title: Characterization of the highly active polyhydroxyalkanoate synthase of Chromobacterium sp. strain USM2.
abstract: The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.
authors.forename: Kesaven
authors.initials: K
authors.lastname: Bhubalan
authors.forename: Jo-Ann
authors.initials: JA
authors.lastname: Chuah
authors.forename: Fumi
authors.initials: F
authors.lastname: Shozui
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Brigham
authors.forename: Seiichi
authors.initials: S
authors.lastname: Taguchi
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Sinskey
authors.forename: Chokyun
authors.initials: C
authors.lastname: Rha
authors.forename: Kumar
authors.initials: K
authors.lastname: Sudesh
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002208:Caproates / Q000378:metabolism; D002861:Chromobacterium / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010421:Pentanoic Acids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D002208:Caproates; D004269:DNA, Bacterial; D010421:Pentanoic Acids; D011994:Recombinant Proteins; C045051:3-hydroxyhexanoic acid; C013056:beta-hydroxyvaleric acid; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D020155:3-Hydroxybutyric Acid