Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Identification and Analysis of Putative Polyhydroxyalkanoate Synthase (PhaC) in Pseudomonas fluorescens

  • Lim, Ju Hyoung (Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST)) ;
  • Rhie, Ho-Gun (Department of Biology, Kyung Hee University) ;
  • Kim, Jeong Nam (Department of Microbiology, College of Natural Sciences, Pusan National University)
  • Received : 2018.03.08
  • Accepted : 2018.05.01
  • Published : 2018.07.28
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Abstract

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.

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References

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