• KSBB Journal
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• v.31 no.1
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• pp.27-32
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• 2016

Several CoA transferases from Clostridium beijerinckii, C. perfringens and Klebsiella pneumoniae were examined for biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) in recombinant Escherichia coli XL1-Blue strain. The CB3819 gene and the CB4543 gene from C. beijerinckii, the pct gene from C. perfringens and the pct gene from K. pneumoniae, which encodes putative CoA transferase gene, respectively, was co-expressed with the Pseudomonas sp. MBEL 6-19 phaC1437 gene encoding engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) to examine its activity for the construction of key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli XL1-Blue expressing the phaC1437 gene and CB3819 gene synthesized poly(3-hydroxybutyrate) [P(3HB)] homopolymer to the P(3HB) content of 60.5 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 3-hydroxybutyrate. Expression of the phaC1437 gene and CB4543 gene in recombinant E. coli XL1-Blue also produced P(3HB) homopolymer to the P(3HB) content of 51.2 wt% in the same culture condition. Expression of the phaC1437 gene and the K. pneumoniae pct gene in recombinant E. coli XL1-Blue could not result in the production of PHAs in the same culture condition. However, the recombinant E. coli XL1-Blue expressing the phaC1437 gene and the C. perfringens gene could produce poly(3-hydroxybutyrate-co-lactate [P(86.4mol%3HB-co-13.7 mol%LA) up to the PHA content of 10.6 wt% in the same culture condition. Newly examined CoA transfereases in this study may be useful for the construction of engineered E. coli strains to produce PHA containing novel monomer such lactate.

• KSBB Journal
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• v.29 no.6
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• pp.443-447
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• 2014

Biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) was examined in recombinant Escherichia coli W strain from sucrose. The Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene, which encode engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) and engineered C. propionicum propionyl-CoA transferase ($Pct_{Cp}$), respectively, were expressed in E. coli W to construct key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli W expressing the phaC1437 gene and the pct540 gene could synthesize P(3HB-co-13mol%LA) up to the polymer content of 31.3 wt% when it was cultured in chemically defined MR medium containing 20 g/L of sucrose and 2 g/L of sodium 3-hydroxybutyrate. When Ralstonia eutropha phaAB genes were additionally expressed to provide 3-hydroxybutyrate-CoA (3HB-CoA) from sucrose, P(3HB-co-16mol%LA) could be synthesized from sucrose as a sole carbon source without supplement of sodium 3-hydroxybutyrate in culture medium, but the PHA content was decreased to 12.2 wt%. The molecular weight of P(3HB-co-16 mol%LA) synthesized in E. coli W using sucrose as carbon source were $1.53{\times}10^4$ ($M_n$) and $2.78{\times}10^4$ ($M_w$), respectively, which are not different from those that have previously been reported by other recombinant E. coli strains. Engineered E. coli strains developed in this study should be useful for the production of lactate-containing PHAs from sucrose, one of the most abundant and least expensive carbon sources.

• Bulletin of the Korean Chemical Society
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• v.22 no.8
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• pp.872-876
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• 2001

Topological changes caused by the alkaline and enzymatic attacks of solution-grown, chain-folded lamellar crystals (SGCs) of poly[(R)-3-hydroxybutyrate] P(3HB) have been studied in order to investigate the chain-folding structure in P(3HB) crystal regions. NaOH and an extracellular PHB depolymerase purified from Alcaligenes faecalis T1 were used for alkaline and enzymatic hydrolysis, respectively. The measurements were performed on crystals attached to a substrate which is inactive to degradation mediums. Both alkaline and enzymatic attacks lead to a breakup of the lamellar crystals along the crystallographic b-axis during initial erosion. Since hydrolysis preferentially occurs in amorphous regions, this morphological result reflects relatively loosely packed chains in core parts of lamellar crystals. Additionally, it was supported by the ridge formation along the b-axis in the lamellar crystals after thermal treatment at a low temperature because of the thermally sensitive nature of the loosely packed chains in lamellar crystals. However, the alkaline hydrolysis accompanied the chain erosions or scissions in quasi-regular folded lamellar surfaces due to smaller size of alkaline ions in comparison to the enzyme, resulting in the decrease of molecular weight.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.537-544
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• 1998

The cultivation conditions of transformant Alcaligenes eutrophus AER5 harboring cloned phbC gene for mass production of poly (3-hydroxybutyrate-3-hydroxyvalerate)[P(3HB-3HV)] containing high molar fraction of 3-hydroxyvalerate (3-HV) were investigated. In two-stage batch cultivation, transformant accumulated P(3HB-3HV) containing 52.2 mol% of 3HV compared to 30 mol% of parent strain A. eutrophus H16. The increased 3-HV molar fraction was due to the amplified activity of PHB synthase participating in condensation of 3-HB and 3-HV. To increase efficiency of P(3HB-3HV) accumulation, fructose was added along with precursor compound valerate, and total cell mass and P(3HB-3HV) concentrations remarkably increased, but not 3-HV molar fraction. The effect of magnesium ion showed that P(3HB-3HV) concentration and 3-HV molar fraction were significantly increased upto 6.1 g/L and 71.3 mol% at 0.01 g/L of MgSO$_4$, respectively. The efficiency of several pH adjuster, NaOH, NaOH and (NH$_4$)$_2$SO$_4$, and NH$_4$OH, on total cell mass, p(3HB-3HV) concentration, and 3-HV molar fraction was also compared. To overcome the disadvantage of two-stage cultivation, one-stage intermittent fed-batch cultivation was attempted, such a way 10.0 g/L of fructose was supplied for cell growth at initial 36 hr and then 10.0 g/L of valerate and 5.0 g/L of fructose were applied to induce the accumulation of P(3HB-3HV), consequently, 10.4 g/L of P(3HB-3HV) with 38 mol% of 3-HV fraction could be obtained after 72 hr. These results can be used for elucidating cultivation strategy for mass production of P(3HB-3HV) containing high 3-HV molar fraction using transformant A. eutrophus AER5 harboring cloned phbC gene.

• Journal of Microbiology and Biotechnology
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• v.3 no.2
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• pp.123-128
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• 1993

The polyesters (polyhydroxyalkanoates; PHAs) production capability in a two-step cultivation of Alcaligenes eutrophus H16(ATCC 17699) was investigated by using various organic carbon sources. The carbon sources used included linear $C_2~C_10$ monocarboxylic acids, $C_3~C_10$ dicarboxylic acids, crotonic acid, and several linear vicinal and $\omega$-diols. The polyesters synthesized were characterized by 500 MHz $^1 H-NMR$ spectroscopy, intrinsic viscosity$[\eta]$ measurement in chloroform and differential scanning calorimetry (DSC). The PHAs synthesis data showed that the use of C-odd ($C_3, C_5, and C_7$) monocarboxylic acids resulted in poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(P(3HB-co-3HV) (3HV content ranging 40 to 70 mol%) while the use of $C_9$ substrate gave the copolyester containing only 4 mol% of 3HV. All culture products obtained on $C_3$~C$_{10}$ dicarboxylic acids gave exclusively P(3HB). 500 MHz $^1 H-NMR$ analysis showed that all polyesters synthesized generally contained 1~2 mol% 3HV even for the unrelated substrates such as the carboxylic acids with even number of carbon. When $\alpha, \omega$-diols with even number of carbon were used as substrates, 4-hydroxybutyrate(4HB) was inserted into the polyester chain composed of P(3HB-co-4HB). Vicinal diols were generally not utilized by the bacterium for polyester production.n.

• Proceedings of the Polymer Society of Korea Conference
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• 2006.10a
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• pp.352-352
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• 2006

Corynebacterium glutamicum, which is well known as an amino acid fermentation bacterium, has been used as a producer of poly(3-hydroxybutyrate) [P(3HB)]. P(3HB) was synthesized in recombinant C. glutamicum harboring the expression plasmid vector with a strong promoter for cell surface protein gene derived from C. glutamicum and P(3HB) biosynthetic gene operon derived from Ralstonia eutropha. The expression of P(3HB) synthase gene was detected by enzyme activity assay. Intracellular P(3HB) was microscopically observed as inclusion granules and its content was calculated to be 22.5 % (w/w) with molecular weight of $2.1{\times}10^{5}$ and polydispersity of 1.63.

• Journal of Environmental Science International
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• v.11 no.12
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• pp.1315-1320
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• 2002

Since the enzymatic degradation of microbial poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (P(3HB-co-3HV)) initially occurs by a surface erosion process, a degradation behavior could be controlled by the change of surface property. In order to control the rate of enzymatic degradation, plasma gas discharge and blending techniques were used to modify the surface of microbial P(3HB-co-3HV). The surface hydrophobic property of P(3HB-co-3HV) film was introduced by CF$_3$H plasma exposure. Also, the addition of small amount of polystyrene as a non-degradable polymer with lower surface energy to P(3HB-co-3HV) has been studied. The enzymatic degradation was carried out at 37 $^{\circ}C$ in 0.1 M potassium phosphate buffer (pH 7.4) in the presence of an extracellular PHB depolymerase purified from Alcaligenes facalis T1. Both results showed the significant retardation of enzymatic erosion due to the hydrophobicity and the enzyme inactivity of the fluorinated- and PS-enriched surface layers.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.901-910
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• 2006

Poly(3-hydroxybutyrate) [P(3HB)] is a microbial polyester intracellularly accumulated as distinct granules in numerous microorganisms as an energy and carbon storage material. Recombinant Escherichia coli harboring the heterologous P(3HB) biosynthesis genes accumulates large amounts of P(3HB) granules, yet the granule-associated proteins have not been identified. Therefore, this study reports on an analysis of the P(3HB) granule-associated proteome in recombinant E. coli. Fiye proteins out of 7 spots identified were found to be involved in functions of translation, heat-stress responses, and P(3HB) biosynthesis. Two of the major granule-associated proteins, IbpA/B, which are already known to bind to recombinant proteins forming inclusion bodies in E. coli, were further analyzed. Immunoblotting and immunoelectron microscopic studies with IbpA/B antibodies clearly demonstrated the binding and localization of IbpA/B to P(3HB) granules. IbpA/B seemed to play an important role in recombinant E. coli producing P(3HB) by stabilizing the interface between the hydrophobic P(3HB) granules and the hydrophilic cytoplasm. Thus, IbpA/B were found to act like phasins in recombinant E. coli, as they are the major proteins bound to the P(3HB) granules, affect the morphology of the granules, and reduce the amount of cytosolic proteins bound to the P(3HB) granules.

• KSBB Journal
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• v.19 no.4
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• pp.327-330
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• 2004

An inducible expression system of poly[(R)-3-hydroxybutyrate] (PHB) depolymerization was established in metabolically engineered Escherichia coli with the PHB biosynthesis genes. The Ralstonia eutropha PHB depolymerase gene was cloned in a vector system containing the PHB biosynthesis genes and expressed under inducible promoter. Recombinant E. coli harboring the PHB biosynthesis genes and depolymerase gene was first cultured for the accumulation of PHB, and then the depolymerase was expressed resulting in the degradation of accumulated PHB into (R)-3-hydroxybutyric acid (R3HB). R3HB could be produced with the concentration of 7.6 g/L in flask culture. Two different PHB biosynthesis genes from Alcaligenes latus and R. eutropha were compared for the production of R3HB. This strategy can be used for the production of enantiomerically pure (R)-hydroxycarboxylic acids with high concentration.

• Journal of Microbiology and Biotechnology
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• v.3 no.2
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• pp.115-122
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• 1993

Poly(3-hydroxybutyrate)(P(3HB)) accumulation under nutrient-rich condition with various amounts of $(NH_4)_2 SO_4$ was systematically investigated. The results of the electron-microscopy and the solvent extraction showed that the P(3HB) accumulation is unavoidable even under nutrient-rich condition. This indicates that in a two-step culture of Alcaligenes eutrophus H16, the researches should be careful in interpreting the data of polyhydroxyalkanoates(PHAs) accumulation in terms of the carbon-source fed in the second step because the two-step culture product contains the P(3HB) produced under nutrient-rich condition. The polyester production capability in a two-step batch culture of A. eutrophus H16(ATCC 17699) was also investigated using various saccharides and their derivatives such as glucose, fructose, gluconic acid, glucaric acid, sorbitol, lactose, galactose, and mannose. The polyesters synthesized were characterized by 500 MHz$^{1}H-NMR$ spectroscopy, intrinsic viscosity$[\eta]$ measurement in chloroform and differential scanning calorimetry(DSC). 500 MHz $^{1}H-NMR$ analysis showed that all polyesters synthesized generally contained 1~2 mol% of 3HV. Another finding is that the glucose utilization can be increased by changing the autoclaving procedure of the substrate to enhance the P(3HB) production yield up to 46 wt% of P(3HB) in dry cells.