• 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.22 no.8
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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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• v.13 no.2
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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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• 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.

• KSBB Journal
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• v.11 no.2
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• pp.201-210
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• 1996

The production of polyhydroxyalkanoate(PHA) from glucose by batch and fed-batch culture of Pseudomonas sp. HJ was studied. In batch culture using fermentor, 400 rpm of agitalion speed, 2 vvm of aeration rate, 18 hours of inoculum age, and 5% (vlv) of inoculum size were optimal. PHA production was not increased by deficiency of oxygen. In a batch culture, the final call mass was $6.251g/\ell$, and PHA content was 20% of dry cell weight. In a constant feeding fed-batch culture, cell mass increased to $33.24g/\ell$, and PHA content reached 48.9% of dry cell weight. In an intermittent feeding fed-batch culture, cell mass increased to $37.89g/\ell$, and PHA content reached 53.5% of dty cell weight.

• Korean Journal of Microbiology
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• v.36 no.2
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• pp.155-160
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• 2000

Pseudomnas sp. HJ-2 is capable of producing a rubber-elastic polyhydroxyalkanoate (PHA) consisting of 3- hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyheptanoate (3HHp) from heptanoic acid as the sole carbon source. The polyester produced was a blend of poly(3HB-co-3HV) and poly(3HHp). Although the mixing of poly(3HHp) fraction to poly(3HB-co-3HV) resulted in a decrease of modulus, the sole fraction of poly(3HB-co-3HV) with a high molar fraction of 3HV was shown to be an elastomer with the maximum percent strain of 740%. The biomass yield and the PHA synthesis were relatively high when the initial heptanoic acid concentration was 40 mM, and were significantly decreased when the substrate concentration exceeded 50 mM. The accumulation of PHA was stimulated by deficiency of nitrogen and phosphorus in the medium. The PHA contents and its monomeric compositions were greatly affected by pH and oxygen transfer rate. At pH 7.5, poly(3HB-~0.38% 3HV) was produced from heptanoic acid and a mixture of 95% 3HHp and 5% 3HV was produced at pH 8.0. Increased conten1 of 3HHp in the polyesters with lhe increasing oxygen transfer rate by agitation speed a1 a fixed aeration rate was observed.

• 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.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.144-151
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• 2002

For elucidating the relationship between the biosynthetic pathways for polyhydroxyslkanoates (PHAs) and 5-aminolevulinic acid (ALA), culture conditions for the production of these two biomaterials by Rhodopseudomonas sp. KCTC 1437 were investigated. Of the carbon substrates tested, acetic acid was the best carbon source for cell growth and PHA biosynthesis. When succinic acid was added as a co-substrate into culture medium, cell growth and PHA production were greatly increased up to 2.5 g/ι and 73% of dry cell weight, respectively. The PHA obtained from the carbon substrates tested was homopolyester of 3-hydroxybutyrate, while valeric acid was only effective for the production of copolyester consisting of 3-hydroxybutyrate and 3-hydroxyvalerate. Anaerobic light culture condition was better for PHA production and cell growth than anaerobic dark or aerobic dark culture condition. The organism was capable of synthesizing ALA when glycine and succinic acid were added to the culture medium. ALA was produced to ca.400 mg/ι when levulinic acid, soccinic acid, and glycine were repeatedly added with a reductant (sodim thioglycolate). However, the presence of glycine, levulinic acid and sodium glycolate inhibited the cell growth and the conversion of carbon substrates to PHA. From these results it is apparent that the production yields of PHA and ALA could not be increased simultaneously because the optimal conditions for the production of PHA and ALA are opposed to each other.

• The Microorganisms and Industry
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• v.18 no.1
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• pp.23-27
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• 1992

전분이 함유된 플ㄹ스틱 및 광분해성 플라스틱은 제외하고 순수하게 생분해되는 생분해성 고분자의 시장은 세계적으로 년간 140만톤으로 추정되고 있다. 이 중에서 생물공학과 직접적으로 관계가 있는 것은 polylactide계 polymer의 원료가 되는 lactic acid, pullulan과 같은 polysaccharide 그리고 PHA와 같은 polyester등으로서 본고에서는 PHA(polyhydroxyalkanoate)를 중심으로 생산공정이 어떻게 개발되어 오고 있는가 간단히 고찰하고자 한다. 이러한 생산공정의 원리는 pullulan 및 xanthan gum과 같은 타 생물고분자의 경우에도 유사하게 적용될 수 있다.

• Korean Journal of Microbiology
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• v.52 no.4
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• pp.455-462
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• 2016

Pseudomonas aeruginosa P-5 is an unusual organism capable of synthesizing polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyvalerate (3HV) and medium-chain-length (MCL) 3-hydroxyalkanoate (3HA) monomer units when C-odd alkanoic acids are fed as the sole carbon source. Evaluation of the substrate chain-length specificity of two P. aeruginosa P-5 PHA synthases ($PhaC1_{P-5}$ and $PhaC2_{P-5}$) by heterologous expression of $PhaC1_{P-5}$ and $PhaC2_{P-5}$ genes in Pseudomonas putida GPp104 revealed that $PhaC2_{P-5}$ incorporates both 3HV and MCL 3HAs into PHA, whereas $PhaC1_{P-5}$ favors only MCL 3HAs for polymerization. In order to obtain $PhaC2_{P-5}$ mutants with altered substrate specificity, site-specific mutagenesis for $PhaC2_{P-5}$ was conducted. Amino acid substitutions of $PhaC2_{P-5}$ at two positions (Ser326Thr and Gln482Lys) were very effective for synthesizing copolymers with a higher 3HV fraction. When recombinant P. putida GPp104 harboring double mutated $phaC2_{P-5}$ gene ($phaC2_{P-5}QKST$) was grown on nonanoic acid, 2.5-fold increase of copolymer content with 3.8-fold increase of 3HV fraction was observed. The $phaC2_{P-5}QKST$-containing Ralstonia eutropha PHB-4 supplemented with valeric acid also produced copolymers consisting of 3HV and 3-hydroxyheptanoate with a high 3HV fraction. These results suggest that recombinants containing $phaC2_{P-5}QKST$ could be useful for production of new PHA copolymers with improved material properties.