Role of phaD in accumulation of medium-chain-length poly(3-hydroxyalkanoates) in Pseudomonas oleovorans

Pseudomonas oleovorans is capable of producing poly(3-hydroxyalkanoates) (P HAs) as intracellular storage material. To analyze the possible involvement of phaD in medium-chain-length (MCL) PHA biosynthesis, we generated a phaD knockout mutant by homologous recombination, Upon disruption of the phaD g ene, MCL PHA polymer accumulation was decreased. The PHA granule size was r educed, and the number of granules inside the cell was increased, Furthermo re, mutant cells appeared to be smaller than wild-type cells. Investigation of MCL PHA granules revealed that the pattern of granule-associated protei ns was changed and that the predominant protein PhaI was missing in the mut ant. Complementation of the mutant with a phaD-harboring plasmid partially restored the wild-type characteristics of MCL PHA production and fully rest ored the granule and cell sizes. Furthermore, PhaI was attached to the gran ules of the complemented mutant. These results indicate that the phaD gene encodes a protein which plays an important role in MCL PEW biosynthesis. Ho wever, although its main effect seems to be the stabilization of MCL PHA gr anules, we found that the PhaD protein is not a major granule-associated pr otein and therefore might act by an unknown mechanism involving the PhaI pr otein.