[Synthesis of medium-chain-length polyhydroxyalkanoate (mcl PHA) in type I PHA synthase negative mutant of Aeromonas...

Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase gene (phaC) only accumulate copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx), abbreviated as PHBHHx, from lauric acid as sole carbon source but not from glucose. The gene encoding type I PHA synthase was interrupted by insertion of a chloramphenicol resistance gene (Cm). Conjugation of suicide plasmid pFH10 transformed A. hydrophila CGMCC 0911 into a recombinant organism with the disrupted type I PHA synthase gene (phaC:: Cm) , through an in vivo homologous recombination process, type I phaC of A. hydrophila genome was replaced by the disrupted phaC, and Cm gene was integrated into the genome of A. hydrophila, resulting in type I phaC-negative mutant, which was proved by DNA sequencing. Results of GC analysis showed that this mutant could not accumulate PHBHHx again but accumulate medium-chain-length (mcl) PHA from lauric acid or glucose as carbon source, clearly indicating the existence of another type I PHA synthase in the wild type A. hydrophila. It will play its function and accumulate mcl PHA only when type I PHA synthase was inactivated. into the genome of A. hydrophila, resulting in type I phaC-negative mutant, which was proved by DNA sequencing. Results of GC analysis showed that this mutant could not accumulate PHBHHx again but accumulate medium-chain-length (mcl) PHA from lauric acid or glucose as carbon source, clearly indicating the existence of another type II PHA synthase in the wild type A. hydrophila. It will play its function and accumulate mcl PHA only when type I PHA synthase was inactivated.