Pseudomonassp. phDV1 is a polyhydroxyalkanoate (PHA) producer. The presence of the endogenous PHA depolymerase (phaZ) responsible for the degradation of the intracellular PHA is one of the main shortages in the bacterial production of PHA. Further, the production of PHA can be affected by the regulatory protein phaR, which is important in accumulating different PHA-associated proteins. PHA depolymerase phaZ and phaR knockout mutants ofPseudomonassp. phDV1 were successfully constructed. We investigate the PHA production from 4.25 mM phenol and grape pomace of the mutants and the wild type. The production was screened by fluorescence microscopy, and the PHA production was quantified by HPLC chromatography. The PHA is composed of Polydroxybutyrate (PHB), as confirmed by1H-nuclear magnetic resonance analysis. The wildtype strain produces approximately 280 μg PHB after 48 h in grape pomace, while the phaZ knockout mutant produces 310 μg PHB after 72 h in the presence of phenol per gram of cells, respectively. The ability of the phaZ mutant to synthesize high levels of PHB in the presence of monocyclic aromatic compounds may open the possibility of reducing the costs of industrial PHB production.