• Korean Journal of Microbiology
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• v.34 no.4
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• pp.219-224
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• 1998

The cyclic metabolism of poly(3-hydroxyhutyrate-co-3-hydroxyvalerate) synthesized from glucose by Alcaligenes sp. SH-69 in the presence or absence of new carbon substrate was investigated. In batch culture, the content and weight average molecular weight of the copolymer already stored in the cell decreased rapidly when there was no other carbon source available. After the depletion of carbon source, the amount of high molecular weight copolymer decreased more rapidly than that of low molecular weight copolymer, and as a result, average molecular weight distribution shifted to the lower value. The addition of a mixture of glucose and levulinic acid when the initial carbon substrate, glucose, was nearly depleted supported the continual increase in cell mass and the accumulation of poly(3HB-co-3HV) with high molar fraction of 3HV. However, solvent fractionation of the polymer with acetone revealed the degradation of pre-existing polyhydroxyalkanoale (PHA) in parallel with the synthesis of PHA from new carbon substrate. Even though PHAs obtained from each substrate alone were the copolymer of 3HB and 3HV, it was found that the polymer accumulated in the cells grown by sequential feeding was mainly physical mixture of two poly(3HB-co-3HV) copolymers containing different molar fractions of 3HV.

• Journal of Microbiology
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• v.42 no.4
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• pp.346-352
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• 2004

Poly(3-Hydroxybutyrate-co­3-Hydroxyvalerate), poly(3HB-co-3HV), copolyesters with a variety of 3HV contents (ranging from 17 to $60\;mol\%$) were produced by Alcaligenes sp. MT-16 grown on a medium containing glucose and levulinic acid in various ratios, and the effects of hydrophilicity and crystallinity on the degradability of the copolyesters were evaluated. Measurements of thermo-mechanical pro­perties and Fourier-transform infrared spectroscopy in the attenuated total reflectance revealed that the hydrophilicity and crystallinity of poly(3HB-co-3HV) copolyesters decreased as 3HV content in the copolyester increased. When the prepared copolyester film samples were non-enzymatically hydrolysed in 0.01 N NaOH solution, the weights of all samples were found to have undergone no changes over a period of 20 weeks. In contrast, the copolyester film samples were degraded by the action of extra­cellular polyhydroxybutyrate depolymerase from Emericellopsis minima W2. The overall rate of weight loss was higher in the films containing higher amounts of 3HV, suggesting that the enzymatic degra­dation of the copolyester is more dependent on the crystallinity of the copolyester than on its hydro­philicity. Our results suggest that the degradability characteristics of poly(3HB-co-3HV) copolyesters, as well as their thermo-mechanical properties, are greatly influenced by the 3HV content in the copoly­esters.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.321-324
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• 2001

Two recombinant Escherichia coli strains, GCSC6576 harboring a plasmid pSYL107 containing the Ralstonia eutropha polyhyclroxyalkanoate (PHA) biosynthesis genes and a fadR atoC mutant LS5218 harboring a plasmid pJC4 containing the Alcaligenes latus PHA biosynthesis genes were compared for their ability to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from whey as the sale carbon source. With the pH-stat fed-batch culture of E. coli LS5218, 、 ,ve obtained a cell concentration, a P(3HB-co-3HV) concentration. a P(3HB-co-3HV) content, and a 3HV fraction of 31.8 g/L, 10.6 g/L, 33.4 wt%. and 6.26 mol%, respectively at 39 h.

• Journal of Adhesion and Interface
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• v.7 no.2
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• pp.19-25
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• 2006

Since the enzymatic degradation of microbial poly(hydroxylalkanoate)s (PHAs), such as poly[(R)-3-hydroxybutyrate] and poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] initially occurs by a surface erosion process, their degradation behaviors can be controlled by the change of surface property. In order to control the rate of enzymatic degradation, plasma modification technique was applied to change the surface property of microbial PHAs. The surface hydrophobic and hydrophilic properties of PHA films were introduced by $CF_3H$ and $O_2$ plasma exposures, respectively. 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. The results showed that the significant retardation of initial enzymatic erosion of $CF_3H$ plasma-treated PHAs was observed due to the hydrophobicity and the enzyme inactivity of the fluorinated surface layers while the erosion rate of $O_2$ plasma-treated PHAs was not accelerated.

• 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.

• Journal of Life Science
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• v.32 no.7
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• pp.510-522
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• 2022

In this study, to biosynthesize PHA with properties more similar to polypropylene, a Bacillus sp. EMK-5020 strain that biosynthesized poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was isolated from soil. Bacillus sp. EMK-5020 strain biosynthesized PHBV containing 1.3% 3-hydroxyvalerate (3HV) using reducing sugar contained in Makgeolli lees enzymatic hydrolysate (MLEH) as a single carbon source. As the amount of propionic acid, which was added as a second carbon source, increased, the content of 3HV also increased. PHBV containing up to 48.6% of 3HV was synthesized when 1.0 g/l of propionic acid was added. Based on these results, the strain was cultured for 72 hr in a 3 l fermenter using reducing sugar in MLEH (20 g/l) and propionic acid (1 g/l) as the main and secondary carbon sources, respectively. As a result, 6.4 g/l DCW and 50 wt% of PHBV (MLEH-PHBV) containing 8.9% 3HV were biosynthesized. Through gel permeation chromatography and thermogravimetric analysis, it was confirmed that the average molecular weight and the decomposition temperature of MLEH-PHBV were 152 kDa and 273℃, respectively. In conclusion, the Bacillus sp. EMK-5020 strain could biosynthesize PHBV containing various 3HV fractions when MLEH and propionic acid were used as carbon sources, and PHBV-MLEH containing 8.9% 3HV was confirmed to have higher thermal stability than standard PHBV (8% 3HV).

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1330-1336
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• 2005

Polyhydroxyalkanoic acid (PHA) inclusion bodies were analyzed in situ by $^{13}C$-nuclear magnetic resonance ($^{13}C$-NMR) spectroscopy. The PHA inclusion bodies studied were composed of poly(3-hydroxybutyrate) or poly(3hydroxybutyrate-co-4-hydroxybutyrate), which was accumulated in Hydrogenophaga pseudoflava, and medium-chain-length PHA (MCL-PHA), which was accumulated in Pseudomonas fluorescens BM07 from octanoic acid or 11-phenoxyundecanoic acid (11-POU). The quantification of the $^{13}C$-NMR signals was conducted against a standard compound, sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). The chemical shift values for the in vivo NMR spectral peaks agreed well with those for the corresponding purified PHA polymers. The intracellular degradation of the PHA inclusions by intracellular PHA depolymerase(s) was monitored by in vivo NMR spectroscopy and analyzed in terms of first-order reaction kinetics. The H. pseudoflava cells were washed for the degradation experiment, transferred to a degradation medium without a carbon source, but containing 1.0 g/l ammonium sulfate, and cultivated at $35^{\circ}C$ for 72 h. The in vivo NMR spectra were obtained at $70^{\circ}C$ for the short-chain-length PHA cells whereas the spectra for the aliphatic and aromatic MCL-PHA cells were obtained at $50^{\circ}C\;and\;80^{\circ}C$, respectively. For the H. pseudoflava cells, the in vivo NMR kinetics analysis of the PHA degradation resulted in a first-order degradation rate constant of 0.075/h ($r^{2}$=0.94) for the initial 24 h of degradation, which was close to the 0.050/h determined when using a gas chromatographic analysis of chloroform extracts of sulfuric acid/methanol reaction mixtures of dried whole cells. Accordingly, it is suggested that in vivo $^{13}C$-NMR spectroscopy is an important tool for studying intracellular PHA degradation in terms of kinetics.

• 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.

• KSBB Journal
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• v.13 no.2
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• pp.187-194
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• 1998

For mass production of polyhydroxyalkanoates (PHA), high cell density cultures of Alcaligenes eutrophus by fed-batch culture under phosphate-limitation condition has been investigated. PHA accumulation by the regulation by the regulation of initial phosphate concentration could be automatically induced, and high density cell culture above 200 g/L also could be successfully produced. The production of Poly-$\beta$-hydroxybutyrate (PHB) and dry cell weight increased with increasing the initial phosphate concentration. When the initial concentrations of phosphate were in the ranges of 1.5~4.5 g-PO$_4$/L, PHB and dry cell weight obtained were 83~266 g/L and 61~216 g/L, respectively, and PHB productivity was in the ranges of 1.35~3.10 g/L.h. When a mixture of glucose and propionic acid is used as carbon sources, poly(3-hydroxybutyrate-co-poly-3-hydroxyvalerate), P(3HB-co-3HV), could be also successfully produced under phosphate limitation condition. When the mole ratio of propionic acid to glucose in the feeding solution is 0.22, a final dry cell weight of 150 g/L and a P(3-HB-co-3HV) of 90 g/L were produced. Morphological changes and size distribution of PHB granules synthesized in A. eutrophus under phosphate-limitation condition are determined by TEM during the course of fed-batch. Mean granule diameters of PHB produced are in the range of 0.36~0.39 $\mu$m, and mean cell size was elongated from 0.54~0.59 $\mu$m$\times$ 1.3~1.5 $\mu$m to 0.83~0.89 $\mu$m $\times$2.0~2.3 $\mu$m. Phosphate concentration in media did not affect size distribution of PHB granule and cell.

• Journal of Microbiology
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• v.39 no.1
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• pp.79-82
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• 2001

The influence of levulinic acid (LA) on the production of copolyester consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) by Ralstonia eutropha was investigated. Addition of LA into the culture medium greatly increased the molar fraction of 3HV in the copolyester, indicating that LA can be utilized as a precursor of 3HV. In shake flask culture, the 3HV content in the copolyester increased from 7 to 75 mol% by adding 0.5 to 4.0 g/L LA to the medium containing fructose syrup as a main carbon source. A maximal copolyester concentration of 3.6 g/L (69% of dry cell weight) was achieved with a 3HV content of 40 mo1% in a jar fermentor culture containing 4.0 g/L of LA. When LA (total concentration, 4 g/L) was added repeatedly into a fermentor culture to maintain its concentration at a low level, the copolyester content and the 3HV yield from LA reached up to 85% of dry cell weight and 5.0 g/g, respectively, which were significantly higher than those when the same concentration of the LA was supplied al1 at once. The present results indicated that LA is more effective than propionate or valerate as a cosubstrate fur the production of copolyesters with varying molar fractions of 3HV by R. eutropha.