• Journal of Microbiology and Biotechnology
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• v.4 no.2
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• pp.146-150
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• 1994

Production of poly-$\beta$-hydroxybutyrate (PHB) in fed-batch fermentation was studied. Utilization of carbon for PHB biosynthesis was investigated by using feeding solutions with different ratios of carbon to nitrogen (C/N). It was observed that at a high C/N ratio carbon source was more preferably utilized for PHB accumulation while its consumption for cellular metabolism appeared to be more favored at a low C/N value. A high cell concentration (184 g/l) was achieved when ammonium hydroxide solution was fed to control the pH, which was also utilized as the sole nitrogen source. For the mass production of PHB, two-stage fed-batch operations were carried out where PHB accumulation was observed to be stimulated by switching the ammonium feeding mode to the nitrogen limiting condition. A large amount of PHB (108 g/l) was obtained with cellular content of 80% within 50 hrs of operation.

• KSBB Journal
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• v.10 no.2
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• pp.137-142
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• 1995

When Escherichia coli W, which is able to utilize sucrose as a carbon source, harboring a high-copy-number plasmid (pSYL105) containing the Alcaligenes eultrophus polyhydroxyalkanoate(PHA) biosynthetic genes was cultured in a defined medium, the final poly(3-hydroxybutyric acid), PHB, concentration obtained was as low as $0.21g/\ell$. Ten different complex nitrogen sources were, therefore, examined for their ability to enhance PHB synthesis when supplemented to a defined medium. Addition of tryptone, casamino acids, casein hydrolysate, or soy bean hydrolysate enhanced PHB synthesis most significantly, resulting in more than 10 times higher PHB concentration compared with that obtained in a defined medium. Furthermore, PHB yield on sucrose was also increased by more than a 10 fold by the addition of these complex nitrogen sources, which suggested that PHB might be efficiently produced by the recombinant E. coli W(pSYL105) using sucrose as a carbon source.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.229-236
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• 1997

A gene, $phbC_{2.4.1}$ encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring $phbA,B_{AC}$ in pRK415, which code for ${\beta}$-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of $phbC_{2.4.1}$ in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [$H^{+}$]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of $phbC_{2.4.1}$ with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SaiI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring $phbA,B_{AC}$. The promoter (s) of the $phbC_{2.4.1}$ were localized within a 340-bp DNA region upstream of the $phbC_{2.4.1}$ start codon according to heterologous expression analysis.

• Journal of the Korea Organic Resources Recycling Association
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• v.11 no.4
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• pp.49-56
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• 2003

The strain Ralstonia eutopha JMP134 (formerly Alcaligenes eutrophus JMP134) can degrade trichloroethylene(TCE) through a chromosomal phenol-dependent pathway. The phenol hydroxylase was previously found to be a single responsible enzyme for TEC degradation. Here, we demonstrate that a recombinant bacterium, R. eutopha AEK301, one of Tn5-induced mutants of JMP134 containing a recombinant plasmid pYK3011, degrades TCE in the absence of inducer, phenol and in the presence of various carbon sources. Complete removal of TCE ($50{\mu}M$) was observed in minimal medium containing only 0.05% ethanol as a carbon source within 24 hours. The bacterium removed $200{\mu}M$ of TCE to below detectable level within two days under non-selective pressure. When TCE concentration was increased up to $400{\mu}M$, the degradation had been continued until two days, then ceased with removal of 70% of detectable TCE.

• KSBB Journal
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• v.10 no.5
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• pp.533-539
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• 1995

An autolytic system based on a thermally inducible phage lambda, λHL1, has been applied for the recovery of poly(3-hydroxybutyrate) [PHB] from a recombinant Escherichia coli XL1-Blue, harbouring a plasmid (pSYL105) containing the Alcaligenes eutrophus PHB biosynthesis genes. The lytic capability ofλHL1 was evaluated in flask culture for both lysogens, XL1-Blue (λHL1) and XL1-Blue (λHL1, pSYL105). When the optical density of culture at 600nm(OD600) reached 0.2, cell lysis was induced by increasing the temperature from $30^{\circ}C$ to $42^{\circ}C$. Most cells of XL1-Blue ($\lambda$HL1) were lysed by the autolytic system in an hour after the thermal induction, while the lytic efficiency was slightly lower for XLl-Blue (λHL1, pSYL105). The existence of pSYL105 in cells seemed to inhibit, to some extent, the lytic capability of λHL1 even at low PHB content. The lylic efficiency remarkably decreased as the induction was delayed to allow PHB accumulation. When a chemical induction using 2% (v/v) chloroform was introduced after an hours of thermal induction, we could obtain a good lytic efficiency.