[논문]Identification and Analysis of Putative Polyhydroxyalkanoate Synthase (PhaC) in Pseudomonas fluorescens

Lim, Ju Hyoung; Rhie, Ho-Gun; Kim, Jeong Nam

doi:10.4014/jmb.1803.03006

Identification and Analysis of Putative Polyhydroxyalkanoate Synthase (PhaC) in Pseudomonas fluorescens 원문보기

Journal of microbiology and biotechnology, v.28 no.7, 2018년, pp.1133 - 1140

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)

Abstract ▼ AI-Helper

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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제안 방법

1). Confirmation that P. fluorescens KLR101 possesses an intrinsic PHA biosynthesis pathway was carried out by amplification and sequencing analysis of the 540-bp internal region of the phaC g ene encoding PHA synthase [10] (data not shown). Members of the genus Pseudomonas are well-known medium-chainlength (mcl)–PHA producers [27].
Gas chromatographic analysis was performed using an HP-Innowax crosslinked PEG column (60 m × 0.32 mm × 0.25 μm) with the HP 6890 GC system (Agilent, USA).
fluorescens KLR101 was constructed using a Lambda DASH kit (Stratagene, USA). Plaque hybridization was performed with the 573-bp PstI fragment of the phbC gene from Ralstonia eutropha [15] by using a Genius kit (Boehringer Mannheim, USA). A 5.

대상 데이터

Some phaC2 genes have been reported to have their own –35/–10 promoters [33, 35], which is considered as bicistronicity, offering flexibility in response to environmental changes. In order to determine whether or not ORF2 is a gene for PHA production, a functional expression system using the K. aerogenes KC2671 (pUMS) strain, which is unable to produce PHA, was employed. When in vivo expression of the 5.

이론/모형

The enzyme assay was performed based on the off-line Ellman method [22-26] using 0.5 mM DL-β-hydroxybutyryl-CoA (Sigma, USA) as a substrate.

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