In vivo assay system of the polymerase as a key enzyme for PHA biosynthesis

Polymerase is a key enzyme involved in the biosynthesis of polyhydroxybutyrate (PHB), a well-known bacterial biodegradable polyester. In this study, we have established an in vivo assay system to analyze mutational effects of Ralstonia eutropha polymerase (termed PhbCRe) on the level of PHB accumulation in recombinant strains of Escherichia coli. This in vitro evolution system consists of polymearse chain reaction (PCR) random mutagenesis and two assay procedures, plate assay using a PHB staining dye and high-pressure liquid chromatographic assay based on the conversion reaction of PHB to crotonic acid. The distribution pattern of the PHB accumulation level of the mutant population using 378 clones arbitrarily selected, suggested that the present level of PhbCRe is high and well optimized. It is noteworthy that many of the amino acid substitutions affecting the PHB accumulation occurred in the conserved positions or regions within an ‘ / hydrolase fold’ which is commonly found among hydrolytic enzymes. Based on the good correlation with the level of PHB accumulation, activity estimation of the PhbCRe could be efficiently achieved by monitoring the level of PHB accumulation using the in vivo assay system established here.