• Journal of Microbiology and Biotechnology
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• v.8 no.5
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• pp.540-542
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• 1998

Five microorganisms capable of degrading an aromatic medium-chain-length polyhydroxyalkanoate ($PHA_{MCL}$), poly(3-hydroxy-5-phenylvalerate) (PHPV), were isolated from wastewater-treatment sludge. Among the isolates, JS02 showed degrading activity consistantly during several transfers. The isolate JS02 could hydrolyze another aromatic MCL copolyester, poly(3-hydroxy-5-phenoxyvalerate-co-3-hydroxy-7-phenoxyheptanoate), ［P(5POHV-co-7POHH)］, and other short-chain-length PHAs ($PHA_{SCL}) such as poly(3-hydroxybutyrate) ［P3(HB)］, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) ［P(3 HB-co-4 HB)］, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) ［P(3HB-co-3HV)］ with relatively low activity. The culture supernatant of JS02 showed hydrolyzing activity for the p-nitrophenyl esters of fatty acids.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.206-215
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• 2005

Polyhydroxyalkanoates (PHAs) are homo or hetero polyesters of (R)-hydroxyalkanoates accumulated in various microorganisms under growth-limiting condition in the presence of excess carbon source. They have been suggested as biodegradable substitutes for chemically synthesized polymers. Recombinant Escherichia coli is one of the promising host strains for the economical production of PHAs, and has been extensively investigated for the process development. The heterologous PHA biosynthetic pathways have been established through the metabolic engineering and inherent metabolic pathways of E. coli have been redirected to supply PHA precursors. Fermentation strategies for cultivating these recombinant E. coli strains have also been developed for the efficient production of PHAs. Nowadays, short-chain-length (SCL) PHAs are being re-invited due to its improved mechanical properties and possible applications in the biomedical area. In this article, recent advances in the development of metabolically engineered E. coli strains for the enhanced production of SCL-PHAs are reviewed. Also, medical applications of SCL-PHAs are discussed.

• 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.

• Korean Journal of Microbiology
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• v.48 no.3
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• pp.200-206
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• 2012

A bacterial strain capable of synthesizing polyhydroxyalkanoates (PHAs) with an unusual pattern of monomer units was isolated from activated sludge using the enrichment culture technique. The organism, identified as Pseudomonas aeruginosa P-5, produced polyesters consisting of 3-hydroxyvalerate and medium-chain-length (MCL) 3-hydroxyalkanoate monomer units when $C_{-odd}$ alkanoic acids such as nonanoic acid and heptanoic acid were fed as the sole carbon source. Solvent fractionation experiments using chloroform and hexane revealed that the 3-hydroxyalkanoate monomer units in these polyesters were copolymerized. The molar concentration of 3-hydroxyvalerate in the polyesters produced were significantly elevated up to 26 mol% by adding 1.0 g/L valeric acid as the cosubstrate. These copolyesters were sticky with low degrees of crystallinity. The PHA synthase genes were cloned, and the deduced amino acid sequences were determined. P. aeruginosa P-5 possessed genes encoding MCL-PHA synthases (PhaC1 and PhaC2) but lacked the short-chain-length PHA synthase gene, suggesting that the MCL-PHA synthases from P. aeruginosa P-5 are uniquely active for polymerizing (R)-3-hydroxyvaleryl-CoA as well as MCL (R)-3-hydroxyacyl-CoAs.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.847-853
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• 1999

A Pseudomonas sp. strain that is capable of utilizing dicarboxylic acids as a sole carbon source was isolated from activated sludge by using the enrichment culture technique. This organism accumulated polyhydroxyalkanoates (PHAs) with an unusual pattern of monomer units that depends on the carbon sources used. Polyhydroxybutyrate (PHB) homopolyester was synthesized from glucose or small $C_{-even}$ alkanoic acids, such as butyric acid and hexanoic acid. Accumulation of PHB homopolyester was also observed in the cells grown on $C_{-odd}$ dicarboxylic acids, such as heptanedioic acid and nonanedioic acid as the sole carbon sources. In contrast, a copolyester consisting of 6 mol% 3-hydroxybutyrate (3HB) and 94 mol% 3-hydroxyvalerate (3HV) was produced with a PHA content of as much as 36% of the cellular dry matter. This strain produced PHAs consisting both of the short-chain-length (SCL) and the medium-chain-length (MCL) 3-hydroxyacid units when heptanoic acid to undecanoic acid were fed as the sole carbon sources. Most interestingly, polyester consisting of significant amount of relevant fractions, 3HB, 3HV, and 3-hydroxyheptanoate (3HHp), was accumulated from heptanoic acid. According to solvent fractionation experiments, the polymer produced from heptanoic acid was a blend of poly(3HHp) and of a copolyester of 3HB, 3HV, and 3HHp units. The hexane soluble fractions contained only 3HHp units while the hexane-insoluble fractions contained 3HB and 3HV units with a small amount of 3HHp unit. The copolyester was an elastomer with unusual mechanical properties. The maximum elongation ratio of the copolyester was 460% with an ultimate strength of 10 MPa, which was very different from those of poly(3HB-co-3HV) copolyesters having similar compositions produced from other microorganisms.

• 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.

• Journal of Microbiology and Biotechnology
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• v.32 no.1
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• pp.110-116
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• 2022

Polyhydroxyalkanoates (PHAs) are emerging as alternatives to plastics by replacing fossil fuels with renewable raw substrates. Herein, we present the construction of engineered Escherichia coli strains to produce short-chain-length PHAs (scl-PHAs), including the monomers 4-hydroxyvalerate (4HV) and 3-hydroxyvalerate (3HV) produced from levulinic acid (LA). First, an E. coli strain expressing genes (lvaEDABC) from the LA metabolic pathway of Pseudomonas putida KT2440 was constructed to generate 4HV-CoA and 3HV-CoA. Second, both PhaAB enzymes from Cupriavidus necator H16 were expressed to supply 3-hydroxybutyrate (3HB)-CoA from acetyl-CoA. Finally, PHA synthase (PhaC_Cv) from Chromobacterium violaceum was introduced for the subsequent polymerization of these three monomers. The resulting E. coli strains produced four PHAs (w/w% of dry cell weight): 9.1 wt% P(4HV), 1.7 wt% P(3HV-co-4HV), 24.2 wt% P(3HB-co-4HV), and 35.6 wt% P(3HB-co-3HV-co-4HV).

• Journal of Microbiology and Biotechnology
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• v.17 no.12
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• pp.2018-2026
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• 2007

A bacterium, Pseudomonas aeruginosa BM114, capable of accumulating a blend of medium-chain-length (MCL)- and short-chain-length (SCL)-polyhydroxyalkanoic acid (PHA), was isolated. Salicylic acid (SA), without being metabolized, was found to specifically inhibit only the accumulation of MCL-PHA without affecting cell growth. An addition of 20 mM SA selectively inhibited the accumulation of MCL-PHA in decanoate-grown cells by 83% of the control content in one-step cultivation, where overall PHA accumulation was inhibited by only ${\sim}11%$. Typically, the molar monomer-unit ratio of the PHA for 25 mM decanoate-grown cells changed from 46:4:25:25 (=[3-hydroxybutyrate]:[3-hydroxycaproate]: [3-hydroxyoctanoate]:[3-hydroxydecanoate]) at 0 mM SA (dry cell wt, 1.97 g/l; PHA content, 48.6 wt%) to 91:1:4:4 at 20 mM SA (dry cell wt, 1.85 g/l; PHA content, 43.2 wt%). Thus, the stimulation of SCL-PHA accumulation was observed. Growth of P. aeruginosa BM114 on undecanoic acid also produced a PHA blend composed of 47.4% P(3HB-co-3-hydroxyvalerate) and 52.6% P(3-hydroxyheptanoate-co-3-hydroxynonanoate-co-3-hydroxyundecanoate). Similar to the case of even-carboxylic acids, SA inhibited the accumulation of only MCL-PHA, but stimulated the accumulation of SCL-PHA. For all medium-chain fatty acids tested, SA induced a stimulation of SCL-PHA accumulation in the BM114 strain. SA could thus be used to suppress only the formation of MCL-PHA in Pseudomonas spp. accumulating a blend of SCL-PHA and MCL-PHA.