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 PhaCCs 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.

Mutations Derived from the Thermophilic Polyhydroxyalkanoate Synthase PhaC Enhance the Thermostability and Activity of PhaC from Cupriavidus necator H16

“…strain USM2, PhaC Cs , which revealed a homology of 46% with PhaC H16 and exhibited a specific activity of 238 Ϯ 98 U/mg at 30°C (3). This is the highest specific activity reported for a purified PHA synthase (3). In this study, the chimera PhaC H16␤ has a specific activity of 172 Ϯ 17.8 U/mg at 30°C.…”

Heterologous Expression of Various PHA Synthase Genes in Rhodospirillum rubrum

“…In these studies, the heterologous expression of pBBR1MCS-2::phaC C. sp in the PHB negative strain R. eutropha PHB -4 resulted in a maximum PHB accumulation, and in vivo enzyme activity tests showed a high activity of 2462 ± 80 U g -1 . This was about 8-fold higher than the PHA synthase activity from R. eutropha H16 46 . Additionally, the heterologous expression of phaC C. sp.…”

“…This PHA synthase was studied intensively in previous studies 45,46 . In these studies, the heterologous expression of pBBR1MCS-2::phaC C. sp in the PHB negative strain R. eutropha PHB -4 resulted in a maximum PHB accumulation, and in vivo enzyme activity tests showed a high activity of 2462 ± 80 U g -1 .…”

Effect of Storage Time and Concentration of Used Cooking Oil on Polyhydroxyalkanoates (PHAs) Production by Cupriavidus necator H16

“…Polyhydroxyalkanoates (PHAs) are biopolymers that has gained widespread interest, since their properties are similar to petrochemical plastics (Bhubalan et al 2011). They can be synthesized by a wide range of different grampositive and gram-negative bacteria.…”