title: Discovery of a new polyhydroxyalkanoate synthase from limestone soil through metagenomic approach.
abstract: PHA synthase (PhaC) is the key enzyme in the production of biodegradable plastics known as polyhydroxyalkanoate (PHA). Nevertheless, most of these enzymes are isolated from cultivable bacteria using traditional isolation method. Most of the microorganisms found in nature could not be successfully cultivated due to the lack of knowledge on their growth conditions. In this study, a culture-independent approach was applied. The presence of phaC genes in limestone soil was screened using primers targeting the class I and II PHA synthases. Based on the partial gene sequences, a total of 19 gene clusters have been identified and 7 clones were selected for full length amplification through genome walking. The complete phaC gene sequence of one of the clones (SC8) was obtained and it revealed 81% nucleotide identity to the PHA synthase gene of Chromobacterium violaceum ATCC 12472. This gene obtained from uncultured bacterium was successfully cloned and expressed in a Cupriavidus necator PHB(-)4 PHA-negative mutant resulting in the accumulation of significant amount of PHA. The PHA synthase activity of this transformant was 64 ± 12 U/g proteins. This paper presents a pioneering study on the discovery of phaC in a limestone area using metagenomic approach. Through this study, a new functional phaC was discovered from uncultured bacterium. Phylogenetic classification for all the phaCs isolated from this study has revealed that limestone hill harbors a great diversity of PhaCs with activities that have not yet been investigated.
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.
authors.forename: Yen Teng
authors.initials: YT
authors.lastname: Tai
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.
authors.forename: Choon Pin
authors.initials: CP
authors.lastname: Foong
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.
authors.forename: Nazalan
authors.initials: N
authors.lastname: Najimudin
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia; Centre for Chemical Biology, Universiti Sains Malaysia, 11800 Penang, Malaysia. Electronic address: ksudesh@usm.my.
authors.forename: Kumar
authors.initials: K
authors.lastname: Sudesh
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000302:isolation & purification*; D002119:Calcium Carbonate; D002861:Chromobacterium / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D056186:Metagenomics; D010802:Phylogeny; D012987:Soil / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D012987:Soil; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D002119:Calcium Carbonate
keywords: Genome walking; Limestone; Metagenomic; PHA synthase gene; Polyhydroxyalkanoate; Uncultured bacterium