Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaC AR for the Biosynthesis of Poly[( R )-3-hydroxybutyrate- co -6-hydroxyhexanoate].

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaC _AR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaC _AR were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P(3HB- co -6HHx) synthesis in Escherichia coli . As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P(3HB- co -6HHx) decreased as the 6HHx fraction increased.