Production of P(3HB-co-3HHx) with Controlled Compositions by Recombinant Cupriavidus necator Re2058/pCB113 from Renewable Resources

Mixtures of crude palm kernel oil (CPKO) and oil palm tree trunk sap (OPTS) as carbon sources for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) by the recombinant Cupriavidus necator strain Re2058/pCB113 were investigated. Shake flask cultures using CPKO as the sole carbon source produced 5.82 g/L of cell dry weight (CDW) with 73 wt% of PHA content and 44 mol% of 3-hydroxyhexanoate (3HHx). When the total CPKO concentration in the cultures was increased from 2.5 to 7.5 g/L, a general pattern of decreasing 3HHx molar fraction (from 42 to 27 mol%) was observed in the resulting copolymer. In order to produce biopolymer with properties that resemble commodity plastics, the 3HHx molar fraction should be lowered to an optimal level of similar to 12-20 mol%. The lowest 3HHx molar fraction (14 mol%) was obtained when a mixture of 4% (w/v) OPTS sugars and 1.5 g/L CPKO was used as the carbon source. The highest molar fraction of 3HHx produced (42 mol%) was observed when the cells were cultivated in 5 g/L of CPKO. H-1-NMR and C-13-NMR analyses confirmed the structure and monomer fraction of the different P(3HB-co-3HHx) polymers. This study has established a method to control the monomer molar fractions in P(3HB-co-3HHx) produced using a mixture of CPKO and sugars from OPTS.