Effect of poly(beta-hydroxybutyrate) accumulation on the stability of a recombinant plasmid in Escherichia coli

Poly(beta-hydroxybutyrate) (PHB) production using a recombinant Escherichia coli VG1 (pTU14) was carried out. PHB granules were gradually biosynthesized in recombinant cells as inclusion bodies and the morphology of cells became linearly inflated with PHB accumulation from 6 h to 36 h of culture. The stability of plasmid pTU14 was inevitably affected by in-cell PHB accumulation. During repeated subcultures under both PHB-accumulating and non-PHB-accumulating conditions, plasmid DNAs of pTU14 were extracted and analyzed after more than 60 generations. The results showed that plasmid pTU14 is structurally stable but segregationally unstable, and that the segregational instability worsened with increasing accumulation of PHB granules. This is due to not only the serious metabolic burden of plasmid replication and gene expression on the host cell, but also the volume effects of PHB granules on the natural random distribution of plasmids during the binary fission of cells.