Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil

Previous studies have demonstrated that heterologous expression of PHA synthase from Aeromonas caviae (PhaC(Ac)), capable of accepting (R)-3-hydroxyacyl-CoA of C(4)-C(7) as substrates, could confer the ability to PHA-negative mutant of Cupriavidus necator PHB(-)4 to synthesize poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from vegetable oils. The mutation point within pha operon in PHB(-)4 was determined to be a single nonsense mutation within the PHA synthase gene (phaC(Cn)), suggesting the much lower beta-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase activities observed in this strain would be a polar effect of the mutation. For further efficient biosynthesis of P(3HB-co-3HHx) copolyester, C. necator wild strain H16 was engineered by homologous recombination targeting the chromosomal phaC(Cn), and the PHA productivity was compared with previous PHB(-)4-derived strain harboring phaC(Ac) on a multi-copy plasmid (PHB(-)4/pJRDEE32d13). A strain H16C(Ac), in which phaC(Cn) was substituted for phaC(Ac) on the chromosome, could produce P(3HB-co-3HHx) from soybean oil with high productivity, but the 3HHx fraction in the accumulated polymer was decreased. Meanwhile, H16 Delta C/pJRDEE32d13, that lost region for the original synthase gene and expresses exochromosomal phaC(Ac), grew and accumulated PHA with similar properties to the PHB(-)4-derived strain. The results of enzyme assay suggested that low beta-ketothiolase activity might be relevant for decrease of growth ability accompanied by increase of 3HHx composition when soybean oil was fed as a sole carbon source.