title: Molecular characterisation of phaCAB from Comamonas sp. EB172 for functional expression in Escherichia coli JM109.
abstract: In this study, PHA biosynthesis operon of Comamonas sp. EB172, an acid-tolerant strain, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA(Co) gene, 1182 bp), acetoacetyl-CoA reductase (phaB(Co) gene, 738 bp) and PHA synthase, class I (phaC(Co) gene, 1694 bp) were identified. Sequence analysis of the phaA(Co), phaB(Co) and phaC(Co) genes revealed that they shared more than 85%, 89% and 69% identity, respectively, with orthologues from Delftia acidovorans SPH-1 and Acidovorax ebreus TPSY. The PHA biosynthesis genes (phaC(Co) and phaAB(Co)) were successfully cloned in a heterologous host, Escherichia coli JM109. E. coli JM109 transformants harbouring pGEM'-phaC(Co)AB(Re) and pGEM'-phaC(Re)AB(Co) were shown to be functionally active synthesising 33 wt.% and 17 wt.% of poly(3-hydroxybutyrate) [P(3HB)]. E. coli JM109 transformant harbouring the three genes from the acid-tolerant Comamonas sp. EB172 (phaCAB(Co)) under the control of native promoter from Cupriavidus necator, in vivo polymerised P(3HB) when fed with glucose and volatile mixed organic acids (acetic acid:propionic acid:n-butyric acid) in ration of 3:1:1, respectively. The E. coli JM109 transformant harbouring phaCAB(Co) could accumulate P(3HB) at 2g/L of propionic acid. P(3HB) contents of 40.9% and 43.6% were achieved by using 1% of glucose and mixed organic acids, respectively.
authors.affiliation: Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Lian-Ngit
authors.initials: LN
authors.lastname: Yee
authors.forename: Jo-Ann
authors.initials: JA
authors.lastname: Chuah
authors.forename: Mei-Ling
authors.initials: ML
authors.lastname: Chong
authors.forename: Lai-Yee
authors.initials: LY
authors.lastname: Phang
authors.forename: Abdul Rahim
authors.initials: AR
authors.lastname: Raha
authors.forename: Kumar
authors.initials: K
authors.lastname: Sudesh
authors.forename: Mohd Ali
authors.initials: MA
authors.lastname: Hassan
mesh_terms: D000101:Acetyl-CoA C-Acetyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D020585:Comamonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon; D011091:Polyesters / Q000378:metabolism; D016415:Sequence Alignment; D000094122:Polyhydroxybutyrates
chemical_list: D000101:Acetyl-CoA C-Acetyltransferase; D000217:Acyltransferases; D000429:Alcohol Oxidoreductases; D001426:Bacterial Proteins; D005947:Glucose; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C023744:acetoacetyl-CoA reductase; C077225:poly(3-hydroxyalkanoic acid) synthase