Using runaway replication to express polyhydroxyalkanoic acid (pha) genes from a novel marine bacterium in enteric bacteria: The influence of temperature and phasins on PHA accumulation

While plastics have revolutionized our world, plastic waste has serious environmental and economic impacts. Polyhydroxyalkanoic acid (PHA) is a bacterial carbon and energy reserve shown to be both biodegradable and biocompatible and could potentially replace conventional plastics. However, cost-effective mass production remains elusive. Bacteria often accumulate PHA as cytoplasmic granules. PHA synthase creates the PHA polymer from acetoacyl-CoA monomers, while phasins are small multifunctional proteins that are found in abundance on the granule surface. The PHA synthase gene from a novel marine isolate, Vibrio B-18 (or B-18), was placed in the presence or absence of an upstream phasin gene in a runaway replication plasmid using polymerase chain reaction (PCR) technology. Plasmid gene expression may be induced chemically or thermally. Overexpression of the PHA genes was demonstrated by SDS-PAGE analysis, and microscopy was used to detect PHA accumulation in three different enteric bacteria (Escherichia coli, Klebsiella aerogenes, and Shigella flexneri). While the B-18 genes were clearly overexpressed at 41°C, PHA accumulation occurred more readily at the lower (30°C) non-inducing temperature regardless of chemical induction if the phasin gene was present. A mutational analysis confirmed the identity of the start codon for the PHA synthase gene and provided evidence supporting the requirement for phasins to allow for PHA accumulation in the recombinant hosts. The findings described in this study confirm the conclusions obtained from related studies from other laboratories and lend support to the importance of including a phasin gene in addition to the basic genes needed for PHA synthesis and accumulation in recombinant enteric bacteria, such as Escherichia coli, Klebsiella aerogenes, and Shigella flexneri