Cloning and characterization of the Pseudomonas sp. 61-3 phaG gene involved in polyhydroxyalkanoate biosynthesis

Pseudomonas sp. 61-3 produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] random copolymer consisting of monomeric units of 4-12 carbon atoms from sugars. The phaG(Ps) gene encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase was cloned from this strain, and homologous expression of this gene under the control of the lac or the native promoter was investigated. Additional copies of the phaG(Ps) gene in Pseudomonas sp. 61-3 led to an increase in both the polyhydroxyalkanoate (PHA) content in the cells and the fraction of medium-chain-length 3HA units in PHA. Disruption of the chromosomal phaG(Ps) gene resulted in an increase in the fraction of the 3HB unit in PHA. The site-directed mutagenesis of the phaG(Ps) gene was carried out to investigate the role of a HX(4)D motif which has been proposed to be related to PhaG activity.