title: Cloning and Characterisation of (R)-3-hydroxyacyl-acyl Carrier Protein-coenzyme A Transferase Gene (phaG) from Pseudomonas sp. USM 4-55.
abstract: The (R)-3-hydroxyacyl-ACP-CoA transferase catalyses the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA derivatives, which serves as the ultimate precursor for polyhydroxyalkanoate (PHA) polymerisation from unrelated substrates in pseudomonads. PhaG was found to be responsible for channelling precursors for polyhydroxyalkanoate (PHA) synthase from a de novo fatty acid biosynthesis pathway when cultured on carbohydrates, such as glucose or gluconate. The phaG gene was cloned from Pseudomonas sp. USM 4-55 using a homologous probe. The gene was located in a 3660 bp Sal I fragment (GenBank accession number EU305558). The open reading frame (ORF) was 885 bp long and encoded a 295 amino acid protein. The predicted molecular weight was 33251 Da, and it showed a 62% identity to the PhaG of Pseudomonas aeruginosa. The function of the cloned phaG of Pseudomonas sp. USM 4-55 was confirmed by complementation studies. Plasmid pBCS39, which harboured the 3660 bp Sal I fragment, was found to complement the PhaG-mutant heterologous host cell, Pseudomonas putida PhaGN-21. P. putida PhaGN-21, which harboured pBCS39, accumulated PHA that accounted for up to 18% of its cellular dry weight (CDW). P. putida PhaGN-21, which harboured the vector alone (PBBR1MCS-2), accumulated only 0.6% CDW of PHA.
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia.
authors.forename: Hasni
authors.initials: H
authors.lastname: Arsad
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia.
authors.forename: Kumar
authors.initials: K
authors.lastname: Sudesh
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia.
authors.forename: Najimudin
authors.initials: N
authors.lastname: Nazalan
authors.affiliation: Department of Biological Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia.
authors.forename: Tengku Sifzizul Tengku
authors.initials: TS
authors.lastname: Muhammad
authors.affiliation: School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia.
authors.forename: Habibah
authors.initials: H
authors.lastname: Wahab
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia.
authors.forename: Mohd
authors.initials: M
authors.lastname: Razip Samian
keywords: Polyhydroxyalkanoate; Pseudomonas sp. USM-455; phaG