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KMID : 0364820160520040455
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Korean Journal of Microbiology
2016 Volume.52 No. 4 p.455 ~ p.462
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Substrate chain-length specificities of polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa P-5
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Woo Sang-Hee
Lee Sun-Hee
Lee Young-Ha
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Abstract
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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 (PhaC1P-5 and PhaC2P-5) by heterologous expression of PhaC1P-5 and PhaC2P-5 genes in Pseudomonas putida GPp104 revealed that PhaC2P-5 incorporates both 3HV and MCL 3HAs into PHA, whereas PhaC1P-5 favors only MCL 3HAs for polymerization. In order to obtain PhaC2P-5 mutants with altered substrate specificity, site-specific mutagenesis for PhaC2P-5 was conducted. Amino acid substitutions of PhaC2P-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 phaC2P-5 gene (phaC2P-5QKST ) was grown on nonanoic acid, 2.5-fold increase of copolymer content with 3.8-fold increase of 3HV fraction was observed. The phaC2P-5QKST-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 phaC2P-5QKST could be useful for production of new PHA copolymers with improved material properties.
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KEYWORD
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Pseudomonas aeruginosa P-5, polyhydroxyalkanoate synthases, site-directed mutagenesis, substrate specificity
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