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

Abstract

Previous studies have demonstrated that heterologous expression of PHA synthase from Aeromonas caviae (PhaCAc), capable of accepting (R)-3-hydroxyacyl-CoA of C4–C7 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 (phaCCn), suggesting the much lower β-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 phaCCn, and the PHA productivity was compared with previous PHB–4-derived strain harboring phaCAc on a multi-copy plasmid (PHB–4/pJRDEE32d13). A strain H16CAc, in which phaCCn was substituted for phaCAc 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, H16ΔC/pJRDEE32d13, that lost region for the original synthase gene and expresses exochromosomal phaCAc, grew and accumulated PHA with similar properties to the PHB–4-derived strain. The results of enzyme assay suggested that low β-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. Key words: poly(hydroxyalkanoates), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), PHA synthase, Cupriavidus necator, vegetable oil.

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

“…It has been established that a single nonsense mutation in phaC1 can dramatically alter PhaA and PhaB activities (23). In our study, deletion of phaB1 increased thiolase activity, likely by increasing expression of phaA.…”

“…Previous work in which PHA biosynthetic genes were overexpressed in R. eutropha indicated that increases in synthase activity do not affect the rate of PHB accumulation (15,17,23). These results imply that the ␤-ketothiolase and/or reductase reactions limit flux through the PHB pathway.…”

Elucidation of β-Oxidation Pathways in Ralstonia eutropha H16 by Examination of Global Gene Expression

“…Palm kernel oil is comprised mostly of lauric (C 12:0 ), myristic (C 14:0 ), and oleic acids (13). R. eutropha has been shown to grow on these oils as carbon sources (32).…”

Polyhydroxyalkanoates: Role of Ralstonia eutropha

“…Wild-type R. eutropha produces short chain length (SCL) PHA like PHB [containing only the 3-hydroxybutyrate (3HB) or 4HB monomer], P (HB-co -HV) [containing 3HB and 3-hydroxyvalerate (3HV) or 4HV monomer]. Due to availability of genetic tools and ease of genetic manipulation of R. eutropha, strains are constructed that can produce short chain length (SCL) PHA and mixed chain length (SCL and MCL) PHA polymers 8,9,10,11 . PHAs are the plastic of the future, bioplastics are more sustainable as they degrade rapidly and synthesized from a renewable resource.…”