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TAGUCHI SeiichiGraduate School of Science, Technology and Innovation / Department of Science, Technology and InnovationProfessor
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■ Paper- Extracellular membrane vesicles (MVs) caused by the artificial production of polyhydroxybutyrate (PHB) were previously detected in Escherichia coli. We herein observed MV biogenesis in the mutant strain (-PHB) of the natural PHB producer, Cupriavidus necator H16. This inverse relationship was revealed through comparative electron microscopic ana-lyses of wild-type and mutant strains. Based on these results, we speculate that a physiological relationship exists between MV biogenesis and PHB biosynthesis. Therefore, we propose the potential of MV biogenesis as a fermentative "stress marker" for monitoring the performance of target polymer-producing microbial platforms.2024, Microbes and environments, 39(3) (3), English, Domestic magazineScientific journal
- The lactate-based polyester poly[lactate (LA)-co-3-hydroxybutyrate (3HB)], termed LAHB, is a highly transparent and flexible bio-based polymeric material. There are many unknowns regarding its degradation process in riverine environments, especially the changes in bacterial flora that might result from its degradation and the identities of any LAHB-degrading bacteria. LAHB were immersed in the river water samples (A and B), and LAHB degradation was observed in terms of the weight change of the polymer and the microscopic changes on the polymer surfaces. A metagenomic analysis of microorganisms was conducted to determine the effect of LAHB degradation on the aquatic environment. The bacterial flora obtained from beta diversity analysis differed between the two river samples. The river A water sample showed the simultaneous degradation of LA and 3HB even though the copolymer was LA-enriched, suggesting preferable hydrolysis of the LA-enriched segments. In contrast, only 3HB degraded for the LAHB in the river B water sample. The linear discriminant analysis effect size (LEfSe) analysis revealed 14 bacteria that were significantly increased in the river A water sample during LAHB degradation, suggesting that these bacteria preferentially degraded and assimilated LA-clustering polymers. Our metagenomic analysis provides useful insights into the dynamic changes in microbial communities and LA-clustering polymer-degrading bacteria.Oct. 2023, Polymers, 15(20) (20), English, International magazineScientific journal
- Escherichia coli is a useful platform for producing valuable materials through the implementation of synthetic gene(s) derived from other organisms. The production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] was carried out in E. coli using a set of five other species-derived genes: Pseudomonas sp. 61-3-derived phaC1STQK (for polymerization), Cupriavidus necator-derived phaAB (for 3HB-CoA generation), and Megasphaera elsdenii-derived pct (for LA-CoA generation) cloned into pTV118NpctphaC1ps(ST/QK)AB. Here, we aimed to optimize the expression level and timing of these genes to improve the production of P(LA-co-3HB) and to manipulate the LA fraction by replacing the promoters with various promoters in E. coli. Evaluation of the effects of 21 promoter replacement plasmids revealed that the phaC1STQK-AB operon is critical for the stationary phase for P(LA-co-3HB) production. Interestingly, the effects of the promoters depended on the composition of the medium. In glucose-supplemented LB medium, the dps promoter replacement plasmid resulted in the greatest effect, increasing the accumulation to 8.8 g/L and an LA fraction of 14.1 mol% of P(LA-co-3HB), compared to 2.7 g/L and 8.1 mol% with the original plasmid. In xylose-supplemented LB medium, the yliH promoter replacement plasmid resulted in the greatest effect, with production of 5.6 g/L and an LA fraction of 40.2 mol% compared to 3.6 g/L and 22.6 mol% with the original plasmid. These results suggest that the selection of an appropriate promoter for expression of the phaC1STQK-AB operon could improve the production and LA fraction of P(LA-co-3HB). Here, we propose that the selection of cell-growth phase-dependent promoters is a versatile biotechnological strategy for effective intracellular production of polymeric materials such as P(LA-co-3HB), in combination with the selection of sugar-based carbon sources.Jul. 2023, Microbial cell factories, 22(1) (1), 131 - 131, English, International magazineScientific journal
- Polyhydroxyalkanoate (PHA) synthases (PhaCs) are key enzymes in PHA polymerization. PhaCs with broad substrate specificity are attractive for synthesizing structurally diverse PHAs. In the PHA family, 3-hydroxybutyrate (3HB)-based copolymers are industrially produced using Class I PhaCs and can be used as practical biodegradable thermoplastics. However, Class I PhaCs with broad substrate specificities are scarce, prompting our search for novel PhaCs. In this study, four new PhaCs from the bacteria Ferrimonas marina, Plesiomonas shigelloides, Shewanella pealeana, and Vibrio metschnikovii were selected via a homology search against the GenBank database, using the amino acid sequence of Aeromonas caviae PHA synthase (PhaCAc), a Class I enzyme with a wide range of substrate specificities, as a template. The four PhaCs were characterized in terms of their polymerization ability and substrate specificity, using Escherichia coli as a host for PHA production. All the new PhaCs were able to synthesize P(3HB) in E. coli with a high molecular weight, surpassing PhaCAc. The substrate specificity of PhaCs was evaluated by synthesizing 3HB-based copolymers with 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate, 3-hydroxy-2-methylbutyrate, and 3-hydroxypivalate monomers. Interestingly, PhaC from P. shigelloides (PhaCPs) exhibited relatively broad substrate specificity. PhaCPs was further engineered through site-directed mutagenesis, and the variant resulted in an enzyme with improved polymerization ability and substrate specificity.2023, Frontiers in bioengineering and biotechnology, 11, 1114946 - 1114946, English, International magazineScientific journal
- A new polythioester (PTE), poly(3-mercapto-2-methylpropionate) [P(3M2MP)], and its copolymer with 3-hydroxybutyrate (3HB) were successfully biosynthesized from 3-mercapto-2-methylpropionic acid as a structurally-related precursor. This is the fourth PTE of biological origin and the first to be α-methylated. P(3M2MP) was biosynthesized using an engineered Escherichia coli LSBJ, which has a high molecular weight, amorphous structure, and elastomeric properties, reaching 2600% elongation at break. P(3HB-co-3M2MP) copolymers were synthesized by expressing 3HB-supplying enzymes. The copolymers were produced with high content in the cells and showed a high 3M2MP unit incorporation of up to 77.2 wt% and 54.8 mol%, respectively. As the 3M2MP fraction in the copolymer increased, the molecular weight decreased and the polymers became softer, more flexible, and less crystalline, with lower glass transition temperatures and higher elongations at break. The properties of this PTE were distinct from those of previously biosynthesized PTEs, indicating that the range of material properties can be further expanded by introducing α-methylated thioester monomers.May 2022, Bioengineering (Basel, Switzerland), 9(5) (5), English, International magazineScientific journal
- Membrane vesicles (MVs) are formed in various microorganisms triggered by physiological and environmental phenomena. In this study, we have discovered that the biogenesis of MV took place in the recombinant cell of Escherichia coli BW25113 strain that intracellularly accumulates microbial polyester, polyhydroxybutyrate (PHB). This discovery was achieved as a trigger of foam formation during the microbial PHB fermentation. The purified MVs were existed as a mixture of outer MVs and outer/inner MVs, revealed by transmission electron microscopy. It should be noted that there was a good correlation between MV formation and PHB production level that can be finely controlled by varying glucose concentrations, suggesting the causal relationship in both supramolecules artificially produced in the microbial platform. Notably, the controllable secretion of MV was governed spatiotemporally through the morphological change of the E. coli cells caused by the PHB intracellular accumulation. Based on a hypothesis of PHB internal-pressure dependent envelope-disorder induced MV biogenesis, here we propose a new Polymer Intracellular Accumulation-triggered system for MV Production (designated "PIA-MVP") with presenting a mechanistic model for MV biogenesis. The PIA-MVP is a promising microbial platform that will provides us with a significance for further study focusing on biopolymer capsulation and cross-membrane transportation for different application purposes.Mar. 2022, Scientific Reports, 12(1) (1), 3393 - 3393, English, International magazine[Refereed]Scientific journal
- The biodegradable polyester poly-(R)-3-hydroxybutyrate [P(3HB)] is synthesized by a polymerizing enzyme called polyhydroxyalkanoate (PHA) synthase and accumulates in a wide variety of bacterial cells. Recently, we demonstrated the secretory production of a (R)-3HB oligomer (3HBO), a low-molecular-weight P(3HB), by using recombinant Escherichia coli expressing PHA synthases. The 3HBO has potential value as an antibacterial substance and as a building block for various polymers. In this study, to construct an efficient 3HBO production system, the coexpression of molecular chaperones and a PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4) was examined. First, genes encoding enzymes related to 3HBO biosynthesis (phaRCYB4, phaA and phaB derived from Ralstonia eutropha H16) and two types of molecular chaperones (groEL, groES, and tig) were introduced into the E. coli strains BW25113 and BW25113ΔadhE. As a result, coexpression of the chaperones promoted the enzyme activity of PHA synthase (approximately 2-3-fold) and 3HBO production (approximately 2-fold). The expression assay of each chaperone and PHA synthase subunit (PhaRYB4 and PhaCYB4) indicated that the combination of the two chaperone systems (GroEL-GroES and TF) supported the folding of PhaRYB4 and PhaCYB4. These results suggest that the utilization of chaperone proteins is a valuable approach to enhance the formation of active PHA synthase and the productivity of 3HBO.Feb. 2022, Microorganisms, 10(2) (2), 458 - 470, English, International magazineScientific journal
- Jan. 2022, Frontiers in Bioengineering and Biotechnology: Bioprocess Engineering, EnglishOptimization of Culture Conditions for Secretory Production of 3-Hydroxybutyrate Oligomers using recombinant Escherichia coli[Refereed]Scientific journal
- Dec. 2021, Front. Bioeng. Biotechnol., 9(777265) (777265), 777265 - 777265, English, International magazine[Refereed][Invited]Scientific journal
- Given their ubiquity in modern society, the development of biodegradable and renewably sourced plastics is essential for the creation of an environmentally sustainable society. One of the drawbacks for currently available biodegradable plastics such as poly(l-lactic acid) (PLLA) and polyhydroxyalkanoates (PHAs) is that it is difficult to simultaneously achieve mechanical flexibility and certain crystallization behavior in these materials, which limits their use as replacements for established petroleum-based plastics such as isotactic polypropylene (iPP). Here, we report the synthesis and characterization of a new biodegradable plastic, poly(3-hydroxy-2-methylbutyrate) [P(3H2MB)], which is a member of the bacterial PHA family whose members include an α-methylated monomer unit. Biosynthesis of P(3H2MB) was achieved using recombinant Escherichiacoli expressing an engineered pathway. Biosynthesized P(3H2MB) exhibited the highest melting temperature (197 °C) among the biosynthesized PHAs and improved thermal resistance. It also exhibited improved crystallization behavior and mechanical flexibility nearly equal to those of iPP. The primary nucleation rate of P(3H2MB) was faster than that of P(3HB), and the spherulite morphology of P(3H2MB) was much finer than that of P(3HB). This crystal morphology may result in more rapid crystallization behavior, increased transparency, and enhanced mechanical properties. The superior physical properties of P(3H2MB) have the potential to open new avenues for the production of high-performance biodegradable plastics for replacing petroleum-based bulk commodity plastics.Nature Research, Dec. 2021, NPG Asia Materials, 13(1) (1), EnglishScientific journal
- May 2021, The Journal of General and Applied Microbiology, 67(2) (2), English
- Mar. 2021, Applied Microbiology and Biotechnology, 105(7) (7), 2737 - 2745, EnglishScientific journal
- Polyhydroxyalkanoates (PHAs) are naturally occurring biopolymers produced by microorganisms. PHAs have become attractive research biomaterials in the past few decades owing to their extensive potential industrial applications, especially as sustainable alternatives to the fossil fuel feedstock-derived products such as plastics. Among the biopolymers are the bioplastics and oligomers produced from the fermentation of renewable plant biomass. Bioplastics are intracellularly accumulated by microorganisms as carbon and energy reserves. The bioplastics, however, can also be produced through a biochemistry process that combines fermentative secretory production of monomers and/or oligomers and chemical synthesis to generate a repertoire of biopolymers. PHAs are particularly biodegradable and biocompatible, making them a part of today’s commercial polymer industry. Their physicochemical properties that are similar to those of petrochemical-based plastics render them potential renewable plastic replacements. The design of efficient tractable processes using renewable biomass holds key to enhance their usage and adoption. In 2008, a lactate-polymerizing enzyme was developed to create new category of polyester, lactic acid (LA)–based polymer and related polymers. This review aims to introduce different strategies including metabolic and enzyme engineering to produce LA-based biopolymers and related oligomers that can act as precursors for catalytic synthesis of polylactic acid. As the cost of PHA production is prohibitive, the review emphasizes attempts to use the inexpensive plant biomass as substrates for LA-based polymer and oligomer production. Future prospects and challenges in LA-based polymer and oligomer production are also highlighted.Frontiers Media S.A., Feb. 2021, Frontiers in Bioengineering and Biotechnology, 8(618077) (618077), EnglishScientific journal
- Poly((R)-3-hydroxybutyrate) (P(3HB)) is a polyester that is synthesized and accumulated in many prokaryotic cells. Recently, a new culture method for the secretion of the intracellularly synthesized (R)-3-hydroxybutyrate oligomer (3HBO) from recombinant Escherichia coli cells was developed. In this study, we attempted to produce microbial 3HBO capped with a diethylene glycol terminal (3HBO-DEG) as a macromonomer for polymeric materials. First, we prepared recombinant E. coli strains harboring genes encoding various polyhydroxyalkanoate (PHA) synthases (PhaC, PhaEC or PhaRC) that can incorporate chain transfer (CT) agents such as DEG into the polymer's terminal and generate CT end-capped oligomers. To this end, each strain was cultivated under DEG supplemental conditions, and the synthesis of 3HBO-DEG was confirmed. As a result, the highest secretory production of 3HBO-DEG was observed for the PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4). To evaluate the usability of the secreted 3HBO-DEG as a macromonomer, 3HBO-DEG was purified from the culture medium and polymerized with 4,4'-diphenylmethane diisocyanate as a spacer compound. Characterization of the polymeric products revealed that 3HBO-based polyurethane was successfully obtained and was a flexible and transparent noncrystalline polymer, unlike P(3HB). These results suggested that microbial 3HBO-DEG is a promising platform building block for synthesizing polyurethane and various other polymers.Jan. 2021, International Journal of Biological Macromolecules, 167(15) (15), 1290 - 1296, English, International magazineScientific journal
- Designing sustainable biobased and/or biodegradable plastics opens up opportunities to achieve a low-carbon society and overcome plastic pollution. Bioplastics manufactured from renewable resources are being designed to feature a minimal carbon footprint and complete biodegradability/compostability. Among them, naturally occurring polyhydroxyalkanoates (PHAs) have currently received increasing attention from academia and industry. A symbolic state-of-the-art PHA industry is a rapidly growing market of PHBH(TM)Kaneka polymers that display excellent marine biodegradability. From an academic perspective, there have been several major breakthroughs in the PHA research field starting with the pioneering works of genetically engineered platforms for the production of artificial PHAs. The discovery of a lactate-polymerizing enzyme enabled us to produce lactate-based PHAs in one-pot microbial systems, whereas polylactide and other relevant copolymers are currently synthesized via biological and chemical processes. This proof-of-concept has been implemented in practical integrated bioprocesses for carbon-neutral polymer production starting from renewable raw bioresources. Challengingly, the photosynthetic machinery RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), has also been applied to synthesize glycolate-based copolymers as a CO(2)fixation model in the current project. Such game-changing technologies contribute to realizing a circular bioeconomy through the utilization of CO2. This review presents the current progress in evolving microbial polymerization systems, including the direct secretion of polymerized products and the creation of sequence-regulated polyesters, which have been considered nearly impossible biological events to date.Polyhydroxyalkanoates (PHAs) are biobased and biodegradable materials. The artificial PHAs, such as lactate-based polymers, synthesized by engineered platforms expand the range of physical properties. The artificial polymers with superior properties are produced mainly from CO2-derived biomass using microbial platform with engineered enzymes. The oligomers can be secreted from cells and derivatized into high-molecular-weight polymers through assembling with other segments. The review summaries recent advances in the biosynthesis and biodegradation of artificial PHAs and oligomers.SPRINGERNATURE, Oct. 2020, Polymer Journal, 53(1) (1), 67 - 79, EnglishScientific journal
- Aug. 2020, AMB Express, 10(155) (155), EnglishScientific journal
- The addition effects of polymeric materials on natural environments have been mainly evaluated in terms of the relationships between culturable polymer-degrading microorganisms and/or their secreted enzymes and target materials. In this study, we applied metagenome analysis to better understand the change in unculturable microorganismal biodiversity in the four different river samples by using poly (3-hydroxybutyrate) [P(3HB)], a well-studied biodegradable polymer. An inverse relationship between the number of microorganisms and the weight of P(3HB) was observed for all the river samples, while no changes were observed for nondegradable polyethylene. Based on statistical analysis (Chao1 index), the microbial consortia exhibited reduced diversity and tended to converge to similar microbial communities among three of the river samples. This suggests a tight relationship between the biodegradation of P(3HB) and bacterial consortium structure in the river environments. Interestingly, metagenomic sequences corresponding to bacteria that have already been identified as culturable P(3HB) degraders were detected, suggesting a close relationship between the unculturable community and the culturable community. The metagenomic analysis has provided us useful insights into the dynamic changes in the microbial community in the river samples and is applicable for various combinations of environments and biopolymers.Jun. 2020, Polymer Degradation and Stability, 176(6) (6), EnglishScientific journal
- 2020, Polymer Degradation and StabilityIsolation of poly[D-lactate (LA)-co-3-hydroxybutyrate)]-degrading bacteria from soil and characterization of D-LA homo-oligomer degradation by the isolated strain.[Refereed]
- Nov. 2019, Biotechnol. J., x, English[Refereed]Scientific journal
- Poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] is produced in engineered Escherichia coli harboring the genes encoding an LA-polymerizing enzyme (LPE) and monomer-supplying enzymes. In this study, high cell-density fed-batch jar fermentation was developed using xylose and/or glucose as the carbon source. Fed-batch fermentation was initially performed with 20 g/L sugar during the batch phase for 24 h, and subsequent sugar feeding from 24 to 86 h. The feeding rate was increased in a stepwise manner. When xylose alone was used for cultivation, the cells produced the polymer at 11.6 g/L, which was higher than the 4.3 g/L obtained using glucose as the sole carbon source. However, in the first 24 h the growth in the glucose culture was greater than in the xylose culture. Based on these results, glucose was used for cell growth (at the initial stage) and xylose was used for polymer production (at the feeding stage). As expected, in the glucose/xylose switching fermentation method, polymer production was significantly enhanced, eventually reaching 26.7 g/L. The enhanced polymer production obtained by using xylose was presumably due to overflow metabolism. In fact, during xylose feeding, acetic acid excretion was greater than that in case of the glucose grown culture, suggesting the channeling of the metabolic flux from acetyl-CoA towards polymer production over into the tricarboxylic acid cycle in the xylose-fed cultures. Therefore, this sequential glucose/xylose feed strategy is potentially useful for production of acetyl-CoA derived compounds in E. coli.Jun. 2019, Journal of bioscience and bioengineering, 127(6) (6), 721 - 725, English, Domestic magazine[Refereed]Scientific journal
- Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) generates 2-phosphoglycolate (2PG) as one of the metabolites from the Calvin-Benson-Bassham (CBB) cycle. In this study, we focused on the fact that glycolate (GL) derived from 2PG can be incorporated into the bacterial polyhydroxyalkanoate (PHA) as the monomeric constituent by using the evolved PHA synthase (PhaC1(Ps)STQK). In this study, the function of the RuBisCO-mediated pathway for GL-based PHA synthesis was evaluated using Escherichia coli JW2946 with the deletion of glycolate oxidase gene (Delta glcD) as the model system. The genes encoding RuBisCO, phosphoribulokinase and 2PG phosphatase (PGPase) from several photosynthetic bacteria were introduced into E. coli, and the cells were grown on xylose as a sole carbon source. The functional expression of RuBisCO and relevant enzymes was confirmed based on the increases in the intracellular concentrations of RuBP and GL. Next, PHA biosynthetic genes encoding PhaC1(Ps)STQK, propionyl-CoA transferase and 3-hydroxybutyryl(3HB)-CoA-supplying enzymes were introduced. The cells accumulated poly(GL-co-3HB)s with GL fractions of 7.8-15.1 mol%. Among the tested RuBisCOs, Rhodosprium rubrum and Synechococcus elongatus PCC7942 enzymes were effective for P(GL-co-3HB) production as well as higher GL fraction. The heterologous expression of PGPase from Synechocystis sp. PCC6803 and R. rubrum increased GL fraction in the polymer. These results demonstrated that the RuBisCO-mediated pathway is potentially used to produce GL-based PHA in not only E. coli but also in photosynthetic organisms. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Apr. 2019, Journal of bioscience and bioengineering, 127(8) (8), 12 - 18, English[Refereed]Scientific journal
- Novel lactate (LA)-based polymers containing medium-chain-length 3-hydroxyalkanoates (MCL-3HA) were produced in fadR-deficient Escherichia coli strains from glucose as the sole carbon source. The genes encoding LA and 3-hydroxybutyrate (3HB) monomers supplying enzymes [propionyl-CoA transferase (PCT), d-lactate dehydrogenase (D-LDH), β-ketothiolase (PhaA), and NADPH-dependent acetoacetyl-CoA reductase (PhaB)], MCL-3HA monomers supplying enzymes of Pseudomonas sp. 61-3 were introduced into E. coli LS5218. This resulted in the synthesis of a novel LA-based copolymer, P(LA-co-3HB-co-3HA). 1H-nuclear magnetic resonance (NMR) analysis revealed the composition of P(LA-co-3HB-co-3HA) to be 19.7 mol% LA (C3), 74.9 mol% 3HB (C4), and 5.4 mol% MCL-3HA units of C8 and C10. Furthermore, the recombinant E. coli CAG18497 strain carrying these genes, excluding the phaAB genes, accumulated P(92.0% LA-co-3HA) with a novel monomer composition containing C3, C8, C10, and C12. 13C-NMR analysis showed the existence of LA-3HA sequence in the polymer. The solvent cast film of P(92.0% LA-co-3HA) exhibited transparency similar to poly(lactic acid).Feb. 2019, Journal of bioscience and bioengineering, 127(7) (7), 7 - 11, English, Domestic magazine[Refereed]Scientific journal
- Microbiology Research Foundation, 2019, The Journal of General and Applied Microbiology, 65(4) (4), 204 - 208Scientific journal
- For enhancing the lactate (LA) fraction of poly(lactate-co-3-hydroxybutyrate)s [P(LA-co-3HB)s], an exogenous D-lactate dehydrogenase gene (ldhD) was introduced into Escherichia coli. Recombinant strains of E. coli DH5α, LS5218, and XL1-Blue harboring the ldhD gene from Lactobacillus acetotolerans HT, together with polyhydroxyalkanoate (PHA)-biosynthetic genes containing a lactate-polymerizing enzyme (modified PHA synthase) gene, accumulated the P(LA-co-3HB) copolymer from glucose under microaerobic conditions (100 strokes/min). The LA fraction of copolymers synthesized in the strains of DH5α, LS5218, and XL1-Blue were 19.8, 15.7, and 28.5 mol%, respectively, which were higher than those of the strains without the ldhD gene (<6.7 mol% of LA units). Introduction of the exogenous ldhD gene into E. coli strains resulted in an enhanced LA fraction in P(LA-co-3HB)s.Jan. 2019, The Journal of general and applied microbiology, 65(2) (2), 1 - 5, English, Domestic magazine[Refereed]
- Nov. 2018, Acta crystallographica. Section F, Structural biology communications, 74(Pt 11) (Pt 11), 733 - 740[Refereed]Scientific journal
- In our previous study, artificial polyhydroxyalkanoate (PHA) poly was successfully biosynthesized from racemic 2HB in recombinant Escherichia coli using an engineered PHA synthase, PhaC1Ps(S325T/Q481K). Although P(2HB) has promising material properties, the low level of polymer production was a drawback. In this study, we performed directed evolution of PhaC1Ps towards enhanced P(2HB) accumulation in E. coli by site-directed dual saturation mutagenesis at the positions 477 and 481, which was known for their potential in enhancing natural PHA accumulation. By using a screening on agar plates with Nile red, eight colonies were isolated which produced a greater amount of P(2HB) compared to a colony expressing the parent enzyme PhaC1Ps(S325T/Q481K). Among them, the cells expressing PhaC1Ps(S325T/S477R/Q481G) [ST/SR/QG] accumulated polymer at the highest level (up to 2.9-fold). As seen in PhaC1Ps(ST/SR/QG), glycine and basic amino acid residues (K or R) were frequently found at the two positions of the select mutated enzymes. The enzymatic activity of PhaC1Ps(ST/SR/QG) toward 2HB-CoA was approximately 3-fold higher than that of the parent enzyme. Additionally, expression levels of the select mutated enzymes were lower than the parent. These results indicated that PhaC1Ps mutagenesis at the positions 477 and 481 increased specific activity toward 2HB-CoA and it could result in the enhanced production of P(2HB).Elsevier B.V., Jun. 2018, Journal of Bioscience and Bioengineering, 125(6) (6), 632 - 636, English[Refereed]Scientific journal
- Engineered D-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaCl(ps)STQK) efficiently produces poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB]) copolymer in recombinant Escherichia coli, while synthesizing tiny amounts of poly(lactate) (PLA)-like polymers in recombinant Corynebacterium glutamicum. To elucidate the mechanisms underlying the interesting phenomena, in vitro analysis of PhaCl(ps)STQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaCl(ps)STQK polymerized LA-CoA as a sole substrate. However, the extension of PLA chains completely stalled at a molecular weight of 3000, presumably due to the low mobility of the generated polymer. The copolymerization of these substrates only proceeded with a low concentration of LA-CoA. In fact, the intracellular LA-CoA concentration in P(LA-co-3HB)-producing E. coli was below the detection limit, while that in C. glutamicum was as high as acetyl-CoA levels. Therefore, it was concluded that the mobility of polymerized products and LA-CoA concentration are dominant factors characterizing PLA and P(LA-co-3HB) biosynthetic systems.AMER CHEMICAL SOC, May 2018, Biomacromolecules, 19(7) (7), 2889 - 2895, English[Refereed]Scientific journal
- Lignocellulose-utilizing biorefinery is a promising strategy for the sustainable production of value-added products such as bio-based polymers. Simultaneous consumption of glucose and xylose in Escherichia coli was achieved by overexpression of the gene encoding Mlc, a multiple regulator of glucose and xylose uptake. This catabolite derepression gave the enhancement in the production of poly (15 mol% lactate-co-3-hydroxybutyrate), up to 65% from 50% (wild-type strain) in the cellular contents, of the Mlc-overexpressing strain of E. coli on a mixture of glucose and xylose as carbon sources. Microscopic analysis indicated that the Mlc-overexpressing strain showed the enlargement of cell volume in the presence and absence of polymer production, consequently making an expanded volumetric space available for enhanced polymer accumulation. The enhanced polymer production by the catabolite derepression was also reproducible using the biomass, Miscanthus×giganteus (hybrid Miscanthus), which was cultivated in the farm of Hokkaido University.Apr. 2018, Journal of bioscience and bioengineering, 125(4) (4), 365 - 370, English, Domestic magazine[Refereed]Scientific journal
- Biological polymer synthetic systems, which utilize no template molecules, normally synthesize random copolymers. We report an exception, a synthesis of block polyhydroxyalkanoates (PHAs) in an engineered Escherichia coli. Using an engineered PHA synthase, block copolymers poly were produced in E. coli. The covalent linkage between P(2HB) and P(3HB) segments was verified with solvent fractionation and microphase separation. Notably, the block sequence was generated under the simultaneous consumption of two monomer precursors, indicating the existence of a rapid monomer switching mechanism during polymerization. Based on in vivo metabolic intermediate analysis and the relevant in vitro enzymatic activities, we propose a model in which the rapid intracellular 3HB-CoA fluctuation during polymer synthesis is a major factor in generating block sequences. The dynamic change of intracellular monomer levels is a novel regulatory principle of monomer sequences of biopolymers.American Chemical Society, Feb. 2018, Biomacromolecules, 19(2) (2), 662 - 671, English[Refereed]Scientific journal
- Glycolate (GL)-based polyhydroxyalkanoate (PHA), P[GL-co-3-hydroxybutyrate (3HB)], was characterized with respect to its physical properties and hydrolytic degradability. The copolymers were produced from GL and xylose in recombinant Escherichia coli JW1375 (Delta ldhA) expressing an engineered PHA synthase and monomer supplying enzymes. The GL molar ratio in the copolymer was regulated in the range of 0 to 16 mol % dependent on the concentration of GL supplemented in the medium. Unlike P(3HB) homopolymers which are rigid and opaque, the transparency and elasticity of P(GL-co-3HB) films could be tuned dependent on the GL molar ratio. For example, Young's modulus of the films varied in the range of 1620 to 54 MPa. The hydrothermal treatment of P(GL-co-3HB)s resulted in the generation of water-soluble oligomers, and their concentration was positively correlated with the GL molar ratio in the polymer, indicating that the GL units in the polymer chain promoted the hydrolytic degradation of the polymer. The results of this study demonstrate that the GL molar ratio is a potent determinant for regulating the elasticity and hydrolytic degradability of P(GL-co-3HB).AMER CHEMICAL SOC, Dec. 2017, ACS BIOMATERIALS SCIENCE & ENGINEERING, 3(12) (12), 3058 - 3063, English[Refereed]Scientific journal
- D-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of D-LA and D-3-hydroxybutyrate that are produced and secreted by engineered Escherichia coli grown on glucose. The cells heterologously express LA-polymerizing polyhydroxyalkanoate synthase and monomer-supplying enzymes. In this study, we attempted to identify the D-LAO secretion route in E. con, which is thought to be mediated by intrinsic membrane proteins. To this end, a loss-of-function screening of D-LAO secretion was carried out using 209 single-gene membrane protein deletants, which are involved in the transport of organic compounds. Among the deletants of the outer membrane-associated proteins, Delta ompF and Delta ompG exhibited diminished D-LAOs secretion and elevated intracellular D-LAO accumulation. When the ompF and ompG expression levels were down- and up-regulated with plasmids harboring these genes, the secreted amounts of the D-LAOs were changed in correspondence with their expression levels. These results suggest that porins mediate D-LAOs transport through the outer membrane. In particular, OmpF is likely to be the major porin involved in the spontaneous secretion of D-LAOS due to the high basal expression of ompF in the parental strain. Among the deletants of the inner membrane-associated proteins, the Delta mngA, Delta argT, Delta macA, Delta citA and Delta cpxA strains were selected by the screening. These genes are also candidate transporters related to D-LAO secretion, suggesting the presence of multiple secretion routes across the inner membrane. To the best of our knowledge, this is the first report on the mechanism of the microbial secretion of oligoesters. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Dec. 2017, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 124(6) (6), 635 - 640, English[Refereed]Scientific journal
- Identifying the target molecules of antimicrobial agents is essential for assessing their mode of action. Here, we propose Acquired Resistance induced by Gene Overexpression (ARGO) as a novel in vivo approach for exploring target proteins of antimicrobial agents. The principle of the method is based on the fact that overexpression of the expected target protein leads to reduced sensitivity to the antimicrobial agent. We applied this approach to identify target proteins of the antimicrobial peptide apidaecin, which is specifically effective against Gram-negative bacteria. To this end, a set of overexpression Escherichia coli clones was tested, and peptide chain release factor 1, which directs the termination of translation, was found as a candidate, suggesting that apidaecin inhibits the termination step of translation. This finding was confirmed in vivo and in vitro by evaluating the inhibitory activity of apidaecin towards lacZ reporter gene expression, which is tightly dependent on its stop codon. The results of this study demonstrate that apidaecin exerts its antimicrobial effects partly by inhibiting release factors.NATURE PUBLISHING GROUP, Sep. 2017, SCIENTIFIC REPORTS, 7(1) (1), 12136, English[Refereed]Scientific journal
- D-Lactate (LA)-based oligomers (D-LAOs), consisting of D-LA and D-3-hydroxybutyrate (D-3HB), are biobased compounds which are produced and spontaneously secreted by recombinant Escherichia coli. By supplementing the bacterial cultivation with diethylene glycol (DEG), the oligomers featuring hydroxyl groups at both ends of their structures, the D-LAOs-DEG, can be efficiently biosynthesized. In the present work, we attempted to verify the feasibility of D-LAOs-DEG as building blocks to be assembled into LA-based poly(ester-urethane) via polyaddition reactionwith diisocyanate. The polymeric products were demonstrated by SEC and the urethane bound formation in the polymer was determined by FT-IR analysis, indicating that the polymerization was successfully performed. These results suggested that the one-step biosynthesized D-LAOs-DEG are potential substrates for the synthesis of LA-based poly(ester-urethane) and can be further applied to the synthesis of other LA copolymers.SPRINGER, Sep. 2017, JOURNAL OF POLYMER RESEARCH, 24(10) (10), English[Refereed]Scientific journal
- Aug. 2017, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 254Synthetic biology for the lactate-based polymers and oligomers: Intracellular and secretory production[Refereed]Scientific journal
- (公社)日本生物工学会, Aug. 2017, 日本生物工学会大会講演要旨集, 平成29年度, 189 - 189, Japanese昆虫由来抗菌ペプチドの高活性化と作用機序解明のための新アプローチ
- (公社)日本生物工学会, Aug. 2017, 日本生物工学会大会講演要旨集, 平成29年度, 327 - 327, Japanese抗菌ペプチド・アピデシンおよび高活性変異体の作用機構解析
- Recently, we have succeeded in establishing the microbial platform for the secretion of lactate (LA)-based oligomers (D-LAOs), which consist of D-LA and D-3-hydroxybutyrate (D-3HB). The secretory production of D-LAOs was substantially enhanced by the supplementation of diethylene glycol (DEG), which resulted in the generation of DEG-capped oligomers at the carboxyl terminal (referred as D-LAOS-DEG). The microbial D-LAOs should be key compounds for the synthesis of lactide, an important intermediate for polylactides (PLAs) production, eliminating the costly chemo-oligomerization step in the PLA production process. Therefore, in order to demonstrate a proof-of-concept, here, we attempted to convert the D-LAOS-DEG into lactide via metal-catalyzed thermal depolymerization. As a result, D-LAOS-DEG containing 68 mol% LA were successfully converted into lactide, revealing that the DEG bound to D-LAOs-DEG does not inhibit the conversion into lactide. However, the lactide yield (4%) was considerably lower than that of synthetic LA homooligomers (33%). We presumed that 3HB units in the polymer chain blocked the lactide formation, and therefore, we investigated the LA enrichment in the oligomers. As the results, the combination of an LA-overproducing Escherichia coli mutant (Mid and ApflA) with the use of xylose as a carbon source exhibited synergistic effect to increase LA fraction in the oligomers up to 89 mol%. The LA-enriched D-LAOS-DEG were converted into lactide with greater yield (18%). These results demonstrated that a greener shortcut route for PLA production can be created by using the microbial D-LAOS secretion system. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Aug. 2017, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 124(2) (2), 204 - 208, English[Refereed]Scientific journal
- Poly is a biobased polyester with flexible properties efficiently synthesized by engineered Escherichia coli. Here, we aimed at optimizing the monomeric composition of the copolymer in terms of its flexibility, and elucidating structural features contributing to their mechanical properties. The IA fraction was successfully regulated in the range of 6-66 mol% by combination of metabolic and enzyme engineering approaches. The copolymers with higher LA fraction showed decreasing melting point from 160 to 125 degrees C, but increasing glass transition temperature from 7 to 27 degrees C. Crystallinity of the as-cast film, that was mainly attributed to the crystallization of 3HB unit, also decreased with the increasing LA fraction. Owing to the combined effect of these parameters, the highest elongation to break (approximately 400%), which is comparable to that of polyethylene, was obtained for the copolymers with middle range LA fraction (33 mol%). The high flexibility was maintained in most of the copolymers. Notably, P(21mol%LA-co-3HB) retained its high elongation at break (about 300%) even after storage under ambient condition for 5 months. These results demonstrate that introduction of LA units into the polymer chain effectively and stably inhibited the crystallization of 3HB units. (C) 2017 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Jul. 2017, POLYMER, 122, 169 - 173, English[Refereed]Scientific journal
- Engineered Escherichia coli is a useful platform for production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] from renewable sugars. Here we screened all non-lethal transcription factor deletions of E. coli for efficient production of the polymer. This approach aimed at drawing out the latent potential of the host for efficient polymer production via indirect positive effects. Among 252 mutants from Keio Collection tested, eight mutants (Delta pdhR, Delta cspG, Delta yneJ, Delta chbR, Delta yiaU, Delta creB, Delta ygfI and Delta nanK) accumulated greater amount of polymer (6.2-10.1 g/L) compared to the parent strain E. coli BW25113 (5.1 g/L). The mutants increased polymer production per cell (1.1-1.5-fold) without significant change in cell density. The yield of the polymer from glucose was also higher for the selected mutants (0.34-0.38 g/g) than the parent strain (0.27 g/g). Therefore, the deletions of transcription factors should channel the carbon flux towards polymer production. It should be noted that the screening employed in this study identified beneficial mutants without analyzing causal relationship between the mutation and the enhanced polymer production. This approach, therefore, should be applicable to broad range of fermentation productions. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, May 2017, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 123(5) (5), 535 - 539, English[Refereed]Scientific journal
- Developing Escherichia coli strains that are tolerant to acetate toxicity is important in light of an increased interest in the efficient utilization of lignocellulosic biomass feedstocks for the biosynthesis of value-added products. In this study, four strains known to produce polyhydroxyalkanoates (PHAs) from the typical hemicellulosic sugar xylose were tested for their tolerance to acetate. E. coli RSC10 was found to be tolerant of acetate, both in growth and fermentation studies. In the presence of acetate the strain showed a >2-fold increase in overall yields compared to using xylose alone as the feedstock. More importantly, the strain was found to be able to utilize acetate as a feedstock for biosynthesis of PHAs, with complete depletion of acetate (25 mM) at 9 h when acetate was the sole feedstock. Higher concentrations of acetate showed greater inhibition of fermentation than growth with a reduction of 90% in PHA yields at 100 mM. Additionally, the present work provides data to support the potential of acetate as a modulator for the control of composition of PHAs that incorporate lactate (LA) monomers into the copolymer from hemicellulose derived sugars. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, May 2017, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 123(5) (5), 547 - 554, English[Refereed]Scientific journal
- Here, we report the one-step secretory production of D-lactate (LA)-based oligomers (D-LAOS) by engineered Escherichia coli from glucose. Notably, D-LAOs are spontaneously secreted into the medium without the introduction of exogenous exporters. This study was originated from the finding that a small amount of oligo[LA-co-3hydroxybutyrate(3HB)] was secreted by the cells expressing the D-specific LA-polymerizing enzyme (LPE). To increase DLAOs production, we attempted to increase the frequency of chain transfer (CT) reaction of LPE using CT agents, which bear alcoholic moiety. As the result, addition of diethylene glycol into the culture medium was found to be particularly effective. The highest yield of D-LAOs production of 8.3 +/- 1.5 g L-1 from 20 g L-1 glucose was 57% of the theoretical maximum. Furthermore, we demonstrated the covalent conjugation of diethylene glycol at the carboxyl terminal of D-LAOs by NMR analyses. The secreted D-LAOs have the potential to be used as precursors of lactide, enabling the establishment of a greener shortcut route in the process of polylactides (PLAs) production.AMER CHEMICAL SOC, Mar. 2017, ACS SUSTAINABLE CHEMISTRY & ENGINEERING, 5(3) (3), 2360 - 2367, English[Refereed]Scientific journal
- Polyhydroxyalkanoate (PHA) is a thermoplastic polymer with several advantageous properties, including biomass origin, biocompatibility, and biodegradability. PHA is synthesized in transgenic plants harboring 3 enzymatic genes: phaA, phaB, and phaC (collectively referred to as phaABC). PHA-producing plants exhibit severe growth inhibition that leads to extremely low PHA accumulation when these enzymes are localized in the cytosol. This growth inhibition could be attributed to the deleterious effects of the PHA biosynthetic pathway on endogenous essential metabolites or to PHA cytotoxicity itself. We performed precise morphological observations of phaABC-overexpressing Arabidopsis (ABC-ox), which displayed typical growth inhibition. On growth medium without sucrose, ABC-ox exhibited a pale green phenotype, dwarfism, including small cotyledons and true leaves, and short roots. ABC-ox partially recovered from this growth inhibition when the growth medium was supplemented with 1% sucrose. This recovery was reversed after ABC-ox grown on 1% sucrose medium was transferred to soil. ABC-ox grown on 1% sucrose medium not only demonstrated recovery from growth inhibition but were also the only examined plants with PHA accumulation, suggesting that growth inhibition was not caused by PHA cytotoxicity but rather by a lack of essential metabolites.JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, Mar. 2017, PLANT BIOTECHNOLOGY, 34(1) (1), 39 - 43, English[Refereed]
- Establishment of the regeneratable whole-cell catalyst platform for the production of biobased polymeric materials is a typical topic of synthetic biology. In this commentary, discovery story of a "lactate-polymerizing enzyme" (LPE) and LPE-based achievements for creating a new variety of polyesters with incorporated unnatural monomers are presented. Besides the importance of microbial platform itself is discussed referring to the "ballooning"-Escherichia coli.SPRINGER, Mar. 2017, FRONTIERS OF CHEMICAL SCIENCE AND ENGINEERING, 11(1) (1), 139 - 142, English[Refereed]Scientific journal
- Woody extract-derived hemicellulosic hydrolysate, which was obtained from dissolving pulp manufacturing, was utilized as feedstock for the production of poly(lactate-co-3-hydroxybutyrate) P(LA-co-3HB)] in engineered Escherichia coil. The hydrolysate was composed of mainly xylose and galactose, and contained impurities mainly acetate, which was found to inhibit the polymer synthesis rather than the cell growth. Thus, acetate and other impurities were removed through active charcoal and ion-exchange columns. Using the purified hydrolysate, P(LA-co-3HB) was successfully produced (cell dry weight 8.6 g/L, polymer concentration 5.4 g/L, LA fraction 5.5 mol%, polymer content 62.4%), the amount of which was comparable to that obtained using reagent grade xylose and galactose. Therefore, the hydrolysate from woody extract is considered as an abundant, inexpensive and efficient feedstock applicable to consolidated process for P(LA-co-3HB) production, when the removal of acetic acid was satisfactorily accomplished. (C)2016 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Mar. 2017, PROCESS BIOCHEMISTRY, 54(2) (2), 102 - 105, English[Refereed]Scientific journal
- Thermostable enzymes are required for the rapid and sustainable production of polyhydroxyalkanoate (PHA) in vitro. The in vitro synthesis of PHA using the engineered thermostable synthase PhaC1(SG)(STQK) has been reported; however, the non-thermostable enzymes acetyl-CoA synthetase (ACS) and CoA transferase (CT) from mesophilic strains were used as monomer-supplying enzymes in this system. In the present study, acs and ct were cloned from the thermophilic bacteria Pelotomaculum thermopropionicum JCM10971 and Thermus thermophilus JCM10941 to construct an in vitro PHA synthesis system using only thermostable enzymes. ACS from P. thermopropionicum (ACS(Pt)) and CT from I thermophilus (CTTt) were confirmed to have high thermostability, and their optimal temperatures were around 60 degrees C and 75 degrees C, respectively. The in vitro PHA synthesis was successfully performed by ACS(Pt), CTTt, PhaC1(SG)(STQK), and poly(3-hydroxybutyrate) [P(3HB)] was synthesized at 45 degrees C. Furthermore, the yields of P(3HB) and P(lactate-co-3HB) at 37 degrees C were 1.4-fold higher than those of the in vitro synthesis system with non-thermostable ACS and CT from mesophilic strains. Overall, the thermostable ACS and CT were demonstrated to be useful for the efficient in vitro PHA synthesis at relatively high temperatures. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Dec. 2016, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 122(6) (6), 660 - 665, English[Refereed]Scientific journal
- Consolidated bioprocessing of lignocellulose is an attractive strategy for the sustainable production of petroleum based alternatives. One of the underutilized sources of carbon in lignocellulose is the hemicellulosic fraction which largely consists of the polysaccharide xylan. In this study, Escherichia coli JW0885 (pyruvate formate lyase activator protein mutant, pflA(-)) was engineered to express recombinant xylanases and polyhydroxyalkanoate (PHA)-producing enzymes for the biosynthesis of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] from xylan as a consolidated bioprocess. The results show that E. coli JW0885 was capable of producing P(LA-co-3HB) when xylan was the only feedstock and different feeding and growth parameters were examined in order to improve upon initial yields. The highest yields of P(LA-co-3HB) copolymer obtained in this study occurred when xylan was added during mid-exponential growth after cells had been grown at high shaking speeds (290 rpm). The results showed an inverse relationship between total PHA production and LA-monomer incorporation into the copolymer. Proton nuclear magnetic resonance (H-1 NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analyses corroborate that the polymers produced maintain physical properties characteristic of LA-incorporating PHB-based copolymers. The present study achieves the first ever engineering of a consolidated bioprocessing bacterial system for the production of a bioplastic from a hemicelluosic feedstock. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Oct. 2016, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 122(4) (4), 406 - 414, English[Refereed]Scientific journal
- Poly[(R)-lactate-co-(R)-2-hydroxybutyrate], which is biosynthesized as a random co-polyester by microbial organisms, with various ratios of lactic acid (LA) to hydroxybutyrate monomers has been isothermally crystallized to investigate its thermal properties and crystal structure changes. Differential scanning calorimetry on the isothermally crystallized samples detected endothermic peaks due to the melting of the crystals at all LA unit ratios. Melting temperatures of the copolymers increased from ca. 107 degrees C-146 degrees C as the LA unit ratio increased. Glass transition temperatures also increased from 24 degrees C to 44 degrees C with increasing LA unit ratio. A melting temperature was observed even at LA unit ratios around 50%. Wide-angle X-ray measurements also revealed that co-crystallization occurred at all samples, including at LA unit ratios of ca. 50%. Despite of "jumping" phenomena many co-crystallizable random copolymers having, the lattice constants changed "linearly" from resembling poly[(R)-lactate] to poly [(R)-2-hydroxybutyrate] depending on the LA unit ratio. This unique co-crystallization behavior is discussed in detail. (C) 2016 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Oct. 2016, POLYMER DEGRADATION AND STABILITY, 132(4) (4), 137 - 144, English[Refereed]Scientific journal
- P was produced in engineered Escherichia coli using lignocellulose-derived hydrolysates from Miscanthusxgiganteus (hybrid Miscanthus) and rice straw. Hybrid Miscanthus-derived hydrolysate exhibited no negative effect on polymer production, LA fraction, and molecular weight of the polymer, whereas rice straw-derived hydrolysate reduced LA fraction. These results revealed that P(LA-co-3HB) was successfully produced from hybrid Miscanthus-derived sugars.TAYLOR & FRANCIS LTD, Apr. 2016, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 80(4) (4), 818 - 820, English[Refereed]Scientific journal
- Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZ(Vs)) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(d-LA-co-d-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZ(Vs) for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZ(Af)) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZ(Vs) generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZ(Vs) cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZ(Vs) but also PhaZ(Af) hydrolyzed all of these substrates, namely PhaZ(Af) also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P. The mutated PhaB increased P(3HB) content by three-fold over the control, indicating that the mutant was a versatile tool for P(3HB) production. Additionally, the PhaB-catalyzed reaction was suggested to be a rate-limiting step of P(3HB) biosynthesis in tobacco BY-2 cells.TAYLOR & FRANCIS LTD, Jun. 2015, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 79(6) (6), 986 - 988, English[Refereed]Scientific journal
- Bacterial polyester polyhydroxyalkanoates (PHAs) have been produced in engineered Escherichia coli, which turned into an efficient and versatile platform by applying metabolic and enzyme engineering approaches. The present study aimed at drawing out the latent potential of this organism using genome-wide mutagenesis. To meet this goal, a transposon-based mutagenesis was carried out on E. coli, which was transformed to produce poly (lactate-co-3-hydroxybutyrate) from glucose. A high-throughput screening of polymer-accumulating cells on Nile red-containing plates isolated one mutant that produced 1.8-fold higher quantity of polymer without severe disadvantages in the cell growth and monomer composition of the polymer. The transposon was inserted into the locus within the gene encoding MtgA that takes part, as a non-lethal component, in the formation of the peptidoglycan backbone. Accordingly, the mtgA-deleted strain E. coli JW3175, which was a derivate of superior PHA-producing strain BW25113, was examined for polymer production, and exhibited an enhanced accumulation of the polymer (7.0 g/l) compared to the control (5.2 g/l). Interestingly, an enlargement in cell width associated with polymer accumulation was observed in this strain, resulting in a 1.6-fold greater polymer accumulation per cell compared to the control. This result suggests that the increase in volumetric capacity for accumulating intracellular material contributed to the enhanced polymer production. The mtgA deletion should be combined with conventional engineering approaches, and thus, is a promising strategy for improved production of intracellularly accumulated biopolymers.PUBLIC LIBRARY SCIENCE, Jun. 2015, PLOS ONE, 10(6) (6), e0125163 - e0125163, English[Refereed]Scientific journal
- A new approach at the transcriptional level was applied to lactate-based polyester production. Four sigma factor disruptants, Delta rpoN, Delta rpoS, Delta fliA and Delta fecl, of Escherichia con were used as hosts for poly(lactate-co-3-hydroxybutyrate) production from glucose. Among them, Delta rpoN caused dual positive effects of polymer production, enhanced cellular content and lactate fraction. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Apr. 2015, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 119(4) (4), 427 - 429, English[Refereed]Scientific journal
- The tight crude oil supply and its negative impacts on the environment have accelerated efforts towards the search for alternatives to the conventional petrochemical-derived plastics. Poly lactic acid (PLA), a polymer produced from renewable resources has emerged as a potential substitute to the conventional synthetic petrochemical-derived plastics owing to their biodegradable and bioresorbable nature. Despite these advantages, PLA has had little success as a substitute to the conventional petrochemical-derived plastics because the PLA synthesis; a two-step bio-chemo process where fermentative lactic acid is polymerized using heavy metal catalysts is expensive. Furthermore, the heavy metal catalyst remnants in the polymer hinder the use of PLA for medical devices and food handling packages. To overcome these obstacles, bacteria have been genetically manipulated to synthesize lactate (LA)-based polymers in a single-step metal-free system. This chapter reviews the production of LA-based polymers using a lactate-polymerizing enzyme (LPE). Production of LA-enriched and PLA-like polymers in bacteria from glucose and xylose, metabolic/ genetic engineering for polymer yield enhancement, polymer properties, and the biodegradability of the LA-based polymers will be discussed. Moreover, future perspectives on the synthesis and applications of the LA-based polymers will be mentioned.AMER CHEMICAL SOC, 2015, GREEN POLYMER CHEMISTRY: BIOBASED MATERIALS AND BIOCATALYSIS, 1192, 113 - 131, English[Refereed]International conference proceedings
- Poly is a biobased polyester with semitransparent and flexible properties produced in engineered bacteria carrying an LA-polymerizing enzyme. In this study, we attempted to isolate the P(enriched LA-co-3HB)-degrading bacteria from soil samples in order to identify enzymes with the capacity to degrade this new type of polymer. Among approximately 500 samples, the Gram-negative bacterium 04, which exhibited potent P(enriched LA-co-3HB)-degrading activity, was isolated based on the decrease in the turbidity of culture medium supplemented with emulsified P(67 mol% LA-co-3HB). Based on its 16S rDNA sequence, this isolated bacterium was identified as a member of Variovorax sp.. Next, we attempted to isolate and purify the depolymerase that contributes to the polymer degradation from the culture supernatant of strain C34. The purified enzyme had a molecular mass of 42 kDa and exhibited degradation activity towards the P(67 mol% LA-co-3HB) as well as 3HB homopolymer [P(3HB)], but not LA homopolymers (PDLA and PLLA). On the other hand, a well-characterized depolymerase of P(3HB) derived from Alcaligenes faecalis T1 did not degrade P(67 mol% LA-co-3HB), PDLA or PLLA. This result suggests that the newly isolated depolymerase differs from the P(3HB) depolymerase from A. faecalis. (C) 2014 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Dec. 2014, POLYMER DEGRADATION AND STABILITY, 110, 44 - 49, English[Refereed]Scientific journal
- The biosynthesis of poly(lactic acid) (PLA)-like polymers, composed of >99 mol% lactate and a trace amount of 3-hydroxybutyrate, in engineered Corynebacterium glutamicum consists of two steps; the generation of the monomer substrate lactyl-coenzyme A (CoA) and the polyhydroxyalkanoate (PHA) synthase-catalyzed polymerization of lactyl-CoA. In order to increase polymer productivity, we explored the rate-limiting step in PLA-like polymer synthesis based on quantitative metabolite analysis using liquid chromatography mass spectroscopy (LC-MS). A significant pool of lactyl-CoA was found during polymer synthesis. This result suggested that the rate-limitation occurred at the polymerization step. Accordingly, the expression level of PHA synthase was increased by means of codon-optimization of the corresponding gene that consequently led to an increase in polymer content by 4.4-fold compared to the control. Notably, the codon-optimization did not significantly affect the concentration of lactyl-CoA, suggesting that the polymerization reaction was still the rate-limiting step upon the overexpression of PHA synthase. Another important finding was that the generation of lactyl-CoA was concomitant with a decrease in the acetyl-CoA level, indicating that acetyl-CoA served as a CoA donor for lactyl-CoA synthesis. These results show that obtaining information on the metabolite concentrations is highly useful for improving PLA-like polymer production. This strategy should be applicable to a wide range of PHA-producing systems.BIOMED CENTRAL LTD, Nov. 2014, AMB EXPRESS, 4(1) (1), 83, English[Refereed]Scientific journal
- Sep. 2014, Bioengineered, 5(5) (5), 284 - 287, English[Refereed]Scientific journal
- Engineering of microorganisms to directly utilize plant biomass as a feedstock for the biosynthesis of value-added products such as bioplastics is the aim of consolidated bioprocessing. In previous research we successfully engineered E. coli LS5218 to produce polyhydroxyalkanoates (PHAs) from xylan. In this study we report further genetic modifications to Escherichia coli LS5218 in order to increase the lactic acid (LA) fraction in poly(lactic acid-co-3-hydroxyalkanoate) P(LA-co-HA) copolymers. Deletion of the pflA gene resulted in increased content of LA repeating units in the copolymers by over 3-fold compared with the wild type; however, this increase was offset by reduced yields in cell mass. Additionally, when acetate was used as a feedstock LA monomer incorporation reached 18.5 (mol%), which suggests that acetate can be used as a feedstock for the production of P(LA-co-HA) copolymers by E. coli.LANDES BIOSCIENCE, Sep. 2014, BIOENGINEERED, 5(5) (5), 284 - 287, English[Refereed]Scientific journal
- AMER CHEMICAL SOC, Aug. 2014, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 248, EnglishBiosynthesis, properties, and enzymatic degradation of the 2-hydroxyalkanoate-based polyesters[Refereed]Scientific journal
- SPRINGER, Sep. 2013, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97(18) (18), 8011 - 8021, English[Refereed]Scientific journalP is an aliphatic polyester analogous to poly(lactic acid) (PLA). However, little has been known for its properties because of a high cost of commercially available chiral 2HB as a starting substance for chemical polymer synthesis. In this study, P[(R)-2HB] and P with a new monomer combination were successfully synthesized in recombinant Escherichia coli LS5218 from less-expensive racemic 2HB using an R-specific polyester synthase. The cells expressing an engineered polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 and propionyl-CoA transferase from Megasphaera elsdenii were grown on LB medium containing 2HB and glucose in a shake flask and accumulated up to 17 wt% of P[(R)-2HB] with optical purity of >99.1%. In addition, the same cells cultured in a jar-fermentor produced P(86 mol% 2HB-co-LA) copolymer. Notably, the molecular weights (M-w) of P(2HB) (27000) and P(2HB-co-LA) (39000) were 2- and 3-fold higher than that of P(2HB) previously synthesized by chemical polycondensation. P(2HB) was processed into a transparent film by solvent-casting and it had flexible properties with elongation at break of 173%, which was contrast to the rigid PLA. Regarding mechanical properties, P(2HB-co-LA) was tougher but less stretchy than P(2HB). These results demonstrated that P(2HB) has useful properties and LA units in 2HB-based polymers can act as a controllable modulator of the material properties. In addition, P[(R)-2HB] was efficiently degraded by treatment of Novozym 42044 (lipase) but not Savinase 16L (protease), indicating that the degrading behavior of the polymer was similar to that of P backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaCRe(A510X) mutants, 15 synthesized P (LA-co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaCReA510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The 13C and 1H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA-co-3HB) copolymer with a weight-averaged molecular weight of 3.2 × 105 Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA×-LA triad sequence, suggesting that the polymer synthesized by PhaCRe mutant may not be a random copolymer, but presumably had a block sequence. © 2012 Springer-Verlag.Apr. 2013, Applied Microbiology and Biotechnology, 97(8) (8), 3441 - 3447, English[Refereed]Scientific journalThe dwindling nature of petroleum resources and increased emission of greenhouse gases into the atmosphere have accelerated efforts towards the finding of alternatives to the extensively used petroleum-derived plastics, with a 'green agenda'. Poly lactic acids (PLAs), which are produced from renewable biomass, have gained enormous attention as replacements for the conventional synthetic petroleum-derived plastics due to their biodegradability and bioresorbability. However, the current system of PLA synthesis involves a two-step bio-chemo process, where fermentative lactic acid is polymerized using heavy metal catalysts. The remnants of metal catalysts hinder the PLA application for medical devices and food handling packages. To circumvent these challenges, bacteria have been engineered to produce lactate (LA)-based polyesters in a single-step metal-free system, which is the focus of this review. First, the discovery of a lactate-polymerizing enzyme (LPE) that facilitated the creation of a microbial plastic factory (MPF) for the synthesis of LA-based polyesters will be discussed in detail. Then, approaches for the enrichment of LA fraction in LA-based polyesters including the change of culture conditions, the use of metabolically engineered bacteria and further evolution of LPE are described. Furthermore, the expansion of monomers that could be copolymerized with LA, the properties of LA-based polyesters, the transfer of the LA-based production system into other bacteria resulting into the synthesis of PLA-like polyesters and the engineering of new LPEs, will be highlighted. Finally, the future perspectives of the MPF for the synthesis of LA-based polyesters are discussed. © 2013 American Chemical Society.American Chemical Society, 2013, ACS Symposium Series, 1144, 175 - 197, English[Refereed]International conference proceedingsJan. 2013, Applied Microbiology and Biotechnology, 97(1) (1), 439, English[Refereed]Scientific journal(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by beta-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L-1 (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L-1, with a productivity of 0.22 g L-1 h(-1). The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of C-13 carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.SPRINGER, Jan. 2013, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97(1) (1), 205 - 210, English[Refereed]Scientific journal(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by beta-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L-1 (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L-1, with a productivity of 0.22 g L-1 h(-1). The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of C-13 carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.SPRINGER, Jan. 2013, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97(1) (1), 205 - 210, English[Refereed]Scientific journalThis study describes the biosynthesis of polyhydroxyalkanoate (PHA) containing 5-hydroxyvalerate (5HV) units. Monomer units like 5HV, with variation in length of the backbone segment of the repeat unit, are known to more greatly impact mechanical properties of polymers. Introduction of 5HV units was also aimed at enhancing biodegradability of the material, as it had been found previously that a homopolymer of 5HV could be degraded by lipase. The produced poly[(R)-3-hydroxybutyrate-co-3-hydroxypropionate-co-5-hydroxyvalerate]s [P(3HB-co-3HP-co-5HV)s] with 5HV content ranging from 1 to 32 mol% by the choice of strain, type and concentration of carbon substrate, exhibited low melting temperature (Tm), reduced crystallinity and elastomeric behavior. Enzymatic degradation test confirmed the role of 5HV unit in enabling lipase-mediated degradation of P(3HB-co-3HP-co-5HV)s, as poly[(R)-3- hydroxybutyrate] and poly, having transparent and flexible properties. The recombinant Escherichia coli JW0885 (pflA(-)) expressing LA-polymerizing enzyme (LPE) and monomer supplying enzymes grown on xylose produced a copolymer having a higher LA fraction (34 mol%) than that grown on glucose (26 mol%). This benefit of xylose was further enhanced by combining it with an evolved LPE (ST/FS/QK), achieving a copolymer production having 60 mol% LA from xylose, while glucose gave a 47 mol% LA under the same condition. The overall carbon yields from the sugars to the polymer were similar for xylose and glucose, but the ratio of the LA and 3HB units in the copolymer was different. Notably, the P(LA-co-3HB) yield from xylose (7.3 g l(-1)) was remarkably higher than that of P(3HB) (4.1 g l(-1)), indicating P(LA-co-3HB) as a potent target for xylose utilization. (C) 2012 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Jan. 2013, METABOLIC ENGINEERING, 15, 159 - 166, English[Refereed]Scientific journalNADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along withβ-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutrophawas engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu(Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, thePhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an importantrole in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex withNADPHand acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity. © 2013, American Society for Microbiology. All Rights Reserved.2013, Applied and Environmental Microbiology, 79(19) (19), 6134 - 6139, English[Refereed]Scientific journalAMER CHEMICAL SOC, Aug. 2012, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 244, EnglishMicrobial plastic factory: Synthesis and properties of the new lactate-based and related biopolymers[Refereed]Scientific journalAMER CHEMICAL SOC, Aug. 2012, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 244, EnglishBiosynthesis and properties of advanced biopolymers containing lactate by metabolically engineered bacteria[Refereed]Scientific journal日本生物工学会, Jul. 2012, 生物工学会誌 : seibutsu-kogaku kaishi, 90(7) (7), 411 - 414, JapaneseMicrobial Plastic Factory : ポリ乳酸から多元ポリ乳酸の時代へRecently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 A degrees C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1(SG) and PhaC2(SG)). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1(SG) to evaluate the potential of the resulting protein as a "thermostable LPE". The mutated PhaC1(SG) [PhaC1(SG)(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1(SG)(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).SPRINGER, Apr. 2012, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 94(2) (2), 365 - 376, English[Refereed]Scientific journalRecently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 A degrees C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1(SG) and PhaC2(SG)). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1(SG) to evaluate the potential of the resulting protein as a "thermostable LPE". The mutated PhaC1(SG) [PhaC1(SG)(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1(SG)(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).SPRINGER, Apr. 2012, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 94(2) (2), 365 - 376, English[Refereed]Scientific journalThe first biosynthetic system for lactate (LA)-based polyesters was previously created in recombinant Escherichia coli (Taguchi et al. 2008). Here, we have begun efforts to upgrade the prototype polymer production system to a practical stage by using metabolically engineered Gram-positive bacterium Corynebacterium glutamicum as an endotoxin-free platform. We designed metabolic pathways in C. glutamicum to generate monomer substrates, lactyl-CoA (LA-CoA), and 3-hydroxybutyryl-CoA (3HB-CoA), for the copolymerization catalyzed by the LA-polymerizing enzyme (LPE). LA-CoA was synthesized by D-lactate dehydrogenase and propionyl-CoA transferase, while 3HB-CoA was supplied by beta-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB). The functional expression of these enzymes led to a production of P(LA-co-3HB) with high LA fractions (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.SPRINGER, Mar. 2012, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 93(5) (5), 1917 - 1925, English[Refereed]Scientific journalA rapid and convenient method for the compositional analysis of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC) and alkaline sample pretreatment in a 96-well plate format. The reliability of this system was confirmed by the fact that a mutant with a D171G mutation of Aeromonas caviae PHA synthase (PhaC(Ac)), which gained higher reactivity toward 3-hydroxyhexanoate (3HHx), was selected from the D171X mutant library. Together with D171G mutant, several single mutants showing high reactivity toward 3HHx were isolated by the HPLC assay. These new mutants and double mutants combined with an N149S mutation were used to synthesize P(3-hydroxybutyrate-co-3HHx) in Ralstonia eutropha PHB(-)4 from soybean oil as carbon source, achieving higher levels of 3HHx fraction than the wild-type enzyme. Based on these results, the high-throughput screening system will serve as a powerful tool for exploring new and beneficial mutations responsible for regulating copolymer composition of PHA. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Mar. 2012, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 113(3) (3), 286 - 292, English[Refereed]Scientific journalA rapid and convenient method for the compositional analysis of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC) and alkaline sample pretreatment in a 96-well plate format. The reliability of this system was confirmed by the fact that a mutant with a D171G mutation of Aeromonas caviae PHA synthase (PhaC(Ac)), which gained higher reactivity toward 3-hydroxyhexanoate (3HHx), was selected from the D171X mutant library. Together with D171G mutant, several single mutants showing high reactivity toward 3HHx were isolated by the HPLC assay. These new mutants and double mutants combined with an N149S mutation were used to synthesize P(3-hydroxybutyrate-co-3HHx) in Ralstonia eutropha PHB(-)4 from soybean oil as carbon source, achieving higher levels of 3HHx fraction than the wild-type enzyme. Based on these results, the high-throughput screening system will serve as a powerful tool for exploring new and beneficial mutations responsible for regulating copolymer composition of PHA. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Mar. 2012, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 113(3) (3), 286 - 292, English[Refereed]Scientific journalThe first biosynthetic system for lactate (LA)-based polyesters was previously created in recombinant Escherichia coli (Taguchi et al. 2008). Here, we have begun efforts to upgrade the prototype polymer production system to a practical stage by using metabolically engineered Gram-positive bacterium Corynebacterium glutamicum as an endotoxin-free platform. We designed metabolic pathways in C. glutamicum to generate monomer substrates, lactyl-CoA (LA-CoA), and 3-hydroxybutyryl-CoA (3HB-CoA), for the copolymerization catalyzed by the LA-polymerizing enzyme (LPE). LA-CoA was synthesized by D-lactate dehydrogenase and propionyl-CoA transferase, while 3HB-CoA was supplied by beta-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB). The functional expression of these enzymes led to a production of P(LA-co-3HB) with high LA fractions (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.SPRINGER, Mar. 2012, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 93(5) (5), 1917 - 1925, English[Refereed]Scientific journalAMER CHEMICAL SOC, Mar. 2012, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 243, EnglishDirect conversion of glucose into lactate-based polyesters using engineered Corynebacterium glutamicum as a whole cell catalyst[Refereed]Scientific journalAMER CHEMICAL SOC, Mar. 2012, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 243, EnglishBioconversion of the hemicellulosic sugar, xylose, into lactate-based polyesters using recombinant Escherichia coli[Refereed]Scientific journalThe flavin selectivity of the flavoenzymes is considered to be very strict in terms of the functional expression of the enzyme. However, we found that an FMN-dependent azoreductase from Bacillus sp. B29 exhibited up to 60% of the activity of native AzrA harboring FMN upon the addition of a FAD cofactor. The FAD binding to the apo-form of AzrA was identified by spectrophotometric analysis, and the bound FAD was stably retained in the enzyme molecule without degradation to FMN. On the other hand, no effect of riboflavin on the activity of AzrA was detected and there was no obvious quenching of riboflavin detected with the addition of apoAzrA. By a docking simulation of FAD into the structure of a homolog of AzrA (AzoR from Escherichia coli), we created a FAD-binding model. Taking all of these results together, it is proposed that the isoalloxazine ring of FAD localizes at the same site and plays the same role as that of FMN in AzrA. (C) 2011 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Feb. 2012, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 74(3-4) (3-4), 204 - 208, English[Refereed]Scientific journalBiological Lactate-Polymers Synthesized by One-Pot Microbial Factory: Enzyme and Metabolic EngineeringPoly lactic acid (PLA) is a representative commodity polyester synthesized from renewable resources. The benefits of PLA over the conventional petrochemical-based plastics are; it is biodegradable, it requires less energy to produce and reduces greenhouse gas production. PLA is mainly produced via a two step bio-chemo process where lactate produced by microorganisms is converted into lactides which are then polymerized by metal catalysts to form high molecular weight PLA. In 2008, microbial LA-based polyester production systems were for the first time established as microbial cell factories (MCFs). This paradigm shift is based on a conversion of a two-step chemical process into a single-step MCF. This breakthrough was triggered by the discovery of an engineered LA-polymerizing enzyme (LPE). LPE was discovered among a collection of polyhydroxyalkanoate (PHA) synthase mutants from Pseudomonas sp. 61-3 through a long-term evolutionary engineering study. The MCF has been advanced by combination of further evolution of LPE with metabolic engineering for improved monomer precursor production. This chapter reviews the backgrounds on the establishment of the MCFs for the synthesis of LA-based polyesters to near PLA homopolymer, structures and properties of LPE-catalyzed polymers and challenges of LA-based polymer production system as well as future perspectives on the microbial LA-based polyesters.AMER CHEMICAL SOC, 2012, BIOBASED MONOMERS, POLYMERS, AND MATERIALS, 1105, 213 - 235, English[Refereed]International conference proceedingsPoly[3-hydroxybutyrate-co-3-hydroxyvalerate(3HV)] was produced in recombinant Escherichia coli LS5218 from ruthenium-catalyzed cellulose hydrolysate and propionate. The strain was found to be resistant to 5-hydroxymethylfurfural (5-HMF), which is a major inhibitory byproduct generated in the cellulose hydrolysis reaction. The 3HV fraction was successfully regulated in the range of 5.6-40 mol%. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Jan. 2012, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 113(1) (1), 70 - 72, English[Refereed]Scientific journalGlycolate(GL)-based polyesters were for the first time produced in the recombinant Escherichia coli fatty acid beta-oxidation pathway reinforcing mutant LS5218, using extracellularly added GL as a monomer precursor. Cells expressing a Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase (PhaC1STQK) from Pseudomonas sp. 61-3, propionyl-CoA transferase from Megasphaera elsdenii and enoyl-CoA hydratase from Pseudomonas aeruginosa grown on GL and dodecanoate were found to produce novel copolymers of GL with 3-hydroxyalkanoates (3HAs) (C(4)-C(12)), P(GL-co-3HA), with a weight-average molecular weight of 34,000. The (1)H and (13)C NMR analyses of the copolymer revealed the incorporation of GL units into the polymer chain. This result demonstrates that PhaC1STQK polymerized glycolyl-CoA as a monomer substrate. Additionally, the novel lactate(LA)-based polyester P(LA-co-3HA) was produced by substituting GL with LA, indicating that the method is versatile and allows the production of a variety of biopolymers. (C) 2011 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Dec. 2011, JOURNAL OF BIOTECHNOLOGY, 156(3) (3), 214 - 217, English[Refereed]Scientific journalGlycolate(GL)-based polyesters were for the first time produced in the recombinant Escherichia coli fatty acid beta-oxidation pathway reinforcing mutant LS5218, using extracellularly added GL as a monomer precursor. Cells expressing a Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase (PhaC1STQK) from Pseudomonas sp. 61-3, propionyl-CoA transferase from Megasphaera elsdenii and enoyl-CoA hydratase from Pseudomonas aeruginosa grown on GL and dodecanoate were found to produce novel copolymers of GL with 3-hydroxyalkanoates (3HAs) (C(4)-C(12)), P(GL-co-3HA), with a weight-average molecular weight of 34,000. The (1)H and (13)C NMR analyses of the copolymer revealed the incorporation of GL units into the polymer chain. This result demonstrates that PhaC1STQK polymerized glycolyl-CoA as a monomer substrate. Additionally, the novel lactate(LA)-based polyester P(LA-co-3HA) was produced by substituting GL with LA, indicating that the method is versatile and allows the production of a variety of biopolymers. (C) 2011 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Dec. 2011, JOURNAL OF BIOTECHNOLOGY, 156(3) (3), 214 - 217, English[Refereed]Scientific journalA previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 x 10(5), and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T (g), T (m), and T (d) (10%) were observed at -1.1A degrees C, 158.8A degrees C, and 252.7A degrees C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.SPRINGER, Nov. 2011, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 92(3) (3), 509 - 517, English[Refereed]Scientific journalChemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from Ralstonia eutropha微生物の細胞内で起こるポリエステルの合成反応を詳細に理解するためには,細胞から酵素を単離して試験管内でポリエステルの合成を行うことが有用な手段である。本研究では,試験管内での反応としては初めて,単離精製した酵素を用いて2-ヒドロキシ酪酸の酵素重合に成功した。Nov. 2011, Appl. Microbiol. Biotechnol., 92(3) (3), 509 - 517, English[Refereed]Scientific journalIn order to evaluate the mechanical properties of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] and its correlation with the LA fraction, P(LA-co-3HB)s with a variety of LA fractions were prepared using recombinant Escherichia coli expressing the LA-polymerizing enzyme and monomer supplying enzymes. The LA-overproducing mutant E. coli JW0885 with a pflA gene disruption was used for the LA-enriched polymer production. The LA fraction was also varied by jar-fermentor based fine-regulation of the anaerobic status of the culture conditions, resulting in LA fractions ranging from 4 to 47 mol%. In contrary to the opaque P(3HB) film, the copolymer films attained semitransparency depending on the LA fraction. Young's modulus values of the P(LA-co-3HB) s (from 148 to 905 MPa) were lower than those of poly(lactic acid) (PLA) (1020 MPa) and P(3HB) (1079 MPa). In addition, the value of elongation at break of the copolymer with 29 mol% LA reached 150%. In conclusion, P(LA-co-3HB) s were found to be a comparatively pliable and flexible material, differing from both of the rigid homopolymers. (C) 2011 Elsevier B. V. All rights reserved.ELSEVIER SCIENCE BV, Jul. 2011, JOURNAL OF BIOTECHNOLOGY, 154(4) (4), 255 - 260, English[Refereed]Scientific journalIn order to evaluate the mechanical properties of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] and its correlation with the LA fraction, P(LA-co-3HB)s with a variety of LA fractions were prepared using recombinant Escherichia coli expressing the LA-polymerizing enzyme and monomer supplying enzymes. The LA-overproducing mutant E. coli JW0885 with a pflA gene disruption was used for the LA-enriched polymer production. The LA fraction was also varied by jar-fermentor based fine-regulation of the anaerobic status of the culture conditions, resulting in LA fractions ranging from 4 to 47 mol%. In contrary to the opaque P(3HB) film, the copolymer films attained semitransparency depending on the LA fraction. Young's modulus values of the P(LA-co-3HB) s (from 148 to 905 MPa) were lower than those of poly(lactic acid) (PLA) (1020 MPa) and P(3HB) (1079 MPa). In addition, the value of elongation at break of the copolymer with 29 mol% LA reached 150%. In conclusion, P(LA-co-3HB) s were found to be a comparatively pliable and flexible material, differing from both of the rigid homopolymers. (C) 2011 Elsevier B. V. All rights reserved.ELSEVIER SCIENCE BV, Jul. 2011, JOURNAL OF BIOTECHNOLOGY, 154(4) (4), 255 - 260, English[Refereed]Scientific journalThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 +/- 80 U/g) than that of the synthase from the model strain C. necator (307 +/- 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 +/- 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 +/- 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.AMER SOC MICROBIOLOGY, May 2011, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77(9) (9), 2926 - 2933, English[Refereed]Scientific journalThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 +/- 80 U/g) than that of the synthase from the model strain C. necator (307 +/- 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 +/- 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 +/- 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.AMER SOC MICROBIOLOGY, May 2011, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77(9) (9), 2926 - 2933, English[Refereed]Scientific journalA poly(lactic acid) (PLA)-like terpolyester consisting of 96 mol% lactate (LA). I mol% 3-hydroxybutyrate and 3 mol% 3-hydroxyvalerate was produced in recombinant Escherichia coli LS5218 expressing LA-polymerizing enzyme (LPE). The strain was grown on glucose with a feeding of valerate as the monomer precursor. The glass transition and melting temperatures of the terpolyester were close to those of chemically synthesized poly(L-LA)s (PLLAs) having similar molecular weights. Additionally, a blend of the terpolyester, which was composed entirely of (R)-LA (D-LA) due to the strict enantiospecificity of LPE, with PLLA formed a stereocomplex with higher melting temperature (201.9 degrees C). These results indicate that the biological PLA-like polyester produced via this one-step microbial process has comparable thermal properties to chemically synthesized PLAs. (C) 2011 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Apr. 2011, POLYMER DEGRADATION AND STABILITY, 96(4) (4), 499 - 504, English[Refereed]Scientific journalPolyhydroxybutyrate [P(3HB)] was produced in the transgenic tobacco harboring the genes encoding acetoacetyl-CoA reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha (Cupriavidus necator) with optimized codon usage for expression in tobacco. P(3HB) contents in the transformants (0.2 mg/g dry cell weight in average) harboring the codon-optimized phaB gene was twofold higher than the control transformants harboring the wild-type phaB gene. The immunodetection revealed an increased production of PhaB in leaves, indicating that the enhanced expression of PhaB was effective to increase P(3HB) production in tobacco. In contrast, codon-optimization of the phaC gene exhibited no apparent effect on P(3HB) production. This result suggests that the efficiency of PhaB-catalyzed reaction contributed to the flux toward P(3HB) biosynthesis in tobacco leaves. 2010, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Apr. 2011, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 111(4) (4), 485 - 488, English[Refereed]Scientific journalLipopolysaccharides free P[3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)] production was achieved using recombinant Corynebacterium glutamicum harboring polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha. Cells grown on glucose with feeding of propionate as a precursor of 3HV unit accumulated 8-47 wt% of P(3HB-co-3HV). The 3HV fraction in the copolymer was varied from 0 to 28 mol% depending on the propionate concentrations. (C) 2010 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Apr. 2011, JOURNAL OF BIOTECHNOLOGY, 152(4) (4), 144 - 146, English[Refereed]Scientific journalLipopolysaccharides free P[3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)] production was achieved using recombinant Corynebacterium glutamicum harboring polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha. Cells grown on glucose with feeding of propionate as a precursor of 3HV unit accumulated 8-47 wt% of P(3HB-co-3HV). The 3HV fraction in the copolymer was varied from 0 to 28 mol% depending on the propionate concentrations. (C) 2010 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Apr. 2011, JOURNAL OF BIOTECHNOLOGY, 152(4) (4), 144 - 146, English[Refereed]Scientific journalPolyhydroxybutyrate [P(3HB)] was produced in the transgenic tobacco harboring the genes encoding acetoacetyl-CoA reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha (Cupriavidus necator) with optimized codon usage for expression in tobacco. P(3HB) contents in the transformants (0.2 mg/g dry cell weight in average) harboring the codon-optimized phaB gene was twofold higher than the control transformants harboring the wild-type phaB gene. The immunodetection revealed an increased production of PhaB in leaves, indicating that the enhanced expression of PhaB was effective to increase P(3HB) production in tobacco. In contrast, codon-optimization of the phaC gene exhibited no apparent effect on P(3HB) production. This result suggests that the efficiency of PhaB-catalyzed reaction contributed to the flux toward P(3HB) biosynthesis in tobacco leaves. 2010, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Apr. 2011, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 111(4) (4), 485 - 488, English[Refereed]Scientific journalWiley-VCH, Mar. 2011, Protein Engineering Handbook, Volume 1 & Volume 2, 2, 877 - 914, English[Refereed]In bookA biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] was developed in Escherichia coli and Corynebacterium glutamicum by an acetoacetyl-coenzyme A (CoA) synthase (AACS) recently isolated from terpenoid-producing Streptomyces sp. strain CL190. Expression of AACS led to significant productions of P(3HB) in E. coli (10.5 wt %) and C. glutamicum (19.7 wt %).TAYLOR & FRANCIS LTD, Feb. 2011, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 75(2) (2), 364 - 366, English[Refereed]Cellulose-derived glucose generated using the supported ruthenium catalyst was applied to poly(3-hydroxybutyrate) [P(3HB)] production in recombinant Escherichia coli. By the reaction with the catalyst at 220 degrees C. 15-20 carbon mol% of cellulose was converted into glucose. The hydrolysate also contained byproducts such as fructose, mannose, levoglucosan, oligomeric cellulose, 5-hydroxymethylfurfural (5-HMF), and furfural together with unidentified compounds. Setting the reaction temperature lower (215 degrees C) improved the ratio of glucose to 5-HMF, which was a main inhibiting factor for the cell growth. Indeed, the recombinant E. coli exhibited better performance on the hydrolysate generated at 215 degrees C and accumulated P(3HB) up to 42 wt%, which was the same as the case of the same concentration of analytical grade glucose. The result indicated that the ruthenium-mediated cellulose hydrolysis has a potency as a useful biorefinery process for production of bio-based plastic from cellulosic biomass. (C) 2010 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Feb. 2011, BIORESOURCE TECHNOLOGY, 102(3) (3), 3564 - 3567, English[Refereed]Scientific journalCellulose-derived glucose generated using the supported ruthenium catalyst was applied to poly(3-hydroxybutyrate) [P(3HB)] production in recombinant Escherichia coli. By the reaction with the catalyst at 220 degrees C. 15-20 carbon mol% of cellulose was converted into glucose. The hydrolysate also contained byproducts such as fructose, mannose, levoglucosan, oligomeric cellulose, 5-hydroxymethylfurfural (5-HMF), and furfural together with unidentified compounds. Setting the reaction temperature lower (215 degrees C) improved the ratio of glucose to 5-HMF, which was a main inhibiting factor for the cell growth. Indeed, the recombinant E. coli exhibited better performance on the hydrolysate generated at 215 degrees C and accumulated P(3HB) up to 42 wt%, which was the same as the case of the same concentration of analytical grade glucose. The result indicated that the ruthenium-mediated cellulose hydrolysis has a potency as a useful biorefinery process for production of bio-based plastic from cellulosic biomass. (C) 2010 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Feb. 2011, BIORESOURCE TECHNOLOGY, 102(3) (3), 3564 - 3567, English[Refereed]Scientific journalBiosynthesis of lactate(LA)-based polyester with a 96 mol% LA fraction and its application to stereocomplex formationポリ乳酸には,鏡像関係にあるL体とD体が存在し,その両者を混合すると,耐熱性が向上した複合体を形成することが知られている。しかし,D体のポリ乳酸は効率的な合成法がなく,コストが高いのが問題であった。本研究では,微生物の乳酸ポリマー合成系を用いて,Dポリ乳酸に極めて近いポリマーを合成することに初めて成功し,実際に耐熱性が向上した複合体の合成にも成功した。Feb. 2011, Polym. Degrad. Stabil., 96, 499 - 504, English[Refereed]Scientific journalA biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] was developed in Escherichia coli and Corynebacterium glutamicum by an acetoacetyl-coenzyme A (CoA) synthase (AACS) recently isolated from terpenoid-producing Streptomyces sp. strain CL190. Expression of AACS led to significant productions of P(3HB) in E. coli (10.5 wt %) and C. glutamicum (19.7 wt %).TAYLOR & FRANCIS LTD, Feb. 2011, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 75(2) (2), 364 - 366, English[Refereed]Scientific journalOne-pot Production of Lactate-Based Polyesters Using Engineered Microbes Expressing Lactate-Polymerizing EnzymePoly(lactic acid) (PLA) is the most widespread biobased plastic with excellent transparency. Such biobased plastics are expected to be an alternative to petroleum-derived materials. Although PLA is currently produced from fermentative lactate(LA) via chemical polymerization using heavy metal catalysts, we developed a new bioprocess producing LA-based polyester by one-pot reaction. The breakthrough for establishing this process was the discovery of lactate-polymerizing enzyme (LPE). LPE is capable of synthesizing LA-based polyesters with extremely high enantiomer excess. Moreover, the LA-based copolyesters incorporating PHA constituents showed improved flexibility. Therefore, the microbial LA-based polyester is a potent candidate for an environmental friendly plastic.The Society of Polymer Science, Japan, 2011, Kobunshi Kagaku, 68(5) (5), 271 - 280, JapaneseThere is currently a threat posed by the rapid emergence of antibiotic-resistant pathogens due to the abuse of conventional antibiotics. To overcome such a resistance problem, it is necessary to develop peptide agents with potent antimicrobial activities and high selectivity for target bacterial strains. Conventional approaches, such as the deletion, addition, and replacement of amino acid residues in the template peptide have been employed to alter the properties of antimicrobial peptides (AMPs). In this review, we summarize a recently developed approach, chemical modification, for the improvement of various types of naturally-occurring AMPs. By applying this design strategy to these peptide sequences, new AMPs with enhanced antimicrobial activities and improved selectivity for microorganisms have been successfully generated. This potential strategy should facilitate the creation of novel class of peptide antibiotics with properties suitable for pharmaceutical application.BENTHAM SCIENCE PUBL LTD, Nov. 2010, MINI-REVIEWS IN ORGANIC CHEMISTRY, 7(4) (4), 282 - 289, English[Refereed]Scientific journalA newly isolated mutation (Gl508Leu) and a combination of it with previously discovered beneficial mutations in polyhydroxyalkanoate synthase 1 from Pseudomonas sp. 61-3 were found to enhance the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoate)s in recombinant Escherichia coli.TAYLOR & FRANCIS LTD, Aug. 2010, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 74(8) (8), 1710 - 1712, English[Refereed]Recombinant strains of Ralstonia eutropha and Pseudomonas putida harboring a chimeric polyhydroxyalkanoate (PHA) synthase, which consisted of PHA synthases of Aeromonas caviae and R. eutropha, produced 3-hydroxybutyrate (3HB)-based PHA copolymers comprised of 3-hydroxyhexanoate and 3-hydroxyoctanoate units from dodecanoate (87-97 mol % 3HB), indicating that the chimeric PHA synthase possesses desirable substrate specificity leading to the production of 3HB-rich copolymers.TAYLOR & FRANCIS LTD, Aug. 2010, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 74(8) (8), 1716 - 1718, English[Refereed]Polylactic acid (PLA), a representative bio-based polyester, has been commonly synthesized via a multi-step by chemical process. The current modes of generating PLA involve microbial fermentation of starting material, lactic acid (LA), followed by chemical ring-opening polymerization. Recently, one-pot complete bioprocess for LA-based polyesters has been established as a microbial cell factory (MCF). The concept is a process conversion from the usual chemical factory to the MCF. This new challenge was triggered by discovery of an engineered LA-polymerizing enzyme (LPE). The LPE was found as one of the members of an extensive mutant library that has been created through the long-term evolutionary engineering study of natural biopolyester-synthesizing enzymes. Needless to say, a strategic method of getting the beneficial mutation in the enzyme is of the utmost importance and an essential step towards accomplishing the desired purpose, the acquisition of the LA-polymerizing activity in this case. In this review, the structures and properties of LPE-catalyzed polymerization products will be discussed as well as backgrounds on establishment of the MCFs for synthesis of LA-based polyesters. Also, experimental strategies for enrichment of the LA fraction will be proposed to further advance the prototype of MCF based on the related metabolic pathways. (C) 2010 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Aug. 2010, POLYMER DEGRADATION AND STABILITY, 95(8) (8), 1421 - 1428, English[Refereed]Scientific journalNew lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate (3HB)-co-3-hydroxyhexananoate (3HHx)]s, were produced in recombinant Escherichia coli LS5218 harboring three genes encoding LA-polymerizing enzyme (LPE), propionyl-coenzyme A (CoA) transferase (PCT) and (R)-specific enoyl-CoA hydratase (PhaJ4). When the recombinant LS5218 was grown on glucose with the feeding of butyrate, 3HB-CoA and 3HHx-CoA were supplied, probably via reverse reactions of the beta-oxidation pathway and PhaJ4. LPE copolymerized the two monomers 3HB-CoA and 3HHx-CoA with LA-CoA, which was generated by PCT, to yield the terpolymers. Gas chromatography analysis revealed that the terpolymers consisted of 2.7-34 mol% LA, 38-81 mol% 3HB and 17-33 mol% 3HHx units, which can be varied depending on the butyrate concentration fed in the medium. In addition, H-1-C-13 COSY NMR analysis provided evidence for a linkage between LA and 3HHx units in the polymer. (C) 2010 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Aug. 2010, POLYMER DEGRADATION AND STABILITY, 95(8) (8), 1340 - 1344, English[Refereed]Scientific journalMicrobial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. Two enzyme engineering approaches were applied to improve MTG activity. One is a novel method of rational mutagenesis, called water-accessible surface hot-space region-oriented mutagenesis (WASH-ROM). One hundred and fifty-one point mutations were selected at 40 residues, bearing high solvent-accessibility surface area, within a 15 space from the active site Cys64. Among them, 32 mutants showed higher specific activity than the wild type. The other is a random mutagenesis of the whole region of the MTG gene, coupled with a new plate assay screening system, using Corynebacterium Expression System CORYNEXA (R). This in vivo system allowed us to readily distinguish the change in enzymatic activity by monitoring the intensity of enzymatic reaction-derived color zones surrounding recombinant cells. From the library of 24,000 mutants, ten were finally selected as beneficial mutants exhibiting higher specific activity than the wild type. Furthermore, we found that Ser199Ala mutant with additional N-terminal tetrapeptide showed the highest specific activity (1.7 times higher than the wild type). These various beneficial positions leading to increased specific activity of MTG were identified to achieve further enzyme improvements.SPRINGER, Aug. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 87(6) (6), 2087 - 2096, English[Refereed]Scientific journalNew lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate (3HB)-co-3-hydroxyhexananoate (3HHx)]s, were produced in recombinant Escherichia coli LS5218 harboring three genes encoding LA-polymerizing enzyme (LPE), propionyl-coenzyme A (CoA) transferase (PCT) and (R)-specific enoyl-CoA hydratase (PhaJ4). When the recombinant LS5218 was grown on glucose with the feeding of butyrate, 3HB-CoA and 3HHx-CoA were supplied, probably via reverse reactions of the beta-oxidation pathway and PhaJ4. LPE copolymerized the two monomers 3HB-CoA and 3HHx-CoA with LA-CoA, which was generated by PCT, to yield the terpolymers. Gas chromatography analysis revealed that the terpolymers consisted of 2.7-34 mol% LA, 38-81 mol% 3HB and 17-33 mol% 3HHx units, which can be varied depending on the butyrate concentration fed in the medium. In addition, H-1-C-13 COSY NMR analysis provided evidence for a linkage between LA and 3HHx units in the polymer. (C) 2010 Elsevier Ltd. All rights reserved.ELSEVIER SCI LTD, Aug. 2010, POLYMER DEGRADATION AND STABILITY, 95(8) (8), 1340 - 1344, English[Refereed]Scientific journalA newly isolated mutation (Gl508Leu) and a combination of it with previously discovered beneficial mutations in polyhydroxyalkanoate synthase 1 from Pseudomonas sp. 61-3 were found to enhance the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoate)s in recombinant Escherichia coli.TAYLOR & FRANCIS LTD, Aug. 2010, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 74(8) (8), 1710 - 1712, English[Refereed]Scientific journalRecombinant strains of Ralstonia eutropha and Pseudomonas putida harboring a chimeric polyhydroxyalkanoate (PHA) synthase, which consisted of PHA synthases of Aeromonas caviae and R. eutropha, produced 3-hydroxybutyrate (3HB)-based PHA copolymers comprised of 3-hydroxyhexanoate and 3-hydroxyoctanoate units from dodecanoate (87-97 mol % 3HB), indicating that the chimeric PHA synthase possesses desirable substrate specificity leading to the production of 3HB-rich copolymers.TAYLOR & FRANCIS LTD, Aug. 2010, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 74(8) (8), 1716 - 1718, English[Refereed]Scientific journalThanatin is an antimicrobial peptide with a strong and wide-ranging antimicrobial spectrum, including certain species of fungi and Gram-negative and Gram-positive bacteria. To evaluate the application of thanatin to the generation of disease-resistant plants, we introduced a synthetic thanatin gene into rice. Several transformants that expressed the introduced gene showed significant level of antimicrobial activity. The substances showing antimicrobial activity were partially purified from these transformants and their properties were determined. The molecule with characteristics similar to those of native thanatin on the elution pattern in HPLC analysis had an identical molecular mass to that of native molecule. It should also be noted that the transformant acquired a sufficient level of resistance to the rice blast fungus, Magnaporthe oryzae, presumably due to the repressive activity of thanatin to its initial stage of infection. This result demonstrates that thanatin has antifungal activity for M. oryzae and that the introduction of the thanatin gene into rice is effective in generating a plant resistant to rice blast disease.SPRINGER, Jun. 2010, TRANSGENIC RESEARCH, 19(3) (3), 415 - 424, English[Refereed]Scientific journalIn order to genetically modify microorganisms capable of producing polyhydroxyalkanoate (PHA) biopolymers, a simple and rapid method to prepare freshly plated Pseudomonas cells for transformation via electroporation was developed. This method can be used to transfer both replicative plasmids and linear DNA to knock out genes into the cells. The transformation efficiencies were in the range of >= 10(7) transformants mu g(-1) DNA for replicative plasmids and >= 10(6) transformants mu g(-1) DNA for linear DNA, which are comparable with commercially available competent cells. Furthermore, this transformation procedure can be performed in less than 10 min, saving a great deal of time compared with traditional methods. Knockout mutants of several Pseudomonas species were generated by transformation of linear DNA and these mutations were verified by PCR and analysis of PHA content. (C) 2009 Society of Chemical IndustryJOHN WILEY & SONS LTD, Jun. 2010, JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, 85(6) (6), 775 - 778, English[Refereed]Scientific journalThe gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was 85 A degrees C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 A degrees C and for 1 month at 30 A degrees C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 A degrees C. Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red 88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions.SPRINGER, May 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 86(5) (5), 1431 - 1438, English[Refereed]Scientific journalAzoreductases from Bacillus sp. B29 are NADH-dependent flavoenzymes which contain a flavin mononucleotide (FMN) as a prosthetic group and exist as homodimers composed of 23 kDa subunits. These enzymes catalyze the reductive degradation of various azo compounds by a ping-pong mechanism. In order to determine the structure-function relationship of the azo-dye reduction mechanism, an X-ray crystallographic study of azoreductases was performed. Selenomethionine-labelled AzrA (SeMet-AzrA) and AzrC were crystallized by the hanging-drop vapour-diffusion method. A crystal of SeMet-AzrA diffracted to 2.0 angstrom resolution and was determined to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.9, b = 69.0, c = 105.4 angstrom. The native crystals of AzrC belonged to space group C2, with unit-cell parameters a = 192.0, b = 56.6, c = 105.5 angstrom, beta = 115.7 degrees, and diffracted to 2.21 angstrom resolution.WILEY-BLACKWELL PUBLISHING, INC, May 2010, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 66(5) (5), 503 - 505, English[Refereed]Scientific journalContributing factors for the antimicrobial activity enhancement of N-terminally engineered mutants of cell-penetrating apidaecins were analyzed based on their cell-penetration efficiency. The flow cytometric analysis of the engineered apidaecins labeled with carboxyfluorescein (FAM) revealed their enhanced cell-penetrating efficiencies into Escherichia coli that should be one of key factors causing the enhanced antimicrobial activity. It is noteworthy that, for one mutant, the enhancement in antimicrobial activity (18-fold higher than wild type) was greater than that of cell penetration (5.9-fold), suggesting that the N-terminal mutation may reinforce both interaction with unidentified intracellular target(s) and cell-penetration efficiency. (C) 2010 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Apr. 2010, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 395(1) (1), 7 - 10, English[Refereed]Scientific journalLactate (LA)-polymerizing enzyme (LPE) is a newly established class of polyhydroxyalkanoate (PHA) synthase, which can incorporate LA units into a polymer chain. We previously synthesized P(LA-co-3-hydroxybutyrate)s [P(LA-co-3HB)s] in recombination Escherichia coli using the first LPE, which is the Ser325Thr/Glu481Lys mutant of PHA synthase from Pseudomonas sp. 61-3 [PhaCl(Ps)ST/QK]. In this study, we finely regulated LA fraction in the copolymer by saturated mutations at position 392 (F392X), which corresponds to the activity-enhancing mutations at position 420 of PHA synthase from Ralstonia eutropha. Among the 19 saturated mutants of LPe at position 392, 17 mutants produced P(La-co-3HB)s with various LA fractions (16-45 mol %), whereas PhaCl(Ps)ST/QK produced P(LA-co-3HB) with 26 mol % LA under the same culture condition. In particular, the F392S mutation exhibited the highest LA fraction of 45 mol %, and also increased polymer content (62 wt %) compared with PhaCl(Ps)ST/QK (44 wt %). Combination of the F392S mutant and anaerobic culture conditions, which promote LA production, led to a further increase in LA fraction up to 62 mol%. The P(LA-co-3HB)s with various LA fractions exhibited altered melting temperatures and melting enthalpy depending on their monomer composition. Accordingly, the mutations at position 392 in LPE greatly contributed to fine-tuning of the LA fraction in the copolymers that is useful for regulating LA fraction-dependent thermal properties.AMER CHEMICAL SOC, Mar. 2010, BIOMACROMOLECULES, 11(3) (3), 815 - 819, English[Refereed]Scientific journalLactate (LA)-polymerizing enzyme (LPE) is a newly established class of polyhydroxyalkanoate (PHA) synthase, which can incorporate LA units into a polymer chain. We previously synthesized P(LA-co-3-hydroxybutyrate)s [P(LA-co-3HB)s] in recombination Escherichia coli using the first LPE, which is the Ser325Thr/Glu481Lys mutant of PHA synthase from Pseudomonas sp. 61-3 [PhaCl(Ps)ST/QK]. In this study, we finely regulated LA fraction in the copolymer by saturated mutations at position 392 (F392X), which corresponds to the activity-enhancing mutations at position 420 of PHA synthase from Ralstonia eutropha. Among the 19 saturated mutants of LPe at position 392, 17 mutants produced P(La-co-3HB)s with various LA fractions (16-45 mol %), whereas PhaCl(Ps)ST/QK produced P(LA-co-3HB) with 26 mol % LA under the same culture condition. In particular, the F392S mutation exhibited the highest LA fraction of 45 mol %, and also increased polymer content (62 wt %) compared with PhaCl(Ps)ST/QK (44 wt %). Combination of the F392S mutant and anaerobic culture conditions, which promote LA production, led to a further increase in LA fraction up to 62 mol%. The P(LA-co-3HB)s with various LA fractions exhibited altered melting temperatures and melting enthalpy depending on their monomer composition. Accordingly, the mutations at position 392 in LPE greatly contributed to fine-tuning of the LA fraction in the copolymers that is useful for regulating LA fraction-dependent thermal properties.AMER CHEMICAL SOC, Mar. 2010, BIOMACROMOLECULES, 11(3) (3), 815 - 819, English[Refereed]Scientific journalThe importance of polylactic acid, a representative bio-based polyester, has been established on a worldwide scale in response to emerging global environmental problems such as green house gas emission and limited petroleum consumption. The current methods for generating this bio-based polymer involve biological synthesis and lactic acid (LA) fermentation, followed by chemical ring-opening polymerization. Among the research community working on polyhydroxyalkanoate polyesters, the prospect of direct biological synthesis of LA into a polymeric form is very attractive from the academic and industrial perspectives. In 2008, this challenge was met for the first time by the discovery of an "LA-polymerizing enzyme". Using this novel enzyme, the metabolic engineering approach outlined here provided an entirely new, single organism generation of the polymer. This is a major breakthrough in the field. In this review, we provide an overview of the whole-cell synthesis of LA-containing polyesters in comparison with conventional lipase-catalyzed polymer synthesis in terms of both the concepts and strategies of their synthetic processes.SPRINGER, Jan. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85(4) (4), 921 - 932, English[Refereed]Scientific journalNovel lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate(3HB)-co-3-hydroxyvalerate(3HV)]s (PLBVs), were produced in LA-overproducing mutant, Escherichia coli JW0885, which was found to be a superior host for the efficient production of LA-based polyesters. Recombinant E. coli JW0885 harboring the genes encoding LA-polymerizing enzyme (Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3) and three monomer supplying enzymes [propionyl-CoA transferase, beta-ketothiolase, and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-dependent acetoacetyl-CoA reductase] was aerobically grown on glucose with feeding of propionate as a precursor of 3-hydroxyvaleryl-CoA (3HV-CoA). Gas chromatography and nuclear magnetic resonance (NMR) analyses revealed that polymers accumulated in the cells were composed of LA, 3HB, and 3HV units, thus being identified as terpolymers, PLBVs. In addition, H-1-NMR analysis suggested the existence of LA-3HV sequence in the terpolymer. When 100 mg/l of sodium propionate was added into the medium, 3HV fraction in the terpolymer linearly reached up to 7.2 mol%, while LA fraction was inversely decreased. This phenomenon could be due to the change in metabolic fluxes of lactyl-CoA (LA-CoA) and 3HV-CoA depending on the concentration of propionate fed into the medium.SPRINGER, Jan. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85(4) (4), 949 - 954, English[Refereed]Scientific journalNovel lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate(3HB)-co-3-hydroxyvalerate(3HV)]s (PLBVs), were produced in LA-overproducing mutant, Escherichia coli JW0885, which was found to be a superior host for the efficient production of LA-based polyesters. Recombinant E. coli JW0885 harboring the genes encoding LA-polymerizing enzyme (Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3) and three monomer supplying enzymes [propionyl-CoA transferase, beta-ketothiolase, and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-dependent acetoacetyl-CoA reductase] was aerobically grown on glucose with feeding of propionate as a precursor of 3-hydroxyvaleryl-CoA (3HV-CoA). Gas chromatography and nuclear magnetic resonance (NMR) analyses revealed that polymers accumulated in the cells were composed of LA, 3HB, and 3HV units, thus being identified as terpolymers, PLBVs. In addition, H-1-NMR analysis suggested the existence of LA-3HV sequence in the terpolymer. When 100 mg/l of sodium propionate was added into the medium, 3HV fraction in the terpolymer linearly reached up to 7.2 mol%, while LA fraction was inversely decreased. This phenomenon could be due to the change in metabolic fluxes of lactyl-CoA (LA-CoA) and 3HV-CoA depending on the concentration of propionate fed into the medium.SPRINGER, Jan. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85(4) (4), 949 - 954, English[Refereed]Scientific journal日本バイオプラスチック協会, Nov. 2009, バイオプラジャーナル, 9(3) (3), 18 - 23, JapaneseBP最前線 乳酸ポリマー完全生合成システムの幕開け--新原理に基づく微生物工場の誕生To characterize an exo-beta-1,3-glucanase (ExgP) of an isolated fungal strain with high laminarin degradation activity, identified as Penicillium sp. KH10, heterologous secretory expression of the ExgP was performed in Aspergillus oryzae. Deduced amino acid sequence of the exgP gene possibly consisted of 989 amino acids which showed high sequence similarity to those of fungal exo-beta-1,3-glucanases belonging to the glycoside hydrolase (GH) family 55. Notably, the purified recombinant ExgP showed a single protein peak in the native state (by gel-permeation chromatographic analysis), but showed two protein bands in the denatured state (by SDS-polyacrylamide gel electrophoresis). These two polypeptides exhibited activity in a coexisting state even under reducing conditions, suggesting that non-covalent association of both polypeptides took place. Taken together with the nucleotide sequence information, the ExgP precursor (104 kDa) would be proteolytically processed (cleaved) to generate two protein fragments (42 and 47 kDa) and the processed products (polypeptide fragments) would be assembled each other by a non-covalent interaction. Moreover, one of the matured ExgP polypeptides was N-glycosylated by the post-translational modi. cation. (C) 2009 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Oct. 2009, PROTEIN EXPRESSION AND PURIFICATION, 67(2) (2), 126 - 131, English[Refereed]Scientific journalClass II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1(Ps)) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1(Ps) that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol.%). However, polymer content was often largely decreased (0.2 wt.%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol.% 3HB and 12 wt.%; Matsumoto et al. (2006) Biomacromolecules, 7:2436-2442). In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1(Ps) was performed using single and double mutations at sites 130 and 325 as templates to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1(Ps) mutants were screened based on enhanced P(3HB) content and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol.%) and also polymer content (18 wt.%), indicating that mutation scrambling was effective for obtaining the desired mutants.SPRINGER, Oct. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 84(6) (6), 1117 - 1124, English[Refereed]Scientific journalClass II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1(Ps)) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1(Ps) that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol.%). However, polymer content was often largely decreased (0.2 wt.%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol.% 3HB and 12 wt.%; Matsumoto et al. (2006) Biomacromolecules, 7:2436-2442). In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1(Ps) was performed using single and double mutations at sites 130 and 325 as templates to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1(Ps) mutants were screened based on enhanced P(3HB) content and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol.%) and also polymer content (18 wt.%), indicating that mutation scrambling was effective for obtaining the desired mutants.SPRINGER, Oct. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 84(6) (6), 1117 - 1124, English[Refereed]Scientific journalThe chemically modified thanatins with the methyll group (CH3), ethyl group (C2H5), and normal-octyl group (C8H17) at the side-chain of cysteine residues were synthesized. The octyl group modified form exhibited 8-fold higher antimicrobial activity against Micrococcus luteus than wild type thanatin. It was found that there was an equilateral correlation between antimicrobial activity and side-chain hydrophobicity at the cysteine residues in thanatin.TAYLOR & FRANCIS LTD, Jul. 2009, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 73(7) (7), 1683 - 1684, English[Refereed]We cloned and expressed two genes encoding azoreductase homologes, AzrB and AzrC, from Bacillus sp. B29. Purified recombinant AzrB and AzrC were homodimers with 23kDa identical subunits, and were flavoproteins. NADH was preferred as electron donors for both azoreductases. The azoreductases showed optimal activities at 70 degrees C (AzrB) and 55 degrees C (AzrC), and retained activities up to 55 degrees C (AzrB) and 50 degrees C (AzrC) after incubation for 1 h. Other enzymatic properties, including the substrate specificities of both azoreductases, were also investigated.TAYLOR & FRANCIS LTD, May 2009, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 73(5) (5), 1209 - 1211, English[Refereed]We previously synthesized poly(3-hydroxybutyrate) [P(3HB)] in recombinant Corynebacterium glutamicum, a prominent producer of amino acids. In this study, a two-step cultivation was established for the dual production of glutamate and P(3HB) due to the differences in the optimal concentration of biotin. Glutamate was extracellularly produced first under the biotin-limited condition of 0.3 mu g/L. Production was then shifted to P(3HB) by addition of biotin to a total concentration of 9 mu g/L. The final products obtained were 18 g/L glutamate and 36 wt% of P(3HB). (c) 2008, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Apr. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 107(4) (4), 409 - 411, English[Refereed]Scientific journalShort-chain-length/medium-chain-length (SCL/MCL) polyhydroxyalkanoate (PHA) was produced in the plastids of Arabidopsis thaliana. Phe87Thr (F87T) mutated 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) from Escherichia coli, and Ser325Thr/Gln481Lys (ST/QK) mutated polyhydroxyalkanoate (PHA) synthase (PhaC1) from Joseudomonas sp. 61-3, along with the beta-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB) from Ralstonia eutropha (Cupriavidus necator) genes were introduced into Arabidopsis. The transgenic Arabidopsis produced PHA copolymers composed of monomers consisting of 4-14 carbons. The introduction of the engineered PHA synthase resulted in a 10-fold increase in PHA content compared to plants expressing the wild-type PHA synthase. In addition, expression of the engineered fabH gene in the plastid led to an increase in the amount of the SCL monomer, 3-hydroxybutyrate, incorporated into PHA, and contributed to supply of MCL monomers for PHA production.AMER CHEMICAL SOC, Apr. 2009, BIOMACROMOLECULES, 10(4) (4), 686 - 690, English[Refereed]Scientific journalConsiderable enrichment of the lactate (LA) fraction in Poly has been achieved, reaching up to 47 mol % from the previous 6 mol % [S. Taguchi et al. Proc. Natl. Acad. Sci. U.S.A. 2008,105 (45), 17323-17327], when recombinant Escherichia coli W3110, harboring the LA-polymerizing enzyme gene together with three genes for supplying monomers, lactyl-coenzyme A (CoA), and 3-hydroxybutyryl-CoA, were grown on glucose under anaerobic conditions. The molecular weights of the copolymer were M(n) = 1.5 x 10(4) g/mol and M(w) = 2.0 x 10(4) g/mol, respectively. Notably, alkali ne-hydrolyzed product analysis revealed that only the (R)-enantiomer of LA was incorporated in the copolymer. Furthermore, the triad sequence of LA-LALA was clearly detected in the polymer chain based on NMR analysis. A remarkably contrasting melting temperature was observed between the copolymer obtained here P[(R)-LA-co-(R)-3HB] (157 degrees C) and the chemically synthesized P[(S)-LA-co-(R)-3HB] (105 degrees C) with similar monomer composition.AMER CHEMICAL SOC, Apr. 2009, BIOMACROMOLECULES, 10(4) (4), 677 - 681, English[Refereed]Scientific journalChimeric enzymes composed of polyhydroxyalkanoate (PHA) synthases from Ralstonia eutropha (Cupriavidus necator) (PhaC(Re)) and Aeromonas caviae (PhaC(Ac)) were constructed. PhaC(Re) is known for its potent enzymatic activity among the characterized PHA synthases. PhaC(Ac) has broad substrate specificity and synthesizes short-chain-length (SCL)/medium-chain-length (MCL) PHA. We attempted to create chimeric enzymes inheriting both of the advantageous properties. Among eight chimeras, AcRe 12, with 26% of the N-terminal of PhaCA, and 74% of the C-terminal of PhaC(Re), exhibited comparable P(3-hydroxybutyrate) accumulation as parental enzymes in Escherichia coli JM109. Thus, AcRe12 was applied to SCL/MCL PHA production using E. coli LS5218 as the host. AcRe12 accumulated higher amount of PHA (50 wt %) than the parental enzymes. Furthermore, the PHA consisted of 2 mol % 3-hydroxyhexanoate as well as 3-hydroxybutyrate. Therefore, the chimeric PHA synthase, AcRe 12, inherited the character of both of the parental enzymes and thus exhibits improved enzymatic properties.AMER CHEMICAL SOC, Apr. 2009, BIOMACROMOLECULES, 10(4) (4), 682 - 685, English[Refereed]Scientific journalChimeric enzymes composed of polyhydroxyalkanoate (PHA) synthases from Ralstonia eutropha (Cupriavidus necator) (PhaC(Re)) and Aeromonas caviae (PhaC(Ac)) were constructed. PhaC(Re) is known for its potent enzymatic activity among the characterized PHA synthases. PhaC(Ac) has broad substrate specificity and synthesizes short-chain-length (SCL)/medium-chain-length (MCL) PHA. We attempted to create chimeric enzymes inheriting both of the advantageous properties. Among eight chimeras, AcRe 12, with 26% of the N-terminal of PhaCA, and 74% of the C-terminal of PhaC(Re), exhibited comparable P(3-hydroxybutyrate) accumulation as parental enzymes in Escherichia coli JM109. Thus, AcRe12 was applied to SCL/MCL PHA production using E. coli LS5218 as the host. AcRe12 accumulated higher amount of PHA (50 wt %) than the parental enzymes. Furthermore, the PHA consisted of 2 mol % 3-hydroxyhexanoate as well as 3-hydroxybutyrate. Therefore, the chimeric PHA synthase, AcRe 12, inherited the character of both of the parental enzymes and thus exhibits improved enzymatic properties.AMER CHEMICAL SOC, Apr. 2009, BIOMACROMOLECULES, 10(4) (4), 682 - 685, English[Refereed]Scientific journalConsiderable enrichment of the lactate (LA) fraction in Poly has been achieved, reaching up to 47 mol % from the previous 6 mol % [S. Taguchi et al. Proc. Natl. Acad. Sci. U.S.A. 2008,105 (45), 17323-17327], when recombinant Escherichia coli W3110, harboring the LA-polymerizing enzyme gene together with three genes for supplying monomers, lactyl-coenzyme A (CoA), and 3-hydroxybutyryl-CoA, were grown on glucose under anaerobic conditions. The molecular weights of the copolymer were M(n) = 1.5 x 10(4) g/mol and M(w) = 2.0 x 10(4) g/mol, respectively. Notably, alkali ne-hydrolyzed product analysis revealed that only the (R)-enantiomer of LA was incorporated in the copolymer. Furthermore, the triad sequence of LA-LALA was clearly detected in the polymer chain based on NMR analysis. A remarkably contrasting melting temperature was observed between the copolymer obtained here P[(R)-LA-co-(R)-3HB] (157 degrees C) and the chemically synthesized P in recombinant Corynebacterium glutamicum, a prominent producer of amino acids. In this study, a two-step cultivation was established for the dual production of glutamate and P(3HB) due to the differences in the optimal concentration of biotin. Glutamate was extracellularly produced first under the biotin-limited condition of 0.3 mu g/L. Production was then shifted to P(3HB) by addition of biotin to a total concentration of 9 mu g/L. The final products obtained were 18 g/L glutamate and 36 wt% of P(3HB). (c) 2008, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Apr. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 107(4) (4), 409 - 411, English[Refereed]Scientific journalShort-chain-length/medium-chain-length (SCL/MCL) polyhydroxyalkanoate (PHA) was produced in the plastids of Arabidopsis thaliana. Phe87Thr (F87T) mutated 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) from Escherichia coli, and Ser325Thr/Gln481Lys (ST/QK) mutated polyhydroxyalkanoate (PHA) synthase (PhaC1) from Joseudomonas sp. 61-3, along with the beta-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB) from Ralstonia eutropha (Cupriavidus necator) genes were introduced into Arabidopsis. The transgenic Arabidopsis produced PHA copolymers composed of monomers consisting of 4-14 carbons. The introduction of the engineered PHA synthase resulted in a 10-fold increase in PHA content compared to plants expressing the wild-type PHA synthase. In addition, expression of the engineered fabH gene in the plastid led to an increase in the amount of the SCL monomer, 3-hydroxybutyrate, incorporated into PHA, and contributed to supply of MCL monomers for PHA production.AMER CHEMICAL SOC, Apr. 2009, BIOMACROMOLECULES, 10(4) (4), 686 - 690, English[Refereed]Scientific journalIn our previous paper, we synthesized poly-3-hydroxybutyrate [P(3HB)] by using the water-organic-solvent two-phase reaction system (TPRS), in which (R)-3-hydroxybutyrylCoA [(R)-3HBCoA] was continuously supplied to PHA synthase by the ester exchange reaction between CoA and thiophenol in thiophenyl (R)-3HB using the LA-polymerizing enzyme as a catalyst. The molar ratios of LA in the copolymers were controllable in the range of 0 to 36 mol% by varying the ratio of (R)-LATP and (R)-3HBTP fed into the TPRS. The number-average molecular weight and the polydispersity of P(36 mol%-co-3HB) were 1.1 x 10(4) and 1.4, respectively. This is the first report on the chemo-enzymatic synthesis of P(LA-co-3HB) by a LA-polymerizing enzyme.AMER CHEMICAL SOC, Mar. 2009, MACROMOLECULES, 42(6) (6), 1985 - 1989, English[Refereed]Scientific journalSeven mutant forms of the antibacterial peptide apidaecin with increased activity were created by combinatorial mutagenesis targeted to the three N-terminal amino acid residues that had previously been identified as a nonessential region. An in vitro MIC assay revealed that the amino acid substitutions in the functionally variable region were effective in improving differential activity toward the four gram-negative bacteria tested, while a gram-positive bacterium was unaffected.AMER SOC MICROBIOLOGY, Mar. 2009, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75(5) (5), 1460 - 1464, English[Refereed]Scientific journalSOC POLYMER SCIENCE JAPAN, 2009, POLYMER JOURNAL, 41(3) (3), 237 - 240, English[Refereed]Polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp 61-3 (PhaCl(Ps)) is able to synthesize P(3HB-co-MA), consisting of a 3HB unit and medium-chain-length 3HA units of 6-12 carbon atoms. Expression vectors encoding 76 PhaC1(Ps) mutants with an amino acid replacement at position 130, 325, 477 or 481 were individually introduced into Ralstonia eutropha. The mutant enzyme genes were evaluated in terms of their abilities to synthesize P(3HB-co-3HA) using soybean oil as a carbon source. 20 mutants showed significantly high accumulation levels of PHA exceeding 30 wt.-% and as high as 57 wt.-%. It was found that hydrophobic amino acids at the positions are more likely to enhance accumulation of PHA in R. eutropha.WILEY-V C H VERLAG GMBH, Jan. 2009, MACROMOLECULAR BIOSCIENCE, 9(1) (1), 71 - 78, English[Refereed]Scientific journalPolyhydroxyalkanoate (PHA) synthase from Pseudomonas sp 61-3 (PhaCl(Ps)) is able to synthesize P(3HB-co-MA), consisting of a 3HB unit and medium-chain-length 3HA units of 6-12 carbon atoms. Expression vectors encoding 76 PhaC1(Ps) mutants with an amino acid replacement at position 130, 325, 477 or 481 were individually introduced into Ralstonia eutropha. The mutant enzyme genes were evaluated in terms of their abilities to synthesize P(3HB-co-3HA) using soybean oil as a carbon source. 20 mutants showed significantly high accumulation levels of PHA exceeding 30 wt.-% and as high as 57 wt.-%. It was found that hydrophobic amino acids at the positions are more likely to enhance accumulation of PHA in R. eutropha.WILEY-V C H VERLAG GMBH, Jan. 2009, MACROMOLECULAR BIOSCIENCE, 9(1) (1), 71 - 78, English[Refereed]Scientific journal既存の研究で創出された人工改変型ポリエステル重合酵素は,多様なモノマーを重合することができる。本研究では,単離精製した酵素を用いて反応速度の解析を行い,改変の前後でどのような酵素の性質の変化が起こったかを明らかにした。SOC POLYMER SCIENCE JAPAN, 2009, POLYMER JOURNAL, 41(3) (3), 237 - 240, English[Refereed]Scientific journal日本生物工学会, Nov. 2008, 生物工学会誌 : seibutsu-kogaku kaishi, 86(11) (11), 543 - 543, Japanese新産業創出に挑むキィーエンザイムの顔ぶれPolylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 x 10(5), as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.NATL ACAD SCIENCES, Nov. 2008, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105(45) (45), 17323 - 17327, English[Refereed]Scientific journalPolylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 x 10(5), as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.NATL ACAD SCIENCES, Nov. 2008, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105(45) (45), 17323 - 17327, English[Refereed]Scientific journalSOC FIBER SCIENCE TECHNOLOGY, Nov. 2008, SEN-I GAKKAISHI, 64(11) (11), P365 - P370, JapaneseMicrobial Factory for the Production of BioplasticsScientific journalT cell intracellular antigen-1 (TIA-1), an apoptosis promoting factor, functions as a splicing regulator for the Fas pre-mRNA. TIA-1 possesses three RNA recognition motifs (RRMs) and a glutamine-rich domain. The second RRM (RRM2) is necessary and sufficient for tight, sequence-specific binding to the uridine-rich sequences buried around the 5' splice sites. In the present study, we solved the solution structure of the murine TIA-1 RRM2 by heteronuclear-nuclear magnetic resonance spectroscopy. The TIA-1 RRM2 adopts the RRM fold (beta alpha beta beta alpha beta) and possesses an extra beta-strand between beta 2 and beta 3, which forms an additional beta-sheet with the C-terminal part of beta 2. We refer to this structure as the beta 2-beta 2' beta-loop. Interestingly, this characteristic beta-loop structure is conserved among a number of RRMs, including the U2AF65 RRM2 and the Sex-lethal RRM1 and RRM2, which also bind to uridine-rich RNAs. Furthermore, we identified a new sequence motif in the beta 2-beta 2' beta-loop, the DxxT motif. Chemical shift perturbation analyses of both the main and side chains upon binding to the uridine pentamer RNA revealed that most of the beta-sheet surface, including the beta 2-beta 2' beta-loop, is involved in the RNA binding. An investigation of the chemical shift perturbation revealed similarity in the RNA recognition modes between the TIA-1 and U2AF65 RRMs.AMER CHEMICAL SOC, Jun. 2008, BIOCHEMISTRY, 47(24) (24), 6437 - 6450, English[Refereed]Scientific journalThe spliceosomal protein p14, a component of the SF3b complex in the U2 small nuclear ribonucleoprotein.(snRNP), is essential for the U2 snRNP to recognize the branch site adenosine. The elucidation of the dynamic process of the splicing machinery rearrangement awaited the solution structural information. We identified a suitable complex of human p14 and the SF3b155 fragment for the determination of its solution structure by NMR. In addition to the overall structure Of the complex, which was recently reported in a crystallographic study (typical RNA recognition motif fold beta 1-alpha 1-beta 2-beta 3-alpha 2-beta 4 of p14, and alpha A-beta A fold of the SF3b155 fragment), we identified three important features revealed by the NMR solution structure. First, the C-terminal extension and. the nuclear localization signal of p14 (alpha 3 and alpha 4 in the crystal structure, respectively) were dispensable for the complex formation. Second, the proline-rich segment of SF3b155, following beta A, closely approaches p14. Third, interestingly, the beta 1-alpha 1 loop and the alpha 2-beta 4 beta-hairpin form a positively charged groove. Extensive mutagenesis analyses revealed the functional relevance of the residues involved in the protein-protein interactions: two aromatic residues of SF3b155 (Phe408 and Tyr412) play crucial roles in the complex formation, and two hydrophobic residues (Val414 and Leu415) in SF3b 155 serve as an anchor for the complex formation, by cooperating with the aromatic residues. These findings clearly led to the conclusion that SFb155 binds to p14 with three contact points, involving Phe408, Tyr412, and Val414/Leu415. Furthermore, to dissect the interactions between p 14 and the branch site RNA, we performed chemical-shift-perturbation experiments, not only for the main-chain but also for the side-chain resonances, for several p14-SF3b155 complex constructs upon binding to RNA. These analyses identified a positively charged groove and the C-terminal extension of p14 as RNA-binding sites. Strikingly, an aromatic residue in the beta 1-alpha 1 loop, Tyr28, and a positively charged residue in the alpha 2-beta 4 beta-hairpin, Agr85, are critical for the RNA-binding activity of the positively charged groove. The 7yr28Ala and Arg85Ala point mutants and a deletion mutant of the C-terminal extension clearly revealed that their RNA binding activities were independent of each other. Collectively, this study provides details for the protein-recognition mode of p14 and insight into the branch site recognition.WILEY-LISS, Jun. 2008, PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 71(4) (4), 1617 - 1636, English[Refereed]Scientific journalActivity improvement of an antimicrobial peptide, thanatin, has been achieved up to 4-fold higher than natural original one by site-specific chemical modifications with tert-butyl group at two cysteine residues which form an intramoleular disulfide bridge. The chemically modified thanatin (C11tBu/C18tBu) exhibited improved antimicrobial activity toward Gram-positive bacteria, Micrococcus luteus, whereas lowered activity toward Gram-negative bacteria, Escherichia coli. This finding suggests that disulfide-bridge formation is not only indispensable for exhibition of antimicrobial activity of thanatin but also closely related to the activity specificity towards bacteria. NMR analysis indicates that thanatin acts against Ecoli stereospecifically by taking advantage of its C-terminal beta-hairpin structure, while the activity against M. luteus does not relate to structures and correlates very well to side-chain hydrophobicity. (c) 2008 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2008, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 369(2) (2), 609 - 615, English[Refereed]Scientific journalPolyhydroxyalkanoates (PHAs) composed of a mixture of short-chain-length-medium-chain-length (SCL-MCL) hydroxyacyl monomers are biologically produced polyesters that have properties ranging from thermoplastic to elastomeric, dependent on the molar ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of properties and applications for SCL-MCL PHA copolymers, it is important to develop and characterize novel metabolic pathways for SCL-MCL PHA production. The current study shows that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes enhances the production of SCL-MCL PHA copolymer from both related and nonrelated carbon sources in Escherichia coli LS5218, indicating the flexibility of FabG as a monomer-supplying enzyme for biological PHA production.WILEY-BLACKWELL, Mar. 2008, BIOTECHNOLOGY PROGRESS, 24(2) (2), 342 - 351, English[Refereed]Scientific journalエヌ・ティー・エス, Jan. 2008, 未来材料, 8(1) (1), 56 - 61, Japaneseバイオポリマー合成プロセス研究の新潮流In our previous study, a system for producing poly(3-hydroxybutyrate) [P(3HB)] was established by introducing a polyhydroxyalkanoate (PHA) biosynthetic gene operon (phaCAB(Re)) derived from Ralstonia eutropha into Corynebacterium glutamicum. In this study, two experimental strategies have been applied to improve P(3HB) production in recombinant C glutamicum. One is a codon optimization of the N-terminal-coding region of the PHA synthase (PhaC(Re)) gene focusing on the codon usage preference for the translation system of C. glutamicum. The other is the replacement of wild-type phaC,, with a modified gene encoding a mutation of Gly4Asp (G4D), which enhanced the production of PhaCRe and P(3HB) in Escherichia coli. The introduction of these engineered PHA synthase genes into C glutamicum enhanced the production of PhaC(Re) and P(3HB). Interestingly, we found that these gene modifications also caused increases in the concentration of the translation products of the genes encoding monomer-supplying enzymes, beta-ketothiolase (PhaA(Re)) and acetoacetyl-CoA reductase (PhaB(Re)). This finding prompted us to carry out a gene dosage of phaAB(Re) for a double plasmid system, and the highest production (52.5 wt%) of P(3HB) was finally achieved by combining the gene dosage of phaAB(Re) with codon optimization. The molecular weight of P(3HB) was also increased by approximately 2-fold, as was P(3HB) content. Microscopic observation revealed that the volume of the cells accumulating P(31113) was increased by more than 4-fold compared with the non -P(3HB)-accumulating cells without filamentous morphologenesis observed in E. coli.SOC BIOSCIENCE BIOENGINEERING JAPAN, Dec. 2007, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 104(6) (6), 457 - 463, English[Refereed]Aeromonas caviae polyhydroxyalkanoate synthase (PhaC(Ac)) is an important biocatalyst for the synthesis of practically useful two-component polyhydroxyalkanoate copolymer, poly. In a previous study, two PhaC(Ac) mutants that have a single amino acid substitution of either asparagine 149 by serine (N149S) or aspartate 171 by glycine (D171G) were isolated as higher active enzymes by means of evolutionary engineering. In this study, the synergistic effects of N149S and D171G double mutation (NSDG) in PhaC(Ac) on polyhydroxyalkanoate biosynthesis were investigated in recombinant Ralstonia eutropha. The PhaC(Ac) NSDG mutant showed enhanced incorporation of longer 3-hydroxyalkanoate (3HA) units into the polyhydroxyalkanoate copolymer from octanoate (3HA fraction: 18.5 mol%) and soybean oil (5.4 mol%) as a carbon source. Besides, the NSDG mutant synthesized P(3HB) homopolymer with a very high molecular weight (M(w)=368 x 10(4)) when fructose was used as a carbon source. Thus, a combination of the beneficial mutations synergistically altered enzymatic properties, leading to synthesis of a polyhydroxyalkanoate copolymer with enhanced 3HA fraction and increased molecular weight.WILEY-BLACKWELL, Dec. 2007, FEMS MICROBIOLOGY LETTERS, 277(2) (2), 217 - 222, English[Refereed]Scientific journalPhaP is a major poly-granule-associated protein. Its gene expression is controlled by an autoregulated repressor, PhaR, in Paracoccus denitrificans. The packing force of the P(3HB) granule by PhaP is greatly influenced by the number of PhaP molecules. In this study, the effects of DNA-binding-ability-reduced mutations of PhaR on morphological change in the cellular granule formation of P(3HB) were examined under a transmission electron microscope using an Escherichia coli recombinant system. Microscopic observation indicated that stronger packing of P(3HB) granules took place when the number of PhaP molecules was increased by reduction in the DNA-binding ability of PhaR.TAYLOR & FRANCIS LTD, Jun. 2007, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 71(6) (6), 1572 - 1576, English[Refereed]Amino acid substitutions at two residues downstream from the active-site histidine of polyhydroxyalkanoate (PHA) synthases are effective for changing the composition and the molecular weight of PHA. In this study, saturation mutagenesis at the position Ala505 was applied to PHA synthase (PhaC(Ac)) from Aeromonas caviae to investigate the effects on the composition and the molecular weight of PHA synthesized in Ralstonia eutropha. The copolymer composition and molecular weight of PHA were varied by association with amino acid substitutions. There was a strong relationship between copolymer composition and PHA synthase activity of the cells. This finding will serve as a rationale for producing tailor-made PHAs.WILEY-V C H VERLAG GMBH, Jun. 2007, MACROMOLECULAR BIOSCIENCE, 7(6) (6), 846 - 854, English[Refereed]Scientific journalThe gene coding for an azoreductase, designated as an azrA, was cloned by polymerase chain reaction amplification from the genomic DNA of Bacillus sp. strain B29 isolated from soil. The azrA encoded a protein of 208 amino acids with calculated molecular mass of 22,766 Da. The enzyme was heterologously expressed in Escherichia coli with a strong band of 23 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified recombinant AzrA was a homodimer with a native molecular mass of 48 kDa containing two molecules of flavin mononucleotide (FMN; oxidized). This activity was oxygen insensitive and was nicotinamide adenine dinucleotide (reduced form; NADH) dependent. Recombinant AzrA exhibited a broad pH stability between 6 and 10 with a temperature optimum of 60-80 degrees C. The enzyme cleaved the model azo compound of methyl red [MR, 4'-(dimethylamino)-azobenzene-2-carboxylic acid] into 2-aminobenzoic acid and N, N'-dimethyl-p-phenylenediamine by ping-pong mechanism. The enzyme was not only able to decolorize MR but also able to decolorize sulfonated azo dyes such as Orange I and Acid Red 88.SPRINGER, May 2007, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75(2) (2), 377 - 386, English[Refereed]Scientific journalAntibodies with high affinity for the surface of a solid material would be advantageous in biomaterial science as a protein device. A human antibody fragment that binds to poly(hydroxybutyrate) (PHB), a biodegradable polymer matter, was generated by a phage display system. Clone PH7-3d3 was isolated after several rounds of selection and prepared as a fragment of immunoglobulin variable regions (Fv). The quartz crystal microbalance technique showed that PH7-3d3 Fv completely inhibited PHB enzymatic degradation by competing with PHB depolymerase. Kinetic analysis based on surface plasmon resonance demonstrated that PH7-3d3 Fv bound to the PHB film with an equilibrium dissociation constant of 14 nM. The three-dimensional structure of PH7-3d3 Fv was resolved to 1.7 angstrom, revealing that the complementarity determining regions (CDRs) in the Fv fragment form a relatively flat surface on which uncharged polar and aromatic amino acids are distributed in clusters. The structure of PH7-3d3 Fv was similar to that of PHB depolymerase in the orientation of aromatic residues in the binding sites. Alanine scanning mutagenesis demonstrated that these aromatic residues, especially tryptophan residues in CDRs, were critical in the interaction between PH7-3d3 Fv and PHB. Our results suggest the possible selection of an antibody fragment that binds a material surface in a manner similar to protein - ligand interaction.AMER CHEMICAL SOC, May 2007, BIOCONJUGATE CHEMISTRY, 18(3) (3), 645 - 651, English[Refereed]Scientific journalPhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PRA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly, one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).AMER SOC MICROBIOLOGY, Feb. 2007, JOURNAL OF BACTERIOLOGY, 189(3) (3), 1118 - 1127, English[Refereed]Scientific journalPoly-3-hydroxyalkanoates [P(3HA)s] are biologically produced polyesters that have attracted much attention as biodegradable polymers that can be produced from biorenewable resources. These polymers have many attractive properties for use as bulk commodity plastics, fishing lines, and medical uses that are dependent on the repeating unit structures. Despite the readily apparent benefits of using P(3HA)s as replacements for petrochemical-derived plastics, the use and distribution of P(3HA)s have been limited by their cost of production. This problem is currently being addressed by the engineering of enzymes involved in the production of P(3HA)s. Polyhydroxyalkanoate (PHA) synthase (PhaC) enzymes, which catalyze the polymerization of 3-hydroxyacyl-CoA monomers to P(3HA)s, were subjected to various forms of protein engineering to improve the enzyme activity or substrate specificity. This review covers the recent history of PHA synthase engineering and also summarizes studies that have utilized engineered PHA synthases.SPRINGER, Jan. 2007, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 73(5) (5), 969 - 979, English[Refereed]Scientific journalPoly production by Corynebacterium glutamicum was developed by introducing the phbCAB operon derived from Ralstonia eutropha. P(3HB) synthase activity was detected in this recombinant C glutamicum carrying a cell surface protein gene promoter. Intracellular P(3HB) was microscopically observed as inclusion granules and its content was calculated to be 22.5% (w/w) with a number average molecular weight of 2.1 x 10(5) and a polydispersity of 1.63.SOC BIOSCIENCE BIOENGINEERING JAPAN, Sep. 2006, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 102(3) (3), 233 - 236, English[Refereed]The gene expression for phasins (PhaP), which are predominantly polyhydroxyalkanoates (PHAs) granule-associated proteins, is regulated by a repressor protein of PhaR through the dual binding abilities of PhaR to the target DNAs and the granules. In this study, the binding functions of PhaR to poly homopolymer and P(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] copolymer productions in the recombinants of Escherichia coli. The results indicated that the S477R mutation contributed to a shift in substrate specificity to smaller monomers containing a 3HB unit rather than to an enhancement in catalytic activity. Multiple mutations of S477R with other beneficial mutations, for example, Ser325Cys, exhibited synergistic effects on both an increase in PHA production (from 9 wt % to 21 wt %) and an alteration of substrate specificity. Furthermore, the effects of complete amino acid substitutions at position 477 were characterized in terms of in vivo PHA production and in vitro enzymatic activity. The five mutations, S477Ala(A)/Phe(F)/His(H)/Arg(R)/Tyr(Y), resulted in a shift in substrate specificity to smaller monomer units. The S477Gly(G) mutant greatly enhanced activity toward all different sizes of substrates with carbon numbers ranging from 4 to 12. These results indicated that the residue 477 contributes to both the catalytic activity and substrate specificity of PHA synthase. In recombinant E. coli, the S477A/F/G/H/R/Y mutations consistently led to increases (up to 6 times that of wild-type enzyme) in weight average molecular weights of P(3HB) homopolymers. On the basis of our studies, we created a structural feasibility accounting for the mutational effects on enzymatic activity and substrate specificity of PHA synthase.AMER CHEMICAL SOC, Aug. 2006, BIOMACROMOLECULES, 7(8) (8), 2436 - 2442, English[Refereed]Scientific journalエヌ・ティー・エス, Jul. 2006, 未来材料, 6(7) (7), 44 - 51, Japanese"酵素"が演出するバイオプラスチックの生合成と生分解公益社団法人 化学工学会, 2006, 化学工学会 研究発表講演要旨集, 2006(0) (0), 759 - 759, JapaneseNi-Y型ゼオライトによる水相中希薄チオフェノール高効率除去高分子刊行会, Jan. 2006, 高分子加工, 55(1) (1), 30 - 43, Japaneseバイオプラスチック合成研究の最前線--in vivoとin vitroのクロスオーバーIn this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.AMER CHEMICAL SOC, Jul. 2005, BIOMACROMOLECULES, 6(4) (4), 2126 - 2130, English[Refereed]Scientific journalThe F420S substitution enhances the specific activity of Ralstonia eutropha PHA synthase (PhaC(Re)). We have now carried out site-directed saturation mutagenesis of F420 of PhaC(Re) and, amongst the F420 mutants, the F420S mutant gave the highest poly(3-hydroxybutyrate) (PHB) content. In vitro activity assay showed that the F420S enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. Enhancement of PHB accumulation was achieved by combination of the F420S mutation with a G4D mutation, which conferred high PHB content and high in vivo concentration of PhaC(Re) enzyme. The G4D/F420S mutant gave a higher PHB content and in vivo concentration of PhaC(Re) enzyme than the F420S mutant, while the molecular weight of the PHB polymer of the double mutant was similar to that of the F420S mutant.SPRINGER, May 2005, BIOTECHNOLOGY LETTERS, 27(10) (10), 705 - 712, English[Refereed]Scientific journal3, Mar. 2005, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 50, 262 - 269[Biosynthesis and creation of bioplastics: regulatory mechanism of bioplastic synthesis and creation of novel plastics by enzyme evolutionary engineering].[Refereed]Scientific journal3, Mar. 2005, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 50(3) (3), 262 - 269[Biosynthesis and creation of bioplastics: regulatory mechanism of bioplastic synthesis and creation of novel plastics by enzyme evolutionary engineering].[Refereed]Modification of the type I polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC(Re)) was performed through systematic in vitro evolution in order to obtain improved PhaC(Re) having an enhanced activity of poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli. For the first time, a beneficial G4D N-terminal mutation important for the enhancement of both PHB content in dry cells and PhaC(Re) level in vivo was identified. Site-directed saturation mutagenesis at the G4 position enabled us to identify other mutations conferring similar enhanced characteristics. In addition, the PHB homopolymer synthesized by most G4X single mutants also had higher molecular weights than that of the wild-type. In vitro enzymatic assays of purified G4D mutant PhaC(Re) revealed that the mutant enzyme exhibited slightly lower activity and reaction efficiency compared to the wild-type enzyme.WILEY-V C H VERLAG GMBH, Mar. 2005, MACROMOLECULAR BIOSCIENCE, 5(3) (3), 197 - 206, English[Refereed]Scientific journalIn vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 61-3 (phaC1p,) has been performed to generate highly active enzymes. In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro. Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) [P(3HB)] from glucose, compared to recombinant E. coli harboring the wild-type PHA synthase gene (0.1 wt %). Recombinant E. coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %). The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase. In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme. The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity. Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo.AMER CHEMICAL SOC, Jan. 2005, BIOMACROMOLECULES, 6(1) (1), 99 - 104, English[Refereed]Scientific journalCreation of Eco-friendly Plastics by Biotechnological ApplicationPolyhydroxyalkanoates (PHAs) are bacterial polyesters which can be used as bio-based and biodegradable plastic materials. The development of bioplastics which are produced from renewable carbon sources is a very important step for reducing the emission of carbon dioxide. Since the thermal and physical properties of PHAs are variable depending on their monomer composition, PHAs can be processed to various materials, such as fibers, films and elastomer. Although PHAs are useful and environmentally friendly material, high cost of production has limited widespread use of PHAs. The most proteins involved in PHA biosynthesis, such as PHA synthases, monomer supplying enzymes, coat proteins of PHA inclusion, and their regulators, have been cloned and characterized. PhaR is the regulator of PHA biosynthesis, and has a unique ability to bind to DNA and PHA. Recently, artificially modified PHA biosynthetic enzymes were generated by the in vitro evolutionary approach. The modified enzymes enabled us to increase the PHA content and regulate the monomer composition. The photosynthetic PHA production by the transgenic plants harboring PHA biosynthetic genes has been investigated to reduce the cost of PHA production. The targeting of enzyme into plastid and the application of highly active mutated PHA synthases enhanced PHA accumulation in plants.Japan Oil Chemists' Society, 2005, Oleoscience, 5(11) (11), 523 - 532, Japanese"電気を通す"プラスチックが生まれ,また土に還る"腐る"プラスチック(グリーンプラ)が産声を上げ大きく成長しようとしている。"軽薄短少"の申し子プラスチック製品のおかげで私たちの日常生活は快適になった。しかし,残念ながら多くの石油プラスチックは分解されにくく,じわじわと地球環境に負荷を与えつつある。この問題解決の切り札のひとつが,グリーンプラである。The Society of Polymer Science, Japan, Nov. 2004, Kobunshi, 53(11) (11), 864 - 867, JapaneseJapan Society for Bioscience, Biotechnology, and Agrochemistry, Aug. 2004, Nippon Nōgeikagaku Kaishi, 78(8) (8), 748 - 750, JapaneseIn our previous study, in vitro evolution of type 11 polyhydroxyalkanoate (PHA) synthase (PhaC1(Ps))from Pseudomonas sp. 61-3 yielded eleven mutant enzymes capable of synthesizing homopolymer of (R)-3-hydroxybutyrate [P(3HB)] in recombinant Escherichia coli JM109. These recombinant strains were capable of accumulating up to approximately 400-fold more P(3HB) than strains expressing the wild-type enzyme. These mutations enhanced the ability of the enzyme to specifically incorporate the 3HB-coenzyme A (3HB-CoA) substrate or improved catalytic efficiency toward the various monomer substrates Of C-4 to C-12 (R)-3-hydroxyacyl-CoAs which can intrinsically be channeled by PhaC1(Ps), into P(3HB-co-3HA) copolymerization. In this study, beneficial amino acid substitutions of PhaC1(Ps) were analyzed based on the accumulation level and the monomer composition of P(3HB-co-3HA) copolymers generated by E. coli LS5218 [fadR601 atoC(Con)] harboring the monomer supplying enzyme genes. Substitutions of Set by Thr(Cys) at position 325 were found to lead to an increase in the total amount of P(3HB-co-3HA) accmumulated, whereas 3HB fractions in the P(3HB-co-3HA) copolymer were enriched by substitutions of Gln by Lys(Arg, Met) at position 481. This strongly suggests that amino acid substitutions at positions 325 and 481 are responsible for synthase activity and/or substrate chain-length specificity of PhaC1(Ps),. These in vivo results were supported by the in vitro results obtained from synthase activity assays using representative single and double mutants and synthetic substrates, (R,S)-3HB-CoA and (R,S)-3-hydroxydecanoyl-CoA. Notably, the position 481 was found to be a determinant for substrate chain-length specificity of PhaC1(Ps).AMER CHEMICAL SOC, Mar. 2004, BIOMACROMOLECULES, 5(2) (2), 480 - 485, English[Refereed]Scientific journalBiotechnological studies towards the biosynthesis of polyhydroxyalkanoates (PHAs) biopolyesters have extensively progressed through the development of various metabolic engineering strategies. Historically, efficient PHA production has been achieved using the fermentation technology of naturally occurring PHA-producing bacteria based on external substrate manipulation (1st generation), and subsequent reinforcement with recombinant gene technology (2nd generation). More recently, "enzyme evolution" is becoming the 3rd generation approach for PHA production. A break-through in the chemical synthesis of macromolecules with desirable properties was achieved by the development of prominent chemical catalysts via "catalyst evolution", as represented by a series of Ziegler-Natta catalysts. Thus, one can easily accept the concept that the molecular evolution of the biocatalysts (enzymes) relevant to PHA synthesis will provide us with a chance to create novel PHA materials with high performance. The first trial of an in vitro enzyme evolution in PHA biosynthesis was reported by our group in 2001. The following literature data, as well as our own experimental results devoted to this new approach, have been accumulated over a short time. This review article focuses specifically on the concept and current case studies of the application of "enzyme evolution" to PHA biosynthesis.WILEY-V C H VERLAG GMBH, Mar. 2004, MACROMOLECULAR BIOSCIENCE, 4(3) (3), 145 - 156, English[Refereed]Scientific journalThe polyhydroxyalkanoate (PHA) synthase (PhaC(Da)) from Delftia acidovorans DS-17 (formerly Comamonas acidovorans) has a unique large insertion consisting of 40 amino acid residues in the alpha/beta hydrolase fold region. In order to examine whether this insertion is necessary for enzyme function, we generated a mutant gene where the nucleotides encoding the insertion sequence were deleted [phaC(Da)del(342-381)]. The ability of the mutant PhaC(Da) lacking the insertion sequence to produce PEA in recombinant Escherichia coli JM109 was compared with that of wild-type PhaCDa. The results revealed that the mutant enzyme had approximately one fourth the activity of the wild-type enzyme. However, there was no significant difference in PHA content accumulated in cells harboring either the mutant PhaC(Da) or wild-type PhaC(Da) nor were there any differences in the molecular masses of the produced polymers. Therefore, we have concluded that the characteristic insertion is not indispensable for PHA synthesis. Also, slight cellular proteolysis in E. coli was found specifically for wild-type PhaC(Da) by Western blot analysis. This result prompted us to further examine the proteolytic stability of PhaC(Da) in D. acidovorans. Consequently, it has been suggested that the insertion region of PhaC(Da) is susceptible to cellular proteolysis during accumulation of PEA. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Feb. 2004, FEMS MICROBIOLOGY LETTERS, 231(1) (1), 77 - 83, English[Refereed]Scientific journalPolyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C-3 to C-5, medium-chain-length (MCL) PHAs consist of monomer units of C-6 to C-14, and SCL-MCL PRAs consist of monomers ranging in size from C-4 to C-14. Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PRA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PRA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C-4 to C-10 and subsequently to produce unusual PRA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PRA production.AMER SOC MICROBIOLOGY, Feb. 2004, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 70(2) (2), 999 - 1007, English[Refereed]Scientific journalEleven laboratory-evolved polyhydroxyalkanoate (PHA) synthases which originated from Pseudomonas sp. 61-3 enzyme (PhaC1(Ps)), together with the wild-type enzyme, were applied for PHA synthesis from fructose using Ralstonia eutropha PHB(-)4 as a host strain. The evolved PhaC1(Ps) mutants had amino acid substitution(s) at position 325 and/or position 481. In these mutants, serine-325 (S325) was replaced by cysteine (C) or threonine (T), while glutamine-481 (Q481) was replaced by lysine (K), methionine (M) or arginine (R). All recombinant strains harboring the genes of the evolved PhaC1(Ps) mutants produced a significantly increased amount of PHA (55-68 wt.-%) compared with the one harboring the wild-type gene (49 wt.-%). Particularly, those evolved PhaC1(Ps) mutants having multiple amino acid substitutions showed higher activities for PHA synthesis. Characterization of the PHA by NMR spectroscopy revealed that they were copolymers consisting of (R)-3-hydroxybutyrate (98-99 mol-%) and medium-chain-length comonomers (1-2 mol-%). This study also confirmed that amino acid substitution at position 481 in PhaC1(Ps), led to an increasing molecular weight of PHA. The number-average molecular weight ((M) over bar (n)) of PHA ((M) over bar (n)=240000) synthesized by the evolved PhaC1(Ps) (Q481K) mutant was 4.6-fold greater than that ((M) over bar (n) = 52 000) synthesized by the wild-type enzyme.WILEY-V C H VERLAG GMBH, Feb. 2004, MACROMOLECULAR BIOSCIENCE, 5(2) (2), 112 - 117, English[Refereed]Scientific journalRalstonia eutropha biopolyester synthase (PhaC(Re)) is a key enzyme for catalyzing formation of polyhydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) copolyesters from (R)-3-hydroxybutyryl-CoA (3HB-CoA) and/or (R)-3-hydroxyalkanoyl-CoA (3HA-CoA). Previously, in Escherichia coli, we found a good correlation between the accumulation of PHB and PhaC(Re) activity using seven PhaC(Re) Mutants, all of which exhibited lower activities compared to the wild-type PhaC(Re). In this study, these PhaC(Re) mutants were individually introduced into a PHB-negative mutant strain (PHB(-)4) of R. eutropha, as well as a new activity-revertant (termed E11S5) of a primary mutant E11. Recombinant R. eutropha PHB(-)4 strains harboring PhaC(Re) mutant or the wild-type expression plasmids were cultivated using renewable carbon sources. No parallel relationship in the accumulation levels of PHA (PHB and PHA copolyesters) between recombinant hosts, E. coli JM109 (for PHB) and R. eutropha PHB(-)4 (for PHB or PHA copolyesters) was observed. This suggests, in contrast with the case of E. coli, that the PHA accumulation in R. eutropha is not governed solely by PhaC(Re) activity. Enhanced PHA accumulation was achieved by some mutants (including E11 and E11S5) throughout all cultivation conditions. It is likely that monomer compositional changes of PHA copolyesters generated by recombinants may be attributable to the changed substrate specificities of mutants toward 3HB-CoA and 3HA-CoA monomer substrates channeled from the related metabolic pathways. (C) 2003 Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE SA, Nov. 2003, BIOCHEMICAL ENGINEERING JOURNAL, 16(2) (2), 107 - 113, EnglishScientific journalAeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJ(Ac)) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid P-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJ(Ac) by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJ(Ac) revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C-8) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PRA formation from dodecanoate (C-12) was examined by using the mutated PhaJ(Ac) as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PRA synthase gene from Pseudomonas sp. strain 61-3 (phaC1(Ps)). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C-8) and 3-hydroxydecanoate (C-10) units were incorporated into PRA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.AMER SOC MICROBIOLOGY, Aug. 2003, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 69(8) (8), 4830 - 4836, EnglishScientific journalSmall-size (4-membered) and medium-size (5-, 6-, and 7-membered) unsubstituted lactones as well as unsubstituted macrolides (12 and 13 membered) were subjected to the ring-opening polymerization using the extracellular PHB depolymerase from Alcaligenes faecalis T1 (PhaZ(Afa)). The characteristic reactivities of the lactones were discussed based on a tertiary structure model of the active site of the PhaZAfa. With respect to the ring-size of the lactones, the 4-membered beta-propiolactone and 6-membered delta-valerolactone (delta-VL) showed the highest polymerization activity, and delta-VL seemed to be the upper size limit for the molecular recognition of the narrow active site cleft of PhaZ(Afa). On the other hand, epsilon-caprolactone, 11-undecanolide, and 12-dodecanolide, which showed excellent polymerization activities by lipases, were scarcely polymerized by PhaZ(Afa). This was ascribed to the difference in the recognition sites between PhaZ(Afa) and lipase. In addition, the effect of the substrate-binding domain of PhaZ(Afa) and the enantioselective ring-opening polymerization of (R,S)-beta-butyrolactone ((R,S)-beta-BL) were studied. The substrate-binding domain lacking PhaZAfa showed higher reactivities than PhaZ(Afa) for the polymerization of the lactones and that a significant enantioselectivity was observed at the early stage of the polymerization of (RS)-beta-BL to produce the (R)-enriched optically active poly(3-hydroxybutyrate).AMER CHEMICAL SOC, May 2003, BIOMACROMOLECULES, 4(3) (3), 537 - 543, English[Refereed]Scientific journalMeiji University, Mar. 2003, Bulletin of the Faculty of Agriculture, Meiji University, 134, 41 - 42, JapaneseLaboratory of Microbial EcologyType II synthase (PhaC1p(s)) for polyhydroxyalkanoate (PRA) from Pseudomonas sp. 61-3 was subjected to an in vitro evolution system including PCR-mediated mutagenesis in order to improve the function of PhaC1p(s) in terms of its ability to produce poly(3-hydroxybutyrate) [P(3HB)] in recombinant Escherichia coli. Based on our established in vivo assay system, two positions (Ser325 and Gln481) where mutations provided remarkable increases in P(3HB) synthesis were identified. Saturation mutagenesis at these positions was carried out to explore whether there might be more beneficial sequences for P(3HB) synthesis than those identified in the point mutation library. As a result, five single mutants [S325C (T) and Q481M (K, R)] gave rise to highly enhanced P(3HB) synthesis. Drastically enhanced P(3HB) synthesis (up to 340- to 400-fold the amount of that of the wild type) was further achieved by generation of all five variants of the double mutants combining the codons for residues 325/481. It is feasible that the replacement of Ser (specific for type II synthase) by Thr (specific for type I synthase) at position 325 resulted in acquiring greater P(3HB) synthesis ability as exhibited by type I synthases. The other hot spot, 481, that positively contributes to enhanced P(3HB) synthesis is located adjacent to a His479, a residue that forms a putative catalytic diad that can be inferred by sequence alignment.JAPANESE BIOCHEMICAL SOC, Jan. 2003, JOURNAL OF BIOCHEMISTRY, 133(1) (1), 139 - 145, English[Refereed]Scientific journalThe use of (R)-specific enoyl-coenzyme A (CoA) hydratase (PhaJ) provides a powerful tool for polyhydroxyalkanoate (PHA) synthesis from fatty acids or plant oils in recombinant bacteria. PhaJ provides monomer units for PHA synthesis from the fatty acid P-oxidation cycle. Previously, two phaJ genes (phaJ1(Pa) and phaJ2(Pa)) were identified in Pseudomonas aeruginosa. This report identifies two new phaJ genes (phaJ3(Pa) and phaJ4(Pa)) in P. aeruginosa through a genomic database search. The abilities of the four PhaJ(Pa) proteins and the (R)-3-hydroxyacyl-acyl carrier protein [(R)-3HA-ACP] dehydrases, FabA(Pa) and FabZ(Pa) to supply monomers from enoyl-CoA substrates for PHA synthesis were determined. The presence of either PhaJ1(Pa) or PhaJ4(Pa) in recombinant Escherichia coli led to the high levels of PHA accumulation (as high as 36-41 wt.% in dry cells) consisting of mainly short-(C4-C6) and medium-chain-length (C6-C10) 3HA units, respectively. Furthermore, detailed characterizations of PhaJ1(Pa) and PhaJ4Pa were performed using purified samples. Kinetic analysis revealed that only PhaJ4Pa exhibits almost constant maximum reaction rates (V-max) irrespective of the chain length of the substrates. The assay for stereospecific hydration revealed that, unlike PhaJ1(Pa), PhaJ4(Pa) has relatively low (R)-specificity. These hydratases may be very useful as monomer-suppliers for the synthesis of designed PHAs in recombinant bacteria. (C) 2002 Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE BV, Jan. 2003, INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, 31(4-5) (4-5), 195 - 205, English[Refereed]Scientific journal2003, Biomacromolecules, 2, 541 - 544Correlation between structure of the lactones and substrate specificity in enzyme-catalyzed polymerization for the synthesis of polyesters2003, Biomacromolecules, 2, 541 - 544Correlation between structure of the lactones and substrate specificity in enzyme-catalyzed polymerization for the synthesis of polyesters日本生物工学会, 2003, 生物工学会誌 : seibutsu-kogaku kaishi, 81(7) (7), 269 - 269, Japanese明治大学におけるバイオ研究体制 : 21世紀の明治維新を目指して(東日本支部2003)(BRANCH SPIRIT)日本生物工学会, 2003, 生物工学会誌 : seibutsu-kogaku kaishi, 81(12) (12), 543 - 543, Japanese酵素進化工学によるグリーンプラ生産システムの最適化(北日本支部シンポジウム開催報告)Overexpression and purification of microbial pro-transglutaminase from streptomyces cinnamoneum and in vitro processing by Streptomyces albogriseolus proteasesProcessing and activation of the precursor of an extracellular Streptomyces transglutaminase were achieved by using three Streptomyces proteases (SAM-P20, SAM-P26 and SAM-P45), all of which are widely distributed in Streptomyces. The use of these proteases would allow us to develop a production process for the active form of this enzyme in recombinant bacteria.SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2002, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 94(5) (5), 478 - 481, English[Refereed]Scientific journal12, Nov. 2002, Gan to kagaku ryoho. Cancer & chemotherapy, 29, 2174 - 2177[Significance of peritonectomy for peritonitis carcinomatosis].[Refereed]Scientific journalProcessing and activation of the precursor of an extracellular Streptomyces transglutaminase were achieved by using three Streptomyces proteases (SAM-P20, SAM-P26 and SAM-P45), all of which are widely distributed in Streptomyces. The use of these proteases would allow us to develop a production process for the active form of this enzyme in recombinant bacteria.SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2002, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 94(5) (5), 478 - 481, EnglishScientific journalBased on the metabolic pathways for polyhydroxyalkanoate (PHA) biosynthesis, we succeeded in establishing the recombinant Pseudomonas sp 61-3 strains that synthesize random copolyesters consisting of (R)-3-hydroxybutyrate (3HB) and (R)-medium-chain-length 3-hydroxyalkanoate (mcl-3HA) units, P(3HB-co-3HA), with very high 3HB compositions (up to 94 mol%) from glucose. The mechanical properties of P(94% 3HB-co-3HA) copolyester were very similar to those of low-density polyethylene. We carried out the molecular cloning and characterization of a PhaG(Ps) encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase of Pseudomonas sp 61-3. It was concluded that the PhaGPs gene product is involved in providing mcl-3HA-CoA from glucose in the original strain. Heterologous expression of the PhaGPs gene with the PhaC1(Ps) gene encoding PHA synthase from Pseudomonas sp 61-3 was performed in the PhbC(Re) negative mutant (PHB(-)4) of Ralstonia eutropha. The recombinant PHB-4 strain successfully produced PHA copolyesters consisting of 3HB and mcl-3HA units of 6-12 carbon atoms from sugars. The 3HB fraction in copolyesters was very high (95-97 mol%). The PHA content in the recombinant strain could further be increased by the additional introduction of the PhbAB(Re) genes from R eutropha encoding beta-ketothiolase and NADPH-dependent acetoacetyl-coenzyme A reductase. Moreover, we have established an in vivo assay system to analyze mutational effects of R eutropha synthase (PhbCRe) on the level of PHB accumulation in recombinant strains of Escherichia coli. The activity of the PhbCRe could be efficiently estimated using the in vivo system constructed here, and would be useful for in vitro evolution of PhbCRe. (C) 2002 Society of Chemical Industry.WILEY-BLACKWELL, Oct. 2002, POLYMER INTERNATIONAL, 51(10) (10), 899 - 906, EnglishScientific journalPhasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA, synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.AMER SOC MICROBIOLOGY, Jul. 2002, JOURNAL OF BACTERIOLOGY, 184(14) (14), 3992 - 4002, EnglishScientific journal化学工業社, Jul. 2002, 化学工業, 53(7) (7), 506 - 512, Japaneseバイオプラスチック生産における進化分子工学 (特集 創造科学技術の展望)In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Rc)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant Ell with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.JAPANESE BIOCHEMICAL SOC, Jun. 2002, JOURNAL OF BIOCHEMISTRY, 131(6) (6), 801 - 806, English[Refereed]Scientific journalStreptomyces viridosponis A-914 was screened as a producer of an enzyme to effectively form chiral intermediates of 1,4-dihydropyridine calcium antagonists. The supernatant liquid of the growing culture of this strain exhibited high activity for enantioselective hydrolysis of prochiral 1,4-dihydropyridine diesters to the corresponding (4R) half esters. The responsible enzyme (termed DHP-A) was purified to apparent homogeneity and characterized. Cloning and sequence analysis of the gene for DHP-A (dhpA) revealed that the enzyme was a serine protease that is highly similar in both structural and enzymatic feature to SAM-P45, which is known as a target enzyme of Streptomyces subtilisin inhibitor (SSI), from Streptomyces albogriseolus. In a batch reaction test, DHP-A produced a higher yield of a chiral intermediate of 1,4-dihydropyridine than the commercially available protease P6. Homologous or heterologous expression of dhpA resulted in overproduction of the enzyme in culture supernatants, with 2.4- to 4.2-fold higher specific activities than in the parent S. viridosporus A-914. This indicates that DHP-A is suitable for use in reactions forming chiral intermediates of calcium antagonists and suggests the feasibility of developing DHP-A as a new commercial enzyme for use in the chiral drug industry.AMER SOC MICROBIOLOGY, Jun. 2002, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68(6) (6), 2716 - 2725, EnglishScientific journalBy in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PRA). Aeromonas caviae FA440 synthase, termed PhaCA, was chosen as a good target for evolution, since it can synthesize a PRA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC(Ac) gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) {by LS5218 [fadR601 atoC(Con)] strain}. The expression vector for the phaC(Ac) gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).AMER SOC MICROBIOLOGY, May 2002, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68(5) (5), 2411 - 2419, EnglishScientific journal3, 2002, Japanese Journal of Gastroenterology, 99(3) (3), 282 - 288, JapaneseA case of neuroendocrine carcinoma of the rectum[Refereed]Scientific journal2002, 化学工業(総説), 53(7) (7), 22 - 28バイオプラスチック生産における進化分子工学Polymerase is a central enzyme involved in the biosynthesis of polyhydroxybutyrate (PHB), a well-known bacterial biodegradable polyester. In this study, we have established an in vivo assay system to analyze mutational effects of Ralstonia eutropha polymerase (termed PhbC(Re)) on the level of PHB accumulation in recombinant strains of Escherichia coli. This in vitro evolution system consists of a polymerase chain reaction-mediated random mutagenesis and two assay procedures, a plate assay using a PHB-staining dye and a high-pressure liquid chromatographic assay based on the converting reaction from PHB to crotonic acid. The distribution pattern of the PHB accumulation level of the mutant population using 378 clones arbitrarily selected, suggested that the present level of PhbC(Re) is high and well-optimized. It is noteworthy that many of the amino acid substitutions affecting the PHB accumulation occurred in the conserved positions or regions within an 'alpha/beta hydrolase fold' which is commonly found among hydrolytic enzymes. From a good correlation with the level of PHB accumulation, an activity estimation of the PhbC(Re) would be efficiently achieved by monitoring the level of PHB accumulation using the in vivo assay system established here. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE BV, Apr. 2001, FEMS MICROBIOLOGY LETTERS, 198(1) (1), 65 - 71, EnglishScientific journal2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 71 - 74Investigation of metabolic pathways for biopolyester production2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 77 - 80Molecular characterization of a regulatory protein (PhaR) involved in PHA biosynthesis2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 71 - 74Investigation of metabolic pathways for biopolyester production2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 3 - 6In vivo assay system of the polymerase as a key enzyme for PHA biosynthesis2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 77 - 80Molecular characterization of a regulatory protein (PhaR) involved in PHA biosynthesis2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 3 - 6In vivo assay system of the polymerase as a key enzyme for PHA biosynthesisRecently, a variety of aliphatic polyesters have been synthesized using hydrolases such as lipases and PHB depolymerases, and the reaction mechanism for these enzyme-catalyzed polymerization has been discussed. In this paper, we have studied the involvement of the catalytic amino acid residues of the hydrolase in enzyme-catalyzed polymerization with an extracellular PHB depolymerase from Alcaligenes faecalis T1. A wild-type PHB depolymerase and three kinds of site-specific mutants (catalytic amino acids were substituted) were prepared and their polymerization activities for the ring-opening polymerization of (R)-β-butyrolactone (BL) were compared. BL was polymerized at 80 °C in bulk by the wild-type enzyme to yield polymers consisting of cyclic and linear structures in a high monomer conversion. In contrast, none of the mutant enzymes showed obvious polymerization activity. These results have clearly demonstrated that the catalytic triad is indeed responsible for the enzyme-catalyzed polymerization of BL.2001, Biomacromolecules, 2(2) (2), 541 - 544, EnglishScientific journalPseudomonas sp. 61-3 produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] random copolymer consisting of monomeric units of 4-12 carbon atoms from sugars. The phaGPs gene encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase was cloned from this strain, and homologous expression of this gene under the control of the lac or the native promoter was investigated. Additional copies of the phaGPs gene in Pseudomonas sp. 61-3 led to an increase in both the polyhydroxyalkanoate (PHA) content in the cells and the fraction of medium-chain-length 3HA units in PHA. Disruption of the chromosomal phaGPs gene resulted in an increase in the fraction of the 3HB unit in PHA. The site-directed mutagenesis of the phaGPs gene was carried out to investigate the role of a HX4D motif which has been proposed to be related to PhaG activity.2001, Biomacromolecules, 2(1) (1), 142 - 147, EnglishScientific journal2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 77 - 80Molecular characterization of a regulatory protein (PhaR) involved in PHA biosynthesisFunctional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay systemPreviously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi ct al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum ct al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta -D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion. partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure- function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta -strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.OXFORD UNIV PRESS, Nov. 2000, JOURNAL OF BIOCHEMISTRY, 128(5) (5), 745 - 754, EnglishScientific journalSubstrate specificity analysis of microbial transglutaminase using proteinaceous protease inhibitors as natural model substratesThe substrate specificity of microbial transglutaminase (MTG) from Streptomyces mobaraensis (formerly categorized Streptoverticillium) was studied using a Streptomyces proteinaceous protease inhibitor, STI2, as a model amine-donor substrate. Chemical modification and mutational analysis to address the substrate requirements for MTG were carried out around the putative reactive site region of STI2 on the basis of the highly refined tertiary structure and the solvent accessibility index of Streptomyces subtilisin inhibitor, SSI, a homolog of STI2, The results suggest that the pi reactive center site (position 70 of STI2) for protease subtilisin BPN' or trypsin may be the prime Lys residue that can be recognized by MTG, when succinylated beta-casein. was used as a partner Gin-substrate. It is characteristic in that the same primary enzyme contact region of STI2 is shared by both enzymes, MTG and proteases, For quantitative analysis of the TG reaction, we established an ELISA-based monitoring assay system using an anti-SSI polyclonal antibody highly cross-reactive with STI2, Site-specific STI2 mutants were prepared by an Escherichia coli expression-secretion vector system and subjected to the assay system. We reached several conclusions concerning the nature of the flanking amino acid residues affecting the MTG reactivity of the substrate Lys residue: (i) site-specific mutations from Asn to Lys or Arg at position 69 preceding the amine-donor 70Lys, led to enhanced substrate reactivity; (ii) amino acid replacement at 67Ile with Ser led to higher substrate reactivity, (iii) additive effects were obtained by a combination of the positive mutations at positions 67 and 69 as described above, and (iv) Gly at position 65 might be essential for MTG; reaction. Moreover, the substrate specificity of guinea pig liver tissue transglutaminase (GTG) was compared with that of MTG using STI2 and its mutants, In contrast to MTG, replacement of Gly by Asp at position 65 was the most favorable for substrate reactivity. Also,70Lys appeared not to be a prime amine-donor site for GTG-mediated cross-linking, suggesting a difference in substrate recognition between. MTG and GTG.JAPANESE BIOCHEMICAL SOC, Sep. 2000, JOURNAL OF BIOCHEMISTRY, 128(3) (3), 415 - 425, EnglishScientific journalTo ascertain whether position 131 of a mesophilic protease, subtilisin BPN', is a potential critical site for cold adaptation as screened by evolutionary engineering (S. Taguchi, A. Ozaki, and H. Momose, Appl. Environ. Microbiol. 64:492-95, 1998), a full set of subtilisin BPN' mutants with mutations at position 131 was constructed by site-saturation mutagenesis.; All mutated enzymes were measured for specific activity at 10 degrees C by the quantitative titer microplate assay system using polyclonal antibody against subtilisin BPN' and a synthetic chromogenic substrate. All the mutants exhibited proteolytic activities almost the same as or higher than that of the wild-type enzyme, suggesting that position 131 may. be important for cold adaptation. In comparison with the wild type, purified mutants G131F, G131R, G131M, and G131W were found to acquire proteolytic activities (k(cat)/K-m) at 10 degrees C that were 150, 93, 84, and 50% higher, respectively. In particular, for the G131F mutant, temperature dependency in enzyme activity was shown by an increase in k(cat) and a decrease in K-m. All of these amino acid substitution mutants, G131F, G131R, G131M, and G131W, acquired increased proteolytic activities at 10 degrees C for three different synthetic peptide substrates but no increase in caseinolytic activity. Furthermore, they all conferred thermolability on the enzyme to differing extents in terms of the half-life of enzyme inactivation at 60 degrees C. No significant correlation was found between the amino acids preferred for cold adaptation surveyed here and those present at position 131 of subtilisin of psychrophilic cells naturally occurring in cold environments. Based on these findings, position 131 is a contributor in artificial evolution for acquiring a cold-active character and may not be related to physiological requirements for subtilisin-producing cells living in cold environments. Therefore, saturation mutagenesis would be effective in achieving rapid improvement in protein properties via evolutionary engineering.AMER SOC MICROBIOLOGY, Apr. 2000, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 66(4) (4), 1410 - 1415, EnglishScientific journalTwo Pseudomonas aeruginosa genes, termed phaJ1(Pa) and phaJ2(Pa), homologous to the Aeromonas caviae (R)-specific enoyl-CoA hydratase gene (phaJ(Ac)) were cloned using a PCR technique to investigate the monomer-supplying ability for polyhydroxyalkanoate (PHA) synthesis from beta-oxidation cycle. Two expression plasmids for phaJ1(Pa) and phaJ1(Pa) were constructed and introduced into Escherichia toil DH5 alpha strain. The recombinants harboring phaJ1(Pa) or phaJ2(Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities. that is, specific for short chain-length enoyl-CoA or medium chain-length enoyl-CoA, respectively. In addition, co-expression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14-29 wt% of cell dry weight from dodecanoate as a sole carbon source. It has been suggested that phaJ1(Pa) and phaJ2(Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE BV, Mar. 2000, FEMS MICROBIOLOGY LETTERS, 184(2) (2), 193 - 198, EnglishScientific journalPreviously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi et al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-β-D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colonysize (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structurefunction relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the β-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.Japanese Biochemical Society, 2000, Journal of Biochemistry, 128(5) (5), 745 - 754, EnglishScientific journalSecondary structures and structural fluctuation in a dimeric protein, Streptomyces subtilisin inhibitorBased on the nuclear magnetic resonance assignments of a dimeric protein, Streptomyces subtilisin inhibitor (SSI), microscopic details of secondary structures in solution have been elucidated. The chemical shift index of C-alpha signals, together with information on the hydrogen exchange rates of the backbone amide protons, were used to identify secondary structures. The locations of these secondary structures were found to be different in some critical points from those determined earlier by X-ray crystallography of the crystal. Notably, the beta 3 strand is completely missing and the alpha 2 helix is extended toward the C-terminus. Furthermore, hydrogen exchange experiments of individual peptide NH protons under strongly folding conditions revealed mechanisms of global and local structural fluctuation within the dimeric structure. It has been suggested that the global fluctuation of the monomeric unit occurs without affecting the accompanying monomer, in contrast to the equilibrium thermal unfolding, which is cooperative, Higher protection against hydrogen exchange for residues in part of the beta 4 strand implies that this region might serve as a folding core.JAPANESE BIOCHEMICAL SOC, Nov. 1999, JOURNAL OF BIOCHEMISTRY, 126(5) (5), 859 - 865, EnglishScientific journalA new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which me developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K-m value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V, The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51, In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.OXFORD UNIV PRESS, Oct. 1999, JOURNAL OF BIOCHEMISTRY, 126(4) (4), 689 - 693, EnglishScientific journalKLUWER ACADEMIC PUBL, Jul. 1999, JOURNAL OF BIOMOLECULAR NMR, 14(3) (3), 285 - 286, EnglishScientific journal1999, J. Biochemistry, 126(5) (5), 859 - 865Secondary structures and structural fluctuation in a dimer protein, Streptomyces subtilisin inhibitor1999, J. Biochemistry, 126(5) (5), 859 - 865Secondary structures and structural fluctuation in a dimer protein, Streptomyces subtilisin inhibitorPolyhydroxyalkanoates (PHAs) are accumulated in many gram-positive and gram-negative bacteria as intracellular carbon and energy storage material under nutrient-limited conditions. Bacterial PHAs are expected to become attractive alternatives for petrochemically based plastics, since they are biodegradable thermoplastics. Typical bacterial PHA, poly-3-hydroxybutyrate [P (3HB) or PHB], is highly crystalline, stiff and brittle. The lack of flexibility limits the range of its applications. The copolyester of (R) -3HB and longer chain (R) -3-hydroxyalkanoate, P (3HB-co-3HA), is less crystalline, more ductile, easier to mold and tougher than P (3HB). Based on the PHA biosynthesis mechanism, control of monomer composition in PHA by genetic and metabolic engineering makes it possible to synthesize a copolyester with good properties.Japan Oil Chemists' Society, 1999, Journal of Oleo Science, 48(12) (12), 1353 - 1364, Japanese
A low molecular weight PHB, termed OHB, has been identified as complexes with other biomacromolecules in a wide variety of organisms from bacteria, to plants and animals. The supermolecular complex of OHB with calcium ion-polyphosphate was demonstrated to be a prebiotic calcium ion transport channel and to be closely related to genetic competency for transforming Escherichia coli. Further studies on the physiological functions of OHB including calcium ion channel formation would pioneer the stage for future PHB polymer science.一般社団法人 日本生物物理学会, 1999, 生物物理, 39(0) (0), S31, JapaneseAn open reading frame (termed ORF-PR) encoding a metallothionein-like domain-including protein was found upstream of a previously identified Streptomyces chymotrypsin-type protease gene (sam-P20). Promoter and terminator activities of ORF-PR were detected using the promoterless Streptomyces tyrosinase gene as a reporter gene and expression of ORF-PR was supposed to occur before that of sam-P20 gene. Frameshift mutation anaysis showed that the ORF-PR product might act as a repressive regulator of the sam-P20 gene.TAYLOR & FRANCIS LTD, Dec. 1998, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 62(12) (12), 2476 - 2479, EnglishScientific journalAssay on a high-quality microtiter plate was found to allow for quantitative analysis of bacterial serine protease, subtilisin BPN', and its mutant enzymes which had been genetically engineered to be adapted to low-temperatures (Taguchi ct al., 1998. Appl. Environ. Microbiol. 64, 492-495), by using polyclonal antibody against subtilisin BPN'. The use of polyclonal antibody was crucial in normalizing the number of various different enzyme molecules in culture supernatant samples of recombinant strains of Bacillus subtilis, giving rise to the performance of specific activity assay of the enzymes. Relative activity of each mutant subtilisin BPN' to wild-type enzyme was estimated by monitoring the increased value of absorbance caused by enzymatic hydrolysis of a chromogenic substrate. The relative activity in each enzyme estimated by this method showed good coincidence with that estimated by kinetic parameters, k(cat)/K-m of the purified enzymes. We termed the system as 'ABEA' (antibody-bound enzyme assay). The efficient ABEA system developed here would be useful for the determination of specific activity of other enzymes of interest and provide us versatile applications in the field of evolutionary engineering. (C) 1998 Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE BV, Dec. 1998, JOURNAL OF BIOTECHNOLOGY, 66(2-3) (2-3), 157 - 163, EnglishScientific journalBy combination of size exclusion and reversed-phase chromatography, we have isolated a novel member of insect defensin-type antimicrobial peptides from the entire bodies of bacteria-challenged Formica rufa (hymenoptera, formicidae). The molecular mass of the purified peptide was estimated to be 4120.42 by matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. Sequence analysis revealed that this peptide consisted of 40 amino acid residues with six cysteines engaged in the formation of three intramolecular disulfide bridges. This peptide is unique among the arthropod defensins in terms of the presence of asparatic acid and alanine at position 33 and as C-terminal residue, respectively. In addition, this novel defensin from Formica rufa has the particularity to have no C-terminal extension in contrast to those reported for other hymenoptera defensins. ((C) Societe francaise de biochimie et biologie moleculaire/Elsevier, Paris).EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER, Apr. 1998, BIOCHIMIE, 80(4) (4), 343 - 346, EnglishScientific journalEngineering of a cold-adapted protease by sequential random mutagenesis and a screening systemA cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S, Taguchi, and M. Momose, Appl. Microbiol. Biotechnol. 47:46-51, 1997), The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as the k(cat)/K-m value) 100% higher than that of the wild type at 10 degrees C when N-succinyl-L-Ala- L-Ala-L-Pro-L-Phe-p-nitroanilide was used as a synthetic substrate, This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V72I), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreased K-m value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V72I) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60 degrees C and circular dichroism spectra at 10 degrees C.AMER SOC MICROBIOLOGY, Feb. 1998, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 64(2) (2), 492 - 495, EnglishScientific journalEngineering of a cold-adapted protease by sequential random mutagenesis and a screening systemA cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S. Taguchi, and H. Momose, Appl. Microbiol. Biotechnol. 47:46-51, 1997). The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as the k(cat)/K(m) value) 100% higher than that of the wild type at 10°C when N-succinyl-L-Ala-L-Ala-L- Pro-L-Phe-p-nitroanilide was used as a synthetic substrate. This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V721), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreased K(m) value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V721) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60°C and circular dichroism spectra at 10°C.Feb. 1998, Applied and Environmental Microbiology, 64(2) (2), 492 - 495, EnglishScientific journal1998, J. Biochemistry, 124(4) (4), 804 - 810An endogenous target protease, SAM-P26, of Streptomyces protease inhibitor (SSI): purification, primary structure determination and enzymatic characterization1998, J. Biochemistry, 124(4) (4), 804 - 810An endogenous target protease, SAM-P26, of Streptomyces protease inhibitor (SSI): purification, primary structure determination and enzymatic characterization日本結晶学会, 1998, 日本結晶学会誌, 40(0) (0), 99 - 99, JapaneseWe previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them ''SSI-like proteins'' (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338-4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised, The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the Pi site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site, Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites, It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins.SPRINGER VERLAG, May 1997, JOURNAL OF MOLECULAR EVOLUTION, 44(5) (5), 542 - 551, EnglishScientific journalA gene encoding a homolog of the Streptomyces chymotrypsinlike serine protease, SAM-P20, was identified downstream of the sam-p20 gene and designated SAM-P20D. This gene has two tandem Shine-Dalgarno sequences and two initiation codons. We have established vector systems with the function of tyrosinase gene-bone melanin pigmentation as a reporter for sam-p20D gene expression in Streptomyces coelicolor in order to identify the promoter and terminator activities, Using this system, the sam-p20D gene was suggested to be transcribed monocistronically.TAYLOR & FRANCIS LTD, May 1997, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 61(5) (5), 909 - 913, EnglishScientific journalWe previously isolated three extracellular endogenous enzymes from a Streptomyces albogriseolus mutant strain which were targets of Streptomyces subtilisin inhibitor (SSI) (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In the present study, of the three enzymes the largest one, with a molecular mass of 45 kDa (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), termed SAM-P45, has been characterized in detail. The entire gene encoding SAM-P45 was cloned as an approximately 10-kb fragment from S. albogriseolus S-3253 genomic DNA into an Escherichia coli host by using a shuttle plasmid vector. The amino acid sequence corresponding to the internal region of SAM-P45, deduced from the nucleotide sequence of the gene, revealed high homology, particularly in three regions around the active-site residues (Asp, His, and Ser), with the amino acid sequences of the mature domain of subtilisin-like serine proteases. In order to investigate the enzymatic properties of this protease, recombinant SAM-P45 was overproduced in Streptomyces coelicolor by using a strung SSI gene promoter. Sequence analysis of the SAM-P45 gene and peptide mapping of the purified SAM-P45 suggested that it is synthesized as a large precursor protein containing a large C-terminal prodomain (494 residues) in addition to an N-terminal preprodomain (23 and 172 residues). A high proportion of basic amino acids in the C-terminal prodomain was considered to serve an element interactive with the phospholipid bilayer existing in the C- terminal prodomain, as found in other membrane-anchoring proteases of gram- positive bacteria. It is noteworthy that SAM-P45 was found to prefer basic amino acids to aromatic or aliphatic amino acids in contrast to subtilisin BPN', which has a broad substrate specificity. The hydrolysis by SAM-P45 of the synthetic substrate (N-succinyl-L-Gly-L-Pro-L-Lys-p-nitroanilide) most preferred by this enzyme was inhibited by SSI, chymostatin, and EDTA. The proteolytic activity of SAM-P45 was stimulated by the divalent cations Ca2+ and Mg2+. From these findings, we conclude that SAM-P45 interacts with SSI and can be categorized as a novel member of the subtilisin-like serine protease family.American Society for Microbiology, 1997, Journal of Bacteriology, 179(2) (2), 430 - 438, EnglishScientific journalArtificial cold adaptation of a mesophilic protease, subtilisin BPN', was attempted by means of random mutagenesis of its entire gene coupled with screening of cleared-zone-forming colonies on skim-milk plates at a low temperature. Out of sixty clones screened at 10 degrees C, one mutant enzyme (termed M-15) was found to acquire higher proteolytic activities, specifically dependent on low temperatures ranging from 10 degrees C to 1 degrees C, in comparison with those of the wild-type. DNA sequencing analysis revealed that, by this mutation, the 84th amino acid residue, valine, was substituted by isoleucine, which is located 1.5 nm from the center of the catalytic triad in the tertiary structure of subtilisin. By kinetic analysis of the purified enzyme samples, the higher proteolytic activities of M-15 at low temperatures were found to be due to the decrease in the K-m value. There was no difference in thermostability between the wild-type and mutant enzymes, when tested by heat treatment. Circular dichroism spectra also showed no difference between them at 10 degrees C, indicating that the mutation of V841 had no effect on the secondary structure of subtilisin.SPRINGER VERLAG, Jan. 1997, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 47(1) (1), 46 - 51, EnglishScientific journalA novel member of the subtilisin-like protease family from Streptomyces albogriseolusWe previously isolated three extracellular endogenous enzymes from a Streptomyces albogriseolus mutant strain which were targets of Streptomyces subtilisin inhibitor (SSI) (S. Taguchi, A. Odaka, Y.,Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In the present study, of the three enzymes the largest one, with a molecular mass of 45 kDa (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), termed SAM-P45, has been characterized in detail. The entire gene encoding SAM-P45 was cloned as an approximately 10-kb fragment from S. albogriseolus S-3253 genomic DNA into an Escherichia coli host by using a shuttle plasmid vector. The amino acid sequence corresponding to the internal region of SAM-P45, deduced from the nucleotide sequence of the gene, revealed high homology, particularly in three regions around the active-site residues (Asp, His, and Ser), with the amino acid sequences of the mature domain of subtilisin-like serine proteases. In order to investigate the enzymatic properties of this protease, recombinant SAM-P45 was overproduced in Streptomyces coelicolor by using a strong SSI gene promoter. Sequence analysis of the SAM-P45 gene and peptide mapping of the purified SAM-P45 suggested that it is synthesized as a large precursor protein containing a large C-terminal prodomain (494 residues) in addition to an N-terminal preprodomain (23 and 172 residues). A high proportion of basic amino acids in the C-terminal prodomain was considered to serve an element interactive with the phospholipid bilayer existing in the C-terminal prodomain, as found in other membrane-anchoring proteases of gram-positive bacteria. It is noteworthy that SAM-P45 was found to prefer basic amino acids to aromatic or aliphatic amino acids in contrast to subtilisin] BPN', which has a broad substrate specificity. The hydrolysis by SAM-P45 of the synthetic substrate (N-succinyl-L-Gly-L-Pro-L-Lys-p-nitroanilide) most preferred by this enzyme was inhibited by SSI, chymostatin, and EDTA. The proteolytic activity of SAM-P45 was stimulated by the divalent cations Ca2+ and Mg2+. From these findings, we conclude that SAM-P45 interacts with SSI and can be categorized as a novel member of the subtilisin-like serine protease family.AMER SOC MICROBIOLOGY, Jan. 1997, JOURNAL OF BACTERIOLOGY, 179(2) (2), 430 - 438, EnglishScientific journal日本結晶学会, 1997, 日本結晶学会誌, 39(0) (0), 134 - 134, JapaneseFunctional mapping of amino acid residues responsible for the antibacterial action of apidaecinFunctional mapping was carried out to address the amino acid residues responsible for the activity of the antibacterial peptide apidaecin from the honeybee by an in vivo assay system developed previously. The C-terminal region and many of the proline and arginine residues which are present at high frequency in apidaecin were found to play an important role in its antibacterial activity.Dec. 1996, Applied and Environmental Microbiology, 62(12) (12), 4652 - 4655, EnglishScientific journalFunctional mapping of amino acid residues responsible for the antibacterial action of apidaecinFunctional mapping was carried out to address the amino acid residues responsible for the activity of the antibacterial peptide apidaecin from the honeybee by an in vivo assay system developed previously. The C-terminal region and many of the proline and arginine residues which are present at high frequency in apidaecin were found to play an important role in ias antibacterial activity.AMER SOC MICROBIOLOGY, Dec. 1996, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 62(12) (12), 4652 - 4655, EnglishScientific journalAug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 134 - 134, Japaneseプロテアーゼインヒビター(SIL)をモデル基質とした微生物トランスグルタミナーゼの基質特異性の解析Aug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 734 - 734, Japanese分化微生物のプロテアーゼインヒビターファミリーの分子進化Aug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 719 - 719, Japanese抗菌ペプチド "アピデシン" の進化工学Aug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 414 - 414, Japanese放射菌ズブチリシンインヒビター/SAM-P20複合体のX線結晶構造解析Aug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 414 - 414, Japanese分化微生物のプロテアーゼインヒビターと相互作用する内因性標的酵素遺伝子の発現制御Aug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 414 - 414, Japanese分化微生物のプロテアーゼ・インヒビター間相互作用系 : SSIとSAM-P26の場合The genes coding for the protease inhibitors, SSI and API-2c', have been analyzed by comparing DNA macrorestriction patterns of Streptomyces albogriseolus S-3253 and S. griseoincarnatus KTo-250 with those of inhibitor-deficient mutants. The mutants were found to suffer from chromosomal deletions rather than plasmid loss which resulted in the loss of the relevant genes. Hybridization experiments indicated that the ssi homologs in S. lividans and S. coelicolor A3(2) are located near the end of the linear chromosome.ELSEVIER SCIENCE BV, May 1996, FEMS MICROBIOLOGY LETTERS, 139(1) (1), 37 - 42, EnglishScientific journal社団法人日本農芸化学会, Mar. 1996, 日本農藝化學會誌, 70(0) (0), 276 - 276, Japanese分化微生物のプロテアーゼ・インヒビター間相互作用 : SSIとSAM-P45の場合 : 微生物社団法人日本農芸化学会, Mar. 1996, 日本農藝化學會誌, 70(0) (0), 275 - 275, Japaneseプロテアーゼ・サチライシンの低温適応化 : 微生物社団法人日本農芸化学会, Mar. 1996, 日本農藝化學會誌, 70(0) (0), 105 - 105, Japanese分化微生物のプロテアーゼインヒビターと相互作用する内因性標的酵素遺伝子の発現制御領域 : 微生物Three new proteinaceous inhibitors of trypsin and subtilisin of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family were isolated and purified from culture media of Streptomyces strains; SIL5 from S. fradiae, SIL7 from S. ambofaciens and SIL12 from S. hygroscopicus. Their complete amino-acid sequences were determined by sequence analysis of the intact SIL proteins and peptides obtained by enzymatic digestion of S-pyridylethylated proteins. SIL7 showed high sequence similarity to other Arg-possessing SSI-family inhibitors at the P1 site. SIL12 is unique in having a two-residue insertion in the flexible loop region. Based on the amino-acid sequences of these inhibitors and other SSI-family inhibitors whose sequences have already been determined, the phylogenetic relationship of SSI-family inhibitors and Streptomyces strains was considered. Among about 110 amino-acid residues possessed by SSI-family inhibitors, 28 are completely conserved. The contribution of these conserved residues to the function and stability of the inhibitor molecules is discussed on the basis of the results obtained from mutational analysis of SSI and its crystal structure.ELSEVIER SCIENCE BV, Feb. 1996, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1292(2) (2), 233 - 240, EnglishScientific journal1996, Appl. Environ. Microbiol., 62(1) (1), 4652 - 4655Functional mapping of amino acid residues responsible for antibacterial action of apidaecinAmino acid sequences of protease inhibitors (Streptomyces subtilisin inhibitor-like proteins) widely distributed in Streptomyces were compared to clarify the taxonomic status of three strains of Streptomyces spp., S. coelicolor A3(2), S. lividans 66 and S. coelicolor Muller, which are closely related by conventional taxonomical procedures. The sequence comparison indicated that S. coelicolor A3(2) is distinct from the type strain S. coelicolor Muller, but belongs to the same taxon as S. lividans 66.ELSEVIER SCIENCE BV, Jan. 1996, FEMS MICROBIOLOGY LETTERS, 135(2-3) (2-3), 169 - 173, EnglishScientific journalSTREPTOMYCES SERINE-PROTEASE (SAM-P20) - RECOMBINANT PRODUCTION, CHARACTERIZATION, AND INTERACTION WITH ENDOGENOUS PROTEASE INHIBITORPreviously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI-producing mutant strain (S. Taguchi, A, Odaka, Y, Watanabe, and H. Momose, Appl, Environ, Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter, The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20, From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine-cleavable activity, i,e, a serine protease with broad substrate specificity, For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80 degrees C by the addition of 10 mM calcium ion, The strong stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 x 10(-10) M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one dimer molecule of SSI., The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-PZ0, This reactive site is the same as that toward an exogenous target enzyme, subtilisin BPN'.AMER SOC MICROBIOLOGY, Nov. 1995, JOURNAL OF BACTERIOLOGY, 177(22) (22), 6638 - 6643, EnglishScientific journal社団法人日本農芸化学会, Jul. 1995, 日本農藝化學會誌, 69(0) (0), 319 - 319, Japanese放線菌プロテアーゼインヒビターSSIの内因性標的酵素SAM-P20の諸性質およびSSIとの複合体形成 : 酵素社団法人日本農芸化学会, Jul. 1995, 日本農藝化學會誌, 69(0) (0), 277 - 277, Japaneseランダム突然変異を用いた高活性サチライシンの創成 : 微生物社団法人日本農芸化学会, Jul. 1995, 日本農藝化學會誌, 69(0) (0), 93 - 93, Japanese放線菌菌体外プロテアーゼ遺伝子の分子クローニングおよび構造解析 : 微生物A gene encoding a homolog of the chymotrypsin-like serine protease (SAM-P20), which was isolated as the target enzyme of a protease inhibitor (SSI), was cloned from Streptomyces lividans 66. This gene contained an open reading frame of 1065 nucleotides encoding 354 amino acid residues with a putative prepro portion of 157 amino acid residues. The deduced amino acid sequence of the cloned gene had significant homology to those of members of Streptomyces extracellular chymotrypsin-like protease family. By Southern blot analysis. it was suggested that protease genes of this type are found at a high frequency in Streptomyces. In this sense, we propose to categorize this protease as a member of the 'SAL' series (SAM-P20-like proteases).TAYLOR & FRANCIS LTD, Jul. 1995, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 59(7) (7), 1386 - 1388, EnglishA secretory production system for the active form of transforming growth factor alpha (TGF alpha) was established in Streptomyces lividans using a gene encoding the secretory protease inhibitor, Streptomyces subtilisin inhibitor (SSI). It was demonstrated that deletion of one of the putative dual ssi terminators is effective to extracellularly produce a heterologous polypeptide in a fused form, The recombinant fusion protein, SSI::TGF alpha, was purified to homogeneity by a combination of hydrophobic chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). It was noteworthy that the SSI::TGF alpha hybrid protein exhibited bifunctional activity: the TGF alpha activity for cell growth promotion and the inhibitory activity of SSI. Taken together with the results of analytical gel filtration, these findings strongly indicate that each moiety in the fusion protein correctly folds and the whole hybrid molecule exists in a dimeric form, which results in its bifunctional activity.ELSEVIER SCIENCE BV, Jul. 1995, GENE, 159(2) (2), 239 - 243, EnglishJun. 1995, 日本農芸化学会誌, 69(7) (7), 745 - 747, JapaneseSSI様インヒビター群と真の標的酵素群の相互作用ならびに生理的意義A SUBTILISIN INHIBITOR PRODUCED BY STREPTOMYCES-BIKINIENSIS POSSESSES A GLUTAMINE RESIDUE AT REACTIVE-SITE P1We determined the complete amino acid sequence of a novel subtilisin inhibitor, SIL15, which had been isolated from the culture supernatant of Streptomyces bikiniensis and shown to be a member of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family, and then identified its reactive site, SIL15 is composed of 113 amino acids and exists as a dimer, Compared with other SSI-family inhibitors, SIL15 was found to be unique in that it possesses a Gin residue at the P1 site of the reactive site and has two-residue insertions in two regions, one in the alpha(1)-helix and the other in the flexible loop region near the reactive site, Inhibition of subtilisin BPN' by SIL15 (inhibitor constant, 2.7X10(-11) M) was due to the presence of a Gin residue at the P1 site, which was well consistent with the results obtained for P1-site mutants of SSI and turkey ovomucoid domain 3.OXFORD UNIV PRESS, Mar. 1995, JOURNAL OF BIOCHEMISTRY, 117(3) (3), 609 - 613, EnglishScientific journal1995, Appl. Environ. Microbiol., 61(1) (1), 180 - 186Molecular characterization of a gene encoding extracellular serine protease isolated from a subtilisin itor-deficient mutant of Streptomyces albogriseolus S-32531995, Appl. Environ. Microbiol., 61(1) (1), 180 - 186Molecular characterization of a gene encoding extracellular serine protease isolated from a subtilisin itor-deficient mutant of Streptomyces albogriseolus S-32531995, Actinomycetologica (Review of the SAJ Promotion Awardee's Lecture), 9(2) (2), 216 - 227We determined the complete amino acid sequence of a novel subtilisin inhibitor, SIL15, which had been isolated from the culture supernatant of Streptomyces bikiniensis and shown to be a member of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family, and then identified its reactive site. SIL15 is composed of 113 amino acids and exists as a dimer. Compared with other SSI-family inhibitors, SIL15 was found to be unique in that it possesses a Gln residue at the P1 site of the reactive site and has two-residue insertions in two regions, one in the α1 -helix and the other in the flexible loop region near the reactive site. Inhibition of subtilisin BPN' by SIL15 (inhibitor constant, 2.7 ×10-11 M) was due to the presence of a Gln residue at the P1 site, which was well consistent with the results obtained for P1-site mutants of SSI and Turkey ovomucoid domain 3. © 1995 BY THE JOURNAL OF BIOCHEMISTRY.Oxford University Press, 1995, Journal of Biochemistry, 117(3) (3), 609 - 613, EnglishScientific journalPreviously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI- producing mutant strain (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter. The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20. From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine- cleavable activity, i.e., a serine protease with broad substrate specificity. For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80°C by the addition of 10 mM calcium ion. The strung stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 x 10-10 M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one direct molecule of SSI. The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-P20. This reactive site is the same as that toward an exogenous target enzyme, subtilisin BPN'.American Society for Microbiology, 1995, Journal of Bacteriology, 177(22) (22), 6638 - 6643, EnglishScientific journalMOLECULAR CHARACTERIZATION OF A GENE ENCODING EXTRACELLULAR SERINE-PROTEASE ISOLATED FROM A SUBTILISIN INHIBITOR-DEFICIENT MUTANT OF STREPTOMYCES-ALBOGRISEOLUS-S-3253An extracellular serine protease produced by a mutant, M1, derived from Streptomyces albogriseolus S-3253 that no longer produces a protease inhibitor (Streptomyces subtilisin inhibitor [SSI]) was isolated. A 20-kDa protein was purified by its affinity for SSI and designated SAM-P20. The amino acid sequence of the aminoterminal region of SAM-P20 revealed high homology with the sequences of Streptomyces griseus proteases A and B, and the gene sequence confirmed the relationships. The sequence also revealed a putative amino acid signal sequence for SAM-P20 that apparently functioned to allow secretion of SAM-P20 from Escherichia coli carrying the recombinant gene. SAM-P20 produced by E. coli cells was shown to be sensitive to SSI inhibition.AMER SOC MICROBIOLOGY, Jan. 1995, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 61(1) (1), 180 - 186, EnglishScientific journalA novel serine protease inhibitor SIL8, which was isolated from the culture medium of Streptomyces virginiae and shown to be a member of the Streptomyces subtilisin-inhibitor-like (SIL) inhibitor family by sequence analysis of its amino-terminal region [Taguchi, S., Kikuchi, H., Kojima, S., Kumagai, I., Nakase, T., Miura, K. and Momose, H. (1993) Biosci. Biotech. Biochem. 57, 522-524], is the first SIL inhibitor demonstrated to show marked inhibitory activity toward alpha-chymotrypsin, in addition to strong inhibitory activity toward subtilisin BPN', a common property of inhibitors of the Streptomyces subtilisin inhibitor (SSI) family. In this study, the complete amino acid sequence of SIL8 was determined from the sequence analysis of peptides obtained by specific cleavage at the reactive site and by enzymic digestion. SIL8 was shown to exist as a dimer protein, each subunit of which was composed of 111 amino acids, and to have less than 50% similarity with other SSI-family inhibitors, indicating its most distant relationship to other members of this family. Insertion of two residues was observed in the flexible loop region of SIL8, and amino acid replacements were found not only on the molecular surface but also in the beta-sheet and hydrophobic core, suggesting that packing rearrangements of the side chains may occur in these regions to maintain the tertiary and quaternary structures. The inhibitor constants K-i obtained using synthetic substrates are 92 pM for subtilisin BPN' and 11 nM for alpha-chymotrypsin. The P1 site was identified as methionine, which was in good agreement with the substrate specificity of alpha-chymotrypsin. SSI, which also possesses a methionine residue at the P1 site, inhibits alpha-chymotrypsin poorly (inhibitor constant, 4.0 mu M). Such a difference in the inhibitory properties of SIL8 and SSI toward alpha-chymotrypsin is discussed on the basis of the structures of the inhibitors.SPRINGER VERLAG, Dec. 1994, EUROPEAN JOURNAL OF BIOCHEMISTRY, 226(2) (2), 627 - 632, EnglishScientific journal3 NOVEL SUBTILISIN-TRYPSIN INHIBITORS FROM STREPTOMYCES - PRIMARY STRUCTURES AND INHIBITORY PROPERTIESThree novel proteinaceous inhibitors, which had been identified as ''Streptomyces subtilisin inhibitor-like (SIL) proteins'' and exhibited trypsin inhibition in addition to strong inhibition toward subtilisin BPN', were purified from the culture broth of three Streptomyces strains: SIL10 from S. thermotolerans, SIL13 from S. galbus, and SIL14 from S. azureus. Their primary structures were determined by sequence analysis of intact SIL inhibitors and peptides obtained by enzymatic digestions of S-pyridylethylated SIL inhibitors. These inhibitors were composed of about 110 amino acids and existed as dimer proteins. The reactive site was identified as Lys-Gln for all three inhibitors by sequence analysis of their modified forms in which the reactive-site peptide bond was specifically cleaved by subtilisin BPN' under acidic conditions. Thus, their inhibition toward trypsin and subtilisin BPN' was due to the presence of a Lys residue at the P1 site. Inhibitor constants toward subtilisin BPN' and trypsin were also determined. These inhibitors showed relatively high sequence homology to other SSI-family inhibitors possessing a Lys residue at the P1 site, with amino acid replacements on their molecular surface.JAPANESE BIOCHEMICAL SOC, Nov. 1994, JOURNAL OF BIOCHEMISTRY, 116(5) (5), 1156 - 1163, EnglishScientific journalIN-VIVO MONITORING-SYSTEM FOR STRUCTURE-FUNCTION RELATIONSHIP ANALYSIS OF THE ANTIBACTERIAL PEPTIDE APIDAECINA unique antibacterial peptide derivative found in immune honeybee lymph, apidaecin 1b (AP1), was randomly mutagenized and characterized by a newly established system to analyze in vivo its structure-function relationship. Initially, a high-level expression host-vector system for AP1 in Escherichia coli was constructed by creating a fusion protein with the highly stable Streptomyces subtilisin inhibitor (SSI) molecule. Expression of the SSI-AP1 fusion protein was found to depend on the concentration of the transcriptional inducer isopropyl-beta-D-thio-galactopyranoside (IPTG) and to parallel the degree of growth inhibition of the transformant cells. Subsequently, apidaecin derivatives produced by localized random mutagenesis were screened with this IPTG concentration-controlled in vivo system by monitoring the growth inhibition patterns of the transformant cells. One mutant apidaecin (P9L) that had reduced activity was purified and isolated from the periplasmic fraction of an L. coli transformant. Its antibacterial activity was reduced to one-third of that of wild-type apidaecin. When considered together with the other mutations, it was concluded that several Pro residues, including that at the ninth position, are responsible for expression of the antibacterial action of apidaecin.AMER SOC MICROBIOLOGY, Oct. 1994, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 60(10) (10), 3566 - 3572, EnglishScientific journalProtein proteinase inhibitors showing sequence homology with Streptomyces subtilisin inhibitor (SSI) have been found to be distributed widely in Streptomyces species, and accordingly have been named SSI-like (SIL) proteins. SIL1 from S. cacaoi was the first of these proteins to be isolated and to be given a serial number. To study the structure-function relationship of SIL proteins, we determined the primary structure of SIL1 and measured its inhibitory activities. It was found to be composed of 110 amino acids and to exist in dimer form. The amino-acid sequence of SIL1 was unique among other characterized SIL proteins in having a one-residue deletion in two regions and a three-residue insertion in the flexible loop region. Sequence comparison indicated that SIL1 was distantly related to other members of the SSI family, and that amino-acid replacements had occurred not only on the surface of the SIL1 molecule but also in the β-sheet region. The reactive site of SIL1 was considered to be Arg70-Glu71 from sequence alignment with other SSI-family inhibitors. SIL1 inhibited subtilisin BPN′ strongly with an inhibitor constant (K1 of 2.8·10-11 M, like other members of the SSI family possessing an Arg residue at the P1 site. In contrast, SIL1 exhibited weak inhibition toward trypsin with a Ki value of 5.5 - 10-8M, possibly as a consequence of insertion of the three residues in the flexible loop region near the reactive site. This contrast seems to be due to the difference in the subsite structure of the two proteinases. © 1994.Jul. 1994, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, 1207(1) (1), 120 - 125, EnglishScientific journalPRIMARY STRUCTURE AND INHIBITORY PROPERTIES OF A PROTEINASE-INHIBITOR PRODUCED BY STREPTOMYCES-CACAOIProtein proteinase inhibitors showing sequence homology with Streptomyces subtilisin inhibitor (SSI) have been found to be distributed widely in Streptomyces species, and accordingly have been named SSI-like (SIL) proteins. SIL1 from S. cacaoi was the first of these proteins to be isolated and to be given a serial number. To study the structure-function relationship of SIL proteins, we determined the primary structure of SIL1 and measured its inhibitory activities. It was found to be composed of 110 amino acids and to exist in dimer form. The amino-acid sequence of SIL1 was unique among other characterized SIL proteins in having a one-residue deletion in two regions and a three-residue insertion in the flexible loop region. Sequence comparison indicated that SIL1 was distantly related to other members of the SSI family, and that amino-acid replacements had occurred not only on the surface of the SIL1 molecule but also in the beta-sheet region. The reactive site of SIL1 was considered to be Arg(70)-Glu(71) from sequence alignment with other SSI-family inhibitors. SIL1 inhibited subtilisin BPN' strongly with an inhibitor constant (K-i) of 2.8.10(-11)M, like other members of the SSI family possessing an Arg residue at the P1 site. In contrast, SIL1 exhibited weak inhibition toward trypsin with a K-i value of 5.5.10(-8)M, possibly as a consequence of insertion of the three residues in the flexible loop region near the reactive site. This contrast seems to be due to the difference in the subsite structure of the two proteinases.ELSEVIER SCIENCE BV, Jul. 1994, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1207(1) (1), 120 - 125, EnglishScientific journalIn order to improve a natural enzyme so as to fit industrial purposes, we have applied experimental evolution techniques comprised of successive in vitro random mutagenesis and efficient screening systems. Subtilisin BPN', a useful alkaline serine protease, was used as the model enzyme, and the gene was cloned to an Escherichia coli host-vector system. Primary mutants with reduced activities of below 80% of that of the wild type were first derived by hydroxylamine mutagenesis directly applied to subtilisin gene DNA, followed by screening of clear-zone non-forming transformant colonies cultured at room temperature on plates containing skim-milk. Then, secondary mutants were derived from each primary mutant by the same mutagenic procedure, but screened by detecting transformant colonies incubated at 10 degrees C with clear zones that were greater in size than that of the wild type. One such secondary mutant, 12-12, derived from a primary mutant with 80% activity, was found to gain 150% activity (k(cat)/K-m value) of the wild-type when the mutant subtilisin gene was subcloned to a Bacillus subtilis host-vector system, expressed to form secretory mutant enzyme in the medium, and the activity measured using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as the substrate. When N-succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide was used, 180% activity was gained. Genetic analysis revealed that the primary and secondary mutations corresponded to D197N and G131D, respectively. The activity variations found in these mutant subtilisins were discussed in terms of Ca2+-binding ability. The thermostability was also found to be related to the activity.SPRINGER VERLAG, Apr. 1994, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 41(2) (2), 239 - 244, EnglishScientific journalThree novel proteinaceous inhibitors of serine proteases which had been identified as Streptomyces subtilisin inhibitor-like (SIL) inhibitors were isolated from culture supernatant of Streptomyces; SIL2 from Streptomyces parvulus, SIL3 from Streptomyces coelicolor and SIL4 from Streptomyces lavendulae. They exhibited not only strong inhibitory activity toward subtilisin BPN' but also less strong inhibition of trypsin. Their primary sequences were determined by sequence analysis of peptides obtained by specific cleavage at the reactive site and subsequent proteolytic digestion. Each inhibitor consisted of about 110 amino acids, and was considered to form a dimer. The reactive site of the inhibitors was identified as Arg-Glu for SIL2 and SIL3, and Lys-Leu for SIL4, from sequence analysis of modified forms of the inhibitors produced from the inhibitor-subtilisin complex under acidic conditions. The presence of an arginine/lysine residue at the P1 site was in agreement with their trypsin-inhibition property. Sequence comparison with other members of the Streptomyces subtilisin inhibitor family revealed that amino acid replacements in the three isolated SIL inhibitors were frequently localized on the surface region, and many of the amino acid residues in beta-sheets and the hydrophobic core were highly conserved. Values of the inhibitor constant (K-i) toward subtilisin BPN' and trypsin were also measured, and the differences were discussed on the basis of the determined structures of the inhibitors.WILEY-BLACKWELL, Mar. 1994, EUROPEAN JOURNAL OF BIOCHEMISTRY, 220(3) (3), 911 - 918, EnglishScientific journal1994, Appl. Environ. Microbiol., 60(10) (10), 3566 - 3572In vivo monitoring system for structure-function relationship analysis of the antibacterial peptide ApidaecinThree novel proteinaceous inhibitors, which had been identified as "Streptomyces subtilisin inhibitor-like (SIL) proteins" and exhibited trypsin inhibition in addition to strong inhibition toward subtilisin BPN', were purified from the culture broth of three Streptomyces strains: SIL10 from S. thermotolerans, SIL13 from S.galbus, and SIL14 from S.azureus. Their primary structures were determined by sequence analysis of intact SIL inhibitors and peptides obtained by enzymatic digestions of S-pyridylethylated SIL inhibitors. These inhibitors were composed of about 110 amino acids and existed as dimer proteins. The reactive site was identified as Lys-Gln for all three inhibitors by sequence analysis of their modified forms in which the reactive-site peptide bond was specifically cleaved by subtilisin BPN' under acidic conditions. Thus, their inhibition toward trypsin and subtilisin BPN' was due to the presence of a Lys residue at the P1 site. Inhibitor constants toward subtilisin BPN' and trypsin were also determined. These inhibitors showed relatively high sequence homology to other SSI-family inhibitors possessing a Lys residue at the P1 site, with amino acid replacements on their molecular surface. © 1994 BY THE JOURNAL OF BIOCHEMISTRY.Oxford University Press, 1994, Journal of Biochemistry, 116(5) (5), 1156 - 1163, EnglishScientific journalIn vivo monitoring system for structure-function relationship analysis of the antibacterial peptide apidaecinA unique antibacterial peptide derivative found in immune honeybee lymph, apidaecin 1b (AP1), was randomly mutagenized and characterized by a newly established system to analyze in vivo its structure-function relationship. Initially, a high-level expression host-vector system for AP1 in Escherichia coli was constructed by creating a fusion protein with the highly stable Streptomyces subtilisin inhibitor (SSI) molecule. Expression of the SSI-AP1 fusion protein was found to depend on the concentration of the transcriptional inducer isopropyl-β-D-thio-galactopyranoside (IPTG) and to parallel the degree of growth inhibition of the transformant cells. Subsequently, apidaecin derivatives produced by localized random mutagenesis were screened with this IPTG concentration-controlled in vivo system by monitoring the growth inhibition patterns of the transformant cells. One mutant apidaecin (P9L) that had reduced activity was purified and isolated from the periplasmic fraction of an E. coli transformant. Its antibacterial activity was reduced to one-third of that of wild-type apidaecin. When considered together with the other mutations, it was concluded that several Pro residues, including that at the ninth position, are responsible for expression of the antibacterial action of apidaecin.1994, Applied and Environmental Microbiology, 60(10) (10), 3566 - 3572, EnglishScientific journalSTREPTOMYCES SUBTILISIN INHIBITOR-LIKE PROTEINS ARE DISTRIBUTED WIDELY IN STREPTOMYCETESStreptomyces subtilisin inhibitor-like proteins were found to be distributed widely in streptomycetes by using the combination of the convenient, newly developed plate assay system and an established liquid culture assay. Almost all the strains formerly categorized as Streptoverticillium species produced proteins that exhibited inhibitory activity against both subtilisin BPN' and trypsin. N-terminal regions of three purified proteins showed high structural similarity to those of other previously reported SIL inhibitors.AMER SOC MICROBIOLOGY, Dec. 1993, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 59(12) (12), 4338 - 4341, EnglishA high-level production system in Escherichia coli for an alkaline serine protease inhibitor, termed Streptomyces subtilisin inhibitor (SSI), from S albogriseolus S-3253 was established by replacing the SSI signal sequence with the OmpA signal sequence using the inducible pIN-III-ompA vector. Significant amounts of recombinant SSI, resulting from accurate cleavage of the OmpA signal peptide, were accumulated in the periplasmic space or excreted into the culture medium. The inhibitory activity of the processed protein against subtilisin BPN' was identical with that of authentic SSI. Furthermore, deletion of one of the putative dual terminators (terminator 1) resulted in a 1.9-fold increase in production. This effect on SSI gene expression efficiency was found to be governed mainly at the transcription level.SPRINGER VERLAG, Aug. 1993, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 39(6) (6), 732 - 737, EnglishScientific journalTAYLOR & FRANCIS LTD, Jul. 1993, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 57(7) (7), 1206 - 1207, EnglishStreptomyces are bacteria with a very high chromosomal G + C composition (> 70 mol%) and extremely biased codon usage. In order to investigate the relationship between codon usage and gene expression in Streptomyces, we used ssi (Streptomyces subtilisin inhibitor) as a reporter gene and monitored its secretory expression in S. lividans. In consequence of alteration of the native codons of Leu, Lys and Ser of ssi to minor ones by site-directed mutagenesis, i.e., Leu79-Leu80: CTG-CTC to TTA-TTA, Lys89: AAG to AAA, Ser108-Ser109: TCG-AGC to TCT-TCT, respectively, the production of SSI was reduced remarkably in the case of TTA codons, while it was slightly increased in the case of AAA and almost the same in TCT codons. This conspicuous decrease found for Leu codon replacement was probably due to the low availability of intracellular tRNALeu (UUA), a product of bldA which has been reported to be expressed only during the late stage of growth. © 1993.Mar. 1993, BBA - Gene Structure and Expression, 1172(3) (3), 262 - 266, EnglishScientific journalEFFECT OF A RARE LEUCINE CODON, TTA, ON EXPRESSION OF A FOREIGN GENE IN STREPTOMYCES-LIVIDANSStreptomyces are bacteria with a very high chromosomal G + C composition (> 70 mol%) and extremely biased codon usage. In order to investigate the relationship between codon usage and gene expression in Streptomyces, we used ssi (Streptomyces subtilisin inhibitor) as a reporter gene and monitored its secretory expression in S. lividans. In consequence of alteration of the native codons of Leu, Lys and Ser of ssi to minor ones by site-directed mutagenesis, i.e., Leu79-Leu80:CTG-CTC to TTA-TTA, Lys89 : AAG to AAA, Ser108-Ser109: TCG-AGC to TCT-TCT, respectively, the production of SSI was reduced remarkably in the case of TTA codons, while it was slightly increased in the case of AAA and almost the same in TCT codons. This conspicuous decrease found for Leu codon replacement was probably due to the low availability of intracellular tRNA(Leu)(UUA), a product of bldA which has been reported to be expressed only during the late stage of growth.ELSEVIER SCIENCE BV, Mar. 1993, BIOCHIMICA ET BIOPHYSICA ACTA, 1172(3) (3), 262 - 266, EnglishScientific journalTAYLOR & FRANCIS LTD, Mar. 1993, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 57(3) (3), 522 - 524, EnglishThe contribution of downstream message secondary structure to the production of Streptomyces subtilisin inhibitor (SSI) was investigated in Streptomyces lividans 66 using a cloned gene. By deletion analysis, two inverted repeats located downstream from the stop codon in the SSI gene were found to exhibit a marked effect on the amount and homogeneity of SSI gene transcript and, consequently, the efficiency of SSI productivity.ELSEVIER SCIENCE BV, Mar. 1993, FEMS MICROBIOLOGY LETTERS, 107(2-3) (2-3), 185 - 189, EnglishScientific journal1993, Biochim. Biophys. Acta, 1172(3) (3), 262 - 266Effect of a rare leucine codon, TTA, on expression of a foreign gene in Streptomyces lividans1993, Biochim. Biophys. Acta, 1172(3) (3), 262 - 266Effect of a rare leucine codon, TTA, on expression of a foreign gene in Streptomyces lividansWe attempted to screen a series of Streptomyces subtilisin inhibitor-like (SIL) proteins among several Streptomyces strains by using a highly sensitive assay system established by us. Of six randomly tested strains, four were found to produce SIL inhibitors as their major secreted proteins, suggesting that they might be distributed in a high frequency among this genus. Three inhibitors exhibited inhibition of both subtilisin BPN' and trypsin. Comparison of the amino terminal sequences of these isolated proteins with those of other reported SIL inhibitors revealed that the beta1- and beta2-sheets in SSI were highly conserved.WILEY-BLACKWELL, Dec. 1992, FEMS MICROBIOLOGY LETTERS, 99(2-3) (2-3), 293 - 297, EnglishScientific journalThe value of a heterologous peptide extracellular production system in Streptomyces using a secretory protease inhibitor, was examined. DNA was synthesized encoding apidaecin 1b (AP1), an interesting antibacterial peptide discovered in lymph fluid of the honeybee, and was joined to the Streptomyces subtilisin inhibitor (SSI) gene via a 12-bp nucleotide sequence corresponding to the amino acid sequence specific for cleavage by blood coagulation factor Xa. The fusion protein (SSI-AP1) could be expressed and excreted efficiently into the medium by culturing S. lividans 66 harbouring a plasmid vector constructed for SSI secretion, into which the synthetic DNA was introduced. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and amino acid analysis of the purified SSI-AP1 protided reasonable results of molecular size and composition value. Interestingly, SSI-AP1 protein showed bifunctional activity: inhibitory activity of SSI and antibacterial activity of AP1. The inhibitory activity against Escherichia coli could be also detected after the fusion protein was cleaved by factor Xa. The extracellular production system presented here should provide a useful tool for production, analysis of mode of action, and also for genetic improvement of antimicrobial peptides such as apidaecin. © 1992 Springer-Verlag.Springer-Verlag, Mar. 1992, Applied Microbiology and Biotechnology, 36(6) (6), 749 - 753, EnglishScientific journalEXTRACELLULAR PRODUCTION SYSTEM OF HETEROLOGOUS PEPTIDE DRIVEN BY A SECRETORY PROTEASE INHIBITOR OF STREPTOMYCESThe value of a heterologous peptide extra-cellular production system in Streptomyces using a secretory protease inhibitor, was examined. DNA was synthesized encoding apidaecin 1b (AP1), an interesting antibacterial peptide discovered in lymph fluid of the honeybee, and was joined to the Streptomyces subtilisin inhibitor (SSI) gene via a 12-bp nucleotide sequence corresponding to the amino acid sequence specific for cleavage by blood coagulation factor Xa. The fusion protein (SSI-AP1) could be expressed and excreted efficiently into the medium by culturing S. lividans 66 harbouring a plasmid vector constructed for SSI secretion, into which the synthetic DNA was introduced. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and amino acid analysis of the purified SSI-AP1 provided reasonable results of molecular size and composition value. Interestingly, SSI-AP1 protein showed bifunctional activity: inhibitory activity of SSI and antibacterial activity of AP1. The inhibitory activity against Escherichia coli could be also detected after the fusion protein was cleaved by factor Xa. The extra-cellular production system presented here should provide a useful tool for production, analysis of mode of action, and also for genetic improvement of antimicrobial peptides such as apidaecin.SPRINGER VERLAG, Mar. 1992, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 36(6) (6), 749 - 753, EnglishScientific journal1992, Actinomycetologica (Review of SAJ Invited Lecture), 6(1) (1), 9 - 20A host-vector system was used for the production of Streptomyces subtilisin inhibitor (SSI). The gene fragment encoding SSI was replaced in the vector with the tyrosinase gene of plasmid pIJ702. It was found that the optimal culture conditions for the SSI production by the former system are almost the same as those for the melanin synthesis by the latter system. This fact suggests a convenient method in that the information on the productivity of an indicator host-vector system with regard to the culture conditions can be applied for the optimization of the production of a different material with a similar host-vector system differing in the gene coding for the different product.SOC FERMENTATION BIOENGINEERING, JAPAN, 1992, JOURNAL OF FERMENTATION AND BIOENGINEERING, 73(4) (4), 317 - 318, EnglishTwo sets of the Shine-Dalgarno sequence and the initiation codon (ATG) for translation of a gene encoding the protein SSI (Streptomyces subtilisin inhibitor) were studied in vivo by site-directed mutagenesis. The result shows that each ATG can function as an initiator of translation in either Streptomyces lividans 66 or Escherichia coli. The choice of initiation codon seems dependent on the host strain and is closely related to the processing mechanism of pre-SSI protein. The upstream ATG is presumed to be utilized preferentially giving two cleavage sites in pre-SSI in S. albogriseolus S-3253, the original SSI producer strain.SPRINGER VERLAG, Apr. 1991, MOLECULAR & GENERAL GENETICS, 226(1-2) (1-2), 328 - 331, EnglishJan. 1991, Japanese放線菌プロテアーゼインヒビターSSI遺伝子の情報発現に関する研究Doctoral thesisTo elucidate differences in the mechanism of gene expression between Streptomyces and Escherichia coli, the regulatory region for expression of the gene for a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), was altered to express efficiently in E. coli. This was carried out by inserting a pre-SSI-encoding region downstream of the tac promoter and ribosome-binding site in a multi-copy plasmid. When the resultant plasmid pMKSI161-9 was introduced into E. coli JM105, SSI protein was found to be expressed and secreted into the periplasmic space by Western blot analysis. When introduced into 'leaky' E. coli strains, this protein was detected in the medium as well as in the periplasmic space in bacteria. NH2-terminal sequencing analysis of the SSI purified from E. coli JM105 indicated two processing sites, {A figure is presented} and {A figure is presented}, of pre-SSI. These sites were different from those in Streptomyces albogriseolus S-3253 and Streptomyces lividans 66. The inhibitor activity of the processed protein toward subtilisin BPN′ was almost the same as that of authentic SSI. © 1990.Jul. 1990, BBA - Gene Structure and Expression, 1049(3) (3), 278 - 285, EnglishScientific journal1990, Biochim. Biophys. Acta, 1049(3) (3), 278 - 285Comparison of secretory expression in Escherichia coli and Streptomyces of Streptomyces subtilisin inhibitor (SSI) gene1990, Biochim. Biophys. Acta, 1049(3) (3), 278 - 285Comparison of secretory expression in Escherichia coli and Streptomyces of Streptomyces subtilisin inhibitor (SSI) geneELSEVIER SCIENCE BV, Dec. 1989, GENE, 84(2) (2), 279 - 286, EnglishScientific journalNATURE PUBLISHING CO, Oct. 1989, BIO-TECHNOLOGY, 7(10) (10), 1063 - 1066, EnglishEFFICIENT EXTRACELLULAR EXPRESSION OF A FOREIGN PROTEIN IN STREPTOMYCES USING SECRETORY PROTEASE INHIBITOR (SSI) GENE FUSIONSScientific journalJAPANESE BIOCHEMICAL SOC, Mar. 1989, JOURNAL OF BIOCHEMISTRY, 105(3) (3), 367 - 371, EnglishMOLECULAR-CLONING AND NUCLEOTIDE-SEQUENCE DETERMINATION OF GENE ENCODING STREPTOMYCES SUBTILISIN INHIBITOR (SSI)Scientific journalUsing the gene for a secreted protease inhibitor, Streptomyces subtilisin inhibitor (SSI), we have established a secretory expression system for the products of foreign genes in Streptomyces. A sex pheromone peptide, cADl, of Enterococcus fae- calis was expressed and secreted as a stable complete fusion protein with SSI in amounts comparable to the native SSI in S. lividans 66. Recombinant cADl, isolated from the fusion protein by chemical digestion with BrCN, has the same amino acid composition and sequence, retention time on reverse phase HPLC, molecular mass, and biological activity as authentic cADl. This system should be useful for the efficient expression of other foreign gene products, and as a model for heterologous gene expression in Streptomyces. © 1989 Nature Publishing Group.1989, Nature Biotechnology, 7(10) (10), 1063 - 1066, EnglishScientific journalA gene for Streptomyces subtilisin inhibitor (SSI) from Streptomyces albogriseolus S-3253 was cloned into E. coli plasmid pBR322 using two oligodeoxyribonucleotides corresponding to Asp68 to Pro77 and Asn99 to Gly107 of the protein, respectively. The SSI gene was localized on a 1.8-kbp BglII/SalI fragment. The nucleotide sequence of this 1.8-kbp fragment was determined by the dideoxy sequencing method. The amino acid sequence of the mature SSI coding region derived from the nucleotide sequence determination corresponded exactly to that from protein sequencing analysis. The nucleotide sequence analysis showed the presence of a putative signal peptide comprising 31 amino acids preceding the mature SSI region. The major transcriptional start point was identified to be 60 nucleotides upstream from the putative initiation codon for translation by the primer extension method. The -45 to -25 region upstream from transcriptional start point was quite homologous to that of CTC promoter of Bacillus subtilis. The overall G+C content of this 1.8-kbp fragment was 72%. On the other hand, an extremely high G+C content (96%) was found at the third letter of codons in the SSI coding region. © 1989 BY The Journal of Biochemistry.Oxford University Press, 1989, Journal of Biochemistry, 105(3) (3), 367 - 371, EnglishScientific journalJAPAN ACAD, Jun. 1988, PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES, 64(6) (6), 147 - 149, EnglishScientific journal良書普及会, Jun. 1987, 自治研究, 63(6) (6), p3 - 33, Japanese人権の理念と基本権の実定性-下-良書普及会, May 1987, 自治研究, 63(5) (5), p27 - 53, Japanese人権の理念と基本権の実定性-上-Mar. 1987, Japanese放線菌プロテアーゼインヒビターSSI遺伝子のクローニングと塩基配列決定Master thesis1987, 東京大学工学部紀要A, 64(25) (25), 58 - 59遺伝子工学による放線菌プロテアーゼインヒビターの人工的改変繊維総合研究所, Apr. 1984, 化繊月報, 37(4) (4), p24 - 34, Japanese綿スフ織物業の活性化のために--その現状と課題官業労働研究所, Nov. 1972, 官公労働, 26(11) (11), 16 - 29公務員・公社職員等の政治活動--法規・裁判例から見たその限界第一法規, Aug. 1963, 自治研究, 39(8) (8), 153 - 160地方公務員法46条による措置要求の対象事項および公立学校職員の勤務評定に関する規則等の取消--行政判例研究-98-第一法規, Jul. 1956, 自治研究, 32(7) (7), 69 - 74行政協定の実施に伴う土地等の使用に関する特別措置法第5条による内閣総理大臣の使用の認定における物件の特定
■ MISC- 2023, 日本生物工学会大会講演要旨集, 75thEffects of LAHB degradation in the water environment on bacterial communities
- 2023, 化学と生物, 61(5) (5)メンブレンベシクル創発の新原理 バイオプラスチック合成が”引き金”
- 2023, 日本農芸化学会関東支部講演要旨集(CD-ROM), 2023プロモーター置換法による進化型ポリ乳酸の生産性向上
- 2023, 日本農芸化学会大会講演要旨集(Web), 2023Effect of bacterial flora in river water samples on the degradation of the biodegradable bioplastic LAHB
- 2023, 日本農芸化学会大会講演要旨集(Web), 2023Microbial production of lactate (LA)-based polymers varying LA fractions aimed to optimize mechanical performance upon blending with PLA
- 2023, 日本農芸化学会大会講演要旨集(Web), 2023Attempts to improve production of biodegradable plastic LAHB and control of lactate fraction by modulating gene expression activity in Escherichia coli
- 2023, 日本農芸化学会大会講演要旨集(Web), 2023Molecular breeding of Cupriavidus necator to synthesize lactate-based polymers
- 2022, 明治大学科学技術研究所年報, (63) (63)生合成「多元ポリ乳酸」の高分子量化に有効な要因解明
- 2022, 日本農芸化学会大会講演要旨集(Web), 2022Controllable secretion of membrane vesicle (MV) driven by biopolymer production system
- 2022, 日本農芸化学会大会講演要旨集(Web), 2022Biosynthesis of lactate-based bioplastics by recombinant Cupriavidus necator
- 2022, 日本放線菌学会大会講演要旨集, 36thA real thrill in Bioplastic study: Microorganisms as a key player
- 2022, 生物工学会誌, 100(9) (9)生分解性「多元ポリ乳酸LAHB」の研究ストーリー:乳酸重合酵素誕生・オリゴマー分泌生産発見・膜小胞創発
- 2022, 日本農芸化学会西日本支部大会およびシンポジウム講演要旨集, 2022Cupriavidus necatorを宿主とした乳酸ベースバイオプラスチックの生合成
- 2022, 日本生物工学会大会講演要旨集, 74thBiosynthesis of lactate-based polymers using Cupriavidus necator as a host
- 2021, 日本農芸化学会大会講演要旨集(Web), 2021Influence of Biodegradable Plastic Degradation by Microbial Community Analysis
- 2021, 岩谷直治記念財団研究報告書, 44Microbial synthesis of high-performance biodegradable plastics
- 2021, 日本農芸化学会大会講演要旨集(Web), 2021Biosynthesis of biopolyesters suitable for practical use by recombinant bacteria
- 2021, 明治大学科学技術研究所年報, (62) (62)生合成「多元ポリ乳酸」の高分子量化に有効な要因解明
- 2021, 日本生物工学会大会講演要旨集, 73rdEfficient Extraction of Biopolymer by Escherichia coli with Incomplete Peptidoglycan Synthesis
- 2021, 日本生物工学会大会講演要旨集, 73rdBiosynthesis of high-performance bioplastics by recombinant Cupriavidus necator strains
- 2021, 日本農芸化学会西日本支部大会およびシンポジウム講演要旨集, 2021 (CD-ROM)組換えCupriavidus necatorによる高性能バイオプラスチックの生合成
- 24 Jun. 2020, (公益社団法人) 日本生物工学会, 98(5) (5), 227 - 227, Japanese【随縁随意】ノーベル賞受賞者から香る研究観Report scientific journal
- 公益社団法人 日本農芸化学会, Apr. 2020, 化学と生物, 58(4) (4), 248 - 254, Japanese「多元ポリ乳酸」の生合成と生分解:メカニズム解明の鍵"オリゴマー"[Refereed][Invited]Introduction research institution
- 2020, 日本農芸化学会西日本支部大会およびシンポジウム講演要旨集, 2020 (CD-ROM)組換え微生物による高性能バイオプラスチックの生合成
- 2020, 食品の包装, 51(2) (2)プラスチック問題 バイオプラスチック「多元ポリ乳酸」:生産プロセスから機能部材化・生分解性まで
- 2020, 日本農芸化学会大会講演要旨集(Web), 2020Secretory production of 3-hydroxybutyrate oligomer in recombinant Escherichia coli haboring polyhydoroxyalkanoate synthase derived from Bacillus cereus YB-4
- 2020, 日本農芸化学会大会講演要旨集(Web), 2020Secretory production and structure analysis of microbial oligoesters with diol end groups
- 2020, 日本農芸化学会大会講演要旨集(Web), 2020Microbial secretion of 3-hydroxybutyrate oligomer by polyhydoroxyalkanoate synthases via chain transfer reaction
- 2020, 日本農芸化学会大会講演要旨集(Web), 2020Culture conditions for biosynthesis of novel lactate-based polymers
- Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by a wide range of bacteria, which serve as a promising candidate in replacing some conventional petrochemical-based plastics. PHA synthase (PhaC) is the key enzyme in the polymerization of PHA, and the crystal structures were successfully determined using the catalytic domain of PhaC from Cupriavidus necator (PhaCCn-CAT) and Chromobacterium sp. USM2 (PhaCCs-CAT). Here, we review the beneficial mutations discovered in PhaCs from a structural perspective. The structural comparison of the residues involved in beneficial mutation reveals that the residues are near to the catalytic triad, but not inside the catalytic pocket. For instance, Ala510 of PhaCCn is near catalytic His508 and may be involved in the open-close regulation, which presumably play an important role in substrate specificity and activity. In the class II PhaC1 from Pseudomonas sp. 61-3 (PhaC1Ps), Ser325 stabilizes the catalytic cysteine through hydrogen bonding. Another residue, Gln508 of PhaC1Ps is located in a conserved hydrophobic pocket which is next to the catalytic Asp and His. A class I, II-conserved Phe420 of PhaCCn is one of the residues involved in dimerization and its mutation to serine greatly reduced the lag phase. The current structural analysis shows that the Phe362 and Phe518 of PhaC from Aeromonas caviae (PhaCAc) are assisting the dimer formation and maintaining the integrity of the core beta-sheet, respectively. The structure-function relationship of PhaCs discussed in this review will serve as valuable reference for future protein engineering works to enhance the performance of PhaCs and to produce novel biopolymers.Feb. 2019, Appl. Microbiol. Biotechnol., 103(3) (3), 1131 - 1141, English, International magazine[Refereed][Invited]Introduction scientific journal
- 2019, 日本ゲノム微生物学会年会要旨集, 13thメチオニン代謝が関与するGTP生合成の新規な制御機構の解析
- 2019, 日本農芸化学会大会講演要旨集(Web), 2019メチオニン代謝が関与するGTP生合成の新規な制御機構の解析
- 2019, 日本農芸化学会大会講演要旨集(Web), 2019微生物産生ポリエステルオリゴマーを用いたウレタン材料の合成
- 2019, 日本農芸化学会大会講演要旨集(Web), 2019生分解性プラスチックの河川微生物叢への影響
- 2019, 日本農芸化学会大会講演要旨集(Web), 2019微生物群集解析による生分解性プラスチックの河川への影響
- 2019, 日本生物工学会大会講演要旨集, 71stRibulose 1,5-bisphosphate carboxylase/oxygenase(RubisCO)反応を経由したグリコール酸ポリマー生合成系構築とその応用
- 2019, 日本分子生物学会年会プログラム・要旨集(Web), 42ndメチオニン代謝が関与するGTP生合成の新規な制御機構の解析
- 2019, 日本生物工学会大会講演要旨集, 71stオリゴエステル分泌生産における種々アルコール化合物の添加効果
- 2019, 日本農芸化学会大会講演要旨集(Web), 2019ポリエステル重合酵素を用いたオリゴマーの分泌生産
- 2019, 日本農芸化学会大会講演要旨集(Web), 2019ジオール末端を有するポリエステルオリゴマーの分泌生産および構造解析
- 2019, 日本農芸化学会大会講演要旨集(Web), 2019ジオール末端基を有するポリヒドロキシアルカン酸オリゴマーの分泌生産
- 2019, 日本農芸化学会大会講演要旨集(Web), 2019ルビスコ経路を利用した組換え大腸菌におけるグリコール酸ポリマー生合成系の構築
- 2019, 日本農芸化学会大会講演要旨集(Web), 2019非天然ポリエステル生合成系の開発と配列制御型ポリマーへの展開
- Dec. 2018, Chemistry, 73(12) (12), 17 - 20, JapaneseProgress in evolutionary molecular engineering by introduction of phage display[Refereed][Invited]Introduction scientific journal
- バイオインダストリー協会, Nov. 2018, Bioscience and Industry(Japan Bioindustry Association), 76(6) (6), 462 - 466, JapaneseShortcut process development of polylactide production inspired by discovery of lactate oligomers secretionIntroduction research institution
- 29 Aug. 2018, 高分子学会予稿集(CD-ROM), 67(2) (2), ROMBUNNO.1Y05, Japanese動的配列制御に基づくブロック共重合ポリエステル生合成系の発見とポリマー構造解析
- 2018, 日本卵子学会誌, 3(1) (1)卵胞成長培地に添加するポリビニルピロリドンの分子量が卵の成熟および発生に及ぼす影響
- 2018, 繊維学会予稿集, 73(2) (2)X線繊維回折法によるポリ[(R)-2-ヒドロキシブタン酸]の結晶構造解析
- 2018, 戦略的創造研究推進事業CREST終了報告書(Web), 2018植物バイオマス原料を利活用した微生物工場による新規バイオポリマーの創製および高機能部材化
- 2018, 日本バイオマテリアル学会大会予稿集(Web), 40thポリ乳酸足場材料上の軟骨細胞培養及び分化の評価
- 2018, 化学系学協会北海道支部冬季研究発表会(Web), 2018オリゴ酪酸およびオリゴ乳酸とポリマー分解酵素間の相互作用の分子動力学シミュレーションを用いた検討
- 2018, 繊維学会予稿集, 73(1 (CD-ROM)) (1 (CD-ROM))ポリ[(R)-2-ヒドロキシブタン酸]の結晶構造解析
- 2018, 日本農芸化学会大会講演要旨集(Web), 2018組換え大腸菌による乳酸ベースオリゴマーの分泌生産と高分子合成への応用
- 2018, 日本農芸化学会大会講演要旨集(Web), 2018転写因子欠失株を用いた乳酸ベースポリマー生産大腸菌のリモデリング
- 2018, 日本農芸化学会大会講演要旨集(Web), 2018透明性を有する新規乳酸ベースポリマーの生合成
- 2018, 日本農芸化学会大会講演要旨集(Web), 2018モノマー組成制御された乳酸ベースオリゴマーの分泌生産
- 2018, 日本生物工学会大会講演要旨集, 70thグルコース/キシロース切替え流加法による乳酸ベースポリマー生産組換え大腸菌の高密度培養
- 2018, 高分子学会予稿集(CD-ROM), 67(2) (2)植物バイオマス原料から微生物合成する多元ポリ乳酸の部材化
- 高分子学会, Jun. 2017, 高分子 = High polymers, Japan : polymers, 66(6) (6), 296 - 300, JapaneseFront-Line Polymer Science : Polymers Synthesized from Plant BiomassIntroduction research institution
- 生体触媒である酵素を微生物産生ポリマーの合成と分解に利活用する上での使用原理や運用について解説している。合成では、モノマーの供給経路に関連した酵素の構造・機能・工学について述べている。分解に関しては、機能特に基質特異性を中心にまとめた。03 May 2017, Mini-Reviews in Organic Chemistry, 14(6) (6), 1 - 32, English[Refereed]Introduction research institution
- 2017, 日本生物工学会大会講演要旨集, 69thDirect secretory production of D-lactate oligomers by engineered Escherichia coli: a shortcut in the process of polylactide production
- 2017, 高分子学会予稿集(CD-ROM), 66(2) (2)ポリ乳酸合成プロセス短縮を実現する乳酸オリゴマーの微生物分泌生産系
- 2017, 日本生物工学会大会講演要旨集, 69thAeromonas caviae由来ポリヒドロキシアルカン酸重合酵素のC末端改変とポリマー生産性向上
- 2017, 戦略的創造研究推進事業CREST終了報告書(Web), 2017植物バイオマス原料を利活用した微生物工場による新規バイオポリマーの創製および高機能部材化
- 2017, 繊維学会予稿集, 72(1 (CD-ROM)) (1 (CD-ROM))配向結晶化フィルム及び単結晶を用いたポリ[(R)-2-ヒドロキシブチレート]の結晶構造および高次構造解析
- 2017, 日本生物工学会大会講演要旨集, 69th転写因子欠失株を用いた乳酸ベースポリマー生産大腸菌のリモデリング
- 2017, 日本生物工学会大会講演要旨集, 69thLactyl-CoA重合における乳酸重合酵素の反応機構解析
- 2017, 日本生物工学会大会講演要旨集, 69th組換え大腸菌による新規モノマー組成からなる乳酸ベースポリマーの生合成
- 2017, 日本生物工学会大会講演要旨集, 69thポリ(乳酸-co-3-ヒドロキシ酪酸)フィルムの柔軟性に及ぼす結晶性と熱物性の影響
- 2017, 日本農芸化学会大会講演要旨集(Web), 2017パルプ製造廃液を炭素源とした乳酸ベースポリマーの生産
- 2017, 日本農芸化学会大会講演要旨集(Web), 2017非天然ポリステル合成のための重合酵素の高活性化
- 2017, 日本農芸化学会大会講演要旨集(Web), 2017組換え大腸菌による新規乳酸ベースポリマーの生合成
- 2017, 日本農芸化学会大会講演要旨集(Web), 2017組換え大腸菌による乳酸ポリマー生産のためのキシロースを炭素源とした高密度培養
- 2017, 日本生物工学会大会講演要旨集, 69th抗菌ペプチド・アピデシンおよび高活性変異体の作用機構解析
- 2017, 日本生物工学会大会講演要旨集, 69th昆虫由来抗菌ペプチドの高活性化と作用機序解明のための新アプローチ
- 2017, 化学関連支部合同九州大会・外国人研究者交流国際シンポジウム講演予稿集, 54th新規の生分解性乳酸ベースポリマーの生合成
- プラスチックス・エージ, Jan. 2017, Plastics age, 63(1) (1), 71 - 75, JapaneseMulti-dimensional Polylactic Acid Biosynthesis and Properties/FunctionalityIntroduction research institution
- 日本工業出版, Nov. 2016, Plastics, 67(11) (11), 30 - 33, JapaneseBaloon-type microbial factory for production of bioplastics[Refereed][Invited]Introduction research institution
- 日本バイオプラスチック協会, Aug. 2016, Biopla Journal, 62(2) (2), 18 - 26, JapaneseAdvanced polylactide created by synthetic biology[Refereed][Invited]Introduction research institution
- 加工技術研究会, Apr. 2016, コンバーテック, 44(4) (4), 125 - 127, Japanese乳酸ベースバイオプラスチックの創製と「風船型」微生物工場の誕生 (コンバーティングとホワイトバイオ)Introduction research institution
- The Adhesion Society of Japan, Feb. 2016, Journal of The Adhesion Society of Japan, 52(2) (2), 44 - 49, JapaneseIntroduction research institution
- 2016, 日本農芸化学会大会講演要旨集(Web), 2016Mechanistic analysis of poly(lactate-co-3-hydroxybutyrate) degradation by soil bacterium depolymerase
- 2016, 農業食料工学会年次大会講演要旨, 75thEscherichia coliを用いた乳酸ベースバイオポリマー生産の検討
- 2016, 日本農芸化学会大会講演要旨集(Web), 2016加水分解性を有するグリコール酸ベースポリマーの微生物合成
- 2016, 日本農芸化学会大会講演要旨集(Web), 2016ジャーファメンターを用いた組換えE.coliによる乳酸ベースバイオポリマー生産法の確立
- 2016, 日本農芸化学会大会講演要旨集(Web), 2016バイオマスを利用した非天然バイオプラスチックの生合成
- 2016, 日本農芸化学会大会講演要旨集(Web), 2016組換え大腸菌による乳酸ユニットを含む新規モノマー組成からなる生分解共重合ポリエステルの生合成
- 2016, 日本農芸化学会大会講演要旨集(Web), 2016ゲノム工学を用いた物質生産微生物工場開発の新戦略
- 2016, 化学関連支部合同九州大会・外国人研究者交流国際シンポジウム講演予稿集, 53rd乳酸ユニットを含む新規モノマー組成からなる生分解性プラスチックの生合成
- 2016, ポリマーフロンティア21講演要旨集, 2016(2) (2)酵素進化工学と合成生物学に基づいて創製する環境調和型キラルポリマー
- 2016, 高分子学会北海道支部研究発表会講演要旨集, 50thポリ乳酸およびポリヒドロキシ酪酸単分子溶液の分子動力学シミュレーション:バイオポリエステル分解酵素の機構解明を目指して
- 2016, 高分子学会予稿集(CD-ROM), 65(1) (1)ポリ[(R)-2-ヒドロキシブチレート]の基礎物性,結晶構造および酵素分解性
- 2016, 日本生物工学会大会講演要旨集, 68th脂質系バイオマスからの中鎖ホモポリヒドロキシアルカン酸の生合成とその物性解析
- 2016, 高分子学会予稿集(CD-ROM), 65(2) (2)ポリ(乳酸-co-3-ヒドロキシ酪酸)の結晶化挙動および力学物性におけるモノマー組成の影響
- 2016, 繊維学会予稿集, 71(1 (CD-ROM)) (1 (CD-ROM))ポリ(乳酸-co-3-ヒドロキシ酪酸)ナノファイバーの作製と細胞培養基材への応用
- 2016, 高分子学会予稿集(CD-ROM), 65(2) (2)合成生物学的手法により創製した多元ポリ乳酸の物性および生分解性
- 2016, 日本ゲノム微生物学会年会要旨集, 10thゲノム工学を用いた細胞内蓄積型物質生産に有益な宿主の開発
- 2016, 日本農芸化学会北海道支部講演会講演要旨, 2016木質抽出液の加水分解物を原料とした乳酸ベースポリマー生産
- 2016, 日本農芸化学会北海道支部講演会講演要旨, 2016酵素進化工学による高活性化ポリ(2-ヒドロキシ酪酸)[P(2HB)]重合酵素の創出
- 2016, 日本農芸化学会北海道支部講演会講演要旨, 2016キシロースを炭素源とした乳酸ポリマーを生産する組換え大腸菌の高密度培養
- 2016, 日本農芸化学会北海道支部講演会講演要旨, 2016転写因子欠失株を用いた乳酸ベースポリマー生産大腸菌のリモデリング
- 2016, 日本農芸化学会北海道支部講演会講演要旨, 2016Synthetic biology for creation of novel bioplastics
- The choice of an appropriate microbial host cell and suitable production conditions is crucial for the downstream processing of pharmaceutical- and food-grade products. Although Escherichia coli serves as a highly valuable leading platform for the production of value-added products, like most Gram-negative bacteria, this bacterium contains a potent immunostimulatory lipopolysaccharide (LPS), referred to as an endotoxin. In contrast, Gram-positive bacteria, notably Bacillus, lactic acid bacteria (LAB), Corynebacterium, and yeasts have been extensively used as generally recognized as safe (GRAS) endotoxin-free platforms for the production of a variety of products. This review summarizes the currently available knowledge on the utilization of these representative Gram-positive bacteria for the production of eco- and bio-friendly products, particularly natural polyesters, polyhydroxyalkanoates, bacteriocins, and membrane proteins. The successful case studies presented here serve to inspire the use of these microorganisms as a main-player or by-player depending on their individual properties for the industrial production of these desirable targets.SPRINGER, Nov. 2015, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 99(22) (22), 9349 - 9360, English[Refereed][Invited]Book review
- 25 Aug. 2015, 高分子学会予稿集(CD-ROM), 64(2) (2), ROMBUNNO.1Y08, Japanese乳酸ポリマーを高蓄積する微生物工場の創製とポリマー高機能化
- 08 Jun. 2015, 繊維学会予稿集, 70(1 (CD-ROM)) (1 (CD-ROM)), ROMBUNNO.1B07, Japaneseポリ[(R)‐2‐ヒドロキシブチレート]に対する結晶構造解析の試み
- 12 May 2015, 高分子学会予稿集(CD-ROM), 64(1) (1), ROMBUNNO.2PD108, Japaneseポリ[(R)‐2‐ヒドロキシブチレート]の結晶構造解析
- 12 May 2015, 高分子学会予稿集(CD-ROM), 64(1) (1), ROMBUNNO.2PD104, Japanese様々な新規微生物産性ポリエステルの熱物性と光学特性
- 化学工学会, Apr. 2015, Chemical Engineering, 79(4) (4), 310 - 312, JapaneseMicrobial Plastic Factory : from Petrochemical Industry to Biochemical Industry[Invited]Introduction research institution
- 05 Mar. 2015, 日本農芸化学会大会講演要旨集(Web), 2015, 2B33P04 (WEB ONLY), Japanese組み換え体大腸菌で生産する資源循環型微生物ポリマーの高生産化に関与する新規遺伝子の探索
- 05 Mar. 2015, 日本農芸化学会大会講演要旨集(Web), 2015, 2B33P07 (WEB ONLY), Japanese組換え大腸菌によるグリコール酸ベースポリマー合成とポリマーの物性評価
- 05 Mar. 2015, 日本農芸化学会大会講演要旨集(Web), 2015, 2B33P03 (WEB ONLY), Japanese大腸菌転写制御因子欠失株を用いたP(LA‐co‐3HB)の高生産化
- 05 Mar. 2015, 日本農芸化学会大会講演要旨集(Web), 2015, 2C31A04 (WEB ONLY), Japanese耐酸性乳酸菌Lactobacillus acetotolerans HTの乳酸脱水素酵素遺伝子のクローニングと生分解性共重合ポリエステルの生合成
- 05 Mar. 2015, 日本農芸化学会大会講演要旨集(Web), 2015, 2B33P02 (WEB ONLY), Japanese組換え大腸菌を利用した高光学純度(S)‐3‐ヒドロキシブタン酸の生産
- 05 Mar. 2015, 日本農芸化学会大会講演要旨集(Web), 2015, 2B33P01 (WEB ONLY), Japanese組換え大腸菌を用いた高光学活性(R)‐3‐ヒドロキシブタン酸の生産におけるCoA転移酵素高活性変異体の効果
- 05 Mar. 2015, 日本農芸化学会大会講演要旨集(Web), 2015, 2C31A05 (WEB ONLY), Japanese組換え大腸菌による糖からの生分解性共重合ポリエステルの生合成
- シーエムシー出版, Feb. 2015, Bio industry = バイオインダストリー, 32(2) (2), 26 - 35, JapaneseMicrobial Factory for Lactate-based Polymer Driven by Lactate-polymerizing Enzyme and Function Improvement of Polylactic Acid : Towards Multiple-component PLAIntroduction research institution
- 2015, 日本農芸化学会大会講演要旨集(Web), 2015Production of poly(lactate-co-hydroxybutyrate) from Miscanthus-derived sugars
- 2015, 日本農芸化学会大会講演要旨集(Web), 2015Characterization of the substrate specificity of a poly(enriched lactate-co-3-hydroxybutyrate) depolymerase
- 2015, 高分子学会予稿集(CD-ROM), 64(2) (2)3-ヒドロキシ酪酸および乳酸をモノマーユニットに含む生分解性ポリエステルナノファイバーの作製と特性解析
- 2015, 高分子学会予稿集(CD-ROM), 64(2) (2)配向結晶性フィルム及び単結晶を用いたポリ[(R)-2-ヒドロキシブチレート]の結晶構造解析
- 2015, Institute for Fermentation, Osaka. Research Communications, (29) (29)ポリ乳酸ステレオコンプレックスの高効率生分解システム開発に資するD乳酸ポリマー分解酵素生産菌の探索とカクテル分解酵素製剤開発
- 2015, 日本農芸化学会北海道支部講演会講演要旨, 2015組換え大腸菌を用いたグリコール酸ベースポリマーの生合成および加水分解性評価
- 2015, 日本農芸化学会北海道支部講演会講演要旨, 2015ゲノム変異導入法を用いた資源循環型微生物生産ポリマー高生産化株の創成
- 2015, 日本農芸化学会北海道支部講演会講演要旨, 2015Miscanthus × giganteus hydrolysate as the carbon source for the microbial production of poly(lactate-co-3-hydroxybutyrate)
- 2015, 日本農芸化学会北海道支部講演会講演要旨, 2015乳酸ベースバイオポリマー生産のための最適培養条件の検討
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 218 - 218, Japanese2P-174 The creation of high efficiency bioplastic producing host strain by genome engineering
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 218 - 218, English2P-175 Degradation mechanism of poly(lactate-co-3-hydroxybutyrate) by depolymerase from soil bacterium Variovorax sp. C34
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 218 - 218, Japanese2P-176 Production of lactate-base polymer from herbaceous plant biomass
- 05 Aug. 2014, 日本生物工学会大会講演要旨集, 66th, 68, Japaneseグリコール酸ポリマーの生合成経路の構築
- 05 Aug. 2014, 日本生物工学会大会講演要旨集, 66th, 68, Japanese高分子量2‐ヒドロキシブタン酸ベースポリマーの生合成および物性解析
- 05 Aug. 2014, 日本生物工学会大会講演要旨集, 66th, 67, Japaneseゲノム工学的手法を用いた微生物ポリマー高生産化宿主の創成
- 05 Aug. 2014, 日本生物工学会大会講演要旨集, 66th, 16, Japanese二酸化炭素からプラスチックを合成する人工生命システムの創製―最短の合成ルートは?―
- 05 Aug. 2014, 日本生物工学会大会講演要旨集, 66th, 67, Japanese乳酸ポリマー合成コリネ菌の代謝解析に基づく高生産化
- 05 Aug. 2014, 日本生物工学会大会講演要旨集, 66th, 78, JapaneseLactobacillus acetotolerans HTの乳酸脱水素酵素遺伝子を利用した乳酸ユニットを含む生分解性プラスチックの生合成
- 05 Aug. 2014, 日本生物工学会大会講演要旨集, 66th, 67, Japanese進化工学的手法によるポリヒドロキシアルカン酸合成酵素の乳酸重合能力の強化
- 05 Aug. 2014, 日本生物工学会大会講演要旨集, 66th, 66, Japanese生合成乳酸ベースポリマーの高生産化を目指した宿主大腸菌の遺伝子改変
- 05 Aug. 2014, 日本生物工学会大会講演要旨集, 66th, 67, Japanese組換え大腸菌を用いたグリコール酸ベースポリマーの生合成と酵素分解性
- 09 May 2014, 高分子学会予稿集(CD-ROM), 63(1) (1), ROMBUNNO.2K21, Japanese微生物産生ポリ[(R)‐ラクテート‐co‐(R)‐2‐ヒドロキシブチレート]の結晶性および熱的性質
- 05 Mar. 2014, 日本農芸化学会大会講演要旨集(Web), 2014, 4SY03-6 (WEB ONLY), Japaneseバイオマスから高効率で乳酸プラスチックを生産する微生物工場の開発とポリマー物性評価
- 05 Mar. 2014, 日本農芸化学会大会講演要旨集(Web), 2014, 2A01P07 (WEB ONLY), Japanese微生物を利用したキラルP(2‐ヒドロキシ酪酸)の合成とポリマー解析
- 05 Mar. 2014, 日本農芸化学会大会講演要旨集(Web), 2014, 2A01P08 (WEB ONLY), Japanese植物バイオマス由来の単糖混合物を炭素源としたバイオポリエステルの生産
- 2014, 日本農芸化学会大会講演要旨集(Web), 2014ゲノム変異導入法による微生物ポリマー高生産化宿主の創成
- 2014, 高分子学会予稿集(CD-ROM), 63(2) (2)微生物産生ポリエステルの熱分解特性と紡糸条件に関する研究
- 日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 68 - 68, English1P-202 Enzymatic characterization of a polymer degrading enzyme purified from an isolated bacterium :
- 日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 85 - 85, English2S-Ba02 Microbial plastic factory driven by renewable carbon sources :
- 2014, 繊維学会予稿集, 69(1 (CD-ROM)) (1 (CD-ROM)), ROMBUNNO.2P250, Japaneseポリ[(R)‐2‐ヒドロキシブチレート]の基礎物性評価と構造解析の試み
- The development of synthetic biology has transformed microbes into useful factories for producing valuable polymers and/or their precursors from renewable biomass. Recent progress at the interface of chemistry and biology has enabled the production of a variety of new biopolymers with properties that substantially differ from their petroleum-derived counterparts. This review touches on recent trials and achievements in the field of biopolymer synthesis, including chemo-enzymatically synthesized aliphatic polyesters, wholly biosynthesized lactate-based polyesters, polyhydroxyalkanoates and other unusual bacterially synthesized polyesters. The expanding diversities in structure and the material properties of biopolymers are key for exploring practical applications. The enzyme and metabolic engineering approaches toward this goal are discussed by shedding light on the successful case studies. © 2013 Elsevier Ltd.Dec. 2013, Current Opinion in Biotechnology, 24(6) (6), 1054 - 1060, English[Refereed][Invited]Book review
- 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
- 27 Nov. 2013, 日本農芸化学会北海道支部講演会講演要旨, 2013, 37, Japanese抗菌スペクトルを改変した抗菌ペプチド「アピデシン」特殊変異体の創成とその抗菌活性
- 27 Nov. 2013, 日本農芸化学会北海道支部講演会講演要旨, 2013, 37, Japanese組換えコリネ型細菌によるポリ乳酸様ポリマー生産の増強
- 微生物ポリマーであるポリヒドロキシアルカン酸PHAの主要構成ユニットは3-ヒドロキシ酸を基本骨格に持つ。しかし、新しく開発した乳酸重合酵素は、乳酸をはじめグリコール酸や2-ヒドロキシ酸ブタン酸などの2-ヒドロキシ酸を基本骨格に有する新しいモノマーを取り込んだポリマーの合成が可能になったことを詳述した。Sep. 2013, Appl. Microbiol. Biotechnol., 97(18) (18), 8011 - 8021, English[Refereed][Invited]Introduction scientific journal
- 28 Aug. 2013, 高分子学会予稿集(CD-ROM), 62(2) (2), ROMBUNNO.2X03, Japanese微生物重合系に基づく乳酸ポリマーからの拡張
- 25 Aug. 2013, 日本生物工学会大会講演要旨集, 65th, 238, Japanese2‐ヒドロキシブタン酸ベースポリマーの微生物合成とその物性解析
- 25 Aug. 2013, 日本生物工学会大会講演要旨集, 65th, 237, Japanese非可食性植物バイオマスからのP(3HB)の微生物生産
- 25 Aug. 2013, 日本生物工学会大会講演要旨集, 65th, 237, Japanese糖を利用した組換え大腸菌内でのグリコール酸ベースポリマーの生産
- 25 Aug. 2013, 日本生物工学会大会講演要旨集, 65th, 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
- 25 Aug. 2013, 日本生物工学会大会講演要旨集, 65th, 237, Japanese植物バイオマスを用いた組換え大腸菌による共重合ポリエステルの生産
- 25 Aug. 2013, 日本生物工学会大会講演要旨集, 65th, 237, Japaneseポリヒドロキシアルカン酸重合酵素の機能改変によるポリマー構造制御
- 25 Aug. 2013, 日本生物工学会大会講演要旨集, 65th, 238, Japanese不飽和モノマーを導入した2‐ヒドロキシブタン酸ベースポリマーの生合成とその応用
- 生物史上初めて乳酸が微生物細胞内でポリマーの形で合成された.自然界になかった“乳酸重合酵素”の開発がもたらしたブレークスルーである.これを契機に,伝統的な“乳酸発酵”から“乳酸ポリマー発酵”という新しい概念が誕生することになる.本稿では,「炭素モル濃度」という尺度を導入した「メタボリックケミストリー」の切り口から,両発酵の化学量論的考察を加えた.今後,メタボロミクス分野で議論するうえで一つの化学的指標を与えるであろう.Japan Society for Bioscience, Biotechnology, and Agrochemistry, 01 Jul. 2013, KAGAKU TO SEIBUTSU, 51(7) (7), 448 - 456, JapaneseIntroduction research institution
- 05 Mar. 2013, 日本顕微鏡学会関東支部講演会予稿集, 37th, 41, JapanesePLAを蓄積した大腸菌の透過型電子顕微鏡観察
- 05 Mar. 2013, 日本農芸化学会大会講演要旨集(Web), 2013, 4C12A05 (WEB ONLY), JapaneseRalstonia eutropha由来PHA重合酵素の改変による2‐ヒドロキシブタン酸重合能力の強化
- 05 Mar. 2013, 日本農芸化学会大会講演要旨集(Web), 2013, 4C12A08 (WEB ONLY), JapanesePhaB高活性体を用いた光学活性(R)‐3‐ヒドロキシブタン酸生産の向上
- 05 Mar. 2013, 日本農芸化学会大会講演要旨集(Web), 2013, 4SY06-5 (WEB ONLY), Japanese微生物ポリエステル合成系酵素の機能改変と多元ポリ乳酸の創製
- 05 Mar. 2013, 日本農芸化学会大会講演要旨集(Web), 2013, 4C12A09 (WEB ONLY), Japanese動力学的解析および立体構造に基づいた進化型アセトアセチル‐CoAレダクターゼの高活性化メカニズムの解明
- 05 Mar. 2013, 日本農芸化学会大会講演要旨集(Web), 2013, 4C12A10 (WEB ONLY), Japanese改変型重合酵素による2‐ヒドロキシブタン酸ベースポリマーの生合成と物性解析
- 05 Mar. 2013, 日本農芸化学会大会講演要旨集(Web), 2013, 4C12A12 (WEB ONLY), JapaneseCorynebacterium glutamicumをプラットフォームとしたポリ乳酸様ポリマー合成系の効率化のための代謝工学および培養工学的アプローチ
- 日本バイオプラスチック協会 ; 2007-, 01 Feb. 2013, バイオプラジャーナル, 12(4) (4), 12 - 17, Japaneseバイオプラ最前線 微生物はどこまでのポリエステルを合成できるかIntroduction research institution
- 2013, 日本農芸化学会大会講演要旨集(Web), 2013PCB分解菌によるビフェニルからのポリヒドロキシアルカン酸(PHA)生産
- 2013, 日本応用酵素協会誌, (47) (47)新規モノマー導入乳酸ポリマー創製を目指した酵素進化工学
- 2013, JST News, 2013(Nov) (Nov)CO2を資源活用せよ!植物の新たな可能性を引き出す「微生物工場」でつくる高付加価値バイオプラスチック
- This review touches on recent trials for producing microbial polyhydroxyalkanoates (PHAs), which are used as a bio-based plastic, from renewable and non-edible lignocellulosic biomass. Lignocellulose is composed of cellulose, hemicellulose and lignin, which form a persistent complex. Thus, for the efficient saccharification of lignocellulose, the physical/chemical processes for removing lignin and unstiffening cellulose fibers are required. The obtained sugar is typically a mixture of glucose and xylose, and contains a certain inpurity derived from lignocellulose and/or byproduct generated during pretreatment and subsequent saccharification processes. The microbes used for PHA production need to utilize the mixed sugar and to be resistant to the impurities. This review introduces several examples for addressing this issue. Moreover, an important direction is to design the polymer with better properties. Metabolic and enzyme engineering are powerful tools to biosynthesize various useful polymers from nonrelated sugar carbon sources. In particular, microbial production of lactate-based polymer from xylose is a potent platform. ©2013, The Society of Polymer Science,.Society of Polymer Science, 2013, Kobunshi Ronbunshu, 70(12) (12), 675 - 683, JapaneseBook review
- 01 Nov. 2012, 日本農芸化学会北海道支部講演会講演要旨, 2012, 25, Japanese可変領域への変異導入とその組み合わせによる抗菌ペプチド「アピデシン」の抗菌スペクトル改変
- 01 Nov. 2012, 日本農芸化学会北海道支部講演会講演要旨, 2012, 26, Japaneseセルロース系糖質バイオマスを原料とした高乳酸分率ポリマーの効率的生産
- 01 Nov. 2012, 日本農芸化学会北海道支部講演会講演要旨, 2012, 27, Japanese進化工学的改変による高活性PhaBの速度論的解析とPHB生産性の向上
- 01 Nov. 2012, 日本農芸化学会北海道支部講演会講演要旨, 2012, 26, JapaneseRalstonia eutropha由来ポリヒドロキシアルカン酸重合酵素の改変によるClass I乳酸重合酵素の創出
- 25 Sep. 2012, 日本生物工学会大会講演要旨集, 64th, 27, Japanese乳酸重合活性を有するClass Iポリヒドロキシアルカン酸(PHA)重合酵素の創出
- 25 Sep. 2012, 日本生物工学会大会講演要旨集, 64th, 26, JapaneseCoA転移酵素を用いた大腸菌による3‐ヒドロキシブタン酸の生産
- 25 Sep. 2012, 日本生物工学会大会講演要旨集, 64th, 27, Japanese2‐ヒドロキシブタン酸ベース新奇バイオプラスチックの微生物合成と物性解析
- 25 Sep. 2012, 日本生物工学会大会講演要旨集, 64th, 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
- 05 Sep. 2012, 高分子学会予稿集(CD-ROM), 61(2) (2), ROMBUNNO.1PB118, Japanese熱安定性酵素を用いたポリヒドロキシアルカン酸のin vitro合成
- 日本生物工学会, 25 Jul. 2012, 生物工学会誌 : seibutsu-kogaku kaishi, 90(7) (7), 411 - 414, JapaneseMicrobial Plastic Factory : ポリ乳酸から多元ポリ乳酸の時代へIntroduction research institution
- 05 Mar. 2012, 日本農芸化学会大会講演要旨集(Web), 2012, 2C01P05 (WEB ONLY), JapaneseCorynebacterium glutamicumの細胞表層提示技術を用いたデンプンからのポリヒドロキシブタン酸(PHB)の直接生産
- 05 Mar. 2012, 日本農芸化学会大会講演要旨集(Web), 2012, 2C01P07 (WEB ONLY), Japanese組換え大腸菌による(R)‐3‐ヒドロキシブタン酸の生産
- 05 Mar. 2012, 日本農芸化学会大会講演要旨集(Web), 2012, 2C01P06 (WEB ONLY), JapaneseアセトアセチルCoAレダクターゼ高活性変異体の解析とそれを用いたCorynebacterium glutamicumによるPHB生産の増強
- 05 Mar. 2012, 日本農芸化学会大会講演要旨集(Web), 2012, 2C01P08 (WEB ONLY), Japanese進化工学による新規乳酸重合酵素の探索
- 05 Mar. 2012, 日本農芸化学会大会講演要旨集(Web), 2012, 4A07A01 (WEB ONLY), Japanese抗菌ペプチド「アピデシン」の可変領域への変異導入とその抗菌活性への影響
- 2012, 育種学研究, 14新規シトクロムP450遺伝子CYP72A31はイネにおける除草剤ビスピリバックナトリウム耐性に関与する
- 2012, 日本応用酵素協会誌, (46) (46)酵素・代謝複合改変によって進化する乳酸ポリマー微生物工場の先進研究
- 2012, 生体触媒化学シンポジウム講演要旨集, 16thMicrobial Plastic Factory~生体触媒開発によって実現する多元ポリ乳酸微生物生産システム~
- 2012, 日本農芸化学会大会講演要旨集(Web), 2012組換え大腸菌による乳酸およびグリコール酸ベース新奇バイオプラスチックの生合成と物性解析
- 2012, 日本農芸化学会大会講演要旨集(Web), 2012Biosynthesis of novel lactate(LA)-based polyester incorporating unsaturated monomer unit by using recombinant Escherichia coli.
- 2012, 高分子学会予稿集(CD-ROM), 61(1) (1)バイオで創る多元ポリ乳酸
- 2012, 日本農芸化学会大会講演要旨集(Web), 2012Microbial Plastic Factory: metabolic-enzyme engineering
- 2012, 日本応用酵素協会誌, (46) (46)酵素・代謝複合改変によって進化する乳酸ポリマー微生物工場の先進研究
- 2012, 酵素工学ニュ-ス, (68) (68)Microbial Plastic Factor-酵素工学研究が駆動する多元ポリ乳酸創製-
- 2012, 日本農芸化学会北海道支部講演会講演要旨, 2012大腸菌を用いた高光学純度(R)-3-ヒドロキシブタン酸の効率的生産
- 石油学会, 01 Dec. 2011, ペトロテック = Petrotech, 34(12) (12), 867 - 872, Japanese乳酸ポリマー生産用微生物工場 : 石油原料からバイオマス原料へIntroduction research institution
- 13 Sep. 2011, 高分子学会予稿集(CD−ROM), 60(2 Disk1) (2 Disk1), ROMBUNNO.1PC117, JapanesePseudomonas sp.SG4502由来耐熱性PHA合成酵素と乳酸重合酵素としての利用
- バイオインダストリー協会, 01 Sep. 2011, Bioscience & industry, 69(5) (5), 375 - 379, JapaneseA microbial factory for lactate polymersIntroduction research institution
- 25 Aug. 2011, 日本生物工学会大会講演要旨集, 63rd, 149, Japanese真菌に対する抗菌ペプチドThanatin誘導体の作用評価
- 25 Aug. 2011, 日本生物工学会大会講演要旨集, 63rd, 232, Japanese進化工学的手法による高乳酸分率のポリ(乳酸‐co‐3‐ヒドロキシブタン酸)が合成可能な乳酸重合酵素の探索
- 25 Aug. 2011, 日本生物工学会大会講演要旨集, 63rd, 232, Japanese組換え大腸菌による乳酸およびグリコール酸ベース新奇バイオプラスチックの生合成
- 25 Aug. 2011, 日本生物工学会大会講演要旨集, 63rd, 232, Japanese進化型PhaBによる組換えタバコにおけるPHB生産性の増強
- 25 Aug. 2011, 日本生物工学会大会講演要旨集, 63rd, 231, Japanese進化工学的改変により得られたアセトアセチル‐CoAレダクターゼ高活性変異体の解析
- 05 Mar. 2011, 日本農芸化学会大会講演要旨集, 2011, 22, Japanese抗菌ペプチドThanatin誘導体の酵母様真菌に対する抗菌活性
- 05 Mar. 2011, 日本農芸化学会大会講演要旨集, 2011, 4, JapanesePHA生合成系におけるモノマー供給酵素アセトアセチル‐CoAレダクターゼの高活性変異体の酵素反応解析
- 05 Mar. 2011, 日本農芸化学会大会講演要旨集, 2011, 4, Japanese乳酸ベース共重合体P(lactate‐co‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)の生合成および物性評価
- 05 Mar. 2011, 日本農芸化学会大会講演要旨集, 2011, 21, Japanese抗菌ペプチド「アピデシン」のN末端領域分子デザインによる高活性変異体の作製と抗菌スペクトル評価
- 05 Mar. 2011, 日本農芸化学会大会講演要旨集, 2011, 4, Japanese高活性化PhaBの導入による組換えタバコでのPHB生産性の向上
- 05 Mar. 2011, 日本農芸化学会大会講演要旨集, 2011, 5, Japanese高乳酸分率のポリ(乳酸‐co‐3‐ヒドロキシブタン酸)[P(LA‐co‐3HB)]が合成可能な乳酸重合酵素の探索
- 01 Feb. 2011, 高分子学会北海道支部研究発表会講演要旨集, 45th, 77, Japaneseグルコースを炭素源とした乳酸ベースポリマーの微生物生産
- 01 Feb. 2011, 高分子学会北海道支部研究発表会講演要旨集, 45th, 26, JapanesePoly(lactate‐co‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)の生合成および物性評価
- 2011, 日本応用酵素協会誌, (45) (45)乳酸重合酵素の進化工学によって駆動する乳酸ポリマー微生物工場の開発研究
- 2011, 高分子学会北海道支部研究発表会講演要旨集, 45thEfficient xylose utilization in microbial production of lactate-based polymer
- 2011, 日本応用酵素協会誌, (45) (45)乳酸重合酵素の進化工学によって駆動する乳酸ポリマー微生物工場の開発研究
- 2011, 日本生物工学会大会講演要旨集, 63rdProduction of lactate-based polyesters from xylose in Escherichia coli
- 2011, 日本農芸化学会大会講演要旨集, 2011酵素・代謝複合改変による次世代乳酸ポリマーの創製
- 2011, 日本結晶学会年会講演要旨集, 2011Structures of AzrA, AzrC, and AzrC-substrate complexes
- 2011, 日本応用酵素協会誌, (45) (45)乳酸重合酵素が駆動する乳酸ポリマーの完全生合成プロセス
- 日本生物工学会, 2011, 日本生物工学会大会講演要旨集, 63, 187 - 187, English2Ka02 Biosynthesis of poly(lactate-co-3-hydroxybutyrate) in endotoxin-free Corynebacterium glutamicum expressing lactatepolymerizing enzyme :
- 日本生物工学会, 2011, 日本生物工学会大会講演要旨集, 63, 123 - 123, English2Bp13 Biosynthesis of flexible lactate-based copolymers incorporating 3-hydroxyvalerate unit in recombinant Escherichia coli by feeding of propionate :
- 2011, 日本農芸化学会北海道支部講演会講演要旨, 2011, 8, Japanese組換え大腸菌を利用した(R)‐3‐ヒドロキシ酪酸の生産
- 2011, 日本農芸化学会北海道支部講演会講演要旨, 2011, 9, Japanese乳酸およびグリコール酸ベース新奇バイオプラスチックの微生物生産
- 2011, 日本農芸化学会北海道支部講演会講演要旨, 2011, 8, Japanese高乳酸分率ポリ(乳酸‐co‐3‐ヒドロキシブタン酸)合成のための乳酸重合酵素の進化工学的改変
- 2011, 日本農芸化学会北海道支部講演会講演要旨, 2011, 1, Japaneseアミノ酸残基置換による抗菌ペプチド「アピデシン」の抗菌スペクトルの改変
- One-pot Production of Lactate-Based Polyesters Using Engineered Microbes Expressing Lactate-Polymerizing EnzymePoly (lactic acid) (PLA) is the most widespread biobased plastic with excellent transparency. Such biobased plastics are expected to be an alternative to petroleum-derived materials. Although PLA is currently produced from fermentative lactate (LA) via chemical polymerization using heavy metal catalysts, we developed a new bioprocess producing LA-based polyester by one-pot reaction. The breakthrough for establishing this process was the discovery of lactate-polymerizing enzyme (LPE). LPE is capable of synthesizing LA-based polyesters with extremely high enantiomer excess. Moreover, the LA-based copolyesters incorporating PHA constituents showed improved flexibility. Therefore, the microbial LA-based polyester is a potent candidate for an environmental friendly plastic.SOC POLYMER SCIENCE JAPAN, 2011, KOBUNSHI RONBUNSHU, 68(5) (5), 271 - 280, JapaneseIntroduction research institution
- Activity Improvement of Antimicrobial Peptides by a Chemical Modification Approach: Toward the Creation of Novel Types of Antimicrobial AgentsThere is currently a threat posed by the rapid emergence of antibiotic-resistant pathogens due to the abuse of conventional antibiotics. To overcome such a resistance problem, it is necessary to develop peptide agents with potent antimicrobial activities and high selectivity for target bacterial strains. Conventional approaches, such as the deletion, addition, and replacement of amino acid residues in the template peptide have been employed to alter the properties of antimicrobial peptides (AMPs). In this review, we summarize a recently developed approach, chemical modification, for the improvement of various types of naturally-occurring AMPs. By applying this design strategy to these peptide sequences, new AMPs with enhanced antimicrobial activities and improved selectivity for microorganisms have been successfully generated. This potential strategy should facilitate the creation of novel class of peptide antibiotics with properties suitable for pharmaceutical application.BENTHAM SCIENCE PUBL LTD, Nov. 2010, MINI-REVIEWS IN ORGANIC CHEMISTRY, 7(4) (4), 282 - 289, English[Refereed][Invited]Introduction scientific journal
- 化学工業社, Oct. 2010, ケミカルエンジニヤリング, 55(10) (10), 770 - 777, Japaneseバイオポリマー合成プロセス開発の変遷--化学工場→微生物工場→植物工場Introduction research institution
- 25 Sep. 2010, 日本生物工学会大会講演要旨集, 62nd, 165, Japaneseポリヒドロキシアルカン酸生合成系のモノマー供給酵素acetoacetyl‐CoA reductaseの進化工学的改変
- 25 Sep. 2010, 日本生物工学会大会講演要旨集, 62nd, 166, Japanese組換え大腸菌を用いたポリ(乳酸‐co‐3‐ヒドロキシブタン酸‐co‐3‐ヒドロキシヘキサン酸)の生産
- 25 Sep. 2010, 日本生物工学会大会講演要旨集, 62nd, 178, JapaneseN末分子デザインによる抗菌ペプチド「アピデシン」の高活性体創出
- 25 Sep. 2010, 日本生物工学会大会講演要旨集, 62nd, 178, Japanese抗菌ペプチドThanatinのシステイン部位に導入されたアルキル基の側鎖長が抗菌活性に及ぼす影響
- 25 Sep. 2010, 日本生物工学会大会講演要旨集, 62nd, 165, JapanesePseudomonas sp.61‐3由来PHA重合酵素の新規高活性変異体の取得と機能解析
- Current advances in microbial cell factories for lactate-based polyesters driven by lactate-polymerizing enzymes: toward further creation of new LA-based polyesters乳酸重合活性を新たに獲得した酵素の進化工学の道筋、開発した乳酸重合酵素を活用したポリ乳酸およびそのコポリマーの効率的な合成法について解説した。Aug. 2010, Polym. Degrad. Stabilit., 95(8) (8), 1421 - 1428, English[Refereed]Introduction research institution
- SOC FIBER SCIENCE TECHNOLOGY, Aug. 2010, SEN-I GAKKAISHI, 66(8) (8), P268 - P273, JapaneseParadigm Shift for Synthesis of Lactate Polymer : from Chemical Factory to Microbial FactoryIntroduction research institution
- 15 May 2010, 日本蛋白質科学会年会プログラム・要旨集, 10th, 38, Japanese新奇かつデザイナブルなバイオポリエステル合成のための重合酵素の機能改変
- 05 Mar. 2010, 日本農芸化学会大会講演要旨集, 2010, 249, Japanese領域指定変異による抗菌ペプチド”Apidaecin”変異体の作製とその抗菌活性評価
- 05 Mar. 2010, 日本農芸化学会大会講演要旨集, 2010, 292, Japaneseジャーファーメンターを用いた乳酸ベースポリマーの微生物発酵生産
- 05 Mar. 2010, 日本農芸化学会大会講演要旨集, 2010, 292, Japanese乳酸重合酵素への新規変異導入による乳酸コポリマーの分率制御
- 05 Mar. 2010, 日本農芸化学会大会講演要旨集, 2010, 292, Japaneseルテニウム触媒によるセルロース糖化物を用いたポリヒドロキシブタン酸(PHB)の微生物生産
- 05 Mar. 2010, 日本農芸化学会大会講演要旨集, 2010, 292, Japanese3‐ヒドロキシブタン酸ベースバイオポリマーの組成制御を目指したモノマー供給酵素の進化工学的改変
- Enzymatic and whole-cell synthesis of lactate-containing polyesters: towards the complete biological production of polylactateポリ乳酸の微生物合成ために必要な関連酵素の進化工学や代謝改変による微生物プラットフォームのデザインについて解説している。特に、乳酸ユニットを取り込むコポリマーの合成に、重合酵素の改変、培養通気度、代謝改変の効果に関して詳細に論述した。Feb. 2010, Appl. Microbiol. Biotechnol., 85(4) (4), 921 - 932, English[Refereed]Introduction research institution
- 26 Jan. 2010, 高分子学会北海道支部研究発表会講演要旨集, 44th, 48, Japanese立体選択的乳酸ベースポリマーのバイオ合成と構造解析
- 26 Jan. 2010, 高分子学会北海道支部研究発表会講演要旨集, 44th, 49, Japanese3‐ヒドロキシ吉草酸ユニットを含む新規乳酸ベース3元共重合体の微生物合成
- 2010, 高分子学会予稿集(CD-ROM), 59(1 Disk1) (1 Disk1)ポリエステル重合酵素の基質特異性改変における高速多検体スクリーニング法の評価
- 2010, 日本農芸化学会大会講演要旨集, 2010ラショナル,ランダム変異による微生物トランスグルタミナーゼの活性向上
- 2010, 日本農芸化学会大会講演要旨集, 2010微生物トランスグルタミナーゼのジスルフィド結合導入による耐熱化
- 2010, 生化学水モミを生じる組換え体イネを用いた抗菌物質タナチンの生産
- 2010, 日本生物工学会大会講演要旨集, 62nd共重合組成比を指標として取得した改変型PHA重合酵素の基質特異性評価
- 2010, 日本生物工学会シンポジウム講演要旨集, 2010Improving the monomer-supplying function of FabH for polyhydroxyalkanoate production through site-specific mutagenesis
- 2010, 日本農芸化学会大会講演要旨集, 2010PCB分解菌によるビフェニルからのポリヒドロキシアルカン酸生産
- 2010, 環境工学連合講演会講演論文集, 24thバイオベースポリマー生産のための微生物工場と植物工場
- 2010, 日本生物工学会シンポジウム講演要旨集, 2010, 29, Japanese担持ルテニウム触媒によるセルロース糖化反応を利用したポリヒドロキシブタン酸(PHB)の微生物生産
- 日本バイオプラスチック協会, 01 Nov. 2009, バイオプラジャーナル, 9(3) (3), 18 - 23, JapaneseBP最前線 乳酸ポリマー完全生合成システムの幕開け--新原理に基づく微生物工場の誕生Introduction research institution
- 01 Sep. 2009, 高分子学会予稿集(CD-ROM), 58(2 Disk1) (2 Disk1), ROMBUNNO.1U-18, Japanese乳酸ポリマー生産用微生物工場の開発
- 01 Sep. 2009, 高分子学会予稿集(CD-ROM), 58(2 Disk1) (2 Disk1), ROMBUNNO.3PC149, Japanese乳酸重合酵素によるP(LA‐co‐3HB)の化学酵素合成
- 01 Sep. 2009, 高分子学会予稿集(CD−ROM), 58(2 Disk1) (2 Disk1), ROMBUNNO.3PC149, Japanese乳酸重合酵素によるP(LA‐co‐3HB)の化学酵素合成
- 25 Aug. 2009, 日本生物工学会大会講演要旨集, 61st, 36, Japanese抗菌ペプチドApidaecinの高活性化のための一戦略:細胞内導入量の向上
- 25 Aug. 2009, 日本生物工学会大会講演要旨集, 61st, 62, JapaneseBacillus sp.B29由来アゾ還元酵素基質複合体の結晶構造解析
- 25 Aug. 2009, 日本生物工学会大会講演要旨集, 61st, 36, Japanese抗菌ペプチドThanatinおよびその高活性化学修飾体の細菌に対する作用
- 25 Aug. 2009, 日本生物工学会大会講演要旨集, 61st, 191, Japanese組換え大腸菌を用いた新規乳酸ベース3元共重合体の生産
- シーエムシー出版, Jul. 2009, Bio industry, 26(7) (7), 54 - 62, JapaneseAn engineered enzyme drives microbial factory for lactate polymerIntroduction research institution
- 05 Mar. 2009, 日本農芸化学会大会講演要旨集, 2009, 331, Japanese乳酸ベースポリエステルの微生物発酵生産
- 05 Mar. 2009, 日本農芸化学会大会講演要旨集, 2009, 332, Japanese乳酸ベースポリエステルの生合成における培養条件の検討
- 05 Mar. 2009, 日本農芸化学会大会講演要旨集, 2009, 331, Japanese嫌気条件下における乳酸ベースポリエステルの微生物発酵生産
- 03 Feb. 2009, 高分子学会北海道支部研究発表会講演要旨集, 43rd, 57, Japanese組換え微生物を用いた乳酸ベースポリマーの発酵生産
- 03 Feb. 2009, 高分子学会北海道支部研究発表会講演要旨集, 43rd, 14, Japanese酵素の進化工学を応用した新規バイオポリマー生産系の開発
- The synthesis and degradation of biopolymers are cardinal reactions that take place to maintain homeostasis in biosystems. Fundamental biological reactions can be reproduced in vitro by means of enzyme-mediated processes. Recently, bioprocesses have been extensively adopted to synthesize representative biopolymers, including aliphatic polyesters, e.g., polyhydroxyalkanoate (PHA). In particular, the protein engineering of enzymes involved in PIIA synthesis has made a great impact on the tailor-made synthesis of polymer materials with high performance. Biopolymers can also be synthesized in vitro by enzyme-catalyzed polymerization using synthetic enzymes and depolymerase-mediated reverse reactions. We will describe these advanced topics from the viewpoint of the boundary field between chemistry and biotechnology.BENTHAM SCIENCE PUBL LTD, Feb. 2009, MINI-REVIEWS IN ORGANIC CHEMISTRY, 6(1) (1), 44 - 54, English[Refereed]Book review
- 2009, 日本農芸化学会大会講演要旨集, 2009PHB生合成系酵素群の発現効率化の組換え体タバコにおけるPHB生産性への影響
- 2009, 日本農芸化学会大会講演要旨集, 2009乳酸ポリマー生産用微生物工場の開発
- 2009, 高分子学会予稿集(CD-ROM), 58(1 Disk1) (1 Disk1)Microbial Production of Lactate-based Polyesters
- 2009, 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨, 2009, 15, Japanese嫌気的条件下における乳酸ベースポリマーの微生物発酵生産
- 日本生物工学会, 25 Nov. 2008, 生物工学会誌 : seibutsu-kogaku kaishi, 86(11) (11), 543 - 543, Japanese新産業創出に挑むキィーエンザイムの顔ぶれIntroduction research institution
- The Society of Fiber Science and Technology, Japan, 10 Nov. 2008, Fiber, 64(11) (11), "P - 365"-"P-370", JapaneseIntroduction research institution
- SOC FIBER SCIENCE TECHNOLOGY, Nov. 2008, SEN-I GAKKAISHI, 64(11) (11), P365 - P370, JapaneseMicrobial Factory for the Production of BioplasticsIntroduction research institution
- 05 Mar. 2008, 日本農芸化学会大会講演要旨集, 2008, 42, JapanesePseudomonas sp.61‐3由来PHA重合酵素変異体による多様な分子量をもつPHAの合成
- 29 Jan. 2008, 高分子学会北海道支部研究発表会講演要旨集, 42nd, 39, JapanesePseudomonas sp.61‐3由来PHA重合酵素変異体による組換え大腸菌体内でのPHA共重合体の蓄積率とポリマー分子量の相関
- 2008, 日本乳癌学会学術総会プログラム・抄録集, 16th乳癌診療ガイドライン2008年版改訂の説明 疫学・予防
- 2008, 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨, 2008システイン側鎖化学修飾による抗菌ペプチドThanatinの構造機能相関:疎水結合力と抗菌活性の関係
- 2008, 日本生物工学会大会講演要旨集, 60th抗菌ペプチドApidaecin高活性体取得におけるin vivoアッセイシステムの有効性の評価
- 2008, 生化学選択的スプライシングを調節するTIA-1RRM2の立体構造解析およびRNAの認識
- 日本生物工学会, 2008, 日本生物工学会大会講演要旨集, 20, 154 - 154, Japanese1Fp08 Crystallization and X-ray analysis of an azoreductase from Bacillus sp. B29
- 日本生物工学会, 2008, 日本生物工学会大会講演要旨集, 20, 4 - 4, Japanese1S1p03 Bioplastic Production using Artificially Evolved Enzymes : Bacteria to Plants
- エヌ・ティー・エス, Jan. 2008, 未来材料, 8(1) (1), 56 - 61, Japaneseバイオポリマー合成プロセス研究の新潮流Introduction research institution
- Jul. 2007, エコマテリアル研究会, 5, Japanese遺伝子組換え植物による生分解性プラスチック生産法の開発。
- バイオインダストリー協会, 01 Jul. 2007, Bioscience & industry, 65(7) (7), 334 - 339, JapanesePHA synthase engineering toward super biocatalysts for custom-made biopolymersIntroduction research institution
- 2007, 生化学選択的スプライシングを調節するTIA-1RRM2の立体構造解析およびRNAの認識
- 2007, 高分子学会予稿集(CD-ROM), 56(2 Disk1) (2 Disk1)微生物合成ポリエステルの分子量特性
- 2007, 高分子夏季大学講演要旨集, 53rd「資源循環型バイオプラスチックのバイオナノ融合研究」
- 2007, 日本農芸化学会大会講演要旨集, 2007Pseudomonas sp.61-3由来PHA重合酵素の推定基質ポケット近傍のアミノ酸残基群の配列と機能の相関
- 2007, 日本農芸化学会大会講演要旨集, 2007バイオポリエステルPHB調節タンパク質PhaRのPHB顆粒形態に与える変異効果
- 2007, 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨, 2007バイオポリエステルPHB調節タンパク質PhaRのPHB顆粒形態に与える変異効果
- 日本生物工学会, 2007, 日本生物工学会大会講演要旨集, 19, 125 - 125, Japanese2E17-2 Enhancement of polyhydroxybutyrate (PHB) production by Corynebacterium glutamicum using evolutionary engineering
- 日本生物工学会, 2007, 日本生物工学会大会講演要旨集, 19, 124 - 124, Japanese2E17-1 Correlation between molecular weight of PHA and amino acid residues in hypothetical substrate binding pocket of Pseudomonas sp. 61-3 PHA synthase
- Aug. 2006, International Symposium on Biological polymer 2006, 不明, EnglishProduction of polyhydrpxyalkanoate (PHA) copolymer in transgenic plants and development of plant tissue-specific PHA production system
- エヌ・ティー・エス, Jul. 2006, 未来材料, 6(7) (7), 44 - 51, Japanese"酵素"が演出するバイオプラスチックの生合成と生分解Introduction research institution
- 10 May 2006, 高分子学会予稿集(CD-ROM), 55(1 Disk1) (1 Disk1), 1PE183, Japaneseポリヒドロキシブタン酸(PHB)分解におけるPHB分解酵素基質吸着部位への変異導入効果
- 2006, 日本農芸化学会大会講演要旨集, 2006Corynebacterium glutamicumを用いたバイオプラスチック(PHB)の生産
- 2006, Polymer Preprints, Japan, 55(1) (1)Mutational effects of substrate-binding domain of poly[(R)-3- hydoxybutyrate] (PHB) depolymerase on PHB degradation
- 2006, Polymer Preprints, Japan, 55(2) (2)Evaluation of P(3HB-co-3HA) production by mutated PHA synthases from Pseudomonas sp. 61-3
- 2006, Polymer Preprints, Japan, 55(1) (1)Interactions of a represser protein, PhaR, with polyhydroxyalkanoate thin film
- 2006, Polymer Preprints, Japan, 55(2) (2)Dual binding abilities of a repressor protein, PhaR, to target DNAs and PHAs
- 2006, 高分子学会予稿集(CD-ROM), 55(1 Disk1) (1 Disk1)PHA生合成調節タンパク質PhaRとPHA薄膜フィルムとの相互作用解析
- 2006, 高分子学会予稿集(CD-ROM), 55(2 Disk1) (2 Disk1)脂肪族ポリエステル生合成調節タンパク質PhaRのDNAとポリエステルに対する二元結合能
- 2006, 日本農芸化学会大会講演要旨集, 2006PHBバイオポリエステル生合成調節タンパク質PhaRの機能解析
- 2006, 高分子学会予稿集(CD-ROM), 55(2 Disk1) (2 Disk1)Pseudomonas sp.61-3由来PHA重合酵素変異体のP(3HB-co-3HA)合成能の評価
- 日本生物工学会, 2006, 日本生物工学会大会講演要旨集, 18, 102 - 102, Japanese2F11-5 Composition control of PHA copolymer by altering substrate specificity of Pseudomonas sp. 61-3 PHA synthase
- 高分子刊行会, Jan. 2006, 高分子加工, 55(1) (1), 30 - 43, Japaneseバイオプラスチック合成研究の最前線--in vivoとin vitroのクロスオーバーIntroduction research institution
- 化学工業社, Jun. 2005, 化学工業, 56(6) (6), 468 - 473, JapaneseEnzyme evolutionary engineering and nano-processing for creation of bioplastics with high performanceIntroduction research institution
- 05 Mar. 2005, 日本農芸化学会大会講演要旨集, 2005, 237, Japanese進化工学的手法により改変されたPseudomonas sp.61‐3由来Polyhydroxyalkanoate(PHA)合成酵素(PhaC1)の精製と解析
- 共立出版, Mar. 2005, 蛋白質核酸酵素, 50(3) (3), 262 - 269, Japaneseバイオプラスチックのつくられ方とつくり方--生合成調節機構と酵素進化工学による新規プラスチック創製Introduction research institution
- 2005, 日本生物工学会大会講演要旨集, 2005重合酵素変異体を用いたバイオポリエステル生産の増強
- 2005, Polymer Preprints, Japan, 54(1) (1)Creation of eco-freindly plastics by biotechnology: From microbial production to production in plant
- 2005, Polymer Preprints, Japan, 54(1) (1)Enhanced production of P(3HB-co-3HA) from soybean oil by mutated PHA synthases
- 2005, 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨, 2005大腸菌に高発現させたBacillus sp.B29株由来アゾリダクターゼの精製と諸性質
- 2005, 日本農芸化学会大会講演要旨集, 2005アゾ色素分解細菌Bacillus sp.B29株からのアゾリダクターゼ遺伝子のクローニングと高発現
- 2005, 日本生物工学会大会講演要旨集, 2005Bacillus sp.B29株由来組換えアゾリダクターゼのキャラクタリゼーション
- 2005, 日本分子生物学会年会講演要旨集, 28th3-ketoacyl-ACP synthase III(FabH)遺伝子を用いた遺伝子組換えシロイヌナズナによるポリヒドロキシアルカン酸共重合体の合成
- 2005, 高分子学会予稿集(CD-ROM), 54(1 Disk1) (1 Disk1)変異型重合酵素を用いた大豆油からのP(3HB-co-3HA)生産の増強
- 2005, 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨, 2005PHAバイオポリエステル合成調節タンパク質PhaRの機能解析
- 2005, 日本農芸化学会大会講演要旨集, 2005PHBバイオポリエステル合成調節タンパク質PhaRの機能解析
- 2005, 日本農芸化学会大会講演要旨集, 2005PHB生合成調節タンパク質PhaRのDNAとの相互作用解析
- 日本生物工学会, 2005, 日本生物工学会大会講演要旨集, 17, 107 - 107, Japanese2C10-5 Quantitative functional analysis of a repressor protein (PhaR) related to PHB biosynthesis
- 日本生物工学会, 2005, 日本生物工学会大会講演要旨集, 17, 107 - 107, Japanese2C11-1 Enhancement of 3HB units-incorporating ability of Pseudomonas sp. 61-3 PHA synthase
- 01 Sep. 2004, 高分子学会予稿集(CD−ROM), 53(2 Disk1) (2 Disk1), 3PB178, Japanese人工進化型polyhydroxyalkanoate(PHA) synthaseのkinetic analysis
- 2004, 高分子学会予稿集(CD-ROM), 53(2 Disk1) (2 Disk1)重合酵素変異体を用いたバイオポリエステルの生合成と分子量の変化
- 2004, 日本生物工学会大会講演要旨集, 2004PHAバイオポリエステル合成調節タンパク質PhaRの機能解析
- 2004, 高分子学会予稿集(CD-ROM), 53(2 Disk1) (2 Disk1)タイプII PHA重合酵素への変異導入とポリエステル分子量への影響
- 2004, 日本農芸化学会大会講演要旨集, 2004バイオポリエステル生合成に関与するリプレッサータンパク質の機能解析:ランダム変異による機能マッピング
- 日本生物工学会, 2004, 日本生物工学会大会講演要旨集, 16, 122 - 122, Japanese2B11-5 Composition control of PHA copolymer by molecular evolutionary engineering of Pseudomonas sp. 61-3 PHA synthase
- 日本生物工学会, 2004, 日本生物工学会大会講演要旨集, 16, 145 - 145, Japanese3C10-5 Enhancement of polyhydroxyalkanoates (PHA) production in transgenic Arabidopsis thaliana by the highly active mutated PHA synthase generated by in vitro evolution
- Ralstonia eutropha biopolyester synthase (PhaC(Re)) is a key enzyme for catalyzing formation of polyhydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) copolyesters from (R)-3-hydroxybutyryl-CoA (3HB-CoA) and/or (R)-3-hydroxyalkanoyl-CoA (3HA-CoA). Previously, in Escherichia coli, we found a good correlation between the accumulation of PHB and PhaC(Re) activity using seven PhaC(Re) Mutants, all of which exhibited lower activities compared to the wild-type PhaC(Re). In this study, these PhaC(Re) mutants were individually introduced into a PHB-negative mutant strain (PHB(-)4) of R. eutropha, as well as a new activity-revertant (termed E11S5) of a primary mutant E11. Recombinant R. eutropha PHB(-)4 strains harboring PhaC(Re) mutant or the wild-type expression plasmids were cultivated using renewable carbon sources. No parallel relationship in the accumulation levels of PHA (PHB and PHA copolyesters) between recombinant hosts, E. coli JM109 (for PHB) and R. eutropha PHB(-)4 (for PHB or PHA copolyesters) was observed. This suggests, in contrast with the case of E. coli, that the PHA accumulation in R. eutropha is not governed solely by PhaC(Re) activity. Enhanced PHA accumulation was achieved by some mutants (including E11 and E11S5) throughout all cultivation conditions. It is likely that monomer compositional changes of PHA copolyesters generated by recombinants may be attributable to the changed substrate specificities of mutants toward 3HB-CoA and 3HA-CoA monomer substrates channeled from the related metabolic pathways. (C) 2003 Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE SA, Nov. 2003, BIOCHEMICAL ENGINEERING JOURNAL, 16(2) (2), 107 - 113, EnglishIntroduction research institution
- Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJ(Ac)) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid P-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJ(Ac) by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJ(Ac) revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C-8) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PRA formation from dodecanoate (C-12) was examined by using the mutated PhaJ(Ac) as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PRA synthase gene from Pseudomonas sp. strain 61-3 (phaC1(Ps)). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C-8) and 3-hydroxydecanoate (C-10) units were incorporated into PRA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.AMER SOC MICROBIOLOGY, Aug. 2003, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 69(8) (8), 4830 - 4836, EnglishIntroduction research institution
- 2003, 高分子学会予稿集, 52(5) (5)Comamonas acidovorans由来バイオポリエステル重合酵素が有するユニーク配列の機能解析
- 2003, 高分子学会予稿集, 52(14) (14)分子進化工学的手法によるPseudomonas sp.61-3由来PHA合成酵素の機能改変
- 2003, 高分子学会予稿集, 52(14) (14)進化工学的手法によって改質したPseudomonas sp.61-3由来ポリエステル重合酵素によるバイオポリエステル生産性の増強
- 2003, 日本生物工学会大会講演要旨集, 2003進化工学によるPseudomonas sp.61-3由来PHA合成酵素の機能改変(2)
- 2003, 生体機能関連化学シンポジウム講演要旨集, 18thGeneration and characterization of anti-poly betahydroxybutyrate (PHB) fragments on the basis of phage display system.
- 2003, 日本生物工学会大会講演要旨集, 2003ポリヒドロキシアルカン酸生合成における3-ケトアシル-ACPシンターゼIIIの改変
- 2003, 高分子学会予稿集, 52(14) (14)ファージ提示系によるpoly hydroxybutyrate(PHB)結合抗体の作製,機能評価
- 2003, 日本分子生物学会年会プログラム・講演要旨集, 26thバイオポリエステル生合成に関与するレプレッサータンパク質の機能解析:ランダム変異による機能マッピング
- 日本生物工学会, 2003, 日本生物工学会大会講演要旨集, 15, 67 - 67, JapaneseIn Vitro Analysis of Altered Substrate Specificity of Polyhydroxyal-kanoate (PHA) synthases which were generated by Means of PCR-mediated mutagenesis of PHA synthase gene
- 2003, Biomacromolecules, 2, 541 - 544Correlation between structure of the lactones and substrate specificity in enzyme-catalyzed polymerization for the synthesis of polyestersIntroduction research institution
- 2003, Biomacromolecules, 2, 541 - 544Correlation between structure of the lactones and substrate specificity in enzyme-catalyzed polymerization for the synthesis of polyestersIntroduction research institution
- 2003, J. Biochemistry, 133(1) (1), 139 - 145Introduction research institution
- The use of (R)-specific enoyl-coenzyme A (CoA) hydratase (PhaJ) provides a powerful tool for polyhydroxyalkanoate (PHA) synthesis from fatty acids or plant oils in recombinant bacteria. PhaJ provides monomer units for PHA synthesis from the fatty acid P-oxidation cycle. Previously, two phaJ genes (phaJ1(Pa) and phaJ2(Pa)) were identified in Pseudomonas aeruginosa. This report identifies two new phaJ genes (phaJ3(Pa) and phaJ4(Pa)) in P. aeruginosa through a genomic database search. The abilities of the four PhaJ(Pa) proteins and the (R)-3-hydroxyacyl-acyl carrier protein [(R)-3HA-ACP] dehydrases, FabA(Pa) and FabZ(Pa) to supply monomers from enoyl-CoA substrates for PHA synthesis were determined. The presence of either PhaJ1(Pa) or PhaJ4(Pa) in recombinant Escherichia coli led to the high levels of PHA accumulation (as high as 36-41 wt.% in dry cells) consisting of mainly short-(C4-C6) and medium-chain-length (C6-C10) 3HA units, respectively. Furthermore, detailed characterizations of PhaJ1(Pa) and PhaJ4Pa were performed using purified samples. Kinetic analysis revealed that only PhaJ4Pa exhibits almost constant maximum reaction rates (V-max) irrespective of the chain length of the substrates. The assay for stereospecific hydration revealed that, unlike PhaJ1(Pa), PhaJ4(Pa) has relatively low (R)-specificity. These hydratases may be very useful as monomer-suppliers for the synthesis of designed PHAs in recombinant bacteria. (C) 2002 Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE BV, Jan. 2003, INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, 31(4-5) (4-5), 195 - 205, EnglishIntroduction research institution
- Processing and activation of the precursor of an extracellular Streptomyces transglutaminase were achieved by using three Streptomyces proteases (SAM-P20, SAM-P26 and SAM-P45), all of which are widely distributed in Streptomyces. The use of these proteases would allow us to develop a production process for the active form of this enzyme in recombinant bacteria.SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2002, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 94(5) (5), 478 - 481, EnglishIntroduction research institution
- Based on the metabolic pathways for polyhydroxyalkanoate (PHA) biosynthesis, we succeeded in establishing the recombinant Pseudomonas sp 61-3 strains that synthesize random copolyesters consisting of (R)-3-hydroxybutyrate (3HB) and (R)-medium-chain-length 3-hydroxyalkanoate (mcl-3HA) units, P(3HB-co-3HA), with very high 3HB compositions (up to 94 mol%) from glucose. The mechanical properties of P(94% 3HB-co-3HA) copolyester were very similar to those of low-density polyethylene. We carried out the molecular cloning and characterization of a PhaG(Ps) encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase of Pseudomonas sp 61-3. It was concluded that the PhaGPs gene product is involved in providing mcl-3HA-CoA from glucose in the original strain. Heterologous expression of the PhaGPs gene with the PhaC1(Ps) gene encoding PHA synthase from Pseudomonas sp 61-3 was performed in the PhbC(Re) negative mutant (PHB(-)4) of Ralstonia eutropha. The recombinant PHB-4 strain successfully produced PHA copolyesters consisting of 3HB and mcl-3HA units of 6-12 carbon atoms from sugars. The 3HB fraction in copolyesters was very high (95-97 mol%). The PHA content in the recombinant strain could further be increased by the additional introduction of the PhbAB(Re) genes from R eutropha encoding beta-ketothiolase and NADPH-dependent acetoacetyl-coenzyme A reductase. Moreover, we have established an in vivo assay system to analyze mutational effects of R eutropha synthase (PhbCRe) on the level of PHB accumulation in recombinant strains of Escherichia coli. The activity of the PhbCRe could be efficiently estimated using the in vivo system constructed here, and would be useful for in vitro evolution of PhbCRe. (C) 2002 Society of Chemical Industry.WILEY-BLACKWELL, Oct. 2002, POLYMER INTERNATIONAL, 51(10) (10), 899 - 906, EnglishIntroduction research institution
- バイオインダストリ-協会, 01 Sep. 2002, Bioscience & industry, 60(9) (9), 30 - 31, Japanese"Green plastics"created by evolutionary enzyme-engineering
- Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA, synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.AMER SOC MICROBIOLOGY, Jul. 2002, JOURNAL OF BACTERIOLOGY, 184(14) (14), 3992 - 4002, EnglishIntroduction research institution
- In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Rc)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant Ell with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.JAPANESE BIOCHEMICAL SOC, Jun. 2002, JOURNAL OF BIOCHEMISTRY, 131(6) (6), 801 - 806, EnglishIntroduction research institution
- Streptomyces viridosponis A-914 was screened as a producer of an enzyme to effectively form chiral intermediates of 1,4-dihydropyridine calcium antagonists. The supernatant liquid of the growing culture of this strain exhibited high activity for enantioselective hydrolysis of prochiral 1,4-dihydropyridine diesters to the corresponding (4R) half esters. The responsible enzyme (termed DHP-A) was purified to apparent homogeneity and characterized. Cloning and sequence analysis of the gene for DHP-A (dhpA) revealed that the enzyme was a serine protease that is highly similar in both structural and enzymatic feature to SAM-P45, which is known as a target enzyme of Streptomyces subtilisin inhibitor (SSI), from Streptomyces albogriseolus. In a batch reaction test, DHP-A produced a higher yield of a chiral intermediate of 1,4-dihydropyridine than the commercially available protease P6. Homologous or heterologous expression of dhpA resulted in overproduction of the enzyme in culture supernatants, with 2.4- to 4.2-fold higher specific activities than in the parent S. viridosporus A-914. This indicates that DHP-A is suitable for use in reactions forming chiral intermediates of calcium antagonists and suggests the feasibility of developing DHP-A as a new commercial enzyme for use in the chiral drug industry.AMER SOC MICROBIOLOGY, Jun. 2002, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68(6) (6), 2716 - 2725, EnglishIntroduction research institution
- By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PRA). Aeromonas caviae FA440 synthase, termed PhaCA, was chosen as a good target for evolution, since it can synthesize a PRA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC(Ac) gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) {by LS5218 [fadR601 atoC(Con)] strain}. The expression vector for the phaC(Ac) gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).AMER SOC MICROBIOLOGY, May 2002, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68(5) (5), 2411 - 2419, EnglishIntroduction research institution
- 2002, 高分子学会予稿集, 51(14) (14)P(3HB)分解酵素基質吸着ドメインの機能解析
- 2002, 高分子学会予稿集, 51(5) (5)バイオポリエステル合成におけるモノマー供給酵素の解析 シュードモナス属細菌が有する4つの(R)-特異的エノイルCoAヒドラターゼの比較
- 日本生物工学会, 2002, 日本生物工学会大会講演要旨集, 14, 90 - 90, Japanese571 Alteration of chain length substrate specificity of an Aeromonas caviae (R)-specific enoyl-CoA hydratase through site-directed mutagenesis
- 日本生物工学会, 2002, 日本生物工学会大会講演要旨集, 14, 79 - 79, Japanese525 Alternation of substrate specificity of PHA synthase from Pseudomonas sp. 61-3 by in vitro molecular evolutionary engeneering
- 2002, 化学工業(総説), 53(7) (7), 22 - 28バイオプラスチック生産における進化分子工学Introduction research institution
- Polymerase is a central enzyme involved in the biosynthesis of polyhydroxybutyrate (PHB), a well-known bacterial biodegradable polyester. In this study, we have established an in vivo assay system to analyze mutational effects of Ralstonia eutropha polymerase (termed PhbC(Re)) on the level of PHB accumulation in recombinant strains of Escherichia coli. This in vitro evolution system consists of a polymerase chain reaction-mediated random mutagenesis and two assay procedures, a plate assay using a PHB-staining dye and a high-pressure liquid chromatographic assay based on the converting reaction from PHB to crotonic acid. The distribution pattern of the PHB accumulation level of the mutant population using 378 clones arbitrarily selected, suggested that the present level of PhbC(Re) is high and well-optimized. It is noteworthy that many of the amino acid substitutions affecting the PHB accumulation occurred in the conserved positions or regions within an 'alpha/beta hydrolase fold' which is commonly found among hydrolytic enzymes. From a good correlation with the level of PHB accumulation, an activity estimation of the PhbC(Re) would be efficiently achieved by monitoring the level of PHB accumulation using the in vivo assay system established here. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE BV, Apr. 2001, FEMS MICROBIOLOGY LETTERS, 198(1) (1), 65 - 71, EnglishIntroduction research institution
- 05 Mar. 2001, 日本農芸化学会誌, 75, 512 - 512, Japanese細胞型進化工学による低温適応化プロテアーゼの分子育種
- 2001, 日本農芸化学会誌, 75(1) (1)生分解性プラスチックの開発の現状と展望 遺伝子組換え微生物によるPHAの合成
- 2001, 日本化学会講演予稿集, 79th(2) (2)Selection of human antibody fragments specific for human EPO receptor extracellular domain and poly(beta-hydroxybutyrate).
- 2001, 高分子学会予稿集, 50(5) (5)ポリヒドロキシブタン酸重合酵素遺伝子のランダム変異による微生物ポリマー生産への影響
- 2001, 高分子学会予稿集, 50(14) (14)ポリヒドロキアルカン酸重合酵素のランダム変異によるポリマー生産への影響
- 2001, 日本生物工学会大会講演要旨集, 2001ポリヒドロキシアルカン酸合成に関与する制御因子PhaRの解析
- 2001, 高分子学会予稿集, 50(5) (5)Aeromonas caviae由来ポリエステルグラニュール結合タンパク質(PhaP)の解析と組換え株によるポリエステル生合成
- 2001, 高分子学会予稿集, 50(14) (14)モノマー供給酵素のタンパク質工学
- 2001, 高分子学会予稿集, 50(14) (14)生分解性ポリエステル合成に関与する制御蛋白質の解析
- 2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 71 - 74Investigation of metabolic pathways for biopolyester productionIntroduction research institution
- 2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 3 - 6In vivo assay system of the polymerase as a key enzyme for PHA biosynthesisIntroduction research institution
- 2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 77 - 80Molecular characterization of a regulatory protein (PhaR) involved in PHA biosynthesisIntroduction research institution
- 2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 71 - 74Investigation of metabolic pathways for biopolyester productionIntroduction research institution
- 2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 3 - 6In vivo assay system of the polymerase as a key enzyme for PHA biosynthesisIntroduction research institution
- Recently, a variety of aliphatic polyesters have been synthesized using hydrolases such as lipases and PHB depolymerases, and the reaction mechanism for these enzyme-catalyzed polymerization has been discussed. In this paper, we have studied the involvement of the catalytic amino acid residues of the hydrolase in enzyme-catalyzed polymerization with an extracellular PHB depolymerase from Alcaligenes faecalis T1. A wild-type PHB depolymerase and three kinds of site-specific mutants (catalytic amino acids were substituted) were prepared and their polymerization activities for the ring-opening polymerization of (R)-β-butyrolactone (BL) were compared. BL was polymerized at 80 °C in bulk by the wild-type enzyme to yield polymers consisting of cyclic and linear structures in a high monomer conversion. In contrast, none of the mutant enzymes showed obvious polymerization activity. These results have clearly demonstrated that the catalytic triad is indeed responsible for the enzyme-catalyzed polymerization of BL.2001, Biomacromolecules, 2(2) (2), 541 - 544, EnglishIntroduction research institution
- Pseudomonas sp. 61-3 produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] random copolymer consisting of monomeric units of 4-12 carbon atoms from sugars. The phaGPs gene encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase was cloned from this strain, and homologous expression of this gene under the control of the lac or the native promoter was investigated. Additional copies of the phaGPs gene in Pseudomonas sp. 61-3 led to an increase in both the polyhydroxyalkanoate (PHA) content in the cells and the fraction of medium-chain-length 3HA units in PHA. Disruption of the chromosomal phaGPs gene resulted in an increase in the fraction of the 3HB unit in PHA. The site-directed mutagenesis of the phaGPs gene was carried out to investigate the role of a HX4D motif which has been proposed to be related to PhaG activity.2001, Biomacromolecules, 2(1) (1), 142 - 147, EnglishIntroduction research institution
- 2001, RIKEN Review (Focused on Ecomolecular Science Research), 42, 77 - 80Molecular characterization of a regulatory protein (PhaR) involved in PHA biosynthesisIntroduction research institution
- Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay systemPreviously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi ct al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum ct al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta -D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion. partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure- function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta -strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.OXFORD UNIV PRESS, Nov. 2000, JOURNAL OF BIOCHEMISTRY, 128(5) (5), 745 - 754, EnglishIntroduction research institution
- Substrate specificity analysis of microbial transglutaminase using proteinaceous protease inhibitors as natural model substratesThe substrate specificity of microbial transglutaminase (MTG) from Streptomyces mobaraensis (formerly categorized Streptoverticillium) was studied using a Streptomyces proteinaceous protease inhibitor, STI2, as a model amine-donor substrate. Chemical modification and mutational analysis to address the substrate requirements for MTG were carried out around the putative reactive site region of STI2 on the basis of the highly refined tertiary structure and the solvent accessibility index of Streptomyces subtilisin inhibitor, SSI, a homolog of STI2, The results suggest that the pi reactive center site (position 70 of STI2) for protease subtilisin BPN' or trypsin may be the prime Lys residue that can be recognized by MTG, when succinylated beta-casein. was used as a partner Gin-substrate. It is characteristic in that the same primary enzyme contact region of STI2 is shared by both enzymes, MTG and proteases, For quantitative analysis of the TG reaction, we established an ELISA-based monitoring assay system using an anti-SSI polyclonal antibody highly cross-reactive with STI2, Site-specific STI2 mutants were prepared by an Escherichia coli expression-secretion vector system and subjected to the assay system. We reached several conclusions concerning the nature of the flanking amino acid residues affecting the MTG reactivity of the substrate Lys residue: (i) site-specific mutations from Asn to Lys or Arg at position 69 preceding the amine-donor 70Lys, led to enhanced substrate reactivity; (ii) amino acid replacement at 67Ile with Ser led to higher substrate reactivity, (iii) additive effects were obtained by a combination of the positive mutations at positions 67 and 69 as described above, and (iv) Gly at position 65 might be essential for MTG; reaction. Moreover, the substrate specificity of guinea pig liver tissue transglutaminase (GTG) was compared with that of MTG using STI2 and its mutants, In contrast to MTG, replacement of Gly by Asp at position 65 was the most favorable for substrate reactivity. Also,70Lys appeared not to be a prime amine-donor site for GTG-mediated cross-linking, suggesting a difference in substrate recognition between. MTG and GTG.JAPANESE BIOCHEMICAL SOC, Sep. 2000, JOURNAL OF BIOCHEMISTRY, 128(3) (3), 415 - 425, EnglishIntroduction research institution
- To ascertain whether position 131 of a mesophilic protease, subtilisin BPN', is a potential critical site for cold adaptation as screened by evolutionary engineering (S. Taguchi, A. Ozaki, and H. Momose, Appl. Environ. Microbiol. 64:492-95, 1998), a full set of subtilisin BPN' mutants with mutations at position 131 was constructed by site-saturation mutagenesis.; All mutated enzymes were measured for specific activity at 10 degrees C by the quantitative titer microplate assay system using polyclonal antibody against subtilisin BPN' and a synthetic chromogenic substrate. All the mutants exhibited proteolytic activities almost the same as or higher than that of the wild-type enzyme, suggesting that position 131 may. be important for cold adaptation. In comparison with the wild type, purified mutants G131F, G131R, G131M, and G131W were found to acquire proteolytic activities (k(cat)/K-m) at 10 degrees C that were 150, 93, 84, and 50% higher, respectively. In particular, for the G131F mutant, temperature dependency in enzyme activity was shown by an increase in k(cat) and a decrease in K-m. All of these amino acid substitution mutants, G131F, G131R, G131M, and G131W, acquired increased proteolytic activities at 10 degrees C for three different synthetic peptide substrates but no increase in caseinolytic activity. Furthermore, they all conferred thermolability on the enzyme to differing extents in terms of the half-life of enzyme inactivation at 60 degrees C. No significant correlation was found between the amino acids preferred for cold adaptation surveyed here and those present at position 131 of subtilisin of psychrophilic cells naturally occurring in cold environments. Based on these findings, position 131 is a contributor in artificial evolution for acquiring a cold-active character and may not be related to physiological requirements for subtilisin-producing cells living in cold environments. Therefore, saturation mutagenesis would be effective in achieving rapid improvement in protein properties via evolutionary engineering.AMER SOC MICROBIOLOGY, Apr. 2000, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 66(4) (4), 1410 - 1415, EnglishIntroduction research institution
- Two Pseudomonas aeruginosa genes, termed phaJ1(Pa) and phaJ2(Pa), homologous to the Aeromonas caviae (R)-specific enoyl-CoA hydratase gene (phaJ(Ac)) were cloned using a PCR technique to investigate the monomer-supplying ability for polyhydroxyalkanoate (PHA) synthesis from beta-oxidation cycle. Two expression plasmids for phaJ1(Pa) and phaJ1(Pa) were constructed and introduced into Escherichia toil DH5 alpha strain. The recombinants harboring phaJ1(Pa) or phaJ2(Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities. that is, specific for short chain-length enoyl-CoA or medium chain-length enoyl-CoA, respectively. In addition, co-expression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14-29 wt% of cell dry weight from dodecanoate as a sole carbon source. It has been suggested that phaJ1(Pa) and phaJ2(Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE BV, Mar. 2000, FEMS MICROBIOLOGY LETTERS, 184(2) (2), 193 - 198, EnglishIntroduction research institution
- 2000, 日本生物工学会大会講演要旨集, 2000Pseudomonas sp.61-3のポリヒドロキシアルカン酸生合成系遺伝子phaGのクローニング及び解析
- 2000, 日本生物工学会大会講演要旨集, 2000ポリエステル合成細菌(R)-特異的エノイルCoAヒドラターゼ遺伝子の単離とその応用
- 2000, 放射線化学討論会講演要旨集, 43rdLET Effects on Behavior of Excited States of Alkane and Polystyrene Studied by Ion Beam Pulse radiolysis.
- 2000, 高分子学会予稿集, 49(5) (5)新規脂肪酸分解系酵素遺伝子の単離およびバイオポリエステル生合成への応用
- 2000, 日本農芸化学会関東支部講演要旨集, 2000(Oct) (Oct)遺伝子組換え微生物によるPHAの合成
- 2000, 高分子学会予稿集, 49(14) (14)ポリエステル合成細菌由来(R)-特異的エノイルCoAヒドラターゼの解析
- 日本生物工学会, 2000, 日本生物工学会大会講演要旨集, 12, 3 - 3, JapaneseCharacterization of polyester granule associated protein (PhaP) from Aeromonas caviae
- 日本生物工学会, 2000, 日本生物工学会大会講演要旨集, 12, 394 - 394, JapaneseCold-adaptation of a mesophilic protease subtilisin BPN' by experimental evolution system
- Secondary structures and structural fluctuation in a dimeric protein, Streptomyces subtilisin inhibitorBased on the nuclear magnetic resonance assignments of a dimeric protein, Streptomyces subtilisin inhibitor (SSI), microscopic details of secondary structures in solution have been elucidated. The chemical shift index of C-alpha signals, together with information on the hydrogen exchange rates of the backbone amide protons, were used to identify secondary structures. The locations of these secondary structures were found to be different in some critical points from those determined earlier by X-ray crystallography of the crystal. Notably, the beta 3 strand is completely missing and the alpha 2 helix is extended toward the C-terminus. Furthermore, hydrogen exchange experiments of individual peptide NH protons under strongly folding conditions revealed mechanisms of global and local structural fluctuation within the dimeric structure. It has been suggested that the global fluctuation of the monomeric unit occurs without affecting the accompanying monomer, in contrast to the equilibrium thermal unfolding, which is cooperative, Higher protection against hydrogen exchange for residues in part of the beta 4 strand implies that this region might serve as a folding core.JAPANESE BIOCHEMICAL SOC, Nov. 1999, JOURNAL OF BIOCHEMISTRY, 126(5) (5), 859 - 865, EnglishIntroduction research institution
- A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which me developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K-m value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V, The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51, In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.OXFORD UNIV PRESS, Oct. 1999, JOURNAL OF BIOCHEMISTRY, 126(4) (4), 689 - 693, EnglishIntroduction research institution
- KLUWER ACADEMIC PUBL, Jul. 1999, JOURNAL OF BIOMOLECULAR NMR, 14(3) (3), 285 - 286, EnglishIntroduction research institution
- Seiwa University, Jun. 1999, The Seiwa journal of law & politics, 6(1) (1), 1 - 13, JapaneseHuman Dignity and Decision of Death
- 1999, 高分子学会予稿集, 48(13) (13)Comamonas acidovorans株のPHA生合成系遺伝子の取得,解析,およびその応用
- 1999, 高分子学会予稿集, 48(13) (13)ポリエステル生合成における(R)-特異的エノイルCoAヒドラターゼの役割
- 1999, 高分子学会予稿集, 48(13) (13)Pseudomonas sp.61-3のポリヒドロキシアルカン酸生合成系遺伝子の解析
- 1999, 高分子学会予稿集, 48(13) (13)組換えRalstonia eutrophaにおけるポリエステル合成と生合成系酵素活性の影響
- 日本生物工学会, 1999, 日本生物工学会大会講演要旨集, 11, 188 - 188, JapaneseAnalysis of polyhydroxyalkanoate biosynthetic genes of Pseudomonas sp. 61-3 and biosynthesis of copolyesters with novel monomer compositions.
- 日本生物工学会, 1999, 日本生物工学会大会講演要旨集, 11, 187 - 187, JapaneseCloning, functional analysis and application of the polyhydroxyalkanoate synthase gene of Comamonas acidovorans DS-17.
- An open reading frame (termed ORF-PR) encoding a metallothionein-like domain-including protein was found upstream of a previously identified Streptomyces chymotrypsin-type protease gene (sam-P20). Promoter and terminator activities of ORF-PR were detected using the promoterless Streptomyces tyrosinase gene as a reporter gene and expression of ORF-PR was supposed to occur before that of sam-P20 gene. Frameshift mutation anaysis showed that the ORF-PR product might act as a repressive regulator of the sam-P20 gene.TAYLOR & FRANCIS LTD, Dec. 1998, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 62(12) (12), 2476 - 2479, EnglishIntroduction research institution
- Assay on a high-quality microtiter plate was found to allow for quantitative analysis of bacterial serine protease, subtilisin BPN', and its mutant enzymes which had been genetically engineered to be adapted to low-temperatures (Taguchi ct al., 1998. Appl. Environ. Microbiol. 64, 492-495), by using polyclonal antibody against subtilisin BPN'. The use of polyclonal antibody was crucial in normalizing the number of various different enzyme molecules in culture supernatant samples of recombinant strains of Bacillus subtilis, giving rise to the performance of specific activity assay of the enzymes. Relative activity of each mutant subtilisin BPN' to wild-type enzyme was estimated by monitoring the increased value of absorbance caused by enzymatic hydrolysis of a chromogenic substrate. The relative activity in each enzyme estimated by this method showed good coincidence with that estimated by kinetic parameters, k(cat)/K-m of the purified enzymes. We termed the system as 'ABEA' (antibody-bound enzyme assay). The efficient ABEA system developed here would be useful for the determination of specific activity of other enzymes of interest and provide us versatile applications in the field of evolutionary engineering. (C) 1998 Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE BV, Dec. 1998, JOURNAL OF BIOTECHNOLOGY, 66(2-3) (2-3), 157 - 163, EnglishIntroduction research institution
- An endogenous target protease, SAM-P26, of Streptomyces protease inhibitor (SSI): Primary structure, enzymatic characterization, and its interaction with SSIWe have been focusing on the potent involvement of the molecular interaction between a protease and a protease inhibitor in the physiological or morphological regulation of Streptomyces cells producing them [Taguchi et al. (1995) J, Bacteriol. 177, 6638-6643; Suzuki et al. (1997) J. Bacteriol. 179, 430-438]. In this study, an extracellular protease, termed SAM-PEG, was isolated as a target of endogenous protease inhibitor (SSI) from the culture medium of an SSI non-producing mutant strain derived from Streptomyces albogriseolus S-3253, Complete amino acid sequence determination revealed that SAM-PEG is identical to a protein encoded by the SAM-P20D gene, which was previously found to be located downstream of the gene for SAM-PEG, another target protease of SSI. Based on the sequence homology, SAM-P26 was categorized as a member of the chymotrypsin family like SAM-PBO. Sequence similarity between SAM-P26 and SAM-P20 was immunologically demonstrated by Western blot analysis using anti-SAM-P20 antiserum. The molecular mass (26 kDa) of SAM-PEG estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was much higher than that calculated from the amino acid sequence of SAM-P26 (18,376.8 Pa) and that of the S-pyridylethylated form (18,808.4 Dal of SAM-P26 determined by Matrix-assisted Laser Desorption/Ionization-Time of Flight/Mass Spectrometry. Analytical gel-filtration analysis revealed that SARI-PEG exists as a monomer (18.8 kDa) in the native state. The results as to substrate specificity and inhibitor sensitivity indicated SAM-PEG exhibits chymotrypsin-like activity. For the proteolytic activity, the optimal pH was 10.5 and the optimal temperature was 60 degrees C. The complex formation of SAM-PEG with SSI was confirmed by native-PAGE analysis.JAPANESE BIOCHEMICAL SOC, Oct. 1998, JOURNAL OF BIOCHEMISTRY, 124(4) (4), 804 - 810, EnglishIntroduction research institution
- By combination of size exclusion and reversed-phase chromatography, we have isolated a novel member of insect defensin-type antimicrobial peptides from the entire bodies of bacteria-challenged Formica rufa (hymenoptera, formicidae). The molecular mass of the purified peptide was estimated to be 4120.42 by matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. Sequence analysis revealed that this peptide consisted of 40 amino acid residues with six cysteines engaged in the formation of three intramolecular disulfide bridges. This peptide is unique among the arthropod defensins in terms of the presence of asparatic acid and alanine at position 33 and as C-terminal residue, respectively. In addition, this novel defensin from Formica rufa has the particularity to have no C-terminal extension in contrast to those reported for other hymenoptera defensins. ((C) Societe francaise de biochimie et biologie moleculaire/Elsevier, Paris).EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER, Apr. 1998, BIOCHIMIE, 80(4) (4), 343 - 346, EnglishIntroduction research institution
- Engineering of a cold-adapted protease by sequential random mutagenesis and a screening systemA cold-adapted protease subtilisin was successfully isolated by evolutionary engineering based on sequential in vitro random mutagenesis and an improved method of screening (H. Kano, S, Taguchi, and M. Momose, Appl. Microbiol. Biotechnol. 47:46-51, 1997), The mutant subtilisin, termed m-63, exhibited a catalytic efficiency (expressed as the k(cat)/K-m value) 100% higher than that of the wild type at 10 degrees C when N-succinyl-L-Ala- L-Ala-L-Pro-L-Phe-p-nitroanilide was used as a synthetic substrate, This cold adaptation was achieved with three mutations, Val to Ile at position 72 (V72I), Ala to Thr at position 92 (A92T), and Gly to Asp at position 131 (G131D), and it was found that an increase in substrate affinity (i.e., a decreased K-m value) was mostly responsible for the increased activity. Analysis of kinetic parameters revealed that the V72I mutation contributed negatively to the activity but that the other two mutations, A92T and G131D, overcame the negative contribution to confer the 100% increase in activity. Besides suppression of the activity-negative mutation (V72I) by A92T and G131D, suppression of structural stability was observed in measurements of activity retention at 60 degrees C and circular dichroism spectra at 10 degrees C.AMER SOC MICROBIOLOGY, Feb. 1998, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 64(2) (2), 492 - 495, EnglishIntroduction research institution
- Seiwa University, Jun. 1997, The Seiwa journal of law & politics, 4(1) (1), 1 - 12, JapaneseSome New Condiderations related to the Article on Human Dignity
- We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them ''SSI-like proteins'' (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338-4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised, The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the Pi site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site, Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites, It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins.SPRINGER VERLAG, May 1997, JOURNAL OF MOLECULAR EVOLUTION, 44(5) (5), 542 - 551, EnglishIntroduction research institution
- A gene encoding a homolog of the Streptomyces chymotrypsinlike serine protease, SAM-P20, was identified downstream of the sam-p20 gene and designated SAM-P20D. This gene has two tandem Shine-Dalgarno sequences and two initiation codons. We have established vector systems with the function of tyrosinase gene-bone melanin pigmentation as a reporter for sam-p20D gene expression in Streptomyces coelicolor in order to identify the promoter and terminator activities, Using this system, the sam-p20D gene was suggested to be transcribed monocistronically.TAYLOR & FRANCIS LTD, May 1997, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 61(5) (5), 909 - 913, EnglishIntroduction research institution
- Artificial cold adaptation of a mesophilic protease, subtilisin BPN', was attempted by means of random mutagenesis of its entire gene coupled with screening of cleared-zone-forming colonies on skim-milk plates at a low temperature. Out of sixty clones screened at 10 degrees C, one mutant enzyme (termed M-15) was found to acquire higher proteolytic activities, specifically dependent on low temperatures ranging from 10 degrees C to 1 degrees C, in comparison with those of the wild-type. DNA sequencing analysis revealed that, by this mutation, the 84th amino acid residue, valine, was substituted by isoleucine, which is located 1.5 nm from the center of the catalytic triad in the tertiary structure of subtilisin. By kinetic analysis of the purified enzyme samples, the higher proteolytic activities of M-15 at low temperatures were found to be due to the decrease in the K-m value. There was no difference in thermostability between the wild-type and mutant enzymes, when tested by heat treatment. Circular dichroism spectra also showed no difference between them at 10 degrees C, indicating that the mutation of V841 had no effect on the secondary structure of subtilisin.SPRINGER VERLAG, Jan. 1997, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 47(1) (1), 46 - 51, EnglishIntroduction research institution
- A novel member of the subtilisin-like protease family from Streptomyces albogriseolusWe previously isolated three extracellular endogenous enzymes from a Streptomyces albogriseolus mutant strain which were targets of Streptomyces subtilisin inhibitor (SSI) (S. Taguchi, A. Odaka, Y.,Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In the present study, of the three enzymes the largest one, with a molecular mass of 45 kDa (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), termed SAM-P45, has been characterized in detail. The entire gene encoding SAM-P45 was cloned as an approximately 10-kb fragment from S. albogriseolus S-3253 genomic DNA into an Escherichia coli host by using a shuttle plasmid vector. The amino acid sequence corresponding to the internal region of SAM-P45, deduced from the nucleotide sequence of the gene, revealed high homology, particularly in three regions around the active-site residues (Asp, His, and Ser), with the amino acid sequences of the mature domain of subtilisin-like serine proteases. In order to investigate the enzymatic properties of this protease, recombinant SAM-P45 was overproduced in Streptomyces coelicolor by using a strong SSI gene promoter. Sequence analysis of the SAM-P45 gene and peptide mapping of the purified SAM-P45 suggested that it is synthesized as a large precursor protein containing a large C-terminal prodomain (494 residues) in addition to an N-terminal preprodomain (23 and 172 residues). A high proportion of basic amino acids in the C-terminal prodomain was considered to serve an element interactive with the phospholipid bilayer existing in the C-terminal prodomain, as found in other membrane-anchoring proteases of gram-positive bacteria. It is noteworthy that SAM-P45 was found to prefer basic amino acids to aromatic or aliphatic amino acids in contrast to subtilisin] BPN', which has a broad substrate specificity. The hydrolysis by SAM-P45 of the synthetic substrate (N-succinyl-L-Gly-L-Pro-L-Lys-p-nitroanilide) most preferred by this enzyme was inhibited by SSI, chymostatin, and EDTA. The proteolytic activity of SAM-P45 was stimulated by the divalent cations Ca2+ and Mg2+. From these findings, we conclude that SAM-P45 interacts with SSI and can be categorized as a novel member of the subtilisin-like serine protease family.AMER SOC MICROBIOLOGY, Jan. 1997, JOURNAL OF BACTERIOLOGY, 179(2) (2), 430 - 438, EnglishIntroduction research institution
- Functional mapping of amino acid residues responsible for the antibacterial action of apidaecinFunctional mapping was carried out to address the amino acid residues responsible for the activity of the antibacterial peptide apidaecin from the honeybee by an in vivo assay system developed previously. The C-terminal region and many of the proline and arginine residues which are present at high frequency in apidaecin were found to play an important role in ias antibacterial activity.AMER SOC MICROBIOLOGY, Dec. 1996, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 62(12) (12), 4652 - 4655, EnglishIntroduction research institution
- The genes coding for the protease inhibitors, SSI and API-2c', have been analyzed by comparing DNA macrorestriction patterns of Streptomyces albogriseolus S-3253 and S. griseoincarnatus KTo-250 with those of inhibitor-deficient mutants. The mutants were found to suffer from chromosomal deletions rather than plasmid loss which resulted in the loss of the relevant genes. Hybridization experiments indicated that the ssi homologs in S. lividans and S. coelicolor A3(2) are located near the end of the linear chromosome.ELSEVIER SCIENCE BV, May 1996, FEMS MICROBIOLOGY LETTERS, 139(1) (1), 37 - 42, EnglishIntroduction research institution
- Three new proteinaceous inhibitors of trypsin and subtilisin of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family were isolated and purified from culture media of Streptomyces strains; SIL5 from S. fradiae, SIL7 from S. ambofaciens and SIL12 from S. hygroscopicus. Their complete amino-acid sequences were determined by sequence analysis of the intact SIL proteins and peptides obtained by enzymatic digestion of S-pyridylethylated proteins. SIL7 showed high sequence similarity to other Arg-possessing SSI-family inhibitors at the P1 site. SIL12 is unique in having a two-residue insertion in the flexible loop region. Based on the amino-acid sequences of these inhibitors and other SSI-family inhibitors whose sequences have already been determined, the phylogenetic relationship of SSI-family inhibitors and Streptomyces strains was considered. Among about 110 amino-acid residues possessed by SSI-family inhibitors, 28 are completely conserved. The contribution of these conserved residues to the function and stability of the inhibitor molecules is discussed on the basis of the results obtained from mutational analysis of SSI and its crystal structure.ELSEVIER SCIENCE BV, Feb. 1996, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1292(2) (2), 233 - 240, EnglishIntroduction research institution
- Amino acid sequences of protease inhibitors (Streptomyces subtilisin inhibitor-like proteins) widely distributed in Streptomyces were compared to clarify the taxonomic status of three strains of Streptomyces spp., S. coelicolor A3(2), S. lividans 66 and S. coelicolor Muller, which are closely related by conventional taxonomical procedures. The sequence comparison indicated that S. coelicolor A3(2) is distinct from the type strain S. coelicolor Muller, but belongs to the same taxon as S. lividans 66.ELSEVIER SCIENCE BV, Jan. 1996, FEMS MICROBIOLOGY LETTERS, 135(2-3) (2-3), 169 - 173, EnglishIntroduction research institution
- STREPTOMYCES SERINE-PROTEASE (SAM-P20) - RECOMBINANT PRODUCTION, CHARACTERIZATION, AND INTERACTION WITH ENDOGENOUS PROTEASE INHIBITORPreviously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI-producing mutant strain (S. Taguchi, A, Odaka, Y, Watanabe, and H. Momose, Appl, Environ, Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter, The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20, From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine-cleavable activity, i,e, a serine protease with broad substrate specificity, For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80 degrees C by the addition of 10 mM calcium ion, The strong stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 x 10(-10) M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one dimer molecule of SSI., The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-PZ0, This reactive site is the same as that toward an exogenous target enzyme, subtilisin BPN'.AMER SOC MICROBIOLOGY, Nov. 1995, JOURNAL OF BACTERIOLOGY, 177(22) (22), 6638 - 6643, EnglishIntroduction research institution
- OXFORD UNIV PRESS UNITED KINGDOM, Sep. 1995, PROTEIN ENGINEERING, 8(9) (9), 29 - 29, EnglishStudies on the structure-function relationship of Streptomyces protease inhibitors proved by using artificial and natural mutantsSummary international conference
- A gene encoding a homolog of the chymotrypsin-like serine protease (SAM-P20), which was isolated as the target enzyme of a protease inhibitor (SSI), was cloned from Streptomyces lividans 66. This gene contained an open reading frame of 1065 nucleotides encoding 354 amino acid residues with a putative prepro portion of 157 amino acid residues. The deduced amino acid sequence of the cloned gene had significant homology to those of members of Streptomyces extracellular chymotrypsin-like protease family. By Southern blot analysis. it was suggested that protease genes of this type are found at a high frequency in Streptomyces. In this sense, we propose to categorize this protease as a member of the 'SAL' series (SAM-P20-like proteases).TAYLOR & FRANCIS LTD, Jul. 1995, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 59(7) (7), 1386 - 1388, EnglishIntroduction scientific journal
- A secretory production system for the active form of transforming growth factor alpha (TGF alpha) was established in Streptomyces lividans using a gene encoding the secretory protease inhibitor, Streptomyces subtilisin inhibitor (SSI). It was demonstrated that deletion of one of the putative dual ssi terminators is effective to extracellularly produce a heterologous polypeptide in a fused form, The recombinant fusion protein, SSI::TGF alpha, was purified to homogeneity by a combination of hydrophobic chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). It was noteworthy that the SSI::TGF alpha hybrid protein exhibited bifunctional activity: the TGF alpha activity for cell growth promotion and the inhibitory activity of SSI. Taken together with the results of analytical gel filtration, these findings strongly indicate that each moiety in the fusion protein correctly folds and the whole hybrid molecule exists in a dimeric form, which results in its bifunctional activity.ELSEVIER SCIENCE BV, Jul. 1995, GENE, 159(2) (2), 239 - 243, EnglishIntroduction scientific journal
- A SUBTILISIN INHIBITOR PRODUCED BY STREPTOMYCES-BIKINIENSIS POSSESSES A GLUTAMINE RESIDUE AT REACTIVE-SITE P1We determined the complete amino acid sequence of a novel subtilisin inhibitor, SIL15, which had been isolated from the culture supernatant of Streptomyces bikiniensis and shown to be a member of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family, and then identified its reactive site, SIL15 is composed of 113 amino acids and exists as a dimer, Compared with other SSI-family inhibitors, SIL15 was found to be unique in that it possesses a Gin residue at the P1 site of the reactive site and has two-residue insertions in two regions, one in the alpha(1)-helix and the other in the flexible loop region near the reactive site, Inhibition of subtilisin BPN' by SIL15 (inhibitor constant, 2.7X10(-11) M) was due to the presence of a Gin residue at the P1 site, which was well consistent with the results obtained for P1-site mutants of SSI and turkey ovomucoid domain 3.OXFORD UNIV PRESS, Mar. 1995, JOURNAL OF BIOCHEMISTRY, 117(3) (3), 609 - 613, EnglishIntroduction research institution
- 1995, Actinomycetologica (Review of the SAJ Promotion Awardee's Lecture), 9(2) (2), 216 - 227Introduction research institution
- MOLECULAR CHARACTERIZATION OF A GENE ENCODING EXTRACELLULAR SERINE-PROTEASE ISOLATED FROM A SUBTILISIN INHIBITOR-DEFICIENT MUTANT OF STREPTOMYCES-ALBOGRISEOLUS-S-3253An extracellular serine protease produced by a mutant, M1, derived from Streptomyces albogriseolus S-3253 that no longer produces a protease inhibitor (Streptomyces subtilisin inhibitor [SSI]) was isolated. A 20-kDa protein was purified by its affinity for SSI and designated SAM-P20. The amino acid sequence of the aminoterminal region of SAM-P20 revealed high homology with the sequences of Streptomyces griseus proteases A and B, and the gene sequence confirmed the relationships. The sequence also revealed a putative amino acid signal sequence for SAM-P20 that apparently functioned to allow secretion of SAM-P20 from Escherichia coli carrying the recombinant gene. SAM-P20 produced by E. coli cells was shown to be sensitive to SSI inhibition.AMER SOC MICROBIOLOGY, Jan. 1995, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 61(1) (1), 180 - 186, EnglishIntroduction research institution
- A novel serine protease inhibitor SIL8, which was isolated from the culture medium of Streptomyces virginiae and shown to be a member of the Streptomyces subtilisin-inhibitor-like (SIL) inhibitor family by sequence analysis of its amino-terminal region [Taguchi, S., Kikuchi, H., Kojima, S., Kumagai, I., Nakase, T., Miura, K. and Momose, H. (1993) Biosci. Biotech. Biochem. 57, 522-524], is the first SIL inhibitor demonstrated to show marked inhibitory activity toward alpha-chymotrypsin, in addition to strong inhibitory activity toward subtilisin BPN', a common property of inhibitors of the Streptomyces subtilisin inhibitor (SSI) family. In this study, the complete amino acid sequence of SIL8 was determined from the sequence analysis of peptides obtained by specific cleavage at the reactive site and by enzymic digestion. SIL8 was shown to exist as a dimer protein, each subunit of which was composed of 111 amino acids, and to have less than 50% similarity with other SSI-family inhibitors, indicating its most distant relationship to other members of this family. Insertion of two residues was observed in the flexible loop region of SIL8, and amino acid replacements were found not only on the molecular surface but also in the beta-sheet and hydrophobic core, suggesting that packing rearrangements of the side chains may occur in these regions to maintain the tertiary and quaternary structures. The inhibitor constants K-i obtained using synthetic substrates are 92 pM for subtilisin BPN' and 11 nM for alpha-chymotrypsin. The P1 site was identified as methionine, which was in good agreement with the substrate specificity of alpha-chymotrypsin. SSI, which also possesses a methionine residue at the P1 site, inhibits alpha-chymotrypsin poorly (inhibitor constant, 4.0 mu M). Such a difference in the inhibitory properties of SIL8 and SSI toward alpha-chymotrypsin is discussed on the basis of the structures of the inhibitors.SPRINGER VERLAG, Dec. 1994, EUROPEAN JOURNAL OF BIOCHEMISTRY, 226(2) (2), 627 - 632, EnglishIntroduction research institution
- 3 NOVEL SUBTILISIN-TRYPSIN INHIBITORS FROM STREPTOMYCES - PRIMARY STRUCTURES AND INHIBITORY PROPERTIESThree novel proteinaceous inhibitors, which had been identified as ''Streptomyces subtilisin inhibitor-like (SIL) proteins'' and exhibited trypsin inhibition in addition to strong inhibition toward subtilisin BPN', were purified from the culture broth of three Streptomyces strains: SIL10 from S. thermotolerans, SIL13 from S. galbus, and SIL14 from S. azureus. Their primary structures were determined by sequence analysis of intact SIL inhibitors and peptides obtained by enzymatic digestions of S-pyridylethylated SIL inhibitors. These inhibitors were composed of about 110 amino acids and existed as dimer proteins. The reactive site was identified as Lys-Gln for all three inhibitors by sequence analysis of their modified forms in which the reactive-site peptide bond was specifically cleaved by subtilisin BPN' under acidic conditions. Thus, their inhibition toward trypsin and subtilisin BPN' was due to the presence of a Lys residue at the P1 site. Inhibitor constants toward subtilisin BPN' and trypsin were also determined. These inhibitors showed relatively high sequence homology to other SSI-family inhibitors possessing a Lys residue at the P1 site, with amino acid replacements on their molecular surface.JAPANESE BIOCHEMICAL SOC, Nov. 1994, JOURNAL OF BIOCHEMISTRY, 116(5) (5), 1156 - 1163, EnglishIntroduction research institution
- IN-VIVO MONITORING-SYSTEM FOR STRUCTURE-FUNCTION RELATIONSHIP ANALYSIS OF THE ANTIBACTERIAL PEPTIDE APIDAECINA unique antibacterial peptide derivative found in immune honeybee lymph, apidaecin 1b (AP1), was randomly mutagenized and characterized by a newly established system to analyze in vivo its structure-function relationship. Initially, a high-level expression host-vector system for AP1 in Escherichia coli was constructed by creating a fusion protein with the highly stable Streptomyces subtilisin inhibitor (SSI) molecule. Expression of the SSI-AP1 fusion protein was found to depend on the concentration of the transcriptional inducer isopropyl-beta-D-thio-galactopyranoside (IPTG) and to parallel the degree of growth inhibition of the transformant cells. Subsequently, apidaecin derivatives produced by localized random mutagenesis were screened with this IPTG concentration-controlled in vivo system by monitoring the growth inhibition patterns of the transformant cells. One mutant apidaecin (P9L) that had reduced activity was purified and isolated from the periplasmic fraction of an L. coli transformant. Its antibacterial activity was reduced to one-third of that of wild-type apidaecin. When considered together with the other mutations, it was concluded that several Pro residues, including that at the ninth position, are responsible for expression of the antibacterial action of apidaecin.AMER SOC MICROBIOLOGY, Oct. 1994, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 60(10) (10), 3566 - 3572, EnglishIntroduction research institution
- PRIMARY STRUCTURE AND INHIBITORY PROPERTIES OF A PROTEINASE-INHIBITOR PRODUCED BY STREPTOMYCES-CACAOIProtein proteinase inhibitors showing sequence homology with Streptomyces subtilisin inhibitor (SSI) have been found to be distributed widely in Streptomyces species, and accordingly have been named SSI-like (SIL) proteins. SIL1 from S. cacaoi was the first of these proteins to be isolated and to be given a serial number. To study the structure-function relationship of SIL proteins, we determined the primary structure of SIL1 and measured its inhibitory activities. It was found to be composed of 110 amino acids and to exist in dimer form. The amino-acid sequence of SIL1 was unique among other characterized SIL proteins in having a one-residue deletion in two regions and a three-residue insertion in the flexible loop region. Sequence comparison indicated that SIL1 was distantly related to other members of the SSI family, and that amino-acid replacements had occurred not only on the surface of the SIL1 molecule but also in the beta-sheet region. The reactive site of SIL1 was considered to be Arg(70)-Glu(71) from sequence alignment with other SSI-family inhibitors. SIL1 inhibited subtilisin BPN' strongly with an inhibitor constant (K-i) of 2.8.10(-11)M, like other members of the SSI family possessing an Arg residue at the P1 site. In contrast, SIL1 exhibited weak inhibition toward trypsin with a K-i value of 5.5.10(-8)M, possibly as a consequence of insertion of the three residues in the flexible loop region near the reactive site. This contrast seems to be due to the difference in the subsite structure of the two proteinases.ELSEVIER SCIENCE BV, Jul. 1994, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1207(1) (1), 120 - 125, EnglishIntroduction research institution
- In order to improve a natural enzyme so as to fit industrial purposes, we have applied experimental evolution techniques comprised of successive in vitro random mutagenesis and efficient screening systems. Subtilisin BPN', a useful alkaline serine protease, was used as the model enzyme, and the gene was cloned to an Escherichia coli host-vector system. Primary mutants with reduced activities of below 80% of that of the wild type were first derived by hydroxylamine mutagenesis directly applied to subtilisin gene DNA, followed by screening of clear-zone non-forming transformant colonies cultured at room temperature on plates containing skim-milk. Then, secondary mutants were derived from each primary mutant by the same mutagenic procedure, but screened by detecting transformant colonies incubated at 10 degrees C with clear zones that were greater in size than that of the wild type. One such secondary mutant, 12-12, derived from a primary mutant with 80% activity, was found to gain 150% activity (k(cat)/K-m value) of the wild-type when the mutant subtilisin gene was subcloned to a Bacillus subtilis host-vector system, expressed to form secretory mutant enzyme in the medium, and the activity measured using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as the substrate. When N-succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide was used, 180% activity was gained. Genetic analysis revealed that the primary and secondary mutations corresponded to D197N and G131D, respectively. The activity variations found in these mutant subtilisins were discussed in terms of Ca2+-binding ability. The thermostability was also found to be related to the activity.SPRINGER VERLAG, Apr. 1994, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 41(2) (2), 239 - 244, EnglishIntroduction research institution
- Three novel proteinaceous inhibitors of serine proteases which had been identified as Streptomyces subtilisin inhibitor-like (SIL) inhibitors were isolated from culture supernatant of Streptomyces; SIL2 from Streptomyces parvulus, SIL3 from Streptomyces coelicolor and SIL4 from Streptomyces lavendulae. They exhibited not only strong inhibitory activity toward subtilisin BPN' but also less strong inhibition of trypsin. Their primary sequences were determined by sequence analysis of peptides obtained by specific cleavage at the reactive site and subsequent proteolytic digestion. Each inhibitor consisted of about 110 amino acids, and was considered to form a dimer. The reactive site of the inhibitors was identified as Arg-Glu for SIL2 and SIL3, and Lys-Leu for SIL4, from sequence analysis of modified forms of the inhibitors produced from the inhibitor-subtilisin complex under acidic conditions. The presence of an arginine/lysine residue at the P1 site was in agreement with their trypsin-inhibition property. Sequence comparison with other members of the Streptomyces subtilisin inhibitor family revealed that amino acid replacements in the three isolated SIL inhibitors were frequently localized on the surface region, and many of the amino acid residues in beta-sheets and the hydrophobic core were highly conserved. Values of the inhibitor constant (K-i) toward subtilisin BPN' and trypsin were also measured, and the differences were discussed on the basis of the determined structures of the inhibitors.WILEY-BLACKWELL, Mar. 1994, EUROPEAN JOURNAL OF BIOCHEMISTRY, 220(3) (3), 911 - 918, EnglishIntroduction research institution
- STREPTOMYCES SUBTILISIN INHIBITOR-LIKE PROTEINS ARE DISTRIBUTED WIDELY IN STREPTOMYCETESStreptomyces subtilisin inhibitor-like proteins were found to be distributed widely in streptomycetes by using the combination of the convenient, newly developed plate assay system and an established liquid culture assay. Almost all the strains formerly categorized as Streptoverticillium species produced proteins that exhibited inhibitory activity against both subtilisin BPN' and trypsin. N-terminal regions of three purified proteins showed high structural similarity to those of other previously reported SIL inhibitors.AMER SOC MICROBIOLOGY, Dec. 1993, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 59(12) (12), 4338 - 4341, EnglishIntroduction scientific journal
- A high-level production system in Escherichia coli for an alkaline serine protease inhibitor, termed Streptomyces subtilisin inhibitor (SSI), from S albogriseolus S-3253 was established by replacing the SSI signal sequence with the OmpA signal sequence using the inducible pIN-III-ompA vector. Significant amounts of recombinant SSI, resulting from accurate cleavage of the OmpA signal peptide, were accumulated in the periplasmic space or excreted into the culture medium. The inhibitory activity of the processed protein against subtilisin BPN' was identical with that of authentic SSI. Furthermore, deletion of one of the putative dual terminators (terminator 1) resulted in a 1.9-fold increase in production. This effect on SSI gene expression efficiency was found to be governed mainly at the transcription level.SPRINGER VERLAG, Aug. 1993, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 39(6) (6), 732 - 737, EnglishIntroduction research institution
- TAYLOR & FRANCIS LTD, Jul. 1993, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 57(7) (7), 1206 - 1207, EnglishIntroduction scientific journal
- EFFECT OF A RARE LEUCINE CODON, TTA, ON EXPRESSION OF A FOREIGN GENE IN STREPTOMYCES-LIVIDANSStreptomyces are bacteria with a very high chromosomal G + C composition (> 70 mol%) and extremely biased codon usage. In order to investigate the relationship between codon usage and gene expression in Streptomyces, we used ssi (Streptomyces subtilisin inhibitor) as a reporter gene and monitored its secretory expression in S. lividans. In consequence of alteration of the native codons of Leu, Lys and Ser of ssi to minor ones by site-directed mutagenesis, i.e., Leu79-Leu80:CTG-CTC to TTA-TTA, Lys89 : AAG to AAA, Ser108-Ser109: TCG-AGC to TCT-TCT, respectively, the production of SSI was reduced remarkably in the case of TTA codons, while it was slightly increased in the case of AAA and almost the same in TCT codons. This conspicuous decrease found for Leu codon replacement was probably due to the low availability of intracellular tRNA(Leu)(UUA), a product of bldA which has been reported to be expressed only during the late stage of growth.ELSEVIER SCIENCE BV, Mar. 1993, BIOCHIMICA ET BIOPHYSICA ACTA, 1172(3) (3), 262 - 266, EnglishIntroduction research institution
- TAYLOR & FRANCIS LTD, Mar. 1993, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 57(3) (3), 522 - 524, EnglishIntroduction scientific journal
- The contribution of downstream message secondary structure to the production of Streptomyces subtilisin inhibitor (SSI) was investigated in Streptomyces lividans 66 using a cloned gene. By deletion analysis, two inverted repeats located downstream from the stop codon in the SSI gene were found to exhibit a marked effect on the amount and homogeneity of SSI gene transcript and, consequently, the efficiency of SSI productivity.ELSEVIER SCIENCE BV, Mar. 1993, FEMS MICROBIOLOGY LETTERS, 107(2-3) (2-3), 185 - 189, EnglishIntroduction research institution
- We attempted to screen a series of Streptomyces subtilisin inhibitor-like (SIL) proteins among several Streptomyces strains by using a highly sensitive assay system established by us. Of six randomly tested strains, four were found to produce SIL inhibitors as their major secreted proteins, suggesting that they might be distributed in a high frequency among this genus. Three inhibitors exhibited inhibition of both subtilisin BPN' and trypsin. Comparison of the amino terminal sequences of these isolated proteins with those of other reported SIL inhibitors revealed that the beta1- and beta2-sheets in SSI were highly conserved.WILEY-BLACKWELL, Dec. 1992, FEMS MICROBIOLOGY LETTERS, 99(2-3) (2-3), 293 - 297, EnglishIntroduction research institution
- EXTRACELLULAR PRODUCTION SYSTEM OF HETEROLOGOUS PEPTIDE DRIVEN BY A SECRETORY PROTEASE INHIBITOR OF STREPTOMYCESThe value of a heterologous peptide extra-cellular production system in Streptomyces using a secretory protease inhibitor, was examined. DNA was synthesized encoding apidaecin 1b (AP1), an interesting antibacterial peptide discovered in lymph fluid of the honeybee, and was joined to the Streptomyces subtilisin inhibitor (SSI) gene via a 12-bp nucleotide sequence corresponding to the amino acid sequence specific for cleavage by blood coagulation factor Xa. The fusion protein (SSI-AP1) could be expressed and excreted efficiently into the medium by culturing S. lividans 66 harbouring a plasmid vector constructed for SSI secretion, into which the synthetic DNA was introduced. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and amino acid analysis of the purified SSI-AP1 provided reasonable results of molecular size and composition value. Interestingly, SSI-AP1 protein showed bifunctional activity: inhibitory activity of SSI and antibacterial activity of AP1. The inhibitory activity against Escherichia coli could be also detected after the fusion protein was cleaved by factor Xa. The extra-cellular production system presented here should provide a useful tool for production, analysis of mode of action, and also for genetic improvement of antimicrobial peptides such as apidaecin.SPRINGER VERLAG, Mar. 1992, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 36(6) (6), 749 - 753, EnglishIntroduction research institution
- 1992, 蛋白質 核酸 酵素, 37(3) (3)Extracellular Production System of Foreign Protein in Streptomyces.
- 1992, Actinomycetologica (Review of SAJ Invited Lecture), 6(1) (1), 9 - 20Introduction research institution
- A host-vector system was used for the production of Streptomyces subtilisin inhibitor (SSI). The gene fragment encoding SSI was replaced in the vector with the tyrosinase gene of plasmid pIJ702. It was found that the optimal culture conditions for the SSI production by the former system are almost the same as those for the melanin synthesis by the latter system. This fact suggests a convenient method in that the information on the productivity of an indicator host-vector system with regard to the culture conditions can be applied for the optimization of the production of a different material with a similar host-vector system differing in the gene coding for the different product.SOC FERMENTATION BIOENGINEERING, JAPAN, 1992, JOURNAL OF FERMENTATION AND BIOENGINEERING, 73(4) (4), 317 - 318, EnglishIntroduction scientific journal
- Two sets of the Shine-Dalgarno sequence and the initiation codon (ATG) for translation of a gene encoding the protein SSI (Streptomyces subtilisin inhibitor) were studied in vivo by site-directed mutagenesis. The result shows that each ATG can function as an initiator of translation in either Streptomyces lividans 66 or Escherichia coli. The choice of initiation codon seems dependent on the host strain and is closely related to the processing mechanism of pre-SSI protein. The upstream ATG is presumed to be utilized preferentially giving two cleavage sites in pre-SSI in S. albogriseolus S-3253, the original SSI producer strain.SPRINGER VERLAG, Apr. 1991, MOLECULAR & GENERAL GENETICS, 226(1-2) (1-2), 328 - 331, EnglishIntroduction scientific journal
- ELSEVIER SCIENCE BV, Jul. 1990, BIOCHIMICA ET BIOPHYSICA ACTA, 1049(3) (3), 278 - 285, EnglishCOMPARISON OF SECRETORY EXPRESSION IN ESCHERICHIA-COLI AND STREPTOMYCES OF STREPTOMYCES SUBTILISIN INHIBITOR (SSI) GENEIntroduction research institution
- ELSEVIER SCIENCE BV, Dec. 1989, GENE, 84(2) (2), 279 - 286, EnglishIntroduction research institution
- NATURE PUBLISHING CO, Oct. 1989, BIO-TECHNOLOGY, 7(10) (10), 1063 - 1066, EnglishEFFICIENT EXTRACELLULAR EXPRESSION OF A FOREIGN PROTEIN IN STREPTOMYCES USING SECRETORY PROTEASE INHIBITOR (SSI) GENE FUSIONSIntroduction research institution
- JAPANESE BIOCHEMICAL SOC, Mar. 1989, JOURNAL OF BIOCHEMISTRY, 105(3) (3), 367 - 371, EnglishMOLECULAR-CLONING AND NUCLEOTIDE-SEQUENCE DETERMINATION OF GENE ENCODING STREPTOMYCES SUBTILISIN INHIBITOR (SSI)Introduction research institution
- JAPAN ACAD, Jun. 1988, PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES, 64(6) (6), 147 - 149, EnglishIntroduction research institution
- 1987, 東京大学工学部紀要A, 64(25) (25), 58 - 59遺伝子工学による放線菌プロテアーゼインヒビターの人工的改変Introduction research institution
- 官業労働研究所, Nov. 1972, 官公労働, 26(11) (11), 16 - 29公務員・公社職員等の政治活動--法規・裁判例から見たその限界Introduction research institution
- 第一法規, Aug. 1963, 自治研究, 39(8) (8), 153 - 160地方公務員法46条による措置要求の対象事項および公立学校職員の勤務評定に関する規則等の取消--行政判例研究-98-Introduction research institution
- 第一法規, Jul. 1956, 自治研究, 32(7) (7), 69 - 74行政協定の実施に伴う土地等の使用に関する特別措置法第5条による内閣総理大臣の使用の認定における物件の特定Introduction research institution
- Single work, NTS出版, Apr. 2021, Japanese「バイオリアクターのスケールアップと物質生産事例集」:乳酸ポリマーP(LAHB)の微生物生産Scholarly book
- Joint work, 2章1節 酵素進化工学によって創出される多様なバイオポリマー、3編1節 進化分子工学のケーススタディー:産業酵素と抗菌ペプチド, シーエムシー, Dec. 2019, 287-295,319-329, Japanese, 本人担当部分の概要:進化工学の典型例として、細胞内合成・蓄積するPHAを取り上げ、各種要素技術を駆使して効率的な微生物生産する道筋を論理的に解説している。進化工学の典型例として、産業酵素と抗菌ペプチドを取り上げ、各種要素技術を駆使して効率的な微生物生産する方法を解説している。非天然型ポリヒドロキシアルカン酸の分解性とその評価方法Scholarly book
- Single work, 包装食品技術協会誌「食品の包装」 特集:バイオプラスチック, Dec. 2019, Japanese多元ポリ乳酸:生分解性バイオマスプラスチックの食品包装材への展開Scholarly book
- Joint work, エヌティーエス, Dec. 2019, Japanese「オリゴマー」が鍵:バイオプラスチックの生合成と生分解Scholarly book
- Joint work, 2章1節 酵素進化工学によって創出される多様なバイオポリマー、3編1節 進化分子工学のケーススタディー:産業酵素と抗菌ペプチド, シーエムシー, Dec. 2019, 287-295,319-329, Japanese, 本人担当部分の概要:進化工学の典型例として、細胞内合成・蓄積するPHAを取り上げ、各種要素技術を駆使して効率的な微生物生産する道筋を論理的に解説している。進化工学の典型例として、産業酵素と抗菌ペプチドを取り上げ、各種要素技術を駆使して効率的な微生物生産する方法を解説している。多元ポリ乳酸の合成/分解の交差点:「オリゴマー」Scholarly book
- Single work, 2章1節 酵素進化工学によって創出される多様なバイオポリマー、3編1節 進化分子工学のケーススタディー:産業酵素と抗菌ペプチド, シーエムシー, Nov. 2019, 287-295,319-329, Japanese, 本人担当部分の概要:進化工学の典型例として、細胞内合成・蓄積するPHAを取り上げ、各種要素技術を駆使して効率的な微生物生産する道筋を論理的に解説している。進化工学の典型例として、産業酵素と抗菌ペプチドを取り上げ、各種要素技術を駆使して効率的な微生物生産する方法を解説している。「多元ポリ乳酸」生合成の新展開:オリゴマー分泌発見 によるプロセス革新Scholarly book
- Single work, 2章1節 酵素進化工学によって創出される多様なバイオポリマー、3編1節 進化分子工学のケーススタディー:産業酵素と抗菌ペプチド, CMC, Apr. 2019, 287-295,319-329, Japanese, 本人担当部分の概要:進化工学の典型例として、細胞内合成・蓄積するPHAを取り上げ、各種要素技術を駆使して効率的な微生物生産する道筋を論理的に解説している。進化工学の典型例として、産業酵素と抗菌ペプチドを取り上げ、各種要素技術を駆使して効率的な微生物生産する方法を解説している。Development and Applications of Natural Antibacterial and Antifungal AgentsScholarly book
- Joint work, Springer, Apr. 2019, EnglishSynthesis of Polyesters III: Acyltransferase as CatalystScholarly book
- Joint work, Chapter 13 Microbial Plastic Factory: Synthesis and Properties of the New Lactate-Based Biopolymers, Nature Springer Press, Apr. 2019, 175-197, English, バイオベースのモノマー、ポリマー、材料について最新知見を網羅的に取り上げている本人担当部分の概要:最近開発した乳酸ベースポリマーの微生物生産法について解説している。特に、乳酸モノマーの供給系確立、重合酵素の創出について重点的に述べている本人担当部分の共著者:J. M. Nduko, K. Matsumoto , S. TaguchiMicrobial production and properties of LA-based polymers and oligomers from renewable feedstock: Biosynthesis of Polymers from Renewable Biomass: Production of Materials from Sustainable Biomass Resources: Biofuels and Biorefineries book series (BIOBIO, volume 9) pp 361-390(持続可能バイオマス資源からの材料生産)Scholarly book
- Joint work, Chapter 13 Microbial Plastic Factory: Synthesis and Properties of the New Lactate-Based Biopolymers, Nature Springer Press, Apr. 2019, 175-197, English, バイオベースのモノマー、ポリマー、材料について最新知見を網羅的に取り上げている本人担当部分の概要:最近開発した乳酸ベースポリマーの微生物生産法について解説している。特に、乳酸モノマーの供給系確立、重合酵素の創出について重点的に述べている本人担当部分の共著者:J. M. Nduko, K. Matsumoto , S. TaguchiMicrobial production and properties of LA-based polymers and oligomers from renewable feedstock: Biosynthesis of Polymers from Renewable Biomass: Production of Materials from Sustainable Biomass Resources: Biofuels and Biorefineries book series (BIOBIO,Scholarly book
- Joint work, Chapter 13 Microbial Plastic Factory: Synthesis and Properties of the New Lactate-Based Biopolymers, American Chemical Society, Nov. 2018, 175-197, English, バイオベースのモノマー、ポリマー、材料について最新知見を網羅的に取り上げている本人担当部分の概要:最近開発した乳酸ベースポリマーの微生物生産法について解説している。特に、乳酸モノマーの供給系確立、重合酵素の創出について重点的に述べている本人担当部分の共著者:J. M. Nduko, K. Matsumoto , S. TaguchiMicrobial secretion system of lactate-based oligomers and its application: Green Polymer Chemistry: New Products, Processes, and Applications (ACS Symposium Series Vol. 1310, pp 41-60(グリーンポリマーケミストリー:生体触媒と材料III)Scholarly book
- Joint work, Chapter 13 Microbial Plastic Factory: Synthesis and Properties of the New Lactate-Based Biopolymers, Wiley Blackwell (John Wiley & Sons), May 2017, 175-197, English, バイオベースのモノマー、ポリマー、材料について最新知見を網羅的に取り上げている本人担当部分の概要:最近開発した乳酸ベースポリマーの微生物生産法について解説している。特に、乳酸モノマーの供給系確立、重合酵素の創出について重点的に述べている本人担当部分の共著者:J. M. Nduko, K. Matsumoto , S. TaguchiBioengineering of Polyhydroxyalkanoates: Encyclopedia of Polymer Science and Technology(ポリマーの科学と技術に関する百科辞典)Scholarly book
- Joint work, Organic Chemistry, May 2017, EnglishEnzymes catalyzing the synthesis and degradation of beta-linked biopolymers and their applications, Biodegradable polymers: recent developments and new perspectivesScholarly book
- Single work, 第5章 昆虫由来抗菌ペプチドの進化工学的高活性化, CMC, May 2017, 287-295,319-329, Japanese, 本人担当部分の概要:抗菌ペプチドのカテゴリー・昆虫由来の抗菌ペプチドの特色から構造機能相関・作用機序・進化工学について詳細に解説。, ISBN: 9784781312453Scholarly book
- Joint work, 第2章 微生物の生体成分利用 第9節 生活に浸透する微生物ポリマーの威力, 文永堂出版, Jul. 2016, 93-9, Japanese, 従来の微生物学,遺伝子工学,生物工学などにゲノム科学や生物情報学を加えた分野横断的な複合科学で,微生物の機能や能力を基盤として開発された利用法とその実例を紹介。本人担当部分の概要:応用微生物学に関する多種多様な分野のうち、脂肪酸からなるバイオポリエステルを対象にその生合成法、構造・物性、機能応用にまで言及している。応用微生物学(第3版)Scholarly book
- Joint work, Chapter 6 Protein Engineering of Enzymes Involved in Bioplastic Metabolism, Springer Berlin Heidelberg, Dec. 2015, 133-165, English, 各産業界で活躍している有用酵素のタンパク質工学実例集的解説書。本人担当部分の概要:最近開発した乳酸ベースポリマーの微生物生産法について解説している。特に、乳酸モノマーの供給系確立、重合酵素の創出について重点的に述べている。本人担当部分の共著者:H. Hiraishi, S. TaguchiBiosynthesis of polymers - Encyclopedia of Polymeric Nanomaterials(ポリマーの生合成 高分子ナノ材料の百科辞典)Scholarly book
- Joint work, Chapter 13 Microbial Plastic Factory: Synthesis and Properties of the New Lactate-Based Biopolymers, American Chemical Society, Nov. 2015, 175-197, English, バイオベースのモノマー、ポリマー、材料について最新知見を網羅的に取り上げている本人担当部分の概要:最近開発した乳酸ベースポリマーの微生物生産法について解説している。特に、乳酸モノマーの供給系確立、重合酵素の創出について重点的に述べている本人担当部分の共著者:J. M. Nduko, K. Matsumoto , S. TaguchiGreen polymer chemistry: Biobased Materials and Biocatalysis (ACS Symposium Series Vol. 1192(グリーンポリマーケミストリー:生体触媒と材料III)Scholarly book
- Joint work, Caister Academic Press, May 2015, English, コリネバクテリウムグルタミカムのシステムバイオロジーとバイオテクノロジーに焦点を当てまとめられた。特にプロテオミクス、代謝解析、代謝工学などを利用した、有機酸やアルコール、ポリエステルの物質生産などや外来タンパク質の分泌生産などのトピックを紹介した。本人担当部分の概要:最近開発した乳酸ベースポリマーの表記微生物生産法について解説している。特に、乳酸モノマーの供給系確立、重合酵素の創出について重点的に述べている。本人担当部分の共著者:Y. Song, J. M. Nduko, K. Matsumoto , S. TaguchiCorynebacterium glutamicum: From systems biology to biotechnological applications(コリネバクテリウムグルタミカム:システムバイオロジーから生物工学的応用まで)Scholarly book
- Joint translation, 第7章 タンパク質の機能と進化, 西村書店, Mar. 2015, 209-256, English, 原著名:Biochemistry (4th Edition)Christopher K. Mathews, Kensal E. van Holde, Dean R. Appling, Spencer J Anthony-Cahill 「カラー 生化学第4版」Scholarly book
- Joint work, 微生物ポリエステル, 丸善出版, Jan. 2014, Japanese化学便覧 応用化学編 第7編Dictionary or encycropedia
- Joint work, Chapter 13 Microbial Plastic Factory: Synthesis and Properties of the New Lactate-Based Biopolymers, American Chemical Society, Nov. 2013, 175-197, English, バイオベースのモノマー、ポリマー、材料について最新知見を網羅的に取り上げている本人担当部分の概要:最近開発した乳酸ベースポリマーの微生物生産法について解説している。特に、乳酸モノマーの供給系確立、重合酵素の創出について重点的に述べている本人担当部分の共著者:J. M. Nduko, K. Matsumoto , S. TaguchiGreen polymer chemistry: Biocatalysis and materials II (ACS Symposium Series Vol. 1144(グリーンポリマーケミストリー:生体触媒と材料Ⅱ)Scholarly book
- Joint work, 2章1節 酵素進化工学によって創出される多様なバイオポリマー、3編1節 進化分子工学のケーススタディー:産業酵素と抗菌ペプチド, エヌ・ティ・エス, Oct. 2013, 287-295,319-329, Japanese, 本人担当部分の概要:進化工学の典型例として、細胞内合成・蓄積するPHAを取り上げ、各種要素技術を駆使して効率的な微生物生産する道筋を論理的に解説している。進化工学の典型例として、産業酵素と抗菌ペプチドを取り上げ、各種要素技術を駆使して効率的な微生物生産する方法を解説している。進化分子工学—高速分子進化によるタンパク質・核酸の開発Scholarly book
- Joint work, Chapter 6 Protein Engineering of Enzymes Involved in Bioplastic Metabolism, INTECH, May 2013, 133-165, English, 各産業界で活躍している有用酵素のタンパク質工学実例集的解説書。本人担当部分の概要:最近開発した乳酸ベースポリマーの微生物生産法について解説している。特に、乳酸モノマーの供給系確立、重合酵素の創出について重点的に述べている。本人担当部分の共著者:H. Hiraishi, S. TaguchiProtein Engineering - Technology and Application(タンパク質工学 技術と応用)Scholarly book
- Joint work, Polyhydroxyalkanoate, CMC publishing, Feb. 2013, 71-85, English, バイオポリマー材料全体を俯瞰し、各種カテゴリーを創設している包括的成書。本人担当部分の概要:微生物産生バイオポリマーの生合成、構造決定、機能物性などの専門家が共同して執筆している新しいタイプの成書。本人担当部分の共著者:T. Iwata, T. Tsuge, S. Taguchi, H. Abe, T. TanakaHandbook of R&D information Japan, Bio-based polymers(バイオベースポリマー)Scholarly book
- Joint work, Part III 細胞システム工学:第9章 バイオポリエステルの生産システム工学, 化学同人, Jan. 2013, 177-204, Japanese, 生命を構成する分子の高機能化・新機能創発を進化原理に基づいて実現する「生命システム工学」のすべてがわかる一冊.究極のゴールである「細胞の創成」に向けた研究の基礎から最前線までを解説。本人担当部分の概要:本人が編集を務め、冒頭のはじめにからポリエステルの微生物生産工学について詳細に解説している。特にポリマーの効率生産に有効な代謝工学や酵素工学についてその戦略から実践までを網羅した。本人担当部分の共著者:松本謙一郎,田口精一生命システム工学(DOJIN BIOSCIENCE SERIES)Scholarly book
- Joint work, Biological Lactate-Polymers Synthesized by One-Pot Microbial Factory: Enzyme and Metabolic Engineering, ACS Publications, Aug. 2012, 213-235, English, バイオベースのモノマー、ポリマー、材料について最新知見を網羅的に取り上げている。本人担当部分の概要:最近開発した乳酸ベースポリマーの微生物生産法について解説している。特に、乳酸モノマーの供給系確立、重合酵素の創出について重点的に述べている。本人担当部分の共著者:J. M. Nduko, K. Matsumoto, S. TaguchiBiobased Monomers, Polymers, and Materials(ACS Symposium Series Volume 1105)(バイオベースのモノマー、ポリマーと材料)Scholarly book
- Joint work, 第17章 微生物工場による「多元ポリ乳酸」創製のための合成生物工学, シーエムシー出版, Apr. 2012, 157-182, Japanese, 微生物などの産業・工業利用に特化した合成生物学を「合成生物工学」とし,その先進的で創造的な研究内容を展開している研究を紹介本人担当部分の概要:合成生物工学の典型例として、細胞内合成・蓄積するPHAを取り上げ、各種要素技術を駆使して効率的な微生物生産する道筋を論理的に解説している。本人担当部分の共著者:渡辺剛志、越智杏奈、田口精一合成生物工学の隆起―有用物質の新たな生産法構築をめざして―Scholarly book
- Joint work, Volume 9 Polymers in biology and medicine, 9.09 Poly(hydroxyalkanoate)s, Elsevier, Mar. 2012, 157-182, English, 生体関連高分子の合成機構と物性および医学的応用について概説した。本人担当部分の概要:多種多様なバイオポリマーのうち、PHAに焦点を当ててその生合成から物性・、機能に至るまでのフルコースを平易に解説している。本人担当部分の共著者:S. Taguchi, T. Iwata, H. Abe, Y. DoiPolymer Science: A Comprehensive Reference, Polymers in biology and medicine(高分子科学:包括的なレファレンス、生物学と医学における高分子)Scholarly book
- Others, 化学同人, 2012, Japanese, ISBN: 9784759815061Others
- Others, 三共出版, 2012, Japanese, ISBN: 9784782706718Others
- Joint work, 第1章 新しい植物由来ポリマー・材料の開発、10節 微生物を用いた乳酸ポリマーのワンステップ重合法, サイエンス&テクノロジー出版, Dec. 2011, 74-80, Japanese, 「植物由来ポリマーは石油由来樹脂に敵わない」を覆す!をコンセプトにまとめたものであり、植物由来原料の利用技術について、最先端の技術進歩を材料開発と用途開発の両面からまとめた。本人担当部分の概要:植物バイオマス由来原料を利活用した、バイオポリマーの微生物生産プロセス開発の歴史・変遷について要素秘術開発を軸に解説している。本人担当部分の共著者:越智杏奈,渡辺剛志,田口精一Scholarly book
- Joint work, 第2章 微生物の生体成分利用 第9節 生活に浸透する微生物ポリマーの威力, 三共出版, Dec. 2011, 93-9, Japanese, 従来の微生物学,遺伝子工学,生物工学などにゲノム科学や生物情報学を加えた分野横断的な複合科学で,微生物の機能や能力を基盤として開発された利用法とその実例を紹介。本人担当部分の概要:応用微生物学に関する多種多様な分野のうち、脂肪酸からなるバイオポリエステルを対象にその生合成法、構造・物性、機能応用にまで言及している。微生物機能学 ―微生物リソースと遺伝子リソース―Scholarly book
- Joint work, 第7章 バイオプロダクトと新プラットフォーム形成、12. 乳酸ポリマーのワンポット微生物合成, シーエムシー出版, Dec. 2010, 237-245, Japanese, 脱石油の新しい産業構造を構築していくためのマイルストーンを明示し、化石燃料と決別して持続可能な未来型の循環型社会への移行に必要な「ものづくり」について詳述。本人担当部分の概要:石油に代替するバイオマス資源を有効利用した、バイオポリマーの微生物合成について解説している。再生可能な糖質や油脂を利用したポリマー合成用微生物発酵について述べている。エコバイオリファイナリー―脱石油社会へ移行するための環境ものづくり戦略―Scholarly book
- Joint work, 第 5 編機能材料 第6章 バイオポリマーPHAと酵素分子進化工学, エヌ・ティー・エス, Apr. 2010, 920-925, Japanese, 酵素の基礎のみならず、新規酵素の研究開発、そして各種応用技術にいたる道標となるためにまとめられた。本人担当部分の概要:ポリヒドロキシアルカン酸の微生物細胞内で酵素合成されることの基礎と応用について解説している。特に進化工学的展開について技術的な解説に重点を置いた。酵素利用技術大系Scholarly book
- Joint work, 第 7 編 酵素を使うII―産業応用、 第26章 バイオプラスチックの合成・調節・分解に関わる酵素群の科学と工学, シーエムシー出版, Mar. 2009, 257-271, Japanese, 産業酵素の実際や最新の酵素化学、新しい用途開発についてまとめた。本人担当部分の概要:微生物ポリエステル合成に関与する酵素群の、代謝合成経路における特質や工学的応用に関して解説している。産業酵素の応用技術と最新動向Scholarly book
- Joint work, Natural polyester-related proteins: Structure, function, evolution and engineering, WILEY-VCH, Nov. 2008, 877-914, English, 蛋白質の工学と設計に関する初めての包括的な参考図書で、急速な発展を遂げている当分野における最新の概念・方法・応用を網羅し、今後の可能性についてまとめた。本人担当部分の概要:微生物ポリエステルPHAの生合成に関連する酵素群に焦点を当て、それらの構造・機能・進化・エンジニアリングについて解説している。特に、タンパク質工学と進化工学の戦略と実践について詳細に述べている。Protein Engineering Handbook(タンパク質工学ハンドブック)Scholarly book
- Joint work, 第5章 化成品素材の生産―バイオポリエステル―, シーエムシー出版, Jun. 2008, 244-253, Japanese, ホワイトバイオテクノロジーの基本である「微生物によるものづくり」に焦点をあて、そのアウトプットである生成物を中心に解説。本人担当部分の概要:化学法で合成される従来の化学品素材を、生物機能を利用したプロセスで合成することの取組みを、微生物ポリエステルを例に紹介している。本人担当部分の共著者:S. Taguchi, T. Tsuge微生物によるものづくり ―化学法に代わるホワイトバイオテクノロジーの全て―Scholarly book
- Joint work, 第2編 バイオプラスチック創製のためのバイオテクノロジーからのアプローチ 1. 酵素系, エヌ・ティー・エス, Apr. 2008, 79-90, Japanese, バイオプラスチックの開発状況および利用動向について解説。主として、ポリ乳酸の高性能化・高機能化のための技術について詳解。本人担当部分の概要:従来の化学法によるポリ乳酸の合成と対比させ、乳酸ベースポリマーを微生物合成することの意義や、実際の生合成法について平易に解説している。バイオプラスチックの高機能化・再資源化技術Scholarly book
- Single work, [北海道大学大学院工学研究科], 2008Scholarly book
- Joint work, 生分解プラスチック, 東京化学同人, Dec. 2007, JapaneseDictionary or encycropedia
- Others, エヌ・ティー・エス, 2007, Japanese, ISBN: 9784860431068Others
- Single work, [北海道大学大学院工学研究科], 2005Scholarly book
- Others, 朝倉書店, 2003Others
- Others, WILEY-VCH Verlag GmbH, 2002Others
- Others, 工学系基礎教材「ポリマーサイエンス高分子合成」(日本高分子学会編), 2001Others
- Others, 東京化学同人編日本生化学会編「基礎生化学実験法」第4巻核酸・遺伝子実験、第11章遺伝子解析のアラカルト, 2001Others
- Others, プラスチックスエージ, 2000Others
- Others, 共立出版, 2000Others
- Others, 朝倉書店「構造生物学」、シリーズ分子生物学, 1998Others
- Others, 財団法人日産科学振興財団・平成8年度研究成果報告書, 1997Others
- Others, 第3回理大奨励研究助成研究成果報告書, 1997Others
- Others, 丸善株式会社生物工学基礎シリーズ, 1997Others
- Others, 財団法人タカノ農芸化学研究助成財団・平成7年度助成研究報告書, 1997Others
- Others, 日刊工業新聞社, 1996Others
- Others, 岩谷財団研究報告書, 1993Others
- Others, 岩谷財団研究報告書, 1992Others
- Others, SUTBULLETIN、生物実験セミナー, 1992Others
- Others, SUTBULLETIN、生物実験セミナー, 1992Others
- Others, SUTBULLETIN、私の研究, 1992Others
- Others, SUTBULLETIN、科学教養講座, 1992Others
- Others, 培風館生物工学実験書(日本生物工学会編), 1992Others
- Others, 蛋白質核酸酵素(共立出版臨時増刊)、蛋白質工学の進展, 1992Others
- Others, 平成2年度理大研究助成金研究実績報告書, 1991Others
- Technical Seminar, Apr. 2019, Japanese, International conferenceSynthetic biology for bioplastics = Biosynthesis x Polymer Chemistry (Invited Lecture)[Invited]Oral presentation
- 10th Edition of International Conference on Biofuels and Bioenergy, Barcelona, Spain, Mar. 2019, English, International conferenceBiodegradable and biocompatible polymers biosynthesized by sustainable integrated bioprocess (Plenary Lecture)[Invited]Oral presentation
- 第5回 生命理工オープンイノベーションハブ(LiHub)、東京工業大学大岡山キャンパス、蔵前会館、ロイヤルブルーホール, Dec. 2018, Japanese, International conference多元ポリ乳酸を細胞培養基材とした軟骨組織形成(招待講演)[Invited]Oral presentation
- 群馬大学(招待講演)、群馬大学総合研究棟303教室(S303), Nov. 2018, Japanese, International conference環境生体に調和するバイオプラスチックの合成生物学と機能部材化(招待講演)[Invited]Oral presentation
- BIT’s 9th World Gene Conversion-2018, Singapore, Nov. 2018, English, International conferenceBiodegradable and biocompatible polymers biosynthesized by microbial platform from renewable feedstock (Invited Lecture)[Invited]Oral presentation
- International Symposium on Biological Polymers 2018 (ISBP2018), Beijing, China, Oct. 2018, English, International conferenceIntegrated production process, processing, and secretion of lactate-PHA (Keynote Lecture)[Invited]Oral presentation
- 第59回高分子討論会、北海道大学高等教育機能開発総合センター, Sep. 2018, English, International conference植物バイオマス原料から微生物合成する多元ポリ乳酸の部材化(招待講演)[Invited]Oral presentation
- JSPS Core-to-Core Program, Bioplastic Global Joint Satellite Symposium in Universiti Sains Malysia, Penang, Malaysia, Jul. 2018, English, International conferenceSynthetic biology for new polyester creation and oligomer secretion (Plenary Lecture)[Invited]Oral presentation
- バイオマスエキスポフォーラム2018、東京ビッグサイト西ホール, May 2018, Japanese, International conferenceバイオマス原料から微生物工場によって作られる環境生体医療用材料、日本発世界を変える新素材〜植物バイオマスからのマテリアル革命「植物バイオマスのマテリアル利用新技術〜JST二酸化炭素資源化研究領域〜」(招待講演)[Invited]Oral presentation
- BESS Conference on Sustainable Production of Molecules 2018, Singapore, May 2018, English, International conferenceSynthetic biology for bio-based polyesters: new polyester creation and oligomer secretion (Plenary Lecture)[Invited]Oral presentation
- 新化学技術推進協会、ライフサイエンス技術部部会/反応分科会講演会、新化学技術推進協会大会議室, Apr. 2018, Japanese, International conferenceSynthetic biology for bioplastics (Invited Lecture)Oral presentation
- 3rd Edition of International Conference and Exhibition on Polymer Chemistry 2018, Vienna, Austria, Mar. 2018, English, International conferenceSynthetic biology for intracellular and secretory production of polymerized enantiopure ester-products in microbial platform (Plenary Lecture)Oral presentation
- 254th ACS National Meeting (Mariott Marquis, Washington DC, USA, Aug. 2017, English, International conferenceSynthetic biology for the lactate-based polymers and oligomers: Intracellular and secretory production (Invited Lecture))[Invited]Oral presentation
- Kick-off Symposium in Osaka University “Japan-South-East Asia Collaboration Hub of Bioplastics Study”, (Osaka University, Osaka, Japan), Jul. 2017, English, International conferenceSynthetic biology for intracellular and secretory production of polymerized ester-products in Escherichia coli (Invited Lecture)[Invited]Oral presentation
- The 6th International Conference on Bio-based Polymers (ICBP2017), Yuan Ze University, Taiwan, May 2017, English, International conferenceCan bacterium secrete the polymerized products? (Keynote Lecture)[Invited]Oral presentation
- National Central University, (National Central University, Taiwan), Feb. 2017, English, International conferenceSynthetic biology for the lactate-based polymers and oligomers from renewable feedstock (Special Lecture)[Invited]Oral presentation
- 平成28年度日本農芸化学会北海道支部第2回講演会(北海道大学農学部、札幌), Nov. 2016, Japanese, International conference新規バイオプラスチック創製のための合成生物学(招待講演)[Invited]Oral presentation
- International Conference on Sustainable Bioplastics, (Melia Alicante Hotel, Alicante, Spain, Nov. 2016, English, International conferenceCharacterization and biodegradation of lactate-based polymer biosynthesized from renewable carbon sources (Invited Lecture)[Invited]Oral presentation
- CUST Seminar (Changchun Univ., Changchun, China), Sep. 2016, English, International conferenceBiosynthesis and properties of eco-friendly polymers (Invited Lecture)[Invited]Oral presentation
- 2016年度 ポリマーフロンティア21(積水化学京都研究所講堂、京都), Jun. 2016, Japanese, International conference酵素進化工学と合成生物学に基づいて創製する環境調和型キラルポリマー(招待講演)[Invited]Oral presentation
- Institute of Chemical and Engineering Sciences, (Jurong Island, Singapore), Apr. 2016, English, International conferenceBiosynthesis and properties of PLA-related polymers (Invited Lecture)[Invited]Oral presentation
- 第一回デザイン生命工学研究会(東京工業大学すずかけ台キャンパス大学会館、神奈川県), Mar. 2016, Japanese, International conference酵素代謝複合改変による「多元ポリ乳酸」生合成システムの創成(招待講演)[Invited]Oral presentation
- The 4th Frontier Chemistry Center International Symposium, (Hokkaido University, Sapporo, Japan), Feb. 2016, English, International conferenceBiosynthesis of glycolate-based polyesters with hydrolytic degradability (Invited Lecture))[Invited]Oral presentation
- 2015 International Chemical Congress of Pacific Basin Societies (Pacifichem2015) (Hawaii Convention Center, Honolulu, Hawaii, USA), Dec. 2015, English, International conferenceAdvances in microbial system for production of PLA-related polymers (Invited Lecture)[Invited]Oral presentation
- エコマテリアル研究会(理化学研究所大河内記念ホール、和光), Oct. 2015, Japanese, International conferenceMicrobial Plastic Factory 開発の進捗:たとえば風船型大腸菌など(招待講演)[Invited]Oral presentation
- BioJapan2015 - World Business Forum - バイオジャパン 2015(パシフィコ横浜・アネックス、横浜), Oct. 2015, Japanese, International conference風船大腸菌でバイオプラスチックを作る(招待講演)[Invited]Oral presentation
- Spiber社、鶴岡、山形, Sep. 2015, Japanese, International conference高性能バイオポリマー微生物生産のための合成生物学研究(招待講演)[Invited]Oral presentation
- Japan-Taiwan Bilateral Polymer Symposium 2015 (JTBPS 2015), (Hokkaido University, Sapporo), Sep. 2015, English, International conferenceBiosynthesis and properties of eco-friendly polymers (Invited Lecture)[Invited]Oral presentation
- 公益社団法人自動車技術会中部支部 第3回技術講習会(刈谷市産業振興センター、刈谷、愛知県), Aug. 2015, Japanese, International conferenceSynthetic biology for bioplastics (Invited Lecture)[Invited]Oral presentation
- 第5回合成生物工学シンポジウム(神戸大学統合研究拠点コンベンションホール、神戸), Aug. 2015, Japanese, International conferenceIntegrated production process for production of multi-PLA from biomass (Invited Lecture)[Invited]Oral presentation
- The 5th International Conference on Bio-based Polymers, (The National University of Singapore (NUS), Faculty of Engineering, Singapore, Jun. 2015, English, National University of Singapore, Singapore, ポリ乳酸および乳酸ポリマーの生合成系は、バイオマスから有用なポリマーマテリアルを一括合成する優れたプラットフォームである。このシステムは、代謝工学および酵素工学の手法を組み合わせて用いることにより、さらに効率的な生産系へと改良することができる。その結果、乳酸ポリマーはこれまでの天然の微生物産生ポリエステルと比較して、有意に高い収率で合成することができた。, International conferenceBiosynthetic PLA-related polymers improved by the metabolic engineering and enzyme engineering (Plenary Lecture)[Invited]Keynote oral presentation
- The 10th SPSJ International Polymer Conference (IPC 2014), (Epochal Tsukuba, Tsukuba, Japan, Dec. 2014, English, Tsukuba International Congress Center, 改変型重合酵素を発現する微生物工場は、乳酸、グリコール酸、2-ヒドロキシ酪酸などの非天然ユニットを含むポリエステルを合成できる。合成されるポリマーの組成や分子量は、使用する微生物、酵素、炭素源などによって制御される。したがって適切な制御を行うことにより、透明性や柔軟性などをコントロールし、さまざまなバイオマテリアルを合成することができる。, International conferenceProduction of unusual microbial polyesters (Invited Lecture)[Invited]Keynote oral presentation
- 5th Symposium on Academic Exchange and Collaborative Research, Nov. 2014, English, ETH, Zurich, Switzerland, エンジニア微生物は、再生可能なバイオマス資源を有用なプラスチックへと変換する能力を有する。組換え微生物で改変型重合酵素を発現させることで、乳酸などの非天然ユニットを含むポリエステルを合成できる。合成されるポリマーの組成や分子量は、使用する微生物、酵素、炭素減などによって制御される。, International conferenceEngineering of bacteria and pathways toward efficient plastic production from renewable feedstock (Invited Lecture)[Invited]Keynote oral presentation
- 2nd International Conference on Bioinspired and Biobased Chemistry & Materials, (Nice, France), Oct. 2014, English, Negresco Palace hotel, Nice, France, Microbial plastic factoryは、プラスチックを生産するための合成装置を組み込んだ工場のように機能する微生物細胞であり、再生可能なバイオマスを炭素源としてバイオポリマーを合成することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も変化する。したがって、適切な制御を行うことにより、透明性や柔軟性などをコントロールできる。, International conferenceMicrobial plastic factory: New lactate-based and related biopolymers (Invited Lecture)[Invited]Keynote oral presentation
- ISBP 2014 – International Symposium on Biopolymers, Sep. 2014, English, Mendes Plaza Hotel, Santos, Brazil, 改変型重合酵素遺伝子を導入した微生物工場は、乳酸、グリコール酸、2-ヒドロキシ酪酸などの非天然ユニットを含むポリエステルを合成できる。合成されるポリマーの組成や分子量は、使用する微生物、酵素、炭素源などによって制御される。これを利用して透明性や柔軟性などのポリマー物性をコントロールできる。このようにして合成される非天然バイオポリマーについてどのようなアプリケーションが考えられるかを論じた。, International conferenceBiosynthesis of polyesters consisting of 2-hydroxyalkanoates and their applications (Invited Lecture)[Invited]Keynote oral presentation
- 第66回日本生物工学会大会シンポジウム, Sep. 2014, Japanese, 札幌コンベンションセンター, 二酸化炭素を有用な化成品に変換することを考える場合、まずバイオマスをリグのセルロースなどの糖質バイオマスへと変換し、ついで微生物発酵により、低分子化合物を合成することが考えられる。生産効率を高めるため、できるだけ短い合成ルートを設計することが望ましい。そのための戦略と、最近の試みについて紹介する。, Domestic conference二酸化炭素からプラスチックを合成する人工生命システムの創成 -最短の合成ルートは?-(招待講演)[Invited]Invited oral presentation
- 第8回日本-韓国バイオマスシンポジウム, Sep. 2014, English, 札幌コンベンションセンター, 再生可能なバイオマス資源を有用なプラスチックへと変換する方法として、エンジニア酵素を発現する微生物工場を用いる方法がある。改変型重合酵素を発現する組換え微生物は、乳酸などの非天然ユニットを含むポリエステルを合成できる。合成されるポリマーの組成や分子量は、使用する微生物、酵素、炭素減などによって制御された。, International conferenceMicrobial plastic factory driven by renewable carbon sources (Invited Lecture)[Invited]Keynote oral presentation
- 248th ACS National Meeting and Exposition, Aug. 2014, English, The Hilton San Francisco Union Square, San Fransisco, USA, ポリ-2-ヒドロキシアルカン酸は、エンジニア酵素により合成される新規バイオマテリアルである。本ポリマーを合成するための代謝設計、得られたポリマーの物性について紹介する。さらに、これらのポリマーが自然環境に存在する微生物によって、分解されうるかどうか、その可能性と評価方法について述べた。, International conferenceBiosynthesis, properties and enzymatic degradation of poly(2-hydroxyalkanoates) (Invited Lecture)[Invited]Keynote oral presentation
- 日本工学アカデミー北海道・東北地区札幌講演会, Aug. 2014, Japanese, 北海道大学, 糖や脂質などのバイオマスは、二酸化炭素が光合成により固定化されたものであるから、バイオマス由来プラスチックは、二酸化炭素から合成されたといえる。このような環境低負荷方のマテリアルを効率よく生産するための、最近の試みとその成果について紹介する。, Domestic conference二酸化炭素の資源化を目指した環境バイオテクノロジー:バイオの力でプラスチックを!(招待講演)[Invited]Invited oral presentation
- International Symposium for Green-Innovation Polymers & The 13th Symposium of the Research Center for Highly Environmental and Recyclable Polymers, Mar. 2014, English, Ohmicho Kouryu Plaza, Kanazawa, 進化工学的に作成した乳酸重合酵素を発現する組換え大腸菌等の微生物を用いると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も自在に制御できた。, International conferenceBiosynthesis and properties of lactate-based and related polymers from renewable carbon sources (Invited Lecture)[Invited]Keynote oral presentation
- International Symposium for Green-Innovation Polymers & The 13th Symposium of the Research Center for Highly Environmental and Recyclable Polymers, (Ohmicho Kouryu Plaza, Kanazawa), Mar. 2014, English, Ohmicho Kouryu Plaza, Kanazawa, 進化工学的に作成した乳酸重合酵素を発現する組換え大腸菌等の微生物を用いると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も自在に制御できた。, International conferenceBiosynthesis and properties of lactate-based and related polymers from renewable carbon sources (Invited Lecture)[Invited]Oral presentation
- 日本農芸化学会2014年度大会シンポジウム, Mar. 2014, Japanese, 明治大学, 乳酸重合酵素を発現する大腸菌は、細胞内で乳酸を重合して乳酸ポリマーを合成することができる。この非天然ポリマーは、合成条件を最適化することにより、天然のポリエステルを超える収率で合成される。ポリマーの合成効率に関与する因子と、得られたポリマーの物性について論ずる。, Domestic conferenceバイオマスから高効率で乳酸プラスチックを生産する微生物工場の開発とポリマー物性評価(招待講演)[Invited]Invited oral presentation
- Joint Symposium on Environmental Science 2013 – Bridging Finland and Japan -, Nov. 2013, English, University of Helsinki, Finland, 微生物にエンジニア酵素を駆使した代謝経路を構築することにより、再生可能なバイオマスを炭素源としてバイオポリマーを合成することができる。石油を原料とせずにプラスチックを合成できることから、持続可能性の高い社会の実現に貢献できる。とくに乳酸はグルコースからの変換効率が高いため、高収率でポリマーを得ることができる。, International conferenceMicrobial plastic factory toward sustainable industry[Invited]Keynote oral presentation
- ICBP2013-International Conference on Biobased Polymers, Sep. 2013, English, Hanyang Institute of Technology, Seoul, Korea, 改変型重合酵素を発現する微生物工場は、乳酸、グリコール酸、2-ヒドロキシ酪酸などの非天然ユニットを含むポリエステルを合成できる。合成されるポリマーの組成や分子量は、使用する微生物、酵素、炭素源などによって制御される。したがって、性質の異なる酵素群を駆使することにより、透明性や柔軟性などのポリマー物性をコントロールできた。, International conferenceProperties of newly biosynthesized lactate-based polymers and related polymers[Invited]Keynote oral presentation
- 第26回 植物脂質シンポジウム, Sep. 2013, Japanese, 北海道大学, 微生物のポリエステル合成系はバイオマスからプラスチックを合成できる有用なシステムであるが、ポリエステルと脂質の構造類似性から、本ポリエステルの合成系は進化的には脂肪酸の合成系から派生したものとだと考えられている。それでは、植物のようなポリエステルを生産しない生物の脂肪酸代謝経路も同様にポリエステル合成に転用できるだろうか?本講演では最近の試みについて紹介する。, Domestic conference微生物産生ポリエステルの植物合成 -植物の脂質代謝系はポリエステル合成に利用できるか-[Invited]Invited oral presentation
- Institute of Chemical and Engineering Sciences, May 2013, English, Forum Room, Singapore, 組換え大腸菌等の微生物で乳酸重合酵素を発現させると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も変化する。したがって、適切な制御を行うことにより、透明性や柔軟性などをコントロールできた。, International conferenceMicrobial factory –Lactate-based polymers–[Invited]Keynote oral presentation
- Singapore Catalysis Society – 6th Annual Forum, May 2013, English, Biopolis, Singapore, 人工進化により創出した乳酸重合酵素を発現する組換え大腸菌等の微生物を用いると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も自在に制御できる。, International conferenceMicrobial plastic factory – Lactate-based polymers production[Invited]Keynote oral presentation
- 日本農芸化学会2013年度大会シンポジウム, Mar. 2013, Japanese, 東北大学, 乳酸重合酵素を発現する組換え微生物は、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。この合成経路は微生物産生ポリエステルの生合成系を拡張したものなり、これにより得られるポリマーを3-ヒドロキシ酪酸をはじめとする天然のモノマーユニットとのハイブリッドポリマーである多元ポリ乳酸の多様性と将来について考える。, Domestic conference微生物ポリエステル合成系酵素の機能改変と多元ポリ乳酸の創製[Invited]Invited oral presentation
- 協和発酵バイオ株式会社技術研究所講演会, Mar. 2013, Japanese, 防府, バイオマスは再生可能な炭素源であるが、複雑な混合物であり、これを効率的に有用物質に変換するためには、微生物変換は強力なツールとなる。さらに、遺伝子組換え、酵素工学の技術を組み合わせることにより、天然では合成されない化合物の生合成も可能になる。ここでは、バイオポリエステルを例にその手法と課題について論じる。, Domestic conferenceモノ作りのための合成生物学 微生物ポリマーのケース(他)[Invited]Invited oral presentation
- 産業技術総合研究所関西センター, Nov. 2012, Japanese, 神戸, 乳酸発酵で得られる乳酸を化学的に重合させるとポリ乳酸が得られる。これに対し、乳酸重合酵素を発現する組み換え大腸菌は、一段階の培養で乳酸ポリマーを合成できる。本系は、バイオプロセスでありながら、従来の化学プロセスに匹敵する能力を有する。このように、近年の合成生物学の進歩に伴い、化学とバイオの境界はあいまいになっている。, Domestic conferenceMicrobial Plastic Factory ‐バイオと化学のクロスオーバー‐[Invited]Invited oral presentation
- 第34回生体触媒化学シンポジウム、生体触媒化学学会, Nov. 2012, Japanese, 富山県民会館, ある種の微生物が有する細胞内にポリエステルを合成・蓄積する能力を、進化工学的に作成した乳酸重合酵素を用いて拡張すると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。この合成経路は微生物産生ポリエステルの生合成系を拡張したものなので、得られるポリマーを3-ヒドロキシ酪酸をはじめとする天然のモノマーユニットとの共重合体、多元ポリ乳酸、の合成が可能になる。この新しいシステムで生み出される多様な新規バイオポリマーについて展望する。, Domestic conferenceMicrobial Plastic Factory ‐生体触媒開発によって実現する多元ポリ乳酸微生物生産システム‐[Invited]Invited oral presentation
- International Conference on Bioinspired and Biobased Chemistry & Materials, Oct. 2012, English, Nice, France, 乳酸重合酵素を発現する組換え大腸菌等の微生物を用いると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。本システムを応用すると、さらに2-ヒドロキシ酪酸やグリコール酸などの非天然ポリエステルを生合成することも可能になる。適切な代謝の制御を行うことにより、透明性や柔軟性などをコントロールできた。, International conferenceMicrobial plastic factory: New lactate-based and related biopolymers[Invited]Keynote oral presentation
- ISBP 2012, International Symposium on Biopolymers, Oct. 2012, English, Pulluman Reef Hotel, Queensland, Australia, ある種の微生物はポリエステルを合成する能力を有するが、これに人工的な改変により機能を拡張した重合酵素を発現させると、乳酸、グリコール酸、2-ヒドロキシ酪酸などの非天然ユニットを含むポリエステルを合成できる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も自在に制御できた。, International conferenceExpanding microbial polyesters: syntheses and properties[Invited]Keynote oral presentation
- 特別セミナー, Oct. 2012, Japanese, 東京理科大学, ポリ乳酸は再生可能なバイオマスから合成される環境低負荷型のプラスチックであるが、乳酸発酵と化学重合を組み合わせたプロセスで合成されている。これに対し、乳酸重合酵素を発現する組み換え大腸菌は、一段階の培養で乳酸ポリマーを合成できる。本系は、バイオプロセスでありながら、従来の化学プロセスに匹敵する能力を有する。このように、近年の合成生物学の進歩に伴い、化学とバイオの境界はあいまいになっている。, Domestic conferenceMicrobial Plastic Factory ‐バイオと化学のクロスオーバー‐[Invited]Invited oral presentation
- International Union of Materials Research Sciences-International Conference on Electronic Materials 2012 (IUMRS-ICEM 1012), Yokohama, Japan, Sep. 2012, English, Pacifico Yokohama, 天然のポリエステル合成系を改変したエンジニア合成経路は、乳酸、グリコール酸、2-ヒドロキシ酪酸などの非天然ユニットを含むポリエステルを合成できる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も変化する。したがって、適切な制御を行うことにより、透明性や柔軟性などをコントロールできた。, International conferenceCreation of bio-based polymers based on biological systems (Plenary Lecture)[Invited]Keynote oral presentation
- ACS National Meeting in Philadelphia, Aug. 2012, English, PA , USA, 改変型重合酵素を発現する微生物工場は、乳酸、グリコール酸、2-ヒドロキシ酪酸などの非天然ユニットを含むポリエステルを合成できる。合成されるポリマーの組成や分子量は、使用する微生物、酵素、炭素減などによって制御される。一方、目的のポリマーを合成するためには、得られたポリマーの構造および物性を精密に評価することも重要な項目となる。, International conferenceBiosynthesis and precise characterization of bio-based polymers containing 2-hydroxyalkanoates: lactate, glycolate and 2-hydroxybutyrate[Invited]Keynote oral presentation
- 高分子学会年次大会, May 2012, Japanese, パシフィコ横浜, 省資源で環境の汚染の少ない環境調和型材料の開発が求められている。改変型重合酵素遺伝子を導入した微生物工場は、再生可能なバイオマスを炭素源としてバイオポリマーを合成することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も変化する。したがって、適切な制御を行うことにより、透明性や柔軟性などをコントロールできる。, Domestic conferenceバイオを駆使した環境調和型プラスチックの創製:微生物生産から植物生産へ[Invited]Invited oral presentation
- 高分子学会年次大会, May 2012, Japanese, パシフィコ横浜, 省資源で環境の汚染の少ない環境調和型材料の開発が求められている。改変型重合酵素遺伝子を導入した微生物工場は、再生可能なバイオマスを炭素源としてバイオポリマーを合成することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も変化する。したがって、適切な制御を行うことにより、透明性や柔軟性などをコントロールできる。, Domestic conferenceバイオを駆使した環境調和型プラスチックの創製:微生物生産から植物生産へ[Invited]Invited oral presentation
- International and Academia Cooperation Material and Chemical Research Laboratories,, Mar. 2012, English, Taiwan, 再生可能なバイオマスを炭素源として有用なバイオマテリアルを細胞内で一括合成するMicrobial plastic factoryの開発戦略について紹介する。微生物の遺伝コードを書き換えることにより、エンジニア酵素を発現させ新たな代謝経路を構築することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も自在に制御できた。, International conferenceMicrobial plastic factory: from DNA to materials[Invited]Keynote oral presentation
- 第7回農芸化学会研究企画賞受賞者最終報告会、2012年度産学官学術交流委員会フォーラム, Mar. 2012, Japanese, 京都女子大学, 天然のある種の微生物は細胞内にポリエステルを合成・蓄積する能力を有する。このシステムを進化工学的に作成した乳酸重合酵素を用いて改変すると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。この合成経路は微生物産生ポリエステルの生合成系を拡張したものなので、天然のモノマーユニットとの共重合体にすることも可能である。この新しいシステムで生み出される多様な新規バイオポリマーについて展望する。, Domestic conference“乳酸バイオポリマー”生産用微生物工場の開発研究[Invited]Invited oral presentation
- 日本農芸化学会2012年度大会シンポジウム, Mar. 2012, Japanese, 京都女子大学, 微生物の中には、高温高圧などの通常生物の生存に適さない環境でも生育できるものがある。このような微生物には、たとえば耐熱性酵素など、通常の生物が持たない特殊な能力を有しているものが多い。これらの能力は、産業的な応用の観点からも貴重なリソースとなる。このような特殊微生物について、実例を交えて紹介する。, Domestic conference特殊環境下における微生物の潜在能力とその応用[Invited]Invited oral presentation
- セルロース学会北海道・東北支部セミナー, Feb. 2012, Japanese, 北海道大学, 乳酸重合酵素を発現する大腸菌は、細胞内で乳酸を重合して乳酸ポリマーを合成することができる。この非天然ポリマーは、驚くべきことに、天然のポリエステルを超える収率で合成される。しかし、効率よくポリマーを合成できるようになるためには、いくつものクリアするべき課題があった。乳酸重合酵素の発見から、ポリマー材料の創製までの道のりを紹介する。, Domestic conference多元ポリ乳酸創製のための合成生物学 *試行錯誤のあれこれ*[Invited]Invited oral presentation
- 第8回よこはまバイオマス研究会講演会, Oct. 2011, Japanese, 理研横浜研究所, 人工進化により創出した乳酸重合酵素遺伝子を導入した組換え大腸菌は、細胞内で乳酸を重合してポリマー化できる。この合成経路は微生物産生ポリエステルの生合成系を拡張したものなので、得られるポリマーを3-ヒドロキシ酪酸をはじめとする天然のモノマーユニットとの共重合体にすることも可能である。この新しいシステムで生み出される多様な新規バイオポリマーについて展望する。, Domestic conference微生物と植物によるポリエステルの合成:ポリ乳酸から多元ポリ乳酸へ[Invited]Invited oral presentation
- ICBP2011- The 3rd International Conference on Bio-based polymers 2011, Oct. 2011, English, Peking University, China, 微生物にエンジニア酵素である乳酸重合酵素を発現させると、乳酸ポリマーを生合成できるが、本システムを応用すると、さらに2-ヒドロキシ酪酸やグリコール酸などをモノマーユニットとする非天然ポリエステルを生合成することも可能になる。これらのポリマーは、これまでに知られている天然の微生物産生ポリエステルとは異なる物性を示すことから、より多様なバイオマテリアルの創製に貢献できる。, International conferenceAdvanced microbial polymer factory for incorporating new monomers: lactate, glycolate and 2-hydroxybutyrate[Invited]Keynote oral presentation
- 第8回よこはまバイオマス研究会講演会, Oct. 2011, Japanese, 理研横浜研究所, 人工進化により創出した乳酸重合酵素遺伝子を導入した組換え大腸菌は、細胞内で乳酸を重合してポリマー化できる。この合成経路は微生物産生ポリエステルの生合成系を拡張したものなので、得られるポリマーを3-ヒドロキシ酪酸をはじめとする天然のモノマーユニットとの共重合体にすることも可能である。この新しいシステムで生み出される多様な新規バイオポリマーについて展望する。, Domestic conference微生物と植物によるポリエステルの合成:ポリ乳酸から多元ポリ乳酸へ[Invited]Invited oral presentation
- BioJapan2011, Oct. 2011, Japanese, パシフィコ横浜, ポリエステル合成系を発現する微生物は、糖や油などのバイオマスを炭素源としてプラスチックを合成する微生物工場として機能する。一方、微生物のポリエステル合成遺伝子群を導入した植物は、二酸化炭素から直接プラスチックを合成する生産系になりうる。これらの合成系の動作原理とその長所短所について紹介する。, Domestic conference微生物工場と植物工場によるバイオポリマー生産[Invited]Invited oral presentation
- 「細胞を創る研究会」 4, Oct. 2011, English, 千里ライフサイエンスセンター・ライフホール, Microbial plastic factoryは、プラスチックを生産するための合成装置を組み込んだ工場のように機能する微生物細胞である。このシステムを進化工学的に作成した乳酸重合酵素を用いて改変すると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も自在に制御できる。, Domestic conferenceMicrobial plastic factory: enzyme engineering and metabolic engineering[Invited]Invited oral presentation
- ICMAT 2011- International Conference on Materials for Advanced Technology, Jun. 2011, English, Suntec Singapore and the Pan Pacific Singapore Hotel, Singapore, 乳酸重合酵素を発現する組換え大腸菌等の微生物を用いると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。一方化学合成のポリ乳酸は、乳酸発酵で得た乳酸を化学的に重合して得られる。この二つのポリマーの合成方法、および得られるポリマーの違いについて論じた。, International conferenceBiological lactate-polymers synthesized by one-pot microbial factory: How different from chemical polylactate?[Invited]Keynote oral presentation
- JBA新資源生物変換研究会シンポジウム, Jun. 2011, Japanese, 神戸大学, ポリ乳酸は再生可能なバイオマスから合成される環境低負荷型のプラスチックであるが、乳酸発酵と化学重合を組み合わせたプロセスで合成されている。これに対し、乳酸重合酵素を発現する組み換え大腸菌は、一段階の培養で乳酸ポリマーを合成できる。すなわち、本システムを用いると再生可能バイオマスからプラスチック材料を一気通貫で合成できる。, Domestic conference再生可能資源から高性能バイオポリマーを“一気通貫”合成する微生物工場の開発[Invited]Invited oral presentation
- 241st ACS National Meeting & Exposition, ,, Mar. 2011, English, Anaheim Convention Center & Area Hotels, California, USA, 大腸菌等の微生物に乳酸重合酵素を発現させると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も変化する。したがって、適切な制御を行うことにより、透明性や柔軟性などをコントロールできる。一段階の培養で合成することができた。, International conferenceBiological lactate-polymers synthesized by one-pot microbial factory: Enzyme and metabolic engineering[Invited]Keynote oral presentation
- 日本農芸化学会2012年度大会シンポジウム, Mar. 2011, Japanese, 京都女子大学, ある種の微生物は細胞内にポリエステルを合成・蓄積する能力を有する。このシステムを進化工学的に作成した乳酸重合酵素を用いて改変すると、グルコースなどの再生可能なバイオマスを炭素源として、有用な物性を有する乳酸ポリマーを一段階の培養で合成することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も自在に制御できる。, Domestic conference代謝・酵素複合改変による次世代乳酸ポリマーの創製[Invited]Invited oral presentation
- Technical Seminar on Bio-Technology in Japan and the Netherlands, Feb. 2011, English, 神戸大学, Microbial plastic factoryとは、微生物の細胞を、プラスチックを生産するための合成装置を組み込んだ工場のように機能させる技術を指す。微生物にエンジニア酵素を駆使した代謝経路を構築することにより、再生可能なバイオマスを炭素源としてバイオポリマーを合成することができる。使用する酵素や代謝経路の種類により、ポリマーの化学構造を制御することができ、それに応じてポリマーの物性も変化する。したがって、適切な制御を行うことにより、透明性や柔軟性などをコントロールできた。, International conferenceMicrobial plasticfactory: from enzyme-metabolic engineering to polymer properties[Invited]Keynote oral presentation
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Research (Exploratory), Tokyo University of Agriculture, 28 Jun. 2019 - 31 Mar. 2023Biosynthesis of chiral polymer with stereochmical reversion細胞内に合成蓄積する微生物ポリマー(ポリヒドロキシアルカン酸:PHA)は、環境生分解性を有する環境循環材料として認識されている。PHAの材料特性を考える上で、光学純度は極めて重要な要因である。PHAを構成するモノマーユニットは、全て立体「R体」であり、光学純度の極めて高いキラルポリマーであることが特徴である。最も標準的なPHAポリマーであるP(3HB)は、(R)-3HB-CoA(補酵素A)モノマーを前駆体として、重合酵素により合成され、100%eeのR体から構成される。 今年度は、細胞内におけるS体 3HB-CoAモノマーの供給フラックスの強化とS体モノマー認識に対する重合能力の開発に注力した。評価系として、市販のラセミ体・3HBモノマーを系外から添加し、3HB→3HB-CoAを触媒するため、外来のCoA転移酵素(PCT)を大腸菌に遺伝子導入し、P(3HB)ホモポリマーの合成の可否を指標に判断した。関連酵素の立体化学認識は、PCTは緩やかで重合酵素は厳密であることは知られている。PCTを組換え発現させたサンプルを用いて作製した抗体はインビボで使用可能できることを今回確認できた。(S)体の3HB-CoAは、(R)体の3HB-CoAと同様に定法に従って化学合成した。機能的に発現させたPCT組換えタンパク質を使用して、両鏡像異性体のモノマー基質に対して相対的反応性比を評価したところ、(R)体の3HB-CoAに対して高い反応性を示したが、微弱ながら(S)体の3HB-CoAに対しても反応性を示すことが判明した。一方、インビトロの重合系にて3HB-CoAの両鏡像異性体に対して、使用した重合酵素の活性試験を行ったところ、本重合反応が代謝上律速になることが示唆された。現在、プレートアッセイで陽性のクローンを取得する体制に入っているが、今までのところ、まだポジティブな結果は得られていない。
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Hokkaido University, 01 Apr. 2017 - 31 Mar. 2020Creation of new biodegradability controlled plastics based on the degradation mechanismMarine pollution caused by petroleum-derived plastics has become a global concern. Among them, biodegradable plastics that can be decomposed in the natural environment into water and carbon dioxide are attracting attention. In this research project, we developed a newly developed bioplastic, lactic acid copolymer [P (LA-co-3HB)], which is a polymer molecule composed of lactic acid (LA) and 3-hydroxybutanoic acid (3-HB). And the degradability by environmental microorganisms was evaluated. First, we investigated the microbial production method of lactic acid copolymer and established a culture method with controlled lactic acid content. The obtained copolymer were formed into a film to evaluate the degradation in the environment, and a phylogenetic analysis of the environmental microorganisms involved in the degradation was performed.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Hokkaido University, 01 Apr. 2014 - 31 Mar. 2017Efficient production of high optical pure hydroxy acid based on the regeneration of coenzyme AOptical purity of building block are essential for the chemical synthesis of bioactive compounds, such as antibiotics and pheromone. Chemical synthesis of optically active compounds are not so easy because of no optical selective synthesis. We developed basic technology of the biosynthesis method for optically active [R]-3-hydroxybutylate (3HB) with high purity using engineered microbiological system. We have newly developed the quantifying analytical method for intermediate compounds of [R}-3HB biosynthesis and found that the improvement of the flux to Acetyl-CoA is effective to produce [R]-3HB production. On the basis this results Escherichi coli JM109 was selected as a high glucose consumption strains. Using this strain the concentrations of glucose and acetate into the medium was optimized. Productivity of high optically pure [R]-3HB was archived to 15g/l in optimized culture condition. In addition newly cultivation technology was futher developed for the efficient production.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Hokkaido University, 01 Apr. 2014 - 31 Mar. 2017Microbial production of elastic polymers with ordered structureMicrobial polyesters are useful material, which are produced from renewable biomass. For the practical use of the material, polymer properties are critical factors. It is well-known that microbial copolyesters are random polymer. However, we found a condition that generated copolymer with low randomness. This study addressed optimization of the polymer production, structure and property analysis, and kinetic analysis of relevant enzymes. As the results, the copolymer, in which the monomer composition was regulated, was successfully synthesized with good yield. The copolymers exhibit transparent and flexible properties. The enzymatic activity of relevant enzymes accounted for the intracellular intermediate levels. Overall, the study contributed to the significantly improved the efficiency of the polymer production system, and provided insight into the mechanism of the polymer synthesis.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 01 Apr. 2014 - 31 Mar. 2016Evolutionary engineering of microbial enzyme that regulates the monomer composition in lactate-based polymerMicrobial synthetic system for lactate (LA)-based polymer, as an advanced member of microbial polyester (polyhydroxyalkanoate) family, was already established by development of lactate-polymerizing enzyme. In order to regulate the monomer composition in P(LA-co-3-hydroxybutyrate (3HB)) copolymer, I have applied evolutionary engineering to NADPH-dependent acetoacetyl-CoA reducatase (PhaB) through the plate assay-based enzyme evolution program. As the result, I obtained two beneficial PhaB enzymes with increased activities that can also increased microbial polymer production. Acquired beneficial properties of these evolved P(3HB) enzymes can be accounted for by flexibility increase in the substrate-related region based on the tertiary structure bearing two domains.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 01 Apr. 2012 - 31 Mar. 2014Evolutionary engineering based creation of monomer-supplying enzyme that allows the microbial synthesis of new monomer-incorporated polymersThis study is based on the microbial system for production of the new type polymers by polymerizing enzyme with altered substrate specificity. So far the most fascinating achievement is the first incorporation of lactic acid as a monomer into the polymer through engineering the polymerase. Upon the project, the reactivity of CoA-transferase towards new monomers susch as lactic acid should be a key for the synthesis of monomer CoA forms. Glycolic acid is a second target monomer bearing 2-OH. Through engineering the CoA transferase as well as polymerase, the incorporation of glycolic acid into the polymeric back bone was demonstrated by using the microbial system carrying engineered enzymes. The fraction of glycolic acid was increased depending on the increase in the glycolic acid concentration.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Hokkaido University, 01 Apr. 2011 - 31 Mar. 2014Establishment of the next generation microbial factory for lactate-based polymers by utilizing biomassWe have constructed the microbial system for the lactate-based polymers by using our developed lactate-polymerizing enzyme. The copolymers have been synthesized by achieving the range control of lactate fraction, from 0 to 50 mol% in Escherichia coli and 50 to nearly 100 mol% in Corynebacterium glutamicum. Among them, lactate polymer with 30 mol% lactate fraction was produced up to 11 g/L. In terms of polymer properties, copolymers gained flexibility with increasing lactate fraction compared to the rigid hopolymers such as PLA. Also, microbial polymers were demonstrated to be synthesized by using the enzymatically hydrolyzed sugars from the plant biomass.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Hokkaido University, 2011 - 2013Development of microbial factory for bioplastic production adapting inedible biomass hydrolysatesLignocellulosic biomass is mainly composed of glucose and xylose as the major sugars. The use of glucose for the biopolymer production has been demonstrated to be efficient whereas, the use of xylose is inefficient. However efficient utilization of both sugars is essential. In this study, methods for the efficient utilization of xylose were developed and enhanced to obtain high productivities of biopolymers. For P(LA-co-3HB) production lactic acid supplementation into the cell contributed to the inprovement of polymer yield from xylose. In addition xylose was found to be superiorb to glucose in giving higher LA fraction in the copolymer. The uptake of xylose by GatC which is galactitol transporter also effective to produce the copolymer. The approach for high yield production of biopolymers in combination with the lignocellulosic-derived mix sugars have a potential for the establishment of biorifinary for the production of variety of biopolymers.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 2010 - 2011Creation of biocatalyst capable of biosynthesizing lactate-based polymers with highly regulated chiralityCurrently microbial production system for lactate(LA) polymers with high performances has been developed. A break-through of this system was a discovery of "LA-Polymerizing Enzyme(LPE)". LPE was created through the study on the engineering of microbial polymer PHA synthase. LA-based polymer was synthesized by highly enatiomeric monomers including R-LA in an one-pot manner in Escherichia coli by introduction of the LPE gene from glucose. I tried to establish the system for synthesis of the polymer incorporating S-LA monomer based on the evolutionary engineering of CoA-transferase and LPE. As a resiult, LA fraction in the polymer has been enriched up to 47% from 6% by combination of metabolic and enzyme engineering. Furthermore, LA-based polymers exhibited distinguishable properties from the counterpart homopolymers, PLA and PHB.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Hokkaido University, 2008 - 2010Establishment of the all biosynthetic system for new monomers-contained biopolymers as a basis for enzyme evolutionary engineeringMicrobial production system for lactate (LA) polymers with high performances has been established. This new microbial factory can be driven by renewable biomass resources. A break-through of this system was a discovery of "LA-Polymerizing Enzyme (LPE)". LPE was created through the study on the engineering of microbial polymer PHA synthase. LA-based polymer was synthesized as an one-pot manner in Escherichia coli by introduction of the LPE gene from glucose. Also, LA fraction in the polymer has been enriched up to 47% from 6% by combination of metabolic and enzyme engineering. Furthermore, LA-based polymers exhibited distinguishable properties from the counterpart homopolymers, PLA and PHB.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Hokkaido University, 2005 - 2007Prroduction System for Carbon Recyclable Green Plastics Based on Enzyme Evolutionary EngineeringPolyhydroxyalakanoates (PHAs) form a class of natural polyesters that many microorganisms in the environment accumulate in the form of intracellular granules to store carbon and reducing equivalents. The wide variety of monomers yields PHAs with diverse material properties that depend on polymer composition. Besides the practical applications of PHAs as bioplastics that are biodegradable and made from renewable resources, from the standpoint of an academic metabolic pathway engineer. PHAs are model compounds for metabolic engineering. The primary aims of the metabolic engineering of PHAs include controlling different factors that determine polymer material properties, such as monomeric compositions, molecular weight and copolymer microstructure, as well as optimizing yield. Pathway engineering for PHA production offers the opportunity to synthesize novel polymers with desirable properties in low-cost, high-productivity fermentations. Most recently, enzyme evolution is becoming the new approach for PHA production. A break-through in the chemical synthesis of macromolecules with desirable properties was achieved by the development of prominent chemical catalysts via "catalyst evolution". Thus, one can easily accept the concept that the molecular evolution of the biocatalysts (enzymes) relevant to PHA synthesis will provide us with a chance to create novel PHA materials with high performance. The enzyme evolution-aided PHA production system be involved in the carbon ecosystem. In this study, I demonstrated the case studies of enzyme engineering for optimization of productivity and regulation of monomeric composition of PHA. Rational molecular design was applied to monomer supplying enzyme based on three dimensional structure of PhaJ and evolutionary engineering was applied to PHA synthase based on functional mapping. A huge amount of library of enzyme mutants, created through engineering of these key enzymes for PHA biosynthesis, can provide tailor-made biopolymers with diverse properties suitable for wide-range applications. Transferring mutants to the plant system has been performed.
- 日本学術振興会, 科学研究費助成事業, 基盤研究(C), 東京大学, 2006 - 2006次世代バイオベースプラスチックの創成とナノ構造制御による高機能化有限化石資源の有効利用と環境保全の観点から、新たに開発すべき環境循環型プラスチックは、単に自然環境中で分解されることにとどまらず、糖や植物油などの再生産可能資源(バイオマス)から生産できるバイオベースプラスチックでなければならない。さらに、環境循環型高分子の観点から、バイオベースプラスチックと生分解性プラスチックの両方の特性および機能を有する高分子材料の基礎、開発、実用化研究が今後最も重要な研究課題になると考えられる。 本基盤研究(C)では、「次世代バイオベースプラスチックの創成」を研究する特定領域研究を発足させるために、世界的研究動向と研究ニーズを調査し、この調査研究に基づいて個別研究および共同研究を企画し、研究組織を作り上げることを目的とした。 平成18年9月15日に、理化学研究所にて本研究に携わる13名が各々のこれまでの研究成果と今後の研究計画を発表し、本研究に関心を持つ一般の研究者・技術者からの意見を収集した。さらに、本企画調査研究への補助金交付決定後ただちに第1回全体会議を東京で開催し、平成19年度新規特定領域研究への申請を行うことを決定した。そのために各研究分担者が、担当する研究分野の世界動向と研究ニーズを調査した。さらに、数回の班長会議および全体会議を行い、追加メンバーの承認、統一テーマの設定、共同研究の開拓、特定領域としての意義等を検討した。 これらの検討をふまえ、平成19年度発足を目指した特定領域研究「新規高性能バイオベースポリマーの創成と高機能化」(領域代表:岩田忠久)を申請した。
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 2002 - 2004Oprimization of production system for bioplastics by enzyme evolutionary engineeringBiotechnological studies for biosynthesis of polyhydroxyalkanoates (PHAs) biopolyester have been extensively progressed through the development of various metabolic engineering strategies involving fermentation technology of naturally occurring PHA-producing bacteria based on external substrate manipulation (1st generation), subsequently reinforced with recombinant gene technology (2nd generation). Most recently, "enzyme evolution" is becoming the 3rd generation approach for PHA production. Break-through in chemical synthesis of macromolecules with desirable properties was achieved by development of prominent chemical catalysts, "catalyst evolution", as represented by a series of Zigler-Natta catalysts. Thus, one can easily accept the concept that molecular evolution of the biocatalysts (enzymes) relevant to PHA synthesis will provide us a chance to create novel PHA materials with high-performance. The first trial of the in vitro enzyme evolution in PHA biosynthesis was reported by our group in 2001. The following literature data as well as our own experimental results devoted to this new approach have been gradually accumulated for a short time. This research project focused specifically on the concept and current case studies of the application of "enzyme evolution" to PHA biosynthesis.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Science University of Tokyo, 1998 - 1999Cold adaptation of enzymes by means of evolutionary and protein engineeringWe have, so far, obtained a variety of cold-adapted proteases from a mesophilic serine protease, subtilisin BPN', by means of evolutionary engineering. In 1998, we focused our attention to a mutation point of G131D in m-63, one of these evolvants, and random amino acid substitution was carried out for this point. Among 19 amino acid substitutions, Phe gave the highest activity. A mutation scrambling system was constructed, in the same year, for combining various mutations selected as those contributed to the cold adaptation. In 1999, DNA shuffling system was also constructed. Using these evolutionary systems, besides random mutagenesis, we will be able to increase the possibility of evolution toward cold adaptation. Finally, we applied our experimental evolution system also to α-amylase, one of industrially important enzymes other than protease. As the result, several cold-adapted α-amylase evolvants were obtained. This indicates that our system so far constructed were universally useful not only for cold adaptation of proteases, but also for that of other industrially and environmentally important enzymes.
- 日本学術振興会, 科学研究費助成事業, 奨励研究(A), 東京理科大学, 1998 - 1999分化微生物プロテアーゼインヒビターと多様な酵素群とのコミュニケーション(1) SAM-P20遺伝子の上流には、メタロチオネイン様蛋白質をコードし得る読み枠が存在し、その機能をフレームシフトにより解析したところ、SAM-P20遺伝子の発現を負に制御することが推定された。またSAM-P20遺伝子の下流には、それと相同な遺伝子が見つかり、独立に転写されることも明らかとなった。またこの遺伝子産物は、26kDaの内因性標的プロテアーゼSAM-P26と同一であることが、塩基配列分析とペプチドマッピングの結果から明らかとなった。 (2) 45 kDaの内因性標的プロテアーゼSAM-P45は、110kDaの前駆体として生合成され、菌体外へ分泌することが推定された。一次構造から本プロテアーゼは、サチライシンファミリーの新規メンバーであることがわかった。基質特異性は、トリプシン類似で活性発現にCa^<2+>を要求する。成熟領域に続いて膜へのアンカー能力をもつと思われるプロ領域が存在し、いわゆるプロホルモンプロセシング酵素フリンをはじめとするKex2型プロテアーゼに類すると考えられた。 (3) 近隣結合法と最大結合法によってSIL蛋白質群の分子系統解析をおこなったところ、それぞれの生産菌自体の系統関係と相関のあることが判明した。また阻害反応中心におけるアミノ酸置換頻度は平均的で、哺乳類血漿中のプロテアーゼインヒビターでいわれている強いDarwinian selectionは働いていないと結論された。
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Science University of Tokyo, 1995 - 1997Experimental evolution of apidaecin of honey bee origin as a model of antimicrobial peptidesThis project concerns "apidaecin" of honey bee origin, a model of antimicrobial peptides.It is composed of l8 amino acid residues and has a potential of being applied to the protection agianst pathogenic infection to plants and animals, and also applied to the food preservative. The aim of this project is to develop experimental evolution systems for the above-mentioned purposes.The research results obtained were as follows. First, we succeeded in obtaining many apidaecin mutants with lower activities by combining localized random mutagenesis for the apidaecin gene and originally developed in vivo assay-screening systems. By using these mutants, functional mapping became possible.indicating, e.g.the significant roles of the C-terminal region and Pro・Arg residues of apidaecin molecule. The relationship between the in vivo and in vitro assay systems and usefulness of the ampicillin screening for obtaining higher active apidaecin mutants were also verified (the first year). Next, in the second year.we carried out some preparative studies, e.g., trials to improve the microbial production of apidaecin.to obtain higher active apidaecin mutants, and also to clarify the mechanism of antimicrobial action. In the third year.based on the results obtained in the past two years, we succeeded in obtaining the higher active apidaecin mutants by chemically synthesizing apidaecin genes which brought about randomized amino acid residues not so essential for the antimicrobial action. Further, we tried to apply all the same systems as have been developed so far to an antimicrobial peptide other than apidaecin : thanatin of schield bug origin, which is effective against not only gramnegative and -positive bacteria but also fungi. As the result, we could obtain many thanatin mutants, and thus verify that our experimental evolution systems can be universally useful for improving any antimicrobial peptide.
- 日本学術振興会, 科学研究費助成事業, 奨励研究(A), 東京理科大学, 1996 - 1996単純分化微生物のプロテアーゼ・インヒビター間コミュニケーション当初の研究実施計画にしたがい、まずSSI様インヒビター(SIL)生産菌の培養上清より精製単離したSILタンパク質の全一次構造決定と阻害特性の解析を行った。その結果、SSIの機能構造を形成する上で重要と考えられた部分は高度に保存されており、機能構造領域を浮き彫りにした。SILインヒビターの構造相同性を利用し、最大節約法と近隣結合法を用いて作成した進化系統樹は、生産菌自身の系統関係と正の相関があった。次にSSI消失変異株を取得したところ、胞子形成能の低下、生育速度の低下、プロテアーゼ生産の増大など多面的形質の変化が走査型電顕によって観察された。そこで、SSIと相互作用する菌体外の内因性標的プロテアーゼをアフィニティーカラムにて複数単離した。その一つ、SAM-P20の遺伝子クローニング、組換え発現を行った。構造/活性から、SAM-P20はキモトリプシン属の新メンバーであることがわかった。また、アルカリ領域、高温領域でも活性を有するユニークな性質を示した。SAM-P20のプロテアーゼ活性は明らかにSSIによって阻害を受けることがインビトロ実験において明らかとなった。SAM-P45遺伝子も同様の戦略により単離することに成功した。配列分析の結果から、本プロテアーゼは約110kDaから成る巨大前駆体が生合成され、少なくとも3段階のプロセシングを経て活性化されることが推定された。今後は、SAM-P20あるいはSAM-P45とSSIとの複合体形成の詳細な解析を実施する予定である。さらに、他の内因性標的酵素についても同様の研究を推進していくことを考えている。
- 日本学術振興会, 科学研究費助成事業, 奨励研究(A), 東京理科大学, 1995 - 1995単純分化モデル放線菌におけるプロテアーゼ・インヒビター相互作用系当初の研究実施計画にしたがい、まず約20種のSSI様インヒビター(SIL)生産菌の培養上清より精製単離したSILタンパク質の全一次構造決定と阻害特性の解析を行った。その結果、SSIの機能構造を形成する上で重要と考えられた部分は高度に保存されていることがわかった。また、標的プロテアーゼの基質特異性と阻害反応部位近傍の配列との間に強い相関性のあることがわかった。さらに、インヒビターの構造相同性と既知の生産菌種間の近縁関係と良い対応があったので、SILインヒビターの構造相同性を利用し、最大節約法と近隣結合法を用いて進化系統樹を作成した。 次に、SSI消失変異株を取得したところ、胞子形成能の低下、生育速度の低下、プロテアーゼ生産の増大など多面的形質の変化が観察された。そこで、SSIと相互作用する菌体外の内因性標的プロテアーゼをアフィニティーカラムにて複数単離した。その一つ、SAM-P20の遺伝子クローニング、塩基配列決定を行った。その結果、推定アミノ酸配列が既知のS.griseus proteaseAおよびBのそれらと高い相同性のあることがわかった。また、組換え大量生産により得た完全精製SAM-P20の酵素化学的解析を詳細に行った。特筆すべきは、基質特異性がキモトリプシン様である上に、トリプシン様の性質も合わせ持つ点である。また、SAM-P20のプロテアーゼ活性は明らかにSSIによって阻害を受けることがインピトロ実験において明らかとなっった。今後は、SAM-P20とSSIとの複合体形成の詳細な解析を実施する予定である。他の内因性標的酵素についても同様の研究を推進していくことを考えている。
- 日本学術振興会, 科学研究費助成事業, 奨励研究(A), 東京理科大学, 1994 - 1994SSI様インヒビター群と真の標的酵素群の相互作用ならびに生理的意義当初の研究実施計画にしたがい、まず約25種のSSI様インヒビター(SIL)生産菌の培養上清より精製単離したSILタンパク質の部分的あるいは全体的な一次構造決定と阻害特性の解析を行った。その結果、SSIの機能構造を形成する上で重要と考えられた部分は高度に保存されていることがわかった。一方、アミノ酸置換・挿入・欠失が分子表面(特にフレキシブルループ)で認められ、抗SSI抗体に対する交差反応性の低さと合致していた。また反応部位近傍の配列に差異があり、トリプシンやキモトリプシンに対して阻害能を示すものもあった。すなわち、標的プロテアーゼの基質特異性とこの領域との間に強い相関性のあることがわかった。また、インヒビターの構造相同性と生産菌種間の近縁関係と良い対応があったので、SILインヒビターが進化系統樹作成の生化学的な指標になると考えられた。そこで、最大節約法により進化系統樹を作成したところ、反応部位にリジン残基を有するインヒビターが初期に多く存在していたことが判明した。 次に、SSI消失変異株を取得したところ、胞子形成能の低下、生育速度の低下、プロテアーゼ生産の増大など多面的形質の変化が観察された。そこで、SSIと相互作用する菌体外プロテアーゼ(SAM-P20と命名)をアフィニティーカラムにて単離し、その遺伝子クローニング、塩基配列決定を行った。その結果、推定アミノ酸配列が既知のS.griseus proteaseAおよびBのそれらと高い相同性のあることがわかった。また、プレプロ型の前駆体として分泌生産されることが推定された。培養初期に出現するSAM-P20mRNAの鎖長は約2600塩基と長く、発現制御遺伝子とオペロンを成していることが推測され現在解析を進めている。SSIの“真の標的酵素"SAM-P20の過剰生産が実現すると、本来のSILインヒビター群の生理的意義の解明に迫れると考えている。
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for General Scientific Research (C), Science University of Tokyo, 1992 - 1994Genetic Studies on Cold-adapted EnzymesFor the future biotechnological application, it is important to develop microbial enzymes which are highly active at low temperatures such as below 15゚C.Two approaches were taken for this purpose : (1) screening of natural bacterial strains possessing cold-adapted enzyme activities of interest, (2) genetic adaptation of a pre-exsisted enzyme to lower temperatures by means of experimental evolution. 1. In the first year of research term, "Approach 1" was tried. We screened candidates of cold-adapted protease- and lactase-producing bacteria in water and soil samples from various points of Antarctica, Lake Baikal and Japan. By an improved screening method, several strains with high activities at 10゚C were obtained for both model enzymes out of each 50-80 candidate strains. However, these high activities were not specific for low temperature. 2. In the second year, we constructed a novel experimental evolution system for "Approach 2", using a typical Bacillus protease "subtilisin" as a model enzyme. This system is characterized by localized and random mutagenesis for primary (activity decrease) and secondary (activity increase) mutations and selection using E.coli host-vector system. Ca.30 evolvants were obtained, one of which possessed 1.5 times k_
/Km value of that of the wild-type enzyme at various temperatures including 10゚C.3. In the third year, the experimental evolution system was improved for obtaining subtilisin evolvants which are more cold-adapted than, or of different types from those obtained in the second year. As the result, many such evolvants were successfully obtained ; e.g., one evolvant showed ca.70% increase in activity at 10゚C compared with the wild-type enzyme, and another evolvant acquired a novel type of cold-adaptation (the activity increase was specific for low temperature). - 日本学術振興会, 科学研究費助成事業, 奨励研究(A), 東京理科大学, 1993 - 1993放線菌に高頻度出現するSSI様プロテアーゼヒビターの生理的意義の解明当初の研究実施計画にしたがい、まず約20種のSSI様インヒビター(SIL)生産菌より精製単離したSILタンパク質の部分的あるいは全体的な一次構造決定を行った。その結果、(1)beta1〜5シートによって形成される構造ドメイン部分、(2)29Arg-113Pheの塩結合、(3)2カ所のS-S結合、(4)15Thr(Ser)-90Argの静電的相互作用部分、(5)2量体形成に関与する13Val、(6)反応部位を後方から支える99Asn、(7)反応部位近傍のP2'Phe(Tyr)-P4'Proのパッキング部分など、SSIの機能構造を形成する上で重要と考えられた部分は高度に保存されていることがわかった。一方、アミノ酸置換・挿入・欠失が分子表面(特にフレキシブルループ)で認められ、それはSIL1とSIL8に顕著であった。また反応部位近傍の配列に差異があり、トリプシンやキモトリプシンに対して阻害能を示すものもあった。すなわち、標的プロテアーゼの基質特異性とこの領域との間に強い相関性のあることがわかった。また、インヒビターの構造相同性と生産菌種間の近縁関係と良い反応があったので、SILインヒビターが進化系統樹作成の生化学的な指標になると考えている。 次に、SSI非生産変異細胞が生産増大するようになったプロテアーゼのうちSSIと相互作用するもの(SAM-P20と命名)の精製・単離を試み、N末端20残基の配列が既知のS.griseus proteaseAおよびBのそれらと高い相同性のあることがわかった。また、クローン化したSAM-P20遺伝子構造から本タンパク質がプレプロ型の前駆体として分泌生産されることが推定された。培養初期に出現するSAM-P20mRNAの鎖長は約2600塩基と長く、発現制御遺伝子とオペロンを成していることが推測され現在解析を進めている。SSIの“真の標的酵素"SAM-P20の過剰生産が実現すると、本来のSILインヒビター群の生理的意義の解明に迫れると考えている。