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

Abstract

Polyhydroxyalkanoates are attractive alternatives to traditional plastics. However, although polyhydroxybutyrate (PHB) is produced in large quantities by <i>Cupriavidus necator</i> 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-<i>co</i>-3-hydroxypropionate) (poly(3HB-<i>co</i>-3HP). To achieve this, a pathway was designed to enable <i>C. necator</i> H16 to convert β-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 <i>Chromobacterium</i> 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 β-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 <i>phaA</i> and <i>phaB</i>1 genes of the <i>phaCAB</i> operon on 3HP molar fraction were investigated. A <i>phaAB1</i> double knockout resulted in a copolymer containing 91 mol % 3HP without the need for cysteine supplementation.