin is generally produced using protein expression in Escherichia coli. In the present study, the secretory production of streptavidin was carried out using Streptomyces lividans as a host. Results: In this study, we used the gene encoding native full-length streptavidin, whereas the core region is generally used for streptavidin production in Escherichia coli. Tetrameric streptavidin composed of native full-length streptavidin monomers was successfully secreted in the culture supernatant of Streptomyces lividans transformants, and had specific biotin binding affinity as strong as streptavidin produced by Escherichia coli. The amount of Sav using Streptomyces lividans was about 9 times higher than using Escherichia coli. Surprisingly, streptavidin produced by Streptomyces lividans exhibited affinity to biotin after boiling, despite the fact that tetrameric streptavidin is known to lose its biotin binding ability after brief boiling. Conclusion: We successfully produced a large amount of tetrameric streptavidin as a secretory-form protein with unique thermotolerance.BIOMED CENTRAL LTD, Jan. 2015, MICROBIAL CELL FACTORIES, 14(1) (1), 5, English[Refereed]Scientific journal
- We here describe a unique beta-D-glucosidase (BGL; Blon_0625) derived from Bifidobacterium longum subsp. infantis ATCC 15697. The Blon_0625 gene was expressed by recombinant Escherichia coli. Purified recombinant Blon_0625 retains hydrolyzing activity against both p-nitrophenyl-beta-D-glucopyranoside (pNPG; 17.3 +/- 0.24 U mg(-1)) and p-nitrophenyl-beta-D-xylopyranoside (pNPX; 16.7 +/- 0.32 U mg(-1)) at pH 6.0, 30 degrees C. To best of our knowledge, no previously described BGL retains the same level of both pNPGase and pNPXase activity. Furthermore, Blon_0625 also retains the activity against 4-nitrophenyl-alpha-L-arabinofranoside (pNPAf; 5.6 +/- 0.09 U mg(-1)). In addition, the results of the degradation of phosphoric acid swollen cellulose (PASC) or xylan using endoglucanase from Thermobifida fusca YX (Tfu_0901) or xylanase from Kitasatospora setae KM-6054 (KSE_59480) show that Blon_0625 acts as a BGL and as a beta-D-xylosidase (XYL) for hydrolyzing oligosaccharides. These results clearly indicate that Blon_0625 is a multi-functional glycoside hydrolase which retains the activity of BGL, XYL, and also alpha-L-arabinofuranosidase. Therefore, the utilization of multi-functional Blon_0625 may contribute to facilitating the efficient degradation of lignocellulosic materials and help enhance bioconversion processes. Copyright (C) 2014 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, Jan. 2015, ENZYME AND MICROBIAL TECHNOLOGY, 68, 10 - 14, English[Refereed]Scientific journal
- Effective secretion of green fluorescent protein (GFP) was investigated by the screening signal sequences for GFP secretion in Aspergillus oryzae. GFP production in A. oryzae was evaluated using fusions with signal sequences from Taka-amylase A (TAA), glucoamylase A, glucoamylase S, and triacylglycerol lipase. The TAA signal sequence promoted the highest protein secretion of GFP. Fusing this signal sequence with an N-terminal 28-amino acid region (N28 fragment) from the Rhizopus oryzae lipase signal sequence increased protein secretion. In addition, using multiple copies of this signal sequence, instead of the N28 fragment, also induced protein secretion. These results show that using multiple signal sequences or combining a signal sequence with the N28 fragment can be used to improve heterogeneous protein secretion in A. oryzae. (C) 2014 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Jul. 2014, PROCESS BIOCHEMISTRY, 49(7) (7), 1078 - 1083, English[Refereed]Scientific journal
- We demonstrated direct utilization of xylooligosaccharides using beta-xylosidase-displaying Escherichia coll. After screening active beta-xylosidases, BSU17580 from Bacillus subtilis or Tfu1616 from Thermobifida fusca YX, were successfully displayed on the E. coli cell surface using Blc or HdeD as anchor proteins, and these transformants directly assimilated xylooligosaccharides as a carbon source. The final OD 600 in minimal medium containing 2% xylooligosaccharides was 1.09 (after 12 h of cultivation) and 130 (after 40 h of cultivation). We then constructed an E. coli strain displaying both beta-glucosidase and beta-xylosidase. beta-glucosidase- and beta-xylosidase-displaying E. coli was successfully grown on a 1% cellobiose and 1% xylooligosaccharides mixture, and the OD 600 was 1.76 after 10 h of cultivation, which was higher and reached faster than that grown on a glucose/xylose mixture (1.20 after 30 h of cultivation).AMER CHEMICAL SOC, Jul. 2014, ACS SYNTHETIC BIOLOGY, 3(7) (7), 446 - 453, English[Refereed]Scientific journal
- We demonstrated direct utilization of xylooligosaccharides using beta-xylosidase-displaying Escherichia coll. After screening active beta-xylosidases, BSU17580 from Bacillus subtilis or Tfu1616 from Thermobifida fusca YX, were successfully displayed on the E. coli cell surface using Blc or HdeD as anchor proteins, and these transformants directly assimilated xylooligosaccharides as a carbon source. The final OD 600 in minimal medium containing 2% xylooligosaccharides was 1.09 (after 12 h of cultivation) and 130 (after 40 h of cultivation). We then constructed an E. coli strain displaying both beta-glucosidase and beta-xylosidase. beta-glucosidase- and beta-xylosidase-displaying E. coli was successfully grown on a 1% cellobiose and 1% xylooligosaccharides mixture, and the OD 600 was 1.76 after 10 h of cultivation, which was higher and reached faster than that grown on a glucose/xylose mixture (1.20 after 30 h of cultivation).AMER CHEMICAL SOC, Jul. 2014, ACS SYNTHETIC BIOLOGY, 3(7) (7), 446 - 453, English[Refereed]Scientific journal
- To develop cost-effective systems for d-lactate production, here, the effect of high-cell density cultivation of metabolically engineered Lactobacillus plantarum on d-lactate production was evaluated. A xylose-assimilating strain of L. plantarum was anaerobically cultured with mixed sugars (glucose and xylose) as substrates. Compared to undiluted nutrient-rich de Man, Rogosa, and Sharpe (MRS) medium, d-lactate production by cultivating in 10-fold diluted MRS (0.1 MRS) medium or normal saline solution was 89.7 and 81.3 %, respectively. Notably, the xylose consumption rate was comparable in the three cultures, whereas the glucose consumption rate decreased by 18.3 and 26.1 % in 0.1 MRS medium and normal saline solution, respectively, resulting in a reduction of the d-lactate production rate. The d-lactate productivity in high-cell density cultivation was proportional to the initial cell concentrations. The use of a two-step cultivation process involving growing and resting cells in a single bioreactor revealed that the ratio of the glucose and xylose consumption rates (based on grams consumed) in resting cell conditions was 1.88, whereas that in growing conditions was 2.58. Cultivation of L. plantarum in growing conditions for 24 h produced 73.2 g/l d-lactate with the yield of 0.90 g/g, whereas cells cultivation under resting cell conditions in a saline solution for 24 h produced 68.7 g/l d-lactate with the yield of 0.93 g/g. In total, 141.9 g/l d-lactate was produced after 48 h cultivation, a value that represents the highest reported concentration of d-lactate produced from mixed sugars to date. Our findings contribute to the cost-effective, large-scale production of d-lactate.SPRINGER, Jun. 2014, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 98(11) (11), 4911 - 4918, English[Refereed]Scientific journal
- Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA and LA-based polymers production using several kinds of genetically modified microorganisms, such as lactic acid bacteria, Escherichia coli, Corynebacterium glutamicum, and yeasts. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass have been significantly improved, as well as LA-based polymers. The drawbacks as well as improvements of LA production by fermentation are discussed.Wiley Blackwell, Apr. 2014, Bioprocessing of Renewable Resources to Commodity Bioproducts, 353 - 380, English[Refereed]In book
- We produced organic acids, including lactate and succinate, directly from soluble starch under anaerobic conditions using high cell-density cultures of Corynebacterium glutamicum displaying α-amylase (AmyA) from Streptococcus bovis 148 on the cell surface. Notably, reactions performed under anaerobic conditions at 35 and 40°C, which are higher than the optimal growth temperature of 30°C, showed 32% and 19%, respectively, higher productivity of the organic acids lactate, succinate, and acetate compared to that at 30°C. However, α-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30°C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30°C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated.Dec. 2013, AMB Express, 3(1) (1), 72 - 72, English, International magazine[Refereed]Scientific journal
- The present study reports the design of a novel bioanode to deeply oxidize glucose in an enzymatic biofuel cell (EFC). This enzymatic glucose cell utilizes three co-immobilized enzymes: NAD-dependent glucose dehydrogenase (GDH), NAD(P)(+)-dependent gluconate-5-dehydrogenase (Ga5DH), and diaphorase (DI). Glucose is oxidized to gluconate by NAD-dependent GDH, gaining two electrons per glucose; the gluconate obtained as a by-product is oxidized at the C5 carbon to 5-keto-gluconate by Ga5DH. Operation of our bioanode enabled the oxidation of glucose in two stages, resulting in the gain of four electrons. The three-enzyme EFC provides a maximum power density of 10.51 +/- 1.72Wcm(-2), which is about 1.6 times higher than the maximum power density of an EFC using a bioanode based on the co-immobilization of two enzymes (GDH and DI). Our results hold promise for increasing the current density of EFCs, and for application in glucose biosensor.WILEY-V C H VERLAG GMBH, Dec. 2013, FUEL CELLS, 13(6) (6), 960 - 964, English[Refereed]Scientific journal
- Background: Protein production as secretory-form is a powerful tool in industrial enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. In order to expand the amount of secreted protein, signal peptide sequences, which encourage protein secretion from inside cell to extracellular environment, are one of the most significant factors. In this study, we focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomass-degradation enzymes derived from Thermobifida fusca YX and S. lividans. Results: Three candidate signal peptides were isolated and evaluated for their protein secretion ability using beta-glucosidase derived from T. fusca YX, which is a non-secreted protein, as a model protein. Using S. lividans xylanase C signal peptide, the amount of produced the beta-glucosidase reached 10 times as much as that when using Streptomyces cinnamoneus phospholipase D signal peptide, which was identified as a versatile signal peptide in our previous report. In addition, the introduction of the beta-glucosidase fused to xylanase C signal peptide using two kinds of plasmid, pUC702 and pTYM18, led to further protein secretion, and the maximal level of produced the beta-glucosidase increased up to 17 times (1.1 g/l) compared to using only pUC702 carrying the beta-glucosidase fused to S. cinnamoneus phospholipase D signal peptide. Conclusion: In the present study, we focused on signal peptide sequences derived from biomass degradation enzymes, which are usually secreted into the culture supernatant, and screened for signal peptides leading to effective protein secretion. Using the signal peptides, the hyper-protein secretion system was successfully demonstrated for the cytoplasmic beta-glucosidase.BIOMED CENTRAL LTD, Oct. 2013, MICROBIAL CELL FACTORIES, 12(1) (1), 88, English[Refereed]Scientific journal
- In this study, the water-retaining cyclic amino acid ectoine was produced from a variety of sugars, including glucose, xylose, cellobiose, and glucose/xylose mixture using engineered Halomonas elongata. When grown on xylose as the sole carbon source, H. elongata produced 333 mmol/kg fresh cell weight (FW) of ectoine, which was 1.4-fold higher than that produced from glucose. To improve ectoine production, an ectD deficient H. elongata mutant was constructed. The engineered H. elongata produced 377 mmol/kg FIN of ectoine from a glucose/xylose mixture. Ectoine was also produced from rice straw hydrolysate. These results show that H. elongata can produce ectoine from a variety of sugars derived from lignocellulosic biomass and thus has tremendous potential as a host for producing useful compounds from biomass resources. (C) 2013 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Aug. 2013, BIORESOURCE TECHNOLOGY, 142, 523 - 529, English[Refereed]Scientific journal
- We constructed beta-glucosidase (BGL)-displaying Corynebacterium glutamicum, and direct l-lysine fermentation from cellobiose was demonstrated. After screening active BGLs, Sde1394, which is a BGL from Saccharophagus degradans, was successfully displayed on the C. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. The optical density at 600 nm of BGL-displaying C. glutamicum grown on cellobiose as a carbon source reached 23.5 after 48 h of cultivation, which was almost the same as that of glucose after 24 h of cultivation. Finally, Sde1394-displaying C. glutamicum produced 1.08 g/l of l-lysine from 20 g/l of cellobiose after 4 days of cultivation, which was about threefold higher than the amount of produced l-lysine using BGL-secretory C. glutamicum strains (0.38 g/l after 5 days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. glutamicum. © 2013 Springer-Verlag Berlin Heidelberg.Aug. 2013, Applied Microbiology and Biotechnology, 97(16) (16), 7165 - 7172, English[Refereed]Scientific journal
- We screened for high-activity endoglucanase (EG) as a first step toward the creation of cellulose-assimilating Streptomyces lividans transformants. EGs derived from Thermobifida fusca YX, Tfu0901, and S. lividans, cellulase B (CelB), were successfully expressed. Genes encoding Tfu0901 or CelB were introduced into S. lividans using the integrative vector pTYM18 and the high-copy-number vector pUC702, and EG activity was detected in the supernatant of each transformant. To achieve coexpression of EG and transglutaminase, the transglutaminase gene was introduced into EG-secreting S. lividans using pUC702. S. lividans coexpressing EG and transglutaminase effectively assimilated phosphoric acid swollen cellulose. The yield of Streptomyces cinnamoneus transglutaminase in the culture supernatant was 7.2 mg/L, which was 18 times higher than that of the control strain. To demonstrate the versatility of our system, we also created an EG-producing S. lividans transformant capable of coexpressing endoxylanase. The EG-secreting S. lividans transformants constructed here can be used to produce other useful compounds through cellulose fermentation.SPRINGER, Jul. 2013, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97(13) (13), 5711 - 5720, English[Refereed]Scientific journal
- The present study reports the design of a novel bioanode to directly utilize starch as a fuel in an enzymatic biofuel cell. The enzymatic fuel cell is based on three enzymes (alpha-amylase, glucoamylase and glucose oxidase). The carbon paste electrode containing these three enzymes and tetrathiafulvalene can both saccharize and oxidize starchy biomass. In cyclic voltammetry, catalytic currents were successfully observed with both glucose and starchy white rice used as a substrate. Finally, a membrane-less white rice/O2 biofuel cell was assembled and the electrochemical performance was evaluated. The three enzyme based electrode was used as a bioanode and an immobilized bilirubin oxidase (derived from Myrothecium verrucaria) electrode was used as a biocathode. The biofuel cell delivered an open circuit voltage of 0.522V and power density of up to 99.0 μWcm(-2). Our results show that a readily available fuel can be used for enzymatic fuel cells, and will lead to new designs.Jun. 2013, New biotechnology, 30(5) (5), 531 - 5, English, International magazine[Refereed]Scientific journal
- Background: p-Hydroxycinnamic acid (pHCA) is an aromatic compound that serves as a starting material for the production of many commercially valuable chemicals, such as fragrances and pharmaceuticals, and is also used in the synthesis of thermostable polymers. However, chemical synthesis of pHCA is both costly and harmful to the environment. Although pHCA production using microbes has been widely studied, there remains a need for more cost-effective methods, such as the use of biomass as a carbon source. In this study, we produced pHCA using tyrosine ammonia lyase-expressing Streptomyces lividans. In order to improve pHCA productivity from cellulose, we constructed a tyrosine ammonia lyase- and endoglucanase (EG)-expressing S. lividans transformant and used it to produce pHCA from cellulose.Results: A Streptomyces lividans transformant was constructed to express tyrosine ammonia lyase derived from Rhodobacter sphaeroides (RsTAL). The transformant produced 786 or 736 mg/L of pHCA after 7 days of cultivation in medium containing 1% glucose or cellobiose as the carbon source, respectively. To enhance pHCA production from phosphoric acid swollen cellulose (PASC), we introduced the gene encoding EG into RsTAL-expressing S. lividans. After 7 days of cultivation, this transformant produced 753, 743, or 500 mg/L of pHCA from 1% glucose, cellobiose, or PASC, respectively.Conclusions: RsTAL-expressing S. lividans can produce pHCA from glucose and cellobiose. Similarly, RsTAL- and EG-expressing S. lividans can produce pHCA from glucose and cellobiose with excess EG activity remaining in the supernatant. This transformant demonstrated improved pHCA production from cellulose. Further enhancements in the cellulose degradation capability of the transformant will be necessary in order to achieve further improvements in pHCA production from cellulose. © 2013 Kawai et al. licensee BioMed Central Ltd.May 2013, Microbial Cell Factories, 12(1) (1), 45, English[Refereed]Scientific journal
- Streptomyces mobaraensis transglutaminase (MTG) is one of the most useful transglutaminases due to its rather broad substrate specificity and independence of Ca2+. To achieve efficient production of active-form MTG using Streptomyces lividans as a host, we created three vector constructs consisting of the signal peptide sequence (pld signal) derived from the phospholipase D gene of Streptomyces cinnamoneus, prepro-domain of S. cinnamoneus transglutaminase, and the sequence encoding mature MTG, and then generated three over-expressing S. lividans strains. We successfully demonstrated that S. lividans can be used as a host for the efficient production of mature, active-form MTG. © 2013 Elsevier B.V.May 2013, Biochemical Engineering Journal, 74, 76 - 80, English[Refereed]Scientific journal
- Metabolic pathway engineering of cyanobacteria for the production of industrially important chemicals from atmospheric CO2 has generated interest recently. Here, we engineered Synechocystis sp. PCC 6803 to produce lactic acid using a lactate dehydrogenase (ldh) gene from various lactic acid-producing bacteria, Lactococcus lactis (ldhB and ldhX), Lactobacillus plantarum (ldhL and ldh), and Lactobacillus rhamnosus (ldhL). The lactic acid was secreted outside the cell using a transporter (lldp) gene from L. plazztarum. Expression of each ldh in Synechocystis sp. PCC6803 was ascertained by reverse-transcriptase polymerase chain reaction. Five transformants led to the production of L-lactic acid. Coexpression of lldp with ldhB from L. plantarum or ldhL from L. rhamnosus led to the secretion of lactic acid into the medium at concentration of 0.17 +/- 0.02 or 0.14 +/- 0.02 mm after 18 d of cultivation.TAYLOR & FRANCIS LTD, May 2013, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77(5) (5), 966 - 970, English[Refereed]Scientific journal
- Metabolic pathway engineering of cyanobacteria for the production of industrially important chemicals from atmospheric CO2 has generated interest recently. Here, we engineered Synechocystis sp. PCC 6803 to produce lactic acid using a lactate dehydrogenase (ldh) gene from various lactic acid-producing bacteria, Lactococcus lactis (ldhB and ldhX), Lactobacillus plantarum (ldhL and ldh), and Lactobacillus rhamnosus (ldhL). The lactic acid was secreted outside the cell using a transporter (lldp) gene from L. plazztarum. Expression of each ldh in Synechocystis sp. PCC6803 was ascertained by reverse-transcriptase polymerase chain reaction. Five transformants led to the production of L-lactic acid. Coexpression of lldp with ldhB from L. plantarum or ldhL from L. rhamnosus led to the secretion of lactic acid into the medium at concentration of 0.17 +/- 0.02 or 0.14 +/- 0.02 mm after 18 d of cultivation.TAYLOR & FRANCIS LTD, May 2013, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77(5) (5), 966 - 970, English[Refereed]Scientific journal
- Here, we demonstrate display of beta-glucosidase (BGL) on the surface of Schizosaccharomyces pombe cells using novel anchor proteins. A total of four candidate anchor proteins (SPBC21D10.06c, SPBC947.04, SPBC19C7.05, and SPBC359.04c) were selected from among almost all of S. pombe membrane proteins. The C-terminus of each anchor protein was genetically fused to the N-terminus of BGL, and the fusion protein was expressed using S. pombe as a host. The highest cell surface-associated BGL activity (107 U/10(5) cells was achieved with SPBC359.04c serving as the anchor, followed by SPBC947.04 (44 U/10(5) cells) and SPBC21D10.06c (38 U/10(5) cells). S. pombe displaying BGL with SPBC359.04c as an anchor showed the highest growth on 2 % cellobiose (10.7 x 10(7) cells/mL after 41 h of cultivation from an initial density of 0.1 x 10(7) cells/mL). Additionally, culturing BGL-displaying S. pombe in medium containing cellobiose as the sole carbon source did not affect protein expression, and ethanol fermentation from cellobiose was successfully demonstrated using BGL-displaying S. pombe. This is the first report describing a cell surface display system for the functionalization of S. pombe.SPRINGER, May 2013, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97(10) (10), 4343 - 4352, English[Refereed]Scientific journal
- We synthesized several surfactant-like ligands and prepared affinity membranes by introducing them into porous polymeric membranes using the thermally induced phase separation method. The ligands (nitrilotriacetate, iminodiacetate, and glutathione) were successfully displayed on the surfaces of cellulose diacetate membranes. Membranes functionalized with nitrilotriacetate and glutathione captured and released hexahistidine-tagged enhanced green fluorescent protein (His-tag GFP) and glutathione S-transferase (GST) selectively under appropriate conditions. The affinity membranes also enabled highly selective purification of target proteins (GFP and GST) from cell lysates. The protein-binding capacity was 15 mu g/cm(2) for His-tag GFP and 13 mu g/cm(2) for GST. The application-specific membranes described in this work will aid high-throughput screening and high-throughput analysis of recombinant proteins. (c) 2012 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Mar. 2013, ANALYTICAL BIOCHEMISTRY, 434(2) (2), 269 - 274, English[Refereed]Scientific journal
- 2013, Colloid and Polymer Science, 291(1) (1)Scientific journal
- Size-controllable TiO2 nanosheets with highly exposed {001} facets were synthesized by a hydrothermal method. The particle sizes ranged from 25 nm to submicrometres by carefully adjusting the F/Ti molar ratio. TiO2 nanosheets smaller than 100 nm have higher photocatalytic activity and are highly stable in degradation of organic dyes.ROYAL SOC CHEMISTRY, 2013, RSC ADVANCES, 3(42) (42), 19268 - 19271, English[Refereed]Scientific journal
- Direct polyhydroxybutyrate (PHB) production from starch was for the first time achieved using engineered Corynebacterium glutamicum expressing PHB biosynthetic genes and displaying a-amylase on its cell surface. The engineered strain accumulated 6.4 wt% PHB from starch which was higher than that obtained from glucose (4.9 wt%). (C) 2012, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Jan. 2013, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 115(1) (1), 12 - 14, English[Refereed]Scientific journal
- A novel multi-cellulase conjugate assembled on a double-stranded DNA scaffold, a DNA-(endoglucanase)(n) conjugate, exhibited unique hydrolytic activity toward crystalline cellulose (Avicel) depending on the cellulase/DNA ratio on the DNA-based artificial cellulosome.ROYAL SOC CHEMISTRY, 2013, CHEMICAL COMMUNICATIONS, 49(62) (62), 6971 - 6973, English[Refereed]Scientific journal
- In this study, we demonstrate the one-step production of cadaverine (1,5-diaminopentane) from cellobiose using an Escherichia coli strain displaying beta-glucosidase (BGL) on its cell surface. L-lysine decarboxylase (CadA) derived from E. coli and BGL from Thermobifida fusca YX (Tfu0937) fused to the anchor protein Blc from E. coli were co-expressed using E. coli as a host. The expression of CadA was confirmed by Western blotting and BGL activity on the cell surface was evaluated using pNPG as a substrate. Growth on cellobiose as the sole carbon source was also achieved. The OD600 value of the BGL and CadA co-expressing strain was 8.0 after 48 h cultivation, which is higher than that obtained by growth on glucose (5.4 after 48 h cultivation). The engineered strain produced cadaverine from cellobiose more effectively than from glucose: 6.1 mM after 48 h from 28 g/L of consumed cellobiose, vs. 3.3 mM from 20 g/L of consumed glucose.BIOMED CENTRAL LTD, 2013, AMB EXPRESS, 3(1) (1), 1 - 7, English[Refereed]Scientific journal
- We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V-HH) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V-HH with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V-HH reached 73.8 mg/1 in a Sakaguchi flask. The V-HH fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V-HH fragment was specifically recognized and could bind to the EGFR with a high affinity.SPRINGER, Oct. 2012, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 96(1) (1), 81 - 88, English[Refereed]Scientific journal
- Bioconjugates are valuable tools in many fields, including protein engineering and environmental and therapeutic research. Chemical methods are commonly used to synthesize protein-protein and protein-functional molecule bioconjugates because they permit easy tethering through covalent bonds. However, chemical methods often produce heterogeneous products and lead to degradation of protein activity due to random modifications. Recently, a number of techniques for modifying proteins or synthesizing bioconjugates have been reported, including more sophisticated chemical modification methods, utilization of noncovalent affinity, and protein splicing. Enzymatic methods in particular have attracted much attention due to the substrate specificity of enzymes, which enables site-specific tethering of proteins to other proteins or functional molecules. Here, we discuss newly developed methods for protein modification and bioconjugate synthesis that exploit the properties of acyltransferases, ligases, and other enzymes.WILEY-BLACKWELL, Sep. 2012, BIOTECHNOLOGY JOURNAL, 7(9) (9), 1137 - 1146, English[Refereed]Scientific journal
- Yeasts are promising hosts for industrial bio-refinery applications. In yeast cell surface displays, functional proteins, such as cellulases or lipases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae is the most commonly used yeast for cell surface display. Engineered yeasts have been utilized for a variety of applications, such as bioethanol production, chemicals synthesis, adsorption of environmental pollutants, and protein evolution. Here, we summarize recent developments in yeast cell surface display techniques for bio-refinery applications, including methods using hosts such as Pichia pastoris, Yarrowia lipolytica, and S. cerevisiae, focusing on the characteristics of anchor proteins and applications.SPRINGER, Aug. 2012, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 95(3) (3), 577 - 591, English[Refereed]Scientific journal
- Efficient bio-production from lignocellulosic biomass is required for the purpose of developing an inexpensive, practical bio-refinery process. As one approach to address this problem, we genetically engineered Escherichia coli to produce isopropanol directly from cellobiose via the cellobiose degradation by Beta-Glucosidase (BGL) on the cell surface. First, we investigated the cellobiose consumption of two E. coli strains with the BGL protein from Thermobifida filsca YX (Tfu0937) fused to the anchor protein Blc (Tfu0937/Blc) using different fusion sites. Next, we introduced the synthetic pathway for isopropanol production into those strains and compared their isopropanol production in the presence of glucose. Based on the results of these assays, TA212/pTA411, which was introduced Tfu-Blc fused protein expression system and the synthetic pathway for isopropanol production, was selected for the directly isopropanol production from cellobiose. TA212/pTA411 produced 69.0 +/- 11.6 mM isopropanol at 21 h of fermentation, whereas TA212/pTA147, which did not introduced the BGL/anchor fused protein but was introduced the synthetic pathway for isopropanol production, showed no cellobiose consumption and no isopropanol production during fermentation. To our knowledge, this is the first report of the production of a bio-product from cellobiose using E. coli. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Jul. 2012, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 114(1) (1), 80 - 85, English[Refereed]Scientific journal
- Modification of proteins with small molecules is a widely used and powerful tool in biological research. Enzymatic approaches are particularly promising because substrate specificity allows for site-specific modification. Sortase A, a transpeptidase from Staphylococcus aureus, cleaves between the T and G residues in the sequence LPXTG, and subsequently links the carboxyl group of the T residue to an amino group of N-terminal glycine oligomers by a native peptide bond. Although Gram-positive bacteria have several kinds of sortases, there are few reports concerning their expression and substrate specificity. Here, we demonstrate site-specific protein modification with primary amine-containing molecules catalyzed by Lactobacillus plantarum sortase. Enhanced green fluorescent protein (EGFP) was employed as a model protein, and an amine-containing biotin molecule was site-specifically conjugated with LPQTSEQ-tagged EGFP. We developed a novel Lactobacillus plantarum sortase that has different substrate specificity compared to Staphylococcus aureus sortase. Amine-directed protein modification was achieved using the Lactobacillus plantarum sortase ''LPQTSEQ'' sequence original recognition tag. Our results demonstrate a promising method for expanding the capabilities of site-specific protein-small molecule modification.WILEY-BLACKWELL, May 2012, BIOTECHNOLOGY JOURNAL, 7(5) (5), 642 - 648, English[Refereed]Scientific journal
- A diploid yeast strain displaying both alpha-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of alpha-amylase was optimized for cell surface display, as there have been no reports of alpha-amylase-displaying yeast. The modified yeast displaying both glucoamylase and alpha-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native alpha-amylase-displaying yeast. Using the glucoamylase and modified alpha-amylase co-displaying diploid strain, we repeated fermentation from 100 g/l of raw starch for 23 cycles without the loss of alpha-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production. (C) 2012 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, May 2012, ENZYME AND MICROBIAL TECHNOLOGY, 50(6-7) (6-7), 343 - 347, English[Refereed]Scientific journal
- A diploid yeast strain displaying both alpha-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of alpha-amylase was optimized for cell surface display, as there have been no reports of alpha-amylase-displaying yeast. The modified yeast displaying both glucoamylase and alpha-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native alpha-amylase-displaying yeast. Using the glucoamylase and modified alpha-amylase co-displaying diploid strain, we repeated fermentation from 100 g/l of raw starch for 23 cycles without the loss of alpha-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production. (C) 2012 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, May 2012, ENZYME AND MICROBIAL TECHNOLOGY, 50(6-7) (6-7), 343 - 347, English[Refereed]Scientific journal
- Background: Benzoic acid is one of the most useful aromatic compounds. Despite its versatility and simple structure, benzoic acid production using microbes has not been reported previously. Streptomyces are aerobic, Gram-positive, mycelia-forming soil bacteria, and are known to produce various kinds of antibiotics composed of many aromatic residues. S. maritimus possess a complex amino acid modification pathway and can serve as a new platform microbe to produce aromatic building-block compounds. In this study, we carried out benzoate fermentation using S. maritimus. In order to enhance benzoate productivity using cellulose as the carbon source, we constructed endo-glucanase secreting S. maritimus. Results: After 4 days of cultivation using glucose, cellobiose, or starch as a carbon source, the maximal level of benzoate reached 257, 337, and 460 mg/l, respectively. S. maritimus expressed beta-glucosidase and high amylase-retaining activity compared to those of S. lividans and S. coelicolor. In addition, for effective benzoate production from cellulosic materials, we constructed endo-glucanase-secreting S. maritimus. This transformant efficiently degraded the phosphoric acid swollen cellulose (PASC) and then produced 125 mg/l benzoate. Conclusions: Wild-type S. maritimus produce benzoate via a plant-like beta-oxidation pathway and can assimilate various carbon sources for benzoate production. In order to encourage cellulose degradation and improve benzoate productivity from cellulose, we constructed endo-glucanase-secreting S. maritimus. Using this transformant, we also demonstrated the direct fermentation of benzoate from cellulose. To achieve further benzoate productivity, the L-phenylalanine availability needs to be improved in future.BIOMED CENTRAL LTD, Apr. 2012, MICROBIAL CELL FACTORIES, 11, 49, English[Refereed]Scientific journal
- A microparticle surface was designed by the unique method incorporating streptavidin biotin affinity and sortase A (SrtA)-catalyzed transpeptidation. Leucine-proline-glutamate-threonine-glycine-tagged streptavidin (Stav-LPETG) was immobilized on the surface using streptavidin biotin affinity, and GGGGG-tagged red fluorescent protein (Gly5-RFP) was conjugated with SrtA. Biotinylated fluorescein isothiocyanate (biotin FITC) was then bound to residual biotin-binding sites in Stav-LPETG. The resulting particles had RFP and FITC immobilized on the surface via Stav-LPETG, and RFP- and FITC-associated fluorescence was observed using fluorescence microscopy. Finally, GGG-tagged glucose oxidase and biotinylated horseradish peroxidase were immobilized on the microparticle surface, resulting in a functional particle capable of detecting glucose. This particle can be repeatedly used and is more sensitive in detecting glucose than particles prepared using chemical modification. Our method provides a simple strategy for site-specific coimmobilization on molecular surfaces and expands the use of protein hybrid devices.AMER CHEMICAL SOC, Feb. 2012, LANGMUIR, 28(7) (7), 3553 - 3557, English[Refereed]Scientific journal
- Bio-nanocapsules (BNCs) are hollow nanoparticles composed of the L protein of hepatitis B virus (HBV) surface antigen (HBsAg), which can specifically introduce genes and drugs into various kinds of target cells. Although the classic electroporation method has typically been used to introduce highly charged molecules such as DNA, it is rarely adopted for proteins due to its very low efficiency. In this study, a novel approach to the preparation of BNC was established whereby a target protein was pre-encapsulated during the course of nanoparticle formation. Briefly, because of the process of BNC formation in a budding manner on the endoplasmic reticulum (ER) membrane, the association of target proteins to the ER membrane with lipidation sequences (ER membrane localization sequences) could directly generate protein-encapsulating BNC in collaboration with co-expression of the L proteins. Since the membrane-localized proteins are automatically enveloped into BNCs during the budding event, this method can be protect the proteins and BNCs from damage caused by electroporation and obviate the need for laborious consideration to study the optimal conditions for protein encapsulation. This approach would be a useful method for encapsulating therapeutic candidate proteins into BNCs. (C) 2011 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Jan. 2012, JOURNAL OF BIOTECHNOLOGY, 157(1) (1), 124 - 129, English[Refereed]Scientific journal
- Transglutaminase from Streptoverticillium cinnamoneum (StvcMTG) was produced using recombinant Streptomyces lividans. When grown on glycerol and xylose as sole carbon sources, S. lividans/StvcMTG produced 360 and 530 mg of StvcMTG per liter, respectively. With starch and xylan, the strain produced 230 and 400 mg of StvcMTG per liter, respectively. Recombinant S. lividans/encP, which expresses phenylalanine ammonia lyase from Streptomyces maritimus, produced 160 mg/L of cinnamic acid from cellulose. These results show that S. lividans can assimilate various carbon sources and produce useful compounds in desirable quantities. (C) 2011 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Jan. 2012, BIORESOURCE TECHNOLOGY, 104, 648 - 651, English[Refereed]Scientific journal
- Task-specific membranes for recombinant proteins with peptide tags were developed by introducing surfactant-like ligands to polymeric membranes, which allowed the simple and rapid purification of target proteins from E. coli cell lysates.ROYAL SOC CHEMISTRY, 2012, RSC ADVANCES, 2(1) (1), 125 - 127, English[Refereed]Scientific journal
- In order to achieve efficient d-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (Delta ldhL1-xpk1::tkt-Delta xpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of Delta ldhL1-xpk1::tkt-Delta xpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-d-lactic acid production was achieved from a mixture of 25 g/l xylose and 75 g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2 g/l of lactic acid was produced with a high yield (0.78 g per gram of consumed sugar) and an optical purity of d-lactic acid of 99.5%. Finally, we successfully demonstrated homo-d-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-d-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.SPRINGER, Oct. 2011, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 92(1) (1), 67 - 76, English[Refereed]Scientific journal
- In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK pathway) that produces both lactic acid and acetic acid and the pentose phosphate pathway (PP pathway) that produces only lactic acid as a final product. Thus a mutant strain that disrupted its phosphokeolase gene (ptk) was constructed. The Delta ptk mutant harboring pXylRAB lacked the PK pathway and produced predominantly lactic acid from xylose via the PP pathway, although its fermentation rate slightly decreased. Further introduction of the transketolase gene (tkt) to disrupted ptk locus led restoration of fermentation rate and this was attributed to enhancement of the PP pathway. As a result, ptk::tkt strain harboring pXylRAB produced 50.1 g/l of L-lactic acid from xylose with a high optical purity of 99.6% and a high yield of 1.58 (moles per mole xylose consumed) that is close to theoretical value of 1.67 from xylose.SPRINGER, Sep. 2011, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 91(6) (6), 1537 - 1544, English[Refereed]Scientific journal
- We demonstrated direct assimilation of cellooligosaccharide using Escherichia coli displaying beta-glucosidase (BGL). BGL from Thermobifida fusca YX (Tfu0937) was displayed on the E. coli cell surface using a novel anchor protein named Blc. This strain was grown successfully on 0.2% cellobiose, and the optical density at 600 nm (OD(600)) was 1.05 after 20 h.AMER SOC MICROBIOLOGY, Sep. 2011, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77(17) (17), 6265 - 6270, English[Refereed]Scientific journal
- We constructed a recombinant industrial Saccharomyces cerevisiae yeast strain OC2-AXYL2-ABGL2-Xyl2 by inserting two copies of the beta-glucosidase (BGL) and beta-xylosidase (XYL) genes, and a gene cassette for xylose assimilation in the genome of yeast strain OC-2HUT. Both BGL and XYL were expressed on the yeast cell surface with high enzyme activities. Using OC2-AXYL2-ABGL2-Xyl2, we performed ethanol fermentation from a mixture of powdered cellulose (KC-flock) and Birchwood xylan, with the additional supplementation of a 30-g/l Trichoderma reesei cellulase complex mixture. The ethanol yield (gram per gram of added cellulases) of the strain OC2-AXYL2-ABGL2-Xyl2 increased approximately 2.5-fold compared to that of strain OC2-Xyl2, which lacked beta-glucosidase and beta-xylosidase activities. Notably, the concentration of additional T. reesei cellulase was reduced from 30 to 24 g/l without affecting ethanol production. The BGL- and XYL-displaying industrial yeast of the strain OC2-AXYL2-ABGL2-Xyl2 represents a promising yeast for reducing cellulase consumption of ethanol fermentation from lignocellulosic biomass by compensating for the inherent weak BGL and XYL activities of T. reesei cellulase complexes.SPRINGER, Sep. 2011, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 91(6) (6), 1553 - 1559, English[Refereed]Scientific journal
- We demonstrate glutamate production from beta-glucan using endoglucanase (EG)-expressing Corynebacterium glutamicum. The signal sequence torA derived from Escherichia coli K12, which belongs to the Tat pathway, was suitable for secreting EG of Clostridium thermocellum using C. glutamicum as a host. Using the torA signal sequence, endoglucanase from Clostridium cellulovorans 743B was successfully expressed, and the secreted EG produced 123 mg of reducing sugar from 5 g of beta-glucan at 30 A degrees C for 72 h, which is the optimal condition for C. glutamicum growth. Subsequently, glutamate fermentation from beta-glucan was carried out with the addition of Aspergillus aculeatus beta-glucosidase produced by recombinant Aspergillus oryzae. Using EG-secreting C. glutamicum, 178 mg/l of glutamate was produced from 15 g of beta-glucan. This is the first report of glutamate fermentation from beta-glucan using endoglucanase-secreting C. glutamicum.SPRINGER, May 2011, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 90(3) (3), 895 - 901, English[Refereed]Scientific journal
- Cinnamic acid production was demonstrated using Streptomyces as a host. A gene encoding phenylalanine ammonia lyase (PAL) from Streptomyces maritimus was introduced into Streptomyces lividans, and its expression was confirmed by Western blot analysis. After 4 days cultivation using glucose as carbon source, the maximal level of cinnamic acid reached 210 mg/L. When glycerol (30 g/L) was used as carbon source, the maximal level of produced cinnamic acid reached 450 mg/L. In addition, using raw starch, xylose or xylan as carbon source, the maximal level of cinnamic acid reached 460, 300, and 130 mg/L, respectively. We demonstrated that S. lividans has great potential to produce cinnamic acid as well as other aromatic compounds.SPRINGER HEIDELBERG, May 2011, JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, 38(5) (5), 643 - 648, English[Refereed]Scientific journal
- Background: Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and beta-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid Saccharomyces cerevisiae strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass. Results: The engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours. Conclusions: We have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzyme-expressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.BIOMED CENTRAL LTD, Apr. 2011, BIOTECHNOLOGY FOR BIOFUELS, 4, English[Refereed]Scientific journal
- Background: Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and beta-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid Saccharomyces cerevisiae strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass. Results: The engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours. Conclusions: We have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzyme-expressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.BIOMED CENTRAL LTD, Apr. 2011, BIOTECHNOLOGY FOR BIOFUELS, 4, 8, English[Refereed]Scientific journal
- Efficient ethanol producing yeast Saccharomyces cerevisiae cannot produce ethanol from raw starch directly. Thus the conventional ethanol production required expensive and complex process. In this study, we developed a direct and efficient ethanol production process from high-yielding rice harvested in Japan by using amylase expressing yeast without any pretreatment or addition of enzymes or nutrients. Ethanol productivity from high-yielding brown rice (1.1 g/L/h) was about 5-fold higher than that obtained from purified raw corn starch (0.2 g/L/h) when nutrients were added. Using an inoculum volume equivalent to 10% of the fermentation volume without any nutrient supplementation resulted in ethanol productivity and yield reaching 1.2 g/L/h and 101%, respectively, in a 24-h period. High-yielding rice was demonstrated to be a suitable feedstock for bioethanol production. In addition, our polyploid amylase-expressing yeast was sufficiently robust to produce ethanol efficiently from real biomass. This is first report of direct ethanol production on real biomass using an amylase-expressing yeast strain without any pretreatment or commercial enzyme addition. (C) 2011 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, Apr. 2011, ENZYME AND MICROBIAL TECHNOLOGY, 48(4-5) (4-5), 393 - 396, English[Refereed]Scientific journal
- Streptavidin is tetrameric protein which has tight and specific biotin binding affinity, and streptavidin modification of proteins or small molecules is widely used for biotechnology tool. Here, we demonstrate site-specific streptavidin-protein conjugation using enzymes. We focused on sortase A, a transpeptidase from Staphylococcus aureus. A streptavidin-tagged LPETG motif (Stav-LPETG) was expressed in Escherichia coli. We achieved soluble streptavidin expression in E. coli without refolding using a cold shock expression system. Then we successfully conjugated Stav-LPETG with pentaglycine-appended green fluorescence protein (Gly5-GFP) or triglycine-appended glucose oxidase (Gly3-GOD) using sortase A. SDS-PAGE analysis showed site-specific tetrameric streptavidin-protein conjugation with the tagged proteins. In addition, the functions of a Stav-GOD conjugate, i.e., biotin-binding and glucose oxidase activity, were significantly higher compared to those of streptavidin-GOD conjugates prepared by chemical modification. (C) 2011 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Mar. 2011, JOURNAL OF BIOTECHNOLOGY, 152(1-2) (1-2), 37 - 42, English[Refereed]Scientific journal
- Cinnamic acid production was demonstrated using Streptomyces as a host. A gene encoding phenylalanine ammonia lyase (PAL) from Streptomyces maritimus was introduced into Streptomyces lividans and its expression was confirmed by western blot analysis. After 4 days cultivation using glucose as a carbon source, the maximal level of cinnamic acid reached 210 mg/L. When glycerol (30 g/L) was used as the carbon source, the maximal level of produced cinnamic acid reached 450 mg/L. In addition, using raw starch, xylose and xylan as the carbon source, the maximal level of cinnamic acid reached 460, 300, 130 mg/L, respectively. We demonstrated that S. lividans has great potential to produce cinnamic acid as well as other aromatic compounds. © 2011 IEEE.2011, 2011 Defense Science Research Conference and Expo, DSR 2011, English[Refereed]International conference proceedings
- 2011, Applied Microbiology and Biotechnology, 92(1), 67-76, EnglishHomo D-lactic acid production from xylose/glucose mixture using xylose-assimilating operon integrated Lactobacillus plantrum[Refereed]Scientific journal
- A ZZ domain (ZZ) and alkaline phosphatase (AP), luciferase (Luc), or glucose oxidase (GOD) were conjugated using Sortase A (SrtA) from Staphylococcus aureus. The specific peptidyl linker for SrtA was genetically used to the C-terminus of ZZ, and the other linker was fused to the N-terminus of AP, Luc, or GOD, respectively. The resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged ZZ and AP, Luc, or GOD were site-specifically conjugated by SrtA through he extra peptidyl linkers in vitro. The SrtA reaction had little influence on either the antibody-binding activity of the ZZ moiety or the enzymatic activity of AP, Luc, or GOD moieties of the conjugates. Additionally, antibody-ZZ-proteins were yielded easily by mixing antibody with ZZ-AP, ZZ-Luc, or ZZ-GOD, allowing their use in an enzyme-linked immunosorbent assay. These results suggest that the enzymatic approach with SrtA facilitates the construction of ZZ-proteins. Furthermore, mixing antibody and ZZ-proteins produces a wide variety of antibody-ZZ-proteins.AMER CHEMICAL SOC, Dec. 2010, BIOCONJUGATE CHEMISTRY, 21(12) (12), 2227 - 2233, English[Refereed]Scientific journal
- The preparation of polyelectrolyte microcapsules by electrospray was investigated. When a polyanionic or polycationic aqueous solution was electrosprayed into an aqueous solution containing a polyelectrolyte with the opposite charge, a spherical interface consisting of a polyelectrolyte complex was formed by electrostatic interaction to produce a microcapsule. Alginate/chitosan microcapsules (similar to 100 mu m) were successfully produced with a narrow diameter distribution (coefficient of variation 4.4%). The diameters of microcapsules were controlled in the range of 80-230 mu m by varying the operating conditions, such as feed rate, working voltage, the distance from needle-to-collector, needle diameter and polyelectrolyte concentrations. We also succeeded in the encapsulation of protein, dextran and a polymeric microsphere within the polyelectrolyte microcapsules with high encapsulation efficiencies (more than 99%). The study of yeast encapsulation reveals that the electrospray technique can encapsulate a physiologically active substrate in the polyelectrolyte microcapsule and maintain its activity. (C) 2010 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Nov. 2010, COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS, 370(1-3) (1-3), 28 - 34, English[Refereed]Scientific journal
- Streptomyces lividans is known to produce large amounts of proteins in culture supernatants. In this report, to expand the secretory expression system with a strong promoter derived from phospholipase D of Streptoverticillium cinnamoneum, we expressed three kinds of proteins: transglutaminase from Sty. cinnamoneum (StvcMTG) and beta-1,4-endoglucanase and beta-glucosidase from Thermobifida fusca YX. The StvcMTG gene was introduced into S. lividans using the shuttle vector pUC702 for Escherichia coli and S. lividans, and high level secretory production of StvcMTG (230 mu g/ml in the culture supernatant) was achieved. The other prokaryotic proteins, beta-1,4-endoglucanase and beta-glucosidase, were also expressed in (His)(6)-tag fused form into culture supernatants and retained high activity. Furthermore, complete purification was achieved by conventional column or affinity column chromatography for each recombinant protein with 1 mg/ml over protein concentration. Three independent proteins were thus successfully expressed and purified, and we expect to use this system for the expression of other valuable heterologous proteins. (C) 2010 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Oct. 2010, PROTEIN EXPRESSION AND PURIFICATION, 73(2) (2), 198 - 202, English[Refereed]Scientific journal
- A novel HER2-targeted carrier was developed using bionanocapsules (BNCs). Bionanocapsules (BNCs) are 100-nm hollow nanoparticles composed of the L-protein of hepatitis B virus surface antigen. An affibody of HER2 was genetically displayed on the BNC surface (Z(HER2)-BNC). For the investigation of binding affinity, Z(HER2)-BNC was incubated with the cancer cell lines SK-BR-3 (HER2 positive), and MDA-MB-231 (HER2 negative). For analysis of HER2 targeting specificity, Z(HER2)-BNC or Z(WT)-BNC (without affibody) was incubated with both SK-BR-3 and MDA-MB-231 cells by time lapse and concentration. For the delivery of encapsulated molecules (calcein), fluorescence of Z(HER2)-BNC mixed with liposomes was also compared with that of Z(WT)-BNC and nude liposomes by incubation with SK-BR-3 cells. As a result, Z(HER2)-BNC-liposome complex demonstrated the delivery to HER2-expressing cells (SK-BR-3) with a high degree of specificity. This indicates that genetically engineered BNCs are promising carrier for cancer treatment. (C) 2010 Elsevier Ltd. All rights reserved.PERGAMON-ELSEVIER SCIENCE LTD, Oct. 2010, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 20(19) (19), 5726 - 5731, English[Refereed]Scientific journal
- We demonstrated ethanolysis of rapeseed oil to produce biodiesel fuel using lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst. We prepared two types of whole-cell biocatalyst: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL) and recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL). Both w-ROL and r-FHL successfully catalyzed the ethanolysis of rapeseed oil, and the fatty acid ethyl ester yield was as high as 79% (w-ROL) or 94% (r-FHL). In the case of r-FHL, the residual monoglycerides (MGs) and diglycerides (DGs) were no more than 0.73 and 0.18%, respectively. In addition, r-FHL could be recycled for the ethanolysis of rapeseed oil, retaining over 85% fatty acid ethyl ester yield by the fifth cycle. r-FHL was revealed to be a promising catalyst for biodiesel production using rapeseed oil and ethanol. (C) 2010 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Sep. 2010, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 66(1-2) (1-2), 101 - 104, English[Refereed]Scientific journal
- To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification-fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 degrees C and 37 degrees C, while the activity of cellulolytic enzymes is highest at around 50 degrees C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus beta-glucosidase on the cell surface, which successfully converts a cellulosic beta-glucan to ethanol directly at 48 degrees C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield ( in grams of ethanol produced per gram of beta-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface.SPRINGER, Sep. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 88(1) (1), 381 - 388, English[Refereed]Scientific journal
- Cancer cell-specific photokilling was successfully demonstrated using antibody-immobilized TiO2 nanoparticles with only 1 J cm(-2) UV irradiation.ROYAL SOC CHEMISTRY, Sep. 2010, MEDCHEMCOMM, 1(3) (3), 209 - 211, English[Refereed]Scientific journal
- A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.SPRINGER, Aug. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 87(5) (5), 1783 - 1789, English[Refereed]Scientific journal
- The co-utilization of sugars, particularly xylose and glucose, during industrial fermentation is essential for economically feasible processes with high ethanol productivity. However, the major problem encountered during xylose/glucose co-fermentation is the lower consumption rate of xylose compared with that of glucose fermentation. Here, we therefore attempted to construct high xylose assimilation yeast by using industrial yeast strain with high beta-glucosidase activity on the cell surface. We first constructed the triple auxotrophic industrial strain OC2-HUT and introduced four copies of the cell-surface-displaying beta-glucosidase (BGL) gene and two copies of a xylose-assimilating gene into its genome to generate strain OC2-ABGL4Xyl2. It was confirmed that the introduction of multiple copies of the BGL gene increased the cell-surface BGL activity, which was also correlated to the observed increase in xylose-assimilating ability. The strain OC2-ABGL4Xyl2 was able to consume xylose during cellobiose/xylose co-fermentation (0.38 g/h/g-DW) more rapidly than during glucose/xylose co-fermentation (0.18 g/h/g-DW). After 48 h, 5.77% of the xylose was consumed despite the co-fermentation conditions, and the observed ethanol yield was 0.39 g-ethanol/g-total sugar. Our results demonstrate that a BGL-displaying and xylose-assimilating industrial yeast strain is capable of efficient xylose consumption during the co-fermentation with cellobiose. Due to its high performance for fermentation of mixtures of cellobiose and xylose, OC2-ABGL4Xyl2 does not require the addition of beta-glucosidase and is therefore a promising yeast strain for cost-effective ethanol production from lignocellulosic biomass.SPRINGER, Aug. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 87(5) (5), 1975 - 1982, English[Refereed]Scientific journal
- We successfully demonstrated batch ethanol fermentation repeated ten times from raw starch with high ethanol productivity. We constructed a yeast diploid strain coexpressing the maltose transporter AGT1, alpha-amylase, and glucoamylase. The introduction of AGT1 allows maltose and maltotriose fermentation as well as the improvement of amylase activities. We also found that alpha-amylase activity during fermentation was retained by the addition of 10 mM calcium ion and that the highest alpha-amylase activity was 9.26 U/ml during repeated fermentation. The highest ethanol productivity was 2.22 g/l/h at the fourth batch, and after ten cycles, ethanol productivity of more than 1.43 g/l/h was retained, as was alpha-amylase activity at 6.43 U/ml.SPRINGER, Jun. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 87(1) (1), 109 - 115, English[Refereed]Scientific journal
- N-linked oligosaccharides or asparagine residues are often involved in G protein-coupled receptor functions. Focusing on Asn13 and Asn26 positioned on N-linked glycosylation motifs in the amino-terminal domain of human somatostatin receptor subtype-5 (hSSTR5), we performed site-directed mutagenesis and evaluated the mutants by using yeast cells as the host strain. This is because analysing the complicated signalling in mammalian cell lines is simplified by the utilization of the monopolistic pheromone signalling pathway in yeast. Western blot analysis and confocal laser scanning microscope observation showed that Asn13 and/or Asn26 mutations had no effects on cell-surface expression of hSSTR5 in yeast. By using an engineered yeast strain of Saccharomyces cerevisiae, which induces the expression of the green fluorescent protein (GFP) reporter gene in response to the agonist-specific signal transduction, it was demonstrated that a single mutation of two asparagine residues attenuated the somatostatin-specific signalling levels, and the double mutant significantly lost the signalling ability. These results clearly show the importance of these asparagine residues in the agonist-specific signalling of hSSTR5, although it was not enough to identify the consequence of oligosaccharides.OXFORD UNIV PRESS, Jun. 2010, JOURNAL OF BIOCHEMISTRY, 147(6) (6), 867 - 873, English[Refereed]Scientific journal
- The yeast Saccharomyces cerevisiae is known as an available host for human G-protein-coupled receptor (GPCR) ligand screening. Although several types of yeast signal sequences (SS) attached with the GPCRs could improve their productivities and facilitate transportation of the GPCRs to the yeast plasma membrane, the effects of additional SS on ligand-specific signalling functions of GPCRs are not reported. Here, we demonstrated the controlling signalling properties by addition of SS using engineered yeast as a host. Prepro and pre regions of alpha-factor and amino-terminal sequence of Ste2 (Ste2N) were used as SS, and somatostatin (SST) receptor subtype-5 (SSTR5) was used as a model GPCR. We also constructed a yeast-based fluorescent assay system for monitoring the activation levels of SSTR5 signalling by a green fluorescent protein (GFP) reporter gene. The production levels and localisation patterns of the SS-attached SSTR5 were more significantly improved than those of wild-type SSTR5. In addition, we successfully controlled the pharmacological efficacy and potency by introducing SS. Among four types of SSTR5 receptors, Ste2N-SSTR5 responded at the lowest ligand concentration. This finding will be informative for constructing optimal yeast-based ligand screening systems to discriminate the cells on the basis of signalling levels.OXFORD UNIV PRESS, Jun. 2010, JOURNAL OF BIOCHEMISTRY, 147(6) (6), 875 - 884, English[Refereed]Scientific journal
- We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and beta-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.WILEY-V C H VERLAG GMBH, May 2010, BIOTECHNOLOGY JOURNAL, 5(5) (5), 449 - 455, English[Refereed]Scientific journal
- Background: The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as beta-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail delta-integration. Results: In cocktail delta-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. Although the total integrated gene copy numbers of cocktail delta-integrant strain was about half that of a conventional delta-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression. Conclusions: To our knowledge, this is the first report on the expression of cellulase genes by delta-integration and optimization of various foreign genes by delta-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.BIOMED CENTRAL LTD, May 2010, MICROBIAL CELL FACTORIES, 9, 32, English[Refereed]Scientific journal
- We have developed a new approach based on the G gamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established G gamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein c subunit (G gamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered G gamma that lacks membrane localization upon deletion of the lipid modi. cation site (G gamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with G gamma(cyto) are expressed. As protein-protein interactions bring G gamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-G gamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced G gamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.WILEY-BLACKWELL PUBLISHING, INC, Apr. 2010, FEBS JOURNAL, 277(7) (7), 1704 - 1712, English[Refereed]Scientific journal
- We used cutinase from the filamentous fungi Aspergillus oryzae to produce dairy flavors. Secretory and displayed forms of cutinase were investigated using salt-free butter, which is composed mostly of triacylglycerides, as the substrate. The secretory form of cutinase, which was produced in recombinant A. oryzae, was suitable for producing butyric acids (16.8 mol%). Also, cutinase displayed on the cell surface of the yeast Saccharomyces cerevisiae as a fusion protein with alpha-agglutinin released butyric acid at a 2.7-fold rate (45.4 mol%) higher than that of the secreted form. Yeasts carrying two copies of cutinase genes into their chromosomes, which were constructed using the HELOH method, released free fatty acids rapidly and showed 2-fold higher lipase activity compared with yeasts carrying one copy of the cutinase gene. (C) 2010 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, Mar. 2010, ENZYME AND MICROBIAL TECHNOLOGY, 46(3-4) (3-4), 194 - 199, English[Refereed]Scientific journal
- We developed a novel strategy for constructing yeast to improve levels of amylase gene expression and the practical potential of yeast by combining delta-integration and polyploidization through cell fusion. Streptococcus bovis alpha-amylase and Rhizopus oryzae glucoamylase/alpha-agglutinin fusion protein genes were integrated into haploid yeast strains. Diploid strains were constructed from these haploid strains by mating, and then a tetraploid strain was constructed by cell fusion. The alpha-amylase and glucoamylase activities of the tetraploid strain were increased up to 1.5- and tenfold, respectively, compared with the parental strain. The diploid and tetraploid strains proliferated faster, yielded more cells, and fermented glucose more effectively than the haploid strain. Ethanol productivity from raw starch was improved with increased ploidy; the tetraploid strain consumed 150 g/l of raw starch and produced 70 g/l of ethanol after 72 h of fermentation. Our strategy for constructing yeasts resulted in the simultaneous overexpression of genes integrated into the genome and improvements in the practical potential of yeasts.SPRINGER, Feb. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85(5) (5), 1491 - 1498, English[Refereed]Scientific journal
- In order to achieve direct fermentation of an optically pure d-lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (a dagger ldhL1) bacterial strain. CelA expression and its degradation of beta-glucan was confirmed by western blot analysis and enzyme assay, respectively. Although the CelA-secreting a dagger ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid, not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed this conversion resulting in the production of 1.27 g/l of D-lactic acid with a high optical purity of 99.5% from a medium containing 2 g/l of cellohexaose. Subsequently, D-lactic acid fermentation from barley beta-glucan was carried out with the addition of Aspergillus aculeatus beta-glucosidase produced by recombinant Aspergillus oryzae and 1.47 g/l of D-lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from beta-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose.SPRINGER, Jan. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85(3) (3), 643 - 650, English[Refereed]Scientific journal
- Here we expand the yeast cell surface display system to display non-natural, functional molecules. The short biotin acceptor peptide (BAP) sequence of biotin ligase from E. coli(BirA) was genetically introduced to the N-terminus of the anchor protein, Flo428. Through co-expression of BAP-fused Flo428 with BirA, biotinylated BAP could be displayed on the yeast cell surface. Subsequent addition of streptavidin-FITC resulted in the display of streptavidin-FITC, and, the display of biotin-FITC was successful using streptavidin as a linker. Our strategy provides a powerful tool for displaying functional molecules on yeast cell surfaces. (c) 2009 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Jan. 2010, JOURNAL OF BIOTECHNOLOGY, 145(1) (1), 79 - 83, English[Refereed]Scientific journal
- Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation; however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA production using several kinds of genetically modified microorganisms, such as LA bacteria, Escherichia coli, Corynebacterium glutamicum, and yeast. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass has been significantly improved. In this review, the drawbacks as well as improvements of LA production by fermentation is discussed.SPRINGER, Jan. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85(3) (3), 413 - 423, English[Refereed]Scientific journal
- The production of optically pure D-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose L-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of D-lactic acid was produced from 50 g/liter of xylose.AMER SOC MICROBIOLOGY, Dec. 2009, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75(24) (24), 7858 - 7861, English[Refereed]Scientific journal
- Bionanocapsule (BNC) is hollow nanoparticle composed of the l-protein of the hepatitis B virus surface antigen. BNC allows targeted delivery of either genes or drugs only to hepatocytes, but not to other cell types. In this study, we attempted to alter the specificity of BNC by insertion of biotin-acceptor peptide (BAP), which is efficiently biotinylated using biotin ligase BirA from Escherichia coli. Using streptavidin as a linker, biotinylated BNC could be display various biotinylated ligands that are otherwise difficult to fuse with BNC, such as antibodies, synthetic peptides and functional molecules. BAP-fused BNC was efficiently biotinylated and effectively displayed streptavidin. Furthermore, we demonstrated that biotinylated BNC was internalized into targeted cells via biotinylated Nanobody displayed on the BNC surface. Biotinylated BNC permit display of diverse ligands, and thus have potential as a versatile carrier for drug delivery to a variety of target cells.OXFORD UNIV PRESS, Dec. 2009, JOURNAL OF BIOCHEMISTRY, 146(6) (6), 867 - 874, English[Refereed]Scientific journal
- We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used alpha-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum.SPRINGER, Sep. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 84(4) (4), 733 - 739, English[Refereed]Scientific journal
- beta-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than P-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides. (C) 2008 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Aug. 2009, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 59(4) (4), 297 - 301, English[Refereed]Scientific journal
- Optically pure D-lactic acid fermentation from arabinose was achieved by using the Lactobacillus plantarum NCIMB 8826 strain whose L-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of D-lactic acid was produced from 50 g/liter of arabinose.AMER SOC MICROBIOLOGY, Aug. 2009, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75(15) (15), 5175 - 5178, English[Refereed]Scientific journal
- A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed beta-glucosidase resisted pepsin digestion compared with secreted, free beta-glucosidase. In SDS-PAGE and Western blotting analysis, the secreted beta-glucosidase was immediately digested within 1 min following SGF treatment, although the displayed beta-glucosidase was stable for more than 60 min following SGF treatment. In addition, the residual activity of secreted beta-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed beta-glucosidase retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology using enzymes can reveal the protease resistance of a protein of interest under various conditions.SPRINGER, Aug. 2009, BIOTECHNOLOGY LETTERS, 31(8) (8), 1259 - 1264, English[Refereed]Scientific journal
- Recent reports on high-speed affinity screening systems for yeast cells using flow cytometry have not been adapted to screening yeast cells that display hydrolyzing enzymes, since the fluorescent molecules which are released from fluoresceinated substrate diffuse into solution after enzymatic reaction. In this research, yeast cells displaying beta-glycosidase were individually captured in micro-sized calcium alginate beads by using the newly developed reverse micelle method to prevent diffusion of hydrolyzed fluorescent substrates. By adopting flow sorting to these captured cells, active cells were successfully enriched about 82-fold from a mixed suspension with negative controls. This system should be a useful method for high-speed screening of yeast cells that display various hydrolyzing enzymes and has potential application to screening randomized libraries of enzyme-displayed yeast cells with higher activities.SPRINGER, Aug. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 84(2) (2), 375 - 382, English[Refereed]Scientific journal
- We constructed a novel cell surface display system to control the ratio of target proteins on the Saccharomyces cerevisiae cell surface, using two pairs of protein-protein interactions. One protein pair is the Z domain of protein A derived from Staphylococcus aureus and the Fc domain of human immunoglobulin G. The other is the cohesin (Coh) and dockerin (Dock) from the cellulosome of Clostridium cellulovorans. In this proposed displaying system, the scaffolding proteins (fusion proteins of Z and Coh) were displayed on the cell surface by fusing with the 3' half of alpha-agglutinin, and the target proteins fused with Fc or Dock were secreted. As a target protein, a recombinant Trichoderma reesei endoglucanase II (EGII) was secreted into the medium and immediately displayed on the yeast cell surface via the Z and Fc domains. Display of EGII on the cell surface was confirmed by hydrolysis of beta-glucan as a substrate, and EGII activity was detected in the cell pellet fraction. Finally, two enzymes, EGII and Aspergillus aculeatus beta-glucosidase 1, were codisplayed on the cell surface via Z-Fc and Dock-Coh interactions, respectively. As a result, the yeast displaying two enzymes hydrolyzed beta-glucan to glucose very well. These results strongly indicated that the proposed strategy, the simultaneous display of two enzymes on the yeast cell surface, was accomplished by quantitatively controlling the display system using affinity binding.AMER SOC MICROBIOLOGY, Jun. 2009, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75(12) (12), 4149 - 4154, English[Refereed]Scientific journal
- The introduction of several kinds of genes into the yeast chromosome is a powerful tool in many fields from fundamental study to industrial application. Here, we describe a general strategy for one-step gene integration and a marker recycling method. Forty base pairs of a short sequence derived from a region adjacent to the HIS3 locus were placed between cell surface displaying beta-glucosidase (BGL) and URA3 marker genes. HIS3 deletion and BGL-URA3 fragment integration were achieved via a PCR fragment consisting of the BGL-URA3 fragment attached to homology sequences flanked by the HIS3 targeting locus. The obtained his3::URA3 disruptants were plated on a 5-FOA plate to select for the URA3 deletion due to repeated sequences at both sides of URA3 gene. In all selected colonies, BGL genes were integrated at the targeted HIS3 locus and URA3 was completely deleted. In addition, introduced BGL was efficiently expressed, and the transformants fermented cellobiose to ethanol effectively. As our strategy creates next transformation markers continuously together with gene integration, this method can serve as a simple and powerful tool for multiple genetic manipulations in yeast engineering.SPRINGER, Jun. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 83(4) (4), 783 - 789, English[Refereed]Scientific journal
- To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay. Moreover, the single-cell analysis revealed that the proportion of cells expressing GFP in the cell cluster reflects the plasmid retention rate; therefore, the FCM analysis of the GFP-expressing population allows the immediate estimation of the plasmid retention rate without the 2- or 3-day incubation required for colony counting. We show that the FCM analysis with GFP reporter is a suitable method to explore the hopeful expression vector and host strain or establish the several expression systems exhibiting the characteristic properties in yeast.OXFORD UNIV PRESS, Jun. 2009, JOURNAL OF BIOCHEMISTRY, 145(6) (6), 701 - 708, English[Refereed]Scientific journal
- The goal of this research was to construct a stable and efficient process for the production of ethanol from raw starch, using a recombinant Saccharomyces cerevisiae. which is productive even under conditions such as non-selection or long-term operation. Three recombinant yeast strains were used, two haploid strains (MT8-1SS and NBRC1440SS) and one diploid strain (MN8140SS). The recombinant strains were constructed by integrating the glucoamylase gene from Rhizopus oryzae fused with the 3'-half of the alpha-agglutinin gene as the anchor protein, and the alpha-amylase gene from Streptococcus bovis, respectively, into their chromosomal DNA by homologous recombination. The diploid strain MN8140SS was constructed by mating these opposite types of integrant haploid strains in order to enhance the expression of integrated amylase genes. The diploid strain had the highest ethanol productivity and reusability during fermentation from raw starch. Moreover, the ethanol production rate of the integrant diploid strain was maintained when batch fermentation was repeated three times (0.67. 0.60, and 0.67 g/l/h in each batch). These results clearly show that a diploid strain developed by mating two integrant haploid strains is useful for the establishment of an efficient ethanol production process. (C) 2009 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, May 2009, ENZYME AND MICROBIAL TECHNOLOGY, 44(5) (5), 344 - 349, English[Refereed]Scientific journal
- In the current study, we report the construction of a novel system for the detection of protein-protein interactions using yeast G-protein signaling. It is well established that the G-protein gamma subunit (G gamma) is anchored to the inner leaflet of the plasma membrane via lipid modification in the C-terminus, and that this localization of G gamma is required for signal transduction. In our system, mutated G gamma (G gamma(cyto)) lacking membrane localization ability was genetically prepared by deletion of the lipid modification site. Complete disappearance of G-protein signal was observed when G gamma(cyto) was expressed in the cytoplasm of yeast cells from which the endogenous G gamma gene had been deleted. In order to demonstrate the potential use of our system, we utilized the Staphylococcus aureus ZZ domain and the Fc portion of human immunoglobulin G (IgG) as a model interaction pair. To design our detection system for protein-protein interaction, the ZZ domain was altered so that it associates with the inner leaflet of the plasma membrane, and the Fc part was then fused to G gamma(cyto). The Fc-G gamma(cyto) fusion protein migrated towards the membrane via the ZZ-Fc interaction, and signal transduction was therefore restored. This signal was successfully detected by assessing growth inhibition and transcription in response to G-protein signaling. Finally, several Z variants displaying affinity constants ranging from 8.0 x 10(3) to 6.8 x 10(8) m(-1) were prepared, and it was demonstrated that our system was able to discriminate subtle differences in affinity. In conclusion, our system appears to be a reliable and versatile technique for detection of protein-protein interactions, and may prove useful in future protein interaction studies.WILEY-BLACKWELL PUBLISHING, INC, May 2009, FEBS JOURNAL, 276(9) (9), 2636 - 2644, English[Refereed]Scientific journal
- In the current study, we report the construction of a novel system for the detection of protein-protein interactions using yeast G-protein signaling. It is well established that the G-protein gamma subunit (G gamma) is anchored to the inner leaflet of the plasma membrane via lipid modification in the C-terminus, and that this localization of G gamma is required for signal transduction. In our system, mutated G gamma (G gamma(cyto)) lacking membrane localization ability was genetically prepared by deletion of the lipid modification site. Complete disappearance of G-protein signal was observed when G gamma(cyto) was expressed in the cytoplasm of yeast cells from which the endogenous G gamma gene had been deleted. In order to demonstrate the potential use of our system, we utilized the Staphylococcus aureus ZZ domain and the Fc portion of human immunoglobulin G (IgG) as a model interaction pair. To design our detection system for protein-protein interaction, the ZZ domain was altered so that it associates with the inner leaflet of the plasma membrane, and the Fc part was then fused to G gamma(cyto). The Fc-G gamma(cyto) fusion protein migrated towards the membrane via the ZZ-Fc interaction, and signal transduction was therefore restored. This signal was successfully detected by assessing growth inhibition and transcription in response to G-protein signaling. Finally, several Z variants displaying affinity constants ranging from 8.0 x 10(3) to 6.8 x 10(8) m(-1) were prepared, and it was demonstrated that our system was able to discriminate subtle differences in affinity. In conclusion, our system appears to be a reliable and versatile technique for detection of protein-protein interactions, and may prove useful in future protein interaction studies.WILEY-BLACKWELL PUBLISHING, INC, May 2009, FEBS JOURNAL, 276(9) (9), 2636 - 2644, English[Refereed]Scientific journal
- Insect cell expression systems are widely used to produce active recombinant proteins. Here, we have developed a high-level expression vector containing a selectable marker for continuous production of recombinant proteins in insect cells. The plasmid, pXIHAbla, developed in this study, established a polyclonal cell line 8 days shorter than pXINSECT-DEST38 and pBmAneo. In addition, pXIHAbla exhibited an approximately fivefold higher average enhanced GFP expression level and approximately a twofold higher bionanocapsule secretion level than pXINSECT-DEST38. Using this plasmid, insect cells that highly express active proteins have been easily established.SPRINGER, May 2009, BIOTECHNOLOGY LETTERS, 31(5) (5), 623 - 627, English[Refereed]Scientific journal
- Bionanocapsule (BNC) is a hollow nanoparticle composed of L-protein of the hepatitis B virus surface antigen. BNC can deliver genes or drugs into specific human hepatocytes, but delivery is limited to hepatocytes. In this study, we attempted to alter the specificity of BNCs by genetically introducing cell-penetrating peptides (CPPs), such as arginine-rich peptides, into BNCs. The CPP-fused BNC was efficiently internalized into various cell lines in a short period without significant cytotoxicity. These results show that CPP-BNC could be applied as an efficient carrier for gene and drug delivery. (C) 2009 Elsevier Ltd. All rights reserved.PERGAMON-ELSEVIER SCIENCE LTD, Mar. 2009, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 19(5) (5), 1473 - 1476, English[Refereed]Scientific journal
- Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 alpha-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli-C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5'-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert l-lysine to cadaverine, as there was no accumulation ofl-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources.SPRINGER, Feb. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82(1) (1), 115 - 121, English[Refereed]Scientific journal
- SOUTHEAST UNIV PRESS, 2009, IFPT'6: PROGRESS ON POST-GENOME TECHNOLOGIES, PROCEEDINGS, 238 - 238, EnglishCONSTRUCTION OF A NOVEL DETECTION SYSTEM FOR PROTEIN-PROTEIN INTERACTIONS USING YEAST G-PROTEIN SIGNALING[Refereed]International conference proceedings
- In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.AMER SOC MICROBIOLOGY, Jan. 2009, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75(2) (2), 462 - 467, English[Refereed]Scientific journal
- Bionanocapsules (BNCs) are nanoparticles with a high biocompatibility composed of the L protein of the hepatitis B virus surface antigen. BNC can deliver bioactive molecules to hepatocytes efficiently and specifically. However, delivery is limited to hepatocytes and incorporation of proteins into BNC is quite troublesome. Here, in order to alter the specificity of BNC and to achieve efficient protein delivery, we developed engineered BNC displaying the ZZ domain of protein A and incorporating enhanced green fluorescent protein (EGFP) inside the particles using an insect cell expression system. The ZZ domain displayed on the surface of BNC binds to anti-epidermal growth factor receptor (EGFR) antibodies, allowing specific delivery of EGFP to HeLa cells. The engineered BNCs are a promising and powerful tool for efficient and cell-specific protein delivery.OXFORD UNIV PRESS, Dec. 2008, JOURNAL OF BIOCHEMISTRY, 144(6) (6), 701 - 707, English[Refereed]Scientific journal
- We constructed a double auxotrophic OC-2 industrial diploid strain of Saccharomyces cerevisiae and introduced 4 copies of cell surface displaying beta-glucosidase (BGL) genes into the chromosome. The engineered OC-2 strain showed 5-fold higher BGL activity compared with the yeast carrying 2 copies of BGL gene and directly produced ethanol from cellobiose.SOC BIOSCIENCE BIOENGINEERING JAPAN, Dec. 2008, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 106(6) (6), 594 - 597, English[Refereed]Scientific journal
- Co-utilization of several sugars, especially xylose and glucose, is essential for economically feasible processes with high ethanol productivity. However, the majorproblem during xylose/glucose co-fermentation is that xylose is used very slowly until after glucose is completely consumed. Here, we demonstrated an effective co-fermentation process using xylose- and cellobiose-assimilating recombinant Saccharomyces cerevisiae. The recombinant yeast is able to consume xylose during xylose/cellobiose co-fermentation as rapidly as during glucose fermentation. After 72 h, 95.6% of xylose was consumed, despite the co-fermentation conditions, and the ethanol yield was 0.358 g-ethanol/g-total sugar. (C) 2008 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, Sep. 2008, ENZYME AND MICROBIAL TECHNOLOGY, 43(3) (3), 233 - 236, English[Refereed]Scientific journal
- Enhancing the sugar uptake ability of the yeast Saccharomyces cerevisiae is a potentially important factor for efficient ethanol production during fermentation of lignocellulosic biomass. Here, we attempted to express a Pichia stipitis gene encoding a sugar transporter, SUT1, in a xylose-assimilating S. cerevisiae strain that expresses xylose reductase, xylosedehydrogenase and xylulokinase. We next investigated xylose fermentation, glucose fermentation and glucose and xylose co-fermentation using the Sut1-expressing S. cerevisiae strain. Expression of Sut1 in xylose-assimilating S. cerevisiae increased both xylose uptake ability and ethanol productivity during xylose fermentation. Moreover, glucose uptake ability and ethanol productivity during glucose fermentation also increased by expressing of Sut1. The yield of ethanol during xylose and glucose co-fermentation by the Sut1-expressing yeast strain (0.44 g/g-consumed sugar) was significantly higher than that of the parental strain (0.39 g/g-consumed sugar). (C) 2008 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, Aug. 2008, ENZYME AND MICROBIAL TECHNOLOGY, 43(2) (2), 115 - 119, English[Refereed]Scientific journal
- Here, we describe a yeast-based fluorescence reporter assay for G protein-coupled receptor (GPCR) signalling using a flow cytometer (FCM). The enhanced green fluorescent protein (EGFP) gene was integrated into the FUS1 locus as a reporter gene. The engineered yeast was able to express the EGFP in response to ligand stimulation. Gene-disrupted yeast strains were constructed to evaluate the suitability of the yeast-based fluorescence screening system for heterologous GPCR. When receptor was expressed by episomal plasmid, the proportion of the signalling-activated cells in response to ligand stimulation decreased significantly. The GPCR-signalling-activated and non-activated cell clusters were individually isolated by analysing the fluorescence intensity at the single-cell level with FCM, and it was found that the plasmid retention rate decays markedly in the non-activated cell cluster. We attributed the loss of plasmid to G1 arrest in response to signalling, and successfully improved the plasmid retention rate by disrupting the FAR1 gene and avoiding cell cycle arrest. Our system will be a powerful tool for the quantitative and high-throughput GPCR screening of yeast-based combinatorial libraries using FCM.OXFORD UNIV PRESS, May 2008, JOURNAL OF BIOCHEMISTRY, 143(5) (5), 667 - 674, English[Refereed]Scientific journal
- The purpose of this research was to develop an infrared spectroscopic technique (Fourier transform infrared spectroscopy with attenuated total reflectance system; FTIR-ATR) for non-invasive measurement of saturated and unsaturated fatty acid compositions in human oral mucosa obtained from three nationalities; Iranian, Vietnamese, and Indonesian. The histogram patterns of fatty acid compositions for three nationalities suggest that the pattern of unsaturated fatty acids were quite different, although the distribution profiles of fatty acid to lipid ratios in FTIR-ATR has a similar normal pattern with small difference in skewness and mode. The second derivative infrared spectra of the mucosal tissues in the wavenumber regions from 1,600 to 1,760 cm(-1) and 2,800 to 3,050 cm(-1) were analyzed with partial least squares (PLS) multivariate regression analysis method. With this analysis method we compared predicted values with the measured values of ten categorized fatty acid compositions, i.e., a(saturated C17 or lower), b(C16:1 + C17:1), c(C18:0), d(C18:1), e(C18:2), f(saturated C20 or longer), g(C20:3 + C20:4), h(C22:1 + C24:1), i(C22:6), j(gamma C18:3). Almost all fatty acid compositions of oral mucosa were well predicted with differences between predicted and measured values within +/- 5% of total, however, errors were relatively larger in minor components such as C22:6 than major components.SPRINGER HEIDELBERG, Apr. 2008, LIPIDS, 43(4) (4), 361 - 372, English[Refereed]Scientific journal
- The purpose of this research was to develop an infrared spectroscopic technique (Fourier transform infrared spectroscopy with attenuated total reflectance system; FTIR-ATR) for non-invasive measurement of saturated and unsaturated fatty acid compositions in human oral mucosa obtained from three nationalities; Iranian, Vietnamese, and Indonesian. The histogram patterns of fatty acid compositions for three nationalities suggest that the pattern of unsaturated fatty acids were quite different, although the distribution profiles of fatty acid to lipid ratios in FTIR-ATR has a similar normal pattern with small difference in skewness and mode. The second derivative infrared spectra of the mucosal tissues in the wavenumber regions from 1,600 to 1,760 cm(-1) and 2,800 to 3,050 cm(-1) were analyzed with partial least squares (PLS) multivariate regression analysis method. With this analysis method we compared predicted values with the measured values of ten categorized fatty acid compositions, i.e., a(saturated C17 or lower), b(C16:1 + C17:1), c(C18:0), d(C18:1), e(C18:2), f(saturated C20 or longer), g(C20:3 + C20:4), h(C22:1 + C24:1), i(C22:6), j(gamma C18:3). Almost all fatty acid compositions of oral mucosa were well predicted with differences between predicted and measured values within +/- 5% of total, however, errors were relatively larger in minor components such as C22:6 than major components.SPRINGER HEIDELBERG, Apr. 2008, LIPIDS, 43(4) (4), 361 - 372, English[Refereed]Scientific journal
- The use of enzymes is a promising approach for site-specific protein modification on living cells owing to their substrate specificity. Herein we describe a general strategy for the site-specific modification of cell surface proteins with synthetic molecules by using Sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of Sortase and an N-terminal triglycine-containing probe results in the site-specific labeling of the tagged protein. We were successful in the C-terminal-specific labeling of osteoclast differentiation factor (ODF) with a biotin- or fluorophore-containing short peptide on the living cell surface. The labeling reaction occurred efficiently in serum-containing medium, as well as serum-free medium or PBS. The labeled products were detected after incabation for 5 min. In addition, site-specific protein-protein conjugation was successfully demonstrated on a living cell surface by the Sortase-catalyzed reaction. This strategy provides 6 powerful tool for cell biology and cell surface engineering.WILEY-V C H VERLAG GMBH, Mar. 2008, CHEMBIOCHEM, 9(5) (5), 802 - 807, English[Refereed]Scientific journal
- The use of enzymes is a promising approach for site-specific protein modification on living cells owing to their substrate specificity. Herein we describe a general strategy for the site-specific modification of cell surface proteins with synthetic molecules by using Sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of Sortase and an N-terminal triglycine-containing probe results in the site-specific labeling of the tagged protein. We were successful in the C-terminal-specific labeling of osteoclast differentiation factor (ODF) with a biotin- or fluorophore-containing short peptide on the living cell surface. The labeling reaction occurred efficiently in serum-containing medium, as well as serum-free medium or PBS. The labeled products were detected after incabation for 5 min. In addition, site-specific protein-protein conjugation was successfully demonstrated on a living cell surface by the Sortase-catalyzed reaction. This strategy provides 6 powerful tool for cell biology and cell surface engineering.WILEY-V C H VERLAG GMBH, Mar. 2008, CHEMBIOCHEM, 9(5) (5), 802 - 807, English[Refereed]Scientific journal
- Magnetic separation provides a relatively quick and easy-to-use method for cell isolation and protein purification. We have developed a rapid and efficient procedure to isolate yeast cells displaying a target polypeptide, namely, the Staphylococcus aureus ZZ domain, which serves as s model for protein interactions and can bind immunoglobulin G (IgG). We optimized selection of ZZ-displaying yeast cells using thermoresponsive magnetic nanoparticles. A model library was prepared by mixing various proportions of target yeast displaying the ZZ domain with control cells. Target cells in the model library that bound to the ZZ-specific binding partner, biotinylated IgG, were selected with biotinylated thermoresponsive magnetic nanoparticles using the biotin-avidin sandwich system. We determined ZZ expression levels and optimized the concentrations of both magnetic nanoparticles and avidin for efficient selection of target cells. After optimization, we successfully enriched the target cell population 4700-fold in a single round of selection. Moreover, only two rounds of selection were required to enrich the target cell population from 0.001% to nearly 100%. Our results suggest that magnetic separation will be useful for efficient exploration of novel protein-protein interactions and rapid isolation of biomolecules with novel functions.WILEY-BLACKWELL, Mar. 2008, BIOTECHNOLOGY PROGRESS, 24(2) (2), 352 - 357, English[Refereed]Scientific journal
- Here, we established a system for displaying heterologous protein to the C terminus of the peptidoglycan-binding domain (cA domain) of AcmA (a major autolysin from Lactococcus lactis). Western blot and flow cytometric analyses revealed that the fusion proteins (cA-AmyA) of the cA domain and alpha-amylase from Streptococcus bovis 148 (AmyA) are efficiently expressed and successfully displayed on the surfaces of L. lactis cells. AmyA was also displayed on the cell surface while retaining its activity. Moreover, with an increase in the number of cA domains, the quantity of cA-AmyA fusion proteins displayed on the cell surface increased. When three repeats of the cA domain were used as an anchor protein, 82% of alpha-amylase activity was detected on the cells. The raw starch-degrading activity of AmyA was significantly higher when AmyA was fused to the C terminus of the cA domain than when it was fused to the N terminus. In addition, cA-AmyA fusion proteins were successfully displayed on the cell surfaces of Lactobacillus plantarum and Lactobacillus casei.AMER SOC MICROBIOLOGY, Feb. 2008, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 74(4) (4), 1117 - 1123, English[Refereed]Scientific journal
- 2008, AIChE Annual Meeting, Conference Proceedings, EnglishNovel cell surface display on lactic acid bacteria and its application to lactic acid production from starchy materialsInternational conference proceedings
- 公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 320 - 320, Japanese
- 公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 944 - 944, Japanese
- 公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 971 - 971, Japanese
- 公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 970 - 970, Japanese
- 公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 990 - 990, Japanese
- 公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 988 - 988, Japanese
- 公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 997 - 997, Japanese
- 公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 399 - 399, Japanese
- 公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 394 - 394, Japanese
- Cytochrome P450 (P450) is an attractive oxygenase due to the diverse catalytic reactions and the broad substrate specificity. Class I P450s require an excess concentration (more than 10 times) of iron-sulfur proteins, which transfer electrons to P450s, to attain the maximum catalytic activity and this requirement is a critical bottleneck for practical applications. Here, we show a site-specific branched fusion protein of P450 with its electron transfer proteins using enzymatic cross-linking with transglutaminase. A branched fusion protein of P450 from Pseudomonas putida (P450cam), which was composed of one molecule each of P450cam, putidaredoxin (Pdx) and Pdx reductase, showed higher catalytic activity (306 min(-1)) and coupling efficiency (99%) than the equimolar reconstitution system due to the intramolecular electron transfer. The unique site-specific branched structure simply increased local concentration of proteins without denaturation of each protein. Therefore, enzymatic post-translational protein manipulation can be a powerful alternative to conventional strategies for the creation of multicomponent enzyme systems with novel proteinaceous architecture.OXFORD UNIV PRESS, Sep. 2007, PROTEIN ENGINEERING DESIGN & SELECTION, 20(9) (9), 453 - 459, English[Refereed]Scientific journal
- Cytochrome P450 (P450) is an attractive oxygenase due to the diverse catalytic reactions and the broad substrate specificity. Class I P450s require an excess concentration (more than 10 times) of iron-sulfur proteins, which transfer electrons to P450s, to attain the maximum catalytic activity and this requirement is a critical bottleneck for practical applications. Here, we show a site-specific branched fusion protein of P450 with its electron transfer proteins using enzymatic cross-linking with transglutaminase. A branched fusion protein of P450 from Pseudomonas putida (P450cam), which was composed of one molecule each of P450cam, putidaredoxin (Pdx) and Pdx reductase, showed higher catalytic activity (306 min-1) and coupling efficiency (99%) than the equimolar reconstitution system due to the intramolecular electron transfer. The unique site-specific branched structure simply increased local concentration of proteins without denaturation of each protein. Therefore, enzymatic post-translational protein manipulation can be a powerful alternative to conventional strategies for the creation of multicomponent enzyme systems with novel proteinaceous architecture. © The Author 2007. Published by Oxford University Press. All rights reserved.Sep. 2007, Protein Engineering, Design and Selection, 20(9) (9), 453 - 459, English[Refereed]Scientific journal
- (公社)日本生物工学会, Aug. 2007, 日本生物工学会大会講演要旨集, 平成19年度, 64 - 64, Japanese酵素を用いた細胞表層膜蛋白質の部位特異的ラベリング技術の開発[Refereed]
- 公益社団法人 化学工学会, 2007, 化学工学会 研究発表講演要旨集, 2007, 410 - 410, Japanese
- 公益社団法人 化学工学会, 2007, 化学工学会 研究発表講演要旨集, 2007, 412 - 412, Japanese
- 公益社団法人 化学工学会, 2007, 化学工学会 研究発表講演要旨集, 2007, 411 - 411, Japanese
- A new split intein-based protein ligation tool that is synthetically accessible and can be used for protein semisynthesis on the cell surface and potentially inside cells has been constructed.ROYAL SOC CHEMISTRY, 2007, CHEMICAL COMMUNICATIONS, 47(47) (47), 4995 - 4997, English[Refereed]Scientific journal
- A new split intein-based protein ligation tool that is synthetically accessible and can be used for protein semisynthesis on the cell surface and potentially inside cells has been constructed.ROYAL SOC CHEMISTRY, 2007, CHEMICAL COMMUNICATIONS, (47) (47), 4995 - 4997, English[Refereed]Scientific journal
- Here, we report the N-terminal glycine (Gly) residue of a target protein can be a candidate primary amine for site-specific protein conjugation catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Gly5-enhanced green fluorescent protein (EGFP) (EGFP with five additional Gly residues at its N-terminus) was cross-linked with Myc-dihydrofolate reductase (DHFR) (DHFR with the myc epitope sequence at its N-terminus) to yield DHFR-EGFP heterodimers. The reactivities of additional peptidyl linkers were investigated and the results obtained suggested that at least three additional Gly residues at the N-terminus were required to yield the EGFP-DHFR heterodimeric form. Site-directed mutagenesis analysis revealed marked preference of MTG for amino acids adjacent to the N-terminal Gly residue involved in the protein conjugation. In addition, peptide-protein conjugation was demonstrated by MTG-catalyzed N-terminal Gly-specific modification of a target protein with the myc epitope peptide. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Apr. 2005, FEBS LETTERS, 579(10) (10), 2092 - 2096, English[Refereed]Scientific journal
- Specific peptidyl linkers that result in the heterodimerization of functional proteins, which is catalyzed by microbial transglutaminase from Streptomyces mobaraensis (MTG), were generated based on a ribonuclease S-peptide using site-directed mutagenesis. The peptidyl linkers designated as Lys-tag and Gln-tag were designed to possess sole reactive Lys or Gln residue that was amenable for selective Lys-Gln cross-linkage of different proteins. Green fluorescent protein variants, ECFP and EYFP, were employed as model proteins, and those Lys- and Gln-tags were fused to the N-termini of ECFP and EYFP, respectively. As a result, we succeeded in solely obtaining the ECFP-EYFP heterodimer without forming multiply cross-linked byproducts. It was found that the reactivity of peptidyl linkers varied according to the type of amino acid to be replaced. Peptidyl linkers with a basic amino acid (Arg) exhibited the highest reactivity in the cross-linking reaction, suggesting the cationic residue substrate preference of MTG. Kinetic analysis utilizing fluorescent resonance energy transfer (FRET), that is only observed upon the heterodimeric ECFP-EYFP conjugation, revealed that the amino acid replacement contributed to the acceleration of cross-linking reactions by increasing catalytic turnover (kcat), rather than substrate binding affinity (Km). Finally, using a ribonuclease S-protein, the manipulation of enzymatic protein cross-linking based on specific S-peptide:S-protein interactions was explored. Since newly designed Lys- and Gln-tags retained binding affinities to the S-protein, the heterodimerization was perfectly restrained by wrapping them with the S-protein. The results suggest the possibility of limited protein conjugation by tuning steric hindrance against the MTG. Tailoring enzymatic posttranslational modifications with either engineering peptidyl substrates or by taking specific peptide-protein interactions into consideration may facilitate the development of a new sequential protein conjugation method for the preparation of multifunctional protein.May 2004, Bioconjugate Chemistry, 15(3) (3), 491 - 497, English[Refereed]Scientific journal
- We found that some kind of peptidyl linkers can work as peptidyl tags for microbial transglutaminase (MTG)-mediated protein heterodimerization. The Myc epitope (the amino acid sequence: EQKLISEEL) and the T7 epitope (MASMTGGQQMG) that were attached to the N-terminus of enhanced green fluorescent protein (EGFP) were found to serve Gln-tags (i.e. peptidyl tags that provide reactive Gln residues for MTG-mediated protein cross-linking). A calmodulin binding peptide (KRRWKKNFIAVSAANRFKKISSSGAL), the V5 epitope (GKPIPNPLLGLD-ST) and the Strep-tag II (WSHPQFEK) that were attached to the N-terminus of EGFP were found to be Lys-tags that provide reactive Lys residues for the enzymatic cross-linking. The specific Gln-Lys cross-linkage was formed through Gln- and Lys-tags by MTG and the major products were EGFP heterodimers. The reactivity of peptidyl linkers containing reactive Lys residue decreased when they were tethered to C-terminus of EGFP, indicating that the microenvironment or steric hindrance affects more the substrate recognition of MTG than their amino acid sequences. It was found that the insertion of a specific peptide sequence (FERQHMDS, a part of ribonuclease S-peptide) into a loop of EGFP could facilitate the protein cross-linking reaction. These results suggest that commercial peptidyl tags can be employed as specific cross-linkers for MTG-mediated site-specific protein ligation.The Society of Chemical Engineers, Japan, 2004, Asian Pacific Confederation of Chemical Engineering congress program and abstracts, 2004, 377 - 377, English
- Sep. 2003, Enzyme and Microbial Technology, 33(4) (4), 492 - 496, English[Refereed]Scientific journal
- We have found that ribonuclease S-peptide can work as a novel peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Enhanced green fluorescent protein tethered to S-peptide at its N-terminus (S-tag-EGFP) appeared to be efficiently cross-linked by MTG. As wild-type EGFP was not susceptible to cross-linking, the S-peptide moiety is likely to be responsible for the cross-linking. A site-directed mutation study assigned Gln15 in the S-peptide sequence as the sole acyl donor. Mass spectrometric analysis showed that two Lys residues (Lys5 and Lys11) in the S-peptide sequence functioned as acyl acceptors. We also succeeded in direct monitoring of the cross-linking process by virtue of fluorescence resonance energy transfer (FRET) between S-tag-EGFP and its blue fluorescent color variant (S-tag-EBFP). The protein cross-linking was tunable by either engineering S-peptide sequence or capping the S-peptide moiety with S-protein, the partner protein of S-peptide for the formation of ribonuclease A. The latter indicates that S-protein can be used as a specific inhibitor of S-peptide-directed protein cross-linking by MTG. The controllable protein cross-linking of S-peptide as a potent substrate of MTG will shed new light on biomolecule conjugation.AMER CHEMICAL SOC, Mar. 2003, BIOCONJUGATE CHEMISTRY, 14(2) (2), 351 - 357, English[Refereed]Scientific journal
- 公益社団法人 化学工学会, 2003, 化学工学会 研究発表講演要旨集, 2003, 674 - 674, Japanese
- 2022, 日本生物工学会大会講演要旨集, 74thCost-effective production of D-lactic acid from corncobs by simultaneous saccharification and fermentation using genetically engineered lactic acid bacteria
- 2020, 化学工学会秋季大会研究発表講演要旨集(CD-ROM), 51st未利用竹資源を活用したを用いたアスタキサンチン及びD-乳酸の微生物生産
- 農林水産・食品産業技術振興協会, 2017, JATAFFジャーナル, 5(3) (3), 33 - 37, Japanese未利用バイオマスからのD-乳酸生産に向けた乳酸菌の代謝工学研究[Refereed]Introduction scientific journal
- Effective degradation of hemicellulose is of utmost importance in a wide variety of applications in bioindustry. Five endoxylanases from different glycoside hydrolase families and microorganisms were tested with an arabinofuranosidase, Araf51A, for the hydrolysis of insoluble wheat arabinoxylan, which is a structural component of hemicellulose. The optimized combination was XynZ/Xyn11A/Araf51A with a loading ratio of 2:2:1, and the value of degree of synergy increased with the increase of Araf51A proportion in the enzyme mixture. Afterwards, selected enzymes were immobilized on commercial magnetic nanoparticles through covalent bonding. Both free and immobilized enzymes showed a similar conversion to reducing sugars after hydrolysis for 48 h. After 10 cycles, approximately 20% of the initial enzymatic activity of both the individual or mixture of immobilized enzymes was retained. A 5.5-fold increase in the production of sugars was obtained with a mixture of enzymes immobilized after 10 cycles in total compared with free enzymes. Importantly, a sustainable synergism between immobilized arabinofuranosidase and immobilized endoxylanases in the hydrolysis of arabinoxylan was demonstrated. (C) 2016 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Oct. 2016, BIOCHEMICAL ENGINEERING JOURNAL, 114, 271 - 278, English
- ELSEVIER SCIENCE BV, Jul. 2016, NEW BIOTECHNOLOGY, 33, S191 - S191, EnglishSummary international conference
- ELSEVIER SCIENCE BV, Jul. 2016, NEW BIOTECHNOLOGY, 33, S194 - S194, EnglishSummary international conference
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 223 - 223, Japanese2P-195 Development of metabolic channeling for efficient production of butanol using Escherichia coli.
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 223 - 223, Japanese2P-196 C-Terminal-oriented Immobilization of Enzymes Using Sortase A-mediated Technique
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 220 - 220, English2P-183 An efficient biomass degradation system by using synergistic action of accessory enzymes
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 178 - 178, Japanese2P-014 Cadaverine production from biomss using Corynebacterium glutamicum
- ELSEVIER SCIENCE BV, Sep. 2014, JOURNAL OF BIOTECHNOLOGY, 185, S52 - S52, EnglishSummary international conference
- (公社)日本医学放射線学会, Feb. 2014, Japanese Journal of Radiology, 32(Suppl.) (Suppl.), 37 - 37, Japanese過酸化チタンナノ粒子による放射線増感の検討
- 日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 251 - 251, Japanese3P-227 Enzymatic degradation of cellulose/hemicellulose with multi-functional beta-glucosidase
- 日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 250 - 250, Japanese3P-224 Study of synergistic effect between xylanases for high efficient degradation of cellulosic biomass
- 日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 251 - 251, Japanese3P-225 Highly-oriented immobilized enzymes using sortase A-mediated transpeptidation
- 日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 134 - 134, Japanese2P-110 Direct cadaverine production from oligosaccharide using Escherichia coli coexpressing beta-glucosidase and beta-xylosidase
- 日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 130 - 130, Japanese2P-094 Analysis of high-yield, high-efficient homo-butanol-fermentative Clostridia
- 日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 134 - 134, Japanese2P-112 Lactic acid production from cellobiose using Schizosaccharomyces pombe displaying beta-glucosidase on the surface
- 日本生物工学会, 2014, 生物工学会誌 : seibutsu-kogaku kaishi, 92(2) (2), 71 - 71, JapaneseDirect isopropanol production from cellobiose by engineered Escherichia coli using a synthetic pathway and a cell surface display system
- 27 Nov. 2013, 日本農芸化学会北海道支部講演会講演要旨, 2013, 38, Japaneseコリネ菌の細胞表層提示技術を用いたデンプンからの乳酸様ポリマーの生産
- 日本生物工学会, 25 Aug. 2013, 日本生物工学会大会講演要旨集, 65th, 238 - 238, Japaneseデンプンを炭素源に用いたポリ乳酸様ポリマーの合成
- 日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 126 - 126, Japanese2P-089 Highly-oriented immobilized cellulase using sortase A-mediated transpeptidation
- 日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 233 - 233, Japanese3P-182 Glucose dehydrogenase and gluconate dehydrogenase co-immobilized bioanode for biofuel cell
- 日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 228 - 228, Japanese3P-161 Direct putrescine production from cellobiose using Escherichia coli displaying beta-glucosidase
- 日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 234 - 234, Japanese3P-183 Oriented co-immobilization of enzymes through sortase A mediated-methodology
- ELSEVIER SCIENCE INC, Nov. 2012, INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS, 84(3) (3), S700 - S700, EnglishNovel Radiosensitization Through Reactive Oxygen Species Generation Using Titanium Peroxide Nanoparticle Compounds Against Pancreas CancerSummary international conference
- 日本生物工学会, 25 Sep. 2012, 日本生物工学会大会講演要旨集, 64th, 27 - 27, Japanese糖質バイオマスから多様なポリエステルを生産するコリネ菌微生物工場の開発
- ELSEVIER SCIENCE BV, Sep. 2012, NEW BIOTECHNOLOGY, 29, S50 - S50, EnglishSummary international conference
- ELSEVIER SCIENCE BV, Sep. 2012, NEW BIOTECHNOLOGY, 29, S97 - S98, EnglishSummary international conference
- ELSEVIER SCIENCE BV, Sep. 2012, NEW BIOTECHNOLOGY, 29, S51 - S51, EnglishSummary international conference
- 日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 234 - 234, Japanese4Hp09 Cellulase and glucose dehydrogenase co-immobilized bioanode for biofuel cell
- 日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 230 - 230, Japanese4Ha05 Construction of biomass-degradation enzymes library from Streptomyces and exploring novel high-level secretion signal
- 日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 156 - 156, Japanese3Hp19 Production of ectoine from biomass using Halomonas elongata
- 日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 153 - 153, Japanese3Hp07 Production of L-Lysine from cellobiose using Corynebacterium glutamicum expressing beta-glucosidase
- 日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 196 - 196, Japanese4Cp07 Highly-oriented co-immobilization of proteins on streptavidin-supported particles
- 日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 208 - 208, Japanese4Dp13 Benzoic acid fermentation from starch and cellulose using the polyketide synthesis pathway of Streptomyces maritimus
- 2012, 日本農芸化学会大会講演要旨集(Web), 2012麹菌Aspergillus oryzaeを用いたラクダ科動物由来一本鎖抗体可変部位VHH分泌生産系の構築
- 日本生物工学会, 2011, 日本生物工学会大会講演要旨集, 63, 69 - 69, Japanese1Kp11 IPA production from cellobiose by E. coli displayed BGL
- 2011, 日本農芸化学会関西支部講演会講演要旨集, 472nd麹菌Aspergillus oryzaeを用いたラクダ由来一本鎖抗体可変部位VHH分泌生産系の構築
- 2011, 日本生物工学会大会講演要旨集, 63rd麹菌Aspergillus oryzaeを用いたラクダ由来一本鎖抗体可変部位VHHの生産
- Nov. 2010, バイオインダストリー, 11, 6-12, Japaneseバイオマスからのバイオ燃料生産に向けた戦略Introduction scientific journal
- Yeast, such as Saccharomyces cerevisiae or Kluyveromyces lactis is appropriate strain for ethanol production or some useful compounds production. Cellulases expressing yeast can ferment ethanol from cellulosic materials; however, the productivity should be increase more and more. To improve and engineer the productivity, the target gene(s) were introduced into yeast genome. Generally, using genetic engineering, increasing integrated gene numbers are increased, the expressed protein ability such as enzymatic activities are also increased. In this mini-review, we focused on the effect of integrated gene copy number and the polyploidy on the productivity such as enzymatic activity and/or product yield.SPRINGER, Oct. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 88(4) (4), 849 - 857, English[Refereed]Book review
- Jul. 2010, 生物工学, 88(7), 333-335, Japanese次世代燃料・化成品原料製造に向けたバイオリファイナリー戦略Introduction scientific journal
- For elucidating protein-protein interactions, many methodologies have been developed during the past two decades. For investigation of interactions inside cells under physiological conditions, yeast is an attractive organism with which to quickly screen for hopeful candidates using versatile genetic technologies, and various types of approaches are now available. Among them, a variety of unique systems using the guanine nucleotide-binding protein (G-protein) signaling pathway in yeast have been established to investigate the interactions of proteins for biological study and pharmaceutical research. G-proteins involved in various cellular processes are mainly divided into two groups: small monomeric G-proteins, and heterotrimeric G-proteins. In this minireview, we summarize the basic principles and applications of yeast-based screening systems, using these two types of G-protein, which are typically used for elucidating biological protein interactions but are differentiated from traditional yeast two-hybrid systems.WILEY-BLACKWELL, May 2010, FEBS JOURNAL, 277(9) (9), 1982 - 1995, English[Refereed]Book review
- 日本生物工学会, 2010, 日本生物工学会大会講演要旨集, 22, 35 - 35, Japanese2P-1106 Over-production of various secretory-form proteins in Streptomyces lividans
- 日本生物工学会, 2010, 日本生物工学会大会講演要旨集, 22, 78 - 78, Japanese3P-1082 Site-specific modification of Streptavidin using Sortase
- 日本生物工学会, 2010, 日本生物工学会大会講演要旨集, 22, 168 - 168, Japanese3P-2082 Development of non-invasive cancer cell killing method based on combination of titanium peroxiside nano-particle and low intensity X-ray irradiation
- 日本生物工学会, 2010, 日本生物工学会大会講演要旨集, 22, 118 - 118, Japanese2P-2047 Direct ethanol production from cellulose by using cellulolytic enzymes expression optimized yeast strain
- 日本生物工学会, 2010, 日本生物工学会大会講演要旨集, 22, 60 - 60, Japanese3P-1011 Construction of the recombinant xylose fermentation yeast with high β-xylosidase activity from OC-2 triple marker strain as a platform
- The dependency on depleting natural resources is a challenge for energy security that can be potentially answered by bioenergy. Bioenergy is derived from starchy and lignocellulosic biomass in the form of bioethanol or from vegetable oils in the form of biodiesel fuel. The acid and enzymatic methods have been developed for the hydrolysis of biomass and for transesterifiaction of plant oils. However, acid hydrolysis results in the production of unnatural compounds which has adverse effects on yeast fermentation. Recent advancements in the yeast cell surface engineering developed strategies to genetically immobilize amylolytic, cellulolytic and xylanolytic enzymes on yeast cell surface for the production of fuel ethanol from biomass. This review gives an insight in to the recent technological developments in the production of bioenergy, i.e, bioethanol using surface engineered yeast. © 2010 Bentham Science Publishers Ltd.2010, Recent Patents on Biotechnology, 4(3) (3), 226 - 234, English[Refereed]Introduction scientific journal
- The Society for Biotechnology, Japan, Nov. 2009, Journal of bioscience and bioengineering, 108(1) (1), S108, EnglishEP-P27 Site-specific streptavidin modification using sortase(Section VII Enzyme and Protein Engineering)
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S50 - S50, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S49 - S49, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S159 - S159, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S27 - S27, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S108 - S108, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S108 - S108, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S50 - S50, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S61 - S62, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S164 - S164, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S96 - S97, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S107 - S108, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S47 - S48, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S48 - S48, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S109 - S109, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S51 - S52, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S51 - S51, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S49 - S49, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S48 - S49, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S52 - S52, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S108 - S108, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S50 - S51, EnglishSummary international conference
- SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S36 - S37, EnglishSummary international conference
- Nov. 2009, 工業材料, 61-64, Japanese生体分子と無機材料を融合した中空バイオナノ粒子による新しいがん治療戦略Introduction scientific journal
- バイオインダストリー協会, 01 Feb. 2009, Bioscience & industry, 67(2) (2), 65 - 67, JapaneseEnzyme-assisted cell surface protein modification
- 日本生物工学会, 2009, 日本生物工学会大会講演要旨集, 21, 170 - 170, Japanese2La01 Efficient ethanol production from raw starch using novel yeast strain
- 日本生物工学会, 2009, 日本生物工学会大会講演要旨集, 21, 172 - 172, Japanese2La11 Development of novel cell-surface display system using porin in Corynebacterium glutamicum
- 日本生物工学会, 2009, 日本生物工学会大会講演要旨集, 21, 173 - 173, Japanese2Lp02 Efficient protein production in recombinant Aspergillus oryzae
- 30 Oct. 2008, バイオイメージング, 17(2) (2), 110 - 111, Japanese特異性改変型バイオナノカプセルを用いたバイオイメージング及びDDSへの応用
- Aug. 2008, 月刊バイオインダストリー, Vol. 25, No. 8, pp. 13-20, Japanese細胞表層提示技術を用いた微生物の高機能化と有用物質生産Introduction scientific journal
- Apr. 2008, 月刊バイオインダストリー, Vol. 25, No. 4, pp. 11-19, Japaneseバイオリファイナリーが求める植物バイオマスIntroduction scientific journal
- 日本生物工学会, 2008, 日本生物工学会大会講演要旨集, 20, 145 - 145, Japanese2Ep19 Efficient Production of Optically Pure D-Lactic Acid from Raw Starch
- 日本生物工学会, 2008, 日本生物工学会大会講演要旨集, 20, 60 - 60, Japanese2Aa08 Efficient ethanol production from raw starch by delta-integrant polyploid fusants
- 日本生物工学会, 2008, 日本生物工学会大会講演要旨集, 20, 60 - 60, Japanese2Aa07 Biodiesel production using whole-cell biocatalyst of Aspergillus oryzae coexpressing Lipases
- 日本生物工学会, 2008, 日本生物工学会大会講演要旨集, 20, 63 - 63, Japanese2Ap01 Biotin-displaying yeast for displaying functional molecule
- 日本生物工学会, 2008, 日本生物工学会大会講演要旨集, 20, 64 - 64, Japanese2Ap05 Fiber weight loss process using enzyme reaction and electron beam irradiation
- Jun. 2007, 未来材料, (6月号) (6月号), 40 - 45, Japaneseタンパク質間相互作用とシグナル伝達解析 −酵母細胞を用いたハイスループット技術の開発−Introduction commerce magazine
- 日本生物工学会, 2007, 日本生物工学会大会講演要旨集, 19, 97 - 97, Japanese1D11-4 Efficient ethanol production process from raw starch by recombinant Saccharomyces cerevisiae
- 日本生物工学会, 2005, 日本生物工学会大会講演要旨集, 17, 220 - 220, Japanese2H10-4 Development of a new technique for the site-specific incorporation of synthetic molecules into membrane-penetrating proteins on a live cell surface
- 日本生物工学会, 2004, 日本生物工学会大会講演要旨集, 16, 104 - 104, Japanese2A10-1 A branched protein mediated electron transfer system
- Joint work, Humana Press, May 2018, EnglishSortase A-Assisted Metabolic Enzyme Ligation in Escherichia coli for Enhancing Metabolic Flux.：Synthetic Biology, Methods in Molecular BiologyScholarly book
- Joint work, シーエムシー出版, Apr. 2018, Japanese, 細胞・生体分子の固定化と機能発現 第3章Sortase Aを用いたタンパク質配向固定化技術の開発Scholarly book
- Joint work, シーエムシー出版, Jan. 2018, Japanese酵母菌・麹菌・乳酸菌の産業応用展開, 第III編 乳酸菌 第2章「乳酸菌の遺伝子操作技術の進展」Scholarly book
- Joint work, Wley, Apr. 2014, EnglishLactic AcidScholarly book
- Joint work, 化学同人, Jun. 2012, Japanese遺伝子工学Scholarly book
- Joint work, シーエムシー出版, Dec. 2010, JapaneseエコバイオリファイナリーScholarly book
- Joint work, シーエムシー出版, Mar. 2010, Japaneseシングルセル解析の最前線 （第３章 細胞内生体分子群の実測定量解析，第4節 「フローサイトメトリーとGFPレポーターによるG蛋白質シグナルのシングルセル解析」）Scholarly book
- Joint work, シーエムシー出版, Apr. 2009, Japanese第二世代バイオ燃料の開発と応用展開Scholarly book
- Single work, シーエムシー出版, Jun. 2008, Japanese微生物によるものづくりScholarly book
- 分裂酵母の代謝改変によるβ-カロテン生産
- 化学工学会第85年会コリネ菌におけるシキミ酸経路の強化
- 化学工学会第85年会大腸菌を用いたメバロン酸生産技術の開発
- 化学工学会第85年会"Parallel Metabolic Pathway Engineering" of Escherichia coli for Shikimate Pathway Derivative Production from Glucose-Xylose Co-substrate
- 酵素工学研究会第８２回講演会, Nov. 2019, 酵素工学研究会, 東京工業大学, Domestic conferenceParallel Metabolic Pathway Engineeringを用いた大腸菌による高収率物質生産
- 酵素工学研究会第８２回講演会, Nov. 2019, 酵素工学研究会, 東京工業大学, Domestic conferenceコリネ菌を用いたバイオマスからの脂肪酸生産技術の開発
- 酵素工学研究会第８２回講演会, Nov. 2019, 酵素工学研究会, 東京工業大学, Domestic conference大腸菌を用いたイソペンテノール生産のための代謝改変
- 酵素工学研究会第８２回講演会, Nov. 2019, 酵素工学研究会, 東京工業大学, Domestic conference分裂酵母の代謝改変によるβ-カロテン生産
- 酵素工学研究会第８２回講演会, Nov. 2019, 酵素工学研究会, 東京工業大学, Domestic conferenceメバロン酸生産 のための新規ターゲット遺伝子の探索
- 酵素工学研究会第８２回講演会, Nov. 2019, 酵素工学研究会, 東京工業大学, Domestic conferenceコリネ菌におけるシキミ酸経路の強化
- 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), Sep. 2019, APCChE, Sapporo Convention CenterThe effect of single gene knockout on mevalonate production by Escherichia coliPoster presentation
- 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), Sep. 2019, English, APCChE, Sapporo Convention Center, International conferenceHigh yield bioproduction of shikimate pathway derivatives by unitization and combination of purpose-specific metabolic pathwaysPoster presentation
- 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), Sep. 2019, English, APCChE, Sapporo Convention Center, International conferenceBeta-carotene Production Using Genome Edited Schizosaccharomyces pombePoster presentation
- 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), Sep. 2019, English, APCChE, Sapporo Convention Center, International conferenceEnhancement of fatty acid productivity using metabolically engineered Corynebacterium glutamicumPoster presentation
- 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), Sep. 2019, English, APCChE, Sapporo Convention Center, International conferenceMetabolic engineering of Escherichia coli for isopentenol productionPoster presentation
- 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), Sep. 2019, English, APCChE, Sapporo Convention Center, International conferenceMetabolic engineering of Escherichia coli for production of p-aminobenzoic acidPoster presentation
- 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), Sep. 2019, English, APCChE, Sapporo Convention Center, International conferenceProduction of shikimic acid using metabolically engineered Corynebacterium glutamicumPoster presentation
- 第71回日本生物工学会大会, Sep. 2019, Japanese, 岡山大学, Domestic conference遺伝子組換え乳酸菌によるコーンコブを原料としたD-乳酸の高濃度・高収率生産Oral presentation
- 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), Jul. 2019, English, APCChE, Sapporo Convention Center, International conferenceMetabolic engineering to improve 1,5-diaminopentane production from cellobiose using BGL-secreting Corynebacterium glutamicumPoster presentation
- 14th International symposium on Biocatalysis and Biotransformations (Biotrans 2019), Jul. 2019, English, University of Groningen, the MartiniPlaza, International conferenceThe effect of gene knockout on mevalonate production orelectricity generation by Escherichia coliPoster presentation
- 14th International symposium on Biocatalysis and Biotransformations (Biotrans 2019), Jul. 2019, English, University of Groningen, the MartiniPlaza, International conferenceMetabolic engineering of Escherichia coli for production of mevalonatePoster presentation
- 14th International symposium on Biocatalysis and Biotransformations (Biotrans 2019), Jul. 2019, University of Groningen, the MartiniPlazaHigh yield bioproduction of shikimate pathway derivatives by"Parallel Metabolic Design"
- Symposium on Biotechnology for Fuels and Chemicals (SBFC), Apr. 2019, Society for Industrial Microbiology and Biotechnology, Hyatt Regency SeattleHigh yield production of muconic acid from glucose by supplementing the carbon source from xylose for cell growth.
- Symposium on Biotechnology for Fuels and Chemicals (SBFC), Apr. 2019, Society for Industrial Microbiology and Biotechnology, Hyatt Regency SeattleEnhancing 3-hydroxypropionic acid production in combination with cell surface display and metabolic engineering of S. pombe
- 化学工学会第84年会, Mar. 2019, Japanese, 芝浦工業大学 豊洲キャンパス, Domestic conferenceコーンコブを原料とした代謝改変乳酸菌による高濃度高収率D-乳酸生産Poster presentation
- 第67回高分子討論会, Sep. 2018, Japanese, 公益社団法人高分子学会, 札幌, Domestic conference酵素反応によりゲルネットワークを精密に合成した金ナノ粒子／DNAハイブリッドゲルOral presentation
- 化学工学会第50回秋季大会, Sep. 2018, Japanese, 鹿児島大学 郡元キャンパス, Domestic conference糖の使い分けによる高収率物質生産技術の開発Poster presentation
- 化学工学会第50回秋季大会, Sep. 2018, Japanese, 鹿児島大学 郡元キャンパス, Domestic conference大腸菌を用いたセロビオースからのメバロン酸生産技術の開発Poster presentation
- 第70回日本生物工学会大会, Sep. 2018, Japanese, 関西大学 千里山キャンパス, Domestic conference遺伝子組換え乳酸菌によるコーンコブを原料とした高収率D-乳酸生産Poster presentation
- 化学工学会第50回秋季大会, Sep. 2018, Japanese, 鹿児島大学 郡元キャンパス, Domestic conferenceコリネ菌を用いたセロビオースからのシキミ酸生産技術の開発Poster presentation
- 第70回日本生物工学会大会, Sep. 2018, Japanese, 関西大学 千里山キャンパス, Domestic conferenceTarget-AID を利用したゲノム編集による高収率ブタ ノール発酵性クロストリジウム属微生物の育種Poster presentation
- 化学工学会第50回秋季大会, Sep. 2018, Japanese, 鹿児島大学 郡元キャンパス, Domestic conferencen-ブタノールを経由した大腸菌利用によるn-ブチルアミン生産技術の開発Poster presentation
- 第70回日本生物工学会大会, Sep. 2018, English, 関西大学 千里山キャンパス, Domestic conferenceMetabolic engineering of Escherichia coli for production of shikimate pathway derivativesPoster presentation
- 化学工学会第50回秋季大会, Sep. 2018, Japanese, 鹿児島大学 郡元キャンパス, Domestic conferenceCorynebacterium glutamicumを用いたセロビオースからのカダベリン生産技術の開発Poster presentation
- The 15th Japan-China-Korea Joint Symposium on Enzyme Engineering, Jun. 2018, English, 京都大学, International conferenceProduction of shikimic acid from cellobiose using Corynebacterium glutamicumPoster presentation
- The 15th Japan-China-Korea Joint Symposium on Enzyme Engineering, Jun. 2018, English, 京都大学, International conferenceProduction of n-butylamine in Escherichia coli by transaminasePoster presentation
- The 15th Japan-China-Korea Joint Symposium on Enzyme Engineering, Jun. 2018, English, 京都大学, International conferenceMetabolic engineering of S. pombe via CRISPR-Cas9 genome editing for 3-hydroxyprpininc acid production from glucose and cellobiosePoster presentation
- The 15th Japan-China-Korea Joint Symposium on Enzyme Engineering, Jun. 2018, English, 京都大学, International conferenceMetabolic engineering of Escherichia coli for production of mevalonate from cellobiosePoster presentation
- The 15th Japan-China-Korea Joint Symposium on Enzyme Engineering, Jun. 2018, English, 京都大学, International conferenceMetabolic design of Escherichia coli for production of shikimate pathway derivativesPoster presentation
- The 15th Japan-China-Korea Joint Symposium on Enzyme Engineering, Jun. 2018, English, 京都大学, International conferenceCadaverine production from cellobiose using Corynebacterium glutamicumPoster presentation
- 化学工学会第83年会, Mar. 2018, Japanese, 関西大学 千里山キャンパス, Domestic conference分裂酵母を用いた有機酸生産技術の開発Poster presentation
- 化学工学会第83年会, Mar. 2018, Japanese, 関西大学 千里山キャンパス, Domestic conference代謝デザイン大腸菌を利用したムコン酸発酵生産Poster presentation
- 化学工学会第83年会, Mar. 2018, Japanese, 関西大学 千里山キャンパス, Domestic conferenceSortaggingを用いた大腸菌代謝酵素の連結Poster presentation
- 酵素工学研究会第７８回講演会, Oct. 2017, Japanese, カレッジプラザ, Domestic conference分裂酵母の代謝改変株を用いた3-ヒドロキシプロピオン生産Poster presentation
- 酵素工学研究会第７８回講演会, Oct. 2017, Japanese, カレッジプラザ, Domestic conference代謝デザイン大腸菌を利用したムコン酸発酵生産Poster presentation
- 酵素工学研究会第７８回講演会, Oct. 2017, Japanese, カレッジプラザ, Domestic conference酵素カスケード反応を用いたヘキサメチレンジアミン生産Poster presentation
- 酵素工学研究会第７８回講演会, Oct. 2017, Japanese, カレッジプラザ, Domestic conferenceトランスアミナーゼを用いた菌体内n-ブチルアミン生産技術の開発Poster presentation
- 酵素工学研究会第７８回講演会, Oct. 2017, Japanese, カレッジプラザ, Domestic conferenceコリネ菌を用いたセルロース系バイオマスからのカダベリン生産技術の開発Poster presentation
- 酵素工学研究会第７８回講演会, Oct. 2017, Japanese, カレッジプラザ, Domestic conferenceグルカル酸生産を志向した大腸菌の代謝改変Poster presentation
- 酵素工学研究会第７８回講演会, Oct. 2017, Japanese, カレッジプラザ, Domestic conferenceSortaggingを用いた代謝チャネリング技術の開発Poster presentation
- 第69回日本生物工学会大会, Sep. 2017, Japanese, 早稲田大学 西早稲田キャンパス, Domestic conference分裂酵母の代謝改変株を用いた3-ヒドロキシプロピオン酸生産Oral presentation
- 第69回日本生物工学会大会, Sep. 2017, Japanese, 早稲田大学 西早稲田キャンパス, Domestic conference大腸菌を用いたムコン酸の高効率発酵生産Poster presentation
- 化学工学会第49回秋季大会, Sep. 2017, Japanese, 化学工学会, 名古屋大学東山キャンパス, Domestic conference酵素ステープラーを用いた代謝酵素の連結による代謝改変技術Poster presentation
- 化学工学会第49回秋季大会, Sep. 2017, Japanese, 化学工学会, 名古屋大学東山キャンパス, Domestic conference酵素カスケード反応を用いたヘキサメチレンジアミン生産Poster presentation
- 化学工学会第49回秋季大会, Sep. 2017, Japanese, 化学工学会, 名古屋大学東山キャンパス, Domestic conferenceトランスアミナーゼを用いた大腸菌内におけるブチルアミン生産Poster presentation
- 第69回日本生物工学会大会, Sep. 2017, Japanese, 早稲田大学 西早稲田キャンパス, Domestic conferenceTarget-AID を利用したゲノム編集による高収率ブタ ノール発酵性クロストリジウム属微生物の育種Oral presentation
- 第69回日本生物工学会大会, Sep. 2017, Japanese, 早稲田大学 西早稲田キャンパス, Domestic conferenceCorynebacterium glutamicumを用いたセルロース系バイオマスからのカダベリン生産技術の開発Poster presentation
- 合成生物工学シンポジウム, Aug. 2017, Japanese, 神戸大学百年記念館六甲ホール, Domestic conference細胞表層工学と代謝工学を用いた物質生産Oral presentation
- バイオ工学シンポジウム, Jul. 2017, Japanese, 大和屋本店 愛媛大学, Domestic conference細胞表層工学と合成代謝経路による物質生産Oral presentation
- SB7.0 The Seventh International Meeting on Synethetic Biology, Jun. 2017, English, University Cultural Center, International conferenceMetabolic engineering of S. pombe via CRISPR-Cas9 genome editing for lactic acid production from glucose and cellobioseOral presentation
- 日本農芸化学2017年度大会, Mar. 2017, Japanese, 日本農芸化学会, 京都女子大学, Domestic conferenceTarget-AID を利用したゲノム編集による高収率ブタ ノール発酵性クロストリジウム属微生物の育種Oral presentation
- the 17th International Biotechnology Symposium and Exhibition (IBS 2016), Oct. 2016, English, IBS, Melbourne, Australia, International conferenceMetabolic engineering for putrescine production from cellobiose by betaglucosidase displaying E. coliOral presentation
- 化学工学会第48回秋季大会, Sep. 2016, Japanese, 化学工学会, 徳島市, Domestic conference分裂酵母におけるマロニルCoAを介した有機酸生合成経路の構築Oral presentation
- 化学工学会第48回秋季大会, Sep. 2016, Japanese, 化学工学会, 徳島市, Domestic conference酵素カスケード反応を用いたヘキサメチレンジアミン生産Oral presentation
- 化学工学会第48回秋季大会, Sep. 2016, Japanese, 化学工学会, 徳島市, Domestic conferenceバイオ燃料電池への応用を志向したストレプトアビジンハイドロゲルの作製Oral presentation
- 第68回日本生物工学会大会, Sep. 2016, Japanese, 日本生物工学会, 富山市, Domestic conferenceSortase A を用いた大腸菌体内での代謝酵素の連結とその影響Oral presentation
- 化学工学会第48回秋季大会, Sep. 2016, Japanese, 化学工学会, 徳島市, Domestic conferenceSortase Aを用いた大腸菌体内での代謝酵素の連結とその影響Oral presentation
- 第68回日本生物工学会大会, Sep. 2016, Japanese, 日本生物工学会, 富山市, Domestic conferenceCRISPR Cas9 systemによる遺伝子組み換えSchizosaccharomyces pombeを用いた有機酸生産Oral presentation
- 第68回日本生物工学会大会, Sep. 2016, Japanese, 日本生物工学会, 富山市, Domestic conferenceCorynebacterium glutamicum を用いたヘミセルロース系バイオマスからのカダベリン生産Oral presentation
- 第68回日本生物工学会大会, Sep. 2016, Japanese, 日本生物工学会, 富山市, Domestic conferenceBGL提示大腸菌に適した代謝改変戦略を用いたセロビオースからのプトレシン生産Oral presentation
- 17th European Congress on Biotechnology (ECB2016), Jul. 2016, English, ECB2016, Kraków; Poland, International conferencePutrescine production from cellobiose by cell surface- and metabolically-engineered E. coliOral presentation
- 17th European Congress on Biotechnology (ECB2016), Jul. 2016, English, ECB2016, Kraków; Poland, International conferenceMetabolic enzyme ligation by sortaggingOral presentation
- 化学工学会第81年会, Mar. 2016, Japanese, Domestic conference異種酵素からなる一次元酵素集合体の設計と協奏効果の検討Oral presentation
- 化学工学会第81年会, Mar. 2016, Japanese, Domestic conferenceβ-グルコシダーゼ提示大腸菌を用いたセロビオースからのプトレシン生産Oral presentation
- 化学工学会第81年会本部大会, Mar. 2016, Japanese, 化学工学会, 福岡市, Domestic conferenceポリメラーゼ連鎖反応を利用したDNA―金ナノ粒子ハイブリッドゲルの作製と特性評価Poster presentation
- 化学工学会第81年会, Mar. 2016, Japanese, Domestic conferenceSorataseAを用いた大腸菌体内での代謝酵素連結技術の開発Oral presentation
- Protein & Antibody Engineering Summit (PEGS EUROPE), Nov. 2015, English, International conferenceStreptavidin Hydrogel as a Scaffold for Protein AssemblyOral presentation
- Protein & Antibody Engineering Summit (PEGS EUROPE), Nov. 2015, English, International conferenceC-terminal-oriented immobilization of enzymes using sortase A-mediated techniqueOral presentation
- 第67回日本生物工学会大会 国際シンポジウム, Oct. 2015, Japanese, Domestic conference効率的なブタノール生産に向けた代謝チャネリング技術の開発Oral presentation
- 第67回日本生物工学会大会 国際シンポジウム, Oct. 2015, Japanese, Domestic conferenceSortase Aを用いた酵素配向固定化微粒子の機能評価Oral presentation
- The 21th Symposium of Yong Asian Biochemical engineer's Communyty, Oct. 2015, English, International conferenceProduction of 2,3-butanediol from cellobiose using Baccillus subtilisOral presentation
- The 21th Symposium of Yong Asian Biochemical engineer's Communyty, Oct. 2015, English, International conferenceDirect cadaverine production from cellobiose using β-gulucosidase displaying Esherichia coilOral presentation
- The 21th Symposium of Yong Asian Biochemical engineer's Communyty, Oct. 2015, English, International conferenceC-terminal-oriented immobilization of enzymes using sortase A-mediated techniqueOral presentation
- 第67回日本生物工学会大会 国際シンポジウム, Oct. 2015, Japanese, Domestic conferenceCorynebacterium glutamicumを用いたバイオマスからのカダベリン生産Oral presentation
- 化学工学会第47回秋季大会, Sep. 2015, Japanese, Domestic conference酵素固定化足場としてのストレプトアビジンハイドロゲルの作成Oral presentation
- 化学工学会第47回秋季大会, Sep. 2015, Japanese, Domestic conference酵素ステープラーを用いた大腸菌体内での代謝酵素の連結Oral presentation
- 化学工学会第47回秋季大会, Sep. 2015, Japanese, Domestic conferenceタンパク質ｰリガンド相互作用を介した一次元セルラーゼ集合体の構築と触媒特性Oral presentation
- 第64回高分子討論会, Sep. 2015, Japanese, 高分子学会, 仙台市, Domestic conferencePCR法を用いた金ナノ粒子を架橋点とするDNAゲルの構築Poster presentation
- 化学工学会第47回秋季大会, Sep. 2015, Japanese, 化学工学会, 札幌市, Domestic conferencePCR増幅による金ナノ粒子を架橋点としたDNAゲルの形成Poster presentation
- 化学工学会第47回秋季大会, Sep. 2015, Japanese, Domestic conferenceCRISPR Cas9 system を用いて代謝改変を行った分裂酵母による有機酸生産技術の開発Oral presentation
- 第61回高分子研究発表会（神戸）, Jul. 2015, Japanese, 高分子学会, 神戸市, Domestic conferenceポリメラーゼ連鎖反応を用いた金ナノ粒子を架橋点とするDNAゲルの形成Poster presentation
- 4th International Frontiers in Polymer Science, May 2015, English, Elsevier, Riva del Garda, International conferenceHydrogel composed of DNA-gold nanoparticle network prepared by polymerase chain reactionPoster presentation
- 化学工学会第80年会, Mar. 2015, Japanese, 化学工学会, 江東区, Domestic conference線菌のタンパク質分泌機構を用いた機能性ストレプトアビジン生産Poster presentation
- 化学工学会第80年会, Mar. 2015, Japanese, 化学工学会, 江東区, Domestic conference枯草菌によるセロビオースからの2,3-ブタンジオール生産Poster presentation
- 化学工学会第80年会, Mar. 2015, Japanese, 化学工学会, 江東区, Domestic conferenceSortase Aを用いた酵素の配向固定化とその評価Oral presentation
- 第66回日本生物工学会大会, Sep. 2014, Japanese, 日本生物工学会, 札幌市, Domestic conference大腸菌を用いたオリゴ糖からのカダべリン生産系の構築Poster presentation
- 第66回日本生物工学会大会, Sep. 2014, Japanese, 日本生物工学会, 札幌市, Domestic conference多機能性ベータグルコシダーゼを用いたセルロース/ ヘミセルロース様基質の糖化Poster presentation
- 化学工学会第46回秋季大会, Sep. 2014, Japanese, 化学工学会, 福岡市, Domestic conference多機能性ベータグルコシダーゼの機能評価とその応用Oral presentation
- 化学工学会第46回秋季大会, Sep. 2014, Japanese, 化学工学会, 福岡市, Domestic conference線菌を用いたバイオマス資源からのスチレン系化合物生産Poster presentation
- 化学工学会第46回秋季大会, Sep. 2014, Japanese, 化学工学会, 福岡市, Domestic conference新規セルラーゼ-ポリマーハイブリッドによる結晶性セルロースの加水分解挙動Poster presentation
- 第66回日本生物工学会大会, Sep. 2014, Japanese, 日本生物工学会, 札幌市, Domestic conference高収率・高生産型クロストリジウム属ホモブタノール生産菌の解析Poster presentation
- 化学工学会第46回秋季大会, Sep. 2014, Japanese, 化学工学会, 福岡市, Domestic conference枯草菌によるバイオマス由来炭素源からの2,3-butanediol生産Oral presentation
- 化学工学会第46回秋季大会, Sep. 2014, Japanese, 化学工学会, 福岡市, Domestic conference化学的前処理バガスの酵素分解におけるキシラナーゼの協奏効果の検討Poster presentation
- 第66回日本生物工学会大会, Sep. 2014, Japanese, 日本生物工学会, 札幌市, Domestic conferenceβ- グルコシターゼ提示分裂酵母を用いた乳酸生産Poster presentation
- 第66回日本生物工学会大会, Sep. 2014, Japanese, 日本生物工学会, 札幌市, Domestic conferenceSortase A を用いた酵素配向固定化粒子の作製Poster presentation
- 化学工学会第46回秋季大会, Sep. 2014, Japanese, 化学工学会, 福岡市, Domestic conferenceBGL提示Corynebacterium glutamicumを用いたセロビオースからのジアミン生産Poster presentation
- EUROPEAN BIOTECHNOLOGY CONGRESS 2014, May 2014, English, European Biotechnology Thematic Network Assosiation, Lecce, Italy, International conferenceCo-assimilation of cellobiose and xylooligosaccharides using E. coli displaying both beta-glucosidase and beta-xylosidase on its cell surfacePoster presentation
- 化学工学会第79年会, Mar. 2014, Japanese, 化学工学会, 岐阜市, Domestic conferenceβグルコシダーゼ提示大腸菌を用いたセロビオースからのカダべリン生産Poster presentation
- 化学工学会第79年会, Mar. 2014, Japanese, 化学工学会, 岐阜市, Domestic conferenceAvidin-biotin相互作用による新規自己集合性人工セルロソームの構築Poster presentation
- Korea-Japan Smart Biodesign Workshop:Technology exchange for green biotechnology, Jan. 2014, English, 東北大学, 仙台市, International conferenceCreation of Escherichia coli Displaying both β-Glucosidase and β-Xylosidase on Its Cell SurfaceOral presentation
- 酵素工学研究会 第70回講演会, Oct. 2013, Japanese, 酵素工学研究会, 文京区, Domestic conference糖の輸送能力強化によるエクトイン高生産Halomonas elongata の創製Poster presentation
- 化学工学会第45回秋季大会, Sep. 2013, Japanese, 化学工学会, 岡山市, Domestic conference放線菌を用いたセルロースからのp-クマル酸生産Poster presentation
- 化学工学会第45回秋季大会, Sep. 2013, Japanese, 化学工学会, 岡山市, Domestic conference放線菌を用いたp-ヒドロキシスチレン生産Poster presentation
- 化学工学会第45回秋季大会, Sep. 2013, Japanese, 化学工学会, 岡山市, Domestic conferenceβ-グルコシダーゼ表層提示 Corynebacterium glutamicumを用いたセロビオースからのリジン生産Poster presentation
- 第65回日本生物工学会大会, Sep. 2013, Japanese, 日本生物工学会, 広島市, Domestic conferenceβ-グルコシダーゼ提示大腸菌を用いたプトレシン生産経路の構築Poster presentation
- 化学工学会第45回秋季大会, Sep. 2013, Japanese, 化学工学会, 岡山市, Domestic conferenceセルロース系バイオマスの酵素分解における協奏効果に関する基礎検討Poster presentation
- 化学工学会第45回秋季大会, Sep. 2013, Japanese, 化学工学会, 岡山市, Domestic conferenceDNA足場上への異種セルラーゼ提示による人工セルロソームの設計Poster presentation
- Enzym Engineering XXII, Sep. 2013, English, 酵素工学研究会, 富山市, International conferenceCo-assimilation of cellobiose and xylooligosaccharides using E. coli displaying both beta-glucosidase and beta-xylosidase on its cell surfacePoster presentation
- The 19th Symposium of Young Asian Biochemical Engineers' Communyty, Aug. 2013, English, 生物工学会, Xinjiang,China, International conferenceCo-assimilation of cellobiose and xylooligosaccharides using E. coli displaying both b-glucosidase andb-xylosidase on its cell surfaceOral presentation
- 9th European Congress of chemical engineering, Apr. 2013, English, European Society of Biochemical Engineering Science, The Hague, Netherland, International conferenceStarchy and Cellulosic Biomass-Powered Enzymatic Biofuel Cells based on Degrading and Oxidizing Enzymes Multi-Immobilized BioanodesPoster presentation
- 9th European Congress of chemical engineering, Apr. 2013, English, European Society of Biochemical Engineering Science, The Hague, Netherland, International conferenceCo-Assimilation of Celloorigosaccharide and Xyloorigosaccharide Using E.coli Displaying both Beta-GlucosidasePoster presentation
- 化学工学会第78年会, Mar. 2013, Japanese, 化学工学会, 豊中市, Domestic conference乳酸菌を用いたオリゴ糖からのD-乳酸生産技術の開発Oral presentation
- 化学工学会第78年会, Mar. 2013, Japanese, 化学工学会, 豊中市, Domestic conferenceβ-グルコシダーゼ発現Corynebacterium glutamicumを用いたセロビオースからのリジン生産Oral presentation
- 化学工学会第78年会, Mar. 2013, Japanese, 化学工学会, 豊中市, Domestic conferenceβグルコシダーゼ提示大腸菌を用いたプトレシン生産経路の構築Oral presentation
- 化学工学会第78年会, Mar. 2013, Japanese, 化学工学会, 豊中市, Domestic conferenceセロビオース資化性分裂酵母を用いた3-ヒドロキシプロピオン酸生産技術の開発Oral presentation
- 化学工学会第78年会, Mar. 2013, Japanese, 化学工学会, 豊中市, Domestic conferenceSortase A を用いた酵素配向固定化微粒子の作製Oral presentation
- 第64回日本生物工学会大会, Oct. 2012, Japanese, 日本生物工学会, 神戸市, Domestic conference放線菌バイオマス分解酵素ライブラリの構築及び新規高分泌シグナルの探索Oral presentation
- 第64回日本生物工学会大会, Oct. 2012, Japanese, 日本生物工学会, 神戸市, Domestic conference糖質バイオマスから多様なポリエステルを生産するコリネ菌微生物工場の開発Oral presentation
- 酵素工学研究会第６８回講演会, Oct. 2012, Japanese, 酵素工学研究会, 文京区, Domestic conference代謝改変によるエクトイン高生産Halomonas elongata の創製Poster presentation
- 第64回日本生物工学会大会, Oct. 2012, Japanese, 日本生物工学会, 神戸市, Domestic conferenceβ- グルコシダーゼ発現Corynebacterium glutamicum を用いたセロビオースからのリジン生産Oral presentation
- 第64回日本生物工学会大会, Oct. 2012, Japanese, 日本生物工学会, 神戸市, Domestic conferenceバイオマス直接利用型電極の開発Oral presentation
- 第64回日本生物工学会大会, Oct. 2012, Japanese, 日本生物工学会, 神戸市, Domestic conferenceストレプトアビジンを介したタンパク質の高配向同時固定化技術の開発Oral presentation
- 第64回日本生物工学会大会, Oct. 2012, Japanese, 日本生物工学会, 神戸市, Domestic conferenceエンドグルカナーゼ分泌生産型放線菌によるセルロースからの安息香酸生産Oral presentation
- 酵素工学研究会第６８回講演会, Oct. 2012, Japanese, 酵素工学研究会, 文京区, Domestic conferenceエンドグルカナーゼ分泌生産型放線菌によるセルロースからの安息香酸生産Poster presentation
- 酵素工学研究会第６８回講演会, Oct. 2012, Japanese, 酵素工学研究会, 文京区, Domestic conferenceSortase A を用いた高配向なタンパク質固定化技術の開発Poster presentation
- 第64回日本生物工学会大会, Oct. 2012, Japanese, 日本生物工学会, 神戸市, Domestic conferenceHalomonas elongata を用いたバイオマスからのエクトイン生産技術の開発Oral presentation
- 化学工学会 第44回秋季大会, Sep. 2012, Japanese, 化学工学会, 東北大学, Domestic conference目的タンパク質のみを特異的に認識する機能性高分子多孔膜の開発Poster presentation
- 化学工学会 第44回秋季大会, Sep. 2012, Japanese, 高分子学会, 仙台市, Domestic conference目的タンパク質のみを特異的に認識する機能性高分子Poster presentation
- 化学工学会第44回秋季大会, Sep. 2012, Japanese, 化学工学会, 仙台市, Domestic conference木質系バイオマス直接利用型電極の開発Oral presentation
- 化学工学会第44回秋季大会, Sep. 2012, Japanese, 化学工学会, 仙台市, Domestic conferenceバイオマス資化性大腸菌を用いたグルカル酸生産技術の開発Oral presentation
- 第58回高分子研究発表会（神戸）, Jul. 2012, Japanese, 高分子学会, 神戸市, Domestic conference特定タンパク質選択性を有する高分子多孔膜の開発Poster presentation
- 第72回分析化学討論会, May 2012, Japanese, 日本分生化学会, 鹿児島市, Domestic conferenceラジカルを発生する金属酸化物粒子に対するXAFSによる非破壊状態分析Oral presentation
- 日本農芸化学会2012年度大会, Mar. 2012, Japanese, 日本農芸化学会, 京都市, Domestic conference麹菌Aspergillus oryzaeを用いたラクダ科動物由来一本鎖抗体可変部位VHH分泌生産系の構築Oral presentation
- 化学工学会第77年会, Mar. 2012, Japanese, 化学工学会, 新宿区, Domestic conferenceセルロースからの高効率一段階エタノール発酵を可能にする高機能酵母の創製Oral presentation
- 日本農芸化学会2012年度大会, Mar. 2012, Japanese, 日本農芸化学会, 京都市, Domestic conferenceエンドグルカナーゼ分泌生産型放線菌を用いたセルロースからの有用タンパク質生産Oral presentation
- 化学工学会第77年会, Mar. 2012, Japanese, 化学工学会, 新宿区, Domestic conferenceエンドグルカナーゼ分泌生産型放線菌によるセルロースからの安息香酸生産Oral presentation
- 化学工学会第77年会, Mar. 2012, Japanese, 化学工学会, 新宿区, Domestic conferenceStreptaivdinを用いた部位特異的な異種タンパク質同時固定化技術の開発Oral presentation
- 化学工学会第77年会, Mar. 2012, Japanese, 化学工学会, 新宿区, Domestic conferenceHalomonas elongataを用いたバイオマスからの高効率エクトイン生産プロセスの開発Oral presentation
- The 2nd i-BioP Asian Symposium, Dec. 2011, English, EBRC, Gyeongbuk、Korea, International conferenceCreation of cellooligosaccharide-assimilating E. coli by displaying active BGL on the cell surface using novel anchor proteinOral presentation
- 第11回糸状菌分子生物学コンファレンス(2011), Nov. 2011, Japanese, 糸状菌分子生物学研究会, 文京区, Domestic conference組換え麹菌生産セルラーゼを用いた広葉樹クラフトパルプの酵素糖化Poster presentation
- 第11回糸状菌分子生物学コンファレンス(2011), Nov. 2011, Japanese, 糸状菌分子生物学研究会, 文京区, Domestic conferenceラクダ由来一本鎖抗体可変部位 VHH の麹菌 Aspergillus oryzae による分泌生産Poster presentation
- 極限環境生物学会2011年度(第12回)年会, Nov. 2011, Japanese, 極限環境生物学会, 長崎市, Domestic conferenceHalomonas elongata を用いたバイオマス由来C5糖からの高効率エクトイン生産プロセスの開発Poster presentation
- 化学工学会第43回秋季大会, Sep. 2011, Japanese, 化学工学会, 名古屋市, Domestic conference放線菌が有する炭素源資化能力を生かしたタンパク質の大量分泌生産Poster presentation
- 第63回日本生物工学会大会, Sep. 2011, Japanese, 日本生物工学会, 小金井市, Domestic conference高効率バイオエタノール製造技術開発- SSCF(同時糖化並行複発酵)のための糖化発酵技術-Oral presentation
- 化学工学会第43回秋季大会, Sep. 2011, Japanese, 化学工学会, 名古屋市, Domestic conference酵素を用いたタンパク質高機能化技術の開発[Invited]Invited oral presentation
- 化学工学会第43回秋季大会, Sep. 2011, Japanese, 化学工学会, 名古屋市, Domestic conferenceβグルコシダーゼを表層提示したセロオリゴ糖資化性大腸菌の創製Poster presentation
- 化学工学会第43回秋季大会, Sep. 2011, Japanese, 化学工学会, 名古屋市, Domestic conferenceバイオマス同時糖化酸化型電極の開発Poster presentation
- 化学工学会第43回秋季大会, Sep. 2011, Japanese, 化学工学会, 名古屋市, Domestic conferenceセルラーゼ発現最適化二倍体酵母による木質系バイオマスからのエタノール発酵Poster presentation
- Enzyme Engineering XXI, Sep. 2011, English, Engineering Conferences International, Colorado. USA, International conferenceCreation of cellooligosaccharide‐assimilating E. coli by displaying active BGL on the cell surface using novel anchor proteinPoster presentation
- 化学工学会第43回秋季大会, Sep. 2011, Japanese, 化学工学会, 名古屋市, Domestic conferenceCorynebacterium glutamicumを用いた高効率なGABA生産技術の開発Poster presentation
- 第63回日本生物工学会大会, Sep. 2011, Japanese, 日本生物工学会, 小金井市, Domestic conferenceBGL 表層提示大腸菌によるセロビオースを基質としたIPA 生産Oral presentation
- 第27回日本DDS学会学術集会, Jun. 2011, Japanese, 日本DDS学会, 文京区, Domestic conferenceバイオナノカプセルを用いた細胞特異的ドラッグデリバリーシステムOral presentation
- 33rd Symposium on Biotechnology for Fuels and Chemicals, May 2011, English, Society for industrial Microbiology, Seattle, USA, International conferenceHomo D-lactic acid fermentation from pentose and xylan by metabolically engineered Lactobacillus plantarumPoster presentation
- 33rd Symposium on Biotechnology for Fuels and Chemicals, May 2011, English, Society for industrial Microbiology, Seattle, USA, International conferenceCinnamic acid production from various carbon sources using phenylalnine ammonia lyase expressing Streptomyces lividansPoster presentation
- 化学工学会第76年会, Mar. 2011, Japanese, 日本化学会, 東京都 小金井市, Domestic conference放線菌を用いたあらゆる炭素源からのケイ皮酸生産Oral presentation
- 日本農芸化学会2011年度大会, Mar. 2011, Japanese, 日本農芸化学会, 京都市, Domestic conference放線菌によるトランスグルタミナーゼの大量分泌生産に向けた生産条件の検討Oral presentation
- 化学工学会第15回学生発表会, Mar. 2011, Japanese, (社)化学工学会, 神戸市, Domestic conference放線菌によるトランスグルタミナーゼの生産Oral presentation
- 化学工学会第15回学生発表会, Mar. 2011, Japanese, (社)化学工学会, 神戸市, Domestic conference放線菌における表層提示技術に関する研究Oral presentation
- 化学工学会第76年会, Mar. 2011, Japanese, 日本化学会, 東京都 小金井市, Domestic conference長期エタノール発酵に向けたアミラーゼ表層提示発現酵母株の創製Oral presentation
- 化学工学会第15回学生発表会, Mar. 2011, Japanese, (社)化学工学会, 神戸市, Domestic conference多様なバイオマスを燃料とした酵素バイオ燃料電池の開発Oral presentation
- 化学工学会第76年会, Mar. 2011, Japanese, 日本化学会, 東京都 小金井市, Domestic conference酵素を用いたタンパク質高機能化技術の開発Oral presentation
- 化学工学会第76年会, Mar. 2011, Japanese, 日本化学会, 東京都 小金井市, Domestic conference過酸化チタンナノ粒子及び低線量X線照射を併用した深部ガンの非侵襲的治療法の開発Oral presentation
- 化学工学会第15回学生発表会, Mar. 2011, Japanese, (社)化学工学会, 神戸市, Domestic conference過酸化チタンナノ粒子及び低線量X線照射を併用した深部ガンの非侵襲的治療法の開発Oral presentation
- 日本農芸化学会2011年度大会, Mar. 2011, Japanese, 日本農芸化学会, 京都市, Domestic conferenceβグルコシダーゼ表層提示によるセロビオース資化性大腸菌の創製Oral presentation
- 化学工学会第76年会, Mar. 2011, Japanese, 日本化学会, 東京都 小金井市, Domestic conferenceβグルコシダーゼを表層提示したセロビオース資化性大腸菌の創製Oral presentation
- 化学工学会第15回学生発表会, Mar. 2011, Japanese, (社)化学工学会, 神戸市, Domestic conferenceランダム変異導入による高活性エンドグルカナーゼの創製Oral presentation
- 化学工学会第76年会, Mar. 2011, Japanese, 日本化学会, 東京都 小金井市, Domestic conferenceバイオマスの直接利用を目指した多機能性電極の開発Oral presentation
- 化学工学会第15回学生発表会, Mar. 2011, Japanese, (社)化学工学会, 神戸市, Domestic conferenceトランスポゾンタギング法による好塩性細菌Halomonas elongata OUT30018株の浸透圧ストレス応答性遺伝子の探索Oral presentation
- 日本農芸化学会2011年度大会, Mar. 2011, Japanese, 日本農芸化学会, 京都市, Domestic conferenceコリネ型細菌を用いたGABAの生産Oral presentation
- 化学工学会第76年会, Mar. 2011, Japanese, 日本化学会, 東京都 小金井市, Domestic conferenceSortaseを用いたStreptavidinの新規修飾技術の開発Oral presentation
- 日本農芸化学会2011年度大会, Mar. 2011, English, 日本農芸化学会, 京都市, Domestic conferenceProduction of enantiomeric pure lactic acid from biomass by cell surface engineered LABOral presentation
- 化学工学会第15回学生発表会, Mar. 2011, Japanese, (社)化学工学会, 神戸市, Domestic conferenceCorynebacterium glutamicumを用いたGABAの高効率生産技術の開発Oral presentation
- The 16th Symposium of Young Asian Biochemical Engineers' Communyty, Nov. 2010, English, (社)化学工学会, 台湾桃園県, International conferenceBiofunctional TiO2 Nanoparticle-mediated Photokilling of Cancer Cells Using UV IrradiationPoster presentation
- 第62回日本生物工学会大会, Oct. 2010, Japanese, (社)日本生物工学会, 宮崎市, Domestic conference放線菌を用いた様々なタンパクの大量分泌系の構築Poster presentation
- 第62回日本生物工学会大会, Oct. 2010, Japanese, (社)日本生物工学会, 宮崎市, Domestic conference過酸化チタンナノ粒子及び低線量X 線照射を併用した深部ガンの非侵襲的治療法の開発Poster presentation
- 第62回日本生物工学会大会, Oct. 2010, Japanese, (社)日本生物工学会, 宮崎市, Domestic conferenceワイン酵母OC-2 トリプルマーカー株をプラットフォームとした高β – グルコシダーゼ活性と高キシロシダーゼ活性を持つキシロース発酵酵母の作製Poster presentation
- 第62回日本生物工学会大会, Oct. 2010, Japanese, (社)日本生物工学会, 宮崎市, Domestic conferenceセルラーゼ発現バランス最適化酵母を用いたセルロースからのエタノール発酵Poster presentation
- 第62回日本生物工学会大会, Oct. 2010, Japanese, (社)日本生物工学会, 宮崎市, Domestic conferenceSortase を用いたStreptavidin の新規修飾技術の開発Poster presentation
- 第62回日本生物工学会大会, Oct. 2010, Japanese, (社)日本生物工学会, 宮崎市, Domestic conferenceAffibody 提示ナノカプセルを用いたHER2 発現癌細胞特異的な薬物送達Poster presentation
- 2010年度日本放線菌学会大会, Sep. 2010, Japanese, 日本放線菌学会, 東京都 江戸川区, Domestic conference放線菌を用いた様々なタンパクの大量分泌系の構築Oral presentation
- 化学工学会第42回秋季大会, Sep. 2010, Japanese, (社)化学工学会, 京都市, Domestic conferenceaffibody提示バイオナノカプセルを用いたタンパク質デリバリーシステムPoster presentation
- RENEWABLE ENERGY 2010, Jun. 2010, English, International Solar Energy Society, 横浜市, International conferenceConstruction of consolidated bioprocesses for production of bio-fuels and chemicals by using cell surface engineered microbial cellsOral presentation
- 化学工学会第75年会, Mar. 2010, Japanese, (社)化学工学会, 鹿児島市, Domestic conference放線菌を用いた様々なタンパクの大量分泌系の構築Oral presentation
- 化学工学会第75年会, Mar. 2010, Japanese, (社)化学工学会, 鹿児島市, Domestic conference非侵襲性がん治療に向けた高機能性二酸化チタンナノ粒子の開発Oral presentation
- 化学工学会第75年会, Mar. 2010, Japanese, (社)化学工学会, 鹿児島市, Domestic conference生デンプンからの効率的な繰返し発酵を達成するアミラーゼ発現新規酵母株の創製Oral presentation
- 日本農芸化学会2010年度大会, Mar. 2010, Japanese, (社)日本農芸化学会, 東京都目黒区, Domestic conference新規リパーゼ発現系を用いたWhole-cell biocatalystによる効率的なバイオディーゼル燃料生産Oral presentation
- 化学工学会第75年会, Mar. 2010, Japanese, (社)化学工学会, 鹿児島市, Domestic conference酵素を用いた抗体結合ドメインー機能性タンパク質調製法の最適化Oral presentation
- 日本農芸化学会2010年度大会, Mar. 2010, Japanese, (社)日本農芸化学会, 東京都目黒区, Domestic conferenceバイオマスからの燃料・化学品生産を目指した細胞工場の創製Oral presentation
- 日本農芸化学会2010年度大会, Mar. 2010, Japanese, (社)日本農芸化学会, 東京都目黒区, Domestic conferenceカクテルδインテグレーション法によるセルラーゼ発現バランス最適化酵母の創製Oral presentation
- 化学工学会第75年会, Mar. 2010, Japanese, (社)化学工学会, 鹿児島市, Domestic conferenceHis-Tagタンパク質特異的精製機能を有する高分子多孔膜の開発Oral presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceSite-specific streptavidin modification using sortasePoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceSite-specific protein modification with functional molecule using novel enzymePoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceIntegrated and energy-saving biodiesel fuel production using fungus whole-cell biocatalystPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceFunctional analysis of mutant human somatostatin receptor using a yeast-based fluorescence reporter assayPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceExpression and signaling analyses of human G protein-coupled receptor in yeastPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEnzyme-mediated site-specific protein modificationOral presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEnzyme-mediated protein immobilization on particlesPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEnzyme-mediated antibody-protein conjugationPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEnzymatic protein conjugation in vivoPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEngineering of endoglucanase expression system for Corynebacterium glutamicumPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEfficient repeated batch fermentation from raw starch by novel yeast expressing transporter gene and amylase genesPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEfficient homo D-lactic acid production from xylosePoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEfficient ethanol production from xylose by mated diploid Saccharomyces cerevisiaePoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEfficient D-lactic acid production from raw starchPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEfficient biodiesel production using whole-cell biocatalyst employing a lipase high expression systemPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceEfficient and practical ethanol production from high yield rice by amylase expressing Saccharomyces cerevisiaePoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceDirect fermentation of cellulosic materials to ethanol using yeast strains codisplaying three types of cellulolytic enzymePoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceDevelopment of novel cell-surface display system of Aspergillus oryzae using chitin binding domainPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceConstruction of a novel detection system for protein–protein interactions using yeast G-protein signalingOral presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceBiodiesel production using whole-cell biocatalyst of Aspergillus oryzae coexpression lipasesPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceAntibody-immobilized TiO2 nanoparticles for cancer therapyPoster presentation
- APBioChEC'09, Nov. 2009, English, APBioChEC'09 Committee, 神戸市, International conferenceAffibody displaying bionanocapsules for HER2 specific drug deliveryPoster presentation
- 第61回日本生物工学会大会, Sep. 2009, Japanese, (社)日本生物工学会, 名古屋市, Domestic conference生デンプンから高効率にエタノールを生産する新規酵母の創製Oral presentation
- 第61回日本生物工学会大会, Sep. 2009, Japanese, (社)日本生物工学会, 名古屋市, Domestic conference癌細胞標的化を目指したAffibody 提示ナノカプセルの開発Oral presentation
- 第61回日本生物工学会大会, Sep. 2009, Japanese, (社)日本生物工学会, 名古屋市, Domestic conference遺伝子組換え麹菌を用いたタンパク質大量分泌生産技術の開発Oral presentation
- 第61回日本生物工学会大会, Sep. 2009, Japanese, (社)日本生物工学会, 名古屋市, Domestic conferencePorin を用いたコリネ菌新規細胞表層提示技術の開発Oral presentation
- The 6th International Forum on Post-Genome Technologies (IFPT'6), Sep. 2009, English, Beijing, China, International conferenceConstruction of a novel detection system for protein-protein interactions using yeast G-protein signalingPoster presentation
- 化学工学会 第74年会, Mar. 2009, Japanese, 化学工学会, 神奈川県横浜市, Domestic conference非侵襲性がん治療のためのタンパク質修飾ナノ粒子の開発Oral presentation
- 化学工学会 第74年会, Mar. 2009, Japanese, 化学工学会, 神奈川県横浜市, Domestic conference新規発現系を用いたWhole-Cell Biocatalystによる効率的なバイオディーゼル燃料生産Oral presentation
- 化学工学会 第74年会, Mar. 2009, Japanese, 化学工学会, 神奈川県横浜市, Domestic conferenceピンポイントDDSを目指したバイオナノカプセルの開発Oral presentation
- 農芸化学会2009年度大会, Mar. 2009, Japanese, 日本農芸化学会, 福岡県福岡市, Domestic conferenceバイオエタノール生産を目指したスーパー酵母の創製Oral presentation
- 化学工学会 第74年会, Mar. 2009, Japanese, 化学工学会, 神奈川県横浜市, Domestic conferenceタンパク質連結酵素を用いた酵素融合タンパク質調製法の開発Oral presentation
- 農芸化学会2009年度大会, Mar. 2009, Japanese, 日本農芸化学会, 福岡県福岡市, Domestic conferenceキシロースからの高効率エタノール生産を目指した宿主酵母株の検討Poster presentation
- 農芸化学会2009年度大会, Mar. 2009, Japanese, 日本農芸化学会, 福岡県福岡市, Domestic conferenceキシロースからの高光学純度D-乳酸生産技術の開発Poster presentation
- 農芸化学会2009年度大会, Mar. 2009, Japanese, 日本農芸化学会, 福岡県福岡市, Domestic conferenceアミラーゼ発現酵母による生デンプンからの効率的な繰り返し発酵プロセスの開発Poster presentation
- 農芸化学会2009年度大会, Mar. 2009, Japanese, 日本農芸化学会, 福岡県福岡市, Domestic conferenceWhole-cell biocatalystを使用したバイオディーゼル燃料生産Poster presentation
- International Workshop HITS 2009, Feb. 2009, English, Tokyo, Japan, International conferenceConstruction of novel detection system for protein-protein interaction using yeastPoster presentation
- 第31回日本分子生物学会年会;第81回日本生化学会大会 合同大会, Dec. 2008, Japanese, 日本分子生物学学会, 日本生化学会, 神戸市, Domestic conference未来バイオ燃料生産用スーパー(CBP)微生物の開発Oral presentation
- 第31回日本分子生物学会年会・第81回日本生化学会大会 合同大会, Dec. 2008, Japanese, 日本分子生物学学会, 日本生化学会, 神戸市, Domestic conference酵母G蛋白質共役型受容体蛍光アッセイシステムによるヒトソマトスタチレセプター細胞外ループドメイン2の変異解析Poster presentation
- 若手フロンティア研究会2008, Dec. 2008, Japanese, 神戸大学研究基盤センター, 神戸市, Domestic conference温度応答性磁性ナノ粒子を用いた迅速かつ高効率なアフィニティー分子の選択法の確立Poster presentation
- 4th Korea-Japan Workshop on Combinatorial Bioengineering, Nov. 2008, English, Association of Combinatorial Bioengineering, Kookmin University, Seoul, International conferenceMethylester Synthesis By Fungi Immobilized BSPs in Ionic LiquidPoster presentation
- 4th Korea-Japan Workshop on Combinatorial Bioengineering, Nov. 2008, English, Association of Combinatorial Bioengineering, Seoul, Korea, International conferenceEfficient and direct ethanol production from raw starch by d-integrant polyploid fusants of Saccharomyces cerevisiaePoster presentation
- 第17回日本バイオイメージング学会, Oct. 2008, Japanese, 日本バイオイメージング学会, 千葉市, Domestic conference特異性改変型バイオナノカプセルを用いたバイオイメージング及びDDSへの応用Oral presentation
- 2008 “Bioseparation for Biorecognition and Bionanotechnology”, Oct. 2008, English, Hanyang University Bionanotechnology BK Program, Inha University Ultra Procesion Bioseparation Technology ERC, Hanyang University Human Sensing System ERC, Ansan, Korea, International conferenceEnzyme-Mediated Site-Specific Protein Conjugation In Vitro and In VivoOral presentation
- 化学工学会 第40回 秋季大会, Sep. 2008, Japanese, 化学工学会, 宮城県仙台市, Domestic conference生デンプンからの高効率D-乳酸生産技術の開発Poster presentation
- 化学工学会 第40回 秋季大会, Sep. 2008, Japanese, 化学工学会, 宮城県仙台市, Domestic conference酵母蛍光レポーターアッセイによるヒトソマトスタチンレセプター変異体の機能解析Poster presentation
- 化学工学会 第40回 秋季大会, Sep. 2008, Japanese, 化学工学会, 宮城県仙台市, Domestic conference酵母でのヒトG蛋白質共役型受容体発現に関する分泌シグナル配列の影響Poster presentation
- 化学工学会 第40回 秋季大会, Sep. 2008, Japanese, 化学工学会, 宮城県仙台市, Domestic conferenceペプチド転移酵素を用いた細胞内タンパク質連結技術の開発Poster presentation
- 化学工学会 第40回 秋季大会, Sep. 2008, Japanese, 化学工学会, 宮城県仙台市, Domestic conferenceセルラーゼ発現酵母によるセルロースからのバイオエタノール生産Poster presentation
- 化学工学会 第40回 秋季大会, Sep. 2008, Japanese, 化学工学会, 宮城県仙台市, Domestic conferenceWhole-cell biocatalystを使用した菜種油からのバイオディーゼル生産Poster presentation
- 第60回 日本生物工学会2008年度大会, Aug. 2008, Japanese, 日本生物工学会, 宮城県仙台市, Domestic conference無蒸煮デンプンから効率的にエタノールを生産する高機能酵母の創製Oral presentation
- 第60回 日本生物工学会2008年度大会, Aug. 2008, Japanese, 日本生物工学会, 宮城県仙台市, Domestic conference生デンプンを原料とした高光学純度D-乳酸の効率的生産技術の開発Oral presentation
- 第60回 日本生物工学会2008年度大会, Aug. 2008, Japanese, 日本生物工学会, 宮城県仙台市, Domestic conference新規酵素を用いたタンパク質への小分子修飾技術Oral presentation
- 第60回 日本生物工学会2008年度大会, Aug. 2008, Japanese, 日本生物工学会, 宮城県仙台市, Domestic conference酵素処理と電子線照射を組み合わせたセルローストリアセテート繊維の減量加工Oral presentation
- 第60回 日本生物工学会2008年度大会, Aug. 2008, Japanese, 日本生物工学会, 宮城県仙台市, Domestic conference酵素を用いた抗体と機能性タンパク質の新規連結法の開発Oral presentation
- 第60回 日本生物工学会2008年度大会, Aug. 2008, Japanese, 日本生物工学会, 宮城県仙台市, Domestic conferenceビオチン提示酵母を用いた新規細胞表層提示システムの開発Oral presentation
- 第60回 日本生物工学会2008年度大会, Aug. 2008, Japanese, 日本生物工学会, 宮城県仙台市, Domestic conferenceビオチン提示バイオナノカプセルを用いるピンポイント薬剤送達システムの開発Oral presentation
- 第60回 日本生物工学会2008年度大会, Aug. 2008, Japanese, 日本生物工学会, 宮城県仙台市, Domestic conferenceバイオディーゼルを目的としたAspergillus oryzae によるリパーゼ共発現系の構築Oral presentation
- 第60回 日本生物工学会2008年度大会, Aug. 2008, Japanese, 日本生物工学会, 宮城県仙台市, Domestic conferenceキチン結合ドメインを用いた麹菌細胞表層提示技術の開発Oral presentation
■ Research Themes
- 日本学術振興会, 科学研究費助成事業, 基盤研究(B), 神戸大学, 01 Apr. 2022 - 31 Mar. 2025わずかひと振りで目的化合物の生産性を向上させる代謝スパイスの開発本年度は、微生物の菌体表層にG6Pと親和性のあるタンパク質を発現させ、G6Pをキャプチャーさせることで菌体内の代謝が代わりシキミ酸経路が強化される現象に着目し、その評価基盤の構築を進めた。シキミ酸経路の強化は経路の最終産物の1つであるフェニルアラニンを指標としてHPLCにて評価する。グルコースおよびG6Pを培養液に添加しても際立った変化は見られず、外部添加からの表層への濃縮は効果がないことが示された。一方で、トランスポーター破壊株ではG6Pキャプチャーの影響は無くなることから、G6Pの膜間での移動が重要であることが確認できた。グルコース以外の糖類で検討したところ、代謝経路に依存する結果が得られた。これは糖を取り込んだあとの代謝物が影響していることを示唆しており、想定しているメカニズム通りに動いている可能性が高まった。また、フェニルアラニン以外にも経路の異なるアミノ酸であるリジン、および合成経路の異なる有機酸を生産する系をベースにした評価系を構築した。これらにおいても上記と同様の検討を行ったところ、こちらは経路により強化する場合と減少する場合があることが新たに判明した。これより、糖取り込み後の代謝物のいずれかが重要な役割を担っていること、および下流経路に存在するであろう別の代謝中間体が、代謝を変化させる働きをしていることが示唆される。しかし、代謝物のみで代謝を劇的に変化することは難しいと考えられるため、酵素もしくはエフェクタータンパク質などの関与が示唆される。これらの新しい因子の探索は次年度以降引きづつき進める。
- 日本学術振興会, 科学研究費助成事業, 基盤研究(A), 神戸大学, Apr. 2020 - Mar. 2023増殖にとらわれずに様々な前駆体を十分量供給できるプラットフォーム微生物群の構築前年度に引き続き増殖制御の基盤構築と重要な前駆体を蓄積する菌株の構築を進めた。キシロースを炭素源とした増殖制御系を野生株および改変株にそれぞれ導入し、炭素源で増殖を評価した。初めは増殖速度の違いは単純に導入した経路の活性に依存すると思われたが、中間体の蓄積量が一致せず、経路の導入により代謝ネットワークが別途活性化されている可能性が示唆された。この可能性のあるポイントをいくつか破壊していくことで増殖経路をスムーズに流す方向性が見出された。次年度はこの最適化を進める。また、野生株に比べるとその増殖能の低下が見られたが、炭素源の取り込みを改善することで回復できる可能性が示された。これについても遺伝子改変によりこれまで抑制されてきた制御系が発現している可能性があり、その解明についても進めている。前駆体においては、アセチルCoAを基本とする各種CoAについて検討を行った。アセチルCoA、マロニルCoA、などのCoA群を評価するためにモデル化合物の生産系を構築した。グルコース及びキシロースを炭素源として増殖と生産をそれぞれ制御し、どのパラメータが鍵となるかを評価できる系を構築した。解糖系の上流の前駆体においてもモデル化合物の生産系を構築し、増殖と生産を別々に制御できる評価系を構築した。物質生産におけるフィードバック阻害などの多数の制御系を解除するとともに、競合経路の破壊や副生成物の削除などを引き続き次年度も進めていく。
- 科学研究費補助金／基盤研究(B), Apr. 2019 - Mar. 2022, Principal investigator糖の使い分けによる高収率物質生産微生物の構築Competitive research funding
- 科学研究費補助金／基盤研究(B), Apr. 2016 - Mar. 2020Competitive research funding
- 科学研究費補助金／若手研究(A), Apr. 2016 - Mar. 2019, Principal investigatorCompetitive research funding
- 科学研究費補助金／基盤研究(B), Apr. 2016 - Mar. 2019Competitive research funding
- 学術研究助成基金助成金／挑戦的萌芽研究, Apr. 2015 - Mar. 2017Competitive research funding
- 国立研究開発法人科学技術振興機構, 未来社会創造事業（探索加速型）, 2017, Principal investigator【未来社会】 細胞表層工学と代謝工学を用いたＰＥＰ蓄積シャーシ株の創製Competitive research funding
- 学術研究助成基金助成金／挑戦的萌芽研究, Apr. 2013 - Mar. 2015Competitive research funding
- 科学研究費補助金／基盤研究(B), Apr. 2012 - Mar. 2015Competitive research funding
- 科学研究費補助金／基盤研究(B), Apr. 2012 - Mar. 2015Competitive research funding
- 研究成果最適展開支援プログラム フィージビリティスタディステージ 探索タイプ, 2013, Principal investigatorA-STEP「バイオマスからワンステップで乳酸ポリマーを直接生産する技術の開発」Competitive research funding
- 研究成果最適展開支援プログラム フィージビリティスタディステージ 探索タイプ, 2012, Principal investigatorA-STEP「バイオマスからワンステップで乳酸ポリマーを直接生産する技術の開発」Competitive research funding
- 学術研究助成基金助成金／挑戦的萌芽研究, 2011Competitive research funding
- 科学研究費補助金／萌芽研究, 2010Competitive research funding
- 科学研究費補助金／若手研究(B), 2009, Principal investigatorCompetitive research funding
- 科学研究費補助金／基盤研究(B), 2008Competitive research funding
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