• 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.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.735-738
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• 2001

Metabolically engineered Escherichia coli strains harboring a plasmid containing a novel artificial polyhydroxyalkanoate (PHA) operon consisting of the Aeromonas PHA biosynthesis related genes and Ralstonia eutropha reductase gene were developed for the production of poly(3-hydroxybutyrate-co-hydroxyhexanoate) [P(3HB-co-3HHx)] from dodecanoic acid. By applying stepwise reduction of dissolved oxygen concentration (DOC) during the fermentation, the final dry cell weight, PHA concentration, and PHA content of 79 g/L, 21.5 g/L, and 27.2 wt%, respectively, were obtained in 40.8 h, which resulted in the PHA productivity of 0.53 g/L/h. The 3HHx fraction slowly increased during the fed-batch culture to reach a final value of 10.8 mol%. The 3HHx fraction in the copolymer could be increased by three fold when the Aeromonas hydrophila orfl gene was co-expressed with the PHA biosynthesis genes.

• 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.

• KSBB Journal
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• v.18 no.5
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• pp.404-407
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• 2003

Two recombinant Escherichia coli strains, GCSC6576 harboring a plasmid pSYL107 containing the Ralstonia eutropha polyhydroxyalkanoate (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. The 3HV fraction could be increased by acetic acid induction and oleic acid supplementation in flask cultures of recombinant E. coli GCSC6576. With the pH-stat fed-batch culture of recombinant E. coli LS5218, we 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%, and 6.26 mol%, respectively in 39 h.

• Microbiology and Biotechnology Letters
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• v.32 no.3
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• pp.243-248
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• 2004

Production of (R)-3-hydroxybutyric acid (R3HB) by fed-batch culture and continuous culture of metabolically engineered Escherichia coli harboring Ralstonia eutropha PHB biosynthesis and depolymerase genes was examined in a 30 1 pilot-scale fermentor. A new stable two-plasmid system, pBRRed containing the R. eutropha PHB depolymerase gene and pMCS 105 containing the R. eutropha PHB biosynthesis genes, was developed. Among a variety of E. coli strains harboring plasmids, recombinant E. coli XL-10 Gold (pBRRed, pMCS105) was able to produce R3HB with the highest efficiency in a batch culture. By the fed-batch culture of recombinant E. coli XL-10 Gold(pBRRed, pMCS 105) in a 30 1 fer-mentor, the final R3HB concentration was 22.4 g/l giving a productivity of 0.97 g/l-h. To produce R3HB to a high concentration with high productivity, a new strategy of fed-batch culture followed by a continuous culture was investigated. The maximum productivity and R3HB concentration were 5.06 g/l-h and 25.3 g/l, respectively. These results show that economical production of R3HB is possible by recombinant E. coli in large scale.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.5
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• pp.237-251
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• 2002

The recent progress on metabolic systems engineering was reviewed based on our recent research results in terms of (1) metabolic signal flow diagram approach, (2) metabolic flux analysis (MFA) in particular with intracellular isotopomer distribution using NMR and/or GC-MS, (3) synthesis and optimization of metabolic flux distribution (MFD), (4) modification of MFD by gene manipulation and by controlling culture environment, (5) metabolic control analysis (MCA), (6) design of metabolic regulation structure, and (7) identification of unknown pathways with isotope tracing by NMR. The main characteristics of metabolic engineering is to treat metabolism as a network or entirety instead of individual reactions. The applications were made for poly-3-hydroxybutyrate (PHB) production using Ralstonia eutropha and recombinant Escherichia coli, lactate production by recombinant Saccharomyces cerevisiae, pyruvate production by vitamin auxotrophic yeast Toluropsis glabrata, lysine production using Corynebacterium glutamicum, and energetic analysis of photosynthesic microorganisms such as Cyanobateria. The characteristics of each approach were reviewed with their applications. The approach based on isotope labeling experiments gives reliable and quantitative results for metabolic flux analysis. It should be recognized that the next stage should be toward the investigation of metabolic flux analysis with gene and protein expressions to uncover the metabolic regulation in relation to genetic modification and/ or the change in the culture condition.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.3
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• pp.196-200
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• 2004

The supra molecular weight poly(〔R〕-3-hydroxybutyrate) (PH B), having a molecular weight greater than 2 million Da, has recently been found to possess improved mechanical properties compared with the normal molecular weight PHB, which has a molecular weight of less than 1 million Da. However, applications for this PHB have been hampered due to the difficulty of its production. Reported here, is the development of a new metabolically engineered Escherichia coli strain and its fermentation for high level production of supra molecular weight PHB. Recombinant E. coli strains, harboring plasm ids of different copy numbers containing the Alcaligenes latus PHB biosynthesis genes, were cultured and the molecular weights of the accumulated PHB were compared. When the recombinant E. coli XL1-Blue, harboring a medium-copy-number pJC2 containing the A. latus PHB biosynthesis genes, was cultivated by fed-batch culture at pH 6.0, supra molecular weight PHB could be produced at up to 89.8 g/L with a productivity of 2.07 g PHB/L-h. The molecular weight of PHB obtained under these conditions was as high as 22 MDa, exceeding by an order of magnitude the molecular weight of PHB typically produced in Ralstonia eutropha or recombinant E. coli.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1408-1415
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• 2008

To produce polyhydroxyalkanoate (PHA) from inexpensive substrates by bacteria, vegetable-oil-degrading bacteria were isolated from a rice field using enrichment cultivation. The isolated Pseudomonas sp. strain DR2 showed clear orange or red spots of accumulated PHA granules when grown on phosphate and nitrogen limited medium containing vegetable oil as the sole carbon source and stained with Nile blue A. Up to 37.34% (w/w) of intracellular PHA was produced from corn oil, which consisted of three major 3-hydroxyalkanoates; octanoic (C8:0, 37.75% of the total 3-hydroxyalkanoate content of PHA), decanoic (C10:0, 36.74%), and dodecanoic (C12:0, 11.36%). Pseudomonas sp. strain DR2 accumulated up to 23.52% (w/w) of $PHA_{MCL}$ from waste vegetable oil. The proportion of 3-hydroxyalkanoate of the waste vegetable-oil-derived PHA [hexanoic (5.86%), octanoic (45.67%), decanoic (34.88%), tetradecanoic (8.35%), and hexadecanoic (5.24%)] showed a composition ratio different from that of the corn-oil-derived PHA. Strain DR2 used three major fatty acids in the same ratio, and linoleic acid was the major source of PHA production. Interestingly, the production of PHA in Pseudomonas sp. strain DR2 could not occur in either acetate- or butyrate-amended media. Pseudomonas sp. strain DR2 accumulated a greater amount of PHA than other well-studied strains (Chromobacterium violaceum and Ralstonia eutropha H16) when grown on vegetable oil. The data showed that Pseudomonas sp. strain DR2 was capable of producing PHA from waste vegetable oil.

• Journal of Microbiology and Biotechnology
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• v.28 no.7
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• pp.1133-1140
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• 2018

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas. In vivo expression of the putative PHA synthase gene ($phaC_{Pf}$) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli (${phaC_{Pf}}^+$, pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an E. coli fadR mutant (${phaC_{Pf}}^+$) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified $PhaC_{Pf}$, suggesting that the putative PHA synthase of P. fluorescens utilizes mainly short-chain-length PHA precursors as a substrate.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.776-784
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• 2019

Polyhydroxybutyrate (PHB), the most well-known polyhydroxyalkanoate, is a bio-based, biodegradable polymer that has the potential to replace petroleum-based plastics. Lignocellulose hydrolysate, a non-edible resource, is a promising substrate for the sustainable, fermentative production of PHB. However, its application is limited by the generation of inhibitors during the pretreatment processes. In this study, we investigated the feasibility of PHB production in E. coli in the presence of inhibitors found in lignocellulose hydrolysates. Our results show that the introduction of PHB synthetic genes (bktB, phaB, and phaC from Ralstonia eutropha H16) improved cell growth in the presence of the inhibitors such as furfural, 4-hydroxybenzaldehyde, and vanillin, suggesting that PHB synthetic genes confer resistance to these inhibitors. In addition, increased PHB production was observed in the presence of furfural as opposed to the absence of furfural, suggesting that this compound could be used to stimulate PHB production. Our findings indicate that PHB production using lignocellulose hydrolysates in recombinant E. coli could be an innovative strategy for cost-effective PHB production, and PHB could be a good target product from lignocellulose hydrolysates, especially glucose.