The potential of plant oils as sole carbon sources for production of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction using the recombinant Cupriavidus necator strain Re2160/pCB113 has been investigated. Various types and concentrations of plant oils were evaluated for efficient conversion of P(3HB-co-3HHx) copolymer. Crude palm kernel oil (CPKO) at a concentration of 2.5 g/L was found to be most suitable for production of copolymer with a 3HHx content of approximately 70 mol%. The time profile of these cells was also examined in order to study the trend of 3HHx monomer incorporation, PHA production and PHA synthase activity. (1)H NMR and (13)C NMR analyses confirmed the presence of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction, in which monomers were not randomly distributed. The results of various characterization analyses revealed that the copolymers containing a high 3HHx monomer fraction demonstrated soft and flexible mechanical properties.
The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.