Advanced Strategies for Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Production: PHA Synthase Homologous Overexpression in the Extremophile Haloferax mediterranei

Bioplastics such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are promising alternatives to conventional plastics. However, the high production cost limits their industrial application. In this study, PHBV production was optimized in <i>Haloferax mediterranei</i> by the homologous overexpression of the key enzyme PHA synthase (PhaEC), resulting in the OE<i>phaEC</i> strain. The growth and PHBV production of OE<i>phaEC</i> compared with the parental strain (HM26) were evaluated in three culture media with different nitrogen sources (KNO₃, NH₄Cl, and casamino acids). The OE<i>phaEC</i> strain exhibited a 20% increase in PHBV production and a 40% increase in 3-hydroxyvalerate monomer (3HV) content in a defined medium with nitrate as a nitrogen source, as determined by GC-MS. Moreover, enzyme activity, measured spectrophotometrically, increased from 2.3 to 3.9 U/mg. Soluble and insoluble protein fractions were analysed to assess the overexpression of PHA synthase. Only PhaE was found in the insoluble protein fraction, where PHBV granules accumulate. Transmission electron microscopy (TEM) images confirmed a higher PHBV content in OE<i>phaEC</i> compared to the parental strain. These results demonstrate that the homologous overexpression of the key enzyme implicated in PHBV biosynthesis can enhance PHBV content, making its production competitive for industrial applications.