Engineering of polyhydroxyalkanoate (PHA) synthase PhaC2Ps of Pseudomonas stutzeri via site-specific mutation for efficient production of PHA copolymers.

The site-specific mutagenesis for PHA synthase PhaC2(Ps1317) from Pseudomonas stutzeri 1317 was conducted for optimizing production of short-chain-length and medium-chain-length polyhydroxyalkanoates (scl-mcl PHA). Recombinant Ralstonia eutropha PHB-4 harboring double mutated phaC2 (Ps1317) gene (phaC2 (Ps) QKST) produced 42 wt.% PHA content in the cell dry weight (CDW) with 93 mol% 3-hydroxybutyrate (HB) as monomer in the PHA copolymer. Compared to that of wild-type phaC2 (Ps1317), the higher PHA content indicated the effectiveness of the specific point mutations for improvement on PhaC2(Ps1317) activity and PHA production. The physical characterization revealed that the PHA produced by the recombinant strain was scl-mcl PHA copolymers with molecular weights and polydispersity reasonable for practical applications. Recombinant R. eutropha PHB-4 containing mutated phaC2 (Ps1317) termed phaC2 (Ps) QKST was demonstrated to be able to produce scl-mcl PHA copolymers consisting of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers covering the carbon chain lengths from C4 to C12 when related substrates were provided. Recombinant R. eutropha PHB-4 containing phaC2PsQKST could be used as a strain for production of copolymers consisting of dominated HB and medium-chain-length 3-hydroxyalkanoates (HA) with better application properties.