• 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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• 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.

• Journal of Microbiology and Biotechnology
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• 제18권7호
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• pp.1290-1297
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• 2008

To investigate the diversities of Accumulibacter phosphatis and its polyhydroxyalkanoate (PHA) synthase gene (phaC) in enhanced biological phosphorus removal (EBPR) sludge, an acetate-fed sequencing batch reactor was operated. Analysis of microbial communities using fluorescence in situ hybridization and 16S rRNA gene clone libraries showed that the population of Accumulibacter phosphatis in the EBPR sludge comprised more than 50% of total bacteria, and was clearly divided into two subgroups with about 97.5% sequence identity of the 16S rRNA genes. PAO phaC primers targeting the phaC genes of Accumulibacter phosphatis were designed and applied to retrieve fragments of putative phaC homologs of Accumulibacter phosphatis from EBPR sludge. PAO phaC primers targeting $G_{1PAO},\;G_{2PAO},\;and\;G_{3PAO}$ groups produced PCR amplicons successfully; the resulting sequences of the phaC gene homologs were diverse, and were distantly related to metagenomic phaC sequences of Accumulibacter phosphatis with 75-98% DNA sequence identities. Degenerate NPAO (non-PAO) phaC primers targeting phaC genes of non-Accumulibacter phosphatis bacteria were also designed and applied to the EBPR sludge. Twenty-four phaC homologs retrieved from NPAO phaC primers were different from the phaC gene homologs derived from Accumulibacter phosphatis, which suggests that the PAO phaC primers were specific for the amplification of phaC gene homologs of Accumulibacter phosphatis, and the putative phaC gene homologs by PAO phaC primers were derived from Accumulibacter phosphatis in the EBPR sludge. Among 24 phaC homologs, a phaC homolog (GINPAO-2), which was dominant in the NPAO phaC clone library, showed the strongest signal in slot hybridization and shared approximately 60% nucleotide identity with the $G_{4PAO}$ group of Accumulibacter phosphatis, which suggests that GINPAO-2 might be derived from Accumulibacter phosphatis. In conclusion, analyses of the 16S rRNA and phaC genes showed that Accumulibacter phosphatis might be phylogenetically and metabolically diverse.

• 미생물학회지
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• 제46권2호
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• pp.219-222
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• 2010

본 연구는 장티푸스 환자에 대한 혈청학적 시험법인 Vi-PHA, $SD^{(R)}$ kit, Widal 시험(O & H)에 대하여 평가를 위해 수행하였다. 2005년부터 2006년까지 수집된 36건의 혈청검체를 대상으로 하였으며, 9건은 배양검사를 통하여 장티푸스균이 검출된 확진환자의(Typhoid fever) 검체이며, 27건은 비장티푸스성 열성환자의(Non-typhoid fever) 검체이다. 시험결과 Vi-PHA는 환자 혈청 9건 중 양성 8건으로 민감도 88.9% (P<0.001; Fisher's exact test), 비장티푸스성 열성환자 27건 중 1건이 양성을 보여 특이도 96.3%로 나타났다. $SD^{(R)}$ kit는 민감도 100% (P<0.001), 특이도 92.6%로 나타났다. Widal (O & H) 시험의 민감도는 각각 88.9% (P=0.001), 100% (P<0.001), 특이도는 77.8%, 70.4%로 나타났다. 민감도는 $SD^{(R)}$ kit와 Widal H 검사법이 가장 높은 것으로 확인되었으며, 특이도는 Vi-PHA가 가장 높게 나타났다. 장티푸스의 효과적인 혈청학적 진단을 위하여 급성 또는 유행지역에서는 특이도가 높은 Vi-PHA로 검사를 수행하고, 장티푸스 비 유행지역과 고위험군에 대하여는 민감도가 높은 Widal H와 $SD^{(R)}$ kit를 적용하는 것이 진단의 유의수준을 높일 수 있을 것으로 판단된다.

• Korean Chemical Engineering Research
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• 제55권3호
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• pp.363-368
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• 2017

메탄올자화균이란 일탄소 화합물인 메탄올을 주탄소원 및 에너지원으로 이용할 수 있는 미생물을 말한다. Methylobacterium extorquens AM1은 serine cycle을 탄소대사경로로 이용하는 메탄올자화균 중에서도 가장 많이 연구가 진행된 균주이다. M. extorquens AM1의 poly 3-hydroxybutyrate (PHB) cycle은 EMCP (ethylmalonyl-CoA pathway), glyoxylate regeneration cycle, TCA cycle과 연결되어 있으며 EMCP 유래 유기산 또는 TCA 유기산을 생산하기 위해서는 PHB cycle로 흐르는 carbon flux의 차단이 필요하다. 이를 위해서 PHB 합성과 acetyl-CoA flux의 조절유전자로 알려져 있는 PhaR 유전자를 markerless gene deletion 방법을 이용해서 M. extorquens AM1에서 knockout했다. 결과적으로, knockout 균주인 ${\Delta}phaR$에서 야생종 대비 확연히 PHB granule이 줄어든 것이 확인되었다. Lag phase가 약 12 h 늦어졌지만, ${\Delta}phaR$은 야생종과 비슷한 세포성장과 메탄올소비 경향을 보임을 확인하였다.

• 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.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.771-774
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• 2001

NADPH has been known as a regulating factor the biosynthesis of polyhydroxyalkanote(PHA), and the flux of NADPH for PHA biosynthesis could be enforced by the amplification of zwf gene encoding glucose 6-phosphate dehydrogenase. The recombinant plasmid pCZWF harboring PHA synthase, phbC from R. eutropha and zwf from E. coli were constructed, and were transformed to R. eutropha by electroporation. The biosynthesis of P(3HB-3HV) copolymer were carried out in transformant R. eutropha through the two-stage cultivation method using valerate as a precursor. The biosynthesis rate and PHA content of transformant R. eutropha harboring pCZWF were increased compared with transformant R. eutropha harboring only phbC. Especially, the molar fraction of 3HV was increased from 68% to 74% due to amplification of zwf gene. And the biosynthesis P(3HB-3HV) and P(3HB-4HB) carried out using propionate and ${\gamma}-butyrolactone$ as a precursor, respectively. But the rate, content, and molar fraction of biosynthesis copolymers were not influenced appreciably. This may be due to the reduced availability of NADPH.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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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.

• 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.

• KSBB Journal
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• 제25권3호
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• pp.239-243
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• 2010

식물성 오일 또는 glycerol로부터 Ralstonia eutropha 여섯 균주의 polyhydroxyalkanoates (PHA) 합성 특성을 조사하였다. 탄소원으로 soybean oil, olive oil, 또는 glycerol 만을 공급시 poly(3-hydroxybutyrate) homopolymer가 생성되었으며, $\gamma$-butyrolactone 또는 pentanoic acid를 함께 공급시 poly(3-hydroxybutyrate-co-4-hydroxybutyrate) 또는 poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer가 각각 합성되었다. 최종 균체농도 및 PHA 함량면에서 최적인 균주는 R. eutropha KCTC 2662로 결정되었으며, 최적 기질인 soybean oil 20 g/L로부터 72 h에 균체농도 1.7~9.2 g/L, PHA 함량 70~92 wt%를 얻을 수 있었다.