Characterization and Properties of G4X Mutants of Ralstonia eutropha PHA Synthase for Poly(3‐hydroxybutyrate) Biosynthesis in Escherichia coli

Abstract

Modification of the type I polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC(Re)) was performed through systematic in vitro evolution in order to obtain improved PhaC(Re) having an enhanced activity of poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli. For the first time, a beneficial G4D N-terminal mutation important for the enhancement of both PHB content in dry cells and PhaC(Re) level in vivo was identified. Site-directed saturation mutagenesis at the G4 position enabled us to identify other mutations conferring similar enhanced characteristics. In addition, the PHB homopolymer synthesized by most G4X single mutants also had higher molecular weights than that of the wild-type. In vitro enzymatic assays of purified G4D mutant PhaC(Re) revealed that the mutant enzyme exhibited slightly lower activity and reaction efficiency compared to the wild-type enzyme. [diagram in text].

Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

“…PhaC Ac was eluted with 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. His-tagged PhaC Re and PhaC Da were expressed in the recombinant Escherichia coli BL21(DE3) strain (Novagen, Madison, WI) with the pET-15b::phaC Re and pET-15b::phaC Da vectors, respectively (16,17). PhaC Re and PhaC Da were purified from the cells using a HisTrap HP column (GE Healthcare), followed by desalting using a HiTrap desalting column with desalting buffer [20 mM Tris-HCl, 150 mM NaCl, 5% (vol) glycerol, and 0.05% (wt) 6-O-(N-heptylcarbamoyl)methyl ␣-D-glucopyranoside (Hecameg), pH 7.5].…”

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

“…For example, in the PHA synthase of C. necator H16 (PhaC H16 ), a G4D mutation increases the protein expression level of PHA synthase (26). An F420S mutation enhances the specific activity of PHA synthase (41).…”

“…An F420S mutation enhances the specific activity of PHA synthase (41). A G4D or F420S mutation results in more PHB accumulation in recombinant E. coli (26,41). Another beneficial mutation, A510D(E), leads PhaC H16 to synthesize a higher-molecular-weight polymer (43).…”

A single-nucleotide substitution in phasin gene leads to enhanced accumulation of polyhydroxyalkanoate (PHA) in <i>Escherichia coli</i> harboring <i>Aeromonas caviae</i> PHA biosynthetic operon

“…Taken together, our observations indicate that the enhanced PHA accumulation could be attributed to the efficient expression of the phaPCJ operon, presumably due to change(s) in transcriptional levels and/or the stability of mRNA transcripts (Normi et al, 2005).…”