Development and validation of an HPLC-based screening method to acquire polyhydroxyalkanoate synthase mutants with altered substrate specificity

A rapid and convenient method for the compositional analysis of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC) and alkaline sample pretreatment in a 96-well plate format. The reliability of this system was confirmed by the fact that a mutant with a D171G mutation of Aeromonas caviae PHA synthase (PhaCAc), which gained higher reactivity toward 3-hydroxyhexanoate (3HHx), was selected from the D171X mutant library. Together with D171G mutant, several single mutants showing high reactivity toward 3HHx were isolated by the HPLC assay. These new mutants and double mutants combined with an N149S mutation were used to synthesize P(3-hydroxybutyrate-co-3HHx) in Ralstonia eutropha PHB−4 from soybean oil as carbon source, achieving higher levels of 3HHx fraction than the wild-type enzyme. Based on these results, the high-throughput screening system will serve as a powerful tool for exploring new and beneficial mutations responsible for regulating copolymer composition of PHA.