Fumi Shozui

Hokkaido University, Sapporo, Hokkaidō, Japan

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Publications (10)30.41 Total impact

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    ABSTRACT: A poly(lactic acid) (PLA)-like terpolyester consisting of 96mol% lactate (LA), 1mol% 3-hydroxybutyrate and 3mol% 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 °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.
    Polymer Degradation and Stability 04/2011; 96(4):499-504. DOI:10.1016/j.polymdegradstab.2011.01.007 · 2.63 Impact Factor
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    ABSTRACT: The 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.
    Applied and Environmental Microbiology 03/2011; 77(9):2926-33. DOI:10.1128/AEM.01997-10 · 3.95 Impact Factor
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    ABSTRACT: New 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 β-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, 1H-13C COSY NMR analysis provided evidence for a linkage between LA and 3HHx units in the polymer.
    Polymer Degradation and Stability 08/2010; DOI:10.1016/j.polymdegradstab.2010.01.029 · 2.63 Impact Factor
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    ABSTRACT: Lactate (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 recombinant Escherichia coli using the first LPE, which is the Ser325Thr/Glu481Lys mutant of PHA synthase from Pseudomonas sp. 61-3 [PhaC1(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 PhaC1(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 PhaC1(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.
    Biomacromolecules 02/2010; 11(3):815-9. DOI:10.1021/bm901437z · 5.79 Impact Factor
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    ABSTRACT: 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.
    Bioscience Biotechnology and Biochemistry 01/2010; 74(8):1716-8. DOI:10.1271/bbb.100242 · 1.21 Impact Factor
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    ABSTRACT: A newly isolated mutation (Gln508Leu) 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.
    Bioscience Biotechnology and Biochemistry 01/2010; 74(8):1710-2. DOI:10.1271/bbb.100224 · 1.21 Impact Factor
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    ABSTRACT: The 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 degrees C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 degrees C and for 1 month at 30 degrees C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 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.
    Applied Microbiology and Biotechnology 12/2009; 86(5):1431-8. DOI:10.1007/s00253-009-2351-7 · 3.81 Impact Factor
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    ABSTRACT: Novel 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, (1)H-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.
    Applied Microbiology and Biotechnology 08/2009; 85(4):949-54. DOI:10.1007/s00253-009-2100-y · 3.81 Impact Factor
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    ABSTRACT: Class 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.
    Applied Microbiology and Biotechnology 07/2009; 84(6):1117-24. DOI:10.1007/s00253-009-2052-2 · 3.81 Impact Factor
  • Polymer Journal 01/2009; 41(3). DOI:10.1295/polymj.PJ2008253 · 1.55 Impact Factor