• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.182-190
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• 2003

A new phaC gene cluster encoding polyhydroxyalkanoate (PHA) synthase I PHA depolymerase, and PHA synthase II was cloned using the touchdown PCR method, from medium-chain length (mcl-) PHA-producing strain Pseudomonas putida KT2440. The molecular structure of the cloned phaCl gene was analyzed, and the phylogenic relationship was compared with other phaCl genes cloned from Pseudomonas species. The cloned phaCl gene was expressed in a recombinant E. coli to the similar level of PHA synthase in the parent strain P. putida KT2440, but no significant amount of mcl-PHA was accumulated. The isolated phaCl gene was re-introduced into the parent strain P. putida KT2440 to amplify the PHA synthase I activity, and the recombinant P. purida accumulated mcl-PHA more effectively, increasing from 26.6 to $43.5\%$. The monomer compositions of 3-hydroxylalkanoates in mcl-PHA were also modified significantly in the recombinant P. putida enforcing the cloned phaCl gene.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.123-129
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• 2021

Polyhydroxyalkanoate (PHA) synthase is a key enzyme for PHA production in microorganisms. The class IV PHA synthase is composed of two subunits: PhaC and PhaR. The PhaR subunit, which encodes the phaR gene, is only present in class IV PHA synthases. Therefore, the phaR gene is used as a biomarker for bacteria that contain a class IV PHA synthase, such as some Bacillus spp. The phaR gene was developed to screen phaR-containing Bacillus spp. The phaR screening method involved two steps: phaR gene amplification by PCR and phaR amplicon detection using a DNA lateral flow assay. The screening method has a high specificity for phaR-containing Bacillus spp. The lowest amount of genomic DNA of B. thuringiensis ATCC 10792 that the phaR screening method could detect was 10 pg. This novel screening method improves the specificity and sensitivity of phaR gene screening and reduces the time and cost of the screening process, which could enhance the opportunity to discover good candidate PHA producers. Nevertheless, the screening method can certainly be used as a tool to screen phaR-containing Bacillus spp. from environmental samples.

• Journal of Microbiology and Biotechnology
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• 제28권7호
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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.

• KSBB Journal
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• 제13권1호
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• pp.96-100
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• 1998

두 개의 프로모터 (trc와 Pp)를 Alcaligenes eutrophus에서 유래된 PHA 오페론에 삽입하여 재조합 대장균에서 분자량이 큰 polyhydroxybutyrate (PHB)를 얻고자 하였다. 두 개의 프로모터는 hydroxybutyric CoA와 PHA 중합반응의 유전자 발현을 각각 독립적으로 제어하기 위해 설계된 것이다 새로운 합성오페론을 포함한 플라즈미드는 E. coli DH5 $\alpha$ 에 transformation 되어 PHB 생산에 이용되었다. 본 실험의 가설로서 PHA 합성오페론의 IPTG에 의한 유도가 없을 경우 낮은 pHA synthase의 활성이 고분자 중합반응의 개시제 농도를 줄여주어 결과적으로 높은 연결수의 고분자를 생성할 것이라는 모델을 세웠다. 실제로 IPTG의 공급이 없는 발효실험을 통해 평균분자량이 $2.5{\times}10^7$ 인 거대 고분자를 얻을 수 있었다. PHA 생합성에 관여는 효소의 활성 분석으로 3-hydroxybutyric CoA의 중합을 촉매하는 효소인 PHA synthase의 활성을 가지고 In vivo에서 분자량이 제어됨을 확인하였다.

• 미생물학회지
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• 제48권3호
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• pp.200-206
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• 2012

활성슬러지로부터 특이한 조성의 polyhydroxyalkanoates (PHAs)를 생합성하는 Pseudomonas aeruginosa P-5를 분리하였다. 이 균주는 nonanoic acid나 heptanoic acid와 같은 홀수개의 탄소수를 가지는 지방산을 단일 탄소원으로 공급해주었을 경우, 3-hydroxyvalerate (3HV)와 medium-chain-length (MCL) 3-hydroxyalkanoates 단위체로 이루어진 공중합체를 생산하였다. 공중합체 내 3HV의 함량은 valeric acid와 같은 보조기질을 공급함으로써 증가시킬 수 있었으며, 2 g/L nonanoic acid와 1 g/L valeric acid로 이루어진 혼합기질로부터 3HV의 함량이 26 mol%에 달하는 공중합체를 얻을 수 있었다. 이러한 공중합체는 결정성이 매우 낮아 점착성 고분자로서의 성질을 보였다. P. aeruginosa P-5 균주는 MCL-PHA synthase 유전자(phaC1, phaC2)를 가지고 있는 반면에 SCL-PHA synthase 유전자는 결여되어 있는 것으로 나타났다. 따라서 P. aeruginosa P-5 균주의 MCL-PHA synthase는 MCL(R)-3-hydroxyacyl-CoAs 뿐만 아니라 (R)-3-hydroxyvaleryl-CoA를 기질로 인지하는 특이한 기질특이성을 갖는 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제22권8호
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• pp.1141-1147
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• 2012

Polyhydroxyalkanoate (PHA) is a class of biodegradable plastics that have great potential applications in the near future. In this study, the micro-biodiversity and productivity of PHA-accumulating bacteria in activated sludge from a domestic wastewater treatment plant were investigated. A previously reported primer set and a self-designed primer set (phaCF1BO/phaCR2BO) were both used to amplify the PHA synthase (phaC) gene of isolated colonies. The new primers demonstrated higher sensitivity for phaC, and combining the PCR results of the two primer sets was able to widen the range of detected genera and raise the sensitivity to nearly 90%. Results showed that 85.3% of the identified bacteria were Gram-negative, with Ralstonia as the dominant genus, and 14.7% were Gram-positive. In addition, Zoogloea and Rhizobium contained the highest amounts of intracellular PHA. It is apparent that glucose was a better carbon source than pentone or tryptone for promoting PHA production in Micrococcus. Two different classes, class I and class II, of phaC were detected from alphaproteobacteria, betaproteobacteria, and gammaproteobacteria, indicating the wide diversity of PHA-accumulating bacteria in this particular sampling site. Simultaneous wastewater treatment and PHA production is promising by adopting the high PHA-accumulating bacteria isolated from activated sludge.

• KSBB Journal
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• 제29권4호
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• pp.244-249
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• 2014

Biosynthesis pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHA) from fatty acid ${\beta}$-oxidation pathway was constructed in recombinant Escherichia coli by introducing the Pseudomonas sp. 61-3 PHA synthase gene (phaC2) and the maoC genes from Pseudomonas putida, Sinorhizobium meliloti, and Ralstonia eutropha. The metabolic link between fatty acid ${\beta}$-oxidation pathway and PHA biosynthesis pathway was constructed by MaoC, which is homologous to P. aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1). When the E. coli W3110 strains expressing the phaC2 gene and one of the maoC genes from P. putida, Sinorhizobium meliloti, and Ralstonia eutropha were cultured in LB medium containing 2 g/L of sodium decanoate as a carbon source, MCL-PHA that mainly consists of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD), was produced. The monomer composition of PHA and PHA contents varied depending on MaoC employed for the production of PHA. The highest PHA content of 18.7 wt% was achieved in recombinant E. coli W3110 expressing the phaC2 gene and the P. putida maoC gene. These results suggest that MCL-PHA biosynthesis pathway can be constructed in recombinant E. coli strains from the b-oxidation pathway by employing MaoC able to supply (R)-3-hydroxyacyl-CoA, the substrate of PHA synthase.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
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• pp.173-182
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• 2005

A three dimensional (3D) model for the catalytic region of Type II Pseudomonas sp. USM 4-55 PHA synthase 1 (PhaC1$_{P.sp\;USM\;4-55}$) from residue 267 to residue 484 was developed. Sequence analysis demonstrated that PhaC1$_{P.sp\;USM\;4-55}$ lacked homology with all known structural databases. PSI-BLAST and HMM Superfamily analyses demonstrated that this enzyme belongs to the ${\alpha}/{\beta}$ hydrolase fold family. Threading approach revealed that the most suitable template to use was the Human gastric lipase (1HLG). The superimposition of the predicted PhaC1$_{P.sp\;USM\;4-55}$ model with the 1HLG template structure covering 86.2% of the backbone atoms showed an RMSD of 1.15 ${\AA}$ The catalytic residues comprising of Cys296, Asp451, His452 and His479 were found to be conserved and were located adjacent to each other. We proposed that the catalytic mechanism involved the formation of two tetrahedral intermediates.

• Journal of Microbiology and Biotechnology
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• 제6권5호
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• pp.309-314
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• 1996

Alcaligenes sp. SH-69 can synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from a single carbon source such as glucose. To clone the phaC gene from Alcaligenes sp. SH-69, a polymerase chain reaction was performed using the oligomers synthesized based on the conserved regions of the phaC genes from other bacteria. A PCR product (550 bp) was partially sequenced and the deduced amino acid sequence was found to be homologous to that of the phaC gene from Alcaligenes eutrophus. Using the PCR fragment Southern blotting of Alcaligenes sp. SH-69 genomic DNA digested with several restriction enzymes was carried out. To prepare a partial genomic library, about 5-Kb genomic DNA fragments digested with EcoRI, which showed a positive signal in the Southern blotting, were eluted from an agarose gel, ligated with pUC19 cleaved with EcoRI, and transformed into Escherichia coli. The partial library was screened using the PCR fragment as a probe and a plasmid, named pPHA11, showing a strong hybridization signal was selected. Restriction mapping of the insert DNA in pPHA11 was performed. Cotransformation into E. coli of the plasmid pPHA11 and the plasmid pPHA21 which has phaA and phaB from A. eutrophus resulted in turbid E. coli colonies which are indicative of PHA accumulation. This result tells us that the Alcaligenes sp. SH-69 phaC gene in the pPHA11 is functionally active in E. coli and can synthesize PHA in the presence of the A. eutrophus phaA and phaB genes.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 1998년도 제38회 춘계학술발표대회
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• pp.47.1-47.1
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• 1998