Biosynthesis of Poly(3HB-co-3HP) with Variable Monomer Composition in Recombinant Cupriavidus necator H16

Polyhydroxyalkanoates are attractive alternatives to traditional plastics. However, although polyhydroxybutyrate (PHB) is produced in large quantities by Cupriavidus necator H16, its properties are far from ideal for the manufacture of plastic products. These properties may be improved through its coproduction with 3-hydroxypropionate (3HP), which leads to the formation of the copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (poly(3HB-co-3HP). To achieve this, a pathway was designed to enable C. necator H16 to convert beta-alanine to 3HP. The initial low levels of incorporation of 3HP into the copolymer were overcome by the overproduction of the native propionyl-CoA transferase together with PHA synthase from Chromobacterium sp. USM2. Following optimization of 3HP incorporation into the copolymer, the molar fraction of 3HP could be controlled by cultivation in medium containing different concentrations of beta-alanine. Between 0 and 80 mol % 3HP could be achieved. Further supplementation with 2 mM cysteine increased the maximum 3HP molar fraction to 89%. Additionally, the effect of deletions of the phaA and phaB1 genes of the phaCAB operon on 3HP molar fraction were investigated. A phaAB1 double knockout resulted in a copolymer containing 91 mol % 3HP without the need for cysteine supplementation.